Context-Specific Function of S6K2 in Th Cell Differentiation

Abstract

The mammalian target of rapamycin (mTOR) is essential for Th cell proliferation and effector differentiation, making the mTOR signaling network an attractive immunomodulatory target for autoimmune-related diseases. Although direct targeting of mTOR complex-1 (mTORC1) with rapamycin can provide clinical benefit, targeting downstream enzymes has the potential to offer more selective immunosuppression. In this study, we evaluated p70 ribosomal protein S6 Kinase 2 (S6K2), a downstream effector of mTORC1, for its role in T cell function and autoimmunity. S6K2 is a direct substrate of mTORC1, with a potential role in Th17 differentiation suggested by biochemical studies. Using a genetic approach with S6K2 knockout mice, we found that S6K2 loss reduces Th17 skewing and increases regulatory T cell differentiation in vitro when cultured in RPMI 1640 media. However, S6K2 was dispensable for Th17 differentiation in IMDM. In an in vivo experimental autoimmune encephalomyelitis model in which rapamycin suppresses disease, S6K2 knockout mice did not exhibit differences in clinical score or Th17 differentiation. These results suggest that S6K2 is dispensable for Th17-driven autoimmunity and highlight how distinct experimental conditions can produce significantly different results in T cell differentiation.

Figure 1

S6K2^−/− mice have normal T cell compartments and CD4⁺ T cells display normal activation marker upregulation. (A) Flow cytometry analysis of CD4⁺ and CD8⁺ populations in the thymus, spleen, and lymph nodes from a representative experiment in (B). (B) Quantitative analysis of CD4⁺, CD8⁺, and CD4⁺CD8⁺ populations in the thymus, spleen, and lymph nodes (numbers indicate millions, mean +/− SEM, n = 3 independent experiments). (C) nTreg populations were analyzed by CD4⁺CD25⁺ expression in the thymus, spleen, and lymph nodes. Average percentage mean (from total CD4⁺ populations) +/− SEM is shown below (n = 3 independent experiments). (D) Flow cytometry analysis of activation markers and cell size (forward scatter; FSC) of WT and S6K2^−/− T cells after 24 hr of stimulation using varying concentrations of anti-CD3 under neutral conditions (n = 3 independent experiments).

Figure 2

S6K2^−/− CD4 T cells have reduced S6 phosphorylation during the first 24 hours of stimulation compared to WT, but have comparable proliferation and cell size under neutral stimulation conditions. (A) Histogram overlays of p-S6 comparing WT and S6K2^−/− CD4 T cells during a time course of neutral stimulation, representative of 3 independent experiments. (B) Left, Graph of normalized average mean fluorescence intensities (MFIs) of p-S6 in 24hr-activated CD4 T cells, * p < 0.05 (n = 5 independent experiments). Error bars indicate SEM. Right, The percentage of p-S6-positive T cells was graphed for 6 independent experiments comparing WT and S6K2^−/− CD4 T cells after 24hr stimulation. The Wilcoxon signed-rank test was used to assess p value. (C) CFSE and p-S6 staining of WT and S6K2^−/− naïve CD4⁺ T cells unstimulated or stimulated for 3 days, representative of 3 independent experiments. (D) CFSE histogram overlay of WT and S6K2^−/− T cells from (C). (E) Cell size histogram overlay WT and S6K2^−/− naïve CD4⁺ T cells stimulated for 24 hours. (F) Average FSC MFI plotted from (E) is shown, * p < 0.05 (n = 3 independent experiements). Error bars indicate SEM.

Figure 3

S6K2 and S6K1 have distinct substrates in activated T cells and have different roles in feedback to Akt. (A) Immunoblot analysis of lysates from WT and S6K2^−/− naïve CD4⁺ T cells unstimulated or stimulated for 24 hours, representative of 3 independent experiments. Rapamycin (+rap) or S6K1 inhibitor (S6Ki) was included during stimulation in the samples indicated. Antibodies used to probe the blots are shown on the left. (B) Analysis of phospho CAD and S6K1 expression from immunoblots, mean +/− SEM, n = 3 independent experiments. *p < 0.05. (C) Intracellular phosphoflow staining of Akt and analysis by normalized MFI, mean +/− SEM, n = 3 independent experiments. *p < 0.05.

Figure 4

Naïve S6K2^−/− CD4⁺ T cells generate fewer Treg and more Th17 cells, with little effect on Th1, Th17, or Treg proliferation. (A) Intracellular staining of IL-17A, FoxP3, and IFNγ in WT and S6K2^−/− T cells stained with CFSE for cell division tracking. Naïve WT or S6K2^−/− CD4⁺ T cells were cultured in RPMI media under indicated conditions for 4 days. Shown plots are representative of data analyzed in (B). (B) Paired analysis of IL-17A and IFNγ positive cells from multiple experiments performed as in (A) (Th17: n = 4 independent experiments, Treg: n = 5 independent experiments). The Wilcoxon signed-rank test was used to assess p value. (C) Histogram overlays of CFSE-labeled T cells from (A). (D) IL-17 and IL-10 cytokine measurements by ELISA from the supernatant of WT and S6K2^−/− T cells cultured in Th17 or Treg skewing conditions for 4 days (mean +/− SEM, n = 3 independent experiments).

Figure 5

S6K2^−/− and WT mice exhibit comparable EAE development. (A) Clinical scores of EAE-induced WT and S6K2^−/− mice (mean +/− SEM, n = 8 for each group). (B) Clinical scores of EAE-induced WT, WT treated with Rap or vehicle (Veh), and S6K2^−/− mice (mean +/− SEM, n = 3 for each group). (C) Graphed analysis of IFNγ and IL-17A positive CD4⁺ T cells in the spinal cord of EAE-induced mice from (B), * p < 0.05 (mean +/− SEM, n = 3). (D) pS6 (MFI and % positive) in CD4⁺ T cells from freshly harvested lymph nodes from 10 day MOG immunized WT and S6K2^−/− mice (mean +/− SEM, n = 3). (E) CD4⁺IL17A⁺ (% positive and MFI) and CD4⁺FoxP3⁺ (% positive) populations in 3 day MOG restimulated splenocytes harvested from the same mice as used in (D) (mean +/− SEM, n = 3).

Figure 6

IMDM rescues Th17 differentiation in S6K2^−/− T cells. (A) Intracellular staining of IL-17A and FoxP3 in WT and S6K2^−/− T cells stained with CFSE. Naïve WT or S6K2^−/− CD4⁺ T cells were cultured under indicated conditions for 4 days in RPMI or IMDM. Plots are representative of 3 independent experiments. (B) Comparative flow cytometric analysis and quantitative analysis for normalized MFI of p-S6 signaling between WT and S6K2^−/− T cells 24 hr of neutral stimulation, *p < 0.05 (mean +/− SEM, n = 3 independent experiments). (C) IL-17 and IL-10 cytokine measurements by ELISA from the supernatant of WT and S6K2^−/− T cells cultured in Th17 or Treg skewing conditions for 4 days (mean +/− SEM, n = 3 independent experiments). (D) Intracellular staining of IL-17A in CFSE-labeled WT and S6K2^−/− T cells cultured in Th17 skewing conditions for 4 days in RPMI, RPMI +FICZ, or IMDM. Plots are representative of 3 independent experiments.