The Contribution of Homeostatically Expanded Donor CD8 T Cells to Host Reconstitution Following Syngeneic Hematopoietic Cell Transplantation

Transcription

1 University of Miami Scholarly Repository Open Access Dissertations Electronic Theses and Dissertations The Contribution of Homeostatically Expanded Donor CD8 T Cells to Host Reconstitution Following Syngeneic Hematopoietic Cell Transplantation Melinda Roskos Keith University of Miami, melinda.roskos@gmail.com Follow this and additional works at: Recommended Citation Keith, Melinda Roskos, "The Contribution of Homeostatically Expanded Donor CD8 T Cells to Host Reconstitution Following Syngeneic Hematopoietic Cell Transplantation" (2008). Open Access Dissertations This Open access is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarly Repository. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of Scholarly Repository. For more information, please contact repository.library@miami.edu.

2 UNIVERSITY OF MIAMI THE CONTRIBUTION OF HOMEOSTATICALLY EXPANDED DONOR CD8 T CELLS TO HOST RECONSTITUTION FOLLOWING SYNGENEIC HEMATOPOIETIC CELL TRANSPLANTATION By Melinda Roskos Keith A DISSERTATION Submitted to the Faculty of the University of Miami in partial fulfillment of the requirement for the degree of Doctor of Philosophy Coral Gables, Florida August 2008

3 UNIVERSITY OF MIAMI A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy THE CONTRIBUTION OF HOMEOSTATICALLY EXPANDED DONOR CD8 T CELLS TO HOST RECONSTITUTION FOLLOWING SYNGENEIC HEMATOPOIETIC CELL TRANSPLANTATION Melinda Roskos Keith Approved: Dr. Robert B. Levy Professor of Microbiology and Immunology Dr. Terri A. Scandura Dean of the Graduate School Dr. Becky Adkins Associate Professor of Microbiology and Immunology Dr. Thomas R. Malek Professor of Microbiology and and Immunology Dr. Eckhard R. Podack Professor of Microbiology and Immunology Dr. Gary I. Kleiner Assistant Professor of Clinical Pediatrics Dr. William R. Drobyski Professor of Medicine Medical College of Wisconsin

4 KEITH, MELINDA ROSKOS (Ph.D., Microbiology and Immunology) The Contribution of Homeostatically Expanded (August 2008) Donor CD8 T Cells to Host Reconstitution Following Syngeneic Hematopoietic Cell Transplantation Abstract of a dissertation at the University of Miami Dissertation supervised by Professor Robert B. Levy Number of pages in text (116) During homeostatic expansion, peripheral T cells proliferate in response to lymphopenia, in the absence of cognate antigen or costimulatory signals. Host CD8 reconstitution following hematopoietic cell transplantation (HCT) involves the de novo-generation of T cells in addition to the homeostatic expansion of mature donor T cells present in the graft and donor lymphocyte infusion as well as host T cells that survive conditioning. Although it is well appreciated that CD8 homeostatic expansion contributes to host CD8 reconstitution following HCT, the factors governing the extent of the contribution by donor CD8 homeostatic expansion to host reconstitution have not been precisely and systematically examined. The ex vivo generation of memory CD8 T cells specific for an epitope of the immunodominant minor histocompatibility antigen H60 was demonstrated. By adapting a previously described culture system designed to generate large numbers of transgenic memory CD8 T cells, memory CD8 cells (TM) specific for a physiological antigen were elicited from a heterogeneous population of CD8 T cells. The ex vivo-generated antigen-specific memory CD8 cells were then expanded under conditions of lymphopenia in ablatively conditioned syngeneic

5 transplant recipients and found to persist greater than 2 months post-transplant. These findings support the notion that the transplantation of small numbers ex vivo-generated memory CD8 cells, specific for a physiologically relevant antigen, can help restore host immune function following HCT. Transplant conditions were found to modulate the contribution of homeostatically expanded donor CD8 TM to the host CD8 compartment. Varying the conditioning intensity, timing of infusion, and infusion dose affected the kinetics of expansion as well as the homeostatic set-point. In my HCT model, delayed infusions of 3 weeks and transplantation of small numbers of donor CD8 T cells resulted in significant contributions to host CD8 T cell compartment. These findings could be applied clinically to enhance the effectiveness of clinical immunotherapy in restoring host immune function post-transplant. Moreover, transplantation of varying doses of donor CD8 TM demonstrated there was maximal donor contribution to host CD8 reconstitution. Finally, the homeostatic expansion, persistence, and function of transgenic memory and naïve CD8 populations were investigated following transplantation into ablatively conditioned syngeneic recipients. Both donor CD8 populations underwent 2 weeks of expansion post-transplant. The naïve CD8 population reproducibly achieved higher homeostatic numbers than the memory CD8 population. By one month post-hct, the transplanted naïve CD8 populations also exhibited a memory CD8 phenotype. Moreover, the CD8 TN TM population mediated an effective response to a primary challenge, comparable to the antigen-experienced memory CD8 population. Thus,

6 regardless of the state of differentiation of the donor CD8 population at the time of transplant, donor CD8 homeostatic expansion contributes a memory CD8 cells population, able to mediating effective immune responses, to host reconstitution.

7 ACKNOWLEDGEMENTS I would like to thank my mentor Dr. Robert Levy. Dr. Levy displayed an amazing ability to balance guidance and direction with encouragement and independence. He has given me an excellent foundation for a career as a physician scientist. I would like to thank the members of my dissertation committee for their helpful questions and suggestions: Dr. Becky Adkins, Dr. Gary Kleiner, Dr. Thomas Malek, and Dr. Eckhard Podack. I would also like to thank Dr. William Drobyski from the Medical College of Wisconsin for his kind participation in my final examination. I thank the members of the Levy lab. Monica Jones provided invaluable technical and administrative assistance. I would like to thank Dr. Alwi Shatry for his assistance in adapting the ex vivo technique to enrich a heterogeneous CD8 population for H60 CD8 TM. Finally, I would like to thank past and present lab members Angela Jones, Alan Hanash, Zach Zimmerman, and Maite Urbieta for making the lab an entertaining and interesting place to work. Finally, I would like to thank my family, in particular my husband, Andy Keith, for all their continued support which seems to have been exponentially needed the weeks leading up my defense. iii

8 TABLE OF CONTENTS List of Figures viii List of Tables...xi List of Abbreviations...xii INTRODUCTION...1 Autologous hematopoietic cell transplantation...1 T cell reconstitution following autologous HCT...3 Specific aims...6 CHAPTER 1: The homeostatic expansion of donor CD8 T cell populations following syngeneic HCT...8 Overview...8 Opportunistic infections following clinical HCT...8 Adoptive cellular immunotherapy for CMV and EBV...10 Development of a culture system to enrich for antigen-specific memory CD8 T cells...12 Results...14 Populations of donor memory and effector CD8 T cells undergo similar homeostatic expansion following syngeneic HCT...14 Both ex vivo-generated and in vivo-derived non-transgeneic CD8 TM populations undergo homeostatic expansion and persist following HCT...19 iv

9 Discussion...25 Homeostatic expansion of effector and memory CD8 T cell populations results in similar contribution, by number, to host CD8 reconstitution...25 Ex vivo-generated CD8 TM populations displays similar homeostatic expansion compared to in vivo-derived CD8 TM population...30 CHAPTER 2: Transplant conditions determine the contribution of homeostatically expanded donor CD8 memory cells to host CD8 compartment following syngeneic HCT...36 Overview...36 The requirement for cytokine in CD8 T cell homeostatic expansion...37 The administration of IL-7 and IL-15 in experimental transplant models...39 The requirement for self-peptide/mhc complexes in CD8 T cell homeostatic expansion...40 Results and Discussion...44 The level of conditioning determines the duration of homeostatic expansion post-transplant...44 The time of infusion of donor CD8 TM determines the kinetics of expansion and the homeostatic plateau...46 v

10 The number of donor CD8 TM transplanted directly affects donor homeostatic expansion...49 Proposed experiments to investigate donor CD8 homeostatic expansion in the absence of CD8 competition...54 The homeostatic expansion of donor CD8 TM can make significant contributions to host CD8 reconstitution...55 Proposed experiments to determine the maximal contribution of homeostatically expanded donor CD8 TM to the host CD8 compartment following syngeneic HCT...58 CHAPTER 3: Can antigen-experienced memory CD8 T cells and memory CD8 cells that develop during homeostatic expansion provide similar immunity following syngeneic HCT...61 Overview...61 Results...64 Transplanted naïve CD8 T cells, undergoing homeostatic expansion in HCT recipients, convert to a memory CD8 phenotype...64 The absence of IL-15 affects the homeostatic expansion but not the phenotypic conversion of the transplanted naïve CD8 population...68 Transplanted CD8 TM and TN TM display similar effector activity in vitro...70 vi

11 Transplanted CD8 TM and TN TM provide similar protective immunity following in vivo tumor challenge...76 Discussion...78 The role of IL-15 in the homeostatic expansion and conversion of naïve CD8 T cells to memory CD8 cells...80 A possible role for CD4 help in the generation of functional CD8 TN TM...84 Future experiments examining the role of CD4 help in the generation of functional CD8 TN TM cells...88 CONCLUDING REMARKS...92 APPENDIX A: Materials and Methods...96 REFERENCES vii

12 LIST OF FIGURES 1.1 Ex vivo-generated OT.I CD8 TE and TM cells exhibited phenotypes characteristic of effector CD8 cells and memory CD8 cells, respectively OT.I CD8 TM and TE undergo similar homeostatic expansion in ablatively conditioned syngeneic transplant recipients Following homoestatic expansion the transplanted CD8 TE and TM populations display a memory CD8 phenotype Both ex vivo-generated and in vivo-derived H60 CD8 memory cells display a memory CD8 phenotype prior to transplantation Similar kinetics and relative expansion were observed for both CD8 H60- tetramer binding TM populations in the splenic and bone marrow compartments of HCT recipients Both ex vivo-generated and in vivo-derived H60 CD8 memory cells retained a memory CD8 phenotype at least two months post-transplant In preliminary experiments to determine the kinetics of EG7 tumor growth following ablative conditioning and syngeneic HCT, the transplantation of OT.I TM was found to slow the growth of tumor The ability of ex vivo-generated and in vivo-derived H60 CD8 memory cells to mediate antigen-specific immunity will be assessed by monitoring the kinetics of graft rejection following transplantation with minormismatched bone marrow.33 viii

13 2.1 The level of conditioning determines the duration of homeostatic expansion post-transplant The kinetics of non-ot.i CD8 T cell reconstitution following HCT was determined The time of donor CD8 TM infusion determines the kinetics of expansion and the homeostatic plateau The dose of donor CD8 TM determines the kinetics and homeostatic numbers of the donor CD8 population following transplant A low dose of donor CD8 TM results in greater relative expansion Transplant conditions determine the contribution of donor CD8 TM to the host CD8 compartment following syngeneic HCT Transplanted CD8 TN undergo a phenotypic conversion to memory CD8 cells during the first month post-transplant The homeostatic expansion of OT.I CD8 TM and TN populations is different following ablative conditioning and syngeneic HCT The absence of IL-15 reduced the homeostatic expansion of both memory and naïve OT.I CD8 populations The absence of IL-15 did not affect the phenotypic conversion of the transplanted TN population OT.I CD8 TN TM exhibited similar cytokine production compared to transplanted OT.I TM following in vitro activation Following in vitro activation, both OT.I TM and TN TM displayed an activated CD8 phenotype: CD62L lo, CD25 hi, CD69 hi 74 ix

14 3.7 Following phenotypic conversion, OT.I TN TM demonstrated cytolytic activity indistinguishable from OT.I TM OT.I TM and TN TM provide similar protective immunity following in vivo tumor challenge The host CD4 T cell compartment is gradually reconstituted following ablative conditioning and HCT. 85 x

15 LIST OF TABLES I. In vivo-derived and ex vivo-generated CD8 populations enriched for H60- tetramer binding cells prior to transplantation into ablatively conditioned syngeneic recipients..22 II. The number of OT.I cells in spleen and draining lymph nodes of transplant recipients immediately before and then one month after challenge with 1x10 6 peptide-pulsed DC 78 xi

16 LIST OF ABBREVIATIONS APC B6 BALB.B BMC CD122 CMV CTL DC EBV FACS GVHD Gy HCT HLA IFNγ IL-2/15Rβ IL-7 IL-7Rα IL-15 IL-15Rα IL-15KO antigen presenting cell C57BL/6 C.B10-H2 b /LilMcdJ bone marrow cells IL-2/15Rβ cytomegalovirus cytolytic T lymphocyte dendritic cell Epstein-Barr virus fluorescence activated cell sorting graft-versus-host disease gray hematopoietic cell transplantation human leukocyte antigen interferon gamma interleukin-2/15 receptor beta chain interleukin-7 interleukin-7 receptor alpha chain interleukin-15 interleukin-15 receptor alpha chain C57BL/6NTac-IL15 tm1imx xii

17 MHC major histocompatibility complex MiHA OT.I TBI minor histocompatibility antigen C57BL/6-Tg(OT-I)-RAG1 tm1mom total body irradiation RAG1 recombination activating gene 1 RAG2 recombination activating gene 2 VZV varicella-zoster virus xiii

18 INTRODUCTION Autologous hematopoietic cell transplantation The first attempts at clinical transplantation were initiated in 1959 by Dr. E. Donnell Thomas to treat leukemia, using high-dose chemotherapy followed by syngeneic marrow transplantation. The first successful allogeneic transplants were performed in the late 1960s. The first successful autologous marrow transplants were performed in the late 1970s. By the mid-late 1980s, the annual number of autologous transplants surpassed that of allogeneic HCT. 1 There are three types of hematopoietic cell translants (HCT), depending on the source of the stem cell graft: allogeneic, syngeneic, and autologous transplants. In allogeneic transplantation, the source of stem cells can come from HLA-identical siblings, other HLA-matched family members, HLA-mismatched family members, and HLA-matched unrelated donors. The best results are achieved with sibling donors who have identical HLA class I and class II determinants. 1,2 Despite HLA matching, allogeneic recipients are at risk for graftv-host disease (GVHD) and graft rejection (i.e., a host-v-graft response), and thus require immunosuppressive therapy. In syngeneic HCT, the donor is an identical twin. There are fewer complications following syngeneic transplantation compared to allogeneic HCT and unlike autologous HCT, the syngeneic graft cannot be contaminated with tumor cells. In autologous transplants, the patient s stem cells are collected, cryopreserved, then, following high doses of chemotherapy or chemoradiotherapy, administered to rescue the patient. (i.e., It 1

19 2 is the treatment for the treatment.) Since the patient s own stem cells are used, he is not at risk for GVHD and does not require immunosuppressive therapy, but he does not have the benefit of a graft-v-tumor response. Furthermore, the patient is at risk for post-transplant tumor relapse due to potentially contaminating tumor cells in the transplant graft. 1 Autologous transplantation is indicated for Hodgkin s disease, non-hodgkin s lymphoma, and neuroblastoma, but it is also been used as therapy for acute myeloid leukemia, multiple myeloma, amyloidosis, and several autoimmune diseases. 3,4 The experiments presented in this dissertation utilize a syngenic murine HCT model as an experimental model for clinical autologous and syngenic HCT. Hematopoietic cell transplantation is possible due to the incredible regenerative potential and homing ability of hematopoietic stem cells. Moreover, the ability of hematopoietic stem cells to survive cryopreservation with relatively little damage has made autologous HCT feasible. 1,5 In autologous transplantation, the source of hematopoietic stem cells comes from the bone marrow or peripheral blood. Hematopoietic stem cells in the bone marrow can be mobilized to the bloodstream with such agents as cyclophosphamide, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, and stem cell factor. Mobilized peripheral blood is the preferred source of hematopoietic stem cells in autologous HCT as its use has been found to speed engraftment, decrease the need for transfusions, and shorten the length of hospital stays. 6

20 3 As stated above, contaminating tumor cells in the graft can contribute to relapse following autologous transplantation. Several methods have been developed to purge the autograft of tumor cells. These methods can be broadly divided into two categories: negative selection and positive selection. In negative selection, chemical agents, such as 4-hydroperoxycyclophosphamide and mafosfamide, monoclonal antibodies with complement, monoclonal antibodies conjugated to magnetic beads or toxin, or incubation with monoclonal antibodies and drugs have all been employed to remove tumor cells. Antibody-coated beads and magnetic sorting systems have been utilized to positively select CD34 + hematopoietic stem cells precursors. 6,7 T cell reconstitution following autologous HCT Following clinical autologous peripheral stem cell transplantation, the number of peripheral T cells returns to normal levels between 2-4 months posttransplant. An inverted CD4/CD8 ratio is observed in the first month and remains low for the first year post-transplant. The number of peripheral CD4 T cells can be reduced for one year or more post-hct. The naïve CD4 T cell subset (CD4 +, CD45RA + ), in particular, is severely reduced and can take up to 2 years to recover. In contrast, the number of CD8 T cells increases more rapidly posttransplant and elevated numbers of CD8 cell have been reported for up to a year. 8,9 T cell reconstitution following autologous transplantation involves the peripheral expansion of mature T cells included in the graft as well as host T cells

21 4 that survived the conditioning regimen, in addition to the de novo-generation of T cells, primarily in the host thymus, from transplanted hematopoietic stem cell precursors and residual host hematopoietic stem cell precursors. 10,11 The ability of mature T cells to expand under conditions of lymphopenia and contribute to host T cell reconstitution has been appreciated for over 20 years. 12 The proliferation of mature T cells present in the graft, rather than the generation of new T cells from hematopoietic stem cell precursors, was found to result in the rapid recovery of T cells, particularly CD8 T cells, following clinical autologous HCT. 13 In murine studies performed by Mackel et al., the expansion of congenic lymph node (LN) cells was examined in thymectomized and thymusbearing recipients following syngeneic HCT. Peripheral expansion of the transplanted LN cells was the predominant source T cell reconstitution in the thymectomized recipients while LN cell expansion and thymic maturation of T cells both contributed to T cell regeneration in thymus-bearing recipients. When the phenotypes of CD4 T cells in the thymus-bearing recipients were examined, CD4 cells derived from the LN infusion were CD45RB - CD44 hi while the CD4 derived de novo were CD45RB + CD44 lo. Thus, it was proposed that in the absence of a functioning thymus, the peripheral expansion of mature T cells dominates T cell reconstitution, resulting in T cells displaying a memory phenotype. However, in thymus-bearing recipients, peripheral T cell expansion and the de novo-generation of T cells both contribute to T cell recovery. Moreover, a functioning thymus is required to reconstitute naïve T cell populations. 14 These experimental findings were subsequently corroborated by

22 5 similar clinical reports. In addition to the rapid recovery of CD8 numbers in recipients of clinical autologous HCT, the mature CD8 T cells displayed an activated 13,15 or memory phenotype 7,16. It has now become well appreciated that T cell reconstitution results from both the peripheral expansion of mature T cells and de novo generation of T cells from T cell precursors. 8,11 The contribution of de novo-generated T cells is not only to restore the overall numbers of T cells but to also restore T cell diversity. The restoration of a full T cell repertoire probably depends on thymic-derived naïve T cells. 11,14 However, the contribution of de-novo generated T cell populations to T cell reconstitution depends on a functioning thymus. Pre-transplant conditioning can severely impair thymic function following transplant. 11 In addition, thymic function diminishes with patient age 11, thus children typically recover T cell immunity by 6 months post-transplant while adults may never achieve a fully competent T-cell repertoire The thymic contribution is further impaired following allogeneic transplantation by GVHD and immunosuppressive therapy. 11 Thus, it not is surprising the contribution of newly derived T cells is not only delayed but also not as significant in older patients. Therefore, the contribution of homeostatically expanded mature T cells becomes more important to the T cell reconstitution of older transplant recipients.

23 6 Specific aims Although it is well appreciated that mature T cells undergo homeostatic expansion and thereby contribute to T cell reconstitution, the specific details governing T cell homeostatic expansion have not been examined precisely and systemically. By elucidating the factors that govern the extent to which donor CD8 homeostatic expansion can contribute to host reconstitution, homeostatic expansion could be used more effectively to help restore immune function following clinical transplantation. For example, the effectiveness of adoptive cellular therapy could be enhanced by a more thorough understanding of how transplant conditions modulate the homeostatic expansion of donor CD8 populations. In addition, the development of adoptive therapy for use as anti-viral prophylaxis would benefit from an examination of the ability of memory and effector CD8 populations to undergo homeostatic expansion and persist posttransplant. Moreover, by determining the contributions of homeostatically expanded memory and naïve CD8 populations to host T cell immunity, subsets of the donor T cell population could either be selected or removed, depending on the ability of the subset to improve host immune function. In this dissertation, the homeostatic expansion, persistence, and function of well-defined donor CD8 T cell populations were investigated in a syngeneic murine HCT model in an effort to address the following questions: (1) What are the contributions of homeostatically expanded naïve, effector, and memory CD8 T cells to the host CD8 compartment?

24 7 (2) Can ex vivo-generated non-transgenic memory CD8 cells be expanded under conditions of lymphopenia and mediate antigen-specific immunity? (3) How do transplant conditions that influence space affect the contributions of homeostatically expanded donor CD8 TM populations? In Chapter 1, the homeostatic expansion and persistence of transgenic memory and effector CD8 populations following syngeneic HCT are determined. In addition, an in vitro culture system for generating memory CD8 T cells specific for a physiologic antigens (i.e., an epitope of an immunodominant minor histocompatibility antigen) is described. The homeostatic expansion and persistence of the ex vivo-generated CD8 TM as well as an in vivo-derived memory CD8 population of the same antigen-specificity are investigated following ablative conditioning and syngeneic HCT. Chapter 2 examines the ability of conditioning intensity, timing of infusion, and infusion dose to modulate the contribution of homeostatically expanded donor CD8 population to host reconstitution. In Chapter 3, the homeostatic expansion of transgenic memory and naïve CD8 populations is examined in wild-type and IL-15-deficient transplant recipients. The ability of the transplanted antigen-experienced memory CD8 population and the memory CD8 population arising from homeostatic expansion to mediate long-term immunity concludes the dissertation.

25 CHAPTER 1: The Homeostatic Expansion of Donor CD8 T Cell Populations Following Syngeneic HCT OVERVIEW Opportunistic infections following clinical HCT Immune deficiency following HCT results in increased susceptibility of transplant recipients to infection. In fact, opportunistic infections are a leading cause of mortality among recipients of allogeneic transplants and a major cause of morbidity among recipients of autologous HCT. 20,21 Recipients of allogeneic transplants are vulnerable to opportunistic infections during every phase of immune reconstitution from the pre-engraftment phase (first 30 days posttransplant) to the post-engraftment phase (30 to 100 days post-transplant) to the late phase (>100 days post-transplant). 22 During the first 30 days posttransplant, infection is commonly caused by Candida species, Aspergillus species, herpes simplex virus reactivation, Staphylococcus epidermidis, facultative Gram-negative bacilli, and gastrointestinal tract Streptocococci species. 20,22 Between 30 and 100 days post-transplant, the most prevalent causes of infection include cytomegalovirus (CMV), Pneumocystis carinii, and Aspergillus species. Greater than 100 days post-transplant, recipients remain at risk for CMV, varicella-zoster virus (VZV), lymphoproliferative disease related to Epstein-Barr virus, and bacterial infections, such as Haemophilus influenzae and Streptococcus pneumoniae. 22,23 Among recipients of allogeneic HCT, those 8

26 9 receiving unrelated grafts have a higher incidence of severe viral infection as well as fatal opportunistic infection following successful engraftment compared to those with HLA-matched sibling donors. 18,24 Although the incidence and severity of infection is greater among allogeneic recipients, recipients of autologous HCT are also at increased risk of infection. 25 The greatest risk of infection following autologous HCT is during the first 30 days post-transplant. 22 Prolonged neutropenia and breaks in the mucocutaneous barriers make autologous recipients particularly susceptible to Candida species, Aspergillus species, and herpes simplex virus reactivation during the pre-engraftment phase. 22 The most frequent causes of viral infection following autologous HCT include CMV and VZV A retrospective study of transplants performed at the Johns Hopkins Oncology Center found the incidence of CMV infection following autologous and allogeneic HCT comparable (45%), although CMV infection was less severe following autologous HCT compared to allogeneic HCT. 30 Both the Johns Hopkins Oncology Center 28 and the University of Minnesota Hospital and Clinical 29 reported an incidence of VZV infection of approximately 25% among autologous HCT recipients. In autologous recipients, VZV infection develops late post-transplant (median onset is the fifth month post-transplant and over 90% of cases are observed by the first year 28 ), and it is not usually fatal. While there are reports of EBV and adenovirus infection in autologous HCT recipients, the incidence is low By 100 days posttransplant, patients of autologous HCT are generally less susceptible to infection than allogeneic HCT recipients. 22 Presumably this difference is due to a more

27 10 rapid recovery of immune function rather than less reactivation of latent infection. 33 Although infection is not as frequent or severe following autologous HCT, recipients are still at increased risk. Thus, infection is a major complication of both autologous and allogeneic HCT and approaches to enhance immune function are thus of clinical importance. Adoptive cellular immunotherapy for CMV and EBV Significant advances have been made in the development of adoptive cellular immunotherapy for the enhancement of anti-viral immunity following clinical HCT. Most of the approaches have focused on the isolation and expansion of human T cells specific for either cytomegalovirus (CMV) or Epstein- Barr virus (EBV). Work by Riddell and colleagues demonstrated that CMV-specific cytotoxic T cells (CTL) clones could be isolated from bone marrow donors, expanded ex vivo, and adoptively transferred to immunodeficient allogeneic HCT recipients, thereby restoring CMV-specific immunity and preventing CMV infection in patients at risk. 34 There was no toxicity associated with the adoptive transfer. Moreover, by utilizing the bulk cultures, the risk of alloreactivity (i.e., graft-versushost disease) was minimized. 35 In addition, these studies also confirmed the requirement for CD4 help to restore long term immune protection. 34,36-39 Peggs and colleagues also developed an in vitro culture system to generate CMV-specific T cells. Polyclonal CMV-specific T cell lines were generated by culturing autologous peripheral blood lymphocytes and monocyte-

28 11 derived dendritic cells (DC) from CMV-seropositive donors with CMV antigen. 35 Beginning on day 10, IL-2 was added to culture every 2 days to stimulate further T cell proliferation. The investigators noted several advantages of this culture system. First, the culture system did not require live virion. Second, detailed knowledge of the antigenic peptides involved was not needed making this system applicable to CMV-seropositive donors of any HLA type. Finally, by using monocyte-derived DC as the APC population, both CD4 and CD8 CMV-specific T cells were generated thus satisfying the requirement for CD4 help. 35,40,41 An EBV-specific CTL line was developed by Rooney and colleagues from donor lymphocytes stimulated with EBV-transformed lymphoblastoid cells derived from donors. IL-2 was added to the cultures 3 times per week, beginning on day 14. Since the requirement for CD4 T cells to maintain and expand effector CD8 cells in vivo is not known, CD4 T cells were purposefully included in the CTL line. There were no toxicities associated with the CTL infusion. EBV-specific CTL infusions, given as prophylaxis, prevented EBV infection in patients at high risk. In patients with EBV reactivation, 3-4 weeks of immunotherapy lowered the viral load to normal range. Most striking, an immunoblastic lymphoma resolved following four EBV-specific CTL infusions. 42,43 Finally, Sun and colleagues developed a culture system to simultaneously expand CMV- and EBV-specific CTL. An EBV-transformed B lymphoblastoid cell line was engineered to express the CMV pp65 peptide (the immunodominant

29 12 viral epitope) for use as an APC population. The resulting polyclonal bispecific CTL demonstrated antigen-specific cytotoxicity against CMV and EBV in a number of in vitro assays. 44 Development of a culture system to enrich for antigen-specific memory CD8 T cells I have developed an ex vivo method for enriching a polyclonal CD8 T cell population for antigen-specific memory CD8 T cells that may be clinically applicable. 45 Memory CD8 T cells (CD8 TM) are attractive for the restoration of immune function because these cells respond more rapidly to lower concentrations of antigen than their naïve counterparts Moreover, upon reexposure to antigen, memory CD8 TM are able to rapidly acquire cytotoxic function and, on a per cell basis, are only slightly slower than effector CD8 cells at eliminating in vivo targets. 50,51 Thus, memory CD8 T cell populations can provide similar in vivo protection compared to effector CD8 populations. In order to effectively utilize ex vivo-generated antigen-specific CD8 TM populations to enhance host immunity, their homeostatic expansion and persistence following syngeneic HCT needs further investigation. During the past several years, the capacity of T cells to expand under conditions of lymphopenia has become well appreciated In studies examining memory CD8 homeostatic expansion, the lymphopenia was either a result of sublethal doses of total body irradiation or a genetic deficiency (i.e., RAG deficient mice) In recent studies examining the homeostatic expansion

30 13 of CD8 T cells post-transplant, polyclonal populations of memory and naïve CD8 T cells have been examined in allogeneic transplant models Earlier work did examine the homeostatic expansion of polyclonal CD8 populations in a syngeneic transplant model as well as naïve transgenic TCR (2C) CD8 T cells in a parent F1 transplant model. 64 However, the homeostatic expansion of antigen-specific donor memory and effector CD8 populations following ablative conditioning and syngeneic HCT has not been systematically and precisely reported to date. Moreover, studies have not been performed directly comparing the homeostatic expansion of different CD8 T cell subsets. Do effector and memory CD8 T cells display similar kinetics of expansion and homeostatic plateau following transplantation? Do in vivo-derived and ex vivo-generated nontransgenic memory CD8 populations undergo similar homeostatic expansion compared to a transgenic memory CD8 population? By addressing these questions, the dissertation studies aimed to precisely characterize the homeostatic expansion of well-defined donor CD8 populations in a model of autologous HCT. In this chapter, findings on the homeostatic expansion of several donor memory CD8 populations following ablative conditioning and syngeneic HCT are presented. The donor CD8 T cell populations included transgenic (OVA-specific) memory and effector CD8 cells as well as non-transgenic (specific for H60 histocompatibility minor antigen) memory CD8 cells either derived by in vivo priming or generated ex vivo. All donor CD8 population underwent similar homeostatic expansion, characterized by a 2-4 week period of expansion

31 14 followed by a homeostatic plateau. Of note, in the absence of antigen, the transgenic effector CD8 population converted to a memory CD8 phenotype by 3 days post-transplant and underwent virtually identical homeostatic expansion compared to the transgenic memory CD8 population. In total, I have demonstrated that populations of transgenic memory and effector CD8 T cells as well as both ex vivo-generated as well as in vivo-derived memory CD8 populations undergo a similar pattern of antigen-independent expansion following transplantation into ablatively conditioned syngeneic recipients. Thus, the transplantation of either memory or effector CD8 T cell populations appear to provide similar contributions by number to the reconstitution of the host CD8 compartment. RESULTS Populations of donor memory and effector CD8 T cells undergo similar homeostatic expansion following syngeneic HCT I was interested in determining whether memory and effector CD8 T cell populations would undergo similar homeostatic expansion in ablatively conditioned HCT recipients. To that end, transgenic T cell receptor OT.I CD8 T cells were selected as the representative memory CD8 (TM) and effector CD8 (TE) populations. Not only could large numbers of highly purified OT.I memory and effector OT.I CD8 populations be obtained quickly, these populations could also be readily detected and monitored in transplant recipients. The memory CD8 population was generated ex vivo from naïve OT.I cells in 5-7 days as previously

32 15 described 45,65,66. Please see Appendix A for a complete description of Materials and Methods. The resultant OT.I TM population displayed a phenotype characteristic of memory CD8 cells, i.e., CD44 +, Ly6C +, CD62L +, CD25 lo (Figure 1.1, bottom panels). The effector CD8 (TE) population was also generated ex vivo. After activating naïve OT.I cells with IL-2 and OVA peptide for 3 days, the OT.I cells expressed an effector CD8 phenotype: CD44 +, Ly6C lo, CD62L lo, CD25 intermed (Figure 1.1, top panels). The homeostatic expansion of these representative CD8 TM and TE populations was then investigated in a syngeneic murine HCT model (Figure 1.2A). By utilizing a syngeneic model, in which neither allo-antigen nor the cognate antigen recognized by the donor CD8 T cell population was present, any antigen-driven proliferation was eliminated, thereby allowing us to focus exclusively on the homeostatic expansion of selected donor CD8 T cell populations. On the day of transplant, 9.0Gy-conditioned animals received T celldepleted (TCD) B6 bone marrow cells and 1.5x10 6 OT.I TM or TE. Homeostatic expansion of the transplanted OT.I populations was determined by assessing CD8 +, Vα5 +, Vβ2 + cell numbers in recipient spleens beginning 3 days post-hct. The numbers of all OT.I populations clearly increased in recipient spleens for the first 14 days post-transplant. The OT.I TM and TE populations consistently achieved a homeostatic set-point between x10 6 cells (Figure 1.2B), representing approximately 40% of host CD8 cells. These steady-state numbers of the transplanted TM and TE populations were maintained as long as 7 and 2

33 16 CD44 LY6C CD62L CD25 OT.I CD8 TE CYC FITC PE PE OT.I CD8 TM CYC FITC PE PE Figure 1.1: Ex vivo-generated OT.I CD8 TE and TM cells exhibited phenotypes characteristic of effector CD8 cells and memory CD8 cells, respectively. Spleen cells from OT.I-Rag1 -/- mice were cultured with 10ng rmil-2 and 0.1nM OVA peptide (SIINFEKL) in 1mL 5% CM for 3 days. The resulting OT.I cells displayed an activated phenotype (CD44 hi, Ly6C lo, CD62L lo, CD25 intermed ) and were utilized as the representative CD8 TE population. To generate memory OT.I cells, OT.I TE were cultured with 300ng rmil-15 in 30mL 5% CM for 2 days. The resulting OT.I CD8 population displayed memory CD8 phenotype:(cd44 hi, Ly6C +, CD62L +, CD25 lo ).

34 17 A. Day -1 Day 0 Post Transplant B. 9.0Gy TBI Number of Splenic OT.I Cells (x10 6 ) TCD B6 Assess OT.I Bone Marrow Numbers and Phenotype + 1.5x10 6 OT.I TM or TE Recipients of OT.I TM Recipients ofot.i TE Days Post-HCT >56 Figure 1.2: OT.I CD8 TM and TE undergo similar homeostatic expansion in ablatively conditioned syngeneic transplant recipients. (A) A syngeneic murine HCT model was utilized to characterize the homeostatic expansion of OT.I TM and TE. One day before transplant, recipients received 9.0Gy TBI. The following day, animals received 2x10 6 TCD B6-wt bone marrow with 1.5x10 6 OT.I TM or TE. Beginning 3 days post-transplant, the number of OT.I cells was determined in recipients spleens by assessing CD8 +, Vβ5 +, Vα2 + cells. (B) The numbers of OT.I TM (diamonds) and TE (triangles) cells in recipient spleens clearly increased in the first 2 weeks post-transplant and both populations reached a steady-state number between x 10 6 cells, representing approximately 40% of the host CD8 cells. TE n = 3-6 animals/time point. TM n = 6-19 animals/time point.

35 18 CD44 LY6C CD62L CD25 OT.I CD8 TE CYC FITC PE PE OT.I CD8 TM CYC FITC PE PE Figure 1.3: Following homeostatic expansion the transplanted CD8 TE and TM populations displayed a memory CD8 phenotype. On day 28 post-transplant, the phenotype of OT.I CD8 cells in recipient spleens was assessed. The phenotype of the transplanted OT.I TE population had converted to a memory CD8 phenotype. The transplanted OT.I TM population retained its memory CD8 phenotype.

36 19 months, respectively, post-transplant (data not shown) indicating that both transplanted CD8 populations could expand and persist in these HCT recipients. One month post-transplant, the phenotypes of the two OT.I populations were examined. The OT.I TM population had retained its memory CD8 phenotype (Figure 1.3, bottom panels). The transplanted OT.I TE converted to a memory CD8 phenotype (Figure 1.3, top panels). In this model, the conversion of OT.I TE to a memory CD8 phenotype occurred within the first 3 days (data not shown) and when examined again 2 months post-transplant, continued to express the same phenotype. Both ex vivo-generated and in vivo-derived non-transgenic CD8 TM populations undergo homeostatic expansion and persist following HCT CD8 cells from B6 animals recognizing the immunodominant minor histocompatibility (MiHA) antigen H60 67,68 were selected to address 2 questions: (1) do non-transgenic, naturally occurring CD8 cells, compromised of more than a single clone, undergo similar homeostatic expansion compared to a transgenic (tg) CD8 TM population and (2) do in vivo-derived CD8 TM undergo similar homeostatic expansion as ex vivo-generated populations? At least one month following in vivo priming, H60-tetramer binding cells expressed a CD8 T cell memory phenotype (i.e. in vivo H60 TM): CD44 hi, Ly6C hi, CD62L +, and CD25 lo (Figure 1.4, top panels). The phenotype of ex vivo-generated H60-tetramer binding CD8 cells after culturing (i.e., ex vivo H60 TM) was that of a memory

37 20 CD8 cell and was similar to the in vivo-derived population (Figure 1.4, bottom panels). To investigate the homeostatic expansion of non-transgenic CD8 memory populations, 9.0Gy-conditioned recipients were transplanted with TCD B6 bone marrow together with a B6 CD8 population enriched for either in vivo or ex vivo H60 TM (Table 1). Homeostatic expansion was assessed by determining the number of H60-tetramer positive CD8 cells in recipient spleen and bone marrow compartments beginning 3 days post-hct (Figure 1.5). To enable comparisons among independent experiments, the relative expansion of the H60 TM population was determined by dividing the number of H60-tetramer binding CD8 cells in each compartment by the number of H60-tetramer binding CD8 cells transplanted. Both in vivo and ex vivo CD8 H60 TM underwent expansion for one month post-transplant as observed in both the splenic and marrow compartments (Figure 1.5 A, B). Although the relative expansion of the in vivo-derived CD8 TM appeared slightly less than the ex vivo derived population in the spleen, these data did not substantially differ (p>0.5). There were also no significant differences in relative expansion in the BM compartment between the two populations. The relative expansion of ex vivo-generated OT.I TM in recipient spleens was comparable to both of these H60 TM populations (data not shown). Thus, regardless of the source of memory population examined, similar numbers of transplanted CD8 cells resulted in similar relative expansion and kinetics.

38 21 CD44 LY6C CD62L CD25 H60 TM Derivation In Vivo CYC FITC FITC FITC Ex Vivo CYC FITC FITC FITC Figure 1.4: Both ex vivo-generated and in vivo-derived H60 CD8 memory cells display a memory CD8 phenotype prior to transplantation. Immediately before transplant, H60-tetramer binding CD8 cells following ex vivo culturing (bottom panel) displayed a memory phenotype similar to in vivo-derived H60 cells from primed animals (top panel). Histograms were generated by collecting about 1000 events in the H60+, CD8+ gate. Pre-transplant histograms are representative 4-5 experiments/group.

39 22 Table I: In Vivo-Derived and Ex Vivo-Generated CD8 Populations Enriched for H60-Tetramer Binding Cells Prior to Transplantation into Ablatively Conditioned Syngeneic Recipients Method of Generation * Experiment Number Total CD8 T Cells Infused (x10 6 ) H Percent of H60-Tetramer Binding CD8 Cells Total Number of CD8 H60 TM Infused (x10 6 ) In Vivo In Vivo In Vivo In Vivo Ex Vivo Ex Vivo Ex Vivo Ex Vivo Ex Vivo *CD8 TM were either in vivo-derived following priming (see Methods) or ex vivo-generated by stimulation with B6 dendritic cells together with H60 peptide (LTFNYRNL) and IL-2 followed by incubation with IL-15 (see Methods). H Recipients (9.0Gy-conditioned) were transplanted with 2x10 6 TCD syngeneic B6 bone marrow together with the total number of CD8 T cells indicated. The number of H60-tetramer binding CD8 cells included within the total CD8 population transplanted are indicated.

40 A B In Vivo Derived Spleen Bone Marrow Ex Vivo Generated Number of H60 TM / Infused Number Spleen Bone Marrow Days Post-HCT Figure 1.5: Similar kinetics and relative expansion were observed for both CD8 H60 TM populations in the splenic and bone marrow compartments of HCT recipients. In vivo-derived CD8 H60-tetramer binding TM underwent homeostatic expansion for approximately one month post-transplant with similar relative expansion observed in both the spleens (solid line) and bone marrow (dashed lines) compartments (A). Ex vivo-generated CD8 H60-tetramer binding TM also underwent expansion for approximately one month in both the spleen and marrow compartments (B). The relative expansion of the ex vivo-generated and in vivo-derived CD8 TM in recipient spleens was not statistically different (p>0.5). In vivo-derived H60 CD8 TM: n = 5-9 recipients / time point in both spleen and bone marrow compartment. Ex vivo-generated H60 CD8 TM: n = 6-10 recipients / time point in spleen and n = 3-6 recipients / time point in bone marrow compartment.

41 24 CD44 LY6C CD62L CD25 H60 TM Derivation In Vivo CYC FITC FITC FITC Ex Vivo CYC FITC FITC FITC Figure 1.6: Both ex vivo-generated and in vivo-derived H60 CD8 memory cells retained a memory CD8 phenotype at least two months post-transplant. Two months post-transplant, H60-tetramer binding CD8 cells, either ex vivo-generated and in vivo-derived, from the peripheral blood of HCT recipients retained a phenotype characteristic of memory CD8 cells. Histograms were generated by collecting about 1000 events in the H60+, CD8+ gate. Post-transplant histograms are representative of 5-8 animals/group.

42 25 To monitor whether the in vivo-derived and ex vivo-generated CD8 H60 TM populations identified in transplant recipients maintained their memory phenotype following HCT, the phenotype of H60-tetramer binding cells was assessed at several time points following transplant. The phenotype of in vivoderived and ex vivo-generated H60 cells from recipient peripheral blood two months post-transplant (Figure 1.6) is representative of the memory phenotype observed beginning a week post-hct. Thus, transplantation of both in vivoderived and ex vivo-generated CD8 H60 TM resulted in the expansion and persistence of memory CD8 populations. DISCUSSION Homeostatic expansion of effector and memory CD8 T cell populations results in similar contribution, by number, to host CD8 reconstitution Many of the adoptive cellular therapies developed to date utilize viralspecific CTL clones and activated T cell lines to enhance the anti-viral immunity of transplant recipients. Having demonstrated the ability to enrich a polyclonal CD8 population for antigen-specific memory CD8 T cells, I asked: Would the state of differentiation affect the contribution of ex vivo-generated antigen-specific CD8 cells to host CD8 T cell reconstitution? The homeostatic expansion of transgenic memory and effector CD8 populations, generated ex vivo, was investigated following ablative conditioning and syngeneic HCT.

43 26 Memory CD8 T cell development is classically characterized by three phases: the activation and expansion of naïve CD8 T cells, the contraction or death of effector CD8 T cells, and the establishment and maintenance of memory CD8 T cells. 69 After antigen encounter, peripheral naïve CD8 T cells undergo a period of activation and proliferation, differentiating into effector CD8 T cells. Effector CD8 cells are able to produce cytokines, including IFNγ and TNFα, as well as cytolytic molecules, such as perforin and granzymes, and traffic to nonlymphoid tissues, where they can mediate the rapid elimination of infectious pathogens. 70 Following antigen clearance, the effector CD8 T population undergoes a massive contraction with only 10% of the effectors CD8 cells surviving and differentiating into long-term memory CD8 T cells. 71,72 Once a naïve CD8 T cell has been activated by antigen and the developmental process has been initiated, antigenic stimulation is no longer required, thus memory CD8 T cell formation can continue in the absence of antigen. 73,74 Thus, I hypothesized that following transplantation into an antigen-free recipient, the OT.I TE population would differentiate into memory CD8 T cells. Indeed, by 3 days post- HCT, the transplanted population of effector OT.I cells did exhibit a memory CD8 phenotype. However, when designing experiments to assess the homeostatic expansion of effector CD8 T cells, it was uncertain whether the effector CD8 population, following transplantation, would undergo contraction before its differentiation into memory CD8 cells. Would an effector CD8 population of 1.5x10 6 OT.I cells contract to approximately 10% of the input number

44 27 immediately post-transplant? If so, would the remaining OT.I cells then undergo homeostatic expansion? How would that homeostatic expansion compare to the transplanted memory CD8 population? In fact, the CD8 TE population underwent no apparent contraction following transplantation. Instead, the transplanted OT.I CD8 TM and TE populations underwent virtually identical homeostatic expansion. Since CD8 TM and TE populations provided similar contributions by number to the reconstitution of the host CD8 compartment, I would like to determine whether the transplanted TM and TE populations will provide similar protective immunity immediately following HCT. To do so, preliminary experiments have been performed to determine the kinetics of tumor growth, in the absence of OT.I cells, following ablative conditioning and syngeneic HCT. Recipients were challenged with either 0.5x10 6 or 1.0x10 6 EG7 tumor cells (EL4 thymoma transfected with OVA) 3 or 6 days before ablative conditioning then transplanted with only TCD syngeneic bone marrow. Tumor growth was monitored post-transplant (Figure 1.7). EG7 cells clearly survived the conditioning regimen as all recipients developed palpable tumor by 2 weeks posttransplant. Moreover, when OT.I CD8 TM were included at the time of transplant, there was a noticeable reduction in tumor growth (Figure 1.7B). Thus, this strategy provides a pre-clinical model in which to assess the ability of CD8 TM and TE populations to provide protective immunity early post-transplant. Experiments are planned to utilize this pre-clinical tumor model to compare the protective immunity of CD8 TM and TE population early posttransplant. Recipients will be challenged with varying numbers of