Tracking total polyclonal CD8 T cell responses in inbred and outbred hosts after infection

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1 University of Iowa Iowa Research Online Theses and Dissertations 2010 Tracking total polyclonal CD8 T cell responses in inbred and outbred hosts after infection Deepa Kumari Rai University of Iowa Copyright 2010 Deepa Kumari Rai This dissertation is available at Iowa Research Online: Recommended Citation Rai, Deepa Kumari. "Tracking total polyclonal CD8 T cell responses in inbred and outbred hosts after infection." MS (Master of Science) thesis, University of Iowa, Follow this and additional works at: Part of the Pathology Commons

2 TRACKING TOTAL POLYCLONAL CD8 T CELL RESPONSES IN INBRED AND OUTBRED HOSTS AFTER INFECTION by Deepa Kumari Rai A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Pathology in the Graduate College of The University of Iowa December 2010 Thesis Supervisor: Assistant Professor Vladimir P. Badovinac

3 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL MASTER'S THESIS This is to certify that the Master's thesis of Deepa Kumari Rai has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Pathology at the December 2010 graduation. Thesis Committee: Vladimir P. Badovinac, Thesis Supervisor Jonathan W. Heusel Kevin L. Legge

4 To my dear parents, brothers Basanta, Hemanta and Nripendra, who kept my spirit up with their unconditional love and support for all these years and who believed in me more than myself. ii

5 ACKNOWLEDGMENTS It is my pleasure to thank all of those who made this thesis successful. I would like to sincerely thank my mentor Dr. Vladimir Badovinac for such an outstanding guidance and continuous support throughout my journey in the graduate school. If it were not for his effort and encouragement, I would not have made this far, so I am truly grateful towards Dr. Badovinac. I would like to thank my thesis committee members Dr. Jonathan Heusel and Dr. Kevin Legge for their valuable input and considerable time in this project. I cannot thank Dr. Thomas Waldschmidt enough for giving me such a great opportunity to pursue graduate studies and for his tremendous contribution, valuable advice and support along the way. I would also like to thank present and past lab members Matthew Martin, Shaniya Khan, Harshini Mehta and Jaime Sabel for giving me company in lab. I am equally thankful to Dr. John Harty and all his lab members for their advices, technical support and resources. Last but not the least, I am grateful to all the members of Department of Pathology, Pathology Learning Center and Pathology Flow Cytometry Facility for continuous help and making my endeavor successful. iii

6 TABLE OF CONTENTS LIST OF TABLES... vi LIST OF FIGURES... vii CHAPTER I. INTODUCTION...1 Ag-specific CD8 T cell responses to acute infection and/or vaccination...1 Memory CD8 T cells...3 Inflammation and the regulation of Ag-specific CD8 T cell responses...5 Detection of Ag-specific CD8 T cell responses...8 Inbred and outbred mice...10 Purpose of study...10 II. CD11a AND CD8α EXPRESSION ON THE SURFACE OF Ag- SPECIFIC EFFECTOR AND MEMORY CD8 T CELLS AFTER INFECTION...14 Introduction...14 Materials and Methods...15 Mice...15 Bacteria and virus infections...15 Peptide-DC immunization...16 Adoptive Transfer Experiments...16 Isolation of lymphocytes from tissues...17 Antibodies and Tetramers...17 Quantification of CD8 T cell responses...18 Statistical analysis...18 Results...19 Changes in the expression of CD11a and CD8α can distinguish naïve from Ag-experienced effector and memory CD8 T cells...19 Changes in the expression of CD11a and CD8α were similar on TCR-tg and on endogenous Ag-specific CD8 T cells after infection...21 Changes in CD11a and CD8α expression on CD8 T cells are controlled by Ag and not by inflammation...23 Functional memory CD8 T cells express high levels of CD11a and low levels of CD8α...25 Changes in CD11a and CD8α expression on CD8 T cells can track Ag-specific effector and memory CD8 T cells in various tissues after LM infection...27 Changes in the expression patterns of CD11a and CD8α can be used to follow Ag-specific CD8 T cell responses after various types of infection...28 Discussion...29 III. MAGNITUDE AND KINETICS OF POLYCLONAL CD8 T CELL RESPONSES IN INBRED MICE AFTER INFECTION...54 iv

7 Introduction...54 Materials and Methods...55 Mice...55 Bacteria and Virus infections...55 Antibodies and Peptides...56 Intracellular cytokine staining...56 Quantification and phenotypic analysis of Ag-specific CD8 T cells...57 Statistical analysis...57 Results and Discussion...57 The magnitude of polyclonal CD8 T cell responses after infection in inbred strains of mice...57 Enumeration of CD8 T cells with defined Ag-specificity among all CD11a high CD8α low CD8 T cells after LM infection...59 The kinetics of newly evoked primary polyclonal CD8 T cell responses can be followed in immune (non-naïve) mice...61 Summary...62 IV. MAGNITUDE AND KINETICS OF POLYCLONAL CD8 T CELL RESPONSES IN OUTBRED MICE AFTER INFECTION...74 Introduction...74 Materials and Methods...75 Mice...75 Bacteria and Virus infections...75 Analysis of bacterial titer after infection...76 Antibodies...76 Quantification and phenotypic analysis of Ag-specific CD8 T cells...76 Statistical analysis...77 Results and Discussion...77 Substantial variability in the magnitude and kinetics of polyclonal CD8 T cell responses after bacterial infection in outbred mice...77 The levels of protection to bacterial re-challenge correlates with the number of CD11a high and CD8α low CD8 T cells...79 Substantial variability in the magnitude and kinetics of CD8 T cell responses after viral infection in outbred mice...80 Summary...81 V. CONCULSIONS AND FUTURE DIRECTIONS...99 Conclusions...99 Future Directions REFERENCES v

8 LIST OF TABLES Table 1. Phenotype of naïve and Ag-specific effector and memory CD8 T cells after infection or vaccination...12 vi

9 LIST OF FIGURES Figure 1. Phenotypic changes on Ag-specific effector CD8 T cells after bacterial infection Increased expression of CD11a and diminished expression of CD8α can distinguish naïve from Ag-experienced memory CD8 T cells Changes in the expression of CD11a and CD8α on OT-I (TCR-tg) and on Ova-specific endogenous CD8 T cells are indistinguishable after infection Changes in the expression of CD11a and CD8α on CD8 T cells are Agdependent Inflammation without Ag does not influence CD11a upregulation and CD8α downregulation on Ag-specific CD8 T cells Immunization under low inflammatory condition induces CD11a upregulation and CD8α downregulation on Ag-specific CD8 T cells Experimental design to determine whether Ag-specific functional memory CD8 T cells can be defined by high CD11a and low CD8α expression High expression of CD11a and low expression of CD8α on CD8 T cells can be used to identify and enrich functional memory CD8 T cells Ag-specific effector and memory CD8 T cells can be tracked by CD11a high and CD8α low expression in various tissues after Listeria infection The levels of expression of CD11a and CD8α can be used to distinguish naïve from Ag-specific effector and memory CD8 T cells in various lymphoid and non-lymphoid tissues after bacterial infection Changes in the expression of CD11a and CD8α can be used to define Agspecific memory CD8 T cell responses after various types of infection Quantifying the magnitude of polyclonal CD8 T cell expansion after bacterial or viral infection in inbred strains of mice based on changes in the expression of CD11a and CD8α Magnitude of Ag-specific CD8 T cell responses varies with the strains of inbred mice as determined by changes in the expression of CD11a and CD8α Contribution of CD8 T cells with defined Ag-specificity among all CD11a high CD8α low CD8 T cells after LM infection in BALB/c mice Characterization of CD11a high CD8α low and CD11a low CD8α high CD8 T cells after LM infection in B6 mice CD11a and CD8α can be used as marker to distinguish Ag-specific CD8 T cells after heterologous challenge in immune mice...72 vii

10 17. Quantifying the magnitude of polyclonal CD8 T cell responses after bacterial infection in outbred mice based on changes in the expression of CD11a and CD8α Characterization of CD11a high CD8α low and CD11a low CD8α high CD8 T cells after bacterial infection in outbred mice Substantial variability in the magnitude of primary LM specific CD8 T cell responses in outbred mice Magnitude of primary LM specific CD8 T cell responses greatly varies within outbred mice and can be determined by the expression of CD11a and CD8α Substantial variability in the kinetics of primary LM-specific CD8 T cell responses in outbred mice The degree of protection to high dose LM re-challenge depends on the number of CD11a high CD8α low CD8 T cells in outbred mice Substantial variability in the magnitude of primary virus-specific CD8 T cell responses in outbred mice Peak of the primary virus-specific CD8 T cell responses and kinetics vary substantially within individual outbred mice as determined by the changes in the expressions of CD11a and CD8α viii

11 1 CHAPTER I INTRODUCTION Ag-specific CD8 T cell responses to acute infection and/or vaccination Cytotoxic antigen (Ag)-specific CD8 T cells are the main components of the adaptive immune response and they are critical in defense against intracellular pathogens as well as in defense against malignancy. Due to diverse repertoire of naïve CD8 T cell precursors the adaptive immune system is able to recognize a wide range of foreign, pathogen-derived Ags. It has been estimated that an adult and non-infected mouse contains ~ 2-4x10 7 naïve CD8 T cells, with a total clonal diversity of at least ~ 2x10 6 unique T cell receptors (TCRs) that ensures exquisitely specific CD8 T cell responses against any given microbial pathogens [1]. There are four distinct phases of CD8 T cell response to infection: activation, proliferation, contraction and memory CD8 T cell generation. Activation (phase I) is initiated by the interaction between mature Ag-presenting MHC class I positive dendritic cells (DCs) and naïve Ag-specific CD8 T cells [2-9]. The number of naïve precursor CD8 T cells for any given epitope specificity is estimated at a very low number (100 to 1000 cells) [1, 10, 11]. These few naïve CD8 T cells circulate between the blood and the secondary lymphoid tissues continuously surveying the Ags (signal 1) displayed by mature DCs [12]. To promote full activation of Ag-specific CD8 T cells, co-stimulatory signals (signal 2) mediated by B7-1 (CD80) and B7-2 (CD86) expressed on mature DC surface are required without which CD8 T cells become anergic [12]. In addition, inflammation associated cytokines (signal 3) are involved to amplify and fine-tune pathogen-specific CD8 T cell responses [6]. However, as several studies collectively report, a brief encounter of naïve CD8 T cells with Ag is sufficient for activating and initiating of full CD8 T cell developmental program [13-17]. Thus, continued display of

12 2 Ag is not required for the ensuing expansion and memory differentiation [13-16]. However, more recent studies suggest that prolonged antigenic stimulation is required for the optimal clonal expansion and memory T cells formation [18, 19]. After the process of activation, expansion (phase II) of Ag-specific CD8 T cells takes place where these cells undergo differentiation process. Depending on types and doses of infection, activated CD8 T cells proliferate in numbers, each precursor dividing more than 13 times, to give rise to effector CD8 T cells [4, 20]. The expansion phase lasts for 5-12 days and at the peak of expansion phase Ag-specific effector CD8 T cells accumulate at > 10,000 fold higher numbers when compared to the initial naïve CD8 T cell precursor numbers [4, 20]. Clonal expansion of Ag-specific CD8 T cells is associated with phenotypic changes and gain in effector function [20, 21]. Effector CD8 T cells downregulate the expression of CD62L and CCR7 to leave secondary lymphoid tissues and migrate throughout the body to defend against pathogen invasion in peripheral tissues [21-23]. These cells acquire antimicrobial functions such as cytolysis (production of cytotoxic molecules granzyme B and perforin) and the ability to rapidly produce cytokines (IL-2, IFN-γ and TNF-α) [4, 20]. Whether or not infection is terminated from the host, effector CD8 T cell population, at the peak of the response, undergoes massive contraction (phase III) in number. It is also referred to as death phase, which eliminates 90-95% of the pathogen (Ag)-specific CD8 T cells. What drives the death of a majority of these effector cells is still a matter of investigations [3, 4]. However, it is demonstrated that the onset and degree of contraction are independent of dose, duration of infection, and magnitude of Ag-specific CD8 T cell expansion, suggesting that early events after infection potentially program contraction phase [24]. Finally, the extent of cell death also determines the size of memory CD8 T cell population [4].

13 3 The development of immunological memory is the central goal of immune responses. Thus, contraction phase is followed by generation of memory CD8 T cells (phase IV). It has been estimated that 5-10% of effector CD8 T cells that survive the contraction phase become memory CD8 T cells [3, 4]. Importantly, the number of memory cells always exceeds the number of naïve CD8 T cell precursors for a given specificity [6]. Memory CD8 T cells can respond to the previously encountered pathogen rapidly and more efficiently and provide improved protection to the host by elevating effector functions (cytotoxicity and cytokine production) [4, 20]. Consequently, these defense mechanisms dramatically decrease the duration of subsequent infection thereby decreasing the spread of pathogens [4, 20]. These pathogen-specific memory CD8 T cells survive in all tissues [22]. Interestingly, the number of memory CD8 T cells that remain after contraction is maintained for the life of the host through a self-renewal process [4]. Infection with lymphocytic choriomeningitis virus (LCMV) has shown to generate memory CD8 T cell pool stable for at least 900 days in mice [25]. Longevity studies in human populations showed that virus-specific memory T cells were detectable up to 75 years after smallpox vaccination [26]. It has been suggested that CD8 T cell memory maintenance is a dynamic process of balanced proliferation and cell death exhibited by homeostatic cytokines IL-7 and IL-15 [27]. However, defining when CD8 T cell memory is established after infection and/or vaccination remains a complicated issue. Memory CD8 T cells Despite much effort focused in characterizing memory CD8 T cells, there still exists a list of unanswered questions. Due to the phenotypic and functional heterogeneity, no single property studied to date can be used to unequivocally identify and define memory CD8 T cells. However, characterization of responding CD8 T cells

14 4 after infection based on phenotypic and functional changes associated with the progression of naïve to memory CD8 T cells have proved to be of great importance. The phenotype of CD8 T cells at naïve, effector and early memory phases based on the expression of well defined surface molecules are summarized in Table 1. At the peak of the expansion after infection, the majority of effector CD8 T cells exhibit phenotypes and functions distinct to that of naïve cells. The recently activated effector cells downmodulate the expression of CD127 (IL-7Rα), CD62L (lymphoid homing molecule), CD27 (co-stimulatory molecule) and upregulate the expressions of CD43 (as detected by mab 1B11), CD11a (a-chain of LFA-1), CD44, KLRG-1 (killer cell lectin-like receptor subfamily G member 1) and granzyme B [3, 21, 28-30]. These phenotypic changes are accompanied by cytokine productions such as interferon-γ (IFN-γ) and tumor necrosis factors (TNFs) as measured by in vitro peptide stimulation assays [3, 21, 28-30]. Importantly, the phenotype of long-term memory CD8 T cells is similar to naïve nonactivated CD8 T cells (as memory CD8 T cells regain the expression of some markers such as CD127 and CD62L [20]) making the memory CD8 T cell characterization on the basis of surface molecule expression more difficult and incomplete. Additional criteria were set to define memory CD8 T cells based on their behavior after re-stimulation. Memory CD8 T cells can undergo vigorous secondary expansion and differentiation in response to pathogens re-challenge or booster immunization resulting in the generation of increased number of secondary memory cells [3, 20]. In addition, the ability to provide increased protection on per-cell basis is another parameter used to distinguish memory from naïve CD8 T cells [31]. When functional memory CD8 T cells develop after infection is still under debate, however, it has been suggested that in B6 mice at least 40 days are required for those cells to acquire memory CD8 T cell qualities [32]. Therefore, the definition of memory CD8 T cells is based solely on functional attributes seemed incomplete as well.

15 5 In the light of several studies, it has been suggested that memory CD8 T cells can be divided into at least two broad lineages based on phenotype, function and location. The earlier studies of memory T cells from human blood [33] describe central memory T cells (Tcm) and effector memory T cells (Tem) characterized by distinct homing capacity and effector function [33]. Tcm express lymphoid homing receptors, CCR7 or CD62L, re-circulate preferentially through secondary lymphoid tissues and lack immediate cytolytic effector function upon reactivation [33]. In contrast, Tem lack lymphoid homing receptors, re-circulate preferentially through nonlymphoid tissues and provide immediate cytolytic function upon re-exposure to the Ag by producing cytotoxic molecules such as granzyme B and perforin [33]. In addition, CCR7 high CD62L high Tcm subsets are able to better persist in hosts and are more efficient in mediating protection due to their increased proliferative potential and higher production of IL-2 compared to CCR7 low CD62L low Tem subsets [34]. Thus, CD62L has been a frequently used marker to define central memory CD8 T cells. However, a recent study from Wirth et al. showed that effector CD8 T cells expressing a non-cleavable mutant form of CD62L gave rise to both effector and central memory CD8 T cells [35]. In addition, knock down of CD62L expression did not preferentially prevent differentiation of effector CD8 T cells into Tcm memory populations [35]. These data suggest that the diversity of memory CD8 T cells may not be limited to just two lineages and that further studies will be required to define the extent to which memory CD8 T cell populations are diverse. Inflammation and the regulation of Ag-specific CD8 T cell responses In order to combat infection efficiently, Ag-specific CD8 T cells are required to expand in numbers substantially. Inflammation plays an important role in regulating the CD8 T cell response against pathogens [6]. Reports from number of studies suggest that

16 6 early after infection, pro-inflammatory cytokines elicit an immediate effect on responding CD8 T cells [6, 12]. Inflammatory cytokines are produced as a result of recognition of pathogen-derived peptides via microbial pathogen recognition receptors present on DCs [12]. These inflammatory cytokines required for the expansion and development of effector and memory generation are called signal 3 [8, 36, 37]. To date, interleukin-12 (IL-12), type I interferons (IFN-αβ), type II interferon (IFN-γ), and IL-21 have been identified as signal 3 molecules [6, 8, 36, 38]. Studies from Schmidt et al. demonstrated using adoptive transfer of TCR-transgenic (TCR-tg) CD8 T cells into IL-12Rβ1-deficient host that in response to peptide immunization and/or infection, IL-12 directly acts on responding CD8 T cells to optimally activate differentiation of effector cells, promote clonal expansion and survival in vitro and in vivo [39]. The enhanced survival by IL-12 is probably atributed to upregulation of antiapoptotic protein Bcl-3 or inhibition of proapoptotic enzyme caspase-3 [40, 41]. Consistently, TCR-Tg CD8 T cells lacking both IL-12 and IFN-αβ receptors have shown reduced expansion and differentiation in comparison to wild type cells after LM infection [6, 8]. Similarly, IFN-γ is shown to act directly on CD8 T cells to promote their expansion after LCMV infection [42]. Importantly, depending on the type of infection various cytokines can act as signal 3 in vivo. For instance, IFN-αβR deficient OT-I cells exhibited only a modest or no defect in expansion and survival after infections with vaccinia virus (VacV), vesicular stomatitis virus (VSV), or LM but a severe defect was observed after LCMV infection [43]. IFN-γ was shown to be dispensable for CD8 T cell expansion after LM infection [44]. On the other hand, large amounts of IFN-αβ but not IL-12 are produced after LCMV infection [45]. Collectively, these findings suggest that the requirement for signal 3 is dependent on the pathogen. Thus, a unique pattern of inflammation provides essential signals for optimal CD8 T cell responses.

17 7 Moreover, the timing and the level of exposure to inflammatory cytokine signaling also influence the response of CD8 T cells to infection. Continuous exposure to IL-12 from the onset of CD8 T cell activation facilitates optimal expansion and survival of responding CD8 T cells. However, 40 hours after CD8 T cell activation, addition of IL-12 still provides improved cytolytic effector function but further delay in IL-12 treatment substantially decreased the lytic function of effector CD8 T cells [46]. Modest downregulation of IFN-αβR mrna is induced early after infection but re-expressed by day 3 after infection whereas IFN-γ is required very early after infection [47]. Once Ag-specific CD8 T cells reach the peak of the response, the contraction phase is initiated [6]. Several groups have reported that the onset of contraction of the CD8 T cell response is independent of the magnitude of expansion, dose and duration of the infection, suggesting that CD8 T cell contraction is also programmed early after T cell activation [17, 48]. IFN-γ has been shown to play a critical role for the contraction of effector CD8 T cells [44]. BALB/c mice deficient in IFN-γ showed substantial reduction in Ag-specific CD8 T cell contraction after LM or LCMV infection [24, 44]. Furthermore, pretreatment of wild type mice with antibiotic prior to LM infection showed substantial reduction in IFN-γ and subsequent reduction in contraction of Ag-specific CD8 T cells [24]. However, CpG ODN [49] injection in antibiotic-pretreated mice restored contraction by inducing inflammatory cytokines (including IFN-γ). Therefore, inflammatory signals appear to play an important role in CD8 T cell homeostasis by ensuing an appropriate contraction of Ag-specific CD8 T cells. Even though signal 3 is important for optimal CD8 T cell expansion and contraction, its requirement for generation or maintenance of memory CD8 T cell populations is still under investigation. Importantly, the extent and duration of inflammation during the early phases of infection influence the rate of memory CD8 T cell generation [50, 51]. A decreased amount of pro-inflammatory cytokines during infection correlated with accelerated generation of memory CD8 T cells [52]. CpG ODN

18 8 injection in the antibiotic treated mice restored inflammation, and also prevented rapid generation of memory CD8 T cells [53]. Consistently rapid differentiation of Ag-specific naïve CD8 T cells to memory was observed after immunization of mice with in vitromatured peptide-loaded DCs [54]. Thus, inflammation plays an important role in regulating all phases of Ag-specific CD8 T cell responses to infection and/or vaccination. Considering the influence of inflammation on Ag-specific CD8 T cell homeostasis following infection, it is highly possible that inflammation could modulate some of the phenotypic characteristics of Ag-non-specific CD8 T cells as well. The extent to which inflammation in the absence of Ags can influence the phenotype of CD8 T cells in vivo will be addressed in detail in the experimental section of this thesis. Detection of Ag-specific CD8 T cell responses A series of recent technological developments have greatly improved detection of Ag-specific CD8 T cell responses in vivo both in humans as well as in various murine models of infection and vaccination. The newly introduced techniques have not only enabled a direct visualization and quantification of Ag-specific CD8 T cells, but also the characterization and functional evaluation of these cells. Several methods of detection have been described which are based on functional (indirect cytokine production) and phenotypic (direct TCR expression) assessment of Ag-specific CD8 T cells [55]. One of the indirect methods currently in use is Enzyme-linked immunospot (ELISPOT) assay. It enumerates Ag-specific CD8 T cells based on IFN-γ production after hours of in vitro stimulation with specific peptide [56]. Intracellular cytokine staining (ICS) is a similar assay in which Ag-specific CD8 T cells were detected during short ex vivo (5-6 hours) incubation in the presence of specific peptide according to their ability to produce cytokines (e.g., IFN-γ and TNF). The advantage of ICS is that cells can be simultaneously stained with antibodies to multiple cytokines, surface markers and

19 9 intracellular proteins before flow cytometry analysis. However this assay is not as sensitive as ELISPOT [57]. In addition, not all Ag-specific CD8 T cells will produce cytokine upon peptide exposure [58] suggesting that total number of CD8 T cells responding to infection/vaccination is higher than detected even in the situation when the Ag-specificity of those cells is known. A novel approach that enabled detection of Ag-specific CD8 T cells based on phenotype is a peptide-major histocompatibility (MHC) class I tetramer (or multimers) staining [59]. This is a direct ex vivo method of CD8 T cell analysis where Ag-specific T cells are assayed on the basis of a structural component such as TCR [55]. CD8 T cells containing TCR that can specifically bind to peptide MHC tetramer can be visualized and characterized by flow cytometry. As with indirect methods, quantifying CD8 T cell responses with this approach requires prior knowledge of the antigenic peptides (epitopes or Ag) together with their restricting MHC Class I allele. Tetramers might also lack the sensitivity when CD8 T cell responses to subdominant epitopes are analyzed and when expression of TCR is downmodulated due to recent Ag-encounter [60, 61]. Due to these reasons, tetramers may not accurately detect all Ag-specific CD8 T cells even in the situation when epitopes are defined. Nonetheless, the introduction of these strategies has dramatically enhanced our ability to evaluate Ag-specific CD8 T cell responses and provided a better understanding of the immune system, all techniques have their own limitations. Thus, our project is aimed at finding an alternative approach for detecting Ag-specific CD8 T cells that could overcome all of the technical limitations and facilitate efficient analysis of all Ag-specific CD8 T cells at various phases of the response after infection.

20 10 Inbred and outbred mice Most of the data related to Ag-specific CD8 T cell responses published to date were obtained from inbred strains of mice. The advantage of using genetically identical siblings that are maintained through brother and sister mating is obvious since unlimited numbers of identical mice are available and the reproducibility of the experimental findings (inside a lab and among different labs) is ensured. On the other hand, while the differences (e.g., in susceptibility or resistance to infection and predominance of Th1 or Th2 cytokine phenotypes) [62-68] observed among the different inbred strains of mice has served as a tool for exploring the mechanisms that control particular biological processes, it is also a reminder that the genetic background of the mice can influence the outcome of the experiments. Therefore, establishing a mouse model in which Agexperienced CD8 T cells of unknown antigen specificity can be analyzed independently of the MHC class I background should allow the analysis of adaptive CD8 T cell immunity in outbred populations [69] which is the main goal of our study. Successful establishment of this model would provide us with a framework through which several critical aspects of anti-bacterial and/or viral specific CD8 T cell responses could be addressed in a genetically diverse outbred population like humans. Purpose of study For the past several decades, continuous effort has been devoted into the study of the adaptive immune system to understand how productive Ag (pathogen)-specific CD8 T cell responses are regulated and maintained [3, 4, 6, 20, 70]. Consequently, a vast amount of information has been gathered that has a potential to contribute tremendously in the development of vaccines against many life threatening human diseases. Since substantial variability in person-to-person immune responses are observed effective translation of findings obtained in tightly controlled laboratory research (mostly

21 11 performed in genetically identical inbred strains of mice) into clinical practice still remains a challenge. Therefore, evaluation of the experimental outcomes generated from studies with inbred animals to outbred populations would largely accelerate the benchto-bedside process. Thus, the main purpose of this project is to define a model where Ag-specific CD8 T cells can be followed in the absence of a priori knowledge of Agspecificity and/or MHC Class I restriction. This includes distinguishing Ag-experienced CD8 T cells from naïve counterparts, followed by detection of the magnitude and kinetics of the total polyclonal Ag-specific CD8 T cell responses to acute infections and/or vaccinations in inbred and outbred mice. The main focuses of this thesis project are as follows: 1. To determine if specific phenotypic changes can distinguish Ag-specific effector and memory CD8 T cells from naïve CD8 T cells after infection and/or vaccination. 2. To quantify total magnitude and analyze kinetics of polyclonal CD8 T cell responses in inbred mice after infection. 3. To analyze the magnitude and kinetics of polyclonal CD8 T cell responses in outbred mice after infection.

22 12 Table 1. Phenotype of naive and Ag-specific effector, and memory CD8 T cells after infection or vaccination.

23 13 In response to infection or vaccination, naïve Ag-specific CD8 T cell precursors undergo an Ag-driven program of expansion, contraction and differentiation into memory CD8 T cells that can be maintained for the life of the host, where naïve to memory CD8 T cell differentiation is accompanied by changes in expression of wide variety of molecules on surfaces of responding cells.

24 14 CHAPTER II CD11A AND CD8α EXPRESSION ON THE SURFACE OF AG-SPECIFIC EFFECTOR AND MEMORY CD8 T CELLS AFTER INFECTION Introduction Activation of naïve Ag-specific CD8 T cells via recognition of pathogen derived Ags displayed by MHC class I molecules on infected cells or on Ag presenting cells (APCs) such as DCs leads to initiation of a program of proliferation and differentiation to generate effector CD8 T cells [3, 4, 6, 7, 12]. A small proportion of the responding effector CD8 T cells live as a memory population and provide a long-term protection against re-infection [3, 20]. This program induces changes in phenotype as well as a gain in function of the responding CD8 T cells [4, 14]. Various technical advances such as peptide-mhc class I tetramer (multimer) and peptide-induced cytokine secretion (ICS and ELISPOT assays) have enabled analysis of magnitude and kinetics of Ag-specific CD8 T cells in response to infection. However, a prior knowledge of specific epitope(s) and MHC class I molecule(s) of CD8 T cells that is required by these techniques limit a potential to characterize all of the CD8 T cells responding to pathogen-derived Ags. Nevertheless, several studies have described CD8 T cells before and after infections as naïve, effector, and memory CD8 T cells based on differential expression of various surface molecules [20, 21, 28-30]. This information suggests an exciting possibility that all CD8 T cells that respond to infection might exhibit a similar phenotypic footprint that will distinguish them from naïve (nonresponding) CD8 T cells. In addition, several studies [71, 72] have suggested that infection-induced inflammation (inflammatory cytokines) might influence and induce phenotypic changes on non-ag-specific CD8 T cells (bystander activation). The contribution, if any, of

25 15 bystander activation on CD8 T cell phenotype in the presence and/or absence of Ag will be also addressed in this chapter. Materials and Methods Mice C57BL/6 (B6; Thy1.2) mice were obtained from National Cancer Institute (NCI; Frederick, MD). B6 Thy1.1 mice were obtained from the Jackson Laboratory (JAX). OT-I (Ova257-specific) and P14 (GP33-specific) transgenic Thy1.1 mice were previously described [73, 74]. Pathogen-infected mice were housed at appropriate bio-safety conditions. Mice were used at 4-6 weeks of age or older. Experiments were performed under Institutional Animal Care and Use Committee (ACURF) approved by the University of Iowa (Iowa City, IA). Bacteria and virus infections The attenuated (acta-deficient) Listeria monocytogenes strains DP-L1942 (Att LM) [75], attenuated LM expressing Ova257 (Att LM-Ova) [76] and attenuated LM expressing GP33 (Att LM-GP33) [74] were resistant to streptomycin and were grown, injected (5 x 10 6 CFU/mouse; i.v.) and quantified, as described [77]. The Armstrong strain of Lymphocytic choriomeningitis virus (LCMV-Arm; 2 x 10 5 PFU/mouse; i.p.) and vaccinia virus expressing GP33 (VacV-GP33; 3 x 10 6 PFU/mouse; i.p.) were used as described [48, 78].

26 16 Peptide-DC immunization Splenic DCs were isolated after subcutaneous injection of B6 mice with 5 x 10 6 B16 cells expressing Flt3L (provided by M. Prlic and M. Bevan, University of Washington, Seattle, WA). When tumors were palpable (5 mm x 5 mm), mice were injected with 2 µg LPS (Sigma-Aldrich) i.v. to mature the DC. Spleens were harvested 16 hours later and were digested with DNase and Collagenase for 20 min at 27 C/7% CO 2 with shaking (120 RPM). Spleen pieces were forced through a nylon cell strainer (70 µm) to generate a single cell suspension, RBC were lysed and splenocytes were resuspended in two parts of 10% FCS RPMI 1640 to one part B16-Flt3L condition medium plus recombinant GM-CSF (1000 µ/ml) plus 2 µm Ova and incubated 2 hours at 27 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. DCs were resuspended in saline and injected ~ 1x10 6 DC per mouse intravenously. Adoptive transfer Experiments Naïve Thy1.1 OT-I or P14 CD8 T cells were obtained from the peripheral blood of naïve TCR-transgenic (TCR-Tg) donors as previously described [79]. Approximately, 500 Thy1.1 OT-I cells were transferred into naïve B6 Thy1.2 recipient mouse. One day later, these mice were infected with indicated strains of LM i.v. or treated with CpG oligonucleotide 1826 (Coley Pharmaceutical Group) (~ 50 µg/mouse; i.p.) or poly(i:c) (~ 200 µg/mouse; i.p.) i.p.. Similarly, ~ 1,000 P14 cells were transferred into naïve B6 Thy1.2 mouse. One day later, these mice were injected with Att LM-GP-33 i.v., LCMV- Arm i.p., or VacV-GP33 i.p.

27 17 For adoptive transfer of memory CD8 T cells, LM immune Thy1.2 mice containing memory CD8 T cells were euthanized at memory time points. Naïve and memory CD8 T cells from spleens were FACS sorted according to their CD11a and CD8α expression. At the same time naïve CD8 T cells were also sorted from naive (noninfected) mice. Sorted populations (naïve or memory) of Thy1.2 CD8 T cells were transferred into naïve Thy1.1 mice. Recipient mice were infected one day later with by Att LM (DP-L1942; ~ 5x10 6 CFU/mouse; i.v.). Isolation of lymphocytes from tissues Samples of blood were obtained by retroorbital puncture at indicated days from Thy1.2 B6 mice that had received a low number of OT-I Thy1.1 cells prior to LM-Ova immunization. Anesthetized mice were then perfused through the left ventricle of the heart with PBS prior to harvesting the organs. Organs were forced through metal strainer with a syringe plunger and the suspension filtered to obtain a single cell suspension from liver, lung, spleen and inguinal lymph nodes. The lymphocytes were washed twice before Thy1.1, CD8 and CD11a staining for FACS analysis. Antibodies and tetramers Monoclonal antibodies of the indicated specificity with appropriate combinations of fluorochromes were used. The following antibodies were from ebioscience (San Diego, CA): CD8 (53-6.7), Thy1.1 (OX-7), CD127 (A7R34), CD62L (MEL-14), CD44 (Pgp-1), CD11a (M17/4), CD27 (LG.7F9), isotype control rat IgG2a (ebr2a), rat IgG2b (KLH/G2b-1-2), and rat IgG1 (ebrg1). The following antibodies are from BD Biosciences (San Diego, CA): CD8 (53-6.7), Thy1.2 (53-2.1) and (CD43 (1B11). MHC

28 18 class I tetramers (K b ) specific for Ova epitope were prepared using published protocols [44, 59, 80] and used as described previously [54]. Quantification of CD8 T cell responses For the quantification of CD8 T cells responding to infection, ~20 µl of blood were obtained from each mouse via retro-orbital bleeding or tail-tip snips at indicated days after infection. For some experiments, spleens were harvested to obtain splenocytes. Spleens were mashed through a metal mesh within a small Petri dish and were filtered through the funnel to collect single cell suspension. Total number of polyclonal CD8 T cells responding to infection or immunization was determined by costaining of CD8α and CD11a and analyzed by FACS. The endogenous Ag-specific CD8 T cells were determined by allophycocyanin (APC)-conjugated tetramer staining as described [54]. TCR-Tg CD8 T cells were deteremined by Thy1 cell surface staining as described [79]. The number of polyclonal endogenous CD8 T cells, Ag-specific CD8 T cells or TCR-Tg CD8 T cells or Ag-specific CD8 T cells was presented as frequency of total PBL or as a frequency of CD8 + T cells. Statistical analysis Statistical significance was assessed using the two-tailed t-test with a confidence interval of > 95%.

29 19 Results Changes in the expression of CD11a and CD8α can distinguish naïve from Ag-experienced effector and memory CD8 T cells Naive CD8 T cells that recognize and respond to pathogen-derived Ag undergo robust proliferation and differentiation, giving rise to Ag-specific effector populations [20]. Once the clonal expansion of effector cells reach their peak (day 5-12 depending on the type of infection), pathogen-specific CD8 T cells decline in number where ~ 90-95% die via apoptosis, leaving behind a small proportion of long-lived memory CD8 T cells [20]. Concurrent with naïve to effector to memory progression, the responding Agspecific CD8 T cells simultaneously modulate the expression of numerous surface molecules [20]. However, reliable phenotypic markers that can distinguish Agexperienced effector and memory CD8 T cells from naïve non-responding CD8 T cells remain to be identified. Thus, initial experiments of this project were aimed at distinguishing Ag-specific CD8 T cells from naïve populations based on phenotypic changes after various types of infection and/or vaccination without a priori knowledge of their Ag-specificity. To test whether phenotypic changes can be used to distinguish recently activated Ag-specific CD8 T cells from naïve counterparts early after infection (effector phase), naïve B6 mice were infected with a sub-lethal dose (0.1LD 50 ) of recombinant strain of attenuated (act A-deficient) Listeria monocytogenes expressing Ova257 epitope (Att LM- Ova) [54, 76, 81]. At day 7 post infection, Ova257-specific CD8 T cells were analyzed in blood using Ova257-specific MHC class I tetramers (K b Ova) (Fig.1A). CD8 T cells were stained with cell surface molecules including CD8α, CD127, CD62L, CD27, CD44, CD43 and CD11a (Fig 1C) and the expressions of these molecules analyzed on Ova257- specific (CD8 + /K b Ova + ) and non-specific (CD8 + /K b Ova - ) CD8 T cells from the same

30 20 infected mice (Fig. 1B-C). CD8 T cells from age and sex matched non-infected naïve mice were used as controls (Fig. 1B-C). The expression of CD127 and CD62L on Ova257-specific CD8 T cells from infected mice were dramatically reduced whereas moderate downregulation of CD27 expression was observed when compared to CD8 T cells from naïve mice at day 7 post infection (Fig. 1C). The expression of CD44, CD43 (detected by 1B11 mab; [81]) and CD11a were substantially increased in Ova257- specific CD8 T cells (Fig. 1C). In addition, all the Ova257-specific CD8 T cells from infected mice showed downregulation of CD8α expression on their surface (Fig. 1B). Interestingly, ~ 20% of the Ova257 non-specific CD8 T cells from infected mice also showed similar patterns of CD127, CD62L and CD27 downregulation and CD44, CD43 and CD11a upregulation as shown by Ova257-specific CD8 T cells (Fig. 1C). Similarly, downregulation of CD8α expression was also observed on some of the CD8 + /K b Ova - T cells (Fig. 1B). Thus, evaluation of changes in the expression patterns of the indicated surface molecules on CD8 + /K b Ova + and CD8 + /K b Ova - T cells suggests that the total number of CD8 T cells responding to LM infection is substantially higher than the number of Ova257-specific CD8 T cells. Moreover, these data suggest that the level of CD8α expression together with changes in the expression of specific cell surface molecules (such as CD127, CD62L, CD44, CD43 and CD11a) can be used to distinguish and enumerate the total number of Ag-specific effector CD8 T cells. It remains unknown whether Ag-specific CD8 T cells retain any of these phenotypic changes (described in Figure 1C) as they progress to memory. Thus, to find a surface marker that can identify Ag-experienced CD8 T cells at any time points after infection (effector and memory phases of the response), K b Ova + CD8 T cells were analyzed at day 50 (memory time point) post infection in the blood using K b Ova + tetramer and various markers used in Fig. 1C (Fig. 2A-B). As previously shown in various infection models [4, 20, 21, 28-30, 82], most of the K b Ova + memory CD8 T cells

31 21 upregulated the expression of CD127 and downregulated the expression of CD43, whereas some but not all of the K b Ova + memory CD8 T cells upregulated the expression of CD62L (Fig. 2C). Importantly, all K b Ova + memory CD8 T cells remained CD44 and CD11a high (Fig. 2C). The elevated expression of CD11a was also observed on some (~ 15%) K b Ova - CD8 T cells (Fig. 2C), suggesting the possibility that CD11a can identify the total Ag-experienced CD8 T cells and might be used to track these cells at any time point after infection. The above results suggest that CD44 and CD11a can be used as markers to enumerate total number of Ag-specific CD8 T cell responding to infection. However, since a relatively high frequency of CD8 T cells from naïve mice were CD44 high (Fig. 1C), changes in CD11a surface expression will be used as a potential marker to identify Ag-experienced CD8 T cells after infection. Additionally, downregulated expression of CD8α was also sustained on K b Ova + memory CD8 T cells as they progress to memory phase (Fig. 2C). Thus, CD8α provides us with additional marker that can be used in conjunction with CD11a to distinguish naïve from Ag-specific effector and memory CD8 T cells. Taken together, data presented in Figures 1 and 2 suggest that changes in the expression of CD11a and CD8α can be used to distinguish total CD8 T cells responding to infection at effector as well as memory phases without a priori knowledge of their Agspecificity. Changes in expression of CD11a and CD8α were similar on TCR-tg and on endogenous Ag-specific CD8 T cells after infection Adoptive transfer of CD8 T cells from T cell receptor transgenic (TCR-Tg) mice into congenic recipients has been an extremely powerful tool for monitoring Ag-specific

32 22 CD8 T cell responses in vivo [3, 52, 83, 84]. In this approach, adoptively transferred defined epitope specific T cells can be tracked into recipient host based on allelic polymorphisms of surface markers like CD90 (Thy1.1) or CD45 [79]. This system allows comparison of wild type or knockout TCR-tg CD8 T cells responding to the same infection in the same host, thus allowing the manipulations of experiments in a controlled manner. Recent studies from several labs including our lab suggest that a physiologic number of TCR-Tg CD8 T cells is required when seeding the recipient to accurately mimic the endogenous CD8 T cell responses of the same specificity after infection [79, 84]. Because the central idea of our study is to reliably differentiate naïve from Agspecific CD8 T cells based on the altered expression of surface molecules, the approach of adoptive transfer of TCR-Tg CD8 T cells is of great importance. This method enables phenotypic analysis of CD8 T cells of known Ag-specificity before and after infection allowing the evaluation of any change (Ag-dependent or independent) associated with infection. Thus, to determine if modulation of CD11a and CD8α expression also occurs in transferred TCR-Tg CD8 T cells, a low number of naïve Thy1.1 OT-I TCR-Tg CD8 T cells (~ 500/mouse) was transferred into naïve B6 Thy1.2 mice. One day after adoptive transfer, the recipient mice were infected with 1x10 7 CFU/mouse of Att LM-Ova (Fig. 3A). At day 7 post infection, the expansion of K b Ova + CD8 T cells were analyzed in the blood of infected mice using tetramer and Thy1 staining and compared with naïve CD8 T cells (Fig. 3B). Increased expression of CD11a and decreased expression of CD8α was observed in ~ 40% of endogenous Ova-non specific CD8 T cells in infected mice in contrast to only ~ 2% of CD8 T cells from naïve (non-infected) mice (Fig. 3B). Interestlingly, >98% of Ova-specific CD8 T cells (both endogenous and TCR-Tg) upregulated CD11a expression and downregulated CD8α expression on their surface suggesting that TCR-Tg CD8 T cells undergo similar phenotypic changes as endogenous CD8 T cells specific for the same epitope. Thus, the approach of adoptive transfer

33 23 system can be utilized to test various aspects of CD8 T cell biology and will be used to further validate whether CD11a and CD8α expression patterns can distinguish naïve from Ag-experienced CD8 T cells. Changes in CD11a and CD8α expression on CD8 T cells are controlled by Ag and not by inflammation Recently, it has been reported that the homeostasis of CD8 T cells, including the rate of memory generation, is influenced by the inflammation present at the time of infection and/or vaccination [3, 6, 85]. It is possible that inflammation (bystander activation) could also influence on the phenotype of CD8 T cells, which could impact our ability to enumerate total number of Ag-specific CD8 T cells responding to the infection/immunization [71, 72]. Moreover, lack of complete identification of antigenic CD8 T cell epitopes against most pathogens, with possible exception of LCMV and vaccinia viruses in B6 mice, further complicates the quantitative analysis of Ag-specific CD8 T cell responses [78, 86-88]. Thus, in order to confirm that CD11a and CD8α can reliably distinguish naïve from Ag-experienced CD8 T cells, it is important to show that increase in CD11a expression and decrease in CD8α expression on Ag-specific CD8 T cells after infection are specific to pathogen-derived Ags and not to inflammation. To address this concern, inflammatory cytokine inducing agents such as CpG or poly(i:c) were used [49, 54, 72]. Naive B6 mice were treated with either ~ 50 µg of CpG (1826) per mouse, or ~ 200 µg of poly(i:c) per mouse (Fig. 4A). Additional group of mice infected with ~5x10 6 CFU of Att LM served as positive controls whereas noninfected mice were used as negative controls (Fig. 4A). At day 6 post infection, blood from all groups was analyzed by co-staining with CD11a and CD8α. ~17% of CD11a high CD8α low CD8 T cells were observed in the blood of mice infected with Att LM (Fig. 4B-C). However, no increase in the frequency of CD11a high CD8α low CD8 T cells

34 24 over naïve controls was observed in the blood (Fig. 4B-C) of mice treated with either CpG or poly(i:c). Thus, inflammation in the absence of Ag stimulation does not influence the changes in expression patterns of CD11a and CD8α on CD8 T cells. To further confirm that inflammation does not play a role in modulating expression of CD11a and CD8α using a known antigenic stimulus, ~ 4x10 5 naïve Thy1.1 TCR-Tg OT-I (Ova257-specific) CD8 T cells were purified from the spleen of naïve Thy1.1 OT-I donors and transferred into groups of naïve B6 Thy1.2 mice. One day later inflammation was induced either in the absence (treatment with CpG, Poly(I:C), or infection with Att LM) or in the presence of Ag (infection with Att LM expressing Ova epitope) (Fig. 5A-B). At day 6 post treatment or infection, analysis was done in the blood and in the spleen by CD8α and Thy1 co-staining. As expected, CpG and poly(i:c) treatments did not increase the frequency of OT-I TCR-Tg Thy1.1 CD8 T cells and did not modulate the expression of CD11a and CD8α (Fig. 5B). Interestingly, substantial increase in the frequency of OT-I TCR-Tg Thy1.1 CD8 T cells was observed in the blood and the spleens of mice infected with Att LM expressing Ova, ~ 99% of which were CD11a high CD8α low (Fig. 5B). In contrast, mice challenged with Att LM that do not express Ova neither showed increase in frequency of OT-I TCR-Tg cells nor modulated the expression of CD11a and CD8α (Fig. 5B). Thus, these data suggest that Ag is required for CD8 T cells to become CD11a high and CD8α low. Finally, the question remained as to whether inflammation in the presence of Ag is required to modulate the expression of CD11a and CD8α on the surface of Ag-specific CD8 T cells. To address this issue, naïve B6 mice were immunized with mature DCs (priming in the low inflammatory environment [6, 85]) coated with Ova257-peptide. At day 7 after DC immunization, Ova-specific CD8 T cells were analyzed in the blood by K b Ova + tetramers and the expression of CD8α and CD11a was determined. Importantly, CD11a upregulation and CD8α downregulation were observed on the surface of K b Ova + CD8 T cells despite the priming in low inflammatory environment (Fig. 6A-B).

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