Regulation of memory CD8 T cell differentiation

Transcription

1 University of Iowa Iowa Research Online Theses and Dissertations Spring 2011 Regulation of memory CD8 T cell differentiation Nhat-Long Lam Pham University of Iowa Copyright 2011 Nhat-Long Lam Pham This dissertation is available at Iowa Research Online: Recommended Citation Pham, Nhat-Long Lam. "Regulation of memory CD8 T cell differentiation." PhD (Doctor of Philosophy) thesis, University of Iowa, Follow this and additional works at: Part of the Immunology of Infectious Disease Commons

2 REGULATION OF MEMORY CD8 T CELL DIFFERENTIATION by Nhat-Long Lam Pham An Abstract Of 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 May 2011 Thesis Supervisor: Professor John T. Harty

3 1 ABSTRACT Antigen-specific CD8 T cells play a critical role in protecting the host from infection by intracellular pathogens including viruses, bacteria and parasites. During the course of an infection, antigen-specific CD8 T cells undergo proliferative expansion to increase in number, which is followed by contraction and generation of a stable pool of long-lived memory cells. Importantly, memory CD8 T cells provide enhanced resistance to re-infection by the same pathogen. Moreover, the number of memory CD8 T cells correlates strongly with the level of protection against re-infection. Therefore, vaccines designed to promote cellular immunity should logically focus on achieving sufficiently high number of these memory cells for protection. Most current vaccines have relied on inducing antibodies to protect the host by neutralizing pathogens or blocking pathogen entry into the cells. However, there is a recognized need to design vaccines that also stimulate a strong CD8 T cell component of the adaptive immune response in addition to antibodies. Importantly, inflammatory cytokines induced by infection or vaccination with adjuvant act directly or indirectly on CD8 T cells to modulate their expansion, contraction and acquisition of memory characteristics. Thus, an understanding of how inflammatory cytokines regulate CD8 T cell memory differentiation may help guide new strategies for rational vaccine design. My studies examine the roles of inflammatory cytokines in regulating CD8 T cell memory differentiation. Specifically, my studies investigate the timing of inflammatory cytokine exposure and the role of type I IFNs and IL-12 in regulating effector/memory CD8 T cell differentiation, and exploiting the cross-presentation pathway to rapidly generate protective CD8 T cell immunity. Specifically, my results indicate that (i) encounter with inflammatory cytokines during the rapid proliferative phase deflects CD8 T cell differentiation away from memory towards a sustained effector program, (ii) that direct signaling by either type I IFNs or IL-12 to the responding CD8 T cells promotes maximal expansion, but none of these cytokines is essential to regulate the

4 2 effector/memory differentiation program, and (iii) cross-priming with either cellassociated antigen or antigen-coated, biodegradable microspheres accelerates CD8 T cell memory development and this approach can be exploited to rapidly generate protective CD8 T cell immunity. Abstract Approved: Thesis Supervisor Title and Department Date

5 REGULATION OF MEMORY CD8 T CELL DIFFERENTIATION by Nhat-Long Lam Pham 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 May 2011 Thesis Supervisor: Professor John T. Harty

6 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL PH.D. THESIS This is to certify that the Ph.D. thesis of Nhat-Long Lam Pham has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Immunology at the May 2011 graduation. Thesis Committee: John T. Harty, Thesis Supervisor Stanley Perlman Vladimir P. Badovinac Kevin L. Legge John D. Colgan Wendy Maury

7 To my parents. To my loving wife, Anh-Thu and my sons, Alan and Aidan. To my brother and his family. ii

8 ACKNOWLEDGMENTS I would like to express my gratitude to many people whose continuous support and encouragement have made this work both possible and enjoyable. I would like to thank all the members of my thesis committee for their incredible support and guidance during this process: Dr. Stanley Perlman, Dr. Vladimir Badovinac, Dr. Kevin Legge, Dr. John Colgan and Dr. Wendy Maury. I am indebted to all the past and present members of the Harty laboratory, who generously offered me their time, technical expertise and insightful advices and created a fun and enjoyable work environment to be in every day: Dr. Vladimir Badovinac, Dr. Noah Butler, Dr. Aaruni Khanolkar, Dr. Jeffrey Nolz, Dr. Nathan Schmidt, Dr. Thomas Wirth, Dr. Kelly Messingham, Dr. Jodie Haring, Rebecca Podyminogin, Lecia Pewe- Epping, Jemmie Hoang, Gabe Starbeck-Miller, and Lisa Hancox. I would like to convey my special thanks to Dr. Noah Butler, Dr. Aaruni Khanolkar, Dr. Jeffrey Nolz, Dr. Nathan Schmidt, Dr. Thomas Wirth, Rebecca Podyminogin, Lecia Pewe-Epping and Jemmie Hoang for their unconditional assistance and friendship. It is my privilege to share this work and journey with them. I also would like to take this opportunity to thank Dr. Vladimir Badovinac for his untiring support and guidance during my graduate training. Most importantly, I am thankful for his friendship both in and out of the laboratory. Several people and laboratories have provided helpful technical assistance and resources for my study: Stacy Hartwig from the Varga Laboratory, Ryan Langlois from the Legge Laboratory, Dr. Joel Kline for the TLR9 -/- mice and Dr. Matthew Mescher from the University of Minnesota for generous gifts of IFNABR -/- mice and IL-12Rβ1 -/- OT-I cells. I am grateful for the critical reviews and astute comments from Dr. Stanley Perlman and Dr. Steve Varga for all the manuscripts prepared for publication. iii

9 I appreciate the help from all members of the Interdisciplinary Immunology Graduate Program and the MSTP, who have been incredibly kind and accommodating: Paulette Scheler, Kathy Dautremont, Leslie Harrington and Elizabeth Snyder. I would like to thank my parents, my brother and his family, and my wife and son for their understanding and unwavering support. Finally, I owe my deepest gratitude and greatest appreciation to my mentor, Dr. John Harty, for his extraordinary mentorship and guidance for this thesis project. None of this work is possible without his patience, dedication and encouragement. iv

10 ABSTRACT Antigen-specific CD8 T cells play a critical role in protecting the host from infection by intracellular pathogens including viruses, bacteria and parasites. During the course of an infection, antigen-specific CD8 T cells undergo proliferative expansion to increase in number, which is followed by contraction and generation of a stable pool of long-lived memory cells. Importantly, memory CD8 T cells provide enhanced resistance to re-infection by the same pathogen. Moreover, the number of memory CD8 T cells correlates strongly with the level of protection against re-infection. Therefore, vaccines designed to promote cellular immunity should logically focus on achieving sufficiently high number of these memory cells for protection. Most current vaccines have relied on inducing antibodies to protect the host by neutralizing pathogens or blocking pathogen entry into the cells. However, there is a recognized need to design vaccines that also stimulate a strong CD8 T cell component of the adaptive immune response in addition to antibodies. Importantly, inflammatory cytokines induced by infection or vaccination with adjuvant act directly or indirectly on CD8 T cells to modulate their expansion, contraction and acquisition of memory characteristics. Thus, an understanding of how inflammatory cytokines regulate CD8 T cell memory differentiation may help guide new strategies for rational vaccine design. My studies examine the roles of inflammatory cytokines in regulating CD8 T cell memory differentiation. Specifically, my studies investigate the timing of inflammatory cytokine exposure and the role of type I IFNs and IL-12 in regulating effector/memory CD8 T cell differentiation, and exploiting the cross-presentation pathway to rapidly generate protective CD8 T cell immunity. Specifically, my results indicate that (i) encounter with inflammatory cytokines during the rapid proliferative phase deflects CD8 T cell differentiation away from memory towards a sustained effector program, (ii) that direct signaling by either type I IFNs or IL-12 to the responding CD8 T cells promotes maximal expansion, but none of these cytokines is essential to regulate the v

11 effector/memory differentiation program, and (iii) cross-priming with either cellassociated antigen or antigen-coated, biodegradable microspheres accelerates CD8 T cell memory development and this approach can be exploited to rapidly generate protective CD8 T cell immunity. vi

12 TABLE OF CONTENTS LIST OF TABLES...x LIST OF FIGURES... xi CHAPTER I. GENERAL INTRODUCTION...1 CD8 T cell biology: A brief historical perspective...1 CD8 T cell development and thymic education...2 Antigen processing and antigen presentation by major histocompatibility (MHC) class I molecules...3 CD8 T cell response to infection or immunization...4 Primary Memory CD8 T cells: Development and differentiation...7 Secondary response and secondary memory CD8 T cells...12 Inflammation and CD8 T cell response...13 Inflammation and memory CD8 T cell differentiation...16 Manipulating CD8 T cell memory...20 Rationale for project...21 II. A DEFAULT PATHWAY OF MEMORY CD8 T CELL DIFFERENTIATION AFTER DENDRITIC CELL IMMUNIZATION IS DEFLECTED BY ENCOUNTER WITH INFLAMMATORY CYTOKINES DURING ANTIGEN-DRIVEN PROLIFERATION...27 Introduction...27 Materials and Methods...28 Mice...28 Dendritic cells...29 Bacteria...29 Adoptive transfer experiments...30 Quantification and phenotypic analysis of antigen-specific T cells...30 Antibodies, peptides, and MHC Class I tetramers...30 Serum cytokine quantification...31 In vivo cytolytic assay...31 Results...31 TLR9 expression by the immunizing DC and responding CD8 T cells is not necessary for CpG ODN to regulate memory differentiation...31 CpG induces inflammatory cytokines with distinct magnitudes and kinetics...34 Encounter with inflammatory cytokines prior to antigen-driven proliferation of CD8 T cells does not prevent accelerated memory generation...35 Inflammatory cytokines exert the greatest impact on proliferating CD8 T cells...38 A default pathway of memory CD8 T cell differentiation is deflected by encounter with inflammatory cytokines...39 Model...41 vii

13 Discussion...42 III. DIFFERENTIAL ROLES OF SIGNAL 3 INFLAMMATORY CYTOKINES IN REGULATING CD8 T CELL EXPANSION AND EFFECTOR/MEMORY DIFFERENTIATION...66 Introduction...66 Materials and Methods...68 Mice...68 Dendritic cells and bacteria...68 Adoptive transfer experiments...68 Generation bone marrow chimeric mice...69 Quantification and phenotypic analysis of antigen-specific T cells...69 Results and Discussion...70 Direct signaling by signal 3 inflammatory cytokines, type I IFNs or IL-12, to CD8 T cells promotes their expansion...70 Neither type I IFNs nor IL-12 direct signaling in CD8 T cells is required for effector differentiation...71 Functional effector/memory differentiation of CD8 T cells is independent of type I IFNs or IL-12 signaling...74 IV. EXPLOITING THE CROSS-PRESENTATION PATHWAY TO RAPIDLY GENERATE PROTECTIVE CD8 T CELL IMMUNITY...89 Introduction...89 Materials and Methods...91 Mice...91 Dendritic cells, recombinant bacteria and viruses...92 Analysis of protective capacity after L. monocytogenes or vaccinia challenge...93 PLGA microspheres, ovalbumin, recombiant hemagglutinin, CpG and peptides...93 Quantification and phenotypic analysis of antigen-specific T-cells...94 Antibodies and MHC Class I tetramers...94 Serum cytokine quantification...95 Measurement of airway resistance...95 Results...95 Cell-associated antigen cross-primes accelerated CD8 T cell memory in vivo...95 Accelerated secondary response to booster immunizations following cross-priming provides enhanced protective immunity...97 Inflammation prevents accelerated memory CD8 T cell generation following cross-priming...99 Autologous peripheral blood mononuclear cells (PBMC) deliver antigen for cross-priming to achieve protective immunity Biodegradable PLGA microspheres serves as universal crosspriming vehicle Cross-priming followed by early boost generates protective heterosubtypic immunity against lethal influenza infection Discussion viii

14 V. DISCUSSION AND FUTURE DIRECTIONS Inflammation and CD8 T cell biology Effector and memory CD8 T cell differentiation: Signal 3 inflammatory cytokines and beyond Accelerated memory CD8 T cell generation: Translating to vaccines REFERENCES ix

15 Table LIST OF TABLES 1. Phenotypic and functional attributes of naïve vs. effector vs. memory antigenspecific CD8 T in mice after infection or vaccination x

16 LIST OF FIGURES Figure 1. Inflammation regulates the kinetics of CD8 T cell response to infection or vaccination with late or early booster immunization Low number OT-I adoptive transfer mimics the endogenous CD8 T cell response after peptide-coated DC immunization in the presence or absence of CpG Similar phenotypes between OT-I and endogenous antigen-specific CD8 T cells following low number OT-I adoptive transfer and peptide-coated DC± CpG immunization or LM infection CpG ODN does not signal directly on transferred DC or T cells but rather induces the inflammatory environment that influences the rate of memory generation following DC immunization CpG-induced inflammatory environment in the host displayed unique cytokine profiles and kinetics Exposure to inflammation prior to antigen-driven expansion of CD8 T cells does not prevent accelerated memory generation after DC immunization Inflammation exerts the greatest impact on memory differentiation during proliferation of CD8 T cells The default pathway of memory CD8 T cell differentiation is deflected by encounter with inflammatory cytokines Antigen-specific CD8 T cells stimulated with either DC or DC+CpG immunization exhibited similar effector functions early Model representing how the timing of inflammation relative to DC immunization influences the rate at which CD8 T cells acquire memory characteristics Type I IFNs and IL-12 serve as signal 3 to promote optimal CD8 T cell expansion Neither type I IFNs nor IL-12 direct signaling to CD8 T cells is required for acquisition of effector phenotype Differential effect of varying CpG amount on both phenotype and functions of antigen-specific CD8 T cells following DC immunization Acquisition of effector phenotype is independent of type I IFNs or IL-12 in polyclonal antigen-specific CD8 T cell response Neither type I IFNs nor IL-12 signaling is required to inhibit accelerated memory differentiation in the presence of CpG-induced inflammation...85 xi

17 16. Type I IFNs or IL-12 signaling in the CD8 T cells was not required for secondary memory development Characterizing K b-/- mova splenocytes and K b-/- mova mice Cross-priming with cell-associated antigen generates functional antigenspecific CD8 T cells with accelerated memory phenotype in vivo Accelerated secondary response to booster infections following cross-priming with cell-associated antigen provides enhanced protective immunity Accelerated response to booster infections following cross-priming with cellassociated antigen generates stable memory pool of antigen-specific CD8 T cell with secondary memory phenotype CpG-induced inflammation prevents accelerated generation of memory CD8 T cells and early prime-boost response following cross-priming with cellassociated antigen Dose-dependent antigen-specific CD8 T cell response following crosspriming with cell-associated antigen and early booster infection Irradiated, Ova-coated syngeneic splenocytes cross prime accelerated memory CD8 T cells that could be boosted early to significantly higher number Irradiated, Ova-coated autologous peripheral blood mononuclear cells (PBMC) cross prime accelerated memory CD8 T cells that could be boosted early to significantly higher number Cross-priming with Ova-adsorbed, biodegradable poly(lactic-co-glycolic) acid microspheres (PLGA) followed by early booster infection quickly generates robust Ova 257 -specific CD8 T cells Amplified secondary response following non-infectious booster regimens in PLGA-Ova-cross-primed mice Cross-priming with avian hemagglutinin H5-adsorbed, biodegradable poly(lactic-co-glycolic) acid microspheres (PLGA) followed by early booster infection generates robust protective memory CD8 T cell immunity against different sub-strain of influenza infections PLGA-H5-cross-primed and early boosted mice have reduced viral titer in the lungs Cross-priming with avian hemagglutinin H5-adsorbed, biodegradable poly(lactic-co-glycolic) acid microspheres (PLGA) followed by early booster infection rapidly generates protective immunity against different sub-strain of influenza infections xii

18 1 CHAPTER I GENERAL INTRODUCTION CD8 T cell biology: A brief historical perspective The nascent field of CD8 T cell biology has evolved immensely during the last 6 decades (1). The pioneering work of Jacques F.A.P. Miller with thymectomized mice in the 1950s and early 1960s illustrated the essential role of the thymus for immunological competence and the existence of another lymphocyte subset, the T lymphocytes, that was different from antibody-producing B lymphocytes (2). In the 1970s, the discovery of two functionally distinct subsets of T lymphocytes based on Ly antigens formally separated CD4 and CD8 T cells (3-5). Although the cytotoxic function of CD8 T cells was well characterized by this time, an intriguing question remained unanswered: how do CD8 T cells recognize and kill their target cells? The landmark discovery of major histocompatibility (MHC) restriction by Rolf Zinkernagel and Peter Doherty in 1974 shed light on this important question (6, 7). In the 1980s, the cloning of the mouse T-cell receptor (TCR) β-chain by Steve Hedrick and Mark Davis and the human TCR β-chain by Tak Mak s group, together with the solving of the crystal structure of MHC Class I by Pamela Bjorkman from Wiley s group, illuminated the intricate interaction between the CD8 T cell and MHC Class I protein (8-12). Around the same time, Alain Townsend et al. discovered that cytotoxic CD8 T cells recognize short peptide fragments derived from influenza virus nucleoprotein (13, 14). The picture of how CD8 T cells recognize foreign antigen loaded on MHC class I would not be complete without the characterization of the CD8 molecule as the co-receptor that binds to the α3 domain of the MHC Class I molecule (15). Over the past few decades, the field of CD8 T cell biology has made numerous great strides from the molecular understanding of specific recognition by CD8 T cells to their development, trafficking, response to pathogens, memory generation and homeostasis. In particular, insights into the process of memory CD8 T cell generation hold the key to rational vaccine design to achieve protective cell-mediated immunity.

19 2 CD8 T cell development and thymic education The αβ-tcr-expressing T cells originate from progenitors that arise from hematopoietic stem cells residing mainly within specialized niches in the bone marrow (16). These progenitors migrate out of the bone marrow and settle into the thymus where they receive instructional signals from thymic epithelial cells and mesenchymal fibroblasts to undergo a complex differentiation process to become mature T lymphocytes (17, 18). Upon entry into the thymus, the progenitors are referred to as double negative (DN) cells due to the lack of expression of both CD4 and CD8 molecules. As the progenitors move from the cortico-medullary junction to the cortex, rearrangement of the TCR β locus is initiated. Successful assembly of TCRβ/invariant pre-tcrα/cd3 complex and signaling through it commit the thymocytes to αβ T lineage development and lead to rearrangement of the TCR α locus and the expression of both CD4 and CD8 co-receptors, thus generating double-positive (DP) thymocytes (19). The DP thymocytes undergo thymic education that consists of positive and negative selection to ensure that cells with functional TCRs that are not self-reactive will emerge from the thymus to circulate as mature T cells. During thymic education, DP thymocytes face three possible fates. If DP thymocytes fail to receive signal through their TCR (either no affinity or very low affinity for self-peptide/mhc complexes), they will die within the thymus by neglect since engaging the TCR provides a vital survival signal (20). On the other hand, DP thymocytes with high affinity for self-peptide/mhc complexes receive strong signals through their TCR and thus undergo clonal deletion, which is the process known as negative selection (21). Although clonal deletion appears to be the main mechanism for eliminating self-reactive T cells, other processes such as anergy have also been described in which self-reactive T cells become hyporesponsive or reactivate the TCR rearrangement program to replace the high-affinity TCRs (21). In contrast, during positive selection, DP thymocytes with moderate affinity for MHC class

20 3 I-restricted self-peptide presented by cortical epithelial cells receive sufficient survival signal through TCR engagement and downregulate CD4 expression to become mature CD8 T cells (20). As the mature CD8 T cells egress from the thymus, they continue to receive survival signals through TCR engagement with self-peptide/mhc class I complexes and through the interleukin-7 receptor for their homeostatic survival (22). Once exiting the thymus, the CD8 T cells circulate throughout the body and populate secondary lymphoid organs, ready to be activated by professional antigen presenting cells such as dendritic cells and to defend the host against microbial infection. Antigen processing and antigen presentation by major histocompatibility (MHC) class I molecules Before naïve CD8 T cells can be primed and activated, they need to engage mature professional antigen-presenting cells (APCs) such as dendritic cells (DCs). DCs comprise a heterogeneous network of migratory and resident cells that survey the environment, capture and process foreign antigens into peptide fragments, and present these antigenic peptides on MHC class I molecules to CD8 T cells (23). Antigen processing and antigen presentation by DCs to prime naïve CD8 T cells has been an active area of intense research since the discovery of DCs by Ralph Steinman and Zanvil Cohn in the 1970s (23-25). Broadly defined, there are two routes by which protein antigen gains access to the MHC class I pathway: endogenously synthesized or exogenously acquired (26). In the classical MHC class I assembly pathway, endogenously synthesized proteins are processed into peptide fragments by the proteasome. These peptide fragments are transported back into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) where they are further processed and loaded onto MHC class I molecules by a multi-component complex. Once loaded in the binding groove, the peptide stabilizes the membrane-bound heavy chain and the soluble β 2 -microglobulin chain to form a peptide-bound MHC class I

21 4 molecule, which is transported to the cell surface (27, 28). The classical pathway plays a dominant role in alerting the immune system, especially CD8 T cells, to most if not all infections by intracellular pathogens. Since most intracellular pathogens, particularly viruses, hijack the host s translation machinery to make their own proteins, pathogenderived antigens gain access efficiently to the host s classical MHC class I assembly pathway (27). In addition to the classical pathway, CD8 T cells can also be primed by exogenously acquired antigen in a process defined as cross-priming, which was first demonstrated by Bevan in 1976 (29). Exogenous protein antigens can be taken up and presented on the MHC class I molecules by DCs in a process defined as crosspresentation (30). Although controversy exists in the molecular details of how exogenously acquired antigens are processed and loaded on MHC class I, it is generally agreed that DCs, especially CD8α + DCs, are more efficient than other cell types at crosspresentation and that cell-associated or particulate antigens are better than soluble antigens as substrates for cross-presentation (31). Both of these pathways have been exploited for vaccine development. For example, the conventional presentation pathway is exploited by DNA vaccination while the cross-presentation pathway is the possible target for subunit vaccines. In addition, vaccines that employed live attenuated viruses such as vaccinia for smallpox or influenza (FluMist ) against seasonal flu potentially target both presentation pathways. CD8 T cell response to infection or immunization CD8 T cells play an essential role in defending the host against intracellular bacteria, viruses and protozoan parasites. In naïve laboratory mice, CD8 T cells with TCRs for any given specificity are present at a very low number, in the range of 10 to 1000 cells (32, 33). The rarity of any single specificity is thought to maximize the diversity of the naïve CD8 T cell repertoire to protect the host against a vast array of potential pathogens (34). In order to combat infection, CD8 T cells with specificity

22 5 against the pathogen must multiply in number and acquire effector functions such as cytotoxic activity and cytokine production. The CD8 T cell response to infection or immunization is initiated with the encounter between naïve CD8 T cells and the mature DC displaying pathogen-derived peptide on MHC class I molecules. Following infection or immunization, DCs acquire and process foreign antigens as a consequence of either direct infection of the DC or phagocytosis of infected cells (35, 36). In addition to antigen acquisition, immature DCs also receive activating signals from ligation of pathogen-associated molecular patterns to toll-like receptors (TLRs) and inflammatory cytokines to differentiate into mature DCs, upregulate co-stimulatory molecules, and migrate to secondary lymphoid organs where encounter with naïve CD8 T cells takes place (37). After receiving signal 1 (TCR:peptide/MHC class I) and signal 2 (co-stimulation), naïve CD8 T cells embark on a differentiation program that can be characterized in 3 distinct phases: expansion, contraction and memory generation and maintenance (38). In addition, pathogen-induced inflammatory cytokines also act directly on the responding CD8 T cells to regulate their expansion and effector differentiation (38, 39). In particular, type I interferons (IFNs) (i.e. IFN-α or IFN-β), interferon-γ (IFN-γ) and IL-12 have been described as critical survival signals (signal 3) for optimal CD8 T cell accumulation during the expansion phase (38, 39). Furthermore, expansion in the numbers of antigen-specific CD8 T cells is coupled with their acquisition of effector functions to combat the infection (Figure 1A). During the expansion phase, antigen-specific CD8 T cells proliferate robustly and accumulate in numbers. At the peak of expansion, the number of antigen-specific CD8 T cells can be greater than 10,000-fold increased compared to the corresponding naïve precursors (38, 40, 41). Concurrent with rapid proliferation, antigen-specific CD8 T cells differentiate into effector cells, downregulate homing molecules (i.e. CD62L and CCR7) to leave the secondary lymphoid organs, and acquire antimicrobial effector functions such as cytolytic activity (i.e. perforin, Granzyme B production) and cytokine production

23 6 (i.e. IFN-γ, TNF-α) to combat the infection (Table 1) (42-44). Although antigen is required to initiate proliferation, antigen persistence is not necessary for full expansion of CD8 T cell numbers (45). A brief encounter with antigen and costimulation is sufficient to set in motion a developmental program that drives CD8 T cell expansion, acquisition of effector function, and memory generation (46). Several studies support this concept of programming the CD8 T cell response (47-49). Using Listeria monocytogenes (LM) infection and antibiotic treatment to terminate the infection early, Mercado et al. demonstrated that both the kinetics and magnitude of Listeria-specific CD8 T cell expansion was not altered by truncating the duration of bacterial infection (47). Using engineered APCs and transgenic OT-I cells (transgenic CD8 T cells specific for H2-K b - restricted SIINFEKL peptide derived from ovalbumin), van Stipdonk and colleagues showed that a brief period of antigenic stimulation is sufficient to drive clonal expansion and acquisition of effector functions by OT-I cells both in vitro and in vivo (48). By combining both in vitro stimulation and adoptive transfer of P14 cells (transgenic CD8 T cells specific for H2-D b -restricted GP 33 peptide derived from LCMV), Kaech et al. also provided evidence that recruitment of naïve transgenic CD8 T cells occurs quickly and that they proliferate and differentiate into memory cells without further antigen stimulation (49). Furthermore, work in our laboratory extended these findings to CD4 T cell responses to LM infection (50). In addition, antigen load, signal strength and duration of antigen presentation potentially influence the magnitude and the functional qualities of the CD8 T cell response but not the differentiation program (51-53). Thus, the concept of programming dissociates the immediate early activation events where antigen is required from the ensuing proliferation and memory differentiation where further antigen stimulus is not necessary (45). Following expansion is the contraction phase where the number of pathogenspecific CD8 T cells declines precipitously. During the contraction phase, 90%-95% of the effector CD8 T cells at the peak of expansion die via apoptosis, leaving behind a

24 7 small fraction that survives and forms the initial memory CD8 T cell pool (34, 54). The contraction in number of effector CD8 T cells following intense expansion is thought to be critical in limiting immunopathology, maintaining homeostasis and preserving flexibility to respond against new pathogens (34). Five to 10% of pathogen-specific CD8 T cells from the peak of expansion survive the contraction phase and initiate the memory pool (45). More importantly, these surviving cells are present at a much higher number, with higher functional avidity, and lower signaling thresholds required for activation than their naïve precursor counterparts with same antigen specificity (38). In addition, memory CD8 T cell number can be stably maintained for the life of the host, at least in experimental animal models (Figure 1A) (54). In humans, both memory CD4 and CD8 T cells specific for vaccinia virus could be detected up to 75 years after immunization in half of the vaccinees who received at least one vaccination (55, 56). Thus, a successful immune response culminates in the generation of memory T cells that are present at a higher number than their corresponding naïve precursors and ready to provide enhanced protection should the host be exposed to the same pathogen again in the future. Primary Memory CD8 T cells: Development and differentiation A productive immune response following an acute infection results in elimination of the pathogen and generation of immunological memory. Memory CD8 T cells are able to respond more efficiently to infection by the same pathogen and thus provide enhanced protection for the host by virtue of their increased numbers and faster elaboration of effector functions (i.e. cytotoxicity and cytokine production) compared to their naïve counterparts (45). This biological outcome serves as the fundamental basis for designing and implementing vaccines that induce immunological memory and protect recipients against many potentially lethal diseases. Consequently, tremendous effort including extensive work from our laboratory has been focused on trying to understand

25 8 how memory CD8 T cells are generated and maintained following infection or vaccination. Studying memory CD8 T cells in vivo at the population level has proved challenging despite the availability of many sophisticated flow cytometric and molecular methodologies. Most, if not all, CD8 T cell immunologists agree that substantial phenotypic and functional heterogeneity is observed in populations of antigen-specific memory CD8 T cells that are examined following infection or immunization. To date, there is no single marker or property that unequivocally identifies and defines memory CD8 T cells (45). Thus, the expression pattern of multiple cell surface molecules coupled with various functional attributes is often examined to delineate naïve versus effector versus memory CD8 T cells. The phenotypic and functional changes that accompany the transition from naïve to effector to memory CD8 T cells are summarized in Table 1. Although incomplete, this table features some of the most common phenotypic and functional attributes that have been used to characterize and identify antigen-specific CD8 T cell at the population level. Furthermore, the memory CD8 T cell pool will be comprised of multiple subpopulations. Thus, subsets within the memory population are also defined and studied by differential expression of cell surface markers (45, 57). One central question in the field of memory CD8 T cell biology is how these cells develop following infection? Currently, there are at least three different models proposed for the memory CD8 T cell developmental pathway (58). Investigating human T cells, Sallusto et al. originally described the two major subsets of memory CD4 T cells based on differential expression of lymphoid homing receptors, CCR7 and CD62L. The CCR7 - CD62L lo subset is defined as effector memory T cells (T EM ) while CCR7 + CD62L hi subset is termed central memory T cells (T CM ) (43). Moreover, the T EM subset is largely excluded from the secondary lymphoid organs and rapidly elaborates effector functions while T CM subset dominates in secondary lymphoid organs and lacks immediate effector function as measured by IFN-γ production (43). In addition, CCR7 - memory CD8 T cells

26 9 also make less IFN-γ and more IL-2 when stimulated using anti-cd3 antibody compared to the CCR7 + cells (43). Since T CM and T EM subsets have seemingly different migratory potential and effector function, research interests have focused on the developmental relationship between these two subsets (57). Using viral infection models in mice, Wherry et al. proposed a linear differentiation model in which naïve CD8 T cells differentiate into effector cells upon antigen encounter, a fraction of which survive the contraction phase to become T EM cells that eventually develop into T CM cells over time (59). Although it is useful to define different memory CD8 T cell subsets based on differential expression of surface molecules and cytotoxicity, it is seemingly inadequate to account for the heterogeneity of memory CD8 T cell populations. Defining different subsets within the memory pool provides a strong argument for further research on the factor(s) and/or condition(s) that regulate the lineage commitment and how to exploit them in vaccine design. However, recent work sheds more light on the complexity of the heterogeneous memory population (45). It is generally accepted that the T CM population has higher secondary proliferative potential compared to the T EM population (57). However, while T CM cells conferred better protective immunity to certain infections (i.e. LCMV) (59), T EM cells appeared to be superior in providing protection against other infections (i.e. sendai virus, vaccinia virus) (60-62). In addition, CCR7 - and CCR7 + transgenic P14 populations following LCMV infection produced similar amounts of IFNγ and exhibited similar cytolytic activity against GP 33 -coated target cells (63). The linear differentiation model is brought into question by a report by Marzo et al. and a study in our laboratory demonstrating that the initial precursor frequency significantly influenced all aspects of CD8 T cell response including memory lineage commitment (64, 65). These two reports raised concern about the use of supraphysiological number of transgenic cells in studies to examine the biology of the CD8 T cell response. An additional layer of complexity was revealed by recent work in our laboratory showing that secondary memory CD62L lo CD8 T cells provided better protection against systemic

27 10 Listeria monocytogenes challenge on a per-cell basis when compared to primary CD62L lo memory CD8 cells (66). Moreover, constitutive expression of a non-cleavable form of CD62L on effector CD8 T cells did not preferentially favor the development of T CM population (67). Conversely, the absence of CD62L expression did not enhance the development of the T EM population (67). Furthermore, the most recent investigation in our laboratory demonstrated that when the same number of either secondary or primary transgenic memory P14 cells were adoptively transferred into a naïve host, the former failed to protect mice from chronic infection by LCMV clone 13 while the latter cleared the virus (under review). These results suggested that focusing on factor(s) or condition(s) that potentially favor the generation of one subset over the other based on phenotypic difference without the knowledge of antigen exposure history, nature of infection (systemic vs. localized; acute vs. chronic), and pathogen may not be the best approach for vaccine design. Chang et al. from Reiner s group proposed a second model for memory CD8 T cell development that described the asymmetric division of naïve CD8 T cells (68). In this model, the division of naïve CD8 T cell following antigenic stimulation into two daughter cells with unequal inheritance of cellular proteins determines their fate. The daughter cell proximal to TCR signaling becomes the effector cell that is characterized with Granzyme B +, IFN-γ +, IL-7Rα lo (CD127) and CD62L lo. In contrast, the daughter cell distal to TCR signaling becomes an early memory precursor that is characterized as Granzyme B -, IFN-γ -, IL-7Rα hi and CD62L hi (68). Although this model offers an attractive mechanism to argue for the formation of memory precursors very early during an immune response, it does not adequately reflect the accounting of memory precursor CD8 T cells, which comprise only 5%-10% of the number at the peak of expansion. Based on the asymmetric division model, the number of memory precursors should equal the number of effector daughter cells. However, the majority of cells found at the peak of expansion exhibit an effector phenotype (34). It is conceivable that the two daughter

28 11 cells generated by asymmetric division have differences in proliferative potential, but this was not addressed by Chang et al. Furthermore, the authors also acknowledged that environmental cues could be essential in determining the choice of cell fate (68). This model also implies that daughter cells inheriting the effector fate would lose the replicative capacity that is characteristic of memory cells. These implications are disputed by the third model of memory CD8 T cell development by Bannard et al. (69). In the third model of memory CD8 T cell development, Bannard et al. speculated that naïve CD8 T cells directly differentiate into T CM or pre-t CM cells with mixed characteristics of both effector and memory cells following activation. The pre-t CM cells can elaborate Granzyme B and IFN-γ that are characteristic of effector cells but retain expression of lymphoid homing molecules such as CD62L and proliferative capacity that are characteristic of central memory T cells. The authors also speculated that other unidentified signals control further differentiation of these pre-t CM cells to commit to short-lived effector CD8 T cells (58). Using a transgenic mouse strain in which enhanced yellow fluorescent protein (EYFP) can be conditionally and irreversibly turned on in cells expressing Granzyme B, Bannard et al. found that EYFP + CD8 T cells are not impaired in secondary replicative potential in a murine model of influenza infection (69). The authors argued that acquisition of effector function as measured by Granzyme B expression by naïve CD8 T cells following activation did not necessarily commit these cells to become senescent effector or T EM cell (58). CD8 T cells that expressed Granzyme B and were permanently marked by EYFP expression were still able to proliferate robustly during secondary challenge (69). As the result, the authors also disagreed with the linear differentiation model (58). The three different models provide a useful conceptual framework for investigating how memory CD8 T cells develop from naïve precursors following antigenic stimulation. Nevertheless, an important issue with respect to vaccine development is how to efficiently achieve protective numbers of memory CD8 T cells.

29 12 While these three models proposed different CD8 T cell developmental pathways, they do not address the rate of memory CD8 generation. For vaccine design to elicit cellmediated immunity, how fast and how efficient the vaccine induces protective numbers of memory cells become the critical parameters to consider. For example, in the cases of viral pandemics or cancer immunotherapy where time is of essence, a vaccine strategy that quickly generates sufficient numbers of memory CD8 T cells to protect the host is highly desirable. Secondary response and secondary memory CD8 T cells The secondary CD8 T cell response to re-infection by the same pathogen is characterized by robust proliferation of antigen-specific CD8 T cells and rapid clearance of the pathogen. This enhanced secondary response is a cardinal hallmark of memory cell function. In contrast to the abundance of literature examining the development and functions of primary memory CD8 T cells, much less is known about the generation and maintenance of secondary or tertiary memory CD8 T cells (34). Even scarcer is information about the behavior of the antigen-specific CD8 T cells that have responded to their cognate antigen for four times or more. This knowledge has tremendous practical relevance since humans are more likely to have different memory cell populations that have been exposed to antigen more than one time either by vaccinations or natural infections. Recent work in our laboratory has started to examine the profile of gene expression in these antigen-specific CD8 T cells that have been stimulated multiple times in vivo (under review). Recent studies from our laboratory and other laboratories begin to shed light on the phenotypic and functional differences between primary and secondary memory CD8 T cells (66, 70-72). Compared to primary memory CD8 T cells, secondary memory CD8 T cells are slow to acquire central memory phenotype and exhibit less basal proliferation (66, 71). In addition, the kinetics of secondary response exhibits faster expansion peak

30 13 and protracted contraction phase compared to primary response (70, 72). Interestingly, our laboratory and others also demonstrated that secondary memory CD8 T cells exhibit sustained effector functions such as Granzyme B expression and protect better against rechallenge with many pathogenic infections on a per cell basis (66, 71). Since many successful regimens require booster immunizations to reach protective memory T cell response (73), a better understanding of secondary CD8 T cell generation, function and maintenance will aid in vaccine development aimed at eliciting protective cell-mediated immunity against specific pathogens. Inflammation and CD8 T cell response A long-standing interest in our laboratory focuses on understanding how inflammatory cytokines regulate memory CD8 T cell differentiation. This knowledge would potentially facilitate vaccine development aimed at manipulating memory CD8 T cell generation. Whereas infection in animal models often generates robust CD8 T cell response, many vaccines, especially subunit vaccines, stimulate weak CD8 T cell responses (45, 73). However, administering vaccines with adjuvant improves both the magnitude of the response and the subsequent immunological memory (74). One property that is shared between an active infection and an adjuvant administration in a host is the generation of pro-inflammatory cytokines. During an infection, the interaction between pathogen and innate arms of the immune response results in the rapid induction of pro-inflammatory cytokines that play a critical role in limiting pathogen replication and maturing professional APC (i.e. upregulation of co-stimulatory molecules and MHC class I and class II) (75). In addition to their established roles as the endogenous danger signals and in APC biology, research in the past decade has demonstrated the direct impact of pro-inflammatory cytokines on all phases of the CD8 T cell response including expansion, contraction and memory generation (38).

31 14 The concept of signal 3 emerges from the identification of pro-inflammatory cytokines that act directly on the responding CD8 T cells to ensure an optimal response (39). This concept proposes that the responding CD8 T cells must also receive and integrate signals from inflammatory cytokines (signal 3) during an infection or vaccination with adjuvant, in addition to signal 1 (antigen) and signal 2 (costimulation). To date, at least four pro-inflammatory cytokines, namely IL-12, type I interferon (IFNαβ), type II interferon (IFN-γ) and most recently IL-21, have been identified to provide signal 3 for optimal CD8 T cell expansion and development of effector function (38, 39). Work from Mescher s group illustrated the role of IL-12, type I IFNs and IL-21 on CD8 T cell clonal expansion and acquisition of effector function in vitro (39). Using artificial APC (microspheres coated with peptide/mhc class I complex and B7 ligands) to stimulate naïve transgenic-tcr CD8 T cells, these studies documented that the addition of IL-12 or type I IFNs or IL-21 promoted clonal expansion and acquisition of effector functions by the responding CD8 T cells (76-79). Accumulating evidence further supports a direct role of IL-12 or type I IFNs in facilitating an optimal CD8 T cell response in experimental models of peptide immunization (80, 81) and infection (82, 83). Experiments using adoptive transfer of transgenic CD8 T cells that lack the receptor for either type I IFNs or type II IFN in a viral infection model provided compelling evidence that direct signaling by these cytokines to responding CD8 T cells is important for their optimal response (82-84). More importantly, the adoptive transfer approach allowed for the dissection of a defect in CD8 T cell expansion following infection. Careful analysis revealed that the receptor-deficient CD8 T cells proliferated, but failed to accumulate due to decreased survival during proliferation when compared to the receptor-sufficient cells (34, 82-84). In addition, type II IFN also played an essential role in promoting optimal expansion of antigen-specific CD8 T cells after viral infection (85). However, memory CD8 T cell generation and maintenance are still detectable or even better in the absence of type I IFNs signaling or IL-12 signaling following viral or bacterial infection,

32 15 respectively (83, 86). Thus, signal 3 seemingly acts as a survival signal to the CD8 T cells during the expansion phase, but is not required for CD8 T cell memory generation (38). Interestingly, the requirement of specific cytokine signaling as crucial signal 3 for optimal CD8 T cell response is dependent on the pathogen. For example, type I IFN signaling in CD8 T cells appeared critical for expansion in LCMV infection model while the absence of IL-12 or IFN-γ signaling resulted in much less severe defect in this model. Furthermore, comparisons between different infections such as LCMV, vaccinia virus, vesicular stomatitis virus and LM while controlling for antigen delivery highlighted the pathogen-specific requirement for signal 3 by responding CD8 T cells (87). It is important to emphasize that analysis of inflammatory cytokine signaling to the responding CD8 T cells is further complicated by the level and timing of expression of many cytokine receptors. For example, type I IFN or IFN-γ receptors are constitutively expressed on naïve CD8 T cells (88-91), whereas as other receptors (such as IL-12 or IL-18 receptors) may be induced by activation (92-95). Furthermore, synergy between inflammatory cytokines and TCR signaling could play an important role in the CD8 T cell response. This synergy has been well documented for CD4 T cells. For example, the IL-12 receptor is undetectable on most resting T cells. Following activation via TCR and co-stimulation, expression of both chains of IL-12 receptor is induced (94, 96). However, under T H 1 polarizing condition, T H 1 cells receive IL-12 and IFN-γ signaling and further upregulate the IL-12Rβ2 chain. On the other hand, under T H 2 polarizing condition, T H 2 cells receive IL-4 signaling and downregulate the IL-12Rβ2 chain and become less responsive to IL-12 (92). Following the expansion phase, a pronounced contraction phase ensues in the CD8 T cell response whether the infection is cleared or not (38). Several studies including those from our laboratory have suggested that contraction of CD8 T cell response is programmed by early events during the infection and is independent of the dose and duration of the infection (97, 98). Interestingly, inflammation has also been

33 16 implicated in the regulation of CD8 T cell contraction. Work in our laboratory provided evidence that the inflammatory cytokine IFN-γ played a crucial role in CD8 T cell contraction (85). IFN-γ-deficient BALB/c mice exhibited substantially reduced contraction resulting in elevated numbers of antigen-specific memory CD8 T cells for at least one year following infection with attenuated LM and LCMV (85). Furthermore, antibiotic pretreatment of BALB/c mice prior to infection with LM substantially reduced systemic IFN-γ and prevented the contraction of antigen-specific CD8 T cells while memory CD8 T cell generation was not impaired (99). Strong evidence has accumulated over the past decade demonstrating the role of inflammatory cytokines in regulating multiple aspects of the CD8 T cell response following infection or vaccination. In particular, an understanding of how inflammation contributes to an immune response provides the rationale and guidance for the development of adjuvants to be employed in conjunction with vaccines (74). However, much less is known about the impact of inflammatory cytokines on memory CD8 T cell differentiation and generation. Since the ultimate goal of vaccine development is to stimulate long-lasting memory cells that confer protection upon re-encounter with the pathogen, an understanding of parameter(s) that influence memory CD8 T cell differentiation is of great importance for rational vaccine design. Inflammation and memory CD8 T cell differentiation Studies on memory CD8 T cell generation revolve around one central question: how do antigen-specific CD8 T cells escape the contraction phase and survive to form the memory compartment? The ability to differentiate between CD8 T cells fated to perish during contraction or survive as memory precursors would provide valuable clues to this question. However, sorting out these two populations has been challenging since no single property unequivocally identifies one versus the other (38). Much effort has been devoted into isolating these two populations. Kaech et al. suggested that expression

34 17 of IL-7Rα chain (CD127) selectively identified effector antigen-specific CD8 T cells that represented the memory precursor population (100). Since interleukin-7 is an essential survival factor for naïve and memory CD8 T cells, a potential hypothesis postulates that expression of CD127 by effector CD8 T cells confers a survival advantage to this population during the contraction phase (101). However, a follow-up study from Kaech s group and study from our laboratory demonstrated that forcing constitutive expression of CD127 on antigen-specific CD8 T cells did not favor their development into memory cells (102, 103). In a subsequent study, Kaech s group proposed that the expression pattern of CD127 and the inhibitory receptor KLRG-1 (killer cell lectin-like receptor subfamily G member 1) on antigen-specific CD8 T cells selectively distinguished the short-lived effector cells (CD127 lo KLRG-1 hi ) from the memory precursors (CD127 hi KLRG-1 lo ) (104). Short-lived effector cells are destined to die during contraction while memory precursors survive to form the memory pool (104). However, previous work in our laboratory showed that although antigen-specific CD8 T cells generated by peptide-coated DC immunization exhibited a CD127 hi KLRG-1 lo phenotype at the peak of expansion, they still underwent normal contraction (105). These data suggested that a CD127 hi KLRG-1 lo phenotype did not confer a selective survival advantage for the antigen-specific CD8 T cells. However, this CD127 hi KLRG-1 lo phenotype correlated well with the ability of these CD8 T cells to expand vigorously to secondary antigen stimulation, a characteristic of memory cells (105). Given the lack of a concrete marker to identify memory cells, our laboratory prefers to define good memory CD8 T cells based on their ability to (1) persist, (2) protect, (3) undergo vigorous expansion following secondary stimulation, and (4) generate higher numbers of memory cells after booster immunization (34, 38, 45, 106). Previous work from our laboratory sheds light on how systemic inflammation regulates the rate of memory CD8 T cell generation. Antibiotic pretreatment in mice followed by bacterial infection dramatically reduced systemic inflammation and resulted

35 18 in antigen-specific CD8 T cells with an accelerated memory phenotype (CD127 hi, CD27 hi, CD43(glycoform) lo ) and function (IL-2 producing and vigorous proliferation following secondary booster challenge) (107). Antibiotic pretreatment reduced substantially both systemic inflammation and bacterial burden, thus potentially altering antigen presentation. However, subsequent studies in our laboratory using peptide-coated bone marrow derived dendritic cells (BMDC) to stimulate a CD8 T cell response addressed the antigen presentation issue and further confirmed the findings that inflammation regulates the rate of memory acquisition by the responding CD8 T cells (105). Immunizing mice with LPS-matured, peptide-loaded BMDC stimulated antigenspecific CD8 T cells that exhibited accelerated acquisition of a memory phenotype (CD127 hi, CD27 hi, CD43(glycoform) lo ) and function (IL-2 producing) as early as day 4 to 6 post immunization. Furthermore, these early memory cells proliferated vigorously in response to different booster regimens resulting in higher numbers of secondary effector cells, secondary memory cells and enhanced protection against pathogen re-challenge (Figure 1B) (105). Interestingly, co-administration of peptide-coated BMDC and CpG ODN 1826, a TLR9 agonist, to induce inflammation, did not change the kinetics and magnitude of CD8 T cell response but prevented the accelerated memory generation when compared to mice receiving only BMDC (Figure 1B) (105). A recent study from Kaech s group proposed that inflammatory signals, specifically IL-12, induced different expression levels of the transcription factor, T-bet, in responding CD8 T cells. Furthermore, differential expression of T-bet determined the fate of CD8 T cells where high T-bet expression drove short-lived effector cells (CD127 lo KLRG-1 hi ) differentiation while low T-bet expression favored memory precursor (CD127 hi KLRG-1 lo ) formation (104). Consistent with this notion, a study from Reiner s group showed that IL-12 positively regulated T-bet expression while repressing Eomes expression in effector CD8 T cells following LM infection (108). These results strongly argued that inflammatory

36 19 cytokines generated during infection or vaccination with adjuvant have a role in regulating memory CD8 T cell differentiation. Given that the ultimate goal of vaccine development is to induce long-lasting protective memory populations, an understanding of how inflammation regulates the rate of memory CD8 T cell generation will provide valuable information for rational design of vaccines. There are several critical questions that need to be addressed, including but not limited to, the timing of inflammatory cytokine exposure to the CD8 T cells and the identity of the inflammatory cytokines that regulate the memory CD8 T cell differentiation process. In particular, the initial exposure of naïve T cells to inflammatory cytokines may occur prior to, during or after their interaction with stimulating DC and it is unknown whether and how the timing of cytokine exposure impacts the CD8 T cell response. My work presented in chapter II of this thesis explores different aspects of systemic inflammatory cytokines in controlling memory CD8 T cell differentiation including whether the inflammatory cytokines act directly on the immunizing DC, the timing of cytokine exposure and the CD8 T cell population that is most impacted by inflammatory cytokines. Inflammatory cytokines such as IL-12, type I IFNs and type II IFN (IFN-γ) have been demonstrated to serve as signal 3 for optimal CD8 T cell response (38, 39). Furthermore, these cytokines have also been implicated in promoting the acquisition of effector function by the responding CD8 T cells, particularly in a cardiac transplant rejection model (39, 109). Previous work in our laboratory suggested that early memory CD8 T cells were not directly generated by DC immunization but rather the transition from effector to memory cells was accelerated in the absence of systemic inflammation (105). CpG ODN 1826 induced several pro-inflammatory cytokines including but not limited to IL-12 and IFNγ and prevented the accelerated memory generation (105). However, it remains unclear whether direct signaling by these signal 3 cytokines on the responding CD8 T cells promotes effector differentiation at the expense of memory

37 20 formation. In chapter III of this thesis, I investigate the role of direct signaling by IL-12 and type I IFNs on effector/memory CD8 T cell differentiation. Manipulating CD8 T cell memory The level of protection against re-infection correlates with increasing numbers of memory CD8 T cells (45, 54). Thus, one strategy of vaccine development may focus on how to maximize the number of memory cells generated. Our laboratory outlines four general approaches to achieve high number of memory CD8 T cells (45): 1. Increasing recruitment of naïve CD8 T cells. 2. Limiting contraction. 3. Booster immunization. 4. Accelerating memory CD8 T cell development. From the vaccine development perspective, increasing antigen dose would improve naïve CD8 T cell recruitment but there is a limit of antigen dosing to reach maximal recruitment. On the other hand, our laboratory has shown that inflammatory cytokines, specifically IFN-γ, potentially played a critical role in regulating contraction (85, 99). However, blocking IFN-γ also compromises CD8 T cell expansion leading to lower memory number. To date, prime-boost vaccination is the best approach to generate a high number of memory cells that provide protective immunity in cases where the initial priming stimulates a weak response (73). Nevertheless, the prime-boost approach has at least two major hurdles, namely an anti-vector immunity and a substantial time interval (several weeks to several months) between priming and boosting to achieve the most robust secondary memory enhancement (45). Furthermore, the long time interval between priming and boosting would be the biggest obstacle in generating protective immunity during a pandemic outbreak or in cancer immunotherapy where time is of the essence.

38 21 Of these four strategies, accelerating memory CD8 T cell development is most amenable to manipulation by modulating the inflammatory environment during CD8 T cell priming as discussed earlier. Vaccines that stimulate early acquisition of memory characteristics by the responding CD8 T cells would also allow for early boosting (i.e. booster immunization after a short time interval, i.e. few days, following priming) of these cells to significantly higher secondary effector and memory numbers. In chapter IV of this thesis, I investigate the cross-presentation pathway as a promising strategy to accelerate memory CD8 T cell development. Both cell-associated antigen and antigencoated biodegradable microspheres are examined as potential priming vehicles to stimulate antigen-specific CD8 T cells that are capable of being boosted within days following priming to rapidly achieve protective immunity. Rationale for project The anamnestic response is a key feature of an adaptive immune response that protects the host against reinfection. In the past century, the expansion of our knowledge of how memory cells are generated and regulated has paved the way for the development of vaccines that protect humans from many life-threatening diseases such as smallpox, polio, hepatitis B, influenza, pneumococcal diseases, meningitis, tetanus and many more including the most recent human papillomavirus vaccine to prevent cervical cancer. Although great strides have been made in the field of CD8 T cell biology, vaccine development aimed at eliciting strong cell-mediated immunity is currently lacking. Most current vaccines have relied on inducing antibodies to protect the host by neutralizing pathogens or blocking pathogen entry into the cells. However, there is a recognized need to design vaccines that also stimulate a strong CD8 T cell component of the adaptive immune response in addition to antibodies (110, 111). Studies in this thesis project aim to better understand how the inflammatory milieu influences the memory CD8 T cell

39 22 differentiation process and investigate an alternative strategy to promote accelerated memory generation. Specifically, the studies in this thesis have three main focuses: 1) How does the timing of inflammatory cytokine exposure influence memory CD8 T cell differentiation? 2) What is the role of signal 3 inflammatory cytokines, type I IFNs and IL-12, in regulating effector/memory CD8 T cell differentiation? 3) Can the cross-presentation pathway be exploited to rapidly generate protective CD8 T cell immunity?

40 Table 1. Phenotypic and functional attributes of naïve vs. effector vs. memory antigenspecific CD8 T in mice after infection or vaccination. 23

41 24 Naïve Effector Memory Phenotypic Attributes CD127 High Low High CD62L High Low Low and High CCR7 High Low Low and High KLRG 1 Low High Low CD43 glycoform Low High Low and High CD11a High Higher Higher CD44 Low High High Functional Attributes IFN γ Low High High IL 2 Low Low Low and High TNF α Low High High Number of antigen specific +/ Persistence +++ +/ +++ Protection +/ Ag specific proliferation +++ +/ +++ Effector functions +/ After infection or vaccination, naïve CD8 T cells undergo robust expansion and acquire effector functions. Following the expansion phase, a fraction of these antigen-specific CD8 T cells survives the contraction to initiate the formation of memory pool. Multiple phenotypic and functional changes on antigen-specific CD8 T cells accompany the transition from naïve to effector to memory cells. This table summarizes some of the most commonly used phenotypic and functional attributes to characterize naïve vs. effector vs. memory CD8 T cells. This table is adapted from the review by Badovinac and Harty (45).

42 25 Figure 1. Inflammation regulates the kinetics of CD8 T cell response to infection or vaccination with late or early booster immunization. In a naïve mouse, CD8 T cells specific for any given epitope are present at a very low number (estimated in the range of 10 to 1000 precursors). (A) Following an infection or vaccination, antigen-specific CD8 T cells undergo rapid and robust proliferation (expansion phase). Independent of pathogen clearance, the contraction phase ensues where 90-95% of antigen-specific CD8 T cells at the peak of expansion die via apoptosis, leaving ~ 5-10% of these cells to initiate the memory pool. The duration of time interval for memory CD8 T cells to exhibit the central phenotype and function depends largely on the pathogen-specific inflammatory environment during priming phase. Upon secondary challenge or booster immunization, these memory CD8 T cells mount an even more vigorous expansion with a protracted contraction and establish a secondary memory pool with higher number of antigen-specific CD8 T cells compared to the primary pool. (B) Infection or DC immunization in the presence of CpG-induced inflammation promotes the acquisition of effector phenotype and function by the responding CD8 T cells. However, these antigen-specific CD8 T cells failed to expand further following early booster immunization at the peak of primary expansion. In contrast, DC immunization in the absence of inflammation accelerates CD8 T cell memory generation. These antigenspecific CD8 T cells respond vigorously to early booster immunization resulting in an increase in secondary memory numbers. (LOD: Limit of detection)

43 26

44 27 CHAPTER II A DEFAULT PATHWAY OF MEMORY CD8 T CELL DIFFERENTIATION AFTER DENDRITIC CELL IMMUNIZATION IS DEFLECTED BY ENCOUNTER WITH INFLAMMATORY CYTOKINES DURING ANTIGEN-DRIVEN PROLIFERATION Introduction Inflammatory cytokines produced by the innate immune system after infection are critical for control of pathogen replication as well as for enhancing host antigenpresenting cell functions. Importantly, these inflammatory cytokines also act directly on pathogen-specific CD8 T cells to modulate all phases of the T cell response (38). Inflammatory cytokine signaling through receptors expressed by CD8 T cells is critical to promote their proliferation, survival and differentiation into effector cells both in vitro and in response to infection (39, 78, 79, 83, 112, 113). Furthermore, IFN-γ produced in response to infection regulates the program of CD8 T cell contraction (99, 114). Recent studies also suggest that inflammatory cytokines such as IL-12 regulate memory CD8 T cell formation through a gradient of T-bet expression (104, 115). Moreover, the rate at which CD8 T cells acquire memory characteristics, such as CD127 and KLRG-1 expression profiles, IL-2 production and the ability to vigorously expand following booster immunization, is also controlled by inflammatory cytokines (105). For example, the acquisition of phenotypic and functional memory CD8 T cell characteristics takes several months after clearance of acute infection (116). In contrast, the time between priming and acquisition of memory characteristics is shortened to days in CD8 T cells responding to dendritic cell (DC) immunization, which induces substantially less inflammation compared to infection (105). Importantly, induction of inflammatory cytokines by administration of TLR-agonists at the time of DC priming prevented the early acquisition of memory characteristics by the responding CD8 T cells (105).

45 28 Naïve CD8 T cells specific for any given peptide-mhc complex constitute a very small fraction of the pre-immune repertoire (32, 33, 117, 118) and these cells are distributed throughout the host blood system and secondary lymphoid organs. Several studies suggest that approximately 2 days after infection or DC immunization are necessary to activate the majority of naïve antigen-specific precursors, whereas production of inflammatory cytokines is initiated within hours after infection (50, 51, 97). Thus, naïve CD8 T cells could be exposed to inflammatory cytokines prior to, during or after their interaction with activating antigen-presenting cells and it is unclear how the timing of cytokine exposure will influence the process of memory differentiation. Furthermore in the case of any pathogen encounter, naïve CD8 T cells that are not specific to that infection would be exposed to the inflammatory cytokines without being stimulated by their cognate antigen. It is unknown how exposure of naïve CD8 T cells to inflammatory cytokines would influence subsequent responses to antigen-stimulation. Here, I document that DC immunization drives CD8 T cells through a transient effector phase to a default pathway of memory differentiation. These results also define a window during the proliferative expansion phase when inflammatory cytokines are capable of influencing the rate that CD8 T cells acquire memory characteristics by sustaining effector differentiation. Thus, the timing of inflammatory cytokine exposure is critical in controlling the rate of memory CD8 T cell differentiation. Materials and Methods Mice C57BL/6 (B6, Thy1.2) were from the National Cancer Institute (Frederick, MD). OT-I (Ova257-specific) transgenic Thy1.1 were previously described (119). TLR9 -/- mice on a B6 background were a generous gift from Dr. Joel Kline (University of Iowa, IA). Pathogen-infected mice were housed at appropriate biosafety conditions. Mice were

46 29 used at 6-10 weeks of age. All experiments were approved by the University of Iowa Institutional Animal Care and Use Committee. Dendritic cells Splenic DC (DC) were isolated after subcutaneous injection of B6 mice with 5x10 6 B16 cells expressing Flt3L (provided by M. Prlic and M. Bevan, University of Washington). When tumors were palpable (5 mm x 5 mm), mice were injected with 2 µg LPS (Sigma) i.v. to mature the DC. Spleens were harvested 16 hours later and were digested with DNase and Collagenase for 20 min at 37 C/7% CO 2 with shaking (120 RPM). Spleen pieces were smashed through a nylon cell strainer (70µm) to generate a single cell suspension, RBC were lysed and splenocytes were resuspended in 2 parts of 10% FCS RPMI-1640 to one part B16-Flt3L conditioned medium + rgm-csf (1000 µ/ml) + 2 µm Ova and incubated 2 hours at 37 C/7%CO 2 with shaking (100 RPM). Spleen cells were washed three times and CD11c+ cells were isolated using anti-cd11c microbeads (Miltenyi Biotec). The purity and activation status of DC were determined by staining for CD11c, CD86 and MHC class II. Routinely, greater than 90% pure CD11c+ DC were obtained and the yield was approximately 15-20x10 6 DC per mouse. DC were resuspended in saline and injected i.v. Bacteria Listeria monocytogenes expressing Ova (LM-Ova) (120) and attenuated (actadeficient) LM-Ova (50) were grown, injected 1x10 5 cfu i.v. per mouse for primary infection or 1x10 7 cfu i.v. per mouse for boosting, respectively, and quantified as described (105).

47 30 Adoptive transfer experiments Thy1.1 OT-I were obtained from the blood of naïve donors and the number of input OT-I was calculated as previously described (64). Approximately 500 Thy1.1 OT-I cells were transferred into each naïve Thy1.2 B6 recipient mouse. These mice were injected with CpG oligonucleotide 1826 (Coley Pharmaceutical Group) intraperitoneally (50 µg) at different time points relative to DC immunization as described (Figure 3). All mice were immunized with ~ 1x10 6 Ova257-coated matured DC at day 0. To measure proliferation, BrdU (Pharmingen) was injected intraperitoneally (2 mg/mouse) and mice were kept on drinking water containing BrdU (0.8 mg/ml) (Sigma) for the indicated intervals. Detection of BrdU incorporation was performed according to the manufacturer s protocol (BrdU Flow Kits, Pharmingen). Quantification and phenotypic analysis of antigen-specific T cells The magnitude of the epitope-specific CD8 T cell response was determined either by intracellular IFN-γ staining (ICS) or tetramer staining as described (97) or by staining for Thy1.1 marker exclusively expressed on transferred TCR-tg cells (64). For analysis of circulating TCR-tg T cells, ~ 100 µl of blood was obtained from each mouse. Phenotypic analysis was performed on Thy1.1 + TCR-Tg CD8 T cells. The number of TCR-Tg T cells (Thy1.1 + CD8) was presented as frequency of total PBL. Antibodies, peptides, and MHC Class I tetramers Antibodies of the indicated specificity with appropriate fluorochromes were used. The following antibodies were from ebioscience (San Diego, CA): IFN-γ (XMG1.2), CD8 (53-6.7), CD127 (A7R34), KLRG1 (2F1), CD11c (N418), CD86 (GL-1), MHC II (I-A/I-E) (M5/ ), isotype control rat IgG2a (ebr2a), rat IgG2b (KLH/G2b-1-2), Armenian Hamster IgG and Golden Syrian Hamster IgG. The following antibodies were

48 31 from BD Pharmingen (San Diego, CA): CD11c (HL3), Thy1.1 (OX-7). The following antibodies were from Caltag (San Diego, CA): anti-human Granzyme B and isotype control mouse IgG1. Ova peptide was described (119). MHC class I tetramers (K b ) specific for Ova prepared using published protocols (121, 122). Serum cytokine quantification Serum (~25µl) was obtained via retro-orbital bleeding at 0, 6, 12 and 48 hours after CpG treatment. Serum IL-12(p70), IL-6, IL-10 and GM-CSF were measured using Bio-Plex Mouse Cytokines Assays (Bio-Rad) and read on the Bio-Rad Bioplex 200 system. In vivo cytolytic assay A mixture of 10x10 6 unpulsed splenocytes labeled with 0.2 µm CFSE and 10x10 6 splenocytes pulsed with 1 µm Ova peptide and labeled with 0.02 µm CFSE was administered i.v. to the indicated groups a well as a control group of naïve mice. 5 hours later, spleens were harvested, and the percentages of CFSE + cells that were CFSE hi and CFSE lo were assessed by flow cytometry. The percent killing was calculated as: 100- (100 x [(% CFSE lo /% CFSE hi )/(% CFSE lo in naïve control group/ % CFSE hi in naïve control group)]). Results TLR9 expression by the immunizing DC and responding CD8 T cells is not necessary for CpG ODN to regulate memory differentiation Injection of CpG ODN at the time of DC priming prevents early acquisition of memory characteristics by the responding CD8 T cells (105). It is unknown if the CpG

49 32 ODN regulate CD8 T cell differentiation by signaling directly through TLR9 expressed by the immunizing DC or the responding CD8 T cells or indirectly through other host cells. In order to address this question I needed an experimental system such as adoptive transfer of TCR- transgenic (TCR-tg) T cells where I could maintain TLR9 expression only in certain cell types. To verify that adoptive transfer of low numbers of OT-I TCR transgenic (TCR-tg) cells specific for the Ova257 peptide cells can serve as a reliable model for endogenous CD8 T cell responses after DC immunization, I transferred ~ 500 naïve Thy1.1 OT-I cells into naïve Thy1.2 C57BL/6 recipient mice and immunized them the next day with ~ 1x10 6 LPS-matured, Ova257-coated splenic dendritic cells (DC) with or without co-injection of CpG ODN 1826, a TLR9 agonist. The DCs were obtained with high purity and they expressed appropriate co-stimulatory (CD86) and activation (MHC Class II) markers (Figure 2A). On day 7 post-dc immunization, I employed peptide-stimulated intracellular cytokine staining (ICS) or direct ex-vivo tetramer or allelic Thy1 marker staining to detect and enumerate both the OT-I and the Ova257- specific endogenous CD8 T cells in the peripheral blood (PBL) and spleen (Figure 2B- D). Consistent with previous findings in our laboratory, the majority of OT-I cells responding to DC immunization exhibited a memory-like phenotype (CD127 hi, KLRG- 1 lo, Granzyme B lo ) at day 7 after immunization (Figure 3A & B). In striking contrast, OT-I cells responding to DC immunization in the presence of heightened inflammation (DC+CpG) or to infection with Listeria monocytogenes expressing the Ova257 peptide (LM-Ova) displayed an effector phenotype (CD127 lo, KLRG-1 hi, Granzyme B hi ) at day 7 after infection (Figure 3A & B) (54, 105, 123). More importantly, this analysis revealed that expression of CD127, KLRG-1 and Granzyme B was quite similar between OT-I and the endogenous Ova257-specific CD8 T cells in each immunization group (Figure 3A & B). Furthermore, similar results were also obtained with different detection methods employed (ICS or direct ex-vivo tetramer and Thy1 staining).

50 33 To determine whether CpG signals directly through TLR9 expressed by the immunizing DC or responding OT-I cells, I analyzed the phenotypes and early booster response of wild-type (wt) OT-I cells that have been adoptively transferred into either wt or TLR9 -/- hosts. The hosts were then immunized with either Ova257- coated wt or TLR9 -/- LPS-matured DC in the presence or absence of in vivo CpG ODN injection (Figure 4A). Both wt and TLR9 -/- DC were recovered with high purity as indicated by CD11c staining post purification and both populations exhibited similar levels of costimulation (CD86) and activation MHC Class II) markers (Figure 4B). Wild-type OT- I cells responding to either wt or TLR9-deficient DC in wt hosts exhibited an accelerated memory phenotype (CD127 hi, KLRG-1 lo ) at day 7 post-immunization that was prevented by injection of CpG ODN in both groups (Figure 4C, top 2 panels). Thus, CpG ODN does not regulate memory CD8 T cell differentiation directly through TLR9 expressed by the immunizing DC. To address a potential role for OT-I expressed TLR9, I repeated the experiment with TLR9-deficient recipients. CpG treatment failed to alter the phenotype of the responding wt OT-I cells at day 7 post-priming with either wt or TLR9 -/- DC in the TLR9 -/- hosts. Since CpG treatment did not alter the phenotype of wt OT-I cells that were primed with TLR9 -/- DC in the TLR9 -/- hosts, I conclude that CpG does not act directly on the responding OT-I cells (Figure 4C, bottom 2 panels). Interestingly, a recent study suggests that CpG ODN treatment influences responding CD8 T cells through their expression of the IL-12 receptor (124). In chapter III of this thesis, I specifically investigated the role of IL-12 signaling directly in antigen-specific CD8 T cells in their effector/memory differentiation. One of the operational and functional hallmarks of memory CD8 T cells is their robust proliferative response upon re-exposure to antigen (34). In the wt recipients, OT-I cells that were primed with either wt DC or TLR9 -/- DC in the absence of CpG-induced inflammation underwent substantial secondary proliferative response to early booster infection at day 7 with acta-deficient LM-Ova (att LM-Ova), consistent with their early

51 34 memory-like phenotype. Here, early booster infection is defined as an infection that is administered at short time interval, i.e. 7 days, following priming. In contrast, OT-I cells that were primed with either wt DC or TLR9 -/- DC in the presence of CpG-induced inflammation failed to respond to early booster infection, consistent with their effector phenotype (Figure 4D). Consistent with their early memory-like phenotype OT-I cells that were primed with wt DC either in the presence or in the absence of CpG in TLR9- deficient hosts underwent vigorous proliferative response after early booster infection resulting in a higher frequency of circulating OT-I in PBL (Figure 4E). Taken together, these data show that CpG ODN does not regulate memory differentiation directly through TLR9 expressed by the immunizing DC or responding CD8 T cells but rather acts indirectly through TLR9 expressed by host cells that do not directly involved in the priming of the CD8 T cells. CpG induces inflammatory cytokines with distinct magnitudes and kinetics CpG ODN stimulation of TLR9 in DC and B cells induces multiple cytokines in the host (125). However, the kinetics and duration of serum cytokine induced by CpG in the context of DC immunization are not defined. Thus, I next evaluated the systemic inflammatory cytokine milieu induced by DC immunization in the presence or absence of CpG ODN treatment. OT-I recipient wt mice were immunized with either DC or DC + CpG at day 0. Sera were obtained at 0, 6, 12, 24 and 48 hrs post DC or DC + CpG immunization for cytokine detection. To ensure that the measured cytokine profiles corresponded with the phenotype and function of the responding OT-I cells, I also analyzed the OT-I cells at day 7 post DC immunization and 5 days following booster infection with att LM-Ova administered on day 7 (Figure 5A). DC immunization alone did not induce detectable serum cytokine responses (Figure 5B). In contrast, DC immunization in the presence of CpG ODN induced multiple cytokines (Figure 5B),

52 35 which exhibited several distinct kinetic profiles. Serum IL-12p70, IL-6, IL-10 and GM- CSF are representative of these different patterns following CpG treatment. Serum IL- 12p70 peaked at 6 hours, remained elevated at 12 hours and declined by 24 to 48 hours after CpG administration while IL-6 also peaked at 6 hours but declined quickly by 12 hours after CpG treatment. Serum IL-10 did not peak until 24 hours and remained elevated up to 48 hours post CpG treatment while serum GM-CSF levels were not elevated in response to CpG ODN (Figure 5B). The different inflammatory environments in the host between DC and DC + CpG immunization clearly influenced the rate of memory differentiation of the responding OT-I cells as reflected by their phenotype and response following early booster infection. Consistent with the serum cytokine data, OT-I cells at day 7 post DC immunization displayed effector (CD127 lo, KLRG-1 hi ) or memory-like phenotype (CD127 hi, KLRG-1 lo ) and function (response to early booster infection) in the presence or absence of CpG ODN co-injection, respectively (Figure 5C & D). Thus, at least some cytokines that are potentially capable of regulating CD8 T cell differentiation were only elevated for a short period of time (6-24hrs) after CpG ODN treatment. Encounter with inflammatory cytokines prior to antigendriven proliferation of CD8 T cells does not prevent accelerated memory generation Two-photon microscopy studies in live animals showed that activation of naïve T cells required DC-T cell interaction that occurred in 3 distinct phases and lasted for more than 20 hrs (126). In addition, another in vivo study demonstrated that the duration of antigen presentation by stimulating DC must be between 30 and 54 hrs to engender the full magnitude of the CD8 T cell response (51). It is likely that some naïve T cells encounter the immunizing DC shortly (hours) after vaccination while other naïve T cells may be initially stimulated relatively late (24-48 hours) after immunization. Due to the

53 36 kinetics of systemic cytokine induction by CpG ODN, naïve CD8 T cells could be exposed to specific inflammatory cytokines prior to, during or after their interaction with stimulating DC. Thus, I next determined the time window when such an inflammatory environment impacts the differentiation process of the responding CD8 T cells. The DC immunization strategy and CpG ODN approach offer the advantage of delivering a constant dose of antigen and costimulation while allowing me to modulate the timing of inflammatory cytokine induction. Induction of inflammatory cytokines was achieved by injection of CpG in the OT- I recipient mice either 2 days before, at the time of (day 0) or 2 days after DC-Ova immunization (Figure 6A). Control OT-I recipient mice were immunized with DC-Ova alone to generate the accelerated memory phenotype or infected with LM-Ova to generate cells with a conventional effector phenotype. At day 6 following immunization or infection, I analyzed the OT-I cells from PBL. Consistent with data obtained from the spleen (Figure 2D and 3A), OT-I cells in the PBL exhibited early memory-like phenotype (CD127 hi, KLRG-1 lo, Granzyme B lo ) at day 6 post DC immunization, but an effector phenotype (CD127 lo, KLRG-1 hi, Granzyme B hi ) at day 6 post LM-Ova infection (Figure 6B, black bar and gray bar, respectively). Interestingly, inflammation induced by CpG ODN 2 days prior to DC immunization did not prevent the responding OT-I cells from acquiring memory-like characteristics at day 6 post immunization. The majority of OT-I cells in this group were CD127 hi, KLRG-1 lo, and Granzyme B lo even though they have been exposed to inflammatory cytokines induced by CpG prior to the encounter with the stimulating DC. In sharp contrast, inflammation induced by CpG at day 0 or day 2 after DC immunization had a significant impact on the phenotypes of these expanding OT-I cells. CD127 expression was down-regulated, KLRG-1 expression was up-regulated and a substantial fraction of OT-I cells expressed the cytolytic effector molecule Granzyme B, consistent with an effector CD8 T cell phenotype (Figure 6B). It should be noted that I routinely observed enhanced CD8 T cell responses (2-4-fold, Figures 2 and 6) when

54 37 CpG ODN were injected at day 0 relative to DC priming. These results are consistent with the potential role of inflammatory cytokines such as IL-12 and type I IFNs to act as signal 3 to promote survival of T cells during expansion (78, 79, 83, 86, 113, 114). However, injection of CpG ODN day +2 relative to DC immunization failed to increase expansion, although this approach had a substantial impact on the phenotype of the responding CD8 T cells. These data suggest that signal 3, promoting T cells survival during proliferation, can be separated from the impact of inflammatory cytokines on the memory differentiation program. I also determined if the timing of cytokine exposure regulated the ability of the stimulated OT-I cells to respond to an early booster immunization. Consistent with their phenotypes, OT-I T cells stimulated by DC underwent substantial secondary proliferative response to booster infection at day 6 with acta-deficient LM-Ova (Figure 6C, left panel), whereas the OT-I T cells in LM-Ova-infected mice failed to expand further in response to the early booster infection (Figure 6C, right panel). While CpG treatment at day 0 or day +2 prevented this early booster-response similar to LM-Ova infection, CpG treatment at day -2 did not prevent this early booster-response (Figure 6C, middle 3 panels). Together, these data demonstrated that exposure of naïve CD8 T cells to inflammatory cytokines prior to their interaction with the stimulating DC did not prevent the accelerated generation of memory CD8 T cells based on both phenotype and function. These results suggested that both the inflammatory cytokine(s) and the duration of inflammation potentially play important roles in regulating the CD8 T cell memory differentiation. Furthermore, inflammatory cytokines must be present during or after the priming phase to influence the rate by which CD8 T cells acquire memory characteristics.

55 38 Inflammatory cytokines exert the greatest impact on proliferating CD8 T cells CD8 T cell priming by mature DC involves TCR and costimulatory molecule signaling that together stimulate proliferation of the activated T cells. Clonal expansion of pathogen-specific CD8 T cells after infection is also associated with differentiation to effector T cells that migrate throughout the body to defend against infection (38, 127). These data suggest a link between proliferation and differentiation, where signals received during proliferation may program patterns of gene expression or epigenetic modifications that are restricted in non-proliferating cells. Thus, I next asked whether inflammatory cytokine signaling influences the rate of CD8 T cell memory characteristic acquisition most drastically during proliferation. To address this possibility, I employed BrdU incorporation to identify the fraction of OT-I cells that were proliferating during 3 defined time periods: day 4-6, day 6-8, and day after DC-Ova immunization in control mice and those that received CpG on the first day of BrdU administration. The expression levels of CD127, KLRG-1 and Granzyme B on the OT-I cells in the presence or absence of CpG treatment were evaluated on the last day of BrdU treatment for each group (Figure 7A). As expected, essentially all (>97%) of the OT-I cells incorporated BrdU between day 4-6 after DC immunization while only a fraction incorporate BrdU between day 6-8, a time interval that consists of the tail end of expansion and initial period of contraction. In contrast, the majority of OT-I cells did not incorporate BrdU between days 12-14, an early memory time point after the contraction phase (Figure 7B). CpG treatment during day 4-6 when all T cells were proliferating resulted in down regulation of CD127 and upregulation of KLRG-1 and Granzyme B on the responding OT-I cells (Figure 7C, left panel). Of note, these alterations are less pronounced than observed when the CpG ODN were administered at day 0 or +2, and the T cells analyzed on day 7, likely due to the shortened interval of analysis in Figure 6.

56 39 CpG ODN treatment during day period, where most OT-I cells did not proliferate, had minimal impact on the expression level of CD127, KLRG-1 and Granzyme B on the OT-I cells (Figure 7C, right panel). Interestingly, CpG treatment resulted in less pronounced CD127 down-regulation and Granzyme B upregulation during the day 6-8 interval when only a fraction of OT-I cells were proliferating (Figure 7C, middle panel). Here, I did not observe dramatic changes in KLRG-1 expression since I restricted my analysis within the 48-hour window following CpG treatment. This short time window may be insufficient to ensure full up-regulation of KLRG-1 following CpG treatment. The day 6-8 interval provided a unique opportunity to examine the impact of CpGinduced inflammation on both proliferating and non-proliferating OT-I populations, which could be clearly identified by their ability to incorporate BrdU (Figure 7B). To directly analyze the effect of inflammation on proliferating and non-proliferating OT-I populations I administered CpG on day 5 post DC immunization to allow the inflammation to take place before initiating BrdU treatment on day 6 through 8. Since the short exposure to CpG-induced inflammatory cytokines had the most significant effect on CD127 expression (Figure 7C), I examined surface expression of this marker on the BrdU+ and BrdU- fractions of the OT-I cells at day 7 and 8. Compared to the DC immunization alone, CpG-induced inflammation resulted in a much more substantial CD127 down-regulation on the BrdU-positive compared to the BrdU-negative fractions (Figure 7D & E). Taken together, these data suggest that CD8 T cell memory differentiation is most susceptible to inflammatory cytokine regulation when these cells are rapidly dividing. A default pathway of memory CD8 T cell differentiation is deflected by encounter with inflammatory cytokines The previous study in our laboratory using supra-physiologic, high-number OT-I adoptive transfers suggested that DC stimulation did not directly generate memory cells

57 40 but rather the responding CD8 T cells exhibited some characteristics of effector populations (CD127 lo, CD43 (glycoform) hi and IL-2 lo ) at early time points (105). Several studies have suggested as genetic or functional evidence that the expression of effector molecules, such as IFN-γ or Granzyme B, indicates that memory T cells have passed though an effector phase (69, 128, 129). One possibility is that inflammatory cytokines are required to initiate the CD8 T cell effector differentiation program. Alternatively, inflammatory cytokines may act to enforce the effector differentiation program initiated by TCR-stimulation and deflect a default pathway of memory CD8 T cell differentiation. To resolve these possibilities in a physiologic setting, I transferred ~ 500 naïve Thy1.1 OT-I cells into Thy1.2 hosts and analyzed both phenotype and functional cytolytic capacity of the OT-I cells at day 4 (~ 90 hrs) following immunization with Ova257- coated DC in the presence and absence of CpG ODN coinjection. The OT-I cells were detectable in the spleens at day 4 and 7 following DC immunization (Figure 8A). Notably, the majority of OT-I cells at day 7 after DC immunization showed a memory phenotype (CD127 hi, KLRG-1 lo and Granzyme B lo ), whereas the OT-I cells at day 7 after DC+CpG immunization showed an effector phenotype (CD127 lo, KLRG-1 hi and Granzyme B hi ) (Figure 8B & C). In contrast, the majority of OT-I cells at day 4 after DC or DC+CpG immunization showed similar effector phenotype, i.e. CD127 lo and similar frequency of cells expressing Granzyme B (Figure 8B & C). KLRG-1 was not upregulated in either population at this early time point. To address the functional capacity of these OT-I cells, I performed a 5-hour in vivo cytolytic assay at day 4 following DC or DC+CpG immunization. Similar small numbers of OT-I were detectable in the spleen in both groups at this early time point, and consistent with their effector phenotype, both groups displayed similar in vivo killing capacity (Figure 8E & F), regardless of whether they were primed and/or expanded in the presence or absence of CpG-induced inflammatory cytokines. Similar proportion of OT-I cells from DC or DC+CpG mice at day 4 after immunization also produced IFN-γ after a short in vitro

58 41 stimulation (Figure 9A & B). Taken together, these results suggest that CD8 T cells responding to DC immunization in the absence of inflammatory cytokines go through a transient effector phase, as demonstrated by both phenotype and function, but then undergo a default pathway rapidly leading to the expression of the phenotypic and functional characteristics of long-term memory populations. However, encounter with inflammatory cytokines deflects this default pathway of CD8 T cell memory differentiation towards a sustained effector differentiation program. Therefore, I conclude that inflammatory cytokines are not required to initiate the CD8 T cell effector differentiation program, but they rather serve to enforce this program at the expense of a default CD8 T cell memory differentiation after DC immunization. Model Based on the data from this study, I propose a model to explain how the timing of inflammatory cytokine signaling in CD8 T cells critically controls the rate at which these cells acquire memory characteristics. DC immunization activates naïve CD8 T cells to undergo proliferation and undergo gene expression changes (downregulation of CD127, upregulation of the 1B11 antibody epitope on CD43 (105), increased ability to produce IFN-γ and Granzyme B, this study) that are characteristic of an effector population. However, in the low inflammation environment of DC immunization, this effector phenotype is not stable and the responding CD8 T cells default to a differentiation program where they rapidly acquire the phenotype and functional characteristics of longterm memory populations as they continue to proliferate (Figure 10A). The timing of exposure of inflammatory cytokine signals to the CD8 T cells is critical in controlling the rate of memory generation, since neither exposure of inflammatory cytokines to naïve non-dividing CD8 T cells prior to stimulation nor late exposure of inflammatory cytokines to antigen-experienced CD8 T cells that have ceased rapid division modulates the phenotype and function of the responding CD8 T cells (Figure 10B).

59 42 In contrast, in an acute bacterial or viral infection or DC immunization in the presence of TLR agonist-induced inflammation, heightened inflammation is present during the period of priming and expansion where the responding CD8 T cells are proliferating robustly. Rapidly dividing CD8 T cells are more susceptible to inflammatory cytokine signaling, which promotes and sustains their effector differentiation program and prevents their early acquisition of memory characteristics (Figure 10C). This model provides a framework to address in detail how inflammatory cytokines regulate the molecular pathways (gene expression and epigenetic modifications) that dictate CD8 T cell memory differentiation. Discussion During an immune response, CD8 T cells must integrate a variety of inputs, including the inflammatory signals generated by an infection that ultimately determine their eventual fate (86, 97, 104, 105, 107, 130). Recent studies from our laboratory have demonstrated that reducing or truncating the inflammatory environment in the host during an immune response accelerated the process of CD8 T cell memory differentiation on the population level (105, 107). Thus, the rate of memory CD8 T cell generation is not fixed in time but rather regulated by exposure to inflammatory cytokine signals. Since there exists a strong correlation between the number of memory CD8 T cells and the level of protection, an understanding of how the host environment influences the differentiation of these memory CD8 T cells may suggest ways to favorably manipulate memory generation (45, 131, 132). However, it is unclear when and how the inflammatory signals regulate the memory differentiation process. Here, I define the time window where the inflammatory signals in the host are crucial in controlling the rate of CD8 T cell memory differentiation. These results show that CD8 T cells responding to DC immunization undergo a default pathway of memory differentiation that can be deflected towards sustained effector commitment by encounter with inflammatory

60 43 cytokines. Furthermore, the inflammatory signals exert greatest impact on the actively proliferating CD8 T cell population. The DC immunization model allowed me to dissociate the early priming events involving signal 1 (TCR:peptide-MHC complex) and signal 2 (costimulation) from inflammatory signals that act either directly or indirectly on the responding CD8 T cells to regulate their memory differentiation process. Inflammatory cytokines such as IL-12, type I IFNs, and IFN-γ have been identified to serve as signal 3 for effector CD8 T cell survival during proliferation (38, 39, 78, 79, 83, 113). Although I did observe a greater accumulation of antigen-specific CD8 T cells at the peak of expansion when CpG ODN were co-delivered with the immunizing DCs at day 0, CpG ODN administered at 2 days after DC immunization did not enhance CD8 T cell expansion compared to DC immunization alone (Figure 6C). However, inflammatory cytokines induced by CpG ODN delivered day 2 after DC immunization still blocked the acquisition of early memory phenotype and function by the responding CD8 T cells. These results strongly suggest that inflammatory cytokines not only can play a role as signal 3 but also serve a distinct function to regulate the CD8 T cell memory differentiation process. Identification of the specific cytokines that regulate memory differentiation remains an active area of investigation in our laboratory. A strong inflammatory response accompanying an acute infection is often beneficial to the host to mature antigen-presenting cells, to inhibit pathogen replication, and to promote the development of a robust effector T cell response. However, naïve CD8 T cells that are not specific for this infection will also be exposed to the inflammatory environment generated by the infection itself. Here, I asked whether such exposure might alter differentiation of these cells in response to DC immunization shortly after being exposed to the inflammatory environment. The results in this study show that the inflammatory cytokines do not alter the memory differentiation potential of naïve CD8 T cells nor adversely impact pre-existing unrelated memory CD8 T cells even

61 44 though these cells are potentially exposed to inflammatory environment multiple times during the life of the host. An interesting question that remains is why inflammatory cytokines don t influence the naïve CD8 T cell memory differentiation in response to subsequent antigen-stimulations. It is possible that naïve CD8 T cells do not express the appropriate cytokine receptors and thus do not respond to the inflammatory cytokine(s) until these receptors are up-regulated after antigen stimulation. At least some of the receptors (such as type I IFNR or type II IFNR) for CpG-induced inflammatory cytokines are constitutively expressed (88-91), whereas as other receptors (such as IL12 or IL-18) may be induced by activation (92-95). Moreover, it is also possible that inflammatory cytokine signaling must be integrated with signal 1 and signal 2 in order to ensure the full development of CD8 T cell effector program. Cooperation between inflammatory cytokines and TCR signaling has been well documented in CD4 T cell polarization. For example, while the IL-12 receptor is undetectable on most resting T cells, activation through the T cell receptor and costimulatory interactions induces the transcription and expression of both chains of IL-12 receptor (94, 96). However, IL-12-responsive T H 1 cells further upregulate IL-12Rβ2 chain in response to IL-12 and IFN-γ while T H 2 cells downregulate IL-12Rβ2 chain in response to IL-4 (92). Thus, it seems that naïve, unstimulated T cells or pre-existing memory T cells largely ignore the cytokine milieu produced in response to unrelated pathogens. In this way, only the differentiation of the responding T cells would be shaped by the particular pathogen-specific inflammatory cytokines produced during infection. This scenario would prevent previous infections from skewing subsequent T cell responses, thus preserving flexibility to mount the appropriate responses to new pathogens. How do inflammatory cytokines regulate the delicate balance between effector and memory CD8 T cell differentiation? It must be emphasized that the responding CD8 T cells that are primed and/or expanded in the low inflammatory environment (i.e. DC immunization) underwent a transient effector phase but they acquired phenotypic and

62 45 functional memory features at an accelerated rate. The results in this study showed that inflammatory cytokines exert the greatest impact on vigorously proliferating cells. Thus, I propose that CD8 T cells initially differentiate into effector cells after receiving TCR and costimulatory signals from activated APC but will undergo a default memory differentiation program unless they encounter inflammatory cytokines, which play a critical role in sustaining the effector differentiation program by the responding CD8 T cells. This model predicts that specific inflammatory signals generated during the immune response to infection do not initiate the effector differentiation program but rather enforce and sustain such program to its fullest potential. Moreover, this model incorporates the contribution of inflammatory signals that must be present during the priming and/or expansion phase of the CD8 T cells as a critical regulator of the CD8 T cell memory differentiation. These results are consistent with recent published works suggesting that inflammation regulates the CD8 T cell effector differentiation program (39, 104, 113). CD8 T cells that are primed and/or expanded in the presence of a heightened inflammatory milieu exhibited an effector phenotype (CD127 lo, KLRG-1 hi, Granzyme B hi ). These cells have been has been characterized as short-lived effector cells (104), which failed to further robustly expand in response to early booster immunization (105, 107). The differentiation of naïve CD4 T cells into polarized effector cells resulted in epigenetic modifications such as demethylation and histone acetylation at several gene loci (133, 134). Similarly, the CD8 differentiation from naïve to effector to memory cells is associated with unique changes in chromatin structure and patterns of gene expression (49, 116, 130, 135). More importantly, such differentiation processes are linked to cell division. The rate of CD8 T cell division during the robust expansion phase has been estimated to be as rapid as 6 hours per cell division (64, 136). During rapid replication, chromatin structures remain in an open configuration to permit access to the replication machinery. The data presented in this study suggest that the proliferating cells are more

63 46 responsive to undergo phenotypic and functional changes induced by inflammatory cytokines than their non-proliferating counterparts. Perhaps, the proliferating cells possess a more permissive chromatin configuration for epigenetic modifications imposed by inflammatory signals. These ideas may explain, in part, why exposure of naïve circulating CD8 T cells that do not proliferate vigorously to inflammatory cytokines prior to their encounter with stimulating DC failed to prevent their early acquisition of memory characteristics. One interesting question arises is whether there also exists a transcriptional master regulator or a transcriptional network whose activity is influenced by inflammatory signals that regulates memory CD8 T cell differentiation. Identifying such a regulator or network will provide valuable insight to the molecular basis of memory CD8 T cell generation. The model system of DC immunization and timed addition of CpG ODN-induced cytokines may prove valuable in dissecting these possibilities. The ultimate goal of the CD8 T cell immune response is to expand in number and differentiate appropriately to fight the inciting pathogen and form memory cells to guard against re-infection. My data shows that interaction of naive CD8 T cells with antigenexpressing mature DC provides sufficient stimulation to drive T cell proliferation and for the responding T cells to acquire an effector phenotype and function. However, in the absence of encounter with inflammatory cytokines, this effector commitment is transient and the responding CD8 T cells default to a differentiation program that rapidly promotes acquisition of memory characteristics as they further proliferate. Encounter with inflammatory cytokines during this rapid proliferative phase efficiently deflects differentiation away from memory towards a sustained effector program. Thus, the inflammatory cytokine response elicited by infection serves as the endogenous danger signals to alert the host to mount an appropriate immune response by shaping the T cell differentiation program. These danger signals participate in the CD8 T cell response not only by maturing professional antigen presenting cells to deliver the activation signals to

64 47 CD8 T cells but also by enforcing the appropriate effector differentiation program to ensure that sufficient resources are devoted to clearance of the inciting pathogen.

65 48 Figure 2. Low number OT-I adoptive transfer mimics the endogenous CD8 T cell response after peptide-coated DC immunization in the presence or absence of CpG. Naïve B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells and were immunized with ~ 1x10 6 Ova coated DC ± CpG (100 µg IP). (A) Recovery of Flt3L-expanded, in vivo LPS stimulated splenic dendritic cells (DC) and their activation profile after isolation and prior to immunization. Frequency (mean ± S.D., N=3) as detected by ICS for IFN-γ (all Ova-specific cells) and Thy1.1 expression (OT-I cells) in the spleen (B) or direct exvivo tetramer staining (all Ova-specific cells) and Thy1.1 expression (OT-I cells) in both the peripheral blood and spleen (C) on day 7 post DC immunization in the presence or absence of CpG. (D) Total number (mean ± S.D., N=3) of endogenous Ova257-specific CD8 T cells and OT-I in the spleen as detected by ICS on day 7 post DC immunization. Data are representative of at least 3 experiments.

66 49

67 50 Figure 3. Similar phenotypes between OT-I and endogenous antigen-specific CD8 T cells following low number OT-I adoptive transfer and peptide-coated DC± CpG immunization or LM infection. Naïve B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells and were immunized with ~ 1x10 6 Ova coated DC ± CpG (100 µg i.p.) or infected with ~1x10 5 cfu LM-Ova. (A) Representative histograms and (B) Cumulative data representing CD127, KLRG-1, and Granzyme B expression on OT-I cells and endogenous Ova257-specific CD8 T cells as detected either by ICS or direct ex-vivo tetramer staining at day 7 following DC immunization in the presence or absence of CpG-induced inflammation or LM-Ova infection. Numbers in histograms represent frequency (mean ± S.D., N=3) of cells that are positive for indicated markers. Shaded histograms represent isotype-control staining. Data are representative of at least 3 experiments. N.D. = not done.

68 51

69 52 Figure 4. CpG ODN does not signal directly on transferred DC or T cells but rather induces the inflammatory environment that influences the rate of memory generation following DC immunization. (A) Experimental design: naïve wt or TLR9 -/- B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells. Mice were immunized with either ~ 1x10 6 Ova coated wt DC or TLR9 -/- DC with or without co-injection of CpG (100 µg IP). OT-I cells from PBL were analyzed on day +7 post immunization or infection. Mice were then challenged with early booster infection with acta-deficient LM-Ova and frequency of OT-I was analyzed 5 days later. (B) Recovery of Flt3L-expanded, in vivo LPS stimulated wt and TLR9 -/- splenic dendritic cells (DC) and their activation profile after isolation and prior to immunization. (C) Percentage (mean ± S.D., N 3) of OT-I in PBL expressing CD127 and KLRG-1 in both wt and TLR9 -/- recipients at day 7 post priming with either wt or TLR9 -/- DC in the presence or absence of CpG co-injection. (D) Frequency of OT-I in PBL in wild-type recipients at day 7 post priming with either wt or TLR9 -/- DC in the presence or absence of CpG and at day 5 following early booster infection with actadeficient LM-Ova. (E) Frequency of OT-I in PBL in TLR9 -/- recipients at day 7 post priming with either wt DC in the presence or absence of CpG and at day 5 following early booster infection with acta-deficient LM-Ova. Data are representative of at least 2 independent experiments.

70 53

71 54 Figure 5. CpG-induced inflammatory environment in the host displayed unique cytokine profiles and kinetics. (A) Experimental design: naïve B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells. Mice were immunized with ~ 1x10 6 Ova coated DC in the presence or absence of CpG co-injection (~100 µg IP). Sera were obtained via retro-orbital bleeding at 6, 12, 24, and 48 hrs after DC immunization. OT-I cells from PBL were analyzed on day +7 post immunization or infection. Mice were then challenged with early booster infection with acta-deficient LM-Ova and frequency of OT-I was analyzed 5 days later. (B) Serum IL- 12(p70), IL-10, IL-6 and GM-CSF from DC and DC + CpG immunization groups were measured using Bio-Plex Mouse Cytokines Assays (Bio-Rad). Serum from naïve B6 mice served to determine the cytokine level at time 0. Data presented as mean ± S.D., N=4. (C) Percentage (mean ± S.D., N=4) of OT-I in PBL expressing CD127 and KLRG- 1 at day 7 post priming with DC in the presence or absence of CpG co-injection. (D) Frequency of OT-I in PBL at day 7 post priming with DC in the presence or absence of CpG and at day 5 following early booster infection with acta-deficient LM-Ova.

72 55

73 56 Figure 6. Exposure to inflammation prior to antigen-driven expansion of CD8 T cells does not prevent accelerated memory generation after DC immunization. (A) Experimental design: naïve B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells at day 3 relative to DC immunization. Inflammation was induced by CpG at day -2, day 0, or day +2 in the indicated groups while other groups did not receive CpG. Day 0 indicates the time when all mice were immunized with ~ 1x10 6 Ova coated DC. An additional control group received ~1x10 5 cfu LM-Ova on day 0. OT-I cells from PBL were analyzed on day +6 post immunization or infection. Half of the mice from each group then received booster infection acta-deficient LM-Ova and the frequency of OT-I was determined at day 4 following boosting. (B) The percentage (mean ± S.D., N=3) of OT-I cells in PBL expressing CD127, KLRG-1, and Granzyme B from the indicated groups of mice treated with CpG at different times. DC immunization and LM-Ova infection served as the controls for low inflammation and high inflammation, respectively. Statistical analysis was performed with student t-test between groups. * = statistically significant (p<0.05); n.s. = not statistically significant (p>.05) (C) OT-I recipient mice immunized with DC-Ova and received no CpG or CpG at day -2, 0, and +2. Control mice were infected with virulent LM-Ova. Mice were boosted with actadeficient LM-Ova on day 6 after DC-Ova immunization or LM-Ova infection. Data presented as frequency of OT-I in PBL on different days. Data are representative of at least 3 independent experiments.

74 57

75 58 Figure 7. Inflammation exerts the greatest impact on memory differentiation during proliferation of CD8 T cells. (A) Experimental design: naïve B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells and were immunized with Ova-coated DC one day later (day 0). BrdU was administered to individual groups of mice at three different time periods: day 4-6, day 6-8, and day CpG was administered for some mice in each group at the beginning of each interval. Control groups received BrdU but no CpG treatment. OT-I cells from the spleen were analyzed at day 6, 8 and 14 from the respective groups. (B) Representative histograms of BrdU incorporation by OT-I cells at various time intervals. Numbers on the right in histograms represent the percentage of OT-I cells incorporated BrdU during the 2-day period. (C) The percentage (mean ± S.D., N=3) of OT-I cells in the spleen expressing CD127, KLRG-1, and Granzyme B from the DC and DC+CpG groups in the three time periods of BrdU treatment (day 4-6, day 6-8, and day 12-14). (D) Representative histograms of isotype staining and CD127 expression on BrdU+ and BrdU- fractions of OT-I cells at day 8 post DC priming either in the presence or absence of CpG administered to the hosts on day 5. Shaded and unshaded histograms represent DC immunization and DC+CpG groups, respectively. Top two panels are from the BrdUnegative OT-I population and bottom two panels are from the BrdU-positive OT-I. Top and bottom (in parentheses) numbers indicates the frequency of OT-I cells expressing CD127 in the absence or presence of CpG, respectively. (E) Normalized percent of reduction in CD127 expression of both BrdU-negative and BrdU-positive OT-I populations is calculated as followed: (100% - ((% CD127 positive OT-I on DC+CpG)/(% CD127 positive OT-I on DC only)) on both day 7 and 8 following DC either in the presence or absence of CpG administered to the hosts on day 5. Statistical analysis was performed with student t-test.

76 59

77 60 Figure 8. The default pathway of memory CD8 T cell differentiation is deflected by encounter with inflammatory cytokines. Naïve B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells. Mice were immunized with ~ 1x10 6 Ova coated DC in the presence or absence of CpG co-injection (~100 µg IP). (A) Analysis of OT-I cells in the spleen from a representative mouse from each group (DC and DC+CpG) at day 4 and day 7 following DC immunization. (B and C) Phenotypic analysis of naïve OT-I cells (day 0) and responding OT-I cells at day 4 and 7 following DC immunization in the presence or absence of CpG ODN coinjection. (B) Representative histograms and (C) and kinetic of CD127, KLRG-1, and Granzyme B expression by OT-I cells. (D) Number of total OT-I cell recovered from the spleen of mice immunized with DC or DC+CpG; n.s. = not statistically significant by student t-test. (E and F) In vivo cytolytic assay: At day 4 following immunization with either DC or DC+CpG, OT-I recipient mice or naïve control mice that received neither OT-I nor DC were injected with target cells consisting of 10x10 6 CFSE hi -labeled B6 splenocytes (no peptide) and 10x10 6 CFSE lo -labeled B6 splenocytes (coated with Ova257 peptide). (E) Representative histograms of CFSE-labeled cells recovered from each group at 5 hours after target cell injection. Number in histogram is percentage of CFSE lo population remained after 5 hours. (F) Percent specific killing calculated by the formula described in Materials and Methods section. Cumulative data are from three mice per group.

78 61

79 62 Figure 9. Antigen-specific CD8 T cells stimulated with either DC or DC+CpG immunization exhibited similar effector functions early. Naïve B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells. Mice were immunized with ~ 1x10 6 Ova coated DC in the presence or absence of CpG co-injection (~100 µg IP). Analysis of OT-I cells in the spleen by ICS from a representative mouse from each group (DC or DC+CpG) at day 4 following DC immunization. (A) Representative dot plot showing OT-I gating and representative histogram showing fraction of OT-I cells expressing IFN-γ. Light gray histogram represents IFN-γ staining of the OT-I cells without peptide stimulation. Gray (filled) and dashed (open) histograms represent IFN-γ staining on the OT-I cells after 5 hrs peptide stimulation from DC and DC+CpG immunization group, respectively. (B) Cumulative data (n = 3 mice per group) of fraction of OT-I cell expressing IFN-γ. (n.s.= not statistically significant by unpaired student t-test, p = 0.08)

80 63

81 64 Figure 10. Model representing how the timing of inflammation relative to DC immunization influences the rate at which CD8 T cells acquire memory characteristics. (A) After DC immunization, responding CD8 T cells go through a transient effector phase but the low inflammatory environment during the robust proliferative expansion phase promotes accelerated acquisition of memory characteristics by the responding CD8 T cells. (B) Either exposure of non-proliferating naïve CD8 T cells to inflammation prior to their interaction with stimulating DC or exposure too inflammation of nonproliferating CD8 T cells after contraction does not prevent early acquisition of memory characteristics or alter existing memory characteristics of the responding CD8 T cells. (C) During an acute infection or DC immunization + TLR agonist, heightened inflammation during the priming and/or robust proliferative expansion sustains the effector differentiation program and prevents early acquisition of memory characteristics by the responding CD8 T cells, resulting in their slow conversion to memory.

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83 66 CHAPTER III DIFFERENTIAL ROLES OF SIGNAL 3 INFLAMMATORY CYTOKINES IN REGULATING CD8 T CELL EXPANSION AND EFFECTOR/MEMORY DIFFERENTIATION Introduction In a naïve host, CD8 T cells specific for any pathogen-derived peptide are present at a very low numbers, estimated in the range between 10 to 1000 cells (32, 33). In order to combat infection, the pathogen-specific CD8 T cells must expand tremendously in number and acquire effector functions. Following an infection, pathogen-induced inflammatory cytokines directly inhibit pathogen replication while providing critical signals to enhance the functions of professional antigen presenting cells in the host. In addition, inflammatory cytokine signaling through receptors expressed by CD8 T cells also modulates all phases of their response including proliferation, survival, effector and memory differentiation (39, 112, 113). In particular, IFN-αβ and IL-12 have been demonstrated to serve as signal 3 for optimal CD8 T cell expansion (38) and these cytokines have also been implicated in differentiation of effector CD8 T cells (39). Inflammatory cytokines clearly play a crucial role in regulating the rate at which responding CD8 T cells acquire memory phenotype and function (34). Priming of CD8 T cells in a low inflammatory environment (i.e. dendritic cell immunization) accelerates memory CD8 T cell development. As early as 6 or 7 days after priming, the antigenspecific CD8 T cells exhibit memory characteristics defined by IL-7Rα (CD127) and KLRG-1 expression profiles (CD127 hi KLRG-1 lo ), IL-2 production and the ability to vigorously expand in response to booster immunization (105). In contrast, the acquisition of phenotypic and functional memory characteristics by CD8 T cells takes several months after clearance of acute infection (116). Consistent with this, dendritic cell immunization in the presence of inflammation also delayed memory CD8 T cell development (105).

84 67 Recent studies suggest that IL-12 signaling directly in CD8 T cells enhances expansion, promotes terminal differentiation (86, 124), and regulates memory formation through a gradient of T-bet expression (104). Thus, signal 3 inflammatory cytokines appeared to regulate multiple aspects of the CD8 T cell response. Interestingly, the specific cytokine serving as the signal 3 depends on the pathogen under investigation (34). For example, type I IFN signaling in CD8 T cells is most critical for numerical expansion in the Lymphocytic choriomeningitis virus (LCMV) infection model, whereas IL-12 serves as the critical signal 3 for CD8 T cells responding to Listeria monocytogenes (LM) infection (83, 87). However, it remains unclear whether these same inflammatory cytokines, which are critical signal 3, also directly regulate effector/memory CD8 T cell differentiation in vivo. Here, I specifically asked whether direct signaling by either type I IFNs or IL-12 to antigen-specific CD8 T cells is required for their acquisition of effector phenotype and function. To address this question, I employed dendritic cell (DC) immunization in two complementary approaches: adoptive transfer of a physiologic number of cytokine receptor-sufficient and receptor-deficient transgenic (Tg) OT-I CD8 T cells that recognize the K b -Ova 257 epitope and bone marrow chimeric mice containing wild-type and receptor-deficient endogenous CD8 T cells. I show that both type I IFNs and IL-12 serve as signal 3 cytokines to promote optimal CD8 T cell expansion after DC immunization in the presence of CpG-induced systemic inflammatory cytokines. However, signaling through neither cytokine receptor as expressed on CD8 T cells is critical for inflammation-induced effector commitment. Thus, these results dissociate the role of type I IFNs and IL-12 as the signal 3 during expansion from the signal(s) that regulate CD8 T cell effector/memory differentiation program.

85 68 Materials and Methods Mice C57BL/6 (CD45.1, Thy1.2) mice were from the National Cancer Institute (Frederick, MD). C57BL/6 (CD45.2, Thy1.1/1.1) and IL-12Rβ2 -/- mice on the B6 background were purchased from the Jackson Laboratory (Bar Harbor, ME). OT-I (Ova257-specific) transgenic Thy1.1/1.2 were previously described (119). IFNABR -/- mice have been previously described (83). IFNABR -/- OT-I (Thy1.2) and IL-12Rβ1 -/- OT-I (Thy1.2) cells were a generous gift from Dr. Matthew F. Mescher (University of Minnesota, Minneapolis, MN). Pathogen-infected mice were housed at appropriate biosafety conditions. Mice were used at 6-10 weeks of age. Experiments were conducted according to federal and institutional guidelines and approved by the University of Iowa Animal Care and Use Committee (Iowa City, IA). Dendritic cells and bacteria Splenic DC (DC) were prepared as previously described in chapter II of this thesis. Attenuated (acta-deficient) Listeria monocytogenes expressing Ova (attlm- Ova) (50) were grown and was injected at 5x10 6 cfu i.v. per mouse for boosting and quantified as described (105). Adoptive transfer experiments Thy1.1/1.2 wild-type OT-I and Thy1.2/1.2 receptor-deficient OT-I cells were obtained from the blood of naïve donors and the number of input OT-I was calculated as previously described (64). Equal number of wild-type and receptor-deficient OT-I were mixed (master mix) and flow cytometry was performed to ensure ~ 1:1 ratio of wild-type to receptor-deficient OT-I cells. Approximately 600 OT-I cells from each type were prepared by dilution from the master mix and transferred into naïve CD45.1 B6 recipient

86 69 mice. All mice were immunized with ~ 1x10 6 Ova257-coated matured DC at day 0 with or without i.p. co-administration of CpG oligonucleotide 1826 (IDT, Iowa City, IA) (100 µg). Generation bone marrow chimeric mice Bone marrow cells were obtained from tibiae and femurs of naïve wild-type B6 (Thy1.1/1.1) and receptor-deficient B6 (Thy1.2/1.2) mice. Following removal of red blood cells, approximately 5x10 6 wild-type cells and 5x10 6 receptor-deficient cells were transplanted to each lethally irradiated (900 Gy) CD45.1 recipient mouse. Reconstitution of donor lymphocytes was verified at 8 weeks following transplantation. All experiments were carried out after 8 weeks of reconstitution. Quantification and phenotypic analysis of antigen-specific T cells The magnitude of the epitope-specific CD8 T cell response was determined either by tetramer staining as described (97) or by staining for the CD45.2 marker that is exclusively expressed on transferred TCR-tg cells (64). Differential expression of Thy1.1 and Thy1.2 allowed for separating wild-type and receptor-deficient TCR-tg cells or transplanted cells. For analysis of circulating TCR-tg T cells, ~ 75 µl of blood was obtained from each mouse. The number of tg OT-I cells or antigen-specific CD8 T cells was presented as frequency of total PBL. In the experiments with bone marrow chimeric mice, staining for CD45.1 was used to distinguish between donor and recipient cells. For phenotypic analysis, blood was pooled from all mice in either DC only or DC+CpG immunization groups for OT-I cells or analyzed individually for bone marrow chimeric mice.

87 70 Results and Discussion Direct signaling by signal 3 inflammatory cytokines, type I IFNs or IL-12, to CD8 T cells promotes their expansion The requirement of direct type I IFNs or IL-12 signaling in CD8 T cells for their optimal expansion depends critically on the inflammatory cytokines produced by the specific pathogen in the study (82, 87, 113). The role of these two cytokines in supporting CD8 T cell expansion in vivo in the non-infectious, DC immunization in the presence of CpG-induced inflammation has not been extensively characterized. To control for potential variations in antigen load and inflammatory environment between recipients, I decided to examine receptor-sufficient and receptor-deficient Tg OT-I CD8 T cell response in the same host. Prior to adoptive transfer, I mixed equal numbers of wt and receptor-deficient OT-I in a master mix and monitored their ratio by flow cytometry to ensure that approximately equal numbers of each type were being transferred (Figure 11A & B). Wild-type (wt) and receptor-deficient OT-I cells exhibit similar naïve phenotypes (CD127 hi and KLRG-1 lo ) (Figure 11C). Approximately 600 wt (CD45.2; Thy1.1/1.2) and 600 receptor-deficient (CD45.2; Thy1.2/1.2) OT-I cells were adoptively co-transferred from the master mix into congenic naïve C57BL/6 hosts (CD45.1). One day after adoptive transfer, mice were immunized with LPS-matured, Ova 257 -coated DC and CpG oligodeoxynucleotide (ODN) 1826, a TLR9 agonist, to induce systemic production of inflammatory cytokines. Following DC immunization, I tracked the frequency of OT-I cells in blood during the primary expansion from day 5 to 7 by analyzing the CD8 + CD cell population and distinguishing wt and receptor-deficient OT-I cells by Thy1.1 staining (Figure 11D). In agreement with the existing literature suggesting that inflammatory cytokines serve as signal 3 for CD8 T cell expansion, OT-I cells deficient for either type I IFN- or

88 71 IL-12-receptor did not expand as robustly as their wild-type counterparts following DC immunization in the presence of CpG-induced inflammatory cytokines (Figure 11E). Furthermore, the differences in CD8 T cell numbers between wt and receptor-deficient OT-I became more pronounced from day 5 to 7 following DC immunization. These results are consistent with the notion that direct type I IFNs or IL-12 signaling to CD8 T cells provides critical survival signals for their accumulation during the proliferative expansion phase (79, 82, 86). Interestingly, the expansion defect was slightly more severe for IL-12β1 -/- OT-I cells than IFNABR -/- OT-I cells when compared to wt OT-I cells (Figure 11E), suggesting a more prominent role of IL-12 as the survival signal in this model (124). Taken together, these results demonstrate an important role for either type I IFNs or IL-12 as signal 3 for optimal CD8 T cell expansion after DC immunization in the presence of CpG-induced inflammatory cytokines. Neither type I IFNs nor IL-12 direct signaling in CD8 T cells is required for effector differentiation DC immunization in the absence of systemic inflammation accelerates the transition from effector to memory differentiation in antigen-specific CD8 T cells (105). However, co-injection of CpG ODN 1826 induces systemic inflammatory cytokines that sustain effector CD8 T cell differentiation and prevent the accelerated progression to memory after DC immunization (105). These findings strongly suggest that inflammatory cytokines regulate memory CD8 T cell development. In addition, the ability of CpG ODN to induce both IL-12p70 and IFN-αβ has been described (137). Thus, DC immunization in the presence or absence of CpG-induced systemic inflammatory cytokines offers a unique opportunity to determine the direct role of type I IFN and IL-12 signaling in regulating effector/memory CD8 T cell differentiation in vivo. To address this question, I examined the phenotype of wt and receptor-deficient Tg OT-I cells from the peripheral blood at day 7 following Ova 257 -coated DC

89 72 immunization in the presence or absence of CpG co-administration. Consistent with previous findings in our laboratory, OT-I cells responding to DC immunization exhibited a memory-like phenotype (CD127 hi, KLRG-1 lo ) at day 7 (105). This was true for both wt and IFNABR -/- OT-I or wt and IL-12Rβ1 -/- OT-I cells responding in the same host (Figure 12A-C). As previously shown, wt OT-I cells responding to DC immunization in the presence of systemic inflammation (DC+CpG) displayed an effector phenotype (CD127 lo, KLRG-1 hi ) at day 7 after immunization. Interestingly, IFNABR -/- OT-I or IL- 12Rβ1 -/- OT-I cells also down-regulate CD127 and up-regulate KLRG-1 expression in the presence of CpG-induced inflammation (Figure 12A-C). Thus, OT-I cells that could not receive either type I IFN or IL-12 signals during expansion still acquired an effector phenotype (CD127 lo, KLRG-1 hi ) that was indistinguishable from wt OT-I cells in the same host. These results demonstrate that direct signaling by either type I IFNs or IL-12 to the responding CD8 T cells was not an absolute requirement for their acquisition of effector phenotype in the presence of CpG-induced systemic inflammation. Hence, although both are required as signal 3 for optimal expansion, neither type I IFNs nor IL- 12 serve as the critical signal to promote and sustain effector differentiation by CD8 T cells. My results differ from a recent study showing that the lack of direct IL-12 signaling promotes CD127 hi KLRG-1 lo phenotype on the responding CD8 T cells in the presence of CpG-induced inflammation (124). This discrepancy may be explained in part by the use of different transgenic CD8 T cells (OT-I vs. P14), experimental approach, or the quality and quantity of inflammatory cytokines induced by CpG 1826 in the studies. I have observed that varying doses of CpG ODN 1826 had differential effects on CD127 and KLRG-1 expression on the responding CD8 T cells and their secondary response potential following early booster infection (Figure 13A & B). In addition, the different results may arise from evaluating wt and receptor-deficient TCR tg cells in the same (my study) or in different hosts (124). In order to resolve these issues, I investigated the

90 73 polyclonal antigen-specific CD8 T cell response in bone marrow chimeric mice containing both cytokine receptor-sufficient and receptor-deficient CD8 T cells. Chimeric mice were immunized with LPS-matured, Ova 257 -coated DC in the presence or absence of CpG-induced inflammation. Following DC immunization, Ova 257 -specific, receptor-deficient and -sufficient CD8 T cell populations can be easily distinguished by Thy1.2 staining on the CD45.1-negative, Ova-tetramer positive CD8 T cells in the same mouse (Figure 14A). Both wt and IFNABR -/- Ova 257 -specific CD8 T cells responding to DC immunization exhibited an accelerated memory phenotype (CD127 hi KLRG-1 lo ) at day 7 post immunization (Figure 14B). In sharp contrast, both wt and IFNABR -/- Ova 257 -specific CD8 T cells displayed an effector phenotype (CD127 lo KLRG-1 hi ) when primed in the presence of CpG-induced inflammation (Figure 14B). Interestingly, I also obtained similar results in WT/IL-12Rβ2 -/- chimeric mice. DC immunization alone promoted early acquisition of a memory phenotype (CD127 hi KLRG- 1 lo ) while CpG-induced inflammation resulted in effector phenotype (CD127 lo KLRG-1 hi ) by both wt and IL-12Rβ2 -/- Ova 257 -specific CD8 T cells at day 7 following immunization (Figure 14B). Moreover, the difference in inflammation-induced CD127 downregulation and KLRG-1 upregulation on both receptor-sufficient and receptor-deficient Ova specific CD8 T cells was statistically significant when compared with DC immunization alone (Figure 14C & D). More importantly, these data with polyclonal, endogenous Ova 257 -specific CD8 T cells recapitulate the earlier data obtained for TCR tg OT-I CD8 T cells. Thus, I conclude that direct signaling by either type I IFNs or IL-12 in the responding CD8 T cells was not required for the acquisition of effector phenotype in the presence of CpG-induced inflammation. Taken together, these results suggest that other yet-to-be identified inflammatory cytokine(s) could fulfill this function or that there is a redundancy in the inflammatory cytokine signaling that promotes effector differentiation of CD8 T cells.

91 74 Functional effector/memory differentiation of CD8 T cells is independent of type I IFNs or IL-12 Due to the substantial phenotypic and functional heterogeneity in populations of antigen-specific CD8 T cells that are examined following infection or immunization, there is no single marker or property that identifies unequivocally and defines memory CD8 T cells (45). Previous work in our laboratory demonstrated that the (CD127 hi KLRG-1 lo ) phenotype correlated well with the ability of antigen-specific CD8 T cells to expand vigorously to secondary antigen stimulation, a functional characteristic of memory cells (105). Therefore, I investigated the secondary response potential in chimeric mice following DC immunization with or without CpG co-injection. At day 7 following Ova 257 -coated DC priming in the presence or absence of CpG co-administration, chimeric mice received booster infection with attlm-ova and the kinetics of Ova 257 -specific CD8 T cell response was monitored in peripheral blood. Consistent with their memory phenotype (CD127 hi KLRG-1 lo ), Ova 257 -specific CD8 T cells in both WT/IFNABR -/- and WT/IL-12Rβ2 -/- chimeric mice that received DC immunization alone underwent a substantial secondary response to early booster infection (Figure 15A). In contrast, Ova 257 -specific CD8 T cells in both chimeric mice that were primed with DC + CpG failed to further expand in response to early booster infection, reflecting their effector phenotype (CD127 lo KLRG-1 hi ) (Figure 15B). More importantly, early booster infection resulted in a higher frequency of Ova 257 -specific memory CD8 T cells 43 days later (day 50 after DC) in both chimeric mice when they were primed in the absence of inflammation (Figure 15A & B). To evaluate the magnitude of the secondary response to booster infection, I determined the fold increase or reduction in the frequency of both receptor-sufficient and receptor-deficient Ova 257 -specific CD8 T in the chimeric mice at day 7+6 (6 days after booster infection) compared to day 7. The booster infection resulted in increased expansion for both wt and IFNABR -/- Ova 257 -specific CD8 T cells following DC

92 75 immunization alone while DC+CpG immunization prevented this secondary expansion (Figure 15C). Similar results were obtained for wt and IL-12Rβ2 -/- Ova 257 -specific CD8 T cells (Figure 15D). Interestingly, DC-primed IFNABR -/- and IL-12Rβ2 -/- Ova specific CD8 T cells did not expand as robustly as their wt counterparts following booster infection (Figure 15C & D, left panels). These results show for the first time an important role of type I IFNs and IL-12 as signal 3 to promote optimal CD8 T cell expansion during secondary responses. Furthermore, compared to their wt counterparts, fewer numbers of IFNABR -/- or IL-12Rβ2 -/- Ova 257 -specific CD8 T cells were detected at day 50 following DC prime and LM boost, possibly reflecting their diminished capacity to accumulate following booster immunization (Figure 16 A-C). These results suggest that type I IFN or IL-12 signaling in the CD8 T cells was not required for secondary memory development. Taken together, these data argue against the model in which either type I IFN or IL-12 signaling directly in the responding CD8 T cells is required to promote their terminal differentiation to effector cells. In this study, I showed that antigen-specific CD8 T cells that failed to receive either type I IFN or IL-12 signaling were still able to acquire an inflammation-induced effector phenotype (CD127 lo KLRG-1 hi ) and did not respond to secondary stimulation, a functional feature of effector CD8 T cells. Furthermore, the results presented here dissociate the role of type I IFNs and IL-12 as the survival signal 3 during expansion from the signal(s) that are required to regulate CD8 T cell effector/memory differentiation program. CpG induces various pro-inflammatory cytokines in addition to type I IFNs and IL-12 (137). Thus, it is possible that a yet to be identified pro-inflammatory cytokine serves as the critical signal that regulates the rate of CD8 T cell memory development. Alternatively, multiple inflammatory cytokines potentially serve a redundant role to enforce the effector CD8 T cell differentiation program. Therefore, elucidating the signal(s) that regulate CD8 T cell memory

93 76 differentiation remains a potential exciting strategy for manipulating memory generation and enhancing protective immunity.

94 77 Figure 11. Type I IFNs and IL-12 serve as signal 3 to promote optimal CD8 T cell expansion. Naïve B6 CD45.1 mice received ~ 600 naïve wt OT-I (Thy1.1/1.2) + ~ 600 receptordeficient OT-I cells and were immunized with ~ 1x10 6 Ova coated DC (i.v.) + CpG (100 µg i.p.) one day later. The ratio of wt and (A) IFNABR -/- or (B) IL-12Rβ1 -/- OT-I cells was monitored prior to adoptive transfer to ensure ~1:1 ratio. (C) Representative profiles of CD127 and KLRG-1 expression on naïve wt or IFNABR -/- OT-I cells prior to adoptive transfer. Similar profiles were obtained for IL-12Rβ1 -/- OT-I cells. (D) Representative dot plot showing gating strategy to distinguish between wt and receptordeficient OT-I cells at day 7 following DC immunization. (E) Kinetics of wt and receptor-deficient OT-I responses from day 5 to 7 after DC+CpG immunization expressed as frequency (mean ± SEM, n=4) of OT-I cells in total peripheral blood leukocytes (PBL). Data are representative of 3 independent experiments.

95 78

96 79 Figure 12. Neither type I IFNs nor IL-12 direct signaling to CD8 T cells is required for acquisition of effector phenotype. Peripheral blood was pooled from mice in either DC or DC+CpG immunization groups. (A) Representative histograms showing CD127 and KLRG-1 expression on wt, IFNABR - /- and IL-12Rβ1 -/- OT-I cells at day 7 following priming. Gray histograms represent isotype controls. Numbers indicated percentage of OT-I positive for the indicated marker. (B) Cumulative data are from 2 independent experiments.

97 80

98 81 Figure 13. Differential effect of varying CpG amount on both phenotype and functions of antigen-specific CD8 T cells following DC immunization. Naïve B6 Thy1.2 mice received ~ 500 naïve Thy1.1 OT-I cells and were immunized with ~ 1x10 6 Ova coated DC ± CpG (200, 50, 25, 10 or 0 µg IP). (A) Cumulative data representing the percent of OT-I cells from PBL expressing CD127 and KLRG-1 at day 7 following DC immunization in the absence (DC only) or presence of different amount of CpG (3 mice per group). (B) On day 7, mice received booster infection with actadeficient LM-Ova and the frequency of OT-I in PBL was determined at day 5 following booster infection.

99 82

100 83 Figure 14. Acquisition of effector phenotype is independent of type I IFNs or IL-12 in polyclonal antigen-specific CD8 T cell response. (A) Representative dot plots showing gating strategy to distinguish between wt and receptor-deficient Ova 257 -specific CD8 T cells at day 7 following DC immunization in bone marrow chimeric mice. Representative histograms (B) and cumulative data of 2 independent experiments (C&D) showing CD127 and KLRG-1 expression on wt, IFNABR -/- and IL-12Rβ1 -/- Ova 257 -specific CD8 T cells at day 7 following DC or DC+CpG immunization. Gray histograms represent isotype controls. Statistical analysis was performed with student t-test between DC & DC+CpG groups. Data are representative of 2 independent experiments.

101 84

102 85 Figure 15. Neither type I IFNs nor IL-12 signaling is required to inhibit accelerated memory differentiation in the presence of CpG-induced inflammation. WT/IFNABR -/- or WT/IL-12Rβ2 -/- chimeric mice were immunized with ~ 1x10 6 Ova coated DC ± CpG (100 µg intraperitoneally) and received booster infections with acta-deficient LM-Ova (~5x10 6 cfu/mouse) seven days later. Kinetics of Ova 257 -specific CD8 T cell response following booster infection expressed as frequency (mean ± SEM, n=6-8, two independent experiments) in total PBL from (A) DC or (B) DC+CpG immunization groups. Fold expansion/reduction is calculated as the ratio of frequency of Ova 257 -specific CD8 T cells in PBL at day 6 post booster infection to the starting frequency prior to booster infection (day 7 post DC immunization) for (C) WT/IFNABR - /- and (D) WT/IL-12Rβ2 -/- chimeric mice.

103 86

104 87 Figure 16. Type I IFNs or IL-12 signaling in the CD8 T cells was not required for secondary memory development. WT/IFNABR -/- or WT/IL-12Rβ2 -/- chimeric mice were immunized with ~ 1x10 6 Ova coated DC ± CpG (100 µg i.p.) and received booster infections with acta-deficient LM-Ova (~5x10 6 cfu/mouse) seven days later. Analysis was done at day 43 after booster infection. (A) Representative plots showing the gating strategy and ratio of WT and receptor-deficient Ova 257 -specific CD8 T cells detected in total PBL. Number showing the percentage of gated population within the total population shown. (B) Cumulative data showing the frequency of Ova 257 -specific CD8 T cells detected in total PBL (mean ± SEM, n=6-8, two independent experiments).

105 88

106 89 CHAPTER IV EXPLOITING THE CROSS-PRESENTATION PATHWAY TO RAPIDLY GENERATE PROTECTIVE CD8 T CELL IMMUNITY Introduction CD8 T cells play a critical role in protecting the host from infection by intracellular pathogens including viruses, bacteria and parasites. During the course of an infection, antigen-specific CD8 T cells undergo proliferative expansion to increase in number, which is followed by contraction and generation of a stable pool of long-lived memory cells (34). Memory CD8 T cells provide enhanced resistance to re-infection by the same pathogen for the host. Moreover, the number of memory CD8 T cells correlates strongly with the level of protection in experimental models of infection (45, 54, 70, 116). Thus, vaccine design to promote cellular immunity should logically focus on achieving a sufficiently high number of memory cells for protection. To date, primeboost immunization remains the most successful approach to generate high level of cellular immunity to a variety of pathogens in both animal models and humans (73, 138). However, most current prime-boost strategies require several months between each immunization to achieve the greatest amplification of immunological memory. Clearly, reducing the time interval between priming and boosting would be beneficial in case of pandemic outbreaks or in immunotherapy of cancer, when time is of the essence. Naïve CD8 T cells are primed by dendritic cells (DC) displaying antigenic peptides in the context of major histocompatibility class I (MHC-I). DC present MHC-Iassociated peptides that are derived from two main sources: endogenously synthesized or exogenously acquired proteins in the process defined as direct presentation or crosspresentation, respectively (29, 139, 140). Activation of naïve CD8 T cells via crosspresentation is thus referred to as cross-priming (29). The cells that express MHC class I molecules are capable of direct presentation to CD8 T cells as targets for lysis (i.e. virus-

107 90 infected cells) and/or as stimulators for priming (i.e. DC). In addition to their welldefined role of activating CD8 T cells via direct presentation, dendritic cells have been extensively studied for their capacity to cross-present to and cross-prime naïve CD8 T cells (31). Numerous types of antigen have been described to enter cross-presentation pathway (26). Nevertheless, cell-associated antigens and particulate antigens are generally considered to be the more efficient substrates for cross-presentation than soluble protein antigen (26, 31, 141). In particular, antigens derived from apoptotic cells ( ) or antigens coupled to iron oxide or latex particles have been implicated in cross-priming of CD8 T cells (147, 148). However, it is unknown how cross-priming of antigen-specific CD8 T cells by either cell-associated antigen or particulate antigen impacts the memory differentiation process in vivo. Successful recruitment and activation of CD8 T cells following acute infection or vaccination will ultimately result in the establishment of a memory pools containing a higher numbers of pathogen-specific CD8 T cells compared to the naïve precursors specific for that pathogen (34). However, in vivo analyses of antigen-specific memory CD8 T cells at the population level demonstrated both phenotypic and functional heterogeneity within memory populations (45, 54, 116, 132). For example, effector memory (T em ) and central memory (T cm ) CD8 T cells exhibit several differences including emergence over time, circulatory pattern, and proliferative capacity following re-exposure to antigen (132, 149). Nevertheless, vigorous secondary expansion in response to rechallenge defines one cardinal attribute of memory CD8 T cells that allows for the greatest amplification of cell number (34). Consequently, vaccine design exploiting the rate of memory CD8 T cell generation and their secondary response potential offers a valuable approach for rapid achievement of protection. Recent studies demonstrated that inflammatory cytokines induced by infection or toll-like receptor agonists controlled the rate at which antigen-specific CD8 T-cell populations acquire characteristics of the memory phenotype (105, 107). Peptide-coated

108 91 dendritic cell (DC) immunization in low inflammation accelerates CD8 T-cell memory generation and secondary response potential to early booster immunization (105). However, autologous DC vaccination can be costly, labor intensive and time consuming for large-scale therapeutic application. Therefore, I sought an alternative strategy to prime accelerated CD8 T-cell memory. In this study, I show that immunization with cellassociated antigen or antigen in particulate formulation with biodegradable microspheres cross-primes CD8 T cells in a low inflammatory environment resulting in accelerated CD8 T cell memory generation. Importantly, the cross-primed CD8 T cells expand robustly in response to early booster immunizations within days of priming. The resulting higher numbers of antigen-specific CD8 T cells following cross-prime and early boost provide greatly enhanced protection against high-dose pathogen rechallenge. In this chapter, early boost is defined as a booster immunization that is administered within short time interval, i.e. 7 days, following cross-priming. Furthermore, this approach also elicits protective heterosubtypic immunity against lethal influenza infection. Thus, the strategy employed in this study could potentially provide an additional route to vaccine design aimed at rapid generation of cell-mediated immunity to combat pandemic outbreaks and support cancer immunotherapy. Materials and Methods Mice C57BL/6 (B6, Thy1.2) and BALB/c (Thy1.2) mice were from the National Cancer Institute (Frederick, MD). Transgenic Act-mOva.K b-/- mice were a generous gift from Dr. Stephen Schoenberger (La Jolla Institute for Immunology and Allergy, La Jolla, CA). Pathogen-infected mice were housed in appropriate biosafety conditions. Mice were between 8-12 weeks of age at the time of the experiment. All experiments were approved by the University of Iowa Institutional Animal Care and Use Committee.

109 92 Dendritic cells, recombinant bacteria and viruses Peptide-coated splenic DC (DC) were prepared as described in Chapter II (150). L. monocytogenes expressing Ova (virlm-ova) (120), attenuated (acta-deficient) LM-Ova (attlm-ova) (50), and attenuated (acta-, IntB-deficient) LM expressing the H- 2K d -restricted influenza epitope, IYSTVASSL (attlm-ha 518 ) (Cerus Corporation, Concord, CA) were grown, injected intravenously via tail vein at the indicated dose per mouse and quantified as described (105). Vaccinia virus expressing the SIINFEKL peptide (VACV-OVA) has been described previously (151); viruses were propagated according to standard protocols and injected i.p. or i.v. into mice at the dose indicated (152). The Armstrong strain of LCMV (2 105 pfu/mouse; i.p.) was used as previously described (85). For influenza infection, BALB/c mice were anesthetized by isofluorane and were infected intranasally (i.n.) with ~ 6.4x10 4 8x10 4 TCID 50 of mouse-adapted A/Puerto Rico/8/34 (H1N1) in 50µl of Iscoves media. Viruses were propagated and stored as previously described (153). For influenza viral titer, lungs from infected mice at day 3 following infection were homogenized and viral titers were determined as previously described (154) by end point dilution assay and expressed as 50% tissue culture infectious dose (TCID 50 ). Briefly, serial 10-fold dilutions of homogenized lungs from influenza virus-infected mice were mixed with 5x10 5 Madin-Darby canine kidney (MDCK) cells in DMEM and incubated at 37 C for 24 h. Then culture supernatants were removed and DMEM containing % L-1-(tosylamido-2-phenyl)ethyl chloromethyl ketone-treated trypsin (Worthington Diagnostics) and penicillin (100U/ml)/streptomycin (100 mg/ml) was added to each well. After 4 days of incubation at 37 C, supernatants were mixed with an equal volume of 0.5% chicken RBC, the agglutination pattern was read, and the TCID 50 values were calculated using the Reed-Muench method.

110 93 Analysis of protective capacity after L. monocytogenes or vaccinia challenge Mice containing Ova-specific CD8 T cells or naïve mice were injected i.v. with virulent LM-Ova (~5 x 10 5 cfu/mouse) or VacV-Ova (~5 x 10 7 pfu/mouse). Three days later, spleens and liver, from infected mice were harvested and analyzed for bacterial content as described previously (155). For the vaccinia plaque assay, the ovaries were obtained, homogenized, subjected to 3 cycles of freeze-thaw and viral titers were quantified using standard plaque assaying on VERO cells as previously described (152). PLGA microspheres, ovalbumin, recombiant hemagglutinin, CpG and peptides Poly(lactic-co-glycolic) acid (PLGA) microspheres with mean diameter of 2.0 µm and density of g/cm 3 (~1.91x10 10 spheres per ml) were purchased from Phosphorex, Inc. (Fall River, MA). Ovalbumin protein from chicken egg white, Grade III, was purchased from Sigma (St. Louis, MO) and dissolved in sterile RPMI 1640 or PBS at 1 mg/ml for coating cells and PLGA microspheres, respectively. Recombinant hemagglutinin protein, H5 (A/Vietnam/1203/2004) was purchased from Protein Sciences Corporation (Meriden, CT) at a concentration of ~0.6 mg/ml. Adsorption of protein onto cells or PLGA microspheres was carried out at 37 0 C by incubating cells or PLGA microspheres with protein solutions with occasional mixing for 1 hour. CpG oligonucleotide 1826 was purchased from IDT (Iowa City, IA), resuspended in clinical grade saline and injected 0.2 ml i.p. (100 µg/mouse) at the indicated times. Synthetic peptides representing defined LCMV epitopes (D b -restricted NP 396 and GP 276 and K b - restricted NP 205 ) as well as Ova epitope were previously described (119, 156).

111 94 Quantification and phenotypic analysis of antigen-specific T-cells The magnitude of the epitope-specific CD8 T cell response was determined either by intracellular IFN-γ staining (ICS) or MHC Class I-peptide tetramer staining as described (97). For analysis of circulating antigen-specific CD8 T cells, ~ 50 µl of blood was obtained from each mouse via retro-orbital bleeding. The number of antigen-specific CD8 T cells was presented as a frequency of the total CD8 T cells in PBL. Antibodies and MHC class I tetramers Antibodies of the indicated specificities conjugated to the appropriate fluorochromes were used. The following antibodies were from ebioscience (San Diego, CA): Thy1.2 (53-2.1), IFN-γ (XMG1.2), CD8 (53-6.7), CD127 (A7R34), IL-2 (JES6-5H4), KLRG-1 (2F1), CD27 (LG.7F9), isotype control rat IgG2a (ebr2a), rat IgG2b, κ, and Golden Syrian Hamster IgG. The following antibodies were from BD Biosciences (San Diego, CA): PD-1 (J43) and Armenian Hamster IgG2, κ. The following antibodies were from Biolegend San Diego, CA): TNF-α (MP6-XT22) and CD43 activationassociated glycoform (1B11). The following antibodies were from Caltag (San Diego, CA): anti-human Granzyme B and isotype control mouse IgG1. Rabbit polyclonal antibody to ovalbumin and normal rabbit IgG isotype control were from Abcam Inc. (Cambridge, MA) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Mouse monoclonal antibody to hemagglutinin, H5 (A/Vietnam/1203/2004) (clone 18E1) and mouse IgG2a, κ, isotype control were from Rockland (Gilbertsville, PA), and BD Pharmingen (San Diego, CA), respectively. FITC-conjugated anti-mouse Ig and FITCconjugated goat anti-rabbit IgG secondary antibodies were from BD Pharmingen and Jackson Immunoresearch Laboratories, Inc. (West Grove, PA), respectively. MHC class I tetramers (K b ) specific for Ova were prepared using published protocols (121, 122). MHC class I tetramers (K d ) specific for HA (IYSTVASSL) were obtained

112 95 from National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility (Atlanta, GA). Serum cytokine quantification Serum (~25µl) was obtained via retro-orbital bleeding at 20 hours after immunizing mice with Ova 257 -coated DC (~10 6 DC/mouse), irradiated K b-/- mova splenocytes (~10 7 cells/mouse), or virlm-ova (~10 5 cfu/mouse). Serum IL-6 and IFN-γ were measured using Bio-Plex Mouse Cytokines Assays (Bio-Rad) and read on the Bio- Rad Bioplex 200 system. Measurement of airway resistance Airway resistance was measured using a whole body plethysmograph (Buxco Electronics) and expressed as Penh values. Baseline Penh values for each mouse were recorded before and on the indicated time points following influenza A/PR/8/34 challenge. Results Cell-associated antigen cross-primes accelerated CD8 T cell memory in vivo Disposal of apoptotic cells by immune cells such as macrophages and dendritic cells prevents overt systemic inflammation in the host and provides a potential mechanism for cross-presentation of antigenic peptides derived from apoptotic cells (146). Therefore, I hypothesized that immunization with irradiated cells containing nonself antigen will cross-prime naive CD8 T cells in a low inflammatory environment, resulting in accelerated memory CD8 T cell differentiation that could be exploited in vaccine development.

113 96 Previous studies demonstrated that cell-associated antigen cross-primed CD8 T cells much more efficiently than soluble antigen (157, 158). In order to evaluate crosspriming of antigen-specific CD8 T cell response in vivo, I took advantage of splenocytes isolated from the Act-mOva.K b-/- transgenic mouse (159). These splenocytes do not express H-2K b molecules, cannot present K b -restricted peptides, and ubiquitously express membrane-bound chicken ovalbumin (Ova) antigen under control of the actin promoter (Figure 17A). I also confirmed the inability of these cells to directly present K b -restricted peptide. Following LCMV Armstrong strain infection, Act-mOva.K b-/- mice failed to generate a CD8 T cell response against K b -restricted NP 205 epitope but mounted a robust CD8 T cell response against two D b -restricted NP 396 and GP 276 epitopes. In contrast, wild-type mice generated a CD8 T cell response to all three epitopes (Figure 17B). Thus, Act-mOva.K b-/- cells provide an ideal source of cell-associated antigen to examine crosspriming of K b -restricted Ova 257 -specific CD8 T cells in C57BL/6 mice. To address this hypothesis, naïve C57BL/6 (B6) mice were immunized with γ- irradiation-induced apoptotic splenocytes isolated from Act-mOva.K b-/- mouse or wt B6 mouse. Immunization with irradiated Act-mOva.K b-/- splenocytes cross-primed detectable Ova 257 -specific CD8 T cells in peripheral blood that peaked at day 7 and contracted to undetectable level in the blood at day 54 (Figure 18A & B). Furthermore, splenic Ova 257 -specific CD8 T cells produced IFN-γ upon ex vivo re-stimulation with Ova 257 peptide, suggesting that they are functional (Figure 18C). Similar to DC immunization, immunizing mice with γ-irradiated Act-mOva.K b-/- splenocytes did not generate overt systemic inflammation (Figure 18D). In contrast, infection with virulent L. monocytogenes expressing the Ova 257 epitope (virlm-ova) resulted in elevated systemic inflammation as measured by serum level of IL-6 and IFN-γ 20 hours after infection (Figure 18D). More importantly, Ova 257 -specific CD8 T cells that were primed with either peptide-coated DC or γ-irradiated Act-mOva.K b-/- splenocytes, exhibited accelerated acquisition of memory features characterized by both phenotype (CD127 hi,

114 97 KLRG-1 lo ) and functional attributes (~35-40% produced IL-2 and were Granzyme B positive after antigen stimulation) at day 7 post immunization. In contrast, Ova specific CD8 T cells that responded to LM-Ova infection showed an effector phenotype (CD127 lo, KLRG-1 hi ) and function (virtually none produced IL-2 while almost all were positive for Granzyme B after antigen stimulation) at day 7 post infection (Figure 18E & F) (54, 105, 123). Thus, similar to DC immunization, cell-associated antigen crossprimed antigen-specific CD8 T cells in a low inflammatory environment resulting in accelerated acquisition of memory phenotype and function by the responding CD8 T cells. Accelerated secondary response to booster immunizations following cross-priming provides enhanced protective immunity One cardinal hallmark of memory CD8 T cells is their robust proliferative response upon re-exposure to antigen (34). Following peptide-coated DC immunization, antigen-specific CD8 T cells exhibited accelerated memory phenotype (Figure 18E & F) and were capable of substantial secondary expansion in response to booster infections as early as day 6 or 7 post-priming (105). To determine whether cross-priming CD8 T cells by cell-associated antigen accelerates their secondary response potential, I administered a range of booster immunizations including virlm-ova, attenuated acta-deficient L. monocytogenes expressing the Ova 257 epitope (attlm-ova) which is ~1000X less virulent than the former, and vaccinia virus expressing the Ova 257 epitope (VacV-Ova) to mice as early as day 7 after they were immunized with either γ-irradiated WT (control group) or Act-mOva.K b-/- splenocytes (Figure 19A). Consistent with their accelerated memory phenotype, Ova 257 -specific CD8 T cells in mice that were immunized with γ-irradiated Act-mOva.K b-/- splenocytes underwent vigorous secondary expansion in response to all three different booster immunizations (cross-prime+boost) compared to primary Ova 257 -

115 98 specific CD8 T responses in control mice (Figure 19B & C). Listeria booster immunizations induced a much more substantial secondary response, resulting in ~ 6 out of 10 circulating CD8 T cells in peripheral blood being specific for a single Ova 257 epitope at the peak of the secondary response (day 5 after booster immunization) (Figure 19B). More importantly, the large numbers of antigen-specific CD8 T cells at the peak of secondary response in all three cross-prime+boost groups remained at a significantly higher numbers at day 47 after the booster immunization (day 54 after priming) compared to control mice that received irradiated WT splenocytes and booster infection (Figure 19C). Similar to DC immunization followed by early booster infection with attlm-ova, the number of memory CD8 T cells in the cross-prime+boost group remained significantly higher for more than 120 days. In contrast, the same booster infection with attlm-ova at day 7 after initial virlm-ova infection did not result in an elevated number of Ova 257 -specific memory CD8 T cells (Figure 20 A & B). In addition, antigen-specific CD8 T cells at 123 days after booster infection consistently exhibited a secondary memory phenotype (CD127 hi, CD43 (1B11) lo, KLRG-1 hi, CD27 not uniformly upregulated) and similar fraction of cells produced IL-2 and TNF-α after antigen stimulation (Figure 20C) (71). These results suggested that cross-prime+boost generated robust, long-lived secondary memory CD8 T cells. Higher numbers of memory CD8 T cells correlate strongly with enhanced immunity to infection by intracellular pathogens (54, 97). Thus, I challenged the immune mice with a lethal dose of virlm-ova to determine the protective capacity. To ensure that anti-listeria immunity did not play a role in protection, I challenged the immune mice that were cross-primed with γ-irradiated Act-mOva.K b-/- splenocytes + boosted with VacV-Ova and examined bacteria burden in the spleen and liver. As expected, mice in cross-prime+boost group were able to clear the bacteria much more efficiently compared to naïve mice and mice immunized with either irradiated Act-mOva.K b-/- splenocytes only

116 99 or irradiated WT splenocytes + VacV-Ova infection (Figure 19D). Taken together, I demonstrated here the proof-of-principle findings that cross-priming by cell-associated antigen generates antigen-specific CD8 T cells with early memory-like phenotype and accelerated secondary response potential. In this chapter, I define the accelerated secondary response potential as the capacity of antigen-specific CD8 T cells to expand vigorously upon antigen re-exposure within a short time interval, ie. 7 days, following cross-priming. Therefore, cross-presentation and cross-priming can be exploited as a potential strategy to shorten the time interval required for amplification of the number of antigen-specific memory CD8 T cells by booster infection to quickly achieve enhanced protective immunity against life-threatening infection by intracellular pathogens. Inflammation prevents accelerated memory CD8 T cell generation following cross-priming After infection or DC immunization in the presence of strong adjuvant, CD8 T cells exhibited effector phenotype and function at the peak of the response (105, 107). Inflammatory cytokines induced by the pathogen or adjuvants promote and sustain the effector differentiation program but limit memory differentiation potential (my study in Chapter II). Similar to peptide-coated DC immunization in the absence of adjuvant, the early memory-like phenotype and accelerated secondary response potential to booster infection observed on antigen-specific CD8 T cells following cross-priming with cellassociated antigen are likely due to the low inflammatory environment in which the CD8 T cells are primed in (Figure 18D) (105). To directly test this hypothesis, I immunized mice with γ-irradiated Act-mOva.K b-/- splenocytes and induced sterile systemic inflammation by administering the TLR9 agonist, CpG oligodeoxynucleotide 1826 (160, 161) on the same day (day 0) or two days after priming (day +2). CpG 1826 induces elevated serum level of several pro-inflammatory cytokines including IL-6, IL-12p70 and IFN-γ with distinct magnitude and kinetics (Figure 5B). In sharp contrast to Ova 257 -

117 100 specific CD8 T cells from mice receiving no CpG, which exhibited early memory-like phenotype (CD127 hi, KLRG-1 lo ), Ova 257 -specific CD8 T cells from mice receiving CpG on either day 0 or day +2 showed an effector phenotype (CD127 lo, KLRG-1 hi ) on day 7 post priming (Figure 21A & B). Consistent with their effector phenotype, Ova 257 -specific CD8 T cells in CpG-treated mice failed to expand robustly following early booster infection and contracted to stable numbers that were not elevated compared to control mice that received γ-irradiated WT splenocytes and a single booster infection. On the other hand, antigen-specific CD8 T cells from mice immunized in the absence of CpG responded vigorously to early booster infection and established a significantly higher and stable number of antigen-specific memory CD8 T cells for more than 100 days (Figure 21C). At least these inflammatory cytokines, IL-12, type I IFNs and IFNγ, have been shown to support survival and accumulation of antigen-specific CD8 T cells (78, 79, 83, 86). Consistent with this notion, CpG treatment at day 0 resulted in greater accumulation of effector Ova 257 -specific CD8 T cells detected on day 7 following cross-priming (Figure 21D). Interestingly, CpG treatment on day +2 did not result in any significant difference in the circulating frequency of antigen-specific CD8 T cells compared to mice receiving no CpG (Figure 21D). However, Ova 257 -specific CD8 T cells in both CpG treated groups failed to be boosted following booster immunization. Thus, the failure of robust expansion in CpG-treated mice following early booster infection cannot be explained solely by the higher numbers of circulating antigen-specific CD8 T cells. Autologous peripheral blood mononuclear cells (PBMC) deliver antigen for cross-priming to achieve protective immunity Having demonstrated that cross-priming with cell-associated antigen followed by early booster infection rapidly generated protective CD8 T cell immunity, I next sought

118 101 to exploit this strategy for therapeutic application. In order to implement this approach in vaccination for humans, it is absolute required that the recipients must receive their own cells in order to prevent rejection and adverse reaction. In the murine model, I asked whether syngeneic cells could serve as carrier for the cross-priming antigen of choice. First, I needed to determine the lowest number of cells required for immunization that can achieve robust boosting effect. Immunizing mice with γ-irradiated Act-mOva.K b-/- splenocytes generates Ova 257 -specific CD8 T cells in a dose-dependent manner. Approximately one million irradiated transgenic cells were sufficient to cross-prime small numbers of antigen-specific CD8 T cells that were detectable in peripheral blood on day 7 after immunization (Figure 22A). More significantly, following early booster infection with attlm-ova, mice primed with ~10 6 irradiated transgenic cells had as many circulating Ova 257 -specific CD8 T cells as those primed with a ten-fold higher number of γ-irradiated Act-mOva.K b-/- splenocytes at both the peak of secondary expansion and a memory time point more than 60 days later (Figure 22B). I next determined whether irradiated syngeneic splenocytes carrying full-length Ova antigen could cross-prime Ova 257 -specific CD8 T cells with early memory phenotype and accelerated secondary response potential to booster infection. Following incubation with naïve C57BL/6 splenocytes, Ova protein was efficiently associated on the cell surface as detected by flow cytometry (Figure 23A). Immunizing mice with ~10 6 γ-irradiated Ova-coated syngeneic splenocytes yielded similar results compared to the same dose of irradiated Act-mOva.K b-/- splenocytes, both of which generated Ova specific CD8 T cells with an early memory phenotype characterized by CD127 hi and KLRG-1 lo (Figure 23B). Consistent with this memory phenotype, early booster infection with virlm-ova at day 7 after priming resulted in substantial secondary expansion and elevated secondary memory level of Ova 257 -specific CD8 T cells compared to mice that received irradiated non-coated syngeneic splenocytes and virlm-ova booster infection (Figure 23C). Thus, irradiated cells associated with antigen could potentially be used as a

119 102 priming vehicle to promote early memory CD8 T cells (defined here as the antigenspecific CD8 T cells that exhibit the characteristic phenotype of long-term memory population) that can be boosted quickly to a significantly higher number. Given the exciting results that I obtained with relatively low number of irradiated syngeneic cells (~10 6 cells) serving as the carrier for the antigen, I sought to extend this strategy as a potential therapeutic application where the recipient s own peripheral blood mononuclear cells (PBMC) coated with the antigen of choice could be used as a priming vehicle. I envisioned this approach having important utility for pandemic viral outbreaks or immunotherapy for cancer where rapid generation of high level of CD8 T cell immunity would be critical for protection. To validate this strategy, I immunized each mouse with γ-irradiated Ova-coated PBMC isolated from its own blood and administered an early booster dose of LM-Ova at day 7 following priming (Figure 24A). Compared to mice that received irradiated PBMC without Ova coating, mice immunized with irradiated Ova-coated PBMC responded vigorously to booster infection with substantial secondary expansion of Ova 257 -specific CD8 T cells and an approximately 12-fold higher memory level more than 60 days later (Figure 24B). To avoid any complication due to anti-listeria immunity, I assessed the protective capacity in the memory mice by challenging them with high dose of VacV-Ova (~5x10 7 pfu i.v.) and examined viral titer in the ovaries obtained three days after viral challenge. Consistent with the elevated level of Ova 257 -specific memory CD8 T cells circulating in the blood, the mice that received cross-prime + early boost regimen demonstrated superior protection against high-dose VacV-Ova challenge compared to naïve mice or mice receiving only LM-Ova booster infection (Figure 24C). None of the mice in this group showed any detectable vaccinia virus in the ovaries at 3 days following viral challenge. Thus, the approach of employing one s own PBMC coated with antigen as the priming vehicle followed by early booster infection could provide a useful strategy to quickly generate protective CD8 T cell immunity.

120 103 Biodegradable PLGA microspheres serve as a universal cross-priming vehicle The strategy of immunizing recipients with their own irradiated PBMC coated with antigen of choice provides an approach for individualizing therapy. However, in case of a pandemic outbreak, rapid deployment of the vaccine would be critical and desirable. Thus, the availability of a vaccine for quick, off-the-shelf usage becomes essential. Exogenous particulate antigen is cross-presented by professional APC to CD8 T cells much more efficiently than soluble antigen, partly due to differential uptake mechanisms and compartmentalization within these cells (162). Particulate formulation of antigen encapsulated in biodegradable particles such as PLGA microspheres or nanospheres has been explored to improve the efficiency of cross-priming CD8 T cells both in vitro and in vivo in the presence of adjuvant ( ). I thus determined whether immunizing mice with a particulate formulation of antigen adsorbed on PLGA microsphere surface without adjuvant would generate antigen-specific CD8 T cells with accelerated secondary response potential. Full-length Ova protein was efficiently adsorbed on the surface of the PLGA microspheres as detected by flow cytometry using FITC-conjugated antibody specific for Ova (Figure 25A). Interestingly, immunizing mice with Ova-coated PLGA microspheres (~10 9 or 10 8 microspheres) did not result in any detectable Ova 257 -specific CD8 T cells circulating in the peripheral blood using tetramer staining compared to control mice that received the same dose of PLGA microspheres coated with bovine serum albumin (BSA) (Figure 25B). However, virlm- Ova booster infection on day 7 following priming dramatically expanded in numbers the Ova 257 -specific CD8 T cell population and resulted in significantly higher circulating memory level in mice that were immunized with Ova-coated PLGA microspheres (Figure 25B). These results suggest that the particulate formulation of Ova-coated PLGA

121 104 microspheres induced cross-priming of Ova 257 -specific CD8 T cells at a low level. However, these antigen-specific CD8 T cells possessed accelerated secondary response potential that allowed them to expand robustly following early booster infection. In addition, the CD8 T cell response to Ova-coated PLGA microspheres appeared to be dose-dependent as mice primed with ~10 7 or ~10 6 microspheres did not show appreciably more boosting relative to the control mice receiving ~10 9 BSA-coated PLGA microspheres (Figure 25B). Thus, the combination of antigen-coated PLGA microspheres as a priming vehicle followed by early booster infection offers an attractive, potentially off-the-shelf approach to rapidly generate a high number of antigen-specific CD8 T cells. Booster immunization with an infectious agent such as recombinant Listeria generates a robust secondary response in mice that have been cross-primed with Ovacoated PLGA microspheres. To ensure that this booster response was not confined to immunization with infectious agents, I also immunized mice that received Ova-coated PLGA microspheres with non-infectious regimens including Ova 257 -coated syngeneic splenocytes in conjunction with CpG or soluble, full length Ova protein with poly(i:c) and α-cd40 monoclonal antibody (167). Both of these non-infectious regimens stimulated a more robust secondary Ova 257 -specific CD8 T cell expansion leading to higher number of memory CD8 T cells in mice that received PLGA-Ova compared to control mice that received PLGA-BSA (Figure 26A & B). Thus, the accelerated secondary response of CD8 T cells in cross-primed mice can be stimulated by infectious and non-infectious booster immunizations. Cross-priming followed by early boost generates protective heterosubtypic immunity against lethal influenza infection Influenza-specific CD8 T cells play a critical role in controlling and eliminating the respiratory infection, especially the highly pathogenic strains of influenza virus (168,

122 ). Furthermore, induction of CD8 T cells specific for conserved or cross-reactive epitopes has been shown to protect the host against different sub-strains of influenza infection in heterosubtypic cell-mediated immunity ( ). Therefore, vaccination to rapidly generate a protective number of influenza-specific CD8 T cells could be beneficial in the case of an influenza pandemic or the emergence of a highly pathogenic influenza strain such as avian H5N1. I thus decided to test the cross-prime + early boost approach to determine their protective capacity against lethal influenza infection in BALB/c mice. I coated PLGA microspheres with full-length, recombinant avian hemagglutinin H5 (A/Vietnam/1203/2004 (H5N1)) that contains the H-2K d -restricted epitope, IYSTVASSL, at position 531. This epitope is identical to the HA 518 -epitope derived from influenza strain A/PR/8/34 (H1N1). The H5 antigen was readily adsorbed on the surface of microspheres as detected by flow cytometry (Figure 27A). Naïve BALB/c mice were immunized with either HA 518 -coated DC (HA 518 peptide = IYSTVASSL) or H5-coated PLGA microspheres and boosted early with attenuated L. monocytogenes expressing the HA 518 epitope (attlm-ha) at day 7 following immunization. Control mice received either CS 252 -coated DC (CS 252 peptide = K d - restricted peptide derived from malaria parasite P. berghei circumsporozoite protein) (173) or BSA-coated PLGA microspheres and were boosted with attlm-ha. Following booster immunization, the HA 518 -specific CD8 T cell population expanded vigorously and a significantly higher circulating number of memory CD8 T cells was established in mice immunized with either HA 518 -coated DC or H5-coated PLGA microspheres compared to control mice. At day 54 following priming, approximately one in ten CD8 T cells circulating in the blood was specific for HA 518 epitope in the prime-early boost mice (Figure 27B & C). To determine the protective capacity of the cross-prime and early boost immunization, I challenged naïve BALB/c mice and immune mice (age-, sex-, and weight-matched) with a lethal dose of a serologically distinct influenza strain, A/PR/8/34

123 106 (H1N1) at day 47 after booster immunization. The majority of naïve mice and 40% of memory mice from the group that received only attlm-ha infection (control immune mice) succumbed to the influenza infection from day 8 to 11 following challenge while all the immune mice from the cross-prime-early boost group survived the infection (Figure 27D & E). Furthermore, immune mice from the cross-prime-early boost group exhibited less severe morbidity compared to both naïve mice and control immune mice as measured by weight loss following the influenza infection (Figure 27 F & G). Protection from mortality and morbidity in the immune mice is likely CD8 T cell-mediated since DC prime/lm boost does not stimulate influenza-specific CD4 or antibody response while antibody against H5, if any, is unlikely to provide a neutralizing effect against H1 of A/PR/8/34 virus. Furthermore, immune mice also lost weight similar to naïve mice during the first 5-6 days before recovering, suggesting that active infection took place in mice from all groups. Interestingly, immune mice that received either DC prime/lm boost or H5-coated PLGA microspheres+lm boost exhibited less morbidity and recovered more quickly compared to the control immune mice. This could be explained in part by the greater frequency of HA 518 -specific CD8 T cells in the prime-early boost mice prior to influenza A/PR/8 challenge (Figure 27 B & C). In addition, consistent with the morbidity results, lung functions as measured by airway resistance were less severely affected and improved faster in the prime-boost immune mice compared to control memory mice (Figure 27H & I). Furthermore, I also documented that the PLGA-H5- cross-primed+attlm-ha early boost immune mice had significantly reduced lung viral titers compared to both naïve and control immune mice 3 days following viral challenge, suggesting that higher numbers of HA 518 -specific memory cells were able to resolve the infection more quickly (Figure 28A). There appeared to be a negative correlation between the frequency of HA 518 -specific CD8 T cells prior to influenza A/PR/8/34 challenge and the lung viral burden (i.e. higher frequency correlates with less viral burden) (Figure 28B).

124 107 Up to this point, I have shown that cross-priming in conjunction with early boosting generates protective memory CD8 T cell immunity. However, the goal of vaccination is to achieve protection quickly. Thus, I wanted to determine whether this immunization approach could quickly provide protection to the host within short time interval following the booster immunization. Naïve BALB/c mice were cross-primed with PLGA-H5 microspheres and boosted with attlm-ha seven days later. Six days after booster immunization, the HA 518 -specific CD8 T cell population had expanded vigorously, reaching a high number of circulating secondary effector cells (~15-30% of all CD8 T cells in the blood) (Figure 29A). On day 7 after booster immunization, these mice were challenged with a lethal dose of influenza strain A/PR/8/34 (H1N1). Three out of four naïve mice succumbed to the lethal viral challenge while all the immunized mice survived. Furthermore, the immunized mice exhibited only slight weight loss and recovered very quickly from the infection (Figure 29B). Consistent with their rapid recovery following viral challenge, these immunized mice showed modest elevations in airway resistance (a measure of lung function) that quickly returned to baseline compared to naïve mice (Figure 29C). These results provide strong evidence that protective immunity can be achieved within a few days after booster immunization. Taken together, these results suggeste that protective heterosubtypic immunity against different sub-strain of influenza virus can be achieved by cross-priming with antigen-coated PLGA microspheres followed by early boosting. Discussion The ultimate goal of any vaccine regimen is to induce protective immunity against the targeted pathogens. More often than not, a single immunization is not sufficient to generate protective immunity against a specific pathogen. Therefore, multiple immunizations (i.e. prime-boost), separated by a substantial time interval, are necessary to amplify the immune response to achieve protection. Shortening the wait time

125 108 between immunizations would be beneficial during pandemic outbreaks where rapid generation of protective immune response is critical in slowing down the spread of disease. In this study, I exploited the cross-presentation pathway followed by early booster immunization to rapidly amplify the CD8 T cell response. Immunization with antigen associated with apoptotic autologous cells or particulate formulations of antigen associated with biodegradable PLGA microspheres induced antigen-specific CD8 T cells with accelerated secondary response potential that could be boosted to a significantly higher number a few days after priming. The elevated numbers of effector and memory CD8 T cells provided enhanced protection against challenge with a high dose of pathogens. These results potentially add a new approach to the existing arsenal of vaccine strategies aimed at fighting pandemic outbreaks and emerging infectious diseases. Immunization with cells isolated from transgenic Act-mOva.K b-/- mice allowed me to exclusively study cross-priming of CD8 T cells. However, one potential confounding factor in this model is the lack of MHC Class I K b on the immunizing cells that could trigger natural killer (NK) cell activity. Using transgenic Act-mOva.K b-/- cells, a recent report suggested that NK-mediated killing of immunized targets induces potent Ova 257 -specific CD8 T cell responses (174). In agreement with the reported results, immunizing mice with irradiated Act-mOva.K b-/- cells resulted in a lower magnitude of expansion of Ova 257 -specific CD8 T cells compared to immunizing with live ActmOva.K b-/- cells. However, I observed that antigen-specific CD8 T cells primed with live Act-mOva.K b-/- cells exhibited an effector phenotype (i.e. CD127 lo, KLRG-1 hi ). Consistent with that notion, NK cell activation potentially triggers inflammatory cytokines including interferons that promote expansion of CD8 T cells as suggested by the recent report (78, 79, 83, 86). I have previously shown that priming of CD8 T cells in the presence of inflammatory cytokines promotes the effector differentiation program at the expense of accelerating memory development (105, 107). Thus, the dichotomy that

126 109 any vaccine design aimed to elicit cell-mediated immunity needs to address is whether a vaccine should prime a robust primary response (i.e. co-administration with adjuvants to induce pro-inflammatory cytokines) with full differentiation to effector phenotype and function and slow development of memory or a primary response with accelerated memory potential that can be effectively boosted at a shorter interval to higher effector and memory CD8 T cell numbers. The former approach requires a substantial time interval after initial immunization to boost the response in case a single immunization is insufficient to induce protective immunity. Therefore, in certain situations such as pandemic outbreak and even tumor immunotherapy, I believe the latter as a strategically better approach to rapidly induce vigorous cell-mediated immunity. Seasonal influenza vaccination that requires annual administration provides effective protection against homologous viral strains by induction of antibodies against viral coat proteins (111). However, antibody-mediated protection is not adequate against serotypically distinct heterologous strains or novel strains. Moreover, antibody-mediated protection could eventually be lost when sufficient mutations are accumulated in the homologous strain to which the vaccine targets due to antigenic drift (110, 111). In addition, the emergence of a highly pathogenic strain such as avian H5N1 or a pandemic strain such as 2009 H1N1 indicates the urgent need for vaccine development that provides protective heterosubtypic immunity (110, 111). Influenza-specific CD8 T cells have been demonstrated to control and limit the progression of severe influenza infection in murine models ( ). In this study, I demonstrated that protective heterosubtypic immunity could be achieved by cross-priming with antigen from a serologically distinct heterologous influenza strain followed by early booster immunization with attenuated L. monocytogenes expressing the conserved CD8 T-cell-epitope. The immunized mice were able to recover faster and did not progress to severe morbidity and eventual mortality compared to naïve mice. Thus, targeting conserved epitopes within the hemagglutinin (HA) molecule provides an attractive option to generate CD8 T cell-mediated

127 110 heterosubtypic immunity. In addition, I am currently investigating other CD8 T-cell conserved epitopes derived from internal proteins common to multiple heterologous viral strains such as nucleoprotein (NP) whose mutation rate is much less frequent. Moreover, rapid generation of effector and memory CD8 T cells against multiple epitopes derived from both HA and NP molecules could potentially increase the breadth of protective heterosubtypic immunity. DC immunization for tumor immunotherapy is currently being investigated in humans with limited success partly due to high cost and intensive labor associated with tailored vaccine preparation for each individual. Thus, although effective, immunization approaches based on DC appear less attractive as a vaccine candidate for mass production. I showed here that a particulate formulation of antigen coupled with biodegradable microspheres elicits antigen-specific CD8 T cells that can be amplified by early booster immunization. The advantage of this strategy includes the use of full-length protein as an antigen that potentially contains more than one MHC class I-restricted epitopes and the possibility of formulating with multiple different antigens. In addition, biodegradable microspheres could be engineered to improve targeting of the antigen payload to dendritic cells for more efficient cross-presentation and cross-priming of CD8 T cells. Furthermore, the booster agents could be easily generated to express the same full-length targeted antigens. Thus, this strategy could theoretically amplify multiple different epitopes in a single prime-boost sequence in a short amount of time. The biocompatible and biodegradable material PLGA has been approved for use in humans (i.e. sutures). In addition, the use of PLGA microspheres or nanospheres as an antigen delivery vehicle offers an attractive option due to their extensive safety record in human clinical applications such as drug delivery ( ). Moreover, Listeria-based booster such as attenuated Listeria or killed-but-metabolically active (KBMA) Listeria is currently being developed and investigated for use as human vaccines (182). Thus, I

128 111 believe that cross-prime-early boost strategy could be readily extended to clinical application in humans.

129 112 Figure 17. Characterizing K b-/- mova splenocytes and K b-/- mova mice. (A) K b expression status on splenocytes from naïve B6, BALB/c or K b-/- mova mice as detected by specific antibody staining for K b molecules and flow cytometry. (B) Detection of Ova protein expression on the cell surface of splenocytes from naïve B6 and K b-/- mova mice as detected by specific antibody staining for Ova protein and flow cytometry. (C) Naïve B6 and K b-/- mova mice were infected with LCMV-Arm (2x10 5 pfu/mouse) and antigen-specific CD8 T cells from the spleen for D b -restricted NP 396 epitope, D b -restricted GP 276 epitope, and K b -restricted NP 205 epitope were detected by ICS assay at day 8 post infection. Representative dot plots show antigen-specific CD8 T cell response for each indicated epitope. Number represents the frequency of antigenspecific CD8 T cells within the CD8+ compartment in the spleen.

130 113

131 114 Figure 18. Cross-priming with cell-associated antigen generates functional antigenspecific CD8 T cells with accelerated memory phenotype in vivo. Naïve C57BL/6 (B6) mice received either ~10 7 irradiated wild-type or K b-/- mova splenocytes i.v. Representative dot plots showing detection of Ova 257 -specific CD8 T cells by tetramer (K b /Ova 257 ) staining from PBL (A) or ICS from spleen (C) at day 7 post priming. (B) Kinetics of Ova 257 -specific CD8 T cell response expressed as frequency (mean ± S.E.M, n=3) of total circulating CD8 positive cells in PBL. (D) Serum IL-6 and IFN-γ at 20 hrs following priming with Ova 257 -coated DC (~10 6 DC/mouse), irradiated K b-/- mova splenocytes (~10 7 cells/mouse), or virulent Listeria monocytogenes expressing Ova (virlm-ova) (~10 5 cfu/mouse). (E) Representative histograms and (F) cumulative data (mean ± S.D., n=3) showing phenotypic and functional status of Ova 257 -specific CD8 T cells at day 7 following Ova 257 -coated DC immunization, irradiated K b-/- mova splenocytes priming, or virlm-ova infection. Numbers in histograms represent frequency of cells that are positive for the indicated markers. Shaded histograms represent isotype-control staining. Data are representative of 2 independent experiments. Unpaired student t-test was used for statistical analysis between indicated groups (p<0.05 indicated statistical significance between the two groups).

132 115

133 116 Figure 19. Accelerated secondary response to booster infections following cross-priming with cell-associated antigen provides enhanced protective immunity. (A) Experimental design: naïve B6 mice were immunized with either ~10 7 irradiated WT or K b-/- mova splenocytes i.v. and challenged with early booster infection at day 7 post priming with virulent LM-Ova (~10 5 cfu/mouse), acta-deficient LM-Ova (attlm-ova) (~10 7 cfu/mouse) or VacV-Ova (~3x10 6 pfu/mouse). At memory time point, naïve or immune mice were challenged with a high dose of virlm-ova (~5x10 5 cfu/mouse). (B) Representative dot plots showing detection of Ova 257 -specific CD8 T cells by tetramer (K b /Ova 257 ) staining from PBL at day 7 post priming and day 5 following early booster infection with different infectious agents as indicated. (C) Kinetics of Ova 257 -specific CD8 T cell response expressed as frequency (mean ± S.E.M, n=3) of total circulating CD8 positive cells in PBL over time following different early booster infections. Numbers in graph (x) represent the fold difference in frequency of Ova 257 -specific CD8 T cells between mice receiving irradiated K b-/- mova and mice receiving wild-type splenocytes. Unpaired student t-test was used for statistical analysis at the indicated time points (p<0.05 indicated statistical significance between the two groups). (D) Bacteria number (mean ± S.D., n=3) in spleen and liver at ~ 65 hours following lethal dose challenge of naïve or immune mice (as indicated at the bottom of graph). Unpaired student t-test was used for statistical analysis on log 10 -transformed data (p<0.05 indicated statistical significance between the indicated groups).

134 117

135 118 Figure 20. Accelerated response to booster infections following cross-priming with cellassociated antigen generates stable memory pool of antigen-specific CD8 T cell with secondary memory phenotype. (A-C) Naïve B6 mice were immunized with Ova 257 -coated DC (~10 6 DC/mouse), irradiated K b-/- mova splenocytes (~10 7 cells/mouse), or virlm-ova (~10 5 cfu/mouse). Mice were challenged with early booster infection 7 days later with attlm-ova (~10 7 cfu/mouse). (A) Representative dot plots showing detection of Ova 257 -specific CD8 T cells by ICS from spleen at day 123 following booster infection. (B) Kinetics of Ova specific CD8 T cell response expressed as total number of Ova 257 -specific CD8 T cells per spleen (mean ± S.E.M, n=3). (C) Representative histograms showing phenotypic and functional status of Ova 257 -specific CD8 T cells at day 123 following booster infection. Numbers in histograms represent frequency of cells that are positive for the indicated markers. Shaded histograms represent isotype-control staining.

136 119

137 120 Figure 21. CpG-induced inflammation prevents accelerated generation of memory CD8 T cells and early prime-boost response following cross-priming with cell-associated antigen. Naïve B6 mice received ~10 7 irradiated K b-/- mova splenocytes with or without coinjection of CpG 1826 (100 µg/mouse intraperitoneally) at the indicated time. Mice were challenged with early booster infection, acta-deficient LM-Ova (~10 7 cfu/mouse), on day 7 following priming. (A) Representative dot plots and (B) cumulative data (mean ± S.E.M, n=3) showing phenotypic status of Ova 257 -specific CD8 T cells at day 7 following irradiated K b-/- mova splenocytes priming. Unpaired student t-test was used for statistical analysis between indicated groups (p<0.05 indicated statistical significance between the two groups). (C) Kinetics of Ova 257 -specific CD8 T cell response expressed as frequency (mean ± S.E.M, n=3) of total circulating CD8 positive cells in PBL over time following early booster infection. Unpaired student t-test was used for statistical analysis at the indicated time points (p<0.005 indicated statistical significance between the group that received no CpG compared to the group that received CpG at day 0 or day +2 or the group that received only booster immunization with acta-deficient LM-Ova). There was no statistically significant difference between groups receiving CpG at day 0 or day +2 at any time point after booster immunization. (D) Frequency of Ova 257 -specific CD8 T cells (mean ± S.E.M, n=3) at day 7 following cross-priming with wild-type or irradiated K b-/- mova splenocytes in the presence or absence of CpG co-administration at day 0 or day +2 as indicated; (n.s. = not statistically significant by student t-test). Background indicates non-specific staining by tetramer (K b /Ova 257 ) on PBL from mice immunized with irradiated WT splenocytes.

138 121

139 122 Figure 22. Dose-dependent antigen-specific CD8 T cell response following crosspriming with cell-associated antigen and early booster infection. Naïve B6 mice received different numbers of irradiated K b-/- mova splenocytes (~10 7, ~10 6, ~10 5 or ~10 4 ) or ~10 7 irradiated WT splenocytes. Mice were challenged with early booster infection, acta-deficient LM-Ova (~10 7 cfu/mouse), on day 7 following priming. (A) Cumulative data showing Ova 257 -specific CD8 T cells expressed as frequency (mean ± S.E.M, n=3 for each group) of total circulating CD8 positive cells in PBL as detected by tetramer (K b /Ova 257 ) staining at day 7 post priming. Background indicates nonspecific staining by tetramer (K b /Ova 257 ) on PBL from mice immunized with irradiated WT splenocytes. (B) Kinetics of Ova 257 -specific CD8 T cell response expressed as frequency (mean ± S.E.M, n=3 for each group) of total circulating CD8 positive cells in PBL over time following early booster infection.

140 123

141 124 Figure 23. Irradiated, Ova-coated syngeneic splenocytes cross prime accelerated memory CD8 T cells that could be boosted early to significantly higher number. Naïve B6 mice were immunized with irradiated, none-coated WT splenocytes, Ovacoated WT splenocytes or K b-/- mova splenocytes (~10 6 cells/mouse). Mice were challenged with early booster infection, virlm-ova (~10 5 cfu/mouse), on day 7 following priming. (A) Detection of Ova protein on the surface of irradiated WT splenocytes after incubation with soluble full-length Ova protein. Shaded and unshaded histograms represent Ova-specific staining and isotype control on splenocytes, respectively. (B) Cumulative data (mean ± S.E.M, n=3) showing phenotypic status of Ova 257 -specific CD8 T cells at day 7 following priming with either irradiated K b-/- mova splenocytes or Ova-coated WT splenocytes. (C) Kinetics of Ova 257 -specific CD8 T cell response expressed as frequency (mean ± S.E.M, n=3 for each group) of total circulating CD8 positive cells in PBL over time following early booster infection with virlm-ova (~10 5 cfu/mouse). Primary response indicates mice group receiving irradiated, nonecoated WT splenocytes and early booster infection 7 days later.

142 125

143 126 Figure 24. Irradiated, Ova-coated autologous peripheral blood mononuclear cells (PBMC) cross prime accelerated memory CD8 T cells that could be boosted early to significantly higher number. (A) Experimental design: PBMC were obtained from individual mouse via retro-orbital bleeding, coated with full length Ova protein in PBS or PBS only after lysis of red blood cells, irradiated and returned to the same mouse. Mice from control group received irradiated autologous PBMC without Ova coating. Mice were challenged with early booster infection at day 7 post priming with virulent LM-Ova (~10 5 cfu/mouse). Ova specific CD8 T cells were detected in PBL by tetramer (K b /Ova 257 ) staining at day 7 post priming and at indicated time points following booster infection. (B) Kinetics of Ova specific CD8 T cell response expressed as frequency (mean ± S.E.M, n=5 for each group) of total circulating CD8 positive cells in PBL over time following early booster infection with virlm-ova (~10 5 cfu/mouse). Unpaired student t-test was used for statistical analysis between non-coated or Ova-coated PBMC groups at indicated times (p<0.05 indicated statistical significance between the two groups). (C) Vaccinia viral titer per ovary pair after three days following a high-dose VacV-Ova challenge (~5x10 7 pfu/mouse i.v.). Naïve or memory mice were challenged with VacV-Ova on day 80 post priming (naïve mice: n=6; mice receiving irradiated non-coat PBMC + virlm-ova: n = 4, mice receiving irradiated Ova-coated PBMC + vir LM-Ova: n=5). Unpaired student t- test was used for statistical analysis on log 10 -transformed data (p<0.05 indicated statistical significance between the indicated groups).

144 127

145 128 Figure 25. Cross-priming with Ova-adsorbed, biodegradable poly(lactic-co-glycolic) acid microspheres (PLGA) followed by early booster infection quickly generates robust Ova 257 -specific CD8 T cells. (A) PLGA microspheres were coated via adsorption with either bovine serum albumin (BSA) as negative control or full-length Ova protein. Detection of adsorbed protein on the surface of PLGA microspheres with either Ova specific antibody by flow cytometry prior to immunizing mice. Shaded histograms represent isotype control staining. (B) Naïve B6 mice were immunized with different doses of Ova-adsorbed PLGA microspheres (~10 9, ~10 8, ~10 7 or ~10 6 ) or BSA-adsorbed PLGA microspheres as control and boosted early at day 7 post priming with virlm-ova (~10 5 cfu/mouse). Kinetics of Ova 257 -specific CD8 T cell response as detected by tetramer staining K b /Ova 257 and expressed as frequency (mean ± S.E.M, n=4 for each group) of total circulating CD8 positive cells in PBL at indicated time points following early booster infection.

146 129

147 130 Figure 26. Amplified secondary response following non-infectious booster regimens in PLGA-Ova-cross-primed mice. Naïve B6 mice were immunized with ~10 9 Ova-adsorbed PLGA microspheres or BSAadsorbed PLGA microspheres as control and boosted early at day 7 post priming with either (A) ~2x10 7 Ova 257 -coated splenocytes + CpG (100 µg i.p.) or (B) 500µg full length Ova protein + poly(i:c) (100 µg) + anti-cd40 mab (clone 1C10) (i.p.). Kinetics of Ova 257 -specific CD8 T cell response as detected by tetramer staining K b /Ova 257 and expressed as frequency (mean ± S.E.M, n=4 for each group) of total circulating CD8 positive cells in PBL at indicated time points following early booster immunizations. Unpaired student t-test was used for statistical analysis between PLGA-BSA and PLGA- OVA groups ( * p<0.05 indicated statistical significance between the two groups).

148 131

149 132 Figure 27. Cross-priming with avian hemagglutinin H5-adsorbed, biodegradable poly(lactic-co-glycolic) acid microspheres (PLGA) followed by early booster infection generates robust protective memory CD8 T cell immunity against different sub-strain of influenza infections. (A) PLGA microspheres were coated via adsorption with either bovine serum albumin (BSA) as negative control or full-length, recombinant hemagglutinin H5 (A/Vietnam/1203/2004) protein. Detection of H5 adsorbed on the surface of PLGA microspheres with H5 specific antibody by flow cytometry prior to immunizing mice. Shaded histograms represent isotype control staining. (B, C) Kinetics of HA 518 -specific CD8 T cell response as detected by tetramer staining, K d /HA 518 (IYSTVASSL), and expressed as frequency (mean ± S.E.M, n=5 for each group) of total circulating CD8 T cells (CD8+Thy1.2+) in PBL at the indicated time points. Naïve BALB/c mice were immunized with (B) ~ 10 6 DC coated with either K d -restricted influenza HA 518 -epitope or K d -restricted Plasmodium bergei circumsporozoite CS 252 -epitope as control or (C) ~10 9 PLGA microspheres coated with recombinant hemagglutinin H5 protein or bovine serum albumin as control. All mice received booster infection with acta, IntB-deficient Listeria monocytogenes expressing the H-2K d -restricited influenza epitope, IYSTVASSL (attlm- HA 518 ) (~10 7 cfu/mouse) on day 7 post priming. (D-I) Naïve BALB/c mice, attlm- HA 518 -primed only mice, and prime-boost mice from (B) or (C) were challenged with a lethal dose (~ 5 LD 50 ) of influenza A/PR/8/34 (H1N1) at day 47 following booster immunization. (D, E) Survival curve after influenza challenge. Numbers on the graph indicated # survival/total #. Mortality is defined as death or loss of more than 30% of starting weight per guidelines of The University of Iowa Institutional Animal Care and Use Committee. Logrank test was used for statistical analysis. (F-G) Morbidity is measured by weight loss and expressed as percent of starting weight. Unpaired student t- test was used for statistical analysis between indicated groups (p<0.05 indicated statistical significance between groups). (H-I) Airway resistance was measured using a whole body plethysmograph (Buxco Electronics) and expressed as Penh values. Baseline Penh values for each mouse were recorded before and on the indicated time points following influenza A/PR/8/34 challenge. Unpaired student t-test was used for statistical analysis between indicated groups (p<0.05 indicated statistical significance between groups).

150 133

151 134 Figure 28. PLGA-H5-cross-primed and early boosted mice have reduced viral titer in the lungs. Naïve BALB/c mice, attlm-ha 518 -primed only mice, and PLGA-H5-cross-primed and early attlm-ha 518 -boosted mice were challenged with a lethal dose (~ 5 LD 50 ) of influenza A/PR/8/34 (H1N1). (A) Viral titer from the lung 3 days after influenza challenge (n=4 per group) (* indicated statistical analysis using unpaired student t-test, p<0.05 is considered statistically significant). (B) Correlation between frequency of HA 518 -specific of circulating PBL CD8 T cells immediately prior to viral challenge and viral burden (TCID 50 ) in the lung at 3 days post challenge. Data points represented individual mouse.

152 135

153 136 Figure 29. Cross-priming with avian hemagglutinin H5-adsorbed, biodegradable poly(lactic-co-glycolic) acid microspheres (PLGA) followed by early booster infection rapidly generates protective immunity against different sub-strain of influenza infections. Naïve BALB/c mice were immunized with ~10 9 PLGA microspheres coated with recombinant hemagglutinin H5 protein. Mice received booster infection with acta, IntBdeficient Listeria monocytogenes expressing the H-2K d -restricited influenza epitope, IYSTVASSL (attlm-ha 518 ) (~10 7 cfu/mouse) on day 7 post priming. (A) Frequency of HA 518 -specific CD8 T cells among circulating PBL CD8 T cells at day 7 post priming and day 6 post boosting (day 7+6) (LOD = Limit of detection). On day 7 post boosting (day 7+7), naïve BALB/c mice (n=4) and cross-prime-boosted mice (n=5) were challenged with a lethal dose (~ 5 LD 50 ) of influenza A/PR/8/34 (H1N1). (B) Morbidity is measured by weight loss and expressed as percent of starting weight. Numbers on the graph indicated # survival/ total #. Student t-test was used for statistical analysis (* indicated statistical significance with p <0.04; indicated statistical significance with p <0.002). (C) Airway resistance was measured using a whole body plethysmograph (Buxco Electronics) and expressed as Penh values. Baseline Penh values for each mouse were recorded before and on the indicated time points following influenza A/PR/8/34 challenge. Student t-test was used for statistical analysis (* indicated statistical significance with p <0.04).

154 137