Title

Organ-Dependent and Epitope-Dependent Repertoire Usage and Apoptosis of Antigen-Specific T Cells in Viral Infections: a Dissertation

Date

April 2004

UMMS Affiliation

Graduate School of Biomedical Sciences, Program in Immunology and Virology

Document Type

Dissertation, Doctoral

Subjects

CD8-Positive T-Lymphocytes; Apoptosis; Lymphocytic choriomeningitis virus; Lymphoid Tissue; Academic Dissertations

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

During virus infections, activation of CD8 T cells takes place in secondary lymphoid organs including spleen and lymph nodes. The kinetics of the T cell response in lymphoid tissues has been clearly studied. However, a large number of virus-specific T cells disseminate into various nonlymphoid tissues. As reservoirs for effector and memory cells, nonlymphoid organs play an important role for defending against infections. T cell responses in nonlymphoid organs may differ from lymphoid organs.

T cell repertoire usage in lymphoid and nonlymphoid tissues was studied in an acute lymphocytic choriomeningitis virus (LCMV)-infected murine model. The hierarchy of CD8 T cell specificities was examined with cytotoxic T lymphocyte (CTL) sodium 51 chromate (51Cr) release assays and intracellular interferon (IFN)γ assays. T cell receptor (TCR) repertoire usage was determined by complementarity determining region (CDR)3 length spectratyping analysis. Both T cell specificity and TCR repertoire usage revealed some similarities and differences between several organs. Within an epitope-specific CD8 T cell population, the TCR repertoire usage was similar in different organs of the same mouse, but highly heterogeneous between individual mice with genetically identical backgrounds.

A very restricted CD4 TCR repertoire was observed in BALB/c mice after secondary respiratory syncytial virus (RSV) infection. Most of the CD4 T cells of BALB/c mice pre-immunized with RSV glycoprotein (GP) predominantly express Vβ14 TCR with discrete oligoclonal CDR3 regions. Depletion of Vβ14 CD4 T cells dramatically reduced immunopathology.

The apoptotic phenotype of LCMV-specific CD8 T cells was studied in various lymphoid and nonlymphoid tissues during acute and memory stages of infections. Peripheral tissues (peritoneal cavity (PEC), fat pad, and lung) reacted with a much lower frequency with the early apoptotic marker Annexin V than those in spleen and lymph nodes. This was not due to a TCR-based selection because similar TCR spectratypes were seen in different organs. Activated lymphoid and nonlymphoid T cells from LCMV GP33 transgenic mice, which have identical TCR α and β chains on all T cells, had differential Annexin V binding. When incubated shortly in vitro, most Annexin V+ T cells rapidly fragmented their DNA and became terminal transferase-mediated dUTP nick end-labeling positive (TUNEL+), while much fewer Annexin V- cells became TUNEL+. Therefore, those Annexin-V+ cells were truly in a pre-apoptotic stage. The differential spontaneous apoptosis in different tissues is independent of several death/survival-related molecules, including Fas/Fas ligand (FasL), turner necrosis factor (TNF)α, interleukin (IL-15), perforin, B cell lymphoma (Bcl)-2 and independent of virus tropism.

I further investigated the significance of the high Annexin V reactivity of lymphoid T cells. Pre-apoptotic cells were prevented from fragmenting their DNA by anti-CD3 or IL-2 stimulation in vitro. However, this pre-apoptotic phenotype precluded generation of memory. Annexin V reactive cells did not give rise to long-lived memory after being transferred into naïve hosts. The pre-apoptotic phenotype is also an intrinsic property of the epitope. Different proportions of apoptotic cells were found in LCMV effector and, memory T cells specific to two different epitopes, nucleoprotein (NP)396 and GP33. Higher Annexin V reactivity of NP396-specific CD8 T cells was independent of virus tropism and duration of encounter with antigen. Higher expression of IL-7R was found in peripheral, Annexin V- and GP33-specific CD8 T cells, indicating that IL-7-dependent signals may inhibit apoptosis.

Nonlymphoid T cells were more resistant than lymphoid T cells to activation-induced cell death (AICD). When stimulated with anti-CD3 in vitro for 40 hours (hr), a significantly reduced number of splenic transgenic T cells were recovered with much higher frequency of Annexin V reactivity and TUNEL staining than transgenic T cells from PEC. Consistent with the finding that Fas and FasL regulates AICD, a much lower expression of Fas and FasL was observed in PEC and lung transgenic T cells than spleen and lymph nodes after short time stimulation. FasL blockage largely increased cell-number recovery and reduced Annexin V and TUNEL staining of spleen transgenic T cells.

Interestingly, the leukocyte environment played an important role of deciding the fate of transgenic T cells. When placing activated spleen transgenic T cells with excess infected PEC cells, spleen transgenic cells rapidly reduced their Annexin V staining and TUNEL staining and were recovered with greater number after stimulation. Vice versa, PEC transgenic T cells became Annexin V and TUNEL positive with lower numbers of cells recovered when placed with excess splenocytes. Less detection of Annexin V+ cells in peripheral tissues was not due to rapid phagocytosis by macrophages, because Cytochalasin D, which can inhibit phagocytosis, did not induce equal amount of pre-apoptotic cells in spleen and PEC. This reduced death in the periphery may contribute to the long-term maintenance of nondividing nonlymphoid memory T cells, enabling them to efficiently function without being driven into apoptosis.

Overall, this study characterizes in detail the different T cell repertoire usage and apoptosis of virus-specific T cells based on their organ localization and specificities and helps to better understand T cell immunity after infections and vaccine design.

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