T Lymphocyte Apoptosis and Memory in Viral Infection: A Dissertation

Abstract

Acute viral infections in humans and mice induce T lymphocyte responses which mediate viral clearance and result in the establishment of immunological memory. The course of an immune response to acute viral infection is associated with an immune deficiency in the lymphocyte compartment. This is usually characterized by the inability of lymphocytes to productively respond to mitogen or recall antigen. This thesis examined the acute lymphocytic choriomeningitis virus (LCMV) infection of the mouse and showed that T lymphocytes isolated from acutely LCMV-infected mice underwent activation-induced apoptosis upon signalling through the T-cell receptor (TcR)-CD3 complex. Kinetic studies demonstrated that this sensitivity to apoptosis directly correlated with the induction of immune deficiency, as measured by impaired proliferation in response to anti-CD3 antibody or to concanavalin A. Cell cycling in interleukin-2 (IL-2) alone stimulated proliferation of LCMV-induced T cells without inducing apoptosis, but preculturing of T cells from acutely-infected mice in IL-2 accelerated apoptosis upon subsequent TcR-CD3 crosslinking. T lymphocytes isolated from mice after the acute infection were less responsive to IL-2, but IL-2 receptor-bearing T cells, presumably memory T cells, responding to IL-2 were primed in each case to die a rapid apoptotic death upon TcR-CD3 crosslinking. These results indicated that virus infection-induced unresponsiveness to T-cell mitogens is in part attributable to apoptosis of the activated lymphocytes and suggest that the sensitization of memory cells by IL-2 and other stimulatory cytokines induced during an acute infection will cause them to die upon antigen recognition, thereby impairing specific responses to nonviral (recall) antigens. The cytotoxic T lymphocyte (CTL) response to acute LCMV infection is characterized by a massive (10-20 fold) expansion of CD8+ cell number, which after clearance of virus declines in number and returns to levels present prior to infection. This thesis documents the presence of high levels of apoptotic lymphocytes in situ in the spleens of mice during the silencing of the immune response to acute LCMV infection. Apoptotic cells were detected by an in situ nucleotidyl transferase (ISNT) assay. Both T and B lymphocytes, as revealed by immunohistochemical analysis, are shown to be dying in vivo, the latter in clusters. A biphasic occurrence of apoptosis during the course of the acute infection was found, with an increase in numbers of apoptotic cells above background at day 3 post-infection, and at day 11 post-infection, a second more pronounced peak coincident with the decline of the CTL response to the infection and with the decrease in total spleen leukocyte number. Apoptosis in vivo was detected in lpr mice lacking Fas expression, a molecule involved in lymphocyte apoptosis. Fas expression thus may not be required for lymphocyte apoptosis in the context of an acute viral infection. Apoptosis in situ and the silencing of the CD8+ T lymphocyte response to acute LCMV infection were unaffected by the enforced lymphocyte-directed expression of Bcl-2, a protein blocking IL-2 deprivation-induced apoptosis of lymphocytes. Experiments aimed at addressing the role of Bcl-2-sensitive apoptotic pathways in the development of viral persistence revealed that high-dose infection of Bcl-2-transgenic mice results in death of the animals. Flow cytometric analysis showed an accumulation of Thy1.2+ T cells in the lungs of these animals, and the air spaces in the lungs were occluded with cellular and fluid infiltrates. These results suggest that the pathology seen in the Bcl-2-transgenic mice upon high-dose infection is perhaps immune response-mediated (an immunopathology). This is consistent with a role for Bcl-2-sensitive pathways of lymphocyte apoptosis in the pathogenesis of persistent LCMV infection. The in situ demonstration of apoptosis in spleens during infection provide direct in vivo evidence for the death of lymphocytes during the recovery from an acute viral infection. This indicates that apoptotic elimination of the population en masse is a mechanism for halting an antiviral immune response upon clearance of virus. Furthermore, the data argue that IL-2 deprivation-driven apoptosis, upon clearance of virus, of the expanded T lymphocyte compartment is not the major mechanism involved in the silencing of the T cell response to acute LCMV infection. Resolution of an acute immune response leads into the generation of longterm immunological memory. Since this thesis focussed on T cell responses in viral infection, it was important to characterize the in vivo state of memory CD8+ T cells. During acute LCMV infection, the majority of the LCMV-specific CTL activity tested immediately ex vivo was mediated by CD8+ L-selectin-Mac-1+ CTL. The L-selectin- population of CD8+ cells elicited during acute infection also carried \u3e99% of the restimulatable CD8+ CTLp to LCMV, and these required added IL-2 for development into effectors in vitro. In contrast to the acute infection, most of the virus-specific CTLp in immune mice were L-selectin+. Examination of CD8+ T cells in LCMV-immune mice revealed that a L-selectin+ blast-sized population of cycling CD8+ cells contained CTLp which developed into effector CTL in the absence of added IL-2. These cells also expressed Mac-1 and IL-2R. Flow cytometric sorting for IL-2R+ and IL-2R-CD8+ cells in the immune animal revealed, by limiting dilution analysis, similar frequencies of CTLp in both populations. In bulk restimulation assays, the CD25+ CTLp did not require added IL-2 for their in vitro development into effectors, whereas the CD25- CTLp did. Hence, the different requirements for CTLp to effector development in vitro reflect qualitative differences in the in vivo state of the CTLp in the various subpopulations. LCMV-specific memory CTLp not requiring added IL-2 for differentiation were also found in the small-sized, non-cycling, CD8+L-selectin- cells. In contrast, the small-sized, non-cycling, CD8+L-selectin+, and CD8+IL-2R- populations also carried CTLp, but these required added IL-2 for development into effector CTL. Hence, T cell memory to LCMV is distributed among various lymphocyte subpopulations in immune animals, and the presence of an activated cycling cell component may account for the stability and long-term perpetuation of antiviral immunological memory. In summary, the susceptibility of activated T lymphocytes to apoptosis probably explains an aspect of virus-induced immune deficiency and allows for the establishment of homeostasis subsequent to the resolution of an acute viral infection