Aberrant CD8+ T-cell responses and memory differentiation upon viral infection of an ataxia-telangiectasia mouse model driven by hyper-activated Akt and mTORC1 signaling

Abstract

Immune system-related pathology is common in ataxia-telangiectasia (A-T) patients and mice that lack the protein kinase, A-T mutated (ATM). However, it has not been studied how ATM influences immune responses to a viral infection. Using the lymphocytic choriomeningitis virus (LCMV) infection model, we show that ATM(-/-) mice, despite having fewer naïve CD8⁺ T cells, effectively clear the virus. However, aberrant CD8⁺ T-cell responses are observed, including defective expansion and contraction, effector-to-memory differentiation, and a switch in viral-epitope immunodominance. T-cell receptor-activated, but not naïve, ATM(-/-) splenic CD8⁺ T cells have increased ribosomal protein S6 and Akt phosphorylation and do not proliferate well in response to IL-15, a cytokine important for memory T-cell development. Accordingly, pharmacological Akt or mammalian target of rapamycin complex 1 (mTORC1) inhibition during T-cell receptor activation alone rescues the IL-15 proliferation defect. Finally, rapamycin treatment during LCMV infection in vivo increases the number of memory T cells in ATM(-/-) mice. Altogether, these results show that CD8⁺T cells lacking ATM have hyperactive Akt and mTORC1 signaling in response to T-cell receptor activation, which results in aberrant cytokine responses and memory T-cell development. We speculate that similar signaling defects contribute to the immune system pathology of A-T, and that inhibition of Akt and/or mTORC1 may be of therapeutic value.

Figure 1

Fewer splenic T cells and altered immunodominance hierarchy in CD8⁺ T cells in ataxia-telangiectasia mutated (ATM)^−/− mice in response to lymphocytic choriomeningitis virus (LCMV) infection. A: Total splenocytes in wild-type ATM^+/+ (black circles in A–D) and ATM^−/− (grey squares in A–D) mice at day 0 (d0), day 8 (d8), and day 41 (d41) post-LCMV infection. B: Total splenic CD8⁺ T cells at d0, d8, and d41 after post-LCMV infection ATM^+/+ and ATM^−/− mice. C: Total splenic CD4 T cells at d0, d8, and d41 post-LCMV infection of ATM^+/+ and ATM^−/− mice. D: Total D^bNP396-CD8+ and D^bGP33-CD8+ T cells at d8 and d41 post-LCMV infection in ATM^+/+ and ATM^−/− mice, respectively. E: Total CD8⁺ T cells in the blood (expressed as the number in 1 × 10⁶ peripheral blood mononuclear cells (PBMCs) in wild-type and ATM^−/− mice at d0, d8, day 15, day 20, day 30, and d41 post-LCMV infection. F: Percentage of D^bNP396-CD8+ and D^bGP33-CD8+ T cells at d0, d8, day 15, day 20, day 30, and d41 post-LCMV infection in wild-type and ATM^−/− mice, respectively. A–C: 5 ATM^+/+ and 5 ATM^−/− mice were analyzed in two separate experiments on d0; the combined data from two separate experiments is shown representing a total of 5 ATM^+/+ and 5 ATM^−/− mice analyzed on d8 postinfection and a total of 7 ATM^+/+ and 8 ATM^−/− mice analyzed on day 41 postinfection from two separate experiments. For blood analyses in data from E and F, 10 ATM^+/+ and 9 ATM^−/− mice were analyzed in two independent experiments. Statistically significant differences are indicated directly or by asterisks, which represent P values from an unpaired t-test of <0.05.

Figure 2

Defective memory CD8⁺ T-cell differentiation ataxia-telangiectasia mutated (ATM)^−/− mice post-LCMV infection. A: Number of viral specific D^bNP396-CD8+, D^bGP33-CD8+ and D^bGP61-CD4⁺ memory T cells in spleens from ATM^+/+ and ATM^−/− mice at day 41 after lymphocytic choriomeningitis virus (LCMV) infection. B: Comparison of median fluorescence intensities (MFI) obtained from fluorescence-activated cell sorting (FACS) analyses of intracellular staining of interferon-γ, tumor necrosis factor-α, and IL-2 of D^bGP33-CD8 T cells from ATM^+/+ and ATM^−/− mice at day 41 postinfection. C: Relative intracellular expression of granzyme B (GzmB) in D^bGP33-CD8+ T cells from in ATM^+/+ and ATM^−/− mice at days 8 and 41 postinfection. The MFI from wild-type cells was given a value of 1.0 for normalization of the results. D: Top row, percent KLRG1^hi, CD127^hi, and CD62L^hi D^bGP33-CD8+ T cells at days 8, 15, 20, 30, and 41 in the blood of LCMV-infected ATM^+/+ (bold black line) and ATM^−/− (dashed gray line) mice. Bottom row, percent KLRG1^hi, CD127^hi, and CD62L^hi D^bGP33-CD8+ T cells at days 8 and 41 post-LCMV infection for ATM^+/+ (black circles) and ATM^−/− (grey squares) mice are shown. Whereas for the blood, the combined data from two separate experiments representing 7 ATM^+/+ and 9 ATM^−/− mice are shown, and for the spleen, the combined data from separate experiments representing 5 pairs of wild-type and ATM^−/− mice on day 8 and 7 wild-type and 9 ATM^−/− mice on day 41 are shown. Statistically significant differences are indicated directly or by asterisks, which represent P values from an unpaired t-test of <0.05.

Figure 3

T-cell receptor (TCR)-activated ataxia-telangiectasia mutated (ATM)^−/− CD8⁺ lymphocytes sustain heightened Akt phosphorylation and exhibit a proliferation defect in response to IL-15 treatment that is rescued by the Akt inhibitor triciribine. A: Experimental scheme used to test the response of ATM^−/− lymphocytes to IL-2 and IL-15, which after TCR activation (using anti-CD3 and anti-CD28 antibodies) skews the cell populations toward effector and memory phenotypes in vitro. B: Number of CD8⁺ T cells after IL-15 (right) or IL-2 (left) treatment of ATM^+/+ and ATM^−/− splenocytes, according to the scheme in A. For culturing in IL-2 and IL-15, the starting CD8⁺ T-cell number was 2.5 × 10⁴ and 1 × 10⁵, respectively. The P value from a two-tailed unpaired t-test is indicated for the IL-15 experiment, and no difference was observed in the IL-2 experiment. C: Western blot analyses of phosphorylated Akt (S473, T308), PTEN (S380/T382/383), S6 (S235/236), AMPK (T172), and ACC (S79) and the corresponding unphosphorylated proteins in splenic CD8⁺ T lymphocytes from wild-type and ATM^−/− mice treated with IL-15 according to the scheme in A. Actin was used as the loading control. Similar results were obtained 3 times from different sets of mice. D: Total CD8⁺ T cells from ATM^+/+ (open bars) or ATM^−/− (filled bars) mice after IL-15 treatment according to the scheme, (A) with (+) or without (−) 10 nmol/L triciribine added at the stage indicated by T (ie, during TCR activation alone or during both TCR activation and IL-15 treatment). (E) Total percent CD8⁺ T-cell death evaluated by propidium iodide (PI)-positive cells after IL-15 treatment according to the scheme (A), with (+) or without (−)10 nmol/L triciribine added at the stage indicated by T. Analysis of cells from ATM^+/+ (open bars) and ATM^−/− (filled bars) mice are shown. D and E: The P value from a two-tailed unpaired t-test for the comparison of ATM^−/− to ATM^+/+ without drug treatment is shown above the brackets, and those for comparisons of drug-treated to untreated within in each group/genotype are as indicated.

Figure 4

Rapamycin treatment during T-cell receptor (TCR) activation rescues the IL-15 proliferation defect in ataxia-telangiectasia mutated (ATM)^−/− splenic CD8⁺ T lymphocytes. A: Western blot analyses (as described in Figure 3C), except the ATM^+/+ and ATM^−/− cells were collected before TCR stimulation (pre-TCR) and 72 hours after TCR activation (post-TCR) (as described in Figure 3A). B: Graphed are the median fluorescence intensities (MFI) from intracellular staining for phosphorylated ribosomal protein S6 (phospho-S6; S235/236) calculated from FACS plots of ATM^+/+ (white bars) and ATM^−/− (gray bars) CD8⁺ T cells before (at 0 hours), and at 24, 48, and 72 hours of TCR activation in vitro. C: Same as B, except the analysis of CD8⁺ T cells on day 8 post-LCMV infection in vivo is shown. The data from 5 ATM^+/+ and 5 ATM^−/− mice were analyzed. D: Total CD8⁺ T cells from ATM^+/+ (open bars) or ATM^−/− (filled bars) after IL-15 treatment, according to the scheme (A), with (+) or without (−) 10 nmol/L rapamycin added at the stage indicated by “R” (ie, during TCR alone or during both TCR activation and IL-15 treatment). E: Total percent CD8⁺ T-cell death evaluated by propidium iodide (PI)-positive cells after IL-15 treatment, according to the scheme (A), with (+) or without (−) 10 nmol/L rapamycin added at the stage indicated by “R”. Analysis of cells from ATM^+/+ (open bars) and ATM^−/− (filled bars) mice are shown. D and E: The P value from a two-tailed unpaired t-test for the comparison of ATM^−/− to ATM^+/+ without drug treatment is shown above the brackets, and those for comparisons of drug treated to untreated within each group/genotype are as indicated.

Figure 5

Rapamycin treatment during lymphocytic choriomeningitis virus infection improves memory CD8⁺ T-cell development in ataxia-telangiectasia mutated (ATM)^−/− mice. A: Percent of KLRG1^hi, CD127^hi, and CD62L^hi D^bGP33-CD8+ T cells in ATM^+/+ and ATM^−/− mice at days 8, 15, and 30 post-LCMV infection with (dashed gray) or without (black line) rapamycin treatment between day −1 to day +8 (indicated by the gray shading). B: Same as in A, except total D^bGP33+ and D^bNP396+ CD8+ T lymphocytes were analyzed. Statistically significant differences are indicated by asterisks, which represent P values from an unpaired t-test of <0.05. PBMCs, peripheral blood mononuclear cells. X-axes denotes days after LCMV infection.