The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin

Abstract

The mechanisms underpinning integration of instructions that program naive CD8+ T cells for effector and/or memory differentiation are not well understood. Herein, we demonstrate that interleukin-12 (IL-12) enhanced and sustained antigen and costimulatory molecule (B7.1)-induced mTOR kinase activity in naive CD8+ (OT-I) T cells via phosphoinositide 3-kinase and STAT4 transcription factor pathways. Blocking mTOR activity by rapamycin reversed IL-12-induced effector functions because of loss of persistent expression of the transcription factor T-bet. Rapamycin treatment of IL-12-conditioned OT-I cells promoted persistent Eomesodermin expression and produced memory cell precursors that demonstrated enhanced sustenance and antigen-recall responses upon adoptive transfer. The memory cell precursors showed greater tumor efficacy than IL-12-conditioned effector OT-I cells. These results identify mTOR as the central regulator of transcriptional programs that determine effector and/or memory cell fates in CD8+ T cells. Targeting mTOR activity offers new opportunities to regulate CD8+ T cell-mediated immunity.

Figure 1. Instructions that program naïve CD8⁺ T cell for heritable type I effector maturation enhances and sustains mTOR activity

(A–B) OT-I cells stimulated with BOK (+/−) IL-12 were evaluated for (A); IFN-γ by ICS and (B); cytolytic activity (Primary–72h post-stimulation, Secondary–24h post-secondary stimulation); ***p<0.0002. (C–E) OT-I cells stimulated with antigen (Ag) (SIINFEKL, 10nM) plus B7.1 (100ug/ml) (Ag/B7.1) (+/−) IL-12 (2ng/ml) were evaluated by ICS at the indicated time points for (C); phosphorylated mTOR (D); phosphorylated S6K and (E); phosphorylated ribosomal S6. For mTOR inhibition, rapamycin (20ng/ml) was added 30 minutes prior to addition of antigen, cytokine. Data are representative of at least three independent experiments with identical outcomes. (Data are presented as mean +/− SEM)

Figure 2. IL-12 enhances antigen induced mTOR activity via PI3K and STAT4

(A–B) OT-I cells stimulated with Ag/B7.1 (+/−) IL-12 and LY294002 (10µM) were evaluated by ICS for (A); phosphorylated Akt at 48h (B); phosphorylated S6K at the indicated time-points. (C) WT or STAT4^−/− OT-I cells stimulated with Ag/B7.1 in the presence or absence of IL-12 were analyzed at the indicated time points for phosphorylated S6K; ***p<0.0001. Experiments shown are representative of three independent experiments with similar outcomes. (Data are presented as mean +/− SEM)

Figure 3. Sustained mTOR activity is essential for heritable type I effector differentiation of CD8⁺ T cells

(A–C) OT-I cells stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin were evaluated at the primary and secondary phase for (A); IFN-γ by ICS; ***p<0.0002, n.s.–not significant (B); cytolytic activity (C); granzyme B expression at 72h by ICS. (D–E) OT-I cells were stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin was added 12 hr post-stimulation to evaluate cells for (D); S6K phosphorylation at 48h (E); IFN-γ production at the primary and secondary phase. Experiments shown are representative of at least three (A, B) and two (CE) independent experiments with similar outcomes. (Data are presented as mean +/− SEM)

Figure 4. IL-12 enhanced mTOR phosphorylation is essential for T-bet determined type I effector maturation of CD8⁺ T cells

(A–C) OT-I cells stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin were evaluated for (A); mRNA for T-bet at the indicated time points by RT-PCR (B); Tbet protein expression at the indicated time-points by ICS (C); T-bet protein expression by ICS before and after antigen recall; **p<0.0035, ***p<0.0005. (D) WT and Tbx21^−/− OT-I cells were stimulated with Ag/B7.1 (+/−) IL-12 and evaluated for IFN-γ production at the primary and secondary phase; ***p<0.0001. (E–F) OT-I cells stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin were transduced with T-bet-ER retroviral vector (+/−) 4-HT (10nM) and evaluated by ICS for (E); T-bet protein expression by ICS (F); IFN-γ at secondary phase (168h). (G–H) OT-I cells stimulated with Ag/B7.1 (+/−) insulin (1 U/ml) and rapamycin were evaluated by ICS for (G); S6K phosphorylation at 48h (H); T-bet expression at 72h. Experiments shown are representative of at least three (A, B, D, E & F) and two (C, G and H) independent experiments with similar outcomes. (Data are presented as mean +/− SEM).

Figure 5. Inhibition of mTOR promotes persistent Eomes expression and phenotypic markers of memory in CD8⁺ T cells

(A–B) OT-I cells stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin were evaluated for (A); mRNA for Eomes at the indicated time points by RT-PCR (B); Eomes protein expression at 72h by ICS; *p<0.03. (C) OT-I cells stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin were transduced with T-bet-ER retroviral vector (+/−) 4-HT (10nM) and evaluated for Eomes protein expression at 96h. (D); OT-I cells stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin were evaluated for CD62L, CD69, KLRG1, CD127 and CD122 expression at 72h (E); Bcl-2 and Bcl-3 mRNA expression at the indicated time points (F–G); OT-I cells stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin for 72h were washed twice and rested for an additional 72h in the presence of (F); IL-7 (10ng/ml); *p<0.02 (G); IL-15 (10ng/ml); *p<0.02 and % cell recovery was calculated at 144h;. Experiments shown are representative of three independent experiments with similar outcomes. (Data are presented as mean +/− SEM).

Figure 6. Inhibition of mTOR enhances memory CD8⁺ T cell generation

OT-I cells (Thy1.1⁺) stimulated with Ag/B7.1 (+/−) IL-12 and rapamycin were harvested at 72h and adoptively transferred (2 × 10⁶ cells) into BL/6 recipients. (A) The absolute number of adoptively transferred OT-I cells in the lymph node; **p<0.0052, spleen; **p<0.0037 and liver; **p<0.0012, **p<0.0011, at 24h post transfer. (B–E) The recipient mice were immunized with IFA-OVA on day 40 post transfer and secondary CD8⁺ T cell responses were measured 3 days later (B); The absolute numbers of adoptively transferred cells before (day 40) and after (day 43) immunization in the spleen. The numbers in parenthesis indicate fold expansion of CD8α⁺/Thy1.1⁺ from day 40 to day 43 (C); absolute numbers of IFN-γ secreting CD8α⁺/Thy1.1⁺ cells in the spleen on day 43; *p<0.01, **p<0.008. The numbers in parenthesis indicate the MFI of IFN-γ expression (D); Granzyme B expression on CD8α⁺/Thy1.1⁺ cells in the spleen on day 43 (E); the in vivo antigen specific cytolysis on day 43. A representative of two independent experiments is shown. (Data are presented as mean +/− SEM).

Figure 7. mTOR inhibition promotes CD8⁺ T cell mediated anti-tumor immunity

(A–B) Naive or 72 h conditioned OT-I cells were adoptively transferred into BL/6 recipients. Mice were inoculated with 2 × 10⁶ E.G7 tumor cells, 24h post adoptive transfer of OT-I cells (A); tumor size (mm³) over time from tumor inoculation (B); Percent of tumor free survival over time from tumor inoculation. A representative of two independent experiments is shown.