A Role for Perforin in Downregulating T-Cell Responses during Chronic Viral Infection

Death of LCMV-infected perf −/− mice is mediated by CD8 T cells. Three groups of mice, wild type (perf +/+; n = 20), perf deficient (perf −/−; n = 14), or CD8-depleted perf −/− (perf −/− plus anti-CD8;n = 11), were infected i.p. with 2 × 10⁵ PFU of LCMV-Armstrong, and their survival was monitored for 30 days. All +/+ mice survived. In contrast, 9 of 20 of the LCMV-infected perf −/− mice died by day 25 postinfection. In vivo depletion of CD8 T cells in LCMV-infected perf −/− mice (perf −/− plus anti-CD8 group) resulted in 100% survival of these mice.

T-cell activation in perf −/− and +/+ mice chronically infected with LCMV clone 13. Spleen cells from uninfected and LCMV clone 13-infected (day 8) perf −/− and +/+ mice were double stained with CD4/CD44 and CD8/CD44. Panel A shows the total numbers of activated (CD44^hi) and naive (CD44^lo) CD8 T cells in the spleen (average of six mice in each group). Note the increased numbers of activated CD8 T cells in the spleens of perf −/− mice despite the similar viral load in both +/+ and −/− mice (see Table 1). Panel B shows the total number of LCMV-specific IFN-γ producing CD8 T cells in the spleen after infection with LCMV-clone 13 (day 8 postinfection). LCMV-specific CD8 T-cell responses were measured by stimulating the spleen cells with LCMV-specific CTL epitope peptides (NP396-404, GP33-41, and GP276-286) and quantitating the number of IFN-γ-producing CD8 T cells by an ELISPOT assay. Panel C shows a representative FACS profile of spleen cells from LCMV clone 13-infected +/+ and perf −/− mice (day 8). Note the higher percentages of both activated CD8 and CD4 T cells in perf −/− mice.

Cell cycle analysis of T cells from LCMV clone 13-infected +/+ and perf −/− mice. Spleen cells from uninfected and LCMV clone 13-infected (day 8) +/+ and −/− mice were stained with CD8/CD44 and CD4/CD44 and then analyzed for DNA content as described in Materials and Methods. Panel A shows the percentage of activated (CD44^hi) CD8 and CD4 T cells in cycle (S+G₂-M phase) in infected +/+ and −/− mice. Note that similar percentages of activated T cells are in cycle in both +/+ and −/− mice. Approximately 5 to 7% of CD44^hi CD8 and CD4 T cells were in cycle in uninfected +/+ and −/− mice (data not shown). In all groups of mice (uninfected and infected +/+ and −/−) <1 to 2% of “naive” CD44^lo CD8 and CD4 T cells were in S+G₂-M phase; >98% of CD44^lo cells were in G₀-G₁ phase (data not shown). Panel B shows the size (forward scatter) of CD8 CD44^hi and CD4 CD44^hi T cells from LCMV clone 13-infected +/+ and −/− mice. Note that “activated” T cells from perf −/− mice contain a higher proportion of large cells.

Proliferative responses and reactivation-induced apoptosis of T cells from perf +/+ and perf −/− mice after anti-CD3 stimulation. Panel A shows the proliferative responses of splenic T cells from perf +/+ and perf −/− mice to restimulation with anti-CD3. Spleen cells (8 × 10⁶ cells/well in 24-well plates) from uninfected perf +/+ and perf −/− mice were initially stimulated for 72 to 96 h with anti-mouse CD3 antibody (1 μg/ml). After this primary round of stimulation, cells were washed, plated at 5 × 10⁵ viable cells/well (96-well plate), and restimulated with anti-mouse CD3 antibody for another 24 h. Cells were pulsed with [³H]thymidine (1 μCi/well) at the time of restimulation. Panel B shows the relative proportions of apoptotic cells among perf +/+ and perf −/− CD8 T cells after restimulation with anti-CD3 as described above. After restimulation, cells were harvested, and the number of apoptotic cells in the culture was determined as described in Materials and Methods.

Upregulation of Fas and TNFR II (p75) expression on activated T cells. Spleen cells from perf +/+ and perf −/− mice were stimulated in vitro with anti-mouse CD3 antibody for 48 h, and the levels of expression of Fas and TNFR II on CD8⁺ and CD4⁺ T cells were analyzed by flow cytometry. Histograms show log fluorescence intensities. Thin and bold lines represent unstimulated and anti-CD3-stimulated T cells, respectively.

Clonal expansion and deletion of Vβ11⁺ T cells after injection with superantigen SEA. Perf +/+ or perf −/− mice were injected i.v. with SEA (10 μg/mouse). Spleen cells were harvested at the indicated times after injection, and the percentages of Vβ11⁺ CD8⁺ (a) and CD4⁺ (b) cells were determined by flow cytometry. Each value is the average of three mice; the standard deviation is indicated by the bars.