Shutdown of an acute T cell immune response to viral infection is mediated by the proapoptotic Bcl-2 homology 3-only protein Bim

Abstract

We used mutant Fas-deficient (lpr) or Bim-deficient mice to investigate the role of the death receptor and Bcl-2-regulated apoptotic pathways in terminating a physiological T cell response to herpes simplex virus infection. In WT and lpr mice CD8+ antigen-specific T cells were deleted after viral clearance. In contrast, the immune response was not terminated in Bim-deficient mice despite viral clearance, and CD8+ antigen-specific T cells accumulated in the spleen. Thus, Bim is dispensable for viral clearance but is necessary for the death of activated T cells when immune responses are terminated. These findings have implications for the therapeutic manipulation of immune responses to infections and immunization.

Fig. 1.

Accumulation of leukocytes and CD8⁺ T cells in Bim-deficient mice. WT, bim^–/–, and lpr mice were injected with HSV-1 in the hind foot. Total leukocyte numbers and numbers of CD8⁺ T cells in draining popliteal lymph nodes (LN) (A and B, respectively) and spleen (C and D, respectively) were quantified by cell counting and immunofluorescent staining with surface marker-specific mAbs at specified time points after infection. Two lymph nodes were harvested from each mouse, and the lymph nodes from three mice of each genotype were pooled to facilitate analysis. Data shown represent numbers of lymph node leukocytes or CD8⁺ T cells per mouse and are mean ± SD for nine WT and nine bim^–/– mice but only for three lpr mice for which SD is not shown. Spleens were not pooled, and data represent mean ± SD for three to seven mice of each genotype.

Fig. 2.

Kinetics of an acute immune response to HSV infection in the draining popliteal nodes and accumulation of HSV-specific CD8⁺ T cells in the spleens of bim^–/– mice. The total numbers of HSV-specific CD8⁺ T cells in draining lymph nodes (LN) of WT, lpr, and bim^–/– mice were quantified at the specified time points before and after HSV infection by using surface staining with K^b-gB_498–505 tetramers (A) or by staining for intracellular IFN-γ in cells that had been restimulated in vitro with HSV gB_498–505 peptide (B). (The gB_498–505 peptide is the major HSV-derived epitope recognized by CD8⁺ T cells in infected C57BL/6 mice.) Lymph nodes from three mice of each genotype were pooled (this was repeated three times in the case of WT and bim^–/– mice, i.e., nine mice). Data points represent average cell numbers (mean ± SD) per mouse. The total numbers of HSV-specific CD8⁺ T cells in spleens of WT, lpr, and bim^–/– mice were quantified at the specified times before and after HSV infection by surface staining with K^b-gB tetramers (C) or by intracellular staining for IFN-γ in cells that had been restimulated in vitro with HSV gB_498–505 peptide (D). Data represent mean ± SD from three to nine mice of each genotype.

Fig. 3.

Rates of in vivo proliferation of HSV-specific CD8⁺ T cells are indistinguishable among WT, lpr, and bim^–/– mice. HSV infected WT, lpr, and bim^–/– mice were pulsed for 20 h with BrdUrd before harvesting organs for analysis. HSV-specific CD8⁺ T cells were FACS-sorted after surface labeling with anti-CD8 mAb plus with K^b-gB_498–505 tetramers and were then stained intracellularly with an anti-BrdUrd mAb to identify cells synthesizing DNA and hence undergoing proliferation. HSV-specific CD8⁺ T cells from WT, lpr, and bim^–/– mice were collected from lymph nodes 4 and 7 days after HSV infection. HSV-specific CD8⁺ T cells from spleen were harvested 7 days after infection (and 20 days after infection only in bim^–/– mice because numbers in WT and lpr animals are too low for analysis). Comparisons are made between control CD8⁺ T cells from uninfected mice (gray lines) and HSV-specific T cells from infected mice (black lines) of the same genotype.

Fig. 4.

Expression of cell surface activation markers on HSV-specific CD8⁺ T cells is indistinguishable among WT, lpr, and bim^–/– mice. CD8⁺ T cells from popliteal nodes (LN) at day 5 (A) or day 7 (B) post-HSV infection and from spleen at day 7 postinfection (C) were stained with HSV gB_498–505 peptide tetramers and either mAbs to CD44 or CD25. Profiles of HSV-specific CD8⁺ T cells (black lines) are plotted with profiles from control CD8⁺ T cells of uninfected mice (gray lines).

Fig. 5.

No differences in viral clearance kinetics and distribution of HSV-specific CD8⁺ T cells to nonlymphoid organs were found among HSV-infected WT, lpr, and bim^–/– mice. (A) Feet from HSV-infected mice were homogenized in medium, and extracts were serially diluted for plaque-forming assays. Data represent arithmetic mean (pair of feet) ± SD of three mice from each genotype. PFU, plaque-forming unit. (B) CD8⁺ T cells were enriched from pooled lungs and livers of three WT and three bim^–/– mice 7 days post-HSV infection. Data represent numbers of HSV-specific CD8⁺ T cells per organ per mouse.

Fig. 6.

HSV-specific CD8⁺ T cells from bim^–/– mice are resistant to cytokine withdrawal. CD8⁺ T cells from lymph nodes (LN) (A) or spleen (B) of HSV-infected WT or bim^–/– mice were cultured in the presence or absence of IL-7. Viability of HSV-specific CD8⁺ T cells was determined by staining with anti-CD8 mAb, anti-TCRVα2 mAb [most HSV-specific CD8⁺ T cells in C57BL/6 mice express Vα2-containing TCR; (33)], and FITC-annexin V. Live cells were considered as being FITC-annexin V^–. Data represent mean ± SD of HSV-specific CD8⁺ T cells from three mice of each genotype.