GSBS Dissertations and Theses

Approval Date

12-10-2013

Document Type

Doctoral Dissertation

Academic Program

Immunology and Microbiology

Department

Pathology

First Thesis Advisor

Raymond M. Welsh, PhD

Keywords

CD8-Positive T-Lymphocytes, Cell Survival, Cell Differentiation, Adaptive Immunity, Immunologic Memory

Subjects

Dissertations, UMMS; CD8-Positive T-Lymphocytes; Cell Survival; Cell Differentiation; Adaptive Immunity; Immunologic Memory

Abstract

Activation and proliferation of antigen-specific T cells is the hallmark of an anti-viral immune response. Effector T cells generated during an immune response are heterogeneous in regards to their ability to populate the memory pool once the immune response has resolved. Initial T cell activation takes place in the lymphoid organs, after which T cells migrate into the non-lymphoid tissues. The presence of memory T cells at non-lymphoid tissue sites has been shown to be critical for protection against secondary virus challenge. Our lab has previously demonstrated that during and after the resolution of the immune response to Lymphocytic choriomeningitis virus (LCMV) CD8+T cells in the nonlymphoid tissues are more resistant to apoptosis than those in the lymphoid organs. This stability of T cells in the non-lymphoid tissues may be critical in ensuring protection against a secondary virus challenge.

Mechanisms regulating tissue-dependent differences in CD8+T cell apoptosis were studied in an acute LCMV infection model. Virus-specific CD8+T cells from lymphoid (spleen, mesenteric lymph nodes (MLN), inguinal lymph nodes (ILN)) and non-lymphoid tissues (peritoneal exudate cells (PEC), fat-pads) were compared for expression of surface antigenic markers known to correlate with a memory phenotype. Non-lymphoid tissues were enriched in IL-7Rhi, KLRG-1lo, CD27hi and CXCR3hi virus-specific CD8+ T cells, and the presence of these antigenic markers correlated with increased memory potential and survival. Transcription factors in addition to cell surface antigens were assessed as correlates of resistance to apoptosis. Virus-specific CD8+T cells in the nonlymphoid tissues were enriched in cells expressing T cell factor-1 (TCF-1), which correlated with increased memory potential and survival. CD8+T cells in the peritoneum of TCF-1-deficient mice had decreased survival during resolution of the immune response to LCMV, suggesting a role for TCF-1 in promoting survival in the non-lymphoid tissues.

As an additional mechanism, I investigated whether apoptosis-resistant CD8+T cells migrate to non-lymphoid tissues and contribute to tissue-dependent apoptotic differences. CXCR3+ CD8+T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into the peripheral tissues. The PECs expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may have recruited the non-apoptotic cells from the lymph nodes. By adoptively transferring splenic T cells into the spleen or PEC environment I showed that the peritoneal environment through a yet undefined factor promoted survival of CD8+T cells. In this study I have elucidated the mechanisms by which CD8+T cells preferentially survive in the non-lymphoid tissues. I found that non-lymphoid tissues were enriched in memory-phenotype CD8+T cells which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosisresistant CD8+T cells may preferentially migrate into the non-lymphoid tissues where the availability of tissue-specific factors may enhance memory cell survival.

Few transcription factors have been identified that regulate CD8+T cell effector-memory differentiation during an immune response. In this thesis, I have also studied the mechanism by which the transcription factor Blimp-1 regulates the generation of effector and memory CD8+T cells. Blimp-1 is known to repress a large number of target genes, and ChIP (chromatin immunoprecipitation) sequencing analysis done by Dr. HyunMu Shin in the lab of Dr. Leslie J. Berg identified CD25 (IL-2Rα) and CD27 as potential targets of Blimp-1. I found that Blimp-1-deficient CD8+T cells had sustained expression of CD25 (IL-2Rα) and CD27 during peak and resolution of the immune response to LCMV. By performing adoptive transfers of CD25hi and CD27hi CD8+T cells I showed that CD25 and CD27 expression on CD8+T cells during resolution of the immune response correlates with enhanced survival. Silencing Il2rα and Cd27 expression reduced the Blimp-1-deficient CD8+T cell response, suggesting that sustained expression of CD25 and CD27 was in part responsible for the enhanced CD8+T cell response seen in the Blimp-1-deficient mice. Furthermore, our collaborator Dr. HyunMu Shin showed that CD25 and CD27 are direct targets of Blimp-1, and that Blimp-1 recruits histone modifying enzymes to Il2rα and Cd27 loci to suppress their expression during the peak of the anti-viral immune response. This study identifies one of the mechanisms by which Blimp-1 regulates the balance between generation of effector and memory CD8+T cells.

In this thesis work I also studied the function of the transcription factor ROG (Repressor of GATA-3) in regulating in vivo T cell responses during both acute and chronic LCMV infection. ROG-deficient mice had increased CD8+T cell responses during an acute LCMV infection. ROG deficiency also led to the generation of memory T cells with an enhanced recall response compared to WT controls. By using LCMV-specific P14+ TCR transgenic ROG-deficient CD8+T cells these defects were shown to be T cell intrinsic. ROG-deficient mice had enhanced CD8+T cell responses and viral clearance during a persistent high dose LCMV Clone 13 infection. During chronic LCMV infection ROG-deficient mice also had increased lung pathology and mortality. The results indicate that ROG negatively regulates T cell responses and memory generation during both acute and chronic LCMV infection.

The studies highlighted in this thesis elucidate the mechanisms promoting CD8+T cell survival in non-lymphoid tissues as well as transcription factormediated regulation of memory CD8+T cell differentiation. Knowledge of this will help us better understand T cell immunity after infections and may eventually help develop better vaccines.

DOI

10.13028/M2D02M

Rights and Permissions

Copyright is held by the author, with all rights reserved.

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