The tumor suppressor Tsc1 enforces quiescence of naive T cells to promote immune homeostasis and function

Abstract

The mechanisms that regulate T cell quiescence are poorly understood. We report that the tumor suppressor Tsc1 established a quiescence program in naive T cells by controlling cell size, cell cycle entry and responses to stimulation of the T cell antigen receptor. Abrogation of quiescence predisposed Tsc1-deficient T cells to apoptosis that resulted in loss of conventional T cells and invariant natural killer T cells. Loss of Tsc1 function dampened in vivo immune responses to bacterial infection. Tsc1-deficient T cells had more activity of the serine-threonine kinase complex mTORC1 but less mTORC2 activity, and activation of mTORC1 was essential for the disruption of immune homeostasis. Therefore, Tsc1-dependent control of mTOR is crucial in actively maintaining the quiescence of naive T cells to facilitate adaptive immune function.

Figure 1. Tsc1 deficiency leads to disrupted homeostasis of peripheral T cell pools

(a) Flow cytometry of thymocytes in wild-type (WT) and Tsc1^−/− mice. Lower panels, numbers of CD4SP and CD8SP thymocytes (n=4–7). (b) Flow cytometry of CD4 and CD8 T cells in the spleens of WT and Tsc1^−/− mice. Lower panels, proportions and absolute numbers of CD4 and CD8 T cells in the spleens of WT and Tsc1^−/− mice (n=4–6). (c) CD62L and CD44 expression on splenic T cells of WT and Tsc1^−/− mice. (d) Flow cytometry of iNKT cells (TCRβ⁺CD1d-PBS57⁺) in the spleens of WT and Tsc1^−/− mice (n≥5). (e) Flow cytometry of CD4 and CD8 T cells in the spleens of mixed BM chimeras. BM stem cells from wild-type (CD45.1⁺) and WT or Tsc1^−/−mice (CD45.2⁺) were mixed at 1:1, and transferred into sublethally irradiated Rag1^−/− mice to generate mixed BM chimeras, followed by analysis at 8 weeks after reconstitution. NS, not significant; * P < 0.005; ** P < 0.0005; *** P < 0.0001. Data are representative of 4 (a–d) and 2 (e) independent experiments.

Figure 2. Tsc1 deletion results in markedly elevated apoptosis of T cells

(a) Caspase activity by FITC-VAD-FMK staining in freshly isolated WT and Tsc1^−/− T cells. Lower panels, proportions of caspase-positive (Casp⁺) T cells (n=7–8). * P <0.005; ** P < 0.0005. (b) Flow cytometry of spleen (upper) and lymph node (lower) cells in the recipient mice (CD45.2⁺) 6 days after adoptive transfer of equal numbers of CD45.1⁺ (spike) cells and WT or Tsc1^−/− donor cells (CFSE-labeled). SPL, spleen; PLN, peripheral lymph nodes. (c) Annexin V and 7-AAD staining in naive WT or Tsc1^−/− T cells cultured with IL-7 in vitro for 3days (left), and expression of IL-7Rα on freshly isolated cells (right). (d) Detection of PI⁺ apoptotic cells after 16 hours of stimulation of CD8 T cells with anti-CD3, anti-CD3-CD28, or PMA-ionomycin. (e) Kinetics of apoptosis (measured by frequency of PI⁺ apoptotic cells) after anti-CD3-CD28 stimulation of CD4 and CD8 T cells. (f) Detection of PI⁺ apoptotic cells in WT and Tsc1^−/− CD8 T cells pretreated with vehicle or caspase inhibitor Z-VAD-FAM for 1 hour and then activated with anti-CD3-CD28 for 10 hours. Although not shown here, similar results were observed in CD4 T cells (d,f). Data are representative of 4 (a,c), 2 (b,e), 5 (d) and 3 (f) independent experiments.

Figure 3. Tsc1-deficient T cells die via the Bcl-2 family-dependent intrinsic apoptotic pathway

(a) Intracellular Bcl-2 expression in WT and Tsc1^−/− T cells. (b) Flow cytometry of CD4 and CD8 T cells in the spleens of WT, Tsc1^−/−, Bcl2-TG, and Tsc1^−/− Bcl2-TG mice. (c) Proportions and absolute numbers of CD4 and CD8 T cells in WT, Tsc1^−/−, Bcl2-TG, and Tsc1^−/−Bcl2-TG spleens (n=3–6). NS, not significant; * P <0.05; ** P < 0.005; *** P< 0.0001. (d,e) Detection of PI⁺ apoptotic cells after 16 hours of stimulation of CD4 (d) and CD8 (e) cells with anti-CD3-CD28. (f) Expression of Bim_EL and Bim_L in naive and anti-CD3-CD28 activated WT and Tsc1^−/− CD4 T cells. Numbers above (Bim_EL) and below (Bim_L) lanes indicate band intensity relative to that of the loading control-actin. (g) ROS production in freshly isolated WT and Tsc1^−/− T cells. Data are representative of 3 (a,d–g) and 4 (b,c) independent experiments.

Figure 4. Tsc1 deficiency causes cell-autonomous loss of quiescence in vivo and hyperactive responses to TCR stimulation

(a) Cell size of freshly isolated WT and Tsc1^−/− T cells. FSC, forward scattering. (b) BrdU staining in splenocytes of WT and Tsc1^−/− mice 16 hours after injection with BrdU (n=3). (c) CD122 and CD44 expression on gated CD8 splenocytes of WT and Tsc1^−/− mice. Right, ratios of CD122⁺/CD122⁻ among CD44^hi populations (n=5–8). (d) CD122 and CD44 expression on gated CD8 splenocytes in the mixed BM chimeras generated as in Fig. 1e. Right, ratios of CD122⁺/CD122⁻ cells among CD44^hi populations of the CD45.2⁺ cells (n=3). (e) Cell size of CD45.2⁺ cells in the mixed BM chimeras. (f) Flow cytometry of WT and Tsc1^−/− naive T cells after stimulation with anti-CD3-CD28 for 16 hours for the measurements of cell size (upper) and ROS production (lower). (g) BrdU staining in naive T cells activated with anti-CD3-CD28 for 20 hours, followed by pulsing with BrdU for 90 min. (h) Expression of activation markers in WT and Tsc1^−/− naive T cells after stimulation with anti-CD3-CD28 for 16 hours. * P <0.05; ** P < 0.01; *** P< 0.0001. Data are representative of 5 (a,c,f), 3 (b,d,e,g), and 4 (h) independent experiments.

Figure 5. Tsc1-dependent gene expression programs

(a) A subset of genes differentially regulated in WT and Tsc1^−/− CD4 T cells activated with TCR for 4 hours (≥ 2-fold difference with false discovery rate <0.1). Triplicate samples were used in the analysis. (b) Real-time PCR analysis of selected genes in WT and Tsc1^−/− CD4 T cells activated for various times. Data are representative of 2 independent experiments.

Figure 6. Loss of T cell quiescence results from inducible deletion of Tsc1 and is independent of cell survival

(a–d) Analyses of WT and Tsc1-CreER mice at 2 weeks after tamoxifen injection, for cell size of freshly isolated T cells (a), BrdU staining in splenocytes of mice 16 hours after injection with BrdU (b), splenic T cell populations (c), and PI⁺ apoptotic cells after 16 hours of anti-CD3-CD28 stimulation of naive T cells (d). (e–h) Analyses of WT and Tsc1^−/− mice expressing the Bcl2 transgene, for expression of CD122 and CD44 on gated CD8 splenocytes (lower left, proportions of total CD44^hi populations; lower right, ratios of CD122⁺/CD122⁻ populations among CD44^hi cells, n=4–6) (e), cell size of freshly isolated T cells (f), and BrdU staining in splenocytes of mice 16 hours after injection with BrdU (g). (h) Flow cytometry of naive T cells after stimulation with anti-CD3-CD28 for 16 hours for the measurements of cell size, ROS production, and expression of CD25 and CD69. NS, not significant; * P = 0.0001. Data are representative of 2 (a–d,h), 4 (e,f), and 3 (g) independent experiments.

Figure 7. Tsc1 regulates mTORC1 and mTORC2 activities, with mTORC1 activation essential to disrupt immune quiescence and homeostasis

(a) Phosphorylation of S6K1, S6 and 4EBP1 in WT and Tsc1^−/− naive CD4 T cells after they were stimulated with anti-CD3-CD28 for various times. (b) Phosphorylation of Akt (Ser473), Foxo1 and Foxo3 in WT and Tsc1^−/−CD4 T cells after they were stimulated with anti-CD3-CD28 for various times. *, non-specific bands. Numbers below lanes (a,b) indicate band intensity relative to that of the loading control β-actin. (c) Cell size of freshly isolated splenocytes after WT and Tsc1^−/− mice were treated with daily injection of rapamycin for a total of 3 or 5 days. (d) Flow cytometry of splenocytes in the recipient mice (CD45.2⁺) 6 days after adoptive transfer of equal numbers of CD45.1⁺ (spike) cells and WT or Tsc1^−/− donor cells (CFSE-labeled) that were purified from mock or rapamycin-treated mice. Similar results were obtained in the recipient lymph nodes (not shown here). (e,f) Naive T cells from rapamycin or mock treated WT and Tsc1^−/− mice were stimulated with anti-CD3-CD28 overnight, followed by measurements of PI⁺ apoptotic cells (e) and cell size (f). Data are representative of 5 (a,b) and 3 (c–f) independent experiments.

Figure 8. Tsc1 deficiency dampens antibacterial immune response in vivo

(a,b) Flow cytometry (a) and proportions and absolute numbers (b) of OVA-reactive tetramer-positive CD8 T cells in WT, Tsc1^−/− and Tsc1^−/− Bcl2-TG mice infected with LM-OVA. (c,d) Flow cytometry (c) and proportions and absolute numbers (d) of OVA-reactive IFN-γ⁺ CD8 T cells in LM-OVA infected mice, detected after OVA stimulation and intracellular cytokine staining. * P <0.005; ** P < 0.001; *** P< 0.0001. Data are representative of 3 independent experiments (n=8–10).