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. 2011 Jun;178(6):2740-51.
doi: 10.1016/j.ajpath.2011.02.022.

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

Anthony D D'Souza  1 , Ian A Parish, Sharen E McKay, Susan M Kaech, Gerald S Shadel
Affiliations

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

Anthony D D'Souza et al. Am J Pathol. 2011 Jun.

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.

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Figures

Figure 1
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 DbNP396-CD8+ and DbGP33-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 DbNP396-CD8+ and DbGP33-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
Figure 2
Defective memory CD8+ T-cell differentiation ataxia-telangiectasia mutated (ATM)−/− mice post-LCMV infection. A: Number of viral specific DbNP396-CD8+, DbGP33-CD8+ and DbGP61-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 DbGP33-CD8 T cells from ATM+/+ and ATM−/− mice at day 41 postinfection. C: Relative intracellular expression of granzyme B (GzmB) in DbGP33-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 KLRG1hi, CD127hi, and CD62Lhi DbGP33-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 KLRG1hi, CD127hi, and CD62Lhi DbGP33-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
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
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
Figure 5
Rapamycin treatment during lymphocytic choriomeningitis virus infection improves memory CD8+ T-cell development in ataxia-telangiectasia mutated (ATM)−/− mice. A: Percent of KLRG1hi, CD127hi, and CD62Lhi DbGP33-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 DbGP33+ and DbNP396+ 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.
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