The tumor suppressor Tsc1 enforces quiescence of naive T cells to promote immune homeostasis and function - PubMed (original) (raw)

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

Kai Yang et al. Nat Immunol. 2011.

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.

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Figures

Figure 1

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

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

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 BimEL and BimL in naive and anti-CD3-CD28 activated WT and _Tsc1_−/− CD4 T cells. Numbers above (BimEL) and below (BimL) 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

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 CD44hi 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 CD44hi 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

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

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 CD44hi populations; lower right, ratios of CD122+/CD122− populations among CD44hi 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

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

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).

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