The Virus-Specific CD4+ T Cell Response During Acute Lymphocytic Choriomeningitis Virus Infection and into Long Term Memory: a Dissertation

Abstract

CD4+ T cells play a central role in immunity. During virus infections, CD4+ T cells provide the necessary help for B cells to secrete anti-viral antibody and may act as effector cells themselves through the secretion of anti-viral cytokines such as IFN-γ and TNF-α. Recent studies in the lymphocytic choriomeningitis virus (LCMV) system have shown that CD4+ T cells are required to maintain the clearance of persistent viral infections as well as maintain virus-specific memory CD8+ cytotoxic T lymphocytes (CTL). Despite these important functions, surprisingly little information exists concerning the longevity, magnitude, and stability of the CD4+ T cell response following a virus infection. This thesis takes advantage of the well-studied LCMV system to address the above issues as well as to examine the role CD4+ T cells play during heterologous virus infections and to determine the fate of CD4+T cells following a high-dose LCMV infection. The cell surface phenotype of the CD4+ T cells was first examined in C57BL/6 mice acutely infected with LCMV. FACS analysis revealed the modulation of several activation markers on CD4+ T cells during an acute infection with LCMV, consistent with an activated cell phenotype. In addition, 25% of the CD4+ T cells were blast-sized by day 7 post-infection (p.i.) even though the total number of CD4+ T cells did not increase in the spleen during the acute infection. Additional studies were performed using CZ-1, a novel monoclonal antibody (mAb) previously generated in our laboratory that defines a sialic acid-dependent CD45RB-associated epitope. Examination of the expression of the CZ-1 antigen on CD4+ T cells following LCMV infection revealed that the blast-sized CD4+ T cells at day 6 p.i. were CZ-1 +. Further cell surface phenotyping showed that those blast cells activated at day 6 p.i. were CD45RB1oCD44hiCD62L-. This contrasts with the CZ-1-CD45RBhiCD441oCD62L+ resting cell population prior to infection. To determine if memory CD4+ T cells continued to express the CZ-1 epitope long after resolution of the LCMV infection, CD4+CZ-1+ and CD4+CZ-1- populations were purified by cell sorting and placed into an in vitro proliferation assay with LCMV-infected antigen-presenting cells (APC). It was found that the CD4+CZ-1+ population contained virtually all of the virus-specific memory. Thus, these studies indicate that the CZ-1 epitope defines a novel activation and memory marker for murine CD4+T cells. Examination of virus-specific cytokine production using ELISPOT assays showed a significant increase in the number of IFN-γ-secreting cells in the spleen during an acute LCMV-infection. CD8+ T cells made up the majority of the IFN-γ-producing cells, but analysis of the cell culture supernatants by ELISA revealed that the CD4+T cells produced more IFN-γ on a per cell basis. No significant increase in IL-4 levels was detected under these experimental conditions. These data suggest that LCMV infection induces primarily a virus-specific Th1 response that is characterized by increased IFN-γ production. No quantitative information was known about the frequency and longevity of the LCMV-specific CD4+ T cell response. Using limiting dilution assays (LDA), I examined the CD4+ T cell precursor (Thp) frequency in C57BL/6 mice infected with LCMV. The virus-specific CD4+ Thp frequency increased from <1/100,000 in uninfected mice to a peak of approximately 1/600 in FACS-purified splenic CD4+ T cell populations by 10 days p.i. with LCMV. After the peak of the response, the CD4+ Thp frequency decreased only about 2-fold per CD4+ T cell to approximately 1/1200 and remained stable into long-term memory. The CD4+ Thp frequency to each of the two known LCMV major histocompatibility complex (MHC) class II-restricted peptides dropped only 2- to 7-fold from the peak of the acute LCMV response into long-term memory. Thus, the CD4+T cell frequencies remain elevated after the acute infection subsides and remain extremely stable throughout long-term immunity. The above results show that LDA can account for +T cells as being virus-specific following LCMV infection. However, using newer, more sensitive assays based on intracellular cytokine production, >20% of the CD4+ T cells secreted IFN-γ after stimulation with phorbol myristic acid and ionomycin during the peak of the acute CD4+ T cell response. In addition, >10% of the CD4+ T cells secreted IFN-γ after stimulation with the LCMV MHC class II-restricted CD4 peptides. Thus, these new sensitive assays reveal a heretofore unappreciated, yet profound antigen-specific CD4+T cell response during LCMV infection. Infection of mice with a series of unrelated viruses, termed heterologous viruses, causes the reduction of memory CD8+ T cells specific to earlier infections. In order to examine the fate of CD4+ T cells under these conditions, I examined cytokine production and followed the CD4+ Thp frequency following heterologous virus infections. Challenge of LCMV-immune mice with vaccinia virus (VV) resulted in a significant increase in both the amount of IFN-γ protein and the frequency of IFN-γ-producing cells in the peritoneal cavity 3 days after infection as compared to control non-immune mice acutely infected with VV or to LCMV-immune mice alone. Intracellular IFN-γ staining revealed that both CD4+ and CD8+ T cells contributed to this increased IFN-γ production. LDA analysis of the LCMV-specific CD4+ Thp frequency following multiple heterologous virus infections or protein antigen immunizations, revealed that the CD4+ Thp frequency remains stable even under conditions that reduce the LCMV-specific CD8+ CTLp frequency. Additional studies using high-dose LCMV Clone 13 demonstrated that, like CD8+ T cells, there is a decline in detectable LCMV-specific CD4+Thp during overwhelming virus infections. The data presented in this thesis help provide a better understanding of the CD4+ T cell response during virus infections. I make several novel observations, including the demonstration that mAb CZ-1 defines a novel activation and memory marker for CD4+ T cells, that the LCMV-specific memory CD4+ Thp frequency remains extremely stable into long-term immunity, and that heterologous virus infections do not disturb the stable memory CD4+ T cell pool following a virus infection. I also provide data using new sensitive assays based on intracellular cytokine production that there is a much more profound antigen-specific CD4+ T cell response during viral infections than has previously been realized. Finally, I provide evidence that the virus-specific CD4+ T cells become unresponsive following a high-dose LCMV Clone 13 infection. Thus, the data presented in this thesis highlight some important similarities and differences between the CD4+ and CD8+ T cell responses during acute viral infections.