Viral immune evasion due to persistence of activated T cells without effector function

Abstract

We examined the regulation of virus-specific CD8 T cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. Our study shows that within the same persistently infected host, different mechanisms can operate to silence antiviral T cell responses; CD8 T cells specific to one dominant viral epitope were deleted, whereas CD8 T cells responding to another dominant epitope persisted indefinitely. These virus-specific CD8 T cells expressed activation markers (CD69(hi), CD44(hi), CD62Llo) and proliferated in vivo but were unable to elaborate any antiviral effector functions. This unresponsive phenotype was more pronounced under conditions of CD4 T cell deficiency, highlighting the importance of CD8- CD4 T cell collaboration in controlling persistent infections. Importantly, in the presence of CD4 T cell help, adequate CD8 effector activity was maintained and the chronic viral infection eventually resolved. The persistence of activated virus-specific CD8 T cells without effector function reveals a novel mechanism for silencing antiviral immune responses and also offers new possibilities for enhancing CD8 T cell immunity in chronically infected hosts.

Figure 1

CD8 T cell effector functions are lost during chronic infection of CD4^−/− mice. (A) Serum virus titers were measured in +/+ (•) and age-matched CD4^−/− (○) mice at various times after infection with LCMV-t1b. The limit of detection is indicated by the dashed line. (B) CTL activity of splenocytes from +/+ (▪, □) or CD4^−/− (♦, ⋄) mice at 90 d after infection with LCMV-t1b was measured after 5 d restimulation in vitro. Target cells were either uninfected (open symbols) or LCMV infected (filled symbols). (C) Limiting dilution analysis was performed using splenocytes from either +/+ (▴) or CD4^−/− (▵) mice at 90 d after infection with LCMV-t1b. The frequency of LCMV-specific cells per spleen is indicated in parentheses. (D) Virus-specific IFN-γ secreting cells were enumerated using single cell ELISPOT assays. Splenocytes were isolated from either +/+ or CD4^−/− mice at 90 d after inoculation with LCMV-t1b.

Figure 2

T cell deletion and functional unresponsiveness are distinct mechanisms for silencing antiviral immune responses. Naive +/+ mice, +/+ immune mice (161 d after LCMV Armstrong infection; 2 × 10⁵ PFU, i.p.), and +/+ or CD4^−/− mice infected with LCMV-t1b (2 × 10⁶ PFU, i.v.) 60 d previously were checked for the physical presence and functional responsiveness of LCMV-specific CD8 T cells. (A–H) LCMV-specific CD8 T cells were visualized using MHC class I tetramers complexed to viral peptides. Cells were costained with anti-CD8–PE, anti-CD44–FITC, and either D^b(GP33–41) or D^b(NP396–404) tetramers conjugated to allophycocyanin. The histograms show gated CD8 lymphocytes, and the percentage of CD8 cells costaining with either D^b(GP33–41) or D^b(NP396–404) are indicated in the corresponding upper right quadrant. Where not shown values are <0.2%. (I–L) Numbers of LCMV-specific IFN-γ producing cells were enumerated using single-cell cytokine ELISPOT assays. The number of splenocytes producing IFN-γ after stimulation with either GP33–41 (black bars) or NP396–404 peptides (hatched bars) are shown (± SD).

Figure 3

Kinetics of LCMV-specific CD8 T cell responses during chronic infection. Splenocytes were prepared from either +/+ or CD4^−/− mice at various days after infection with LCMV-clone 13 (2 × 10⁶ PFU, i.v.). The total numbers of GP33- (□) and NP396- (○) specific CD8 T cells were enumerated by staining with recombinant MHC H-2D^b tetramers, and IFN-γ ELISPOT assays were performed to determine the functional responsiveness of these cells (GP33–41, ▪; NP396–404, •). Serum virus titers were also determined and are represented by the gray area. Mean values are shown for two to four mice at each time point and the limit of detection is represented by the dashed line. Standard deviations were <10%.

Figure 4

Chronic LCMV infection and CD8 T cell unresponsiveness ensue after transient loss of CD4 T cells. +/+ mice were transiently depleted of CD4 T cells by injection with the anti-CD4 antibody, GK1.5, 1 d before and 3 d after infection with LCMV-t1b. (A) Serum virus titers were determined in +/+ (•) and GK1.5 treated (□) +/+ mice at various days after infection. (B) Reconstitution of CD4 T cells in GK1.5-treated mice was determined at 60 d after infection with LCMV-t1b. Splenocytes were costained for CD4 and the activation marker CD44. The percentage of CD4⁺ cells are indicated in the upper quadrants. By this time point, both +/+ and GK1.5-treated mice contained comparable numbers of CD4 T cells. (C) Splenocytes from GK1.5-treated mice were prepared at 60 d after infection with LCMV-t1b and both the total number (measured by tetramer staining) and the number of IFN-γ–secreting (measured by ELISPOT) GP33- or NP396-specific CD8 T cells were determined. Greater than 98.5% of GP33-specific T cells were unresponsive and NP396-specific T cells were not detectable. In A and C the limit of detection is indicated by the dashed line. At least three mice were analyzed at each time point.

Figure 5

GP33-specific CD8 T cells in chronically infected CD4^−/− mice are activated and proliferate, but are refractive to stimulation with PMA and ionomycin. (A) CD69 expression on freshly explanted splenocytes from +/+ and CD4^−/− mice at 78 d after infection was assessed using flow cytometry. (B) IFN-γ production by GP33-specific CD8 T cells after PMA and ionomycin stimulation. Splenocytes were isolated from +/+ and CD4^−/− mice at 108 d after infection with LCMV-t1b. (C) The in vivo proliferation of GP33-specific CD8 T cells in LCMV-t1b infected +/+ and CD4^−/− mice (day 100 after infection) was assessed by in vivo BrdU labeling. Mice were fed BrdU in their drinking water for a total of 8 d. All histograms show D^b(GP33–41)-positive cells and the percentage of GP33-specific T cells within each region is indicated.