New roles for an old cytokine : characterizing how exposure to Il-12 alters human CD4 And CD8 T cell responses

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1 University of Iowa Iowa Research Online Theses and Dissertations Summer 2016 New roles for an old cytokine : characterizing how exposure to Il-12 alters human CD4 And CD8 T cell responses Aldo Fabian Vacaflores Salinas University of Iowa Copyright 2016 Aldo Fabian Vacaflores Salinas This dissertation is available at Iowa Research Online: Recommended Citation Vacaflores Salinas, Aldo Fabian. "New roles for an old cytokine : characterizing how exposure to Il-12 alters human CD4 And CD8 T cell responses." PhD (Doctor of Philosophy) thesis, University of Iowa, Follow this and additional works at: Part of the Immunology of Infectious Disease Commons

2 NEW ROLES FOR AN OLD CYTOKINE: CHARACTERIZING HOW EXPOSURE TO IL-12 ALTERS HUMAN CD4 AND CD8 T CELL RESPONSES by Aldo Fabian Vacaflores Salinas A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Immunology in the Graduate College of The University of Iowa August 2016 Thesis Supervisor: Associate Professor Jon C. D. Houtman

3 Copyright by Aldo Fabian Vacaflores Salinas 2016 All Rights Reserved

4 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL This is to certify that the Ph.D. thesis of PH.D. THESIS Aldo Fabian Vacaflores Salinas has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Immunology at the August 2016 graduation. Thesis Committee: Jon C. D. Houtman, Thesis Supervisor John Harty Gail Bishop Frederick Quelle Steven Varga

5 This dissertation is dedicated to my family who continuously supported me in this amazing journey ii

6 ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest gratitude to my mentor Jon Houtman, for his exceptional guidance and continuous support at both the academic and personal level. His enormous patience and wisdom helped me overcome the frustrations and boundaries that you faced during your graduate education. He taught me how to be a better writer, public speaker, and researcher. His style of mentoring made my time in graduate school very rewarding and enjoyable. I could not image having a better advisor and mentor for my Ph.D. studies. I would also like to thank the members of my committee Dr. John Harty, Dr. Gail Bishop, Dr. Frederick Quelle, and Dr. Steve Varga for their guidance over the years. I would also like to thank the past and present members of the Houtman Lab, Mahmood Bilal, Mikaela Tremblay, Michael Zhang, and Nicole Chapman. These people patiently trained me, helped me with questions, proofread my writing, kept the lab running, discussed and gave me ideas, and made the lab feel like home. We had great discussions both inside and outside the lab. I consider them not only colleagues but great friends as well. My sincerest gratitude goes to the different mentors that I had the pleasure to work with throughout my education. When I first came to the United States with an exchange program to take some undergraduate courses, I had the pleasure to have Alice Soldan and Karyn Fay as the instructors for most of my classes. They patiently trained me, answered my countless questions, and made me love the subject matter. They both iii

7 helped me transfer to the United States to finish my education, they assisted me in improving my career, and trusted me when I needed their support to get into internships and graduate school. My sincere thanks also go to Dr. John Donelson, who allowed me to spend the summer in his laboratory doing research at the University of Iowa. This experience concreted my desire to continue my education and was instrumental for me to get into graduate school. Also, I wish to acknowledge the help provided by a good friend Shalini Suryanarayana, her advices were a great help to achieve my career goals. I am also particularly grateful for the assistance given by my uncle Julio Chiarella who was instrumental for me to finish and succeed in my undergraduate and graduate education. Last but not the least, I would like to thank my family: my parents, my sisters, and my girlfriend Monica for providing me with unfailing support and continuous encouragement throughout my years of study. This accomplishment would not have been possible without them. Thank you. iv

8 ABSTRACT CD4 and CD8 T cells are constantly exposed to inflammatory signals that influence diverse functional outcomes during infections and certain autoimmune disorders. One of the signals controlling CD4 and CD8 T cell functions is the inflammatory cytokine IL-12. Previous studies have focused on how IL-12 regulates CD4 and CD8 T cell functions when present during or after the activation of the T cell receptor (TCR). However, based on murine studies, we have only recently begun to appreciate that exposure to inflammatory signals, driven in part by IL-12, could alter how CD4 and CD8 T cells respond to TCR stimulation. Although intriguing, these studies have left several questions unanswered. Does IL-12 similarly regulate the function of human T cells? If so, what is the exact molecular mechanism by which IL-12 mediates these effects? To address these critical questions, I examined how IL-12 pretreatment altered human CD4 and CD8 T cell responses to subsequent TCR stimulation. In CHAPTER III, I examined how prior exposure to IL-12 alters the responses of human CD4 T cells to subsequent TCR stimulation. Some of my key findings were that IL-12 pretreatment increased the production of IFN-γ, TNF-α, IL-13, IL-4 and IL-10 after TCR stimulation, suggesting that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. Based on the intracellular staining and mrna expression data, we concluded that the IL-12-mediated increased production of a range of cytokines was a consequence of at least two separate mechanisms, increased mrna expression for IFN-γ and increased release of TNF-α, IL-13, IL-4 and IL-10. In CHAPTER IV, we explored the mechanisms by which IL-12 pretreatment altered human CD4 T cell v

9 responses to TCR stimulation. I observed that IL-12 pretreatment increased the phosphorylation of AKT, P38 and LCK following TCR stimulation without altering other TCR signaling molecules, suggesting that this potentially mediates the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that were not transcriptionally regulated was partially driven by increased oxidative metabolism. Collectively my results uncovered a novel function of IL-12 in regulating human CD4 T cell responses; specifically, it enhanced the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism. Then, in CHAPTER V, I examined the effects of IL-12 pretreatment in altering the responses of human CD8 T cells to subsequent TCR stimulation. My key finding was that pretreatment of human CD8 T cells with IL-12 resulted in increased IFN-γ and TNF-α cytokine mrna and protein production following subsequent TCR challenge. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules. Together my results suggest that prior exposure to IL-12 potentiates human CD8 T cell responses to TCR stimulation possibly by altering the activation of TCR signaling pathways. In the end, my results increase our understanding of the physiologic properties of human CD4 and CD8 T cell and provide mechanistic insight into novel functions for IL- 12. My results also provide insights into potential avenues to improve the current uses of IL-12 in therapeutics. vi

10 PUBLIC ABSTRACT T cells are a type of white blood cell that plays a key role in protecting us against infections. Based on their functions, T cells can be divided into two groups called CD4 and CD8 T cells. In order for these cells to function, they need to be activated through their T cell receptor (TCR). In addition, the functions of these cells are regulated by proteins called cytokines. One of these cytokines is named IL-12. How exactly IL-12 regulates the functions of CD4 and CD8 T cells is not completely understood. In order to address this knowledge gap, we examined whether exposing human CD4 or CD8 T cells to IL-12 alters the responses of these cells to TCR stimulation. We found that when the CD4 or CD8 T cells were exposed to IL-12 they had increased activation in response to TCR stimulation. This suggests that IL-12 enhances the responses of both CD4 and CD8 T cells to activation via the TCR. We also explored the mechanism by which IL-12 mediates these effects. In both CD4 and CD8 T cells, we found that exposure to IL-12 altered the activation of several proteins that transmit the TCR signals inside the cell. Also, in CD4 T cells, we found that IL-12 altered pathways involved in the production of energy. These results provide potential mechanisms by which IL-12 regulates the functions of CD4 and CD8 T cells. Our studies help us understand how these cells protect us from infections, and this information could be harnessed in new therapeutic approaches. vii

11 TABLE OF CONTENTS LIST OF FIGURES... xiii LIST OF ABBREVIATIONS... xvi CHAPTER I: GENERAL INTRODUCTION... 1 Introduction to immune system... 1 Cells of the immune system... 2 Cytokines and their receptors... 2 Introduction to T cells... 3 TCR signaling... 4 T cell development... 8 CD8 vs CD4 T cells... 9 Overview of T cells responses Alternative hypothesis for naïve T cell activation and differentiation CD4 T cell subsets and plasticity T cell metabolism Regulation of T cell responses by cytokines and/or inflammatory signals IL-12 history The IL-12 molecule and IL-12 family members The IL-12 receptor and signal transduction Biological functions of IL-12 on T cells Cytokines and inflammatory signals altering T cell responses to subsequent TCR stimulation Conclusions CHAPTER II: MATERIALS AND METHODS Ethics statement Reagents Antibodies Cell lines Human peripheral blood CD4 and CD8 T cell isolation and activation viii

12 Cytokine pretreatment of human CD4 and CD8 T cells Cytokine production measured by ELISA Intracellular cytokine and surface molecule staining Cell viability assays Quantitative Real-time PCR Immunoblotting Seahorse and ECAR/OCR measurements Statistical analysis CHAPTER III: ROLEOF PRIOR EXPOSURE TO IL-12 IN ALTERING HUMAN CD4 T CELL RESPONSES TO TCR STIMULATION Introduction Results Prior exposure to IL-12 selectively potentiates ensuing TCR- induced IFN-γ production in human activated CD4 T cells Short pretreatments with low doses of IL-12 are sufficient to transiently potentiate the TCR-mediated production of IFN-γ Conditioning human activated CD4 T cells with IL-12 does not alter their functional avidity Pretreatment of human activated CD4 T cell with IL-12 does not alter the expression of the activation marker CD69 following TCR stimulation IL-12 exposure does not alter the basal proliferation/survival of human activated CD4 T cells Prior exposure to IL-12 leads to a transient increase in proliferation/survival of human activated CD4 T cells Pretreatment of human activated CD4 T cells with IL-12 enhances the TCR-induced release of a range of cytokines IL-12 increases the number of cells capable of producing IFN-γ, while altering the release of other cytokines via a separate mechanism Human activated CD4 T cells have variable expression of the IL-12R β1and IL-12R β The IL-12 mediated enhancement of cytokine production ix

13 following TCR stimulation is mediated by a function of both transcriptional and post-transcriptional effects Prior exposure to IL-12 alters how cells respond to TCR stimulation similarly whether it s present only before or before and during TCR stimulation Responses of resting peripheral blood CD4 T cells to IL-12 pretreatment Discussion CHAPTER IV: MECHANISM BY WHICH IL-12 PRETREATMENT POTENTIATES HUMAN CD4 T CELLS RESPONSES TO TCR STIMULATION Introduction Results IL-12 exposure does not alter the expression of surface molecules associated with T cell activation The IL-12-mediated increase of cytokine production is not mediated by residual STAT4 synergizing with TCR stimulation signals IL-12 pretreatment enhances the activation of select signaling molecules downstream of the TCR IL-12 pretreated human activated CD4 T cells undergo metabolic reprograming towards oxidative metabolism Exposure to IL-12 does alter glucose consumption or mitochondrial mass in human activated CD4 T cells Exposure to TNF-α does not alter the metabolic profile of human activated CD4 T cells IL-12 mediated enhancement of cytokine secretion following TCR stimulation is partially regulated by an increase in oxidative metabolism Discussion CHAPTER V: PRETREATMENT OF HUMAN CD8 T CELLS WITH IL-12 LEADS TO ENHANCED TCR-INDUCED SIGNALING AND CYTOKINE PRODUCTION 150 Introduction Results x

14 Conditioning human activated CD8 T cells with IL-12 leads to increased production of IFN-γ and TNF-α upon TCR stimulation Characterizing the IL-12 mediated priming of cytokine production Human activated CD8 T cells have variable surface expression of IL-12R β1 and β Prior exposure to IL-12 increases the frequency of cells capable of producing IFN-γ and TNF-α upon TCR stimulation The IL-12-mediated increase of cytokine production is a consequence of increased transcription of cytokines IL-12 exposure does not alter the expression of surface molecules involved in T cell activation The IL-12 mediated enhancement of cytokine production is not due to residual STAT4 synergizing with TCR stimulation signals. 158 IL-12 pretreatment enhances the TCR-induced activation of select MAPK and AKT without altering the activation of more proximal TCR signaling molecules IL-12 pretreatment enhances the levels of SOS1 and increases the activation of MKK3/MKK6 downstream of the TCR Discussion CHAPTER VI: CONCLUSIONS AND FUTURE PERSPECTIVES Summary Prior exposure to IL-12 selectively alters human CD4 and CD8 T cell responses to TCR stimulation Prior exposure to IL-12 regulates human activated CD4 and CD8 T cells in distinct ways Pretreatment of human activated CD4 T cells with IL-12 enhances the TCR-induced release of a range of cytokines Mechanism by which IL-12 pretreatment potentiates human CD4 and CD8 T cell responses to TCR stimulation The IL-12-mediated increase of cytokine production is not mediated by residual STAT4 synergizing with TCR stimulation signals xi

15 IL-12 pretreatment enhances the activation of select signaling molecules downstream of the TCR IL-12 pretreated human activated CD4 T cells undergo metabolic reprograming towards oxidative metabolism Conclusions REFERENCES xii

16 LIST OF FIGURES Figure 1: T cell receptor signal transduction Figure 2: Overview of T cell responses Figure 3: Production of IL-12 and IL-12 signaling pathway Figure 4: Isolation and activation of human CD4 and CD8 T cells Figure 5: Conditioning HuT78 T cells with IL-12 enhances the TCR-induced production of IL Figure 6: Exposure to IL-12 selectively alters how human activated CD4 T cells respond to TCR stimulation Figure 7: Short pretreatments with low doses of IL-12 are sufficient to transiently potentiate the TCR-mediated production of IFN-γ Figure 8: Conditioning CD4 T cells with IL-12 does not alter their functional avidity Figure 9: Prior exposure to IL-12 does not alter the activation phenotype of human activated CD 4 T cells Figure 10: IL-12 exposure does not alter the basal proliferation/survival of human activated CD4 T cells Figure 11: Prior exposure to IL-12 leads to a transient increase in proliferation/ survival of human activated CD4 T cells Figure 12: IL-12 pretreated human CD4 T cells have enhanced production of a range of cytokines following TCR stimulation Figure 13: Prior exposure to IL-12 increases the number of cells capable of producing IFN-γ and enhances the release of other cytokines from the T cell Figure 14: Prior exposure to IL-12 increases the number of cells capable of producing IFN-γ and enhances the release of other cytokines from the T cell Figure 15: Prior exposure to IL-12 increases the number of cells capable of producing IFN-γ and enhances the release of other cytokines from the T cell Figure 16: Prior exposure to IL-12 increases the number of cells capable of producing IFN-γ and enhances the release of other cytokines from the T cell Figure 17: Characterization of the expression of the IL-12 receptor in human CD4 T cells Figure 18: The IL-12-mediated priming of cytokine release is driven by a function of both transcriptional and post-transcriptional effects Figure 19: Prior exposure to IL-12 alters how cells respond to TCR stimulation xiii

17 similarly whether it s present only before or before and during TCR stimulation Figure 20: Responses of resting peripheral blood CD4 T cells to IL-12 pretreatment Figure 21: IL-12 pretreatment does not alter the expression of surface molecules on human activated CD4 T cells Figure 22: The IL-12-mediated priming of cytokine production is not mediated by residual STAT4 synergizing with TCR stimulation signals Figure 23: IL-12 pretreatment increases the activation of select signaling molecules downstream of the TCR Figure 24: IL-12 pretreatment increases the activation of select signaling molecules downstream of the TCR Figure 25: IL-12 pretreatment increases the activation of select signaling molecules downstream of the TCR Figure 26: IL-12 pretreatment does not alter the total protein expression of TCR signaling molecules Figure 27: IL-12 pretreatment does not alter the expression of GAPDH Figure 28: IL-12 pretreated human activated CD4 T cells undergo metabolic reprogramming towards oxidative metabolism Figure 29: IL-12 pretreated human activated CD4 T cells have an increased ability to up-regulate mitochondrial respiration in response to stimulation Figure 30: Glucose consumption and mitochondrial mass in IL-12 pretreated and untreated cells Figure 31: TNF-α pretreatment does not alter metabolic pathways in human activated CD4 T cells Figure 32: The IL-12-mediated enhancement of the secretion of cytokines not transcriptionally regulated is partially driven by an increase in oxidative metabolism Figure 33: Human activated CD8 T cells pretreated with IL-12 have increased IFN-γ and TNF-α production following TCR stimulation Figure 34: IL-12 pretreatment does not alter the proliferation/survival of human activated CD8 T cells Figure 35: Human activated CD8 T cells pretreated with IL-12 have increased IFN-γ and TNF-α production following TCR stimulation Figure 36: The potentiation of TCR-mediated cytokine production by human activated CD8 T cells is transient and requires low physiological doses of IL-12 for at least 24 hours Figure 37: Human activated CD8 T cells have variable surface expression of IL-12R β1 and β Figure 38: Prior exposure to IL-12 increases the frequency of human activated CD8 xiv

18 T cells making IFN-γ and TNF-α upon TCR stimulation Figure 39: The IL-12 pretreatment increases IFN-γ and TNF-α mrna expression after TCR stimulation in human activated CD8 T cells Figure 40: IL-12 pretreatment does not alter the expression of surface molecules on human activated CD8 T cells Figure 41: The IL-12 mediated enhancement of cytokine production in human activated CD8 T cells is not due to residual STAT4 synergizing with TCR signals Figure 42: Prior exposure to IL-12 does not alter the activation of proximal TCR signaling molecules in human activated CD8 T cells Figure 43: IL-12 pretreatment enhances the TCR-induced activation of select MAP kinases and AKT in human activated CD8 T cells Figure 44: IL-12 pretreatment does not alter the total protein levels of P38 in human activated CD8 T cells Figure 45: IL-12 pretreatment increases the expression of SOS1 and the phosphorylation of MKK3/MKK6 downstream of the TCR xv

19 LIST OF ABBREVIATIONS APC ATP BFA CLMF CSK DAG DCs Antigen presenting cell Adenosine triphosphate Brefeldin A Cytotoxic lymphocyte maturation factor C-terminal src kinase Diacylglycerol Dendritic cells EBi3 p40 related protein Epstein-Barr virus induced gene 3 EBV ECAR ELISA ERK FADH 2 FBS FI Epstein Barr Virus Extracellular acidification rate Enzyme-linked immunosorbent assay Extracellular signal-regulated kinase Flavin adenine dinucleotide Fetal bovine serum Fluoresce intensity GADD45 Growth arrest and DNA damage-inducible genes 45 GAPDH Glyceraldehyde 3-phosphate dehydrogenase GLUT1 Glucose transporter 1 JAK IL-12R IMDM IP3 IRB ITAM Janus kinase IL-12 receptor Iscoves Modified Dulbecco s Media Inositol 1,4,5 phosphate Institutional Review Board Immunoreceptor tyrosine-based activation motif xvi

20 LAT LCK LCMV LM LRS MAPK MFI Linker for the activation of T cells Lymphocyte-specific tyrosine kinase Lymphocytic choriomeningitis virus Listeria Monocytogenes Leukocyte reducing system Mitogen-associated protein kinases Mean fluoresce intensity mtorc2 Mammalian target of rapamycin complex 2 NADH NFAT NKSF OCR OXPHOS PAMPS PBMC PBS Nicotinamide adenine dinucleotide Nuclear factor of activated T cells Natural killer stimulating factor Oxygen consumption rate Oxidative phosphorylation Pathogen associated molecular patterns Peripheral blood mononuclear cell Phosphate buffered saline PDK1 Phosphoinositide-dependent kinase 1 PH PI PIP 2 PIP 3 PI3K PKC PRR RA RasGRP SH2 Pleckstrin-homology Propidium iodide Phosphatidylinositol 4,5 phosphate Phosphatidylinositol 3,4,5-triphosphate Phosphoinositide-3-kinase Protein kinase C Pattern recognition receptors Rheumatoid arthritis Ras guanyl nucleotide releasing protein SRC homology 2 domain xvii

21 SHP1 SH2 domain-containing phosphatase 1 SLP-76 Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kda SOS1 Sons of sevenless 1 STAT TCA TCR Tfh Th TLR Treg ZAP-70 Signal transducer and activator of transcription Tricarboxylic acid T cell receptor Follicular helper T cells T helper Toll like receptor Regulatory T cell ζ-associated protein of 70 kda xviii

22 CHAPTER I: GENERAL INTRODUCTION Introduction to the immune system Our bodies are constantly exposed to a plethora of foreign agents from the environment. The majority of these foreign substances are innocuous to our bodies, but some of them are pathogenic. Our bodies have different barriers of protection to cope with possible pathogenic agents that could cause disease. The epithelial surfaces of the body serve as a first line of defense, providing mechanical, chemical, and microbiological barriers to infection. If a pathogenic agent breaches the first line of defense then different components of the immune system are in place to prevent the pathogen from thriving. The different components of the immune system include a variety of cells and molecules that are incredibly effective in providing protection to the host. The components of the immune system have been separated by immunologists into two groups, called innate and adaptive immune responses, due to different characteristics of each response. Both innate and adaptive responses play important roles in the host defense against infection and are also interconnected to each other. Innate responses are available to rapidly fight a wide array of pathogens, relying on the recognition of features that are common among different pathogens. These type of responses occurs immediately after exposure to the pathogen, are not specific for a particular pathogen, and don t lead to the formation of long lasting immunity. In contrast, adaptive immune responses take longer to develop, they are specific to a particular pathogen, and provide long-lasting protection against reinfection with the same pathogen. 1

23 Cells of the immune system Both arms of the immune system (innate and adaptive) are formed by an arsenal of different cells that play diverse roles during immune responses. Some of the common cells that are grouped into the innate arm of the immune system based on their characteristics include the following: monocytes and macrophages, that are known for being exceptional at engulfing and killing microorganisms and also for orchestrating immune responses by secreting proteins that regulate the function of other cells of the immune system [1] Granulocytes, that their primary function is to destroy microorganism by release of granules [1]; dendritic cells (DCs), that are also phagocytic cells but their main role is not to clear pathogens but instead their role is to activate cells of the adaptive immune system called T cells [1]; and NK cells, which can recognize and kill abnormal cells but they are not specific for a particular pathogens [1]. Some of the common cells that are grouped into the adaptive arm of the immune system based on their characteristics are: T cells and B cells, which their primary function is to provide protection to pathogens. Both cell types recognize specific pathogens by their antigen receptors and have the capacity to form memory to specific pathogens [1]. Although DCs are known to provide signals to activate T cells, in certain settings, macrophages and B cells can also fulfill this role. Collectively, these cells that provide signals to activate T cells are also known as antigen presenting cells (APCs) [2, 3]. Cytokines and their receptors Cytokines can be broadly defined as small proteins that are produced by a variety of cells and that affect the behavior of immune and other cells by binding to specific 2

24 receptors. In the immune system, cytokines are known to direct the development, maturation, localization, interactions, activation and life span of the cells. Early during immune responses, cytokines are secreted by different immune and non-immune cells in response to pathogenic agents, and these cytokines are involved in promoting a local inflammatory response that in many different ways helps contain the infection. Cytokines are also involved in adaptive immunity by promoting the mobilization of APCs that will induce the activation and differentiation of T cells and B cells [1]. Besides their involvement in non-specific and specific responses to pathogenic agents, cytokines are involved in normal physiological functions of immune cells such as the homeostatic proliferation of T cells [4]. The overall focus of this thesis is to study the effects of cytokines, particularly IL- 12, in altering the responses of T cells to subsequent activation. Therefore, in the next sections I will discuss in more detail the characteristics of T cells, followed by a discussion of the regulation of T cell responses by cytokines, particularly the cytokine IL- 12. Introduction to T cells The primary signal for activation of T cells is through the ligation of the T cell receptor (TCR). In the next section, I will first start by reviewing in detail the intracellular events that occur upon TCR stimulation leading to T cell activation. Next, I will examine general concepts of T cell biology starting with an introduction to T cell 3

25 development, then giving an overview of T cell responses and the different subsets that mediate these responses, and finally I will discuss emerging topics in T cell metabolism. TCR signaling T cells are activated when the TCR on the T cell recognizes cognate peptide presented on the correct MHC molecules on the surface of an APC. TCR stimulation results in a coordinated activation of a series of signaling molecules that eventually promote the transcription of genes that control the cellular responses. The TCR is made up of two chains (α and β) that vary in each T cell in regions that recognize the antigen. These chains are associated with the CD3 protein complex that contain cytoplasmic domains that mediate signaling [5]. One of the early events detected following TCR stimulation is the activation of the kinases, lymphocyte-specific tyrosine kinase (LCK) and FYN (Fig. 1) [5, 6]. The activities of these kinases are positively regulated by the phosphorylation of tyrosine residues (LCK Y394 and FYN Y417) in their kinase domains and negatively regulated by the phosphorylation on tyrosine residues on their C-terminal tail (LCK Y505 and FYN Y528) by different kinases and phosphatases [7]. Some of the important known regulators of the activation of LCK and FYN are the C-terminal src kinase (CSK) and CD45. Although the precise mechanism regulating the activities of CSK and CD45 are not completely understood, we do know that CSK is a tyrosine kinase that negatively regulates the activation of LCK and FYN by phosphorylating tyrosine residues that inhibit the kinase function (LCK Y505 and FYN Y528) [7]. Also, CD45 is a phosphatase that acts as a positive regulator of the activation of LCK and FYN by 4

26 dephosphorylating the tyrosine residues that inhibit the kinase function (LCK Y505 and FYN Y528) [7]. Activation of LCK and FYN leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) found in the subunits of the TCR. Upon phosphorylation the tyrosine kinase ζ-associated protein of 70 kda (ZAP-70) is recruited to the ITAMs, where it is activated by LCK and FYN upon phosphorylation of its tyrosine 319 (Fig. 1) [5, 6]. Activated ZAP-70 then phosphorylates multiple targets including the adaptor proteins linker for the activation of T cells (LAT) and src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kda (SLP-76). ZAP-70 phosphorylates LAT on several tyrosine residues from which the last four tyrosine sites in the human sequence (Y132, Y171, Y191, and Y226) serve as docking sites for several SH2 domain containing proteins, such as the adaptor proteins GRB2 and Gads, and the phospholipase PLC-γ (Fig. 1) [5, 8, 9]. Once recruited to LAT, PLC-γ is phosphorylated on Y783, which promotes its enzymatic activity [5, 10]. The enzymatic function of activated PLC-γ is to cleave phosphatidylinositol 4,5 phosphate (PIP 2 ) into inositol 1,4,5 phosphate (IP3) and diacylglycerol (DAG), which act as second messengers and promote calcium influx and the activation of downstream kinases, respectively (Fig. 1) [5, 6, 10]. TCR induced calcium influx activates a family of transcription factors called nuclear factor of activated T cells (NFAT) that translocate to the nucleus and promote the activation of many genes, including the gene for the cytokine IL-2 (Fig. 1) [5]. TCR induced DAG diffuses in the plasma membrane and recruits RasGRP (Ras guanyl nucleotide releasing protein), which is a guanine-nucleotide exchange factor that specifically activates Ras (a guanine 5

27 nucleotide-binding protein) by promoting the exchange of GDP for GTP. Activated Ras activates a cascade of serine/threonine kinases that leads to the activation of the mitogenassociated protein kinase (MAPK) extracellular signal-regulated kinase (ERK)1 and ERK2 (Fig. 1) [5, 6]. Activated ERK enters the nucleus and phosphorylates transcription factors resulting in the transcription of numerous genes. TCR-induced DAG also recruits PKC-θ (an isoform of PKC) to the plasma membrane, resulting in the activation of a series of molecules that eventually promote NFκB activation. NFκB then translocates to the nucleus and promotes the transcription of its target genes (Fig. 1) [5, 6]. Upon TCR stimulation the adaptor protein GRB2 also binds to phosphorylated tyrosine residues on LAT and recruits the protein sons of sevenless 1 (SOS1) which is a guanine nucleotide exchange factor that its constitutively bound to GRB2 (Fig. 1) [5, 11]. Once recruited to the plasma membrane SOS1 also activates Ras (along with RasGRP) which as described above modulates the activation of the MAPK ERK1/2 (Fig. 1) [5]. The significance of SOS1 and RasGRP in mediating Ras activation is still under debate [12-14]. However, recent data from our laboratory demonstrated that in the absence of GRB2 the phosphorylation of ERK is moderately abrogated, suggesting that at a minimum GRB2 is needed for optimal activation of ERK1/2 [15]. TCR stimulation also promotes the phosphorylation of SLP-76 on several tyrosine residues (Y113, Y128, Y145) by ZAP-70 [16]. Phosphorylated SLP-76 serves as an adaptor protein and associates with several proteins at the LAT complex, including PLCγ, ITK, and VAV1 (Fig.1) [16]. The recruitment of these proteins and the formation of this multiprotein complex in cooperation with LAT is critical for the transmission of TCR signals [5]. SLP-76, in cooperation with LAT, also directs the recruitment and activation 6

28 of phosphoinositide-3-kinase (PI3K) [17, 18]. Activated PI3K generates phosphatidylinositol 3,4,5-triphosphate (PIP 3 ) from PIP2 [5]. PIP 3 serves as a docking site for proteins with pleckstrin-homology (PH) domains in the plasma membrane such as the phosphoinositide-dependent kinase 1 (PDK1) and AKT (Fig. 1) [5, 19]. At the membrane AKT is phosphorylated on T308 by PDK1 and on S473 by the mammalian target of rapamycin complex 2 (mtorc2). AKT has its maximum catalytic function when phosphorylated on these residues (T308 and S473) [20, 21]. Once activated, AKT has multiple targets that are involved with the regulation of different cellular events related to cellular proliferation, growth, survival and metabolism [5, 19, 21]. The activation of the MAP kinase pathways JNK and P38 are downstream of the LAT and SLP-76 complexes (Fig.1). Although many studies in different cell types have looked at how diverse stimuli promote P38 and JNK activation, how TCR stimulation promotes P38 or JNK activation in T cells remains elusive [22]. There are two proposed mechanisms for TCR-induced P38 activation in T cells, termed the canonical pathway and the alternative pathway. In the canonical pathway, P38 is phosphorylated and activated on T180 and Y182 by the MAP kinases MKK3, MKK4, and MKK6 [22]. The upstream events connecting early TCR signaling and the activation of MKK3, MKK4, and/or MKK6 remain to be fully elucidated [22]. In the alternative pathway, TCR stimulation results in phosphorylation of P38 on residue Y323 which activates the catalytic function of the kinase and promotes auto phosphorylation of T180/Y182 [22, 23]. Interestingly, TCR stimulation of a mutant P38 Y323 still has P38 activation, suggesting that the canonical pathway may predominate in TCR-mediated P38 activation [22]. Activated P38 has many targets including transcription factors and protein kinases 7

29 [22]. Importantly, using inhibitors of P38, several studies have shown that P38 is involved with cytokine secretion following TCR stimulation [22]. We know that JNK is activated on T183 and Y185 by the MAP kinases MKK7 and MKK4 and this is involved in cytokine production and proliferation. However, the mechanisms leading to JNK activation upon TCR ligation and the direct targets of JNK to mediate their functions remain uncharacterized [24, 25]. Importantly, recent data from our laboratory demonstrated that in the absence of GRB2 the phosphorylation of JNK and P38 were moderately abrogated, suggesting that at a minimum GRB2 is needed for optimal activation of these kinases [15]. T cell development T cells, like the other immune cells, originate from stem cells in the bone marrow. Following development, common lymphoid progenitors migrate to the thymus where they finish their maturation. In the thymus, developing T cells (known as thymocytes) rearrange the genes of their TCRs resulting in two distinct lineages of T cells, a major population of αβ and a minor population of γδ T cells [1]. For the purpose of this thesis, we will focus on the αβ T cells and will refer to them simply as T cells. Also, in the thymus, thymocytes bearing the αβ receptors pass through a series of stages and go through a selection process resulting in mature T cells of two distinct subsets, CD4 T cells and CD8 T cells [1]. These cells have a repertoire of TCRs that can recognize a range of different foreign antigens and don t, for the most part, react to self-antigens. Along with the classical CD4 and CD8 T cells subsets, there are other lineages that will not be discussed since they are beyond the scope of this thesis. However, it should be 8

30 noted that the thymus also generates a distinct lineage of CD4 T cells known as natural regulatory T cells (Tregs) [1]. Mature T cells (CD4 and CD8 T cells) that have left the thymus and haven t experienced antigen are termed naïve T cells. Human naïve T cells can be identified by the expression of the longest isoform of the surface molecule CD45 (CD45RA) and/or the lack of expression of the shortest isoform (CD45RO) [26]. These naïve T cells navigate from the blood to secondary lymphoid organs where they constantly survey for antigens and continue to recirculate between the blood and lymph until they encounter antigen. CD8 vs CD4 T cells CD8 T cells recognize peptides presented by APCs in the context of MHC class I molecules and CD4 T cells recognize peptides presented by APCs in the context of MHC class II molecules [1]. MHC class I molecules deliver peptides from the cytosol, such as peptides from viruses, and MHC class II molecules present peptides originating from the vesicular system, such as peptides from bacteria that are phagocytized or taken up by receptor-mediated endocytosis [1]. CD8 T cells are generally important for the defense against intracellular pathogens such as viruses. The canonical effector function of CD8 T cells is to recognize infected cells and kill them. However, they are also known for releasing IFN-γ and TNF-α which contribute to host defense by inhibiting viral replication and by activating macrophages [1, 27]. CD4 T cells are known to differentiate into different subsets with separate functions including activation of cells of the immune system, B cells, CD8 T cells, and non-immune cells, as well as suppression of immune responses. CD4 T cells carry out these functions primarily by secreting a variety of 9

31 cytokines [1, 28]. Although CD4 and CD8 T cell responses are intended to protect the host from pathogenic agents, this response has to be tightly regulated since dysfunctional T cell responses are known to promote or exacerbate autoimmune and other inflammatory diseases. Overview of T cells responses During the course of an infection APCs such as DCs take up the infectious agent and transports them via the lymphatic system to the nearest lymph node or secondary lymphoid organ (Fig. 2). Once DCs reach the local draining lymph nodes, they present antigens derived from the pathogen on their surface bound to MHC class I and class II molecules. DCs also upregulate the expression of various costimulatory surface molecules and cytokines (Fig. 2). Mature naïve T cells that recently left the thymus enter the blood stream and recirculate between the blood and secondary lymphoid organs (Fig. 2). Once the naïve T cells enter the secondary lymph nodes, they make contact with the different DCs that are presenting an array of peptides/mhc molecules on their surfaces (Fig. 2). Each of the naïve T cells has a unique TCR that is responsible for the specificity of each T cell and recognizes peptide antigens only when they are loaded on the appropriate class I or class II MHC molecules. Most of the time the naïve T cells migrating in the lymph nodes will not recognize any peptide presented by the DC and will continue to recirculate between the blood and secondary lymphoid organs [1]. However, when that the naïve T cell encounters a properly activated DC displaying the appropriate peptide/mhc ligand, the naïve T cell becomes activated, starts clonally 10

32 expanding, differentiating into an effector T cell, and migrates back into the circulation (Fig. 2) [1]. The process of initial activation of a naïve T cells by an APC is known as priming. In order to have successful activation of the naive T cells, there are at least three signals that are critical (Fig. 2)[27]. Signal 1, compromises the binding of the TCR with its cognate antigen bound to MHC molecules. Signal 2, which is provided by costimulatory signals, promotes the survival and expansion of the T cells during priming. The best studied signal 2 consists of the B7 family of molecules on the surface of APCs that bind CD28 on the T cell. In addition to Signal 1 and Signal 2, several lines of evidence also suggest that other membrane bound or soluble inflammatory signals play important roles for the successful activation and differentiation of the T cell [27, 29, 30]. This allows the immune system to produce a specialized immune response to the diverse spectrum of pathogens that can be encountered by the host. Some of the most important cytokines that play important roles in the successful activation and differentiation of the T cells will be further described in the sections below. After priming, the antigen-experienced T cells will express a set of surface molecules that will allow them to migrate to the sites of infection (Fig. 2) [27]. At the sites of infection, the antigen experienced T cells that encounter APCs presenting their cognate antigens will execute their effector functions to clear the infection (Fig. 2). After clearing the infection the majority of the activated T cells will die. However, some will differentiate into a pool of memory T cells that have a variety of migratory properties and functions. These cells are able to quickly respond and protect against pathogen reinfection [27, 31]. Some of the memory T cells will become resident memory cells and 11

33 reside in non-lymphoid tissues and others will continuously recirculate through the blood and secondary lymphoid organs [32]. In this thesis I will refer to antigen experienced T cells, either effector or memory, using the terminology activated T cell. Alternative hypothesis for naïve T cell activation and differentiation Traditionally, the model is that the activation, proliferation, and differentiation of T cells, particularly of CD4 T cells, occurs in the lymph nodes as a simultaneous process [28]. Naïve CD4 T cells receiving signal 1 and 2 will commit to different stable lineages depending on the cytokines from the environment that they receive [28]. However, there is accumulating evidence that challenges the current dogma. This alternative hypothesis for naïve T cell activation is called the second touch hypothesis and argues that T cell differentiation occurs in several steps [33]. According to the second touch hypothesis, naïve T cells have an initial encounter with antigen in the lymph nodes. They receive signal 1 and signal 2 from APCs, become activated and start proliferating, and acquire homing receptors that allow them to migrate to the tissue where antigen resides. These activated T cells leave the lymph nodes partially programmed but without being fully committed to a differentiation program. The activated T cells will recirculate through various tissues until they reach sites of inflammation or infection. Here they will encounter APCs presenting antigen in the context of different costimulatory molecules and cytokines. Depending on the cytokine milieu that they experience during the second interaction, they will fully polarize into the different subsets [33]. Evidence from different reports supports this hypothesis. For example, the expression of co-stimulatory receptors and cytokines by macrophages and DCs is different in tissues compared to 12

34 secondary lymphoid organs [34]. Also, only a small fraction of DCs are migratory and therefore it could be hard for a small fraction of cells to represent the cytokine environment of the infected tissue [35]. Also, T-bet, which is the master regulator of Th1 cells, regulates the induction of most homing receptors of other T cell subsets, suggesting that cells acquire homing receptors before full T cell differentiation [36]. This subtle but important change in the model provides a window for primed cells migrating through the circulatory or lymphatic system to be exposed to different cytokines and inflammatory signals before entering the sites of infection, where they fully commit to a differentiation program during their second interaction with APCs. CD4 T cell subsets and plasticity Following priming, naïve CD4 T cells differentiate into distinct lineages with seemingly stable phenotypes and specialized functions [30, 37]. This is a multistep process, driven by signature transcription factors, that results in discreet yet sequential epigenetic changes in the CD4 T cell [38]. The resulting pool of antigen experienced CD4 T cells is very heterogeneous, composed of different subsets with specialized functions [39]. The different CD4 T cell subsets can be classified based on the production of selective signature cytokines and the expression of characteristic lineage-defining transcription factors. Based on this criteria there are five principal subsets of CD4 T cells that have been described: T helper (Th)1, Th2, Th17, Tregs, and follicular helper T cells (Tfh) [30, 39]. Besides these subsets, several new additional subsets have recently been identified but are less defined and therefore will not be discussed in this thesis [39]. Th1, Th2, and Th17 cells, provide protection against different classes of pathogens. Th1 cells 13

35 fight intracellular pathogens and are characterized for producing large amounts of IFN-γ and TNF-α and for expressing the transcription factor T-bet [30]. Th2 cells control extracellular parasites, and they are also known to be involved in allergic diseases. Th2 cells produce IL-4, IL-13, and IL-5 and express the transcription factor GATA3 [30]. Th17 cells are important for immune responses towards fungi and extracellular bacteria. Th17 cells selectively produce IL-17 and express the transcription factor RORγT [30]. Tregs play key roles in preventing unwanted immune responses. These cells can develop directly from thymic selection or can be differentiated in the periphery. Either way, Tregs produce anti-inflammatory cytokines such as IL-10 and express the transcription factor FOXP3 [28, 30]. Finally, Tfh cells provide help to B cells for antibody production and are defined by the expression of the transcription factor Bcl6 and the production of the cytokine IL-21 [28]. The differentiation of the CD4 T cell subsets is largely driven by the set of transcription factors they express and the genes these factors help transcribe. The conventional model for genetic reprograming of the CD4 T cell lineages suggests that the cytokine milieu present during the TCR mediated priming of the cells guides this process [28, 30]. The cytokines that guide the main CD4 T cell subsets in vitro are fairly well defined. Th1 cells are induced when the cytokines IFN-γ and IL-12 are present during priming. When naïve CD4 T cells are activated in the presence of IL-4, they differentiate into Th2 cells. Th17 cells arise when naïve CD4 T cells are activated in the presence of IL-6, TGF-β, IL-21, and IL-23. The requirements for the generation of Tfh cells in vitro are not clearly defined, but we know that IL-6 is important for the development of this subset. Finally, Tregs are produced when naïve T cells are activated in the presence of 14

36 TGF-β alone in the absence of IL-6. Whether the in vitro differentiation signals recapitulate the in vivo differentiation process guided by pathogenic agents is not known. In this regard, Zhu and colleagues, in their review of CD4 T cell differentiation, speculate that unlike the highly polarized cells obtained in vitro, cells differentiated in vivo probably will present partial states of differentiation [30]. Classically, the different subsets have been seen as terminally differentiated lineages. However, there is an increasing recognition that the subsets maintain a certain degree of plasticity and can acquire different properties and functions given appropriate stimulation [30, 37, 38]. For example, helper T cells have been show to change their characteristic cytokine profile and functions depending on the environment that they experience. Human Th1 memory cells exposed to IL-4 in vitro started producing IL-4 in addition to IFN-γ upon TCR stimulation [40]. Similarly, in mice, in vivo primed Th2 cells exposed to IL-12 and IFN-γ start producing IFN-γ in addition to IL-4 [41]. Also, Th1 clones treated with IL-10 started producing IL-10 in addition to IFN-γ [42]. In addition, Th17 and Tregs have been shown to be flexible and acquire different cytokine production capacities depending on the cytokine milieu they experience [37, 38]. More evidence demonstrating the flexibility of CD4 T cell subsets comes from studies showing subsets co-producing the signature cytokines of other subsets and expressing more than one master regulator [38]. For instance, IFN-γ/IL-10 co-producing T cells have been isolated from peripheral blood of healthy humans and FOXP3 Tregs co-expressing T-bet are found when Tregs are exposed to IL-12 [43, 44]. 15

37 T cell metabolism Cells need adenosine triphosphate (ATP) to provide energy for cellular functions. Glucose is one of the critical substrates for ATP generation in T cells and can be used to fuel this process by many integrated pathways [45]. One of these pathways is glycolysis, and involves the conversion of glucose to pyruvate in the cytoplasm. In this pathway, glucose is first transported into the cells via a glucose transporter; for T cells, glucose transporter 1 (Glut1) is the major mechanism for internalization of glucose [46]. Glucose is then catalyzed by a series of enzymes and broken down into two molecules of pyruvate. This process yields a small number of ATP molecules and reduced nicotinamide adenine dinucleotide (NADH) [45, 46]. Pyruvate then has two fates. In one, pyruvate is converted into acetyl-coa which enters the tricarboxylic acid (TCA) cycle resulting in the generation of NADH and reduced flavin adenine dinucleotide (FADH 2 ) [45]. NADH and FADH 2 donate electrons to the electron transport chain to fuel oxidative phosphorylation (OXPHOS) [45]. OXPHOS is an oxygen dependent process that occurs in the mitochondria and generates a high number of ATP molecules [45]. The other fate for pyruvate is to be transformed into lactate, which regenerates NAD+ that can be used for subsequent glycolysis. Besides glucose, cells can also metabolize other substrates to replenish the TCA cycle and fuel OXPHOS to generate ATP, but for the purpose of this thesis we will not discuss this pathway [47]. In the absence of oxygen, cells can only produce ATP from glucose via glycolysis but in some cases cells ferment glucose via glycolysis even when there is sufficient oxygen to fuel OXPHOS; this process is known as aerobic glycolysis [46]. In T cells, the dogma suggests that naïve and memory T cells maintain low rates of glycolysis and 16

38 primarily oxidize glucose derived pyruvate via OXPHOS to generate ATP. Upon activation, effector cells that are proliferating and differentiating into their respective phenotypes primarily switch from an oxidative metabolism to glycolytic metabolism, in which glucose is metabolized to lactate in the cytosol even though there is enough oxygen for OXPHOS (aerobic glycolysis) [45, 46]. However, this paradigm has been challenged recently by studies showing that upon activation CD4 T cells strongly induce oxidative phosphorylation in addition to glycolysis, and by studies showing that inhibition of OXPHOS blunts different T cell effector functions, suggesting that during T cell activation both OXPHOS and glycolysis are upregulated and are important for T cell activation [46, 48]. Interestingly, recent literature has demonstrated that induction with cytokines or inflammatory signals alters metabolic pathways in immune cells and this is mediated by signaling pathways downstream of these receptors. For example, stimulation of the B-cell antigen receptor on B cells and stimulation of different TLRs on dendritic cells promotes increases in glycolytic metabolism in these cells and these effects are driven by the PI3K and AKT pathway [49, 50]. Also, in recently activated memory CD8 T cells, IL-15 was shown to increase mitochondrial biogenesis, which translated into greater capacity to produce energy [51]. Whether exposure to inflammatory cytokines such as IL-12 or TNFα will also alter metabolic pathways in T cells has not been explored yet and will be examined in CHAPTER IV. The data from these reports are interesting since they demonstrate that metabolic pathways are connected to extracellular signals from cell surface receptors by their signaling pathways. 17

39 Interestingly, the alterations in metabolic pathways induced by extracellular signals were shown to regulate cellular effector functions of immune cells, such as cytokine production and proliferation. Everts et al. found that for DCs, LPS-induced production of cytokines IL-6, IL-12 and TNF-α was regulated at the translational level by glycolysis [52]. Mechanistically, LPS induced increased glycolysis and this supported the de novo synthesis of fatty acids that expanded the endoplasmic reticulum and Golgi, which resulted in increased production and secretion of proteins [52]. For T cells, glycolysis was shown to regulate the production of IFN-γ and potentially IL-2. Zheng and colleagues found that stimulating Th1 cells with anti-cd3antibodies and anti-cd28 antibodies in the presence of an inhibitor of glycolysis resulted in decreased IFN-γ and IL-2 production [53]. Similarly, Cham and Gajewski found that in CD8 T cells IFN-γ and partially IL-2 production were inhibited when cells were stimulated (anti-cd3 antibodies and anti-cd28 antibodies) in the presence of an inhibitor of glycolysis [54]. Also, Chang and colleagues found that in CD4 T cells, inhibition of aerobic glycolysis and not OXPHOS abrogated IFN-γ protein levels without altering the mrna levels following challenge with anti-tcr/cd28 antibodies [55]. Furthermore, Renner et al. found that inhibition of glycolysis in T cells, but not OXPHOS, abrogated IFN-γ, TNF-, IL-10, and IL-4 production but did not affect IL-2 production following anti-tcr/cd28 antibody stimulation [56]. Collectively these data demonstrate that exposure to inflammatory signals alters metabolic pathways in immune cells and these changes in metabolic pathways can regulate immune cell functions such as cytokine secretion. 18

40 Regulation of T cell responses by cytokines and/or inflammatory signals During priming, the myriad of cytokines and inflammatory signals that T cells receive from the environment play key roles in influencing the activation, differentiation, and function of these cells. As mentioned above, one of these cytokines is the cytokine IL-12. Since the focus of this thesis is on IL-12, in the next section I will start by further describing the current knowledge of IL-12 and the roles it plays in regulating T cell responses. IL-12 history IL-12 was originally described with the name of natural killer stimulating factor (NKSF) in 1989 [57]. It was found as a factor secreted by Epstein Barr Virus (EBV)- transformed cell lines that could alter some of the biological activities of human T and NK cells, such as IFN-γ production, cytotoxicity, and proliferation. Later IL-12 was also described by another group with the name cytotoxic lymphocyte maturation factor (CLMF), which was also produced by EBV-transformed cell lines and had similar effects to NKSF [58]. After the genes coding for NKSF and CLMF were cloned it was found that both factors were the same cytokine and the unifying name of IL-12 chosen for this cytokine [59, 60]. 19

41 The IL-12 molecule and IL-12 family members IL-12 is a heterodimeric cytokine of 70 kda composed of two subunits, p35 (35kDa) and p40 (40kDa) (Fig. 3). Each of the subunits (IL-12 p35 and IL-12 p40) are encoded on different chromosomes, and co-expression of both of the subunits (IL-12 p70) in the same cell is required for the formation of biologically active IL-12 [60, 61]. Interestingly, the IL-12 p40 subunit is produced in excess over the IL-12 p35 subunit and the secretion of IL-12 p40 monomers and dimers has been described by several groups [60, 62-64]. The physiological role of these IL-12 p40 monomers or dimers remains to be fully elucidated [60]. In addition, different reports have demonstrated that the IL-12 p35 and IL-12 p40 subunits associate with different proteins to form new heterodimeric cytokines (IL-23, IL-27, IL-35) [65-67]. IL-23 is formed by the association of IL-12 p40 with the molecule p19 [65]. IL-27 is formed by the molecule p28 and the p40 related protein Epstein-Barr virus induced gene 3 (EBi3) molecule [66]. IL-35 is formed by the IL-12 p35 subunit and the molecule EBi3 [67]. For this thesis we will focus on the heterodimeric cytokine IL-12 (IL-12 p70). IL-12 is known to be produced by activated inflammatory cells: monocytes, macrophages, neutrophils, DCs and B cells (Fig. 3). In addition, non-immune cells such as keratinocytes, osteoblasts, epithelial and endothelial cells have also been shown to produce some amount of IL-12 [60, 68]. The production of IL-12 by these cells has been shown to be induced in vivo or in vitro by pathogenic organisms such as bacteria (Gram + and Gram -), parasites, viruses, and fungi [60, 61]. The pathogen associated molecular patterns (PAMPS) in these organisms, such as LPS, teichoic acid, peptidoglycan, and bacterial CpG DNA can induce the production of IL-12 by engaging with pattern 20

42 recognition receptors (PRRs) like TLRs in these cells (Fig. 3) [61]. The precise molecular mechanisms by which microbial products induce IL-12 secretion are still an active area of research. In addition, the production of IL-12 by these cells can be regulated by different positive and negative regulators. Cytokines like IFN-γ and surprisingly, IL-4 and IL-13 are enhancers of IL-12 production. Another enhancer of IL-12 secretion is the engagement of the CD40 on DCs or macrophages with CD40 ligand on T cells [61, 69]. In contrast, IL-10 and TGF-β have been shown to suppress IL-12 production. Surprisingly, cytokines that promote inflammation or that have overlapping functions with IL-12, including IFN-α and IFN-β are also negative regulators of IL-12 production [61]. The amount of IL-12 that the T cells will encounter in vivo during infections or at sites of inflammation is hard to determine accurately. Several groups have examined the concentration of IL-12 in human serum in healthy patients with varied results. The concentrations of IL-12 in the healthy patients ranged from 25 pg/ml to 5 ng/ml [70-74]. Also, groups have determined the concentration of IL-12 in mice following challenges with different bacteria or components of bacteria. The average concentration of IL-12 in serum of mice following LPS injection ranged from 8-30 ng/ml, and following bacterial infection was around 6 ng/ml [75-77]. Similarly, intranasal infection of mice with mycobacteria resulted in IL-12 levels of around 2 ng/ml in bronchoalveolar lavage of the lungs and around 5 ng/ml in the serum [78]. While, the studies mentioned above provide us with an estimate of the amount of IL-12 that the T cells will encounter in vivo, we suspect that the localized concentrations of IL-12 near the producing cells is likely to be higher than the amounts found in these studies. 21

43 The IL-12 receptor and signal transduction IL-12 promotes its biological activities by binding to the IL-12 receptor (IL-12R). The IL-12R is composed of two subunits; the IL-12R β1 and β2 (Fig. 3) [79]. Both subunits spontaneously form homodimers and oligomers, which bind IL-12 with low affinity. However, combination of the IL-12R β1 and IL-12R β2 forms high affinity binding sites for IL-12 [60, 79]. The IL-12 p35 subunit interacts with the IL-12R β1 and the IL-12 p40 subunit interact with IL-12R β2 (Fig. 3) [60]. The IL-12R has been detected in activated T cells, NK cells, and DCs [61]. In T cells, the expression of the IL- 12R is highly regulated. Resting T cells have undetectable levels of the IL-12R but after TCR stimulation the transcription and expression of both subunits of the IL-12R are increased [61, 80, 81]. In addition to TCR stimulation, the expression of the IL-12R is upregulated by the cytokines IL-12, IFN-γ, IFNα, TNF-α, and also following CD28 triggering and negatively regulated by IL-4 [61, 69]. In addition, IL-12R is differentially expressed in Th1 and Th2 cells; Th1 cells express both subunits whereas Th2 cells don t express the IL-12R β2 [61]. The regulation of the expression of the IL-12R by cytokines during priming of naïve CD4 T cells seems to be a contributor to Th1 and Th2 differentiation [61]. The IL-12R β1 associates with the Janus kinase (JAK) family member Tyk2 and the IL-12R β2 binds JAK2 (Fig. 3). Upon binding of IL-12 with the IL-12R, these kinases become activated and phosphorylate tyrosine residues in the IL-12R β2 which act as a binding site for the SH2 domain of the signal transducer and activator of transcription (STATs), particularly STAT4 (Fig. 3) [69]. At the receptor STAT4 becomes phosphorylated by JAK2 and forms STAT4 homodimers which translocate to the nucleus 22

44 and regulate gene transcription (Fig. 3) [69]. The STAT4-mediated pathway appears to be one of the critical mediators of the canonical effects of IL-12 as shown by different animal models. For example, STAT4-knockout mice have deficient IL-12-mediated Th1 differentiation and IFN-γ production, which are key outcomes of IL-12 stimulation of T cells [69]. Exactly how IL-12-induced STAT4 activation mediates the effects of IL-12 is not clear, particularly whether STAT4 directly regulates the transcription of the IFNG gene [61]. In addition, to STAT4, STAT1, STAT3, and STAT5 have also been shown to be activated by IL-12 signals; but the roles of these proteins in regulating IL-12 signaling and IL-12 functions remains to be characterized [69]. Besides activating the JAK/STAT pathway, IL-12 has also been shown to activate other pathways. In NK cells, IL-12 was shown to increase LCK activation. How IL-12 activates LCK or the functional role of LCK activation in IL-12 signaling is not known [82]. In T cells, IL-12 signals were shown to quickly promote P38 activation but they do not alter the activation of other MAPK such as JNK or ERK [83-85]. Activation of P38 seems to be important for IL-12 induced IFN-γ production, but the mechanism for crosstalk between IL-12 and P38 remains elusive [69, 85]. Whether or not IL-12 signals activate AKT in T cells is controversial since there are two studies in the literature that examined this with contradictory results [86, 87]. Biological functions of IL-12 on T cells Many groups have examined the effects of IL-12 in altering T cell responses. One of the well-known functions of IL-12 is to promote the differentiation of naïve CD4 T cells into Th1 cells which primarily produce IFN-γ [61]. These effects have been shown 23

45 using recombinant IL-12 in vivo and in vitro, by using neutralizing antibodies for IL-12 and with animals that are genetically deficient in the subunits of IL-12, the IL-12 receptor, or STAT4 [61, 69]. Also, the presence of IL-12 during priming of CD8 T cells in secondary lymphoid organs has been shown to promote strong effector functions and memory development [29]. In addition, several groups have examined whether exposure to IL-12 alters the effector functions of the T cells. IL-12 does not induce cytokine production or proliferation of resting peripheral blood T cells. However, IL-12 synergizes with TCR stimulation and enhances TCR-induced proliferation and IFN-γ production [61]. Finally, IL-12 in combination with IL-18 has been shown to promote IFN-γ production independent of TCR stimulation [88]. Although highly informative, this work has largely examined the effects of IL-12 if it is present either during or following TCR activation. However, during immune responses, antigen experienced T cells will be exposed to IL-12 as they migrate through the blood or lymph and at sites of infection before they are further activated through the TCR in infected and inflamed tissue. As explained in the section below, recent studies have examined how exposure to cytokines or inflammatory signals such as IL-12 alters the responses of T cells to subsequent TCR stimulation. Cytokines and inflammatory signals altering T cell responses to subsequent TCR stimulation After the initial priming, T cells will also be exposed to different cytokines and inflammatory signals as they migrate through the circulatory or lymphatic system before entering the sites of infection, where they will be further activated via TCR induction by 24

46 antigen presenting cells. The role of these different cytokines and inflammatory signals in regulating the functions of activated T cells (effector or memory) is an active area of investigation. For example, IL-2, IL-7, and IL-15 have been shown to play important roles in activated T cells by regulating memory progression and maintenance of memory. Inflammatory cytokines such as IL-12 in combination with IL-18 have been shown to promote effector functions of activated T cells in an antigen independent manner [89-91]. More importantly for the studies done in this thesis, recent reports have demonstrated that prior exposure to different cytokines or inflammatory signals, including IL-12, dynamically regulate how T cells respond to TCR stimulation [92-96]. This idea of cytokines and inflammatory signals altering how T cells respond to subsequent TCR stimulation is fairly new and provides a new mechanism in the regulation of T cell responses by cytokines and inflammatory signals. But as it will be discussed below, it still remains to be fully characterized. In the next part I will review some of the key findings from these reports, highlighting the roles of IL-12 in mediating these effects. In 2013, Deshpande and colleagues published an article in which they explored whether exposure to the homeostatic cytokines IL-7 and IL-15 altered how human T cells respond to TCR stimulation [96]. The rationale for this study was based on previous findings from this group that demonstrated that T cells from patients with rheumatoid arthritis (RA) have increased ERK phosphorylation upon TCR stimulation [97], and from the findings by other groups that showed increased levels of IL-7 and IL-15 cytokines on patients with RA [98, 99]. Therefore, they were interested in examining if there was a connection between exposure to IL-7 and IL-15 and increased responsiveness to ERK activation upon TCR stimulation. 25

47 To test their hypothesis Deshpande et al. exposed human CD4 and CD8 T cells to IL-7, IL-15, or media alone as a control for 24 h, washed the cells, and stimulated them through the TCR using anti-cd3 antibodies [96]. Exposure of human CD4 or CD8 T cells to IL-7 or IL-15 for 24 h resulted in increased activation of ERK after TCR stimulation in comparison to cells treated in media alone, similar to the increased ERK activation that they previously described in T cells from patients with RA [97]. Deshpande et al. then examined the functional consequences of the increased ERK after IL-7 or IL-15 pretreatment and found that pretreated cells have increased expression of the activation markers CD25 and CD69 and proliferation upon subsequent TCR challenge [96]. Additionally, they explored if exposure of human CD4 T cells to IL-7 or IL-15 will overcome the normal tolerance to self-antigens of CD4 T cells [96]. A hallmark of RA is antibodies against citrullinated self-proteins such as vimentin. In fact, T cell epitopes on the citrullinated vimentin that bind HLA-DR4 on the surface of APCs and can activate T cells have been identified [100]. To address their question, they incubated PBMCs of a healthy HLA-DR4 donor with the citrullinated vimentin peptide known to activate T cells following conditioning with IL-7 or IL-15 for 24 h and they assessed proliferation 7 days later. Unconditioned cells have minimal proliferation in response to citrullinated vimentin but cells exposed to IL-7 or IL-15 show strong proliferation in response to the same peptide, suggesting that exposure to IL-7 or IL-15 can lower the threshold of activation of autoreactive T cells to self-antigens. Mechanistically, they found that TCR-induced ERK activation was mediated by increased Ras [5]. Since Ras is activated by RasGRP and/or SOS they suppressed the expression of SOS on human CD4 T cells and examined whether IL-15 pretreatment still 26

48 increases the TCR induced activation of ERK [96]. Interestingly, the IL-15 mediated increase in ERK activation following TCR stimulation is substantially decreased when SOS was inhibited on the human CD4 T cells, suggesting that SOS was mediating the increase of ERK by Ras. The results from Deshpande et al. demonstrate that exposure to IL-7 or IL-15 alters the responses of T cells to TCR stimulation. The results also provide a model by which increased levels of IL-7 or IL-15, seen in patients with RA, could enable the responses of autoreactive T cells to self-antigens that mediate disease progression. Similar to the studies conducted by Deshpande et al., in 2014 our laboratory published a study in which we examined the effects of prior exposure to inflammatory signals, particularly TLR ligands, in altering how human T cells respond to subsequent TCR challenge [95]. The rationale for exploring this question was that previous studies had demonstrated that some TLR ligands, when present during TCR stimulation, can amplify TCR mediated functions; however, no studies explored whether prior exposure to TLR ligands alters the responsiveness of the T cell to TCR stimulation. To address this question, Tremblay et al. conditioned HuT78T cells (a human CD4 T cell line) or human peripheral blood activated T cells (T cells pre-activated under non polarizing conditions) with TLR ligands for 24 h, washed the cells to remove the TLR ligands, and measured TCR-induced cytokine production using plate bound anti-cd3 antibodies [95]. Tremblay et al. found that prior exposure to the TLR5 ligand (flagellin), and not TLR2 or TLR7 ligands, increased the production of IL-2 and IFN-γ after TCR stimulation. Interestingly, pretreatment with flagellin for at least 24 h was required in order to increase the production of cytokines following TCR stimulation. Also, the flagellin-mediated increase 27

49 of cytokine production upon TCR stimulation was transient, lasting only h after removal of the TLR5 ligand. To determine the mechanism by which flagellin pretreatment enhances human T cell responses, Tremblay et al. examined if flagellin (TLR5 ligand) pretreatment affects TCR signaling pathways [95]. Surprisingly, flagellin pretreatment results in reduced TCR-induced activation of LCK, LAT, and calcium flux, but does not alter the activation of ERK. Interestingly, flagellin pretreatment increased the TCR-induced activation of AKT. This study demonstrated that prior exposure to TLR5 transiently increases the production of cytokines upon subsequent TCR stimulation potentially by altering TCR signaling pathways. Collectively these studies clearly show that prior exposure to different cytokines (IL-7 and IL-15) or the inflammatory signal (flagellin) in vitro alters how human T cells respond to subsequent TCR stimulation. In addition to the studies discussed above, recent work using murine models has demonstrated that exposure to inflammatory cytokines in vivo alters how murine CD4 and CD8 T cells respond to subsequent TCR stimulation. Richer and colleagues examined the effects of exposure to inflammatory signals in vivo in altering murine effector and memory CD8 T cell responses to ex vivo re-challenge with antigen [92]. The rationale for exploring this question came from previous reports demonstrating that as an infection progresses, monoclonal populations of pathogen-specific CD8 T cells exhibit increased sensitivity to antigen [101], suggesting that signals from the environment could be responsible for regulating the antigen sensitivity of the cells. To address this possibility, Richer et al. activated a monoclonal population of CD8 T cells specific for OVA peptide (OT-I CD8 T cells), using LPS-matured DCs that were coated with OVA 28

50 peptide, in the presence or absence of an unrelated infection [92]. The unrelated infections used were Listeria monocytogenes (LM) and lymphocytic choriomeningitis virus (LCMV), which promote distinct inflammatory environments but are not a source of antigens for effector OT-I CD8 T cells. Five days after immunization, OT-I CD8 T cells were re-challenged ex vivo with titrating doses of OVA, and the dose of antigen required to stimulate the half maximal T cell response (a measure of antigen sensitivity) was assessed. Interestingly, upon re-stimulation with antigen, OVA-specific effector CD8 T cell responses from LM or LCMV infected mice are more sensitive to lower concentrations of OVA compared to the responses from uninfected mice [102]. Thus, diverse inflammatory cytokines (promoted by the unrelated infections) alter how murine effector CD8 T cells respond to subsequent stimulation. To examine the mechanisms by which pathogen induced-inflammation altered the responses of effector OT-I CD8 T cells to subsequent stimulation, Richer et al. took effector OT-I CD8 T cells at day 5 after immunization in either the presence or the absence of LCMV-induced inflammation, and stimulated them with plate bound anti- CD3 antibody [92]. Upon TCR stimulation using plate bound anti-cd3, effector OT-I CD8 T cells from LCMV immunized mice have higher sensitivity to TCR stimulation compared to effector OT-I CD8 T cells from mice immunized with DC-OVA alone, suggesting that pathogen-induced inflammation altered the sensitivity of the CD8 T cells by altering TCR signaling. Richer et al. then examined the effects of pathogen-induced inflammation on the activation of TCR signaling molecules [92]. They found that pathogen-induced inflammation increases the phosphorylation of ZAP-70, PLC-γ, ERK1/2, and JNK1 in response to TCR stimulation via CD3-antibody crosslinking. 29

51 Interestingly, pathogen-induced inflammation does not alter the TCR-induced activation of P38. In addition, using OT-I CD8 T cells lacking the receptor for IL-12 or type 1 interferons, Richer et al. found that the changes in TCR signaling and sensitivity caused by pathogen-induced inflammation are abrogated [92]. Therefore, IL-12 and/or type 1 interferon signals can directly regulate how murine effector CD8 T cells respond to TCR stimulation. Finally, using a similar system, but with memory CD8 T cells instead of effector CD8 T cells, they found that pathogen-induced inflammatory signals also alter how memory CD8 T cells respond to TCR stimulation by enhancing early TCR signaling [92]. Collectively, these studies demonstrate that murine effector/memory CD8 T cells exposed to pathogen induced-inflammation have enhanced ability to respond to TCR stimulation and this seems to be primarily driven by IL-12 and type I interferons. Similar to the experiments done by Richer et al., Kim and colleagues examined whether the inflammatory environment present during secondary challenge of murine memory CD4 T cells alters how these cells respond to TCR stimulation [94]. To this end, SMARTA T cells, a monoclonal population of CD4 T cells that is specific for an epitope of LCMV, was adoptively transferred into a naïve host. This mouse was then infected with LCMV to generate memory cells. At day 50 following LCMV infection, memory SMARTA CD4 T cells underwent a second adoptive transfer to a naïve uninfected host. Memory SMARTA CD4 T cells were then challenged with LM expressing an MHC class II restricted LCMV epitope in the presence or absence of neutralizing antibodies to IL-12 or type I interferon receptor. Seven days after secondary challenge, secondary effector SMARTA cells were isolated and re-challenged ex vivo with titrating doses of antigen, and the dose of antigen required to stimulate the maximal T cell response was determined 30

52 [94]. Secondary effector SMARTA cells isolated from mice receiving IL-12 neutralizing antibodies have lower antigen sensitivity in comparison to cells from control mice that received PBS. In contrast, secondary effector SMARTA cells isolated from mice receiving blocking antibodies to type 1 interferon receptor have increased antigen sensitivity. Interestingly, type I interferons decrease the antigen sensitivity of secondary effector SMARTA CD4 T cells whereas they increase the antigen sensitivity of effector/memory CD8 T cells. Thus, type 1 interferons may to differently regulate effector/memory CD8 T cell and secondary effector CD4 T cell responses to subsequent TCR stimulation. Likewise, Raue et al. examined the effects of short exposure to IL-12 and IL-18 in vitro in altering how murine memory CD8 T cells respond to TCR stimulation. CD8 T cells were isolated from spleens from LCMV immune mice (>100 days post-infection) and were incubated in vitro with IL-12 and IL-18 or media alone for 5 h and then incubated for 3 days in medium without exogenous cytokines. Cytokine production was then measured after re-stimulation using a B cell line (A20) coated with the NP peptide. They found that after stimulation with antigen, memory CD8 T cells that were pretreated with IL-12 and IL-18 have an increased frequency of IFN-γ and TNF-α double producers and increased production of these cytokines on a per cell basis compared to cells that were treated in media alone. Raue et al. also looked at cytotoxicity by measuring granzyme B expression and by measuring killing of peptide coated target cells. Memory CD8 T cells that were pretreated with IL-12 and IL-18 have increased granzyme B expression and enhanced lysis of peptide coated cells. Thus, brief exposure 31

53 to IL-12 and IL-18 increased the responses of memory CD8 T cells to subsequent challenge. Conclusions The studies discussed above clearly showed that different cytokines or inflammatory signals alter the responses of activated T cells (effector or memory) to TCR stimulation and also provided mechanistic insight into how cytokines or inflammatory signals mediate their effects. Interestingly, cytokines and inflammatory signals alter TCR signaling pathways, and each cytokine or inflammatory signal alters TCR-mediated signaling via a distinct molecular mechanism. In this thesis, I answered some of the many unaddressed questions that remain in the field. Richer et al. and Kim et al., showed that murine effector/memory CD8 or secondary effector CD4 T cells exposed to in vivo inflammatory signals have an altered response to ex vivo re-challenge with antigen that is driven primarily by IL-12 and/or type I interferons. Likewise, Raue et al. showed that murine memory CD8 T cells conditioned with IL-12 and IL-18 in vitro have enhanced cytokine production and cytotoxic activity upon re-stimulation with antigen. Whether inflammatory signals will regulate the function of human T cells similar to the murine T cells remains to be elucidated. Also, how individual inflammatory cytokines or combinations of inflammatory cytokines will alter the responses of human T cells remains unanswered. In addition, the functional consequences of exposing human T cells to inflammatory signals remains to be fully explored. In CHAPTERS III and V I examined these questions using human CD4 and CD8 T cells. From the different inflammatory cytokines tested I found that IL-12 selectively altered the responses of 32

54 human CD4 and CD8 T cells. In CHAPTERS IV and V I explored the precise molecular mechanisms by which IL-12 alters subsequent TCR-mediated responses. 33

55 Figure 1: T cell receptor signal transduction. One of the early events seen upon stimulation of the TCR is the phosphorylation and activation of LCK and FYN. LCK and FYN phosphorylate the ITAMS on the TCR subunits leading to the recruitment and activation of ZAP-70. Activated ZAP-70 phosphorylates several tyrosine residues on LAT and SLP-76, leading to the formation of a multi-protein signaling complex. These complexes regulate PLC-γ activation which promotes Ca 2+ influx and generation of DAG, which in turn promote the activation of the transcription factors NFAT and NFκB. DAG also recruits the guanine-nucleotide exchange factor RasGRP that activates Ras. Activated Ras promotes the activation of the MAPK ERK1/2 which translocates to the nucleus and regulates the transcription of numerous genes. Also upon TCR stimulation GRB2 binds to LAT and recruits the guanine-nucleotide exchange factor SOS1. At the membrane SOS1 activates Ras. SLP-76 in cooperation with LAT recruits and activates PI3K and activated PI3K generates PIP 3, which recruits PDK1 and AKT. At the membrane AKT is activated and regulates multiple cellular events related to proliferation, growth, survival and metabolism. Finally, TCR stimulation also results in the activation of the MAPKs P38 and JNK. P38 is activated by the kinases MKK3, MKK4, and MKK6; and JNK is activated by the kinases MKK4 and MKK7. The direct upstream activators of these kinases are not well characterized. Activated P38 and JNK have many targets including transcription factors and protein kinases. Collectively, the activation of these signaling proteins is critical for the successful activation and function of the T cell. 34

56 PIP 2 DAG IP3 LCK & FYN ZAP-70 RasGRP PKC-θ Ca ++ PIP 2 PIP 3 PI3K MKK 4/7 MKK 3/4/6 Ras NFκB NFAT AKT PDK1 JNK p38 Erk1,2 Cytokine release Receptor upregulation Proliferation/survival Cytotoxicity 35

57 Figure 2: Overview of T cell responses. A) During the course of an infection tissue resident APCs such as DCs take up the infectious agents and transport them via the lymphatic system to the nearest lymph node or secondary lymphoid organs. Once it reaches the local lymph nodes, the DCs will start presenting antigens derived from the pathogen along with co-stimulatory molecules and cytokines. B) Mature naïve T cells are constantly recirculating between the blood and secondary lymphoid organs. In the case that the naïve T cells encounters a properly activated DC displaying the appropriate peptide/mhc ligand, the naïve T cells becomes activated, starts clonally expanding, and differentiates into an effector T cell. In order to have successful activation of the naive T cells, there at least three signals that are critical. Signal 1, compromise the binding of the TCR with its cognate antigen bound to MHC molecules, Signal 2, which are provided by co-stimulatory signals, and Signal 3 provided by different inflammatory signals. C) Activated T cells circulate various tissues including the tissue where the antigen came. At the sites of infection, the antigen experienced T cells that encounter APCs presenting their cognate antigens will execute their effector functions to clear the infection. After clearing the infection the majority of the activated T cells will die. However, some will differentiate into a pool of memory T cells that have a variety of migratory properties and functions. 36

58 A Secondary lymphoid tissue Peripheral Tissue Circulation B Pathogen DC Activated DC Inflammatory cytokines Naïve T cell Activated T cell C Secondary lymphoid Peripheral Tissue Circulation 37

59 Figure 3: Production of IL-12 and IL-12 signaling pathway. IL-12 is primarily produced by monocytes, macrophages, neutrophils, DCs and B cells following induction of pattern recognition receptors (PRRs) by organisms such as bacteria (Gram + and Gram -), parasites, viruses, and fungi. IL-12 is a heterodimeric cytokine of 70 kda composed of two subunits, p35 (35kDa) and p40 (40kDa). IL-12 promotes its biological activities by binding to the IL-12 receptor (IL-12R) which is composed of two subunits the IL-12R β1 and β2. Upon binding of IL-12 with the IL-12R, the kinases JAK2 and Tyk2 become activated and phosphorylate tyrosine residues in the IL-12R β2 which recruits STAT4. At the receptor STAT4 molecules becomes phosphorylated and activated and translocates to the nucleus to regulate gene transcription. The STAT4-mediated pathway appears to be one of the critical mediators of the canonical effects of IL-12. However, other STAT molecules are also activated by IL-12. In addition, other pathways have been shown to be activated by IL-12 like P38, LCK, and possibly AKT. 38

60 Pathogen TLR s DCs, monocytes, macrophages, neutrophils, or B cells IL-12 cytokine p35 p40 T cell Tyk2 Jak2 LCK p38 AKT STAT1,3,4,5 Target genes Th1 differentiation IFN-γ production 39

61 CHAPTER II: MATERIALS AND METHODS This chapter contains information about the most common materials and methods used throughout the dissertation. Ethics statement Blood donors recruited by the DeGowin Blood Center at the University of Iowa Hospitals and Clinics provided informed consent for cells not used for transfusion to be used for research. The consent process was approved by the Institutional Review Board (IRB) for the University of Iowa. Our laboratory was not provided with information about the age, gender, or health status of the donors. However, we requested that donors were between 18 and 55 years, and that they were not taking any anti-viral or antibacterial medications at the time of donation. Because all cells used in these studies were obtained from normally discarded products, the donors approved for the use of their cells in research projects, and the donors were completely de-identified, these studies were exempt from further IRB approval. All human subject studies were in compliance with the Declaration of Helsinki. 40

62 Reagents RPMI 1640, Iscoves Modified Dulbecco s Media (IMDM), L-glutamine, penicillin-streptomycin, and phosphate buffered saline (PBS) were purchased from Gibco/Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Lawrenceville, GA, USA). The Lymphoprep and the human pan CD4 T cell and pan CD8 T cell enrichment kits were purchased from Stem cell Technologies (Vancouver, BC, Canada). PVDF-FL Transfer membranes were bought from Millipore (Billerica, MA, USA). The fixation buffer, the permeabilization wash buffer, the brefeldin A solution, and the monensin were from Biolegend (San Diego, CA, USA) The Fluor-4-AM dye, the magnetic Dynabeads, DyLight680 or 800CW-conjugated secondary antibodies used in dual-color immunoblotting, were bought from Invitrogen/Life Technologies (Grand Island, NY, USA). The CellVue Burgundy cell labeling kit, NEWBLOT PVDF Stripping Buffer, and near-infrared conjugated secondary antibodies used in dual-color immunoblotting were purchased from LI-COR Biosciences (Lincoln, NE, USA). The TMB Peroxidase Substrate Solutions were obtained from KPL (Gaithersburg, MD, USA). Costar 96-well EIA/RIA flat-bottom plates were from Corning Incorporated (Corning, NY, USA). Tissue culture-treated 6 or 24-well plates were from Corning Incorporated or BD (Franklin Lakes, NJ, USA). SEA BLOCK blocking buffer and the SYBR Green master mix from were acquired from Thermo Scientific (Waltham, MA, USA). The annexin V and propidium iodide were from BD (San Jose, CA, USA). The recombinant human IL-2 used in human T cell cultures was obtained from the AIDS Research and Reference Reagent Program, Division 41

63 of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA. The recombinant proteins used as standards for the enzymelinked immunosorbent assay (ELISA) and for the treatments were obtained from R&D Systems Inc (Minneapolis, MN, USA). The SB was from Cayman Chemical (Ann Arbor, MI, USA). The RNeasy Mini Kit and RNase-free DNase set was from Qiagen. All chemicals used in these studies were research grade and were obtained from various sources. Antibodies The following antibodies were used for immunoblotting, cell-surface, and intracellular stains: The anti-lat Y191 and anti-lat from Millipore. The anti-lck py505, and anti-slp-76 py128, anti-il-12rβ1 from BD Biosciences. The anti-plc-γ1 py783, anti-p38 pt180/y182, anti-akt pt308, anti-zap-70 py319, anti-src py416, anti-plc-γ1, anti-lck, anti-slp-76, anti-akt, anti-stat4, anti-stat4 py693, anti- S6 ribosomal protein ps235/236, and anti-erk 1/2 antibodies were purchased from Cell Signaling Technologies. The anti ERK1/2 ptpy185/187 and anti-akt ps473 were from Invitrogen. The anti-gapdh was from Meridian Life Science. The DyLight 800 and DyLight 680 labeled secondary antibodies were obtained from Thermo Scientific. The FITC anti-ifn-γ, Alexa Fluor 647 anti-il-10, Alexa Fluor 647 anti-il-4, Alexa Fluor 647 anti-tnf-α, and APC anti-il-13 all from BioLegend. The anti-cd3, anti-cd4, anti- CD8, anti-il-2, anti- IFN-γ, anti- TNF-α, anti-il-4, anti-il-13, anti-il-10, biotin anti-il- 2, biotin anti- IFN-γ, biotin anti- TNF-α, biotin anti-il-4, biotin anti-il-13, biotin anti- 42

64 IL-10, anti-cd45 RO, anti-cd8 FITC, anti-cd69 PE/Cy5, anti-cd28, anti-cd-2, anti- CD49d, anti-cd11a, FITC anti-cd278, anti-cd150, and DyLight 488 IgG antibodies were obtained from Biolegend. The goat anti-mouse IgG from Southern Biotech, Anti- IL-12 Rβ2 was purchased from R&D Systems. The anti-mouse Ig from Rockland. Cell lines HuT78 T cells were purchased from ATCC (Manassas, VA, USA) and were grown in IMDM (20% FBS, 2 mm l-glutamine, 50 U/ml penicillin, 50μg/ml streptomycin) at 37 C in 5% CO 2. Cells were grown in IMDM to a concentration of 6x10 5 cells/ml. Human peripheral blood CD4 and CD8 T cell isolation and activation Leukocyte reducing system (LRS) cones were provided to investigators at the University of Iowa by the DeGowin blood center. Peripheral blood mononuclear cells (PBMCs) were obtained from these cones by flushing the contents of the cone using isolation buffer (PBS containing 2 mm EDTA and 2% FBS) and then using Hypaque- Ficoll density-gradient separation. Then PBMCs were washed three times with 1X PBS and resuspended in complete RPMI media (RPMI 1640 with 10% FBS, penicillin/streptomycin, and l-glutamine) at 37 C in 5% CO 2 for at least 4 h before further isolation of T cells. For the experiments performed in CHAPTER III and IV CD4 T cells 43

65 were negatively selected from the PBMCs using the human pan CD4 T cell enrichment kit enrichment kit following manufacturer instructions (Stem Cell Technologies). For the experiments in CHAPTER V, CD8 T cells were negatively selected from the PBMCs using the human pan CD8 T cell enrichment kit also from Stem Cell Technologies and following manufacturer instructions. Following CD4 T cell isolation, we examined the purity of the cells by examining CD4 expression and the distribution of naïve/antigen experienced cells by looking at CD45RO. Cells were consistently greater than 99% positive for CD4 and approximately 60% of the cells were positive for CD45RO (Fig. 4), suggesting that the isolation method effectively yielded pure CD4 T cells that consisted of a mix of naïve and antigen experienced populations. We also examined the phenotype of the CD8 T cells following isolation. Cells were around 99% positive for CD8 and around 58% positive for CD45RO, again showing that the isolation yielded pure CD8 T cells populations that are a mix of naïve and antigen experienced cells (Fig. 4). The isolated CD4 and CD8 T cells were then preactivated for 5 days with magnetic Dynabeads (Invitrogen) bound with anti-cd3 (OKT3, BioLegend) and anti-cd28 (CD28.2, BioLegend) antibodies in the presence of 100 U/mL IL-2. During the activation, cells were maintained at concentration of 1.5x10 6 cells/ml by adding complete RPMI and IL-2. This activation method consistently resulted in a population of cells with a phenotype of an antigen experienced T cell (around 90% cells positive for CD45RO) (Fig. 4). Therefore, our system is composed of naïve CD4 T cells primed under non polarizing conditions and recently activated memory CD4 T cells; in this thesis I will refer to these cells as activated T cells. Following activation, the stimulatory signals were removed and the cells were rested for 24 h in complete RPMI before further 44

66 testing. The successful removal of the stimulatory signals (mouse anti-tcr/ mouse anti- CD28 antibodies) was controlled in our cell cultures by staining with anti-mouse conjugated antibodies. As shown in Fig. 4, both the unstained control and the stained cell had similar fluorescence, this lack of fluorescence in the stained cells suggests that the stimulatory signals were effectively removed. Alternatively in CHAPTER III, isolated human peripheral blood CD4 T cells were also used immediately after isolation without preactivation with CD3/CD28 antibodies and IL-2. Cytokine pretreatment of human CD4 and CD8 T cells Activated human CD4 (CHAPTERS III and IV) or activated CD8 T cells (CHAPTER V) were resuspended at 1.5 x 10 6 cells/ml in complete RPMI and treated with or without recombinant cytokines (R&D Systems) for different times and doses. After pretreatment, cells were washed with RPMI 1640 three times to remove the cytokines. Alternatively, in CHAPTER III, HuT78 T cells were resuspended at 1 x 10 6 cells/ml in IMDM and pretreated with or without recombinant IL-12 (50 ng/ml for 24 h). Then cells were washed three times with IMDM to remove the cytokines. Cytokine production measured by ELISA After the different cytokine pretreatments, cells were resuspended at 1 X 10 6 cells/ml in complete RPMI and stimulated with different doses of plate-bound anti-cd3 45

67 antibodies (that induce TCR stimulation) for 24 h. For simplicity we will use the term anti-tcr when referring to anti-cd3 antibodies from now on. For the rest of the experiments in CHAPTERS III and V, pretreated cells were rested for various times before anti-tcr antibody stimulation. For experiments using oligomycin and 2-DG inhibitors in CHAPTER IV, IL-12 pretreated or untreated cells were TCR-stimulated in the presence of oligomycin (2.5 μm) or 2-DG (5 mm). Alternatively, in some experiments in CHAPTER III, IL-12 pretreated cells were TCR stimulated in the presence of IL-12 (50ng/mL). Following TCR stimulation, cell suspensions were collected in 1.5 ml Eppendorf tubes and centrifuged at 13,000 rpm for 1min. Then culture supernatants were collected and protein levels of the cytokines were measured in the culture supernatants in triplicate using standard TMB based ELISA. The cytokine protein levels were measured immediately or after no more than 24 h at -20 C. To account for the variations between human donors and the conditions of independent experiments, in CHAPTER III, data were normalized for each donor as: Fold-increase over no cytokine = (concentration of treated sample maximum concentration of no cytokine sample). Functional avidity in CHAPTERS III and V was determined as follows: Percent maximum response = (concentration of sample maximum concentration of respective treatment) x 100%. Normalized data were fitted to a sigmoidal curve to calculate EC 50. Intracellular cytokine and surface molecule staining For intracellular staining, IL-12 pretreated or untreated cells were resuspended at 1 x 10 6 cells/ml in complete RPMI and stimulated with different doses of plate-bound 46

68 anti-tcr antibodies for 8, 18, or 24 h. Brefeldin A (BFA) or Monensin (BioLegend) were added for the last 6 h of TCR stimulation in most experiments unless otherwise noted. Following TCR stimulation, cells were washed in FACS buffer (PBS, 10% FBS, and 0.05% sodium azide), and then stained for intracellular cytokines per manufacturer s suggestion. Samples were collected using an Accuri C6 flow cytometer. Live lymphocytes were gated based on forward and side scatter. Quadrants markers were set so that the baseline cytokine production of non-tcr stimulated cells have a frequency of less than 1%. Then, the frequencies of cells making cytokines and the median fluoresce intensity (median FI) were determined using the Accuri C6 flow Plus software. For surface molecule staining, cells were washed in FACS buffer and stained on ice with the primary and/or secondary antibodies, followed by FACS analysis. Live lymphocytes were gated based on forward and side scatter. The median FI of each sample was obtained using an Accuri C6 flow cytometer. Cell viability assays Activated human CD4 T cells (CHAPTER III) or CD8 T cells (CHAPTER V) were incubated with or without various cytokines for various times, washed, and stimulated with or without plate bound anti-tcr antibodies. Cell viability was determined before and after TCR stimulation using the trypan blue dye exclusion assay, and live and dead cells were counted using a hemocytometer. Alternatively, IL-12 pretreated and untreated cells (TCR stimulated or unstimulated) were washed in annexin V binding buffer and stained with annexin V and propidium iodide (PI) per 47

69 manufacturer s instructions (BD Biosciences). Annexin V and PI double negative cells were considered as viable cells, annexin V positive but PI negative cells as apoptosis undergoing cells, and annexin V and PI double positive cells as dead cells. Quantitative Real-time PCR Total RNA was isolated with an RNeasy Kit (Qiagen) from IL-12 pretreated and untreated cells at 6 and 18 hours following stimulation with plate-bound anti-tcr antibodies. Single-strand cdna was then synthesized from 1μg of total RNA using the High Capacity cdna Reverse Transcription Kit (Applied Biosystems). Real-time RT- PCR was performed on an Applied Biosystems Model 7000 using SYBR Green PCR master mix (Applied Biosystems) and primers according to the manufacturer s instructions. The expression of mrna was normalized to that of mrna encoding β-actin and quantification of fold induction of treated vs untreated was analyzed by the 2-ΔΔCT method [103]. Immunoblotting To examine STAT4 expression and activation, cells were treated with IL-12 (50 ng/ml) for different times and immunoblotting was performed. To examine the expression and activation of TCR signaling molecules in CHAPTER IV, activated human CD4 T cells were treated with 2 μg/ml of anti-tcr and anti-cd4 antibodies on ice for 48

70 30 minutes. We use anti-tcr antibodies in combination with anti-cd4 antibodies because this provides the best enhancement of proximal and distal TCR signaling [ ]. Cells were then warmed at 37 C for 10 min and crosslinked with 25 μg/ml of antimouse IgG antibodies for various times. Immunoblotting was then performed. To examine the expression and activation of TCR signaling molecules in CHAPTER V, activated human CD8 T cells were treated with 3 μg/ml of anti-tcr antibodies on ice for 30 minutes, then warmed at 37 C for 10 min, and then crosslinked with 25 μg/ml of anti-mouse IgG antibodies for various times. Immunoblotting was then performed. For immunoblotting, the proteins were first separated using SDS-PAGE, then proteins were transferred to PVDF membranes, and membranes were blocked using 50% (v/v) SEA BLOCK buffer diluted in PBS. Membranes were then incubated with one or two primary antibodies of different species overnight at 4 C. Then membranes were washed 2X using PBST (PBS ph 7.2 and 0.1% Tween 20) and incubated with DyLight 680- and DyLight 800-conjugated secondary antibodies for 45 minutes at room temperature. Subsequently, the membranes were washed once with PBST containing 0.05% SDS and twice with PBST alone. The immunoblots were visualized using the LICOR Odyssey Infrared Imager. In some instances, dual-color immunoblots were stripped of their antibodies using NEWBLOT PVDF Stripping Buffer diluted 1:3 in PBS. These blots were incubated for 20 min at room temperature and washed extensively with PBST prior to re-probing with antibodies as described above. The intensity of the immunoblotting bands was determined using the Licor Odyssey v3.0 software. The phospho-protein intensity was normalized to the expression of actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the following formulas: 49

71 (1) Normalized actin or GAPDH = Raw intensity of actin or GAPDH of time point raw intensity of lowest actin or GAPDH value. (2) Normalized intensity at time point = Raw intensity of phospho-protein at time point Normalized actin or GAPDH value at time point. (3) % of the control maximum = (Normalized intensity at time point Normalized intensity of maximum control value) 100% The normalized values were then averaged and expressed as the mean ± s.e.m. as indicated in each figure legend. Seahorse and ECAR/OCR measurements After cytokine pretreatments, cells were resuspended at 3 x 10 5 cells in 125 μl of XF media (DMEM + 25 mm glucose + 1 mm pyruvate + 2 mm glutamine) and were plated onto XF-96 plates previously coated with poly-l-lysine. Optimal cell concentrations were determined based on previous literature [ ]. Cells were then incubated for 30 min at 37 C in a non-co 2 incubator and their metabolic profiles were subsequently examined using an XF-96 Extracellular Flux Analyzer (Seahorse Bioscience) following the manufacturer s suggested protocol. The optimal concentrations of mitochondrial inhibitors were determined experimentally to be: Oligomycin 2.5 μm; FCCP 1.5 μm; Rotenone 5 μm; Antimycin A 5 μm) (data not shown). Mitochondrial respiration and aerobic glycolysis were assessed by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). OCR and ECAR values were normalized to cell numbers as previously described [111]. Each experimental group was 50

72 analyzed using five replicates and repeated in separate donors. Metabolic parameters were then assessed as described before using: (1) Basal respiration = (Basal OCR before adding Oligomycin) (OCR following Rotenone/Antimycin A). (2) Maximal respiratory capacity = (OCR peak rate following FCCP) (OCR following Rotenone/Antimycin A). (3) Basal ECAR= Basal ECAR rate before adding Oligomycin (4) Maximal ECAR= ECAR rate following addition of Rotenone/Antimycin A. OCR and ECAR time courses and metabolic parameters were plotted using GraphPad Prism. Statistical analysis Statistical analysis between the groups was assessed using GraphPad Prism or Microsoft Excel. Specific tests for statistical significance are indicated in the figure legends. Differences were considered significant when p values were below

73 Figure 4: Isolation and activation of human CD4 and CD8 T cells. (A) Human CD4 T cells were isolated from PBMCs. Cells were stained with CD4 and CD45RO antibodies before activation (left), and following 5 day activation with anti-tcr/cd28 antibodies and recombinant IL-2 (right), and analyzed by flow cytometry. (B) Human CD8 T cells were isolated from PBMCs. Cells were stained with CD8 and CD45RO antibodies before activation (left), and following 5 day activation with anti-tcr/cd28 antibodies and recombinant IL-2 (right), and analyzed by flow cytometry. (C) Human CD4 T cells were activated as described above, then stimulatory signals were removed, and cells were left unstained (black line) or stained with anti-mouse fluorescently labeled antibodies (gray line). Then cells were examined by flow cytometry. Data shown in (A-C) are representative plots of two to four different donors. 52

74 A Day 0 Day 5 B Day 0 Day 5 C 53

75 CHAPTER III: ROLE OF PRIOR EXPOSURE TO IL-12 IN ALTERING HUMAN CD4 T CELL RESPONSES TO TCR STIMULATION Introduction Upon encounter with cognate antigen presented by APCs, naïve CD4 T cells proliferate and differentiate into effector and memory subsets. These activated (effector or memory) CD4 T cells then recirculate through various tissues to aid infection clearance and protect against pathogen re-exposure. Emerging literature suggests that T cell differentiation occurs in several steps. Initially, naïve and central memory CD4 T cells are primed in secondary lymphoid organs. These primed but undifferentiated cells are released from the secondary lymphoid organs and migrate to the sites of inflammation. Full CD4 T cell differentiation then occurs at sites of infection and inflammation after they have migrated from the secondary lymphoid organs [33] Furthermore, classically the activated CD4 T cells were thought to differentiate into different subsets that were mutually exclusive and irreversible [37, 112]. However, it is becoming increasingly clear that the activated CD4 T cells are not all terminally differentiated and the majority can actually remain flexible and be re-polarized in different inflammatory environments [37, 40, 112, 113]. Therefore, it is clear that the myriad of inflammatory signals that effector and memory CD4 T cells receive from the environment play key roles in influencing their subsequent responses. One of the signals controlling CD4 T cell function is the inflammatory cytokine IL-12. This cytokine is produced in response to pathogens, but is also present at 54

76 inflammatory sites in human disorders [61, ]. The current paradigm is that IL-12 primarily promotes the differentiation of naïve CD4 T cells into Th1 cells [61]. Also, the presence of IL-12 during priming of T cells in secondary lymphoid organs has been shown to promote strong effector functions and memory development [29]. Finally, IL- 12 enhances TCR-induced proliferation, IFN-γ production and cytotoxicity of T cells [61]. Although highly informative, this work has largely examined the effects of IL-12 if it s present during or following TCR activation. However, during immune responses, activated CD4 T cells will be exposed to IL-12 as they migrate through the blood or lymph and at sites of infection before they are further activated through the TCR in infected and inflamed tissue. The effects of IL-12 in altering the response of activated, non-fully differentiated T cells to subsequent TCR stimulation remains incompletely understood. Recent murine studies, suggest that IL-12 alters the response of activated T cells to subsequent TCR stimulation, suggesting that the modulation of CD4 T cell responses by IL-12 is more complex than simply inducing the differentiation of Th1 cells. In this regard, Richer et al. and Kim et al. demonstrated that murine effector/memory CD8 T cells or secondary effector CD4 T cells exposed to in vivo inflammatory signals, driven primarily by IL-12 and/or type I interferons, have an altered response to ex vivo rechallenge with antigen [92, 94]. In these studies, exposure to IL-12 decreased the dose of antigen required to stimulate the half maximal T cell response (also known as functional avidity). Likewise, murine memory CD8 T cells conditioned with IL-12 and IL-18 in vitro have enhanced cytokine production and cytotoxic activity upon TCR re-stimulation [93]. In addition, previous studies from us and others has demonstrated that prior 55

77 exposure to IL-7, IL-15 or a TLR5 ligand increases the responsiveness of human T cells to TCR stimulation [95, 96]. Collectively these studies suggest that prior exposure to different cytokines or inflammatory signals alters how T cells respond to TCR stimulation. Although these studies provide insight into murine T cell biology there are still key questions that remain to be explored. It is unclear if prior exposure to IL-12 will also alter the responses of activated human CD4 T cells to TCR stimulation. We will examine this knowledge gap further in this chapter. In addition, the precise molecular mechanism by which IL-12 alters subsequent TCR-mediated responses has not been fully elucidated. This question will be addressed in CHAPTER IV. To address these questions, we used a system composed of human peripheral blood CD4 T cells that have been recently activated under non-polarizing conditions which models primed, yet not fully differentiated, human CD4 T cells that are released from the secondary lymphoid organs into circulation. In this chapter, we found that prior exposure to IL-12 primes activated human CD4 T cells to produce a range of cytokines upon TCR re-stimulation. These data suggest that the regulation of CD4 T cell function by IL-12 is more complex than simply driving Th1 differentiation. Instead it seems that IL-12 is continually shaping human CD4 T cell responses in a context-specific manner. Based on our results we propose a model in which IL-12 present in blood, infection sites, and/or at inflammatory sites primes human effector or memory CD4 T cells that are not terminally differentiated, allowing them to respond faster when they encounter their cognate antigen at sites of infection and allowing them to be easily polarized depending upon the cytokine milieu they encounter. 56

78 Results Prior exposure to IL-12 selectively potentiates ensuing TCR-induced IFN-γ production in activated human CD4 T cells Previous studies have demonstrated that exposure to inflammatory cytokines, such as IL-12 and type I interferons, alters the responsiveness of murine effector/memory CD8 T cells and secondary effector CD4 T cells to subsequent activation through the TCR [92, 94]. To determine whether these mechanisms are also involved in regulating the function of human CD4 T cells, we first examined whether conditioning a human CD4 T cell line (HuT78 cells) with IL-12 will alter how these cells respond to TCR stimulation. HuT78 T cells are a CD4 T cell line, derived from a patient with Sézary syndrome [118], that has been previously shown by our lab and others to behave very similar to primary human T cells [106]. Thus, it provides a useful tool and excellent starting point to examine the effects of IL-12 on human T cells. To address the question, HuT78 T cells were exposed to IL-12 for 24 h, then extensively washed to remove the IL-12, and stimulated with plate-bound anti-tcr antibodies for 24 h. Then IL-2 production was determined in the cell cultures by ELISA. As expected, TCR stimulation increased the production of IL-2 in cells conditioned in media alone (No cytokine) (Fig. 5). However, cells pretreated with IL-12 produced significantly more IL-2 in response to TCR stimulation than control cells pretreated in media alone (Fig. 5). These results using a human CD4 T cell line suggest that, similar to the previous murine studies, prior exposure to IL-12 alters how human CD4 T cells respond to TCR stimulation. These experiments were performed by Samantha Freedman who was rotating in our laboratory 57

79 as part of a summer program. As mentioned above, previous studies have shown that murine effector/memory CD8 T cells and secondary effector CD4 T cells exposed to pathogen-induced inflammation have an altered ability to respond to TCR stimulation [92, 94]. Using IL-12 receptor β1 and type 1 interferon receptor deficient cells these studies demonstrated that IL-12 and type 1 interferons are important in mediating these responses. We sought to determine if similar effects will occur in primary activated human CD4 T cells, and if so, which cytokines are important in mediating these effects. In our experimental setup, we consider activated CD4 T cells to be primary human peripheral blood CD4 T cells that were preactivated for 5 days with anti-tcr/cd28 antibodies and recombinant IL-2 (non polarizing conditions) and then rested without stimulatory signals for 24 h before further analysis. Therefore, the cells in our system will be a mix of not fully differentiated effector and memory CD4 T cells. This experimental setup is intended to mimic the in vivo situation of a recently activated, not fully differentiated, effector or memory CD4 T cell entering a site of infection, where it will encounter inflammatory signals and antigen. Activated CD4 T cells were incubated with recombinant IL-12, IFN-γ, IFN-β, TNF-α, IL- 4 or media alone (no cytokine) for 6 h. After pretreatment, cells were extensively washed to remove the cytokines and stimulated with plate-bound anti-tcr antibodies for 24 h, with IFN-γ production assessed as readout for TCR activation. No other additional costimulatory signals were provided in order to determine the effects of the inflammatory cytokine strictly on TCR-induced T cell activation. Pretreatment with IFN-γ, IFN-β, TNF-α, or IL-4 had no significant effect on TCR-induced IFN-γ production in comparison to cells treated in media alone (Fig. 6A, 58

80 6B). In contrast, pretreatment with IL-12 significantly enhanced the TCR-induced production of IFN-γ compared to cells that were not treated with cytokines (Fig. 6A, 6B). IL-12 pretreatment alone did not cause measurable IFN-γ production, suggesting that this factor selectively altered TCR-induced cytokine production (Fig. 6B). Next, we examined the effects of IL-12 when used in combination with the other inflammatory cytokines. No synergistic or antagonist effects on IFN-γ production were seen when IL-12 was used in combination with IFN-γ, IFN-β, or TNF-α (Fig. 6C, 6D). Together, these data show that prior exposure to IL-12 enhances the TCR-induced production of IFN-γ in activated human CD4 T cells. Interestingly, these effects appear selective for IL-12, since pretreatment with other cytokines did not alter how activated human CD4 T cell respond to TCR stimulation. Short pretreatments with low doses of IL-12 are sufficient to transiently potentiate the TCR-mediated production of IFN-γ To more fully characterize these effects, the optimal dose of IL-12 that increases TCR-induced IFN-γ production was determined. To test for this, activated human CD4 T cells were pretreated for 6 h with various doses of IL-12 and then stimulated with platebound anti-tcr antibodies. Pretreatment of cells with doses of ng/ml of IL-12 significantly augmented IFN-γ production in comparison to cells with no IL-12 pretreatment (Fig. 7A, 7B). Next, the duration of IL-12 pretreatment required to increase IFN-γ production was examined. As shown in Fig. 7C and 7D, exposure to IL-12 for at least 1.5 h was required to significantly increase the production of IFN-γ in comparison to cells with no IL-12 pretreatment. The enhancement of IFN-γ production was also seen 59

81 with longer pretreatments (6, 12, 24, 48 h); however, there were no significant differences between any of the times for 1.5 h of pretreatment or greater (Fig. 7C, 7D, and data not shown). This observation also suggests that the effects of IL-12 seen are not due to the presence of residual cytokine during TCR stimulation, since any potential effects from residual IL-12 would be observed at all times of pretreatment. Finally, we examined the duration of the effects after the removal of IL-12. To accomplish this, activated CD4 T cells were rested for various times after treatment with IL-12 before then being stimulated through the TCR. As shown in Fig. 7E and 7F, the ability of IL-12 to enhance IFN-γ production following TCR stimulation was short-lived and lasted for 3-6 h after cessation of IL-12 treatment. Collectively, these experiments show that IL-12 pretreatment transiently increases TCR-induced IFN-γ production after exposure with low doses of IL-12 for at least 1.5 hours. Conditioning activated human CD4 T cells with IL-12 does not alter their functional avidity As stated previously, murine effector/memory CD8 T cells and secondary effector CD4 T cells exposed to inflammatory signals have an altered ability to respond to TCR stimulation [92, 94]. In these studies, the inflammatory cytokines, particularly IL-12, decreased the dose of antigen required to stimulate the maximal T cell response, also known as functional avidity. Therefore, we examined if IL-12 pretreatment will have similar effects in activated human CD4 T cells using an antibody based stimulation. To this end, IL-12 pretreated or untreated cells were stimulated with titrating doses of anti- TCR antibodies and the production of IFN-γ was determined. The functional avidity was 60

82 then determined by calculating the dose of stimulatory antibody needed to induce 50% of maximal IFN-γ production (EC 50 ). We found that IL-12-pretreated cells stimulated with different doses of anti-tcr antibodies had a dose dependent increase in IFN-γ production. The IL-12 mediated increase in IFN-γ production was statistically significant for all doses of anti-tcr antibodies higher than 2 µg/ml (Fig. 8A, 8B). We also found that IL-12 exposure does not alter the functional avidity of activated human CD4 T cells in response to TCR ligation alone (Fig. 8C, 8D). These results demonstrate that the IL-12 mediated increase in IFN-γ production is seen with even low doses of anti-tcr antibodies. Furthermore, in an antibody based stimulation system, IL-12 exposure did not alter the functional avidity of activated human CD4 T cells in response to TCR ligation. Pretreatment of activated human CD4 T cell with IL-12 does not alter the expression of the activation marker CD69 following TCR stimulation TCR stimulation not only results in cytokine production but also results in changes in the phenotype of the cells. One of the early surface molecules that its upregulated following TCR stimulation is CD69, therefore the expression of this surface molecule is used as an activation marker [119]. Since IL-12 pretreatment increased the production of cytokines following TCR stimulation, we examined if IL-12 pretreatment will also alter the activation phenotype of the cells. To this end, activated human CD4 T cells were pretreated for 6 h with IL-12 (50 ng/ml), and then the induction of the activation marker CD69 was assessed by flow cytometry before and after stimulation with plate-bound anti-tcr antibodies. Following TCR stimulation both control and IL- 12 pretreated cells upregulated the expression of CD69 to a similar extent suggesting that 61

83 IL-12 pretreatment did not alter the TCR-induced activation of the cells (Fig. 9). Similarly, both IL-12 pretreated and control cells had similar levels of CD69 expression in cells that were not TCR stimulated, suggesting that IL-12 pretreatment alone was not altering the activation phenotype of the cells (Fig. 9). IL-12 exposure does not alter the basal proliferation/survival of activated human CD4 T cells Previous literature has shown that IL-12 exposure increases the basal proliferation and survival of murine CD4 T cells [87]. In these studies, cell numbers and cell survival were compared between cells exposed to IL-12 or media alone at different times. Based on these results, we first determined if exposure to IL-12 alone will alter the basal proliferation/survival of human CD4 T cells. To this end, activated human CD4 T cells were incubated with or without IL-12 for different times and cell viability/survival were assessed by using a trypan blue dye exclusion assay. Using trypan blue dye exclusion assay, we found that cells treated in media alone and IL-12 pretreated cells had similar viability during the time of our experiments ( h) (Fig. 10A). It is worth noting that after 48 h, cells exposed to IL-12 had a consistent, but not significant, trend towards an increase in cell viability compared to cells treated with medium alone (Fig. 10A). We further characterized the effects of 6 h IL-12 exposure (pretreatment time used in most of our experiments) in cell survival, by assessing apoptosis via staining with annexin V and PI. In this assay, annexin V and PI double negative cells were considered as viable cells, annexin V positive but PI negative cells undergoing apoptosis, and annexin V and PI double positive cells as dead cells. We found that IL-12 exposure for 6 h did not alter the 62

84 frequency of dead cells or cells undergoing apoptosis compared to untreated cells (Fig. 10B). Collectively these data demonstrates that exposure to IL-12 alone does not increase the viability/survival of activated human CD4 T cells. Prior exposure to IL-12 leads to a transient increase in proliferation/survival of activated human CD4 T cells TCR stimulation promotes proliferation of CD4 T cells. Since IL-12 pretreatment increased the production of IFN-γ following TCR stimulation, we determined if IL-12 pretreatment will also alter the TCR-induced proliferation/survival of activated human CD4 T cells. To this end, activated human CD4 T cells were pretreated for 6 h with IL-12 (50 ng/ml), washed, and TCR-stimulated with different doses of anti-tcr antibodies. At different times following TCR stimulation, cell proliferation/survival was assessed by using a trypan blue dye exclusion assay. We found that at 24 and 48 h following TCR stimulation, both control and IL-12 pretreated cells were present in similar numbers (Fig. 11A, 11B). Similarly, both IL-12 pretreated cells and controls had similar cell numbers 72 h following TCR stimulation with doses of 0.5 µg/ml or 6 µg/ml anti-tcr antibodies (Fig. 11C). Interestingly, IL-12 pretreated cells had significantly higher cell numbers than cells treated in media alone following 72 h of TCR stimulation with a dose of 2 µg/ml (Fig. 11C). Finally, after 120 h of TCR stimulation both control and IL-12 pretreated cells had similar cell numbers (Fig. 11D). The fact that IL-12 pretreatment led to increased cell numbers 72 h following TCR stimulation and by 120 h these effects were gone suggest that the effects of IL-12 are transient. The cytokine production data presented so far has been collected using an ELISA. 63

85 A caveat of this technique is that true changes in cytokine production between the groups could be obscured by differences in proliferation/survival between the groups. Therefore, it was possible that the increased release of IFN-γ in the IL-12 pretreated groups was due to differences in cell viability and/or survival. However, the fact that following 6 h IL-12 pretreatment both control and IL-12 pretreated cells had similar apoptosis (Fig. 10B) and that following 24 h TCR stimulation both groups have similar viability (Fig 11A), demonstrates that the increased release of IFN-γ in the IL-12 pretreated group was in fact due to changes in cytokine production and not because of differences in cell viability and/or survival. Pretreatment of activated human CD4 T cells with IL-12 enhances the TCR-induced release of a range of cytokines Our data suggest that prior exposure to IL-12 enhances the TCR-induced production of cytokines and the proliferation/survival of activated human CD4 T cells without altering the activation phenotype based on CD69 expression. We further characterized the effects of IL-12 in altering the TCR-induced production of cytokines. CD4 T cells have the capacity to produce multiple cytokines, including IFN-γ, TNF-α, IL-4, IL-13, and IL-10 [37, 39]. Numerous studies have established that IL-12 induces naive CD4 T cell differentiation into IFN-γ and TNF-α-producing Th1 subsets [30]. Therefore, we examined whether prior exposure to IL-12 will promote activated human CD4 T cells to selectively produce Th1 cytokines or more generally upregulate cytokine production. To test for this, activated human CD4 T cells were pretreated with or without IL-12, and the TCR-induced release of IFN-γ, TNF-α, IL-4, IL-13 and IL-10 was 64

86 measured. Consistent with other reports [40, 120, 121], we observed that TCR engagement led to the production of IFN-γ, TNF- α, IL-13, and IL-10 (~9 ng/ml, ~1 ng/ml, ~1 ng/ml, and ~3.5 ng/ml respectively) and modest amounts of IL-4 (~0.03 ng/ml) in cells pretreated with media alone (Fig. 12A-J). The production of a range of cytokines by the cells in our system was expected since our activated CD4 T cells consists of naïve CD4 T cells primed under non-polarizing conditions and recently activated memory CD4 T cells. Surprisingly, prior exposure to IL-12 not only increased the production of IFN-γ but also enhanced the protein levels of TNF-α, IL-13, IL-4 and IL-10 cytokines at all doses of TCR induction (Fig. 12A-J). This IL-12-mediated potentiation of cytokine production was statistically significant for all cytokines at TCR doses of 6 μg/ml and higher (Fig. 12A-J). These data indicate that IL-12 pretreatment in activated human CD4 T cells does not solely increase Th1-associated cytokines but instead enhances the production of a range of cytokines following TCR stimulation. IL-12 increases the number of cells capable of producing IFN-γ, while altering the release of other cytokines via a separate mechanism In order to determine if IL-12 is mediating its effects by increasing the proportion of cells producing these cytokines or by increasing the amount of cytokines produced on a per-cell basis, TCR-mediated cytokine expression was measured using intracellular staining. The TCR concentration dose used in these studies was 6 μg/ml since this was the lowest TCR dose that gave significant differences in the production of all cytokines. Exposure to IL-12 before TCR stimulation for 18 h resulted in a significant increase in the frequency of cells producing IFN-γ and a consistent increase in the frequency of cells 65

87 producing TNF-α and IL-10 that did not reach statistical significance compared to cells treated in media alone (Fig. 13A, 13B). Surprisingly, the proportion of cells producing IL-4 and IL-13 following TCR stimulation remained the same between both IL-12 pretreated and cells treated in media alone (Fig. 13A, 13B). Furthermore, IL-12 pretreatment resulted in no significant differences in the MFI of IFN-γ, TNF-α, IL-13, IL- 4 and IL-10 on a per-cell basis compared to untreated cells (Fig. 13C). The fact that IL-12 pretreatment increased the TCR-induced release of TNF-α, IL-4, IL-13 and IL-10 into the culture supernatants, but had little to no effect when intracellular levels of these cytokines were measured using BFA which is an inhibitor of protein transport from the endoplasmic reticulum to the Golgi apparatus [122], suggests that IL-12 could mediate its effects by altering the release of cytokines. However, because intracellular staining only provides a snapshot of cytokine production during a short time window, we confirmed our results by measuring intracellular cytokines at different time points and in the presence or absence of different protein secretion inhibitors (BFA, monensin, or no inhibitor). First, we repeated the experiments using monensin (an inhibitor of trans-golgi function) or without adding an inhibitor of protein transport. When we repeated the experiments using monensin or no protein secretion inhibitors, we found a similar pattern of cytokine secretion than the one observed using BFA. Following 18 h of TCR stimulation using monensin or without using any inhibitors, we found that IL-12 pretreatment increased the frequency of cells producing IFN-γ without altering the levels of TNF-α, IL-13, IL-4 and IL-10 in comparison to cells treated in media alone (Fig. 14A, 14B). Similarly, following 18 h of TCR stimulation without using any protein secretion inhibitors, we found that IL-12 pretreatment resulted in no substantial differences in the 66

88 MFI of IFN-γ, TNF-α, IL-13, IL-4 and IL-10 on a per-cell basis compared to untreated cells (Fig. 14A, 14B). Since our previous experiments looked at cytokine production at 18 h post-tcr stimulation, we then examined the production of cytokines at 6 h and 24 h after TCR stimulation. 24 h after TCR stimulation (BFA added the last 6 h) we observed a similar pattern of cytokine secretion than the one observed at 18 h after TCR stimulation (Fig. 15). Interestingly, we found that 6 h post-tcr stimulation both IL-12 pretreated and untreated cells had similar frequencies of cells making cytokines and similar MFIs; and this cytokine secretion profile was observed using BFA, monensin, or no protein transport inhibitors (Fig. 16A-C). Collectively, our data suggest that IL-12 potentiates the production of a range of cytokines following TCR stimulation by at least two mechanisms, one where IL-12 increases the number of cells capable of producing IFN-γ and a second where IL-12 enhances the release of other cytokines from the T cell. Activated human CD4 T cells have variable expression of the IL-12R β1and IL-12R β2 Our intracellular cytokine staining data suggests that, from our heterogenous population of effector and memory CD4 T cells, some cells respond to IL-12 by increasing cytokine production. We examined the cells capable of responding to IL-12 from our population of activated human CD4 T cells by determining the expression of IL- 12 receptor before IL-12 pretreatment. IL-12 receptor is composed of two subunits IL- 12Rβ1 and IL-12Rβ2. Co-expression of both subunits is required for the canonical cellular effects of IL-12 [61]. As a reference, we first measured the expression of IL- 12Rβ1 and IL-12Rβ2 on freshly isolated human CD4 T cells, then at day 3 and day 5 following activation with TCR/CD28/ and IL-2. Day 5 is the timepoint in which all of our 67

89 experiments have previously been performed. Consistent with previous literature [80, 123, 124], we found that a small proportion of freshly isolated CD4 T cells express IL- 12Rβ1 above the level of the unstained control and a small fraction express levels of IL- 12Rβ2 above the level of the unstained control (Fig. 17A). The expression of both IL- 12Rβ1 and IL-12Rβ2 receptors was upregulated at day 3 following activation (Fig. 17BA). Interestingly, at day 5 of activation, almost all cells highly expressed the IL- 12Rβ1 above the level of the unstained control. In addition, at day 5 after activation, we found that the expression of IL-12Rβ2 decreased in comparison to day 3 resulting in only a small proportion of cells expressing the IL-12Rβ2 above the levels of the unstained control, but importantly, the entire curve shifts to the right (Fig. 17C). Therefore, the majority of the cells express a low level of IL-12Rβ2 that is near or below the limit of detection (Fig. 17C). Based on the expression of the IL-12Rβ1 and IL-12Rβ2, our data demonstrate that activated human CD4 T cells are able to respond to IL-12 signals. The IL-12 mediated enhancement of cytokine production following TCR stimulation is mediated by both transcriptional and post-transcriptional effects Our previous data suggested that IL-12 enhances cytokine production by at least two separate mechanisms. For that reason, we examined whether the IL-12 potentiation of cytokine production was a consequence of increased transcription of cytokines. To explore this possibility, total RNA was isolated from untreated and IL-12 treated activated human CD4 T cells before and after TCR ligation and the levels of mrna expression of IFNG, TNF, IL4, IL13 and IL10 were assessed by real time PCR. In the absence of TCR stimulation there were no differences in the mrna levels of the 68

90 cytokines between IL-12 treated and untreated cells (Fig. 18A, 18B), showing that IL-12 does not directly stimulate the production of cytokine mrna. As expected, TCR induction for 6 and 18 h upregulated the mrna levels of IFNG, TNF, IL4, IL13 and IL10 when compared to non-tcr stimulated cells (Fig. 18A, 18B). Moreover, TCR stimulation for 6 and 18 h substantially increased the mrna expression of IFNG (3.5 fold) and modestly increased the mrna expression of IL10 (1.7 fold) in IL-12 pretreated cells in comparison to cells pretreated in media alone (Fig. 18A, 18B). In contrast, the levels of TNF, IL4, and IL13 mrna were not altered in IL-12-treated cells in comparison to untreated cells at 6 and 18 after TCR stimulation (Fig. 18A, 18B). The fact that IL-12 pretreatment increased the TCR-induced mrna expression of IFNG withouth altering the mrna levels of TNF-α, IL-13, or IL-4 is consistent with our previous intracellular staining studies, where IL-12 pretreatment significantly increased the proportion of cells making IFN-γ without altering the levels of TNF-α, IL-13, or IL-4 (Figs ). In contrast, the slightly increased mrna levels of IL-10 in IL-12 pretreated cells is inconsistent with the previous intracellular staining studies in which IL-12 pretreatment resulted in trend towards increased frequency of cells making IL-10 that did not reach statistical significance (Figs ). Collectively, these results suggest that the IL-12- mediated priming of a range of cytokines is a consequence of at least two separate mechanisms, increased mrna expression for IFN-γ and increased release of TNF-α, IL- 13, IL-4 and IL

91 Prior exposure to IL-12 alters how cells respond to TCR stimulation similarly whether it s present only before or before and during TCR stimulation So far our experiments have been tightly focused on examining the effects of prior exposure to IL-12 in altering the responses of human CD4 T cells to TCR stimulation. However, in vivo it is very likely that human CD4 T cells will be exposed to IL-12 not only before TCR stimulation but also during TCR stimulation. Therefore, we compared how IL-12 pretreatment alters TCR responses when it present before TCR stimulation or both before and during TCR stimulation. To this end, activated human CD4 T cells were pretreated for 6 h with or without IL-12 (50 ng/ml), washed, and then TCR stimulated. Some of the IL-12 pretreated cells were also TCR stimulated in the presence of IL-12 (50 ng/ml). IFN-γ and IL-4 were then assessed by ELISA. As expected, IL-12 pretreatment significantly increased TCR-induced IFN-γ and IL-4 production in comparison to cells treated in media alone (Fig. 19A, 19B). Interestingly, the IL-12 mediated increase of IFN-γ and IL-4 was not altered when IL-12 was present during TCR stimulation (Fig. 19A, 19B). These data suggest that prior exposure to IL-12 alters how cells respond to TCR stimulation in the same way whether IL-12 is present before or both before and during TCR stimulation. Responses of resting peripheral blood CD4 T cells to IL-12 pretreatment So far we have been examining the effects of prior exposure to IL-12 in altering the TCR responses of activated human CD4 T cells that consist of recently activated effector and memory CD4 T cells. To further explore the effects of IL-12 in human CD4 T cells, we explored the effects of IL-12 pretreatment in freshly isolated peripheral CD4 70

92 T cells that consists of a mix of naïve and antigen experienced CD4 T cells (Fig. 4A). To this end, freshly isolated human peripheral CD4 T cells were incubated with IL-12 (50 ng/ml) for different times, washed and then stimulated with anti-tcr and anti-cd28 antibodies for 24 h. We found that freshly isolated human peripheral blood CD4 T cells pretreated with IL-12 for different times consistently had a trend towards increased production of IFN-γ in comparison to cells cultured in media alone (Fig. 20A, 20B). However, even though there was a trend of the IL-12 pretreated cells to make more IFN-γ production, this did not reach statistical significance (Fig. 20A, 20B). We found that there was substantial variability in the production of IFN-γ between the donors. Future experiments will determine if resting peripheral blood CD4 T cells respond to IL-12 signals and make more IFN-γ in response to activation in a similar way to activated CD4 T cells. Discussion Previous studies by us and others have demonstrated that prior exposure to IL-7, IL-15 or a TLR5 ligand increases the responsiveness of human T cells to TCR stimulation [95, 96]. In addition, recent studies by Richer at al. and Kim et al. showed that murine effector/memory CD8 T cells or secondary effector CD4 T cells exposed to pathogen induced-inflammation have enhanced ability to respond to TCR stimulation [92, 94]. From the multiple cytokines which compose the inflammatory environment, this response was attributed to be driven by at least IL-12 and type I interferons. Similarly, murine memory CD8 T cells conditioned with IL-12 and IL-18 in vitro have enhanced 71

93 cytokine production and cytotoxic activity upon TCR re-stimulation [93]. Herein, we showed that prior exposure to IL-12 led to a transient increase in the TCR-induced proliferation/survival of the cells but did not alter the expression of the activation marker CD69. Furthermore, we demonstrated that prior exposure to IL-12 results in increased production of a range of cytokines upon TCR re-stimulation. Collectively, these studies highlight underappreciated roles for inflammatory signals in continually shaping TCRmediated responses via context-specific mechanisms. The canonical role for IL-12 in naïve CD4 T cells is the differentiation of Th1 cells; thus we expected IL-12 pretreatment to induce primarily IFN-γ and TNF-α production upon TCR stimulation [30]. To our surprise, IL-12 pretreatment of activated human CD4 T cells led to a general upregulation of cytokine production upon TCR stimulation. Interestingly, the effects of IL-12 pretreatment in enhancing the production of IL-4 and IL-13 were not as robust as for other cytokines. However, the levels of IL-4 and IL-13 produced in our studies are similar to what it has been observed in human exvivo isolated Th2 cells [40, 125]. After polarization, human CD4 T cells remain flexible and can be re-polarized in different inflammatory environments [37, 40]. Furthermore, emerging literature suggests that full CD4 T cell differentiation occurs at sites of infection and inflammation after they have migrated from the secondary lymphoid organs [33]. It is therefore possible that beyond its role in Th1 polarization, IL-12 may also prepare effector/memory CD4 T cells to release all cytokines prior to their final polarization. Similar to our study, the IL-12-mediated production of cytokines other than IFN-γ has been previously reported in T clones, peripheral blood T cells, and tumor reactive T cells [ ]. Likewise, several preclinical studies have shown that IL-12 72

94 administration increases plasma levels of IFN-γ and other cytokines, such as IL-10 [ ]. Our data fit well with these findings and demonstrate that the regulation of CD4 T cells responses by IL-12 is more complex than previously appreciated. The effect of IL-12 on activated human CD4 T cells had several interesting features. First, short-term exposure to IL-12 was sufficient to transiently alter responses to TCR stimulation. The findings in this paper are similar to previous studies where short exposures to IL-12 and IL-18 was sufficient to render murine memory CD8 T cells more responsive to subsequent re-stimulation [93]. In contrast, IL-7, IL-15 or TLR5 ligands required longer 24-hour pretreatments in order to amplify subsequent TCR-induced functions [95, 96]. Because exposure to IL-12 for only 1.5 hours is sufficient to alter TCR stimulation, we speculate that the mechanism by which IL-12 mediates its effects is largely independent of transcriptional activation. Second, doses of 25 ng/ml or higher of recombinant IL-12 were sufficient to significantly alter the responses to TCR stimulation. Based on data from previous studies, the required doses of IL-12 are similar to the in vivo levels produced during an inflammatory immune response [72, 76], suggesting these effects can occur under physiological conditions. Third, the effects of IL-12 were transient and lasted between 3-6 h. Similarly, Deshpande et al. showed that the effects of IL-7 and IL-15 on human T cells only last for 3 hours or less [96]. In contrast, we have shown that a human TLR5 ligand increases TCR-induced cytokine production for hours [95]. The fact that the IL-12 mediated effects on activated human CD4 T cells were short lived suggests that the priming of activated CD4 T cells is tightly regulated to minimize the risk of immunopathology. Fourth, pretreatment of activated human CD4 T cells with IL-12 did not enhance TCR functional avidity, at least using an antibody based 73

95 system to stimulate the TCR. These data are in contrast to previous studies on murine CD8 and CD4 T cells, where IL-12 signals from pathogen induced inflammation enhanced the functional avidity of these cells [92, 94]. These distinctions could be attributed to differences between human and mouse T cell responses or caused by experimental variations in our defined IL-12 treatment versus in vivo inflammation driven by many cytokines. Fifth, in contrast to the previous findings from murine CD8 and CD4 T cells, we found that pretreatment with IFN-β (one of the type I interferons) had no effect in altering activated human CD4 T cell responses. As stated above, these differences could be attributed to differences between human and mouse T cell responses or to differences in experimental approaches. We found that the proportion of cells making TNF-α, IL-10, IL-4 and IL-13, and the MFI of all assessed cytokines was not significantly altered by IL-12 pretreatment. We hypothesized that by using an inhibitor of protein transport from the endoplasmic reticulum to the Golgi apparatus [122], we were incapable of detecting IL-12-driven effects in cytokine release downstream of the inhibitor. However, we found similar results when we repeated the experiments in the absence of inhibitors. Furthermore, since intracellular staining only gives a snapshot of cytokine production of a short time window we examined if IL-12 was altering cytokine secretion at other times. Interestingly, a similar pattern of cytokine production was observed when we examined earlier and later time points of TCR induction (6h and 24h). In support of our findings, IL-12 pretreatment altered the gene expression of IFN-γ, but not TNF-α, IL-4, IL-13 or IL-10, following TCR stimulation. Together, these data suggest that IL-12 mediates its effects by at least 74

96 two mechanisms: increased mrna expression of IFN-γ resulting in more cells releasing this cytokine and increased ability of cells to release TNF-α, IL-4, IL-13 and IL-10. Our findings highlight a critical question: what is the physiological role of the potentiation of cytokine release by IL-12? Naive CD4 T cells are primed and activated in secondary lymphoid tissues. These cells then migrate through the circulatory or lymphatic system before entering into the sites of infection. During this migration, these cells are exposed to IL-12 and other inflammatory signals. Since human CD4 T cells remain capable of differentiating into multiple CD4 T cell lineages after initial priming and activation, we propose a model in which IL-12 present in blood, infection sites, and/or at inflammatory sites alters activated human CD4 T cells that have not terminally differentiated, rendering them capable of producing a range of cytokines upon TCR activation. This allows the cells to respond faster when they encounter their cognate antigen at sites of infection and be easily polarized into CD4 helper T cell subsets depending upon the cytokine milieu they encounter. This function of IL-12 is the likely reason why clinical trials of IL-12 to treat human cancer have shown limited efficacy [132, 133]. IL-12 treatment will not only drive Th1 T cell differentiation, but also enhance the production of Th2 cytokines and suppressive cytokines such as IL-10. In fact, IL-10 levels are increased in patients treated with IL-12 [132, 133]. 75

97 Figure 5: Conditioning HuT78 T cells with IL-12 enhances the TCR-induced production of IL-2. HuT78 T cells were incubated with or without IL-12 (50 ng/ml for 24 h), washed, and stimulated with or without 2 μg/ml of plate bound anti-tcr antibodies for 24 h. IL-2 production was determined by ELISA. Graphs show the mean ±SEM of values from four repeats. Data were statistically compared to cells treated in media alone with a two-tail, unpaired Student s. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 76

98 77

99 Figure 6: Exposure to IL-12 selectively alters how activated human CD4 T cells respond to TCR stimulation. (A-D) Activated human CD4 T cells were left untreated (no cytokine) or treated for 6 h with various cytokines (50 ng/ml). After pretreatment, cells were washed and stimulated with or without 2 μg/ml of plate bound anti-tcr antibodies for 24 h. The production of IFN-γ was determined by ELISA. Results are presented as normalized to the amounts produced by no cytokine (A) and (C) or as absolute values (B) and (D). Graphs show the mean ±SEM of values from three to seven different donors. Data of normalized results (A and C) were statistically compared with a two-tail Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 78

100 A B C D 79

101 Figure 7: Short pretreatments with low doses of IL-12 are sufficient to transiently potentiate the TCR-mediated production of IFN-γ. Activated human CD4 T cells were left untreated (no cytokine) or treated for 6 h with various doses of IL-12 (A and B), or for various times with 50 ng/ml of IL-12 (C and D). After pretreatment, cells were washed and stimulated with or without 2 μg/ml of plate bound anti-tcr antibodies for 24 h. Alternatively, cells were pretreated for 6 h with 50 ng/ml of IL-12, rested for various times and then stimulated with plate bound anti-tcr antibodies (2 μg/ml) for 24 h (E and F). IFN-γ production was determined by ELISA. Results are presented asnormalized to the amounts produced by no cytokine (A), (C), and (D) or as absolute values (B), (D) and (F). Graphs are shown as the mean ±SEM of values from four to seven different donors. Data in (A), (C), and (E) were statistically compared to no cytokine cells using a two-tail Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s. or no symbol=not significant. 80

102 A B C D E F 81

103 Figure 8: Conditioning CD4 T cells with IL-12 does not alter their functional avidity. (A-D) Activated human CD4 T cells were incubated with or without 50 ng/ml IL-12 for 6 h and then stimulated with titrating doses of plate bound anti-tcr antibodies for 24 h. The IFN-γ production was determined by ELISA. Results are presented as normalized values to the amounts produced by no cytokine (A) or absolute values (B). Alternatively, Data were normalized, plotted using GraphPad Prism, and the EC 50 values calculated (C-D). The mean value ± SEM from four to five different donors is shown. Data in (A and D) were statistically compared to no cytokines cells with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 82

104 A B C D 83

105 Figure 9: Prior exposure to IL-12 does not alter the activation phenotype of activated human CD 4 T cells. Activated human CD4 T cells were incubated with or without 50 ng/ml of IL-12 for 6 h and then stimulated with or without different doses of plate bound anti-tcr antibodies for 24 h. Then the expression of CD69 was analyzed using flow cytometry. Data are shown as representative plots of two donors. 84

106 Count anti-tcr 0 μg/ml anti-tcr 0.5 μg/ml anti-tcr 2 μg/ml CD69 85

107 Figure 10: IL-12 exposure does not alter the basal proliferation/survival of activated human CD4 T cells. (A) Activated human CD4 T cells were incubated with IL-12 for different times (50 ng/ml), washed, and then viable cell numbers were determined by trypan blue dye exclusion assay. Graphs show the mean ± SEM values from five separate donors. (B) Activated human CD4 T cells were left untreated or pretreated with 50 ng/ml of IL-12 for 6 h and then stained with annexin V in a buffer containing propidium iodide and analyzed by flow cytometry. Data are shown as representative plots of two donors. Data in (A) were statistically compared to cells treated in media alone (no cytokine) with a two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 86

108 A B 87

109 Figure 11: Prior exposure to IL-12 leads to a transient increase in proliferation/survival of activated human CD4 T cells. Activated human CD4 T cells were incubated with or without IL-12 (50 ng/ml for 6 hours), washed, and stimulated with different doses of plate bound anti-tcr antibodies for 24, 48, 72 and 120 hours. Viable cell numbers were determined by trypan blue dye exclusion assay. Graphs show the mean ± SEM values from five separate donors. Data were statistically compared to cells treated in media alone (no cytokine) with a two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant 88

110 A B C D * 89

111 Figure 12: IL-12 pretreated human CD4 T cells have enhanced production of a range of cytokines following TCR stimulation. Activated human CD4 T cells were left untreated or pretreated with 50 ng/ml of IL-12 for 6 h and then subsequently stimulated with various doses of plate bound anti-tcr antibodies for 24 h. Protein levels of indicated cytokines were assessed by ELISA. Results are presented as normalized to the amounts produced by no cytokine (A), (C), (E), (G), and (I) or absolute values (B), (D), (F), (H), and (J). The mean value ± SEM from four to five different donors is shown. Data in (A), (C), (E), (G), and (I). were statistically compared to no cytokines cells with a twotail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 90

112 A B C D 91

113 Figure 12 continued E F G H I J 92

114 Figure 13: Prior exposure to IL-12 increases the number of cells capable of producing IFNγ and enhances the release of other cytokines from the T cell. Activated human CD4 T cells were left untreated or pretreated with 50 ng/ml IL-12 for 6 h and then subsequently stimulated with 6 μg/ml plate bound anti-tcr antibodies for 18 h with BFA added for the last 6 h. Protein levels of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were determined by analysis of intracellular-cytokine staining. Live lymphocytes were gated based on forward and side scatter. Quadrants were set so the baseline cytokine production of non-tcr stimulated cells was less than 1%. The frequencies and median fluoresce intensities of cytokine expression were then determined. Data are shown as (A) representative plots or (B and C) mean ± SEM of five to nine separate donors. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 93

115 A B C 94

116 Figure 14: Prior exposure to IL-12 increases the number of cells capable of producing IFNγ and enhances the release of other cytokines from the T cell. Activated human CD4 T cells were left untreated or pretreated with 50 ng/ml IL-12 for 6 h and then subsequently stimulated with 6 μg/ml plate bound anti-tcr antibodies for 18 h with Monensin added for the last 5-6 h (A) or no inhibitor (B). Protein levels of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were determined by analysis of intracellular-cytokine staining. Live lymphocytes were gated based on forward and side scatter. Quadrants were set so the baseline cytokine production of non-tcr stimulated cells was less than 1%. The frequencies and median fluoresce intensities of cytokine expression were then determined. Data are shown as mean ± SEM of two separate donors. 95

117 A 18 h anti-tcr stimulation Monensin B 18 h anti-tcr stimulation No inhibitor 96

118 Figure 15: Prior exposure to IL-12 increases the number of cells capable of producing IFNγ and enhances the release of other cytokines from the T cell. Activated human CD4 T cells were left untreated or pretreated with 50 ng/ml IL-12 for 6 h and then subsequently stimulated with 6 μg/ml plate bound anti-tcr antibodies for 24 h with BFA added for the last 5-6 h. Protein levels of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were determined by analysis of intracellular-cytokine staining. Live lymphocytes were gated based on forward and side scatter. Quadrants were set so the baseline cytokine production of non-tcr stimulated cells was less than 1%. The frequencies and median fluoresce intensities of cytokine expression were then determined. Data are shown as mean ± SEM of two separate donors. 97

119 24h anti-tcr stimulation BFA 98

120 Figure 16: Prior exposure to IL-12 increases the number of cells capable of producing IFNγ and enhances the release of other cytokines from the T cell. Activated human CD4 T cells were left untreated or pretreated with 50 ng/ml IL-12 for 6 h and then subsequently stimulated with 6 μg/ml plate bound anti-tcr antibodies for 6 h with BFA (A), Monensin (B), or no inhibitor (C) added for the last 5-6 h. Protein levels of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were determined by analysis of intracellular-cytokine staining. Live lymphocytes were gated based on forward and side scatter. Quadrants were set so the baseline cytokine production of non-tcr stimulated cells was less than 1%. The frequencies and median fluoresce intensities of cytokine expression were then determined. Data are shown as mean ± SEM of two separate donors. 99

121 A 6h anti-tcr stimulation BFA B 6h anti-tcr stimulation Monensin C 6h anti-tcr stimulation No inhibitor 100

122 Figure 17: Characterization of the expression of the IL-12 receptor in human CD4 T cells. IL-12Rβ1 and IL-12Rβ2 expression was measured on freshly isolated human CD4 T cells and at day 3 and day 5 following activation, using flow cytometry. Left: Histograms showing the expression of IL-12 receptor β1 or β2. Black line represents staining with IL-12R antibody and gray line represents staining with isotype-matched control antibody. Right: Plots showing the expression of IL-12 receptor β1 and β2. Live lymphocytes were gated based on forward and side scatter and quadrants were set based on isotype controls. Results are representative of five different donors. 101

123 IL-12Rβ2 IL-12Rβ2 IL- 12R 2 A IL-12Rβ1 IL-12Rβ2 Day 0 IL-12Rβ1 B IL-12Rβ1 IL-12Rβ2 Day 3 IL-12Rβ1 C IL-12Rβ1 IL-12Rβ2 Day 5 IL-12Rβ1 102

124 Figure 18: The IL-12-mediated priming of cytokine release is driven by a function of both transcriptional and post-transcriptional effects. The expression of the mrna levels of IFNG, TNF, IL4, IL13 and IL10 were determined by qpcr in IL-12 pretreated and untreated activated CD4 T cells stimulated with or without 6 μg/ml of plate bound anti-tcr antibodies for 6 and 18 h. Data were normalized to levels of mrna encoding β-actin and presented relative to levels in untreated cells. Results are shown as mean ± SEM of five separate donors. 103

125 A B 104

126 Figure 19: Prior exposure to IL-12 alters how cells respond to TCR stimulation similarly whether it s present only before or before and during TCR stimulation. Activated human CD4 T cells were left untreated or pretreated with 50 ng/ml of IL-12 for 6 h and then subsequently stimulated with plate bound anti-tcr antibodies for 24 h. Some of the IL- 12 pretreated cells were TCR stimulated in the presence of IL-12 (50ng/mL). Cytokine levels were then assessed by ELISA. Data were normalized to the amounts produced by no cytokine. The mean value ± SEM from three different donors is shown. 105

127 A B 106

128 Figure 20: Responses of resting peripheral blood CD4 T cells to IL-12 pretreatment. Fresh isolated human peripheral blood CD4 T cells were left untreated or pretreated with 50 ng/ml of IL-12 for different times, washed and then stimulated with anti-tcr and anti-cd28 antibodies for 24 h. IFN-γ levels were then assessed by ELISA. Results are presented as absolute values (A) or normalized to the amounts produced by no cytokine (B). Graphs are shown as the mean ±SEM of values from four different donors. Data in (B) was statistically compared to no cytokine cells using a two-tail Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s. or no symbol=not significant. 107

129 A B 108

130 CHAPTER IV: MECHANISM BY WHICH IL-12 PRETREATMENT POTENTIATES HUMAN CD4 T CELLS RESPONSES TO TCR STIMULATION Introduction The results in CHAPTER III demonstrated that pretreatment of activated human CD4 T cells with IL-12 altered how cells respond to TCR stimulation. We showed that prior exposure to IL-12 led to a transient increase in the TCR-induced proliferation/survival of the cells but did not alter the expression of the activation marker CD69. Furthermore, we demonstrated that IL-12 pretreatment transiently increased TCRinduced IFN-γ production after exposure to IL-12 for at least 1.5 hours. In addition, we found that IL-12 pretreatment did not solely increase Th1-associated cytokines but instead enhanced the production of a range of cytokines following TCR stimulation. Finally, based on the intracellular staining and mrna expression data, we concluded that the IL-12-mediated increased production of a range of cytokines is a consequence of at least two separate mechanisms, increased mrna expression for IFN-γ and increased release of TNF-α, IL-13, IL-4 and IL-10. In this chapter we examined the potential mechanisms by which prior exposure to IL-12 is altering how activated human CD4 T cells respond to TCR stimulation. Prior studies have shown that TCR-mediated signaling is enhanced in murine CD4 and CD8 T cells exposed to pathogen-induced inflammation [92, 94] and in human T cells pretreated with IL-7, IL-15 or the TLR5 ligand flagellin [95, 96]. Collectively, these studies suggest that prior exposure to cytokines or inflammatory signals alters TCR- 109

131 mediated signaling. Interestingly, each cytokine or inflammatory signal seems to be doing this via a distinct molecular mechanism. Pathogen-induced inflammation increased proximal TCR signaling (ZAP-70, PLC-γ) and the MAPKs ERK1/2 and JNK1/2 without altering the activation of P38 in murine effector/memory CD8 T cells and increased ZAP- 70 and ERK1/2 in murine secondary effector CD4 T cells [92, 94]. In human T cells, IL- 7 and IL-15 mediate their effects by increasing the activation of ERK1/2 following TCR stimulation [96]. Our laboratory observed that prior activation of T cells with a TLR5 ligand enhances TCR-mediated AKT activation, while simultaneously reducing LCK and LAT phosphorylation [95]. Based on these studies, in this chapter we tested the hypothesis that IL-12 pretreatment will alter the activation of signaling molecules downstream of the TCR and this is mediating the increased mrna expression of IFN-γ. Previous reports have demonstrated that brief exposure to inflammatory signals alters the metabolic state of different immune cells. For example, TCR stimulation of T cells, stimulation of surface immunoglobulin on B cells, and TLR stimulation of macrophages and dendritic cells all promote changes in metabolic pathways in these cells and this is mediated by signaling pathways downstream of these receptors [49, 50, 134, 135]. However, no studies have examined whether IL-12 alters metabolic pathways in T cells. Furthermore, previous reports have demonstrated that changes in metabolic pathways can regulate immune cell function like cytokine secretion. Everts et al. found that for DCs, LPS-induced production of cytokines IL-6, IL-12 and TNF-α is regulated at the translational level by glycolysis [52]. For T cells, glycolysis regulates the production of IFN-γ and potentially IL-2. Zheng and colleagues found that stimulating Th1 cells with anti-cd3 and anti-cd28 antibodies in the presence of an inhibitor of glycolysis results in 110

132 decreased IFN-γ and IL-2 production [53]. Similarly, Cham and Gajewski found that in CD8 T cells IFN-γ and partially IL-2 production are inhibited when cells are stimulated with anti-cd3 and anti-cd28 antibodies in the presence of an inhibitor of glycolysis [54]. Also, Chang and colleagues found that in CD4 T cells, inhibition of aerobic glycolysis and not OXPHOS abrogates IFN-γ protein levels without altering the mrna levels following challenge with anti-tcr/cd28 antibodies [55]. Furthermore, Renner et al. found that inhibition of glycolysis in T cells, but not OXPHOS, abrogates IFN-γ, TNF-, IL-10 and IL-4 production but does not affect IL-2 production following anti- TCR/CD28 antibody stimulation [56]. Therefore, in this chapter we examined if IL-12 was altering the metabolic state of the cells and if this was involved in regulating the potentiation of cytokines that were not transcriptionally regulated. We found that IL-12 exposure does not alter the expression of surface molecules associated with T cell activation. Furthermore, we observed that the IL-12-mediated increase of cytokine production following TCR stimulation was not due to residual STAT4 synergizing with TCR signals. Interestingly, we observed that IL-12 exposure altered the TCR induced activation of select TCR signaling molecules. We suspect that the enhanced activation of these molecules is responsible for the increased gene expression of IFN-γ. We also found that IL-12 pretreated cells undergo metabolic reprogramming towards oxidative metabolism, which partially regulates the release of IL-4 and IL-13. Based on our results, we propose a model in which IL-12 exposure primes the production of a range of cytokines upon TCR stimulation by enhancing the activation of select molecules downstream of the TCR, which in turn promote increased gene expression of IFN-γ, and by promoting metabolic reprogramming towards oxidative 111

133 metabolism, which regulates the release of cytokines that were not transcriptionally regulated. Results IL-12 exposure did not alter the expression of surface molecules associated with T cell activation. Surface TCR expression level has been shown to influence T cell responses and TCR signaling [136]. Furthermore, costimulatory and adhesion receptors are known to contribute to T cell activation [ ]. Therefore, we first examined if the potentiation of TCR-induced cytokine production was due to changes in the expression of critical surface molecules. To explore this possibility, activated human CD4 T cells were pretreated for 6 h with IL-12 (50 ng/ml), and then changes in the expression of surface molecules were examined by flow cytometry. We found no significant changes in the expression of the TCR, the co-receptor CD4, the costimulatory receptors CD28, ICOS or SLAM or the adhesion receptors CD2, CD49d, and CD11a after pretreatment with IL-12 (Fig. 21). Our data suggest that overt changes in surface expression of key receptors were not responsible for the effects of IL-12 in altering how activated human CD4 T cells respond to TCR stimulation. 112

134 The IL-12-mediated increase in cytokine production is not mediated by residual STAT4 synergizing with TCR stimulation signals. Following IL-12 stimulation, the Janus kinase-stat signaling pathway is activated and leads to STAT4 phosphorylation, which allows STAT4 to translocate into the nucleus and promote IFNG transcription [61, 69]. STAT4 is considered to be one of the critical mediators of the canonical effects of IL-12 because STAT4-knockout mice have impaired Th1 differentiation and IFN-γ production [69]. Therefore, we examined if the IL-12-mediated increase of transcription and protein levels of cytokines following TCR stimulation was mediated by residual STAT4 synergizing with TCR-induced signaling pathways. To address this possibility, the total expression and the activation of STAT4 were determined before and after IL-12 treatment using quantitative immunoblotting. Treatment with IL-12 significantly increased the phosphorylation of STAT4 (Y693) peaking at min following treatment (Fig. 22A, 22B). STAT4 phosphorylation returned back to near basal levels by the end of the 6 h IL-12 pretreatment, which is the time when we stimulated the cells through the TCR in most of our experiments (Fig. 22A, 22B). The total expression levels of STAT4 decreased significantly following IL-12 stimulation and became almost undetectable by the end of the IL-12 treatment, suggesting that STAT4 was getting degraded upon exposure to IL-12 signals (Fig. 22A, 22B). Since STAT4 activation is similar before and after 6 hours of IL- 12 treatment and STAT4 protein expression is almost undetectable after 6 hours of IL-12 treatment, we conclude that the IL-12 mediated increase in cytokine production upon TCR stimulation is likely not mediated directly by STAT4 alone synergizing with TCR stimulation signals. 113

135 IL-12 pretreatment enhances the activation of select signaling molecules downstream of the TCR. Ligation of the TCR results in the activation of signaling molecules that ultimately promote transcriptional changes at cytokine genes. Prior studies have shown that TCR-mediated signaling was enhanced in murine CD4 and CD8 T cells exposed to pathogen-induced inflammation [92, 94] and in human T cells pretreated with IL-7, IL-15 or the TLR5 ligand flagellin [95, 96]. Collectively, these studies suggest that prior exposure to inflammatory signals alters TCR-mediated signaling. Based on these studies, we tested the hypothesis that IL-12 pretreatment will alter the activation of signaling molecules downstream of the TCR in activated human CD4 T cells. To examine this possibility, activated human CD4 T cells were left untreated or exposed to IL-12 and then stimulated for various times using crosslinked anti-tcr/cd4 antibodies. We used anti- TCR in combination with anti-cd4 for signaling studies because this provides enhanced proximal and distal TCR signaling [104, 105]. Then, we performed quantitative immunoblotting to measure changes in the total expression and phosphorylation of TCRinduced signaling molecules. One of the early events upon TCR activation is the induction of the tyrosine kinases LCK, FYN and ZAP-70 [5]. The activities of these kinases are positively regulated by the phosphorylation of tyrosine residues (LCK Y394 and FYN Y417) in their kinase domains and negatively regulated by the phosphorylation on tyrosine residues on their C-terminal tail (LCK Y505) [7]. To detect changes in LCK Y394and FYN Y417, we used an anti-src py416 antibody, which recognizes all SRC kinases, including LCK and FYN, when they are phosphorylated on their activating sites. We 114

136 found that in cells treated in media alone, LCK Y394, FYN Y417, and LCK Y505 were phosphorylated at the basal state and the phosphorylation of these sites was not altered following TCR stimulation (Fig. 23A, 23B). We also found that IL-12 pretreatment substantially increased the TCR-induced phosphorylation of LCK Y394 and FYN Y417 in comparison to cells treated in media alone (Fig. 23A, 23B). Also, IL-12 pretreatment significantly reduced the phosphorylation of LCK at its inhibitory site Y505 at 2 minutes after TCR stimulation in comparison to cells treated in media alone (Fig. 23A, 23B). Surprisingly, the TCR-induced phosphorylation kinetics of an activating tyrosine on ZAP-70 Y319 was similar between IL-12 treated and untreated groups ((Fig. 23A, 23B). Although surprising, the disconnection between LCK activity and ZAP-70 phosphorylation has been previously observed [140, 141]. Following activation, ZAP-70 phosphorylates several tyrosine residues at the adaptor protein LAT including Y191. Phosphorylated LAT serves as a docking site for different proteins including, the phospholipase PLC-γ and the adaptor protein SLP-76. These proteins are then activated by phosphorylation on Y783 of PLC-γ and Y128 on SLP-76. Activated PLC-γ1 then cleaves PIP 2 into IP3 which is important for calcium influx [5, 11, 142]. Consistent with the lack of increased ZAP-70 phosphorylation, we observed no differences in the TCR-induced phosphorylation of LAT Y191, SLP-76 Y128, or PLC-γ Y783 between control and IL-12 treated cells (Fig. 24A, 24B). Similarly, IL-12 pretreatment did not alter the TCR-induced calcium influx in comparison to cells treated in media alone (Fig. 24C). Finally, the recruitment and activation of proteins at LAT promotes the induction of the mitogen activated protein kinase (MAPK) and AKT pathways, resulting in the 115

137 transcription of target genes that regulate cytokine production [5]. We found that IL-12 pretreated and untreated cells had no difference in the TCR-induced phosphorylation of ERK1/ERK2 T187/Y187 (Fig. 25A, 25B). AKT is phosphorylated on T308 and S473 and the phosphorylation of these residues is correlated with its activity [19]. We observed that IL-12 pretreated cells had a trend towards increased TCR-induced phosphorylation of AKT on T308 and S473 in comparison to cells treated in media alone (Fig. 25A, 25B). We also observed that IL-12 pretreatment led to a significant increase in the phosphorylation of P38 T180/T182 at 2 minutes following TCR stimulation (Fig. 25A, 25B). The IL-12 mediated changes in AKT and P38 phosphorylation occurred only after TCR stimulation, since IL-12 treatment alone did not alter the basal phosphorylation of these molecules (Fig. 25A, 25B). As shown in Fig. 26, the changes in activation of all the signaling molecules were not due to altered total expression of these proteins. To confirm its use as a loading control, we also examined the expression of GAPDH in both IL-12 pretreated and untreated activated human CD4 T cells in all the samples from the donors used in our studies. After quantifying the data of all the donors used in our studies, we found that both IL-12 pretreated and untreated cells have similar expression of GAPDH (Fig. 27). Overall, these findings suggest that prior exposure to IL-12 alters select TCRinduced signaling pathways on activated human CD4 T cells. IL-12 pretreated activated human CD4 T cells undergo metabolic reprograming towards oxidative metabolism. Our previous intracellular staining, mrna expression, and signaling studies suggest that IL-12 pretreatment increases the release of cytokines via increased gene 116

138 transcription and by an unknown post-transcriptional mechanism. Changes in the cellular function of immune cells, like cytokine secretion, have been shown to be post transcriptionally regulated by alterations in metabolic pathways [52, 55]. Therefore, we determined whether IL-12 exposure altered the metabolic state of activated human CD4 T cells. To address this knowledge gap, the metabolic profiles of IL-12 pretreated cells or untreated cells were determined using a metabolic flux analyzer. Mitochondrial respiration and aerobic glycolysis were assessed by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, under basal conditions and after drug-induced mitochondrial stress. Under basal conditions, IL-12 pretreated cells exhibited a trend towards increased basal respiration compared to untreated cells as measured by the OCR; however, the basal respiration was variable between donors and did not reach statistical significance after compiling the data from four different donors (Fig. 28A, 28C). The capacity of treated cells for oxidative metabolism was examined by measuring OCR after addition of oligomycin (an ATP synthase inhibitor that eliminates OCR due to ATP production), FCCP (an uncoupler of ATP synthesis from the electron transport chain (ETC) that reveals the maximum capacity of the mitochondria to use oxidative metabolism), and rotenone plus antimycin A (an inhibitor of complexes I and III that completely shuts down the ETC). IL-12 pretreated cells exhibited a significant increase in the maximal respiratory capacity in comparison to cells treated in media alone as measured by the OCR (Fig. 28A. 28E). In contrast, basal ECAR and maximal ECAR following pharmacological inhibition of mitochondrial respiration were similar in both IL-12 pretreated and untreated cells (Fig. 28B, 28D, 28F). 117

139 We then examined the metabolic profiles of IL-12 pretreated cells or untreated cells after TCR stimulation using the metabolic flux analyzer. As previously reported, TCR stimulation for 6 h and 18 h resulted in an increase in the ECAR and OCR compared to non TCR stimulated cells (Fig. 29A, 29B) [48, 56]. Before TCR stimulation, IL-12 pretreated cells had significant increases in the OCR in comparison to cells treated in media alone (Fig. 29A). In contrast, both IL-12 pretreated and untreated cells had similar ECAR levels before TCR stimulation (Fig. 29B). Interestingly, after 6 and 18 h of TCR stimulation both IL-12 pretreated and untreated cells had similar OCR and ECAR levels (Fig. 29A, 29B). Overall, these findings demonstrate that IL-12 pretreatment results in cells that have more oxidative metabolism and have a greater capacity for oxidative metabolism. Exposure to IL-12 does not alter glucose consumption or mitochondrial mass in activated human CD4 T cells. To further verify the effects of prior exposure to IL-12 in altering metabolic pathways in activated human CD4 T cells, we examined glucose consumption and mitochondrial mass in our cells before and after TCR stimulation. First, glucose consumption was determined in IL-12 pretreated and untreated cells using a fluorescent glucose analog (2-NBDG) that is used as an indicator for glucose uptake. Consistent with our previous data (Fig. 28), we found that both IL-12 pretreated and untreated cells have similar 2-NBDG uptake (Fig. 30A). Next, mitochondrial mass was examined by using a fluorescent probe that accumulates in the mitochondria (MitoTracker). In contrast to our previous data (Fig. 28) showing that IL-12 increases oxidative phosphorylation, we found 118

140 that mitochondrial mass was similar in both IL-12 pretreated and untreated cells (Fig. 30B). Collectively, our data suggest that IL-12 does not alter glucose uptake on activated human CD4 T cells; and the IL-12 mediated increase in oxidative phosphorylation is not mediated by alterations in mitochondrial mass. Exposure to TNF-α does not alter the metabolic profile of activated human CD4 T cells. In CHAPTER III we found that pretreatment only with IL-12, and no other cytokines, altered the responses of activated human CD4 T cells to TCR stimulation. Since IL-12 pretreatment altered oxidative metabolism, we then tested whether these effects were particular for IL-12. To this end, activated human CD4 T cells were exposed to TNF-α or media alone and then their metabolic profiles were determined using a metabolic flux analyzer. As shown in Fig. 31A, 31B, both untreated and TNF-α treated cells had similar ECAR and OCR under basal conditions and after drug-induced mitochondrial stress. These results demonstrate that exposure to other inflammatory cytokines like TNF-α does not alter metabolic pathways. Furthermore, these data suggest that the cytokine induced increases in oxidative metabolism are a particular effect of IL

141 IL-12 mediated enhancement of cytokine secretion following TCR stimulation is partially regulated by an increase in oxidative metabolism. On the basis of these findings, we tested if the IL-12-mediated increase of oxidative metabolism was involved in regulating the priming of cytokines that were not transcriptionally regulated. To this end, IL-12 pretreated cells were TCR-stimulated in the presence of an inhibitor of oxidative metabolism (oligomycin) and cytokine secretion was then measured. Consistent with published work [55, 56], oligomycin was shown to effectively block mitochondrial respiration, as shown by the decrease of OCR in both IL- 12 treated and untreated groups (Fig. 28). As expected, pretreatment with IL-12 resulted in the increase of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 upon TCR stimulation in comparison to cells treated in media alone (Fig. 32A-E). The IL-12 mediated enhancement of IFN-γ, TNF-α, and IL-10 was unaffected by the presence of oligomycin during TCR stimulation (Fig. 32A, 32B, 32E). However, the IL-12-mediated increase of IL-4 and IL-13 were significantly impaired by the presence of oligomycin during TCR stimulation (Fig. 32C, 32D). Notably, the dose of oligomycin used had minimal effects on the production of cytokines by the cells treated in media alone (Fig. 32A-E). These findings suggest that IL-12-mediated increase in oxidative metabolism is involved in regulating the release of IL-4 and IL-13 without altering the release of IFN-γ, TNF-α, and IL

142 Discussion We have begun to characterize the molecular mechanism by which IL-12 potentiates the TCR-mediated production of a range of cytokines. We examined if the effects of IL-12 were mediated by residual STAT4 synergizing with TCR-induced signaling pathways. Interestingly, we found that STAT4 activation was similar before and after 6 hours of IL-12 treatment and STAT4 protein expression was almost undetectable after 6 hours of IL-12 treatment. Therefore, we concluded that the IL-12 mediated increase in cytokine production upon TCR stimulation was likely not mediated directly by STAT4 alone synergizing with TCR stimulation signals. Although we didn t detect STAT4 phosphorylation after 6 hours of treatment, it is still possible that a minor proportion of STAT4 is bound to DNA and is capable of synergizing with TCR signals. Nevertheless, we suspect that STAT4 is not playing a major role in mediating the ability of IL-12 pretreatment to increase the responses of activated human CD4 T cells to TCR stimulation. The reason for this conclusion comes from our previous findings showing that pretreatments with IFN-β did not alter how activated human CD4 T cells respond to TCR stimulation, and from findings in the literature showing that IFN-β activates STAT4 in human T cells [143]. Also, from the fact that STAT4 seems to be degraded following IL-12 exposure and protein expression was almost undetectable at the time when we stimulated the cells through the TCR (after 6 hours of IL-12 treatment). In support of our findings, the degradation of STAT4 following exposure to IL-12 has also been described in the literature [80]. 121

143 We found that IL-12 exposure selectively increased the TCR induced activation of AKT, and P38 without altering the activation of other signaling molecules. IL-12 pretreatment also increased the TCR activation of LCK as shown by the increased TCR induced phosphorylation of the activating sites in LCK and FYN and reduced phosphorylation of LCK at its inhibitory site Y505. In contrast, pathogen-induced inflammation increased proximal TCR signaling (ZAP-70, PLC-γ) and ERK1/2 and JNK1/2 without altering the activation of P38 in murine effector/memory CD8 T cells and increased ZAP-70 and ERK1/2 in murine secondary effector CD4 T cells [92, 94]. Interestingly, in human T cells IL-7 and IL-15 mediated their effects by increasing the activation of ERK1/2 following TCR stimulation [96]. Our laboratory also observed that prior activation of T cells with a TLR5 ligand enhances TCR-mediated AKT activation, while simultaneously reducing LCK and LAT phosphorylation, and without altering ERK [95]. These studies suggest that each inflammatory signal potentiates TCR-mediated signaling via a distinct molecular mechanism. In support of our findings, a previous report has shown that P38 plays an important role in the production of IFN-γ by murine Th1 CD4 T cells in vitro. In this study, the authors demonstrate that blockade of P38 activity inhibits the gene expression of IFN-γ [144]. Furthermore, AKT activation has been shown to have multiple roles on T cell responses including cytokine release [17]. Unpublished data from our laboratory suggests that the presence of an AKT inhibitor (BML 257) reduces TCR-mediated IL-2 production, indicating the critical role that this kinase plays in cytokine production in human T cells. Similar to our findings, previous literature has reported a crosstalk between IL-12 signals and LCK, AKT, and P38. Visconti and colleagues demonstrated 122

144 that IL-12 signals activate P38 and AKT pathways without altering ERK or JNK activation in T cells [83]. Furthermore, in human resting and activated NK cells, IL-12 signals were shown to increase LCK activation [82]. Future studies will have to explore how IL-12 signals alter the activation of LCK, AKT, and P38 following TCR stimulation without altering more proximal TCR signals. Together, our results suggest that the IL-12- mediated increase of P38 and AKT could be responsible for the increased gene expression of IFN-γ. This correlation will be further examined in future studies to determine if the IL-12 mediated changes in TCR signaling are in fact responsible for the increased gene expression of IFN-γ. We also found that IL-12 pretreated cells undergo metabolic reprogramming towards oxidative metabolism which partially regulates the release of IL-4 and IL-13. This suggests that the IL-12 priming of cytokines that are not transcriptionally regulated could be partially attributed to enhanced oxidative metabolism. This seems to be a particular effect of IL-12, since exposure to other inflammatory cytokines like TNF-α did not alter metabolic pathways (Fig. 31). Previous reports have demonstrated that brief exposure to inflammatory signals alters the metabolic state of different immune cells [49, 50, 134, 135]. However, our studies are the first to demonstrate that the cytokine IL-12 promotes metabolic reprograming towards oxidative metabolism, and that oxidative metabolism is involved in the regulation of cellular functions like cytokine secretion. 123

145 Figure 21: IL-12 pretreatment did not alter the expression of surface molecules on activated human CD4 T cells. Activated human CD4 T cells were incubated with or without IL-12 (50 ng/ml for 6 hours). Changes in the expression of surface molecules were assessed by flow cytometry. Plots were gated on live cells and then the MFIs were determined. The mean value ± SEM from three to four different donors is shown. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 124

146 125

147 Figure 22: The IL-12-mediated priming of cytokine production is not mediated by residual STAT4 synergizing with TCR stimulation signals. Activated human CD4 T cells were treated with or without IL-12 (50 ng/ml) for different times, then the phosphorylation of signaling molecules was determined in whole cell lysates by immunoblotting. Data are shown as (A ) representative blots and (B) mean ± SEM of normalized results of four separate donors. Results were normalized to GAPDH and the maximal level of STAT4 expression/activation. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 126

148 A IL-12 Treatment Time (min): B 127

149 Figure 23: IL-12 pretreatment increases the activation of select signaling molecules downstream of the TCR. Activated human CD4 T cells were incubated with or without IL-12 (50 ng/ml for 6 h) and then stimulated with anti-tcr and anti-cd4 antibodies for various times. The phosphorylation of signaling molecules was determined in whole cell lysates by immunoblotting. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A) representative blots and (B) mean ± SEM of normalized results of six different donors. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 128

150 A B 129

151 Figure 24: IL-12 pretreatment increases the activation of select signaling molecules downstream of the TCR. (A and B) Activated human CD4 T cells were incubated with or without IL-12 (50 ng/ml for 6 h) and then stimulated with anti-tcr and anti-cd4 antibodies for various times. The phosphorylation of signaling molecules was determined in whole cell lysates by immunoblotting. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A) representative blots and (B) mean ± SEM of normalized results of six different donors. (C) Activated human CD4 T cells were incubated with or without IL-12 (50 ng/ml for 6 h) and then stimulated with anti-tcr antibodies. Calcium influx curve was measured in real time for 6 minutes by flow cytometry. Data shown is compiled from 3 independent experiments. 130

152 A B C 131

153 Figure 25: IL-12 pretreatment increases the activation of select signaling molecules downstream of the TCR. Activated human CD4 T cells were incubated with or without IL-12 (50 ng/ml for 6 h) and then stimulated with anti-tcr and anti-cd4 antibodies for various times. The phosphorylation of signaling molecules was determined in whole cell lysates by immunoblotting. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A) representative blots and (B) and mean ± SEM of normalized results of six to nine different donors. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 132

154 A B 133

155 Figure 26: IL-12 pretreatment does not alter the total protein expression of TCR signaling molecules. Activated human CD4 T cells were incubated with or without IL-12 (50 ng/ml for 6 hours). Pretreated cells were stimulated with anti-tcr and anti-cd4 antibodies for various times. The total expression of signaling molecules were determined in whole cell lysates by immunoblotting and the results were normalized to GAPDH. Upon normalization, the average of the total expression of the proteins at each time point was calculated. Data are shown as mean value ± SEM of two to six different donors. Results were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 134

156 135

157 Figure 27: IL-12 pretreatment does not alter the expression of GAPDH. Activated human CD4 T cells were incubated with or without IL-12 (50 ng/ml for 6 h). The expression of GAPDH was then assessed in whole cell lysates by immunoblotting. Data are shown as mean value ± SEM of twenty different donors. Results were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 136

158 137

159 Figure 28: IL-12 pretreated activated human CD4 T cells undergo metabolic reprogramming towards oxidative metabolism. Activated human CD4 T cells were treated with or without 50 ng/ml IL-12 for 6 h. Cells were plated onto poly-l-lysine-coated plates and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed at basal state and following addition of indicated compounds. (A) and (B) graphs are representative of four different donors and used to calculate the (C) basal respiration, (D) basal ECAR, (E) maximal respiratory capacity, and (F) maximal ECAR. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 138

160 A B C D E F 139

161 Figure 29: IL-12 pretreated activated human CD4 T cells have an increased ability to upregulate mitochondrial respiration in response to stimulation. Activated human CD4 T cells were treated with or without 50 ng/ml IL-12 for 6 h. Cells were left unstimulated or stimulated with 6 μg/ml plate bound anti-tcr antibodies for 6 and 18 h. Subsequently, cells were plated onto poly-l-lysine-coated plates and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed at basal state. The mean ± SEM of OCR (A) and ECAR (B) values assessed under basal state of 3-4 different donors are shown. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 140

162 A B 141

163 Figure 30: Glucose consumption and mitochondrial mass in IL-12 pretreated and untreated cells. Activated human CD4 T cells were left untreated or pretreated with 50 ng/ml of IL-12 for 6 h. In (A) glucose consumption was assessed using a fluorescent glucose analog that is used as an indicator for glucose uptake (2-NBDG). In (B) mitochondrial mass was examined by using a fluorescent probe that accumulates in the mitochondrial (MitoTracker). Dotted gray line represent unstained control, gray line represents untreated cells, and black line represents IL-12 pretreated cells. Blots are representative of two donors. 142

164 A B Mito Tracker 143

165 Figure 31: TNF-α pretreatment does not alter metabolic pathways in activated human CD4 T cells. Activated human CD4 T cells were treated with or without 50 ng/ml TNF-α for 6 h. Cells were plated onto poly-l-lysine-coated plates and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed at basal state and following addition of indicated compounds. 144

166 A B 145

167 Figure 32: The IL-12-mediated enhancement of the secretion of cytokines not transcriptionally regulated is partially driven by an increase in oxidative metabolism. Activated human CD4 T cells were treated with or without 50 ng/ml of IL-12 for 6 h. Cells were then stimulated with 6 μg/ml plate bound anti-tcr antibodies for 24 h in the presence or absence of oligomycin (2.5 μm). The protein levels of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were determined by ELISA. Results are presented as absolute values or normalized to the amounts produced by no cytokine. Graphs show the mean ±SEM of values from four to six different donors Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 146

168 A B 147

169 Figure 32 continued C D 148

170 Figure 32 continued E 149

171 CHAPTER V: PRETREATMENT OF HUMAN CD8 T CELLS WITH IL-12 LEADS TO ENHANCED TCR-INDUCED SIGNALING AND CYTOKINE PRODUCTION Introduction Activated CD8 T cells (effector or memory) are constantly bombarded with different inflammatory signals that regulate their function. One of these signals is the proinflammatory cytokine IL-12. IL-12 is quickly produced by antigen presenting cells following induction with products from various microorganisms, including bacteria, fungi, intracellular parasites, double stranded RNA, bacterial DNA, and CpG-containing oligonucleotides [61]. IL-12 is also clinically important, where it is found at sites of inflammation in a number of human disorders [61, ]. T cell subsets vary in their responsiveness to IL-12 based on the expression of the IL-12 receptor. Resting CD8 T cells have undetectable levels of the IL-12 receptor. However, the receptor is upregulated in activated CD8 T cells, allowing them to respond to IL-12 signals [61]. Many groups have examined the effects of IL-12 in altering T cell responses. The presence of IL-12 during priming of CD4 T cells promotes the differentiation of naïve CD4 T cells into type 1 T helper (Th1) cells [61]. Also, the presence of IL-12 during priming of CD8 T cells has been shown to promote strong effector functions and memory development [29]. Finally, IL-12 enhances TCR-induced proliferation, IFN-γ production and cytotoxicity of T cells [61]. Although highly informative, this work has largely examined the effects of IL-12 if it s present during or following TCR activation. 150

172 However, during immune responses, activated CD8 T cells will be exposed to IL-12 as they migrate through the blood or lymph and at sites of infection before they are further activated through the TCR in infected and inflamed tissue. How prior exposure to IL-12 alters activated CD8 T cell responses to subsequent TCR activation is not well understood. Several recent studies have shown that prior exposure to cytokines or inflammatory signals alters the responses of T cells to TCR challenge. In this regard, it was demonstrated that prior exposure to IL-7, IL-15 or a TLR5 ligand increases the responsiveness of human T cells to TCR stimulation [95, 96]. Similarly, in CHAPTER III I demonstrated that short exposure of human CD4 T cells to IL-12 enhances the TCRinduced production of a range of cytokines. In addition, exposure to pathogen-induced inflammation was shown to alter the responsiveness of murine effector/memory CD8 T cells and secondary effector CD4 T cells to subsequent activation through the TCR [92-94]. From the multiple cytokines which compose the inflammatory environment, this response was attributed to IL-12 and type I interferons [92, 94]. Collectively, these studies demonstrate that cytokines and/or inflammatory signals alter the function of T cells if they are present before further TCR stimulation, suggesting a new role for IL-12 in the regulation of T cell responses. Although these studies are informative, there are still key questions unanswered. First, whether similar biology will occur in activated human CD8 T cells remains to be addressed. Furthermore, the precise mechanisms by which IL-12 signals could alter T cell function if they are present before TCR activation remain to be fully elucidated. Addressing these knowledge gaps will increase our understanding of the basic properties of human CD8 T cells, which is crucial for clinical 151

173 applications, due to an increasing awareness that human and mice have subtly different mechanisms driving immune function that markedly alter clinical outcomes [ ]. Furthermore, since IL-12 is currently being tested as a therapy for infections and human cancer [132], a better understanding of how IL-12 regulates human T cell functions could provide insights for improving the current uses of IL-12 therapeutics. Results Conditioning activated human CD8 T cells with IL-12 leads to increased production of IFN-γ and TNF-α upon TCR stimulation Previous reports have shown that prior exposure to inflammatory cytokines alters the responses of murine CD4 and CD8 T cells to TCR stimulation [92, 93]. However, whether similar effects occur in activated human CD8 T cells, and which cytokines are important in mediating these effects, remains to be examined. To address this knowledge gap, I used activated human CD8 T cells that were obtained as described in the materials and methods section. Activated CD8 T cells were exposed to different recombinant cytokines or media alone, washed to remove the cytokines, and stimulated through the TCR. Cytokine production was then used as a readout for T cell activation. No other additional costimulatory signals were provided in order to determine the effects of the inflammatory cytokine on secondary TCR-induced T cell activation alone. We found that prior exposure to IFN-γ, IFN-β, IFN-α, TNF-α, IL-6, IL-17, or IL-10 did not alter the TCR-induced production of IFN-γ and TNF-α in comparison to cells treated in media 152

174 alone (Fig. 33A, 33B). In contrast, pretreatment with IL-12 significantly increased the TCR-induced production of IFN-γ and TNF-α compared to control cells (Fig. 33A, 33B). Importantly, this effect was not due to IL-12-driven cytokine production, since IL-12 pretreated cells had undetectable levels of cytokine release in the absence of TCR stimulation (Fig. 33C, 33D). Since the cytokine production assay required 24 hours of stimulation, it was possible that the IL-12 mediated increase in cytokine production was a consequence of increased proliferation/survival of the activated CD8 T cells. To control for this, cell viability was determined in activated human CD8 T cells untreated or pretreated with IL- 12 or other cytokines before and after 24 h TCR stimulation. Similar viable cell numbers were found in IL-12 pretreated and controls cells before and after TCR stimulation (Fig. 34), suggesting that the IL-12 effects on cytokine production were not due to altered cell viability between the groups. During a normal in vivo immune response, activated human CD8 T cells are exposed to a combination of cytokines. Therefore, we then examined whether the IL-12 potentiation of cytokine production was altered when other cytokines were present during pretreatment. As expected, IL-12 pretreatment significantly increased the TCR-induced production of IFN-γ and TNF-α in comparison to control cells. However, pretreatment with IL-12 in combination with other cytokines did not have any agonistic or antagonistic effects on cytokine production (Fig. 35A, 35B). Collectively, our data suggest that activated human CD8 T cells pretreated with IL-12 have increased cytokine production following subsequent TCR challenge. These effects appear to be specific for IL-12, since 153

175 pretreatment with other pro- and anti-inflammatory cytokines did not alter human CD8 T cell responses to TCR stimulation. Characterizing the IL-12 mediated priming of cytokine production To further characterize these effects, we first examined the length of IL-12 pretreatment needed to alter activated human CD8 T cell responses. To address this question, activated human CD8 T cells were pretreated with IL-12 for different times, washed, and stimulated through the TCR. We found that exposing the cells to IL-12 for 6 and 12 h resulted in a consistent, but not statistically significant, increase in the production of IFN-γ following TCR stimulation in comparison to cells treated in media alone (Fig. 36A). In contrast, cells pretreated for 24 and 48 h had significantly increased amounts of IFN-γ production compared to control cells (Fig. 36A). We next examined the responsiveness of activated human CD8 T cells to various doses of IL-12. Activated human CD8 T cells were pretreated with a range of physiological doses of IL-12 [72, 76], washed, and stimulated through the TCR. Following TCR stimulation, we found that pretreatment with doses of 5-70 ng/ml of IL-12 resulted in augmented TCR induced IFN-γ production in comparison to cells treated in media alone; however, there were no significant differences in the potentiation of IFN-γ production between any of the doses (Fig. 36B and data not shown). Next, we examined the duration of the effects after removal of IL-12. Following IL-12 pretreatment, activated human CD8 T cells were rested for various times before being challenged through the TCR. As seen in Fig. 36C, IL-12 pretreated cells stimulated through the TCR immediately after IL-12 pretreatment had a significant increase in the production of IFN-γ in comparison to cells treated in 154

176 media alone. We also observed that the ability of IL-12 to enhance TCR-induced IFN-γ production lasted for h (Fig. 36C). In addition, the effects of IL-12 pretreatment were examined following challenge with different doses of anti-tcr antibodies. As expected, TCR stimulation with low doses of anti-tcr antibodies increased IFN-γ production in untreated cells (Fig. 36D). Interestingly, we saw that the IL-12-pretreated cells stimulated with different doses of anti-tcr antibodies had a dose dependent increase in the production of IFN-γ (Fig. 36D). The IL-12-mediated increase in IFN-γ production was significant for all doses of anti-tcr higher than 0.12 µg/ml (Fig. 36D). Finally, since pathogen induced inflammation decreased the dose of antigen required to stimulate the maximal T cell response (functional avidity) in murine effector/memory CD8 T cells and secondary effector CD4 T cells [92, 94], we examined whether IL-12 exposure will have similar effects on activated human CD8 T cells. To test this, IL-12 pretreated or untreated cells were stimulated with titrated doses of anti-tcr antibodies and the production of IFN-γ was determined. The functional avidity was then determined by calculating the dose of stimulatory antibody needed to induce 50% of maximal IFN-γ production (EC 50 ). We observed that exposure to IL-12 does not alter the functional avidity of activated human CD8 T cells (Fig. 36E, 36F). Collectively these data demonstrate that prior exposure to physiologically-relevant doses of IL-12 for at least 24 h transiently potentiates the TCR-induced production of IFN-γ. Activated human CD8 T cells have variable surface expression of IL-12R β1 and β2 The IL-12 receptor consists of two subunits, IL-12R β1 and β2 [61, 69]. The expression of these subunits in T cells is highly regulated by TCR stimulation and 155

177 cytokines [61, 69]. Previous literature suggests that resting T cells do not express detectable levels of the receptor, but the subunits are upregulated following TCR activation [69]. However, no studies to date have examined the expression of the IL-12 receptor on activated human CD8 T cells. In order to examine the cells capable of responding to IL-12 in our system, we sought to determine the expression of the IL-12 receptor subunits on these cells using flow cytometry. We found that a substantial proportion of activated human CD8 T cells expressed IL-12R β1above the level of the unstained control, suggesting that nearly all the CD8 T cells express surface levels the IL- 12R β1 (Fig. 37). In addition, we found that a small fraction of the activated human CD8 T cells expressed the IL-12 β2 above the level of the unstained control, but importantly, the entire curve shifts to the right (Fig. 37). This suggests that the majority, if not all, of activated human CD8 T cells express a low level of IL-12R β2 on their surfaces that are near or below the limit of detection (Fig. 37). These data indicate that activated human CD8 T cells used in our system respond to IL-12 signals. Prior exposure to IL-12 increases the frequency of cells producing IFN-γ and TNF-α upon TCR stimulation We next wanted to determine if IL-12 pretreatment potentiated the production of IFN-γ and TNF-α by increasing the frequency of cells capable of producing cytokine or by increasing the amount of cytokine produced on a per-cell basis. To explore this, activated human CD8 T cells were left untreated or pretreated with IL-12, and then the intracellular levels of IFN-γ and TNF-α were determined after 6 h of TCR stimulation. At 6 h post TCR stimulation, both IL-12 pretreated and untreated cells had similar 156

178 frequencies of cells producing IFN-γ and TNF-α (Fig. 38A). We then examined the effects of IL-12 pretreatment on cytokine production at later points of TCR stimulation (18 h). In cells treated in media alone, TCR stimulation resulted in a frequency of cells producing IFN-γ or TNF-α alone and a frequency of cells co-producing IFN-γ and TNF (Fig. 38B, 38C). In the IL-12 pretreated cells, TCR stimulation resulted in significantly higher frequencies of cells producing IFN-γ and/or TNF-α in comparison to cells not treated with IL-12 (Fig. 38B, 38C). On a per-cell basis, IL-12 pretreatment resulted in a trend towards increased IFN-γ and TNF-α median FI in comparison to cells treated in media alone, but these effects did not reached statistical significance (Fig. 38D). Overall, these data demonstrate that prior exposure to IL-12 increases the proportion of cells capable of producing cytokines upon TCR re-stimulation. The IL-12-mediated increase in cytokine production is a consequence of increased transcription of cytokine genes We next determined whether the IL-12-mediated increase in IFN-γ and TNF-α was a result of enhanced transcription of cytokine mrna. To explore this possibility, the mrna levels of IFNG and TNF were examined in IL-12 pretreated and untreated cells at 6 and 18 h after TCR stimulation. We found that, in the absence of TCR stimulation, both untreated and IL-12 pretreated cells had similar mrna levels of IFNG and TNF (Fig. 39A, 39B). This is consistent with our results showing that IL-12 pretreatment alone has no effect on cytokine production (Fig.33C, 33D). As expected, in cells pretreated with media alone, TCR stimulation for 6 and 18 h increased mrna levels of IFNG and TNF (Fig. 39A, 39B). However, when cells were pretreated with IL-12, we found that they had 157

179 substantially increased mrna levels of IFNG compared to media treated cells at both 6 and 18 h after TCR stimulation (Fig. 39A, 39B). The TCR-mediated increase in TNF mrna expression at 6 h was not altered in IL-12 pretreated cells in comparison to untreated cells (Fig. 39B). In contrast, IL-12 pretreatment consistently led to a 2.5-fold increase in the mrna levels of TNF at 18 h after TCR stimulation (Fig. 39B). Overall, these data show that IL-12 pretreatment increases TCR-induced IFNG and TNF mrna expression. IL-12 exposure does not alter the expression of surface molecules involved in T cell activation To explore the mechanisms by which IL-12 pretreatment alters the responses of activated human CD8 T cells to TCR stimulation, we first examined whether IL-12 exposure was changing the expression of surface molecules involved in T cell activation. We found that IL-12 exposure does not substantially alter the expression of TCR, CD8, CD49d, CD11a, CD2, ICOS or SLAM (Fig. 40). These results suggest that IL-12 is not mediating its effects via changes in TCR expression or expression of the major adhesion proteins. The IL-12 mediated enhancement of cytokine production is not due to residual STAT4 synergizing with TCR stimulation signals We next explored whether IL-12 pretreatment was altering intracellular signaling events. IL-12 stimulation is known to induce STAT4 phosphorylation, which allows 158

180 STAT4 to translocate into the nucleus and promote IFNG transcription [61, 68]. Since STAT4-knockout mice have impaired Th1 differentiation and IFN-γ production, STAT4 is considered to be one of the primary mediators of the effects of IL-12 [69]. Since IL-12 pretreatment enhanced the transcription and protein levels of IFN-γ and TNF-α after TCR stimulation, we tested the hypothesis that long-term STAT4 phosphorylation following IL-12 treatment synergizes with TCR-induced signaling pathways to promote these effects. To test our hypothesis, we examined the total expression and the phosphorylation levels of STAT4 in untreated and IL-12-treated cells. STAT4 protein levels were reduced over time following IL-12 treatment (Fig. 41A, 41B), while there was a transient increase in STAT4 phosphorylation that peaked at 1 h after exposure and returned to basal levels by 24 h (Fig. 41A, 41C). The lack of overt STAT4 phosphorylation and the marked decreased in STAT4 expression after 24 hours of treatment suggests that the IL-12- mediated increase of IFN-γ after TCR stimulation is not due to residual STAT4 synergizing with TCR signals. IL-12 pretreatment enhances the TCR-induced activation of select MAPK and AKT without altering the activation of more proximal TCR signaling molecules TCR stimulation results in a coordinated activation of a series of signaling molecules that lead to the transcription of cytokine genes. In murine CD4 and CD8 T cells, exposure to pathogen-induced inflammation enhances T cell functions by increasing the activation of TCR signaling molecules [92, 94]. In addition, pretreatment of human T cells with IL-12, IL-7 and IL-15, or the TLR5 ligand bacterial flagellin, which all potentiate TCR-mediated functions, alteres the activation of distinct sets of 159

181 molecules downstream of the TCR [95, 96]. Therefore, inflammatory stimuli that enhance subsequent TCR-induced downstream functions all regulate TCR signaling through distinct mechanisms. Because of this, we explored whether IL-12 pretreatment alters the activation of TCR signaling molecules in activated human CD8 T cells. To test this possibility, activated human CD8 T cells were left untreated or exposed to IL-12, washed, and then stimulated by crosslinking the TCR for various times. Then, changes in the phosphorylation and total expression of TCR-induced signaling molecules were measured using quantitative immunoblotting. It should be noted that based on the stimulation method that we used, cells that did not receive any anti-tcr signals are labeled as No TCR, we have cells that were not crosslinked but that were briefly exposed to anti-tcr antibodies time 0, and cells that were briefly exposed to anti-tcr antibodies followed by crosslinking for various times, are labeled as time 2,5,15,30. The earliest events detected following TCR stimulation are the activation of the kinases, LCK and FYN. The activities of these kinases are positively regulated by the phosphorylation of tyrosine residues (LCK Y394 and FYN Y417) in their kinase domains and negatively regulated by phosphorylation on tyrosine residues in their C-terminal tails (LCK Y505) [7]. To detect changes in the activation of LCK Y394 and FYN Y417, we used an anti-src py416 antibody, which recognizes all SRC kinases, including LCK and FYN, when they are phosphorylated on their activating sites. We found that cells treated in media alone had substantial phosphorylation of activating tyrosine kinases at basal state, and the phosphorylation of these sites remained unaltered following TCR stimulation (Fig. 42A, 42B). We also found that the TCR-induced phosphorylation of LCK Y394 and FYN Y417 was similar between IL-12 pretreated and untreated cells (Fig. 160

182 42A, 42B). We observed that, in cells treated in media alone, the phosphorylation of LCK at its inhibitory residue (LCK Y505) was observable at the basal state and was modestly increased following TCR stimulation (Fig. 42A, 42B). Again, IL-12 pretreatment had no effect on the phosphorylation of LCK at its inhibitory site Y505 after TCR stimulation (Fig. 42A, 42B). This suggests that IL-12 pretreatment does not alter the TCR-induced activation of LCK or FYN. LCK and FYN phosphorylate ITAMs on the CD3 subunits of the TCR. The full phosphorylation of the ITAMs promotes the recruitment of ZAP-70 to the TCR, where it is activated and phosphorylated by LCK on Y319 [5]. Activated ZAP-70 then phosphorylates the adaptor proteins LAT and SLP-76, inducing downstream signaling events. LAT is phosphorylated on four key tyrosine residues that serve as docking sites for several molecules, such as the phospholipase PLC-γ [5, 11, 142]. Once recruited to LAT, PLC-γ is phosphorylated on Y783, leading to its activation and the subsequent release of intracellular calcium and the activation of the protein kinase C (PKC) and MAPK pathways [5, 10]. TCR stimulation also promotes the phosphorylation of SLP-76 on several tyrosine residues [148]. Similarly to LCK and FYN, the TCR-induced phosphorylation of the activating tyrosine on ZAP-70 Y319 was similar between IL-12 treated and untreated cells (Fig. 42A, 42B). We also observed that IL-12 pretreated cells and untreated cells had no difference in the kinetics of phosphorylation of TCR-induced LAT Y191, SLP-76 Y128, or PLC-γ Y783 (Fig. 42C, 42D). Together, these data show that prior exposure to IL-12 does not alter the TCR-induced activation of proximal signaling in activated human CD8 T cells. 161

183 Phosphorylated SLP-76 directs the recruitment and activation of PI3K, which is an important regulator of downstream molecules like AKT [5, 18, 149]. AKT is phosphorylated on T308 and the phosphorylation of this residue is correlated with its activity [19]. We observed that IL-12 pretreated cells had a trend towards increased phosphorylation of AKT T308 in comparison to untreated cells (Fig. 43A, 43B). However, the slight IL-12-mediated increase of AKT T308 phosphorylation was variable between donors and it did not reach statistical significance after compiling the data from four different donors (Fig. 43A, 43B). Downstream of the LAT and SLP-76 complexes is the activation of the MAP kinase pathways. This family is comprised of three groups of kinases: ERK1/ERK2, JNK, and P38. These kinases have multiple substrates that play key roles in a variety of cellular functions, including cytokine secretion [5]. We found that IL-12 pretreated and untreated cells had no difference in the TCR-induced phosphorylation of ERK1/ERK2 pt187/py187 (Fig. 43A, 43B). In addition, we observed that IL-12 pretreated samples had a slight increase in the levels of JNK T183/Y185 phosphorylation compared to control samples that were not significant after compiling multiple donors (Fig. 43A, 43B). Finally, we saw that IL-12 pretreated cells had an overall increase in the TCRinduced phosphorylation kinetics of P38 pt180/t182 in comparison to untreated cells. The basal phosphorylation (No TCR) of P38 pt180/t182 was similar in both control and IL-12 pretreated cells. However, the TCR-induced phosphorylation of P38 pt180/t182 in IL-12 pretreated cells was significantly increased at 0, 2, and 5 minutes in comparison to untreated cells. It should be noted that phosphorylation at time 0 is not considered as basal, because even though cells were not crosslinked at that time point, cells were 162

184 briefly exposed to anti-tcr antibodies. The fact that both untreated and IL-12 pretreated cells have similar P38 activation at basal state suggests that IL-12 is not directly activating the P38 pathway after 24 hours of stimulation. As shown in Fig. 44, the increased activation of P38 was not due to altered total expression of the protein. Collectively, these findings suggest that IL-12 signals significantly increase the TCRinduced activation of P38, and to a lesser extent JNK and AKT, without altering the activation of other more proximal TCR signals. IL-12 pretreatment enhances the levels of SOS1 and increases the activation of MKK3/MKK6 downstream of the TCR Our previous data suggest that IL-12 signals alter the expression or activation of a positive or negative regulator of P38 activation. Although the exact mechanism regulating the activation of MAPK kinases following TCR stimulation remain unclear, TCR activation recruits effector molecules to LAT, such as the son-of-sevenless (SOS) family proteins [5]. The GRB2/SOS1 axis has recently been shown by us and others to control the activation of P38 in human T cells [150, 151]. In addition, the kinases MKK3/MKK6 are known to be direct upstream activators of P38 [22]. To further investigate the crosstalk between IL-12 signals and P38 activation, we determined whether IL-12 pretreatment altered the activation of MKK3/MKK6 and/or the total expression of SOS1. We found that cells treated in media alone had substantial basal phosphorylation of MKK3/MKK6, and this remained relatively unaltered upon TCR stimulation (Fig. 45A, 45B). We also we observed that that upon TCR stimulation, IL-12 pretreated cells had increased phosphorylation of MKK3/MKK6, compared to the control 163

185 cells, that was significant at several time points (Fig. 45A, 45B). The IL-12 mediated enhancement of MKK3/MKK6 was significant at 0, 2, and 15 min after TCR stimulation (Fig. 45A, 45B). We also found that the IL-12 pretreated cells had significantly increased levels of SOS1 before and after TCR stimulation in comparison to control cells (Fig. 45A, 45B). Together, these data suggest that IL-12 pretreated cells have increased levels of SOS1, which upon TCR stimulation, results in increased activation of MKK3/MKK6 and P38. Discussion Numerous studies have examined the effects of IL-12 in regulating T cell responses when it s present during and after TCR stimulation. IL-12 present during priming of CD4 and CD8 T cells plays key roles in the regulation of the responses of these cells [29, 61]. Furthermore, IL-12 acts as a costimulatory signal by enhancing TCRinduced proliferation, IFN-γ production and cytotoxicity [61]. However, we have only recently begun to appreciate that exposure to inflammatory signals like IL-12 could alter how T cells respond to subsequent TCR stimulation. In this regard, Richer et al. and Kim et al. showed that murine effector/memory CD8 T cells or secondary effector CD4 T cells exposed to pathogen induced-inflammation, primarily driven by IL-12 and type I interferons, have enhanced ability to respond to TCR stimulation [92, 94]. Similarly, Raue and colleagues showed that murine memory CD8 T cells conditioned with IL-12 and IL-18 in vitro have enhanced cytokine production and cytotoxic activity upon TCR re-challenge [93]. Finally, in CHAPTER III we discussed the finding that IL

186 pretreatment in human CD4 T cells enhances the production of a range of cytokines following TCR induction. Collectively, these studies suggest that the regulation of T cell responses by IL-12 is more complex than previously appreciated. Beyond its well-studied co-stimulatory effects, prior exposure to IL-12 alters the responsiveness of murine CD4 and CD8 T cells to TCR challenge and enhances the production of multiple cytokines in activated human CD4 T cells. The results reported in this study extend our expanding understanding of the effects of IL-12 on antigen experienced T cells to activated human CD8 T cells. In vivo, recently activated effector or memory CD8 T cells encounter inflammatory signals in the blood and lymph as they migrate from the lymph nodes into sites of infection. In the site of infection, the activated CD8 T cells exposed to inflammatory stimuli receive secondary antigen and/or inflammatory signals that impact their activation. In this study, we mimicked this physiological setting in vitro using human peripheral blood CD8 T cells that have been activated for 5 days with anti- TCR/CD28 antibodies and recombinant IL-2, removed from the priming stimuli, and transiently pretreated with inflammatory signals before being restimulated via the TCR. Interestingly, our data suggest that among different pro and anti-inflammatory cytokines, only IL-12 alters the responses of human CD8 T cells to subsequent TCR stimulation. In contrast to the previous findings from murine CD8 and CD4 T cells, we found that pretreatment with type I interferons (IFN-α or IFN-β) has no effect on subsequent activated human CD8 T cell responses. This could be attributed to differences between human and mouse T cell responses, but could also be derived from differences in the experimental setup. Our results provide novel evidence that prior exposure specifically to 165

187 IL-12 increases the responses of CD8 T cells to TCR stimulation and that there is no synergistic or antagonist effects seen when IL-12 is present in combination with other cytokines. It was possible that the increased release of IFN-γ and TNF-α in the IL-12- pretreated cells was due to residual IL-12 present during TCR stimulation. However, the IL-12-mediated increase of activated human CD8 T cell responses to further TCR stimulation required pretreatments with IL-12 for at least 24 h. These results suggest that the observed effects are not because residual IL-12 is providing co-stimulatory signals upon TCR stimulation, since any potential effects would be observed at all times of pretreatment. The requirement for at least 24 hours of treatment also suggests that effects of IL-12 require transcriptional alterations. In addition, IL-12 pretreatment transiently increased the TCR-induced production of cytokines for h. We speculate that the effects of IL-12 are short lived in order to minimize the risk of immunopathology that would occur with long term enhancement of CD8 T cell function. IL-12 signals are known to promote STAT4 activation, which is thought to mediate IFN-γ transcription. Therefore, it was tempting to predict that following IL-12 pretreatment phosphorylated STAT4 would synergize with TCR stimulation and enhance the production of cytokines following TCR stimulation. Contrary to our hypothesis, we found that IL-12 pretreated cells had little detectable phosphorylation levels of STAT4 after 24 hours of treatment, which is the time when TCR stimulation occurred in the majority of our experiments. Interestingly, we found that STAT4 expression was reduced following IL-12 exposure, suggesting that STAT4 is degraded following IL-12 exposure. Our findings are supported by previous studies showing that IL-12 signaling results in 166

188 STAT4 degradation [80]. Even though there was no overt STAT4 phosphorylation after 24 hours of treatment, it is still possible that a minor proportion of STAT4 bound to DNA and capable of synergizing with TCR signals. However, the fact that IL-12 pretreatment required at least 24 h in order to alter how cells respond to TCR stimulation and that IL- 12 pretreated cells had increased expression of the TCR signaling protein SOS1, suggest that large scale STAT4 signaling is not synergizing with TCR-induced pathways. Instead, IL-12-induced pathways, including STAT4 and other signaling proteins, appear to increase the transcription of positive or negative regulators of TCR signaling molecules such as SOS1. It should be noted that type I interferons are known to activate STAT4 in human T cells [143], and pretreatments with type I interferons did not alter how our human CD8 T cells responded to TCR stimulation. This suggests that STAT4 is not solely mediating the ability of IL-12 pretreatment to increase the responses of human activated CD8 T cells to TCR stimulation. Our results indicate that IL-12 could be mediating its effects by amplifying TCR signaling pathways. We found that IL-12 signals significantly increase the TCR-induced activation of P38, and to a lesser extent JNK and AKT, without altering the activation of other more proximal TCR signals. Interestingly, P38 activation has been shown to have multiple roles on T cell responses, including cytokine release. Even though the effects of IL-12 in altering the activation of AKT and JNK were not statistically significant, it is possible these minor alterations have substantial biological relevance. Several recent reviews have suggested that relatively small perturbations in the activation of proximal signaling proteins can result in extensive changes in downstream functions [152, 153]. Thus, the subtle changes we observe in transient proximal signaling events are capable of 167

189 altering subsequent cellular function. To better determine whether IL-12 alters the activation of AKT, future studies should examine the effects of IL-12 pretreatment on the TCR-induced phosphorylation of AKT S473 (which is correlated with AKT activation) or substrates of AKT such as FoxO1 on threonine 24 and serine 256, FoxO3 on threonine 32 and serine 253, PRAS40 on threonine 246 and TSC2 on threonine1462. Similarly to better determine whether IL-12 modulates the activation of JNK we could examine the phosphorylation of its substrate c-jun. Our results fit well with previous literature from us and others demonstrating that prior exposure to inflammatory signals change the responses of T cells to subsequent TCR stimulation by altering TCR-mediated signaling. We showed in CHAPTER IV that in activated human CD4 T cells, IL-12 exposure selectively increased the TCR-induced activation of LCK, AKT, and P38 without altering the activation of other signaling molecules. In contrast, pathogen-induced inflammation increases proximal TCR-induced ZAP-70, PLC-γ, ERK1/2 and JNK1/2 phosphorylation without altering the activation of P38 in murine effector/memory CD8 T cells and increases ZAP-70 and ERK1/2 phosphorylation in murine secondary effector CD4 T cells [92, 94]. In addition, in human T cells, IL-7 and IL-15 mediate their effects by increasing the activation of ERK1/2 following TCR stimulation [96]. Our laboratory also observed that prior activation of T cells with a TLR5 ligand enhances TCR-mediated AKT activation, while simultaneously reducing LCK and LAT phosphorylation without altering ERK activation [95]. This suggests that individual inflammatory signals that alter subsequent T cell functions mediate this phenomenon by regulating TCR-mediated signaling through distinct mechanisms. 168

190 While examining the mechanism for the increase in TCR-induced P38 activation, we found that IL-12 pretreatment resulted in increased activation of the direct upstream activators of P38 (MKK3/MKK6) and SOS1. Even though the GRB2/SOS1 axis has recently been shown by us and others to control the activation of P38 [150, 151], the mechanism by which this is happening remains unexplored. SOS1 is one of the two Ras guanine nucleotide exchange factors present on T cells that play a key role in regulating Ras activity [5]. Ras in turn is canonically known to mediate the activation of ERK1/ERK2. However, recent studies suggest that SOS1 may mediate the activation of the P38 pathway independently of the enzymatic function of SOS1 [151]. Therefore, we speculate that the IL-12 mediated increased levels of SOS1 could be leading to the increased activation of P38 by an unknown mechanism. Overall, our results suggest that the IL-12 mediated increase in mrna and protein levels of IFN-γ and TNF-α are mediated by increasing the TCR-induced activation of select MAPK and AKT without altering more proximal TCR signals. 169

191 Figure 33: Activated human CD8 T cells pretreated with IL-12 have increased IFN-γ and TNF-α production following TCR stimulation. (A-B) Activated human CD8 T cells were exposed to media alone (no cytokine) or to different recombinant cytokines (50 ng/ml) for 24 h, washed, and then stimulated with 1 μg/ml of plate bound anti-tcr antibodies for 24 h. Alternatively, in (C-D), activated human CD8 T cells were treated with IL-12 (50 ng/ml) or media alone for 24 h. Cells were then washed and stimulated with or without plate bound anti-tcr antibodies for 24 h (1 μg/ml). (A-D) IFN-γ and TNF-α production were determined in the cell culture supernatants by ELISA. Graphs are shown as the mean ±SEM of values from three to seven different donors. Data were statistically compared to no cytokine cells using a two-tail Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s. or no symbol=not significant. 170

192 A B C D 171

193 Figure 34: IL-12 pretreatment does not alter the proliferation/survival of activated human CD8 T cells. Activated human CD8 T cells were incubated with or without various cytokines (50 ng/ml for 24 h), washed, and stimulated with or without 1 μg/ml of plate bound anti-tcr antibodies for 24 h. Viable cell numbers were determined by using the trypan blue dye exclusion assay. Graphs show the mean ± SEM values from five separate donors. Data were statistically compared to cells treated in media alone (no cytokine) with a two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 172

194 173

195 Figure 35: Human activated CD8 T cells pretreated with IL-12 have increased IFN-γ and TNF-α production following TCR stimulation. Human activated CD8 T cells were exposed to media alone (no cytokine) or to different combinations of recombinant cytokines (50 ng/ml) for 24 h, washed, and then stimulated with 1 μg/ml of plate bound anti-tcr antibodies for 24 h. FN-γ and TNF-α production were determined in the cell culture supernatants by ELISA. Graphs are shown as the mean ±SEM of values from three to seven different donors. Data were statistically compared to no cytokine cells using a two-tail Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s. or no symbol=not significant. 174

196 A B 175

197 Figure 36: The potentiation of TCR-mediated cytokine production by activated human CD8 T cells is transient and requires low physiological doses of IL-12 for at least 24 hours. Activated human CD8 T cells were left untreated (no cytokine) or (A) treated for various times with IL-12 (50 ng/ml), (B) exposed for 24 h to various doses of IL-12, or (C) treated with IL-12 for 24 h (50 ng/ml), washed and rested for various times. After treatment, the cells were washed and stimulated with 1 μg/ml of plate bound anti-tcr antibodies for 24 hours. Then IFN-γ production was determined by ELISA. (D-F) Activated human CD8 T cells were left untreated or treated with IL-12 for 24 h (50 ng/ml), washed and immediately stimulated with different doses of plate bound anti-tcr antibodies for 24 hours. IFN-γ production was then determined by ELISA. (E-F) Data were normalized, plotted using GraphPad Prism, and the EC 50 values calculated. Graphs are shown as the mean ±SEM of values from three to five different donors. Data were statistically compared to no cytokine cells using a two-tail Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s. or no symbol=not significant. 176

198 A B C D E F 177

199 Figure 37: Activated human CD8 T cells have variable surface expression of IL-12R β1 and β2. The expression of IL-12R β1 and IL-12R β2 were measured on activated human CD8 T cells using flow cytometry. Representative histograms show the expression of IL-12 receptor β1 or β2 following gating on live lymphocytes (based on forward and side scatter). Black line represents staining with IL-12R antibody and gray line represents unstained controls. Results are representative of three different donors. 178

200 179

201 Figure 38: Prior exposure to IL-12 increases the frequency of activated human CD8 T cells making IFN-γ and TNF-α upon TCR stimulation. Activated human CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/ml). Cells were then washed and stimulated with 1 μg/ml of plate bound anti-tcr antibodies for 6 h (A) or 18 h (B-D) with BFA added for the last 5 h. Intracellular protein levels of IFN-γ and TNFα were determined by flow cytometry. Live lymphocytes were gated based on forward and side scatter. Quadrants were set so the baseline cytokine production of non-tcr stimulated cells was less than 1%. The frequencies and median fluorescence intensities of cytokine expression were then determined. Data are shown as (A-B) representative plots or (C and D) mean ± SEM of four separate donors. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 180

202 A B C D 181

203 Figure 39: The IL-12 pretreatment increases IFN-γ and TNF-α mrna expression after TCR stimulation in activated human CD8 T cells. Activated human CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/ml), washed, and stimulated with or without 1 μg/ml of plate bound anti-tcr antibodies for 6 and 18 h. The expression of IFN-γ and TNF-α mrna was then determined by qpcr at these times. Data were normalized to those of mrna encoding β-actin and presented relative to no cytokine cells. Results are shown as mean ± SEM of three to four separate donors. 182

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205 Figure 40: IL-12 pretreatment does not alter the expression of surface molecules on activated human CD8 T cells. Activated human CD8 T cells were incubated with or without IL-12 (50 ng/ml for 6 h). Changes on the expression of surface molecules were assessed by flow cytometry. Cells were gated on live cells and then the MFIs were determined. The mean value ± SEM from two different donors is shown. Results were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 184

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207 Figure 41: The IL-12 mediated enhancement of cytokine production in activated human CD8 T cells is not due to residual STAT4 synergizing with TCR signals. Activated human CD8 T cells were left untreated or treated with 50 ng/ml of IL-12 for different times. At the indicated times, cells were lysed and immunoblotting for total and phosphorylated STAT4 was performed in whole cell lysates as described in the materials and methods section. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as representative blots (A) and (B-C) mean ± SEM of normalized results of three separate donors. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 186

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209 Figure 42: Prior exposure to IL-12 does not alter the activation of proximal TCR signaling molecules in activated human CD8 T cells. Activated human CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/ml). Cells were then washed and stimulated by crosslinking the TCR. To crosslink, cells were first exposed to 3 μg/ml of anti-tcr antibodies on ice, then warmed at 37 C for 10 min, and subsequently crosslinked for varios times with anti-mouse antibodies. Cells that did not receive any anti-tcr signals, are labeled as No TCR ; cells that were not crosslinked but that were briefly exposed to anti-tcr are labeled as time 0 ; cells that were briefly exposed to anti-tcr followed by crosslinking for various times are labeled as time 2,5,15,30. Then the phosphorylation of signaling molecules was assessed in whole cell lysates by immunoblotting as described in the materials and methods section. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A and C) representative blots and (B and D) mean ± SEM of normalized results of three separate donors. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 188

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211 Figure 42 continued C D 190

212 Figure 43: IL-12 pretreatment enhances the TCR-induced activation of select MAP kinases and AKT in activated human CD8 T cells. Activated human CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/ml). Cells were then washed and stimulated by crosslinking the TCR. To crosslink, cells were first exposed to 3 μg/ml of anti-tcr antibodies on ice, then warmed at 37 C for 10 min, and subsequently crosslinked for varios times with anti-mouse antibodies. Cells that did not receive any anti-tcr signals, are labeled as No TCR ; cells that were not crosslinked but that were briefly exposed to anti-tcr are labeled as time 0 ; cells that were briefly exposed to anti-tcr followed by crosslinking for various times are labeled as time 2,5,15,30. Then, the phosphorylation of signaling molecules was assessed in whole cell lysates by immunoblotting as described in the materials and methods section. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A) representative blots and (B) mean ± SEM of normalized results of three to seven separate donors. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 191

213 A B 192

214 Figure 44: IL-12 pretreatment does not alter the total protein levels of P38 in activated human CD8 T cells. Activated human CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/ml). Cells were then washed and stimulated by crosslinking the TCR. To crosslink, cells were first exposed to 3 μg/ml of anti-tcr antibodies on ice, then warmed at 37 C for 10 min, and subsequently crosslinked for varios times with anti-mouse antibodies. Then, the total expression of P38 was assessed in whole cell lysates by immunoblotting as described in the materials and methods section. Results were normalized to GAPDH, then the average of the total expression of P38 at each time point was calculated. Data are shown as mean ± SEM of normalized results of three separate donors. 193

215 194

216 Figure 45: IL-12 pretreatment increases the expression of SOS1 and the phosphorylation of MKK3/MKK6 downstream of the TCR. Activated human CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/ml). Cells were then washed and stimulated by crosslinking the TCR. To crosslink, cells were first exposed to 3 μg/ml of anti-tcr antibodies on ice, then warmed at 37 C for 10 min, and subsequently crosslinked for varios times with anti-mouse antibodies. Cells that did not receive any anti-tcr signals, are labeled as No TCR ; cells that were not crosslinked but that were briefly exposed to anti-tcr are labeled as time 0 ; cells that were briefly exposed to anti-tcr followed by crosslinking for various times are labeled as time 2,5,15,30. The phosphorylation or total expression of signaling molecules was assessed in whole cell lysates by immunoblotting and the results were normalized to GAPDH. Upon normalization, the average of the total expression of SOS1 at each time point was calculated. Data are shown as (A) representative blots and (B) mean ± SEM of normalized results of three to six separate donors. Data were analyzed with two-tail, unpaired Student s t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant. 195

217 A B 196