Akt and mtor pathways differentially regulate the development of natural and inducible T H 17 cells Jiyeon S Kim, Tammarah Sklarz, Lauren Banks, Mercy Gohil, Adam T Waickman, Nicolas Skuli, Bryan L Krock, Chong T Luo, Weihong Hu, Kristin N Pollizzi, Ming O Li, Jeffrey C Rathmell, Morris J Birnbaum, Jonathan D Powell, Martha S Jordan & Gary A Koretzky Supplementary Figures 1 1
a b 2.1.639 RORγt 1.2 CCR6 9.8 7.8 Foxp3 c Human Umbilical Cord Blood 4.9 99.5 CD8 no stim 7.39e-3 ex vivo stim.262 RORγt.232 CD3 CCR6 Supplementary Figure 1. nt H 17 cells are present in human thymus and umbilical cord blood. (a) Thymocytes from 18-19-week human fetal thymi were stained for RORγt and CCR6 expression. Flow plot are gated on SP cells. (b) Thymocytes from 18-19 week human fetal thymi were stained for IL-17 and Foxp3 following ex vivo stimulation for 5 h with PMA/ionomycin and brefeldin A. Flow plots are gated on SP cells. (c) Cells from umbilical cord blood were isolated and stained for surface markers and intracellular expression of upon ex vivo stimulation for 5 h with PMA/ionomycin and brefeldin A. Gating strategy as shown. Data are representative of at least three independent experiments. Numbers indicate percentages for the gated populations.
Supplementary Figure 2. Effect of Akt inhibitor treatment on thymocyte subsets in FTOC. Thymocytes from d7 of E15-initiated FTOC, cultured for the last 2 days in the presence of indicated concentrations of allosteric Akt inhibitors, were analyzed. Percentage of each thymocyte population is indicated. Data are pooled from two (MK-226) and three (Akti-1/2) independent experiments (n = 4-5; mean ± SEM).
a FTOC + MK-226 µm 1µM.932.81 IL-17 FTOC + MK-226 µm 1µM.949 1.44 Foxp3 b in vitro Th17 differentiation + MK-226 µm.5µm 1µM 35.1 19.2 14 IL-17 Foxp3 8.1 12.9 17.9 Supplementary Figure 3. Allosteric Akt inhibitor MK-226 treatment impairs nt H 17 cell development in FTOC. (a) Cells from d7 of E15-initiated FTOC, cultured for the last 2 days in the presence of indicated concentrations of allosteric Akt inhibitor MK-226, were stimulated with PMA/ionomycin and analyzed. Representative flow plots are gated on SP TCRβ + TCRγδ cells. Graphs show either the percent of IL-17 + or Foxp3 + cells among SP cells pooled from two independent experiments (n = 4; mean ± SEM; P value from twotailed Student s t-test). (b) IL-17 or Foxp3 expression is shown for naïve + T cells activated for 18 h with anti-cd3 plus anti-cd28, followed by 36 h culture with it H 17-polarizing condition in the presence of indicated concentrations of MK-226, and 5 h re-stimulation with PMA/ionomycin with brefeldin A. Representative flow plots are shown. The graph shows pooled data from n = 3 per condition and bars and error bars represent mean ± SEM. *P.5, ***P.1 (one-way ANOVA followed by Dunnett s post-test with µm as control group).
Supplementary Figure 4. Schematic of the Akt pathway.
.193.138.25.865 17.9 IL-22.126 IL-17F.468.231.166 IL-22.633.11 16.7 ARNT cko IL-22.392 IL-17F.654 IL-22 Supplementary Figure 5. IL-22 and IL-17F expression is intact in ARNT-deficient nt H 17 cells. IL-17F and IL-22 expression in thymocytes from and ARNT-cKO mice following ex vivo stimulation is shown. Representative flow plots are gated on SP TCRβ + TCRγδ cells (first and second columns) or + SP TCRβ + TCRγδ cells (third column). Data are from two independent experiments.
Supplementary Figure 6. mrna expression of Akt1 and Akt2 in + T cell subsets and thymocyte populations. Akt1 and Akt2 mrna transcripts from either in vitro differentiated + T cells (T H, T H 1, T H 2, it H 17, it reg ) or sorted thymocytes ( SP, + 4 lo CCR6 ; nt H 17, + 4 hi CCR6 + ; nt reg, + Foxp3 + from Foxp3-GFP reporter mice), relative to β-actin, was determined by real-time PCR. cdna was generated from 3 independently sorted thymic populations or 2 independently generated T H subsets. All samples were run in triplicate; bars and error bars represent mean ± SEM.
a αcd3/αcd28 (min) 5 3 5 3 5 3 pakt (T38) pakt (S473) Akt pfoxo1/3a FoxO1 β-actin b αcd3 αcd28 (min) 5 % of Max αcd3/αcd28 5min 1 8 6 4 3 pakt (T38) 2 pakt (T38) αcd3 αcd28 (min) 5 % of Max αcd3/αcd28 5min 1 8 6 4 2 3 pakt (S473) pakt (S473) Supplementary Figure 7. Akt1 and Akt2 contribute equally to the pool of active Akt in + T cells. (a) Immunoblot analysis of Akt activation, assessed by phosphorylation of p-akt (T38) (n = 2), p-akt (S473) (n = 1) and p-foxo1/3a proteins (n = 2), in,, and + T cells stimulated for 5 min or 3 min with anti-cd3 and anti-cd28. (b) Phospho-flow analysis of Akt activation at indicated residue (T38 and S473) in, and + T cells stimulated for 5 min or 3 min with anti-cd3 and anti-cd28. Data are representative of two independent experiments with n 5 per genotype.
a b Isotype IL-6R c IL-6 p-akt (T38) Akt p-foxo1/3a + + + d % of Max 1 8 6 4 2 IL-6 3min FoxO1 pakt (T38) p-stat3 STAT3 β-actin Supplementary Figure 8. IL-6 does not differentially activate Akt1 versus Akt2. (a) Expression of IL-6R (CD126) in, and + T cells analyzed by flow cytometry. Data are representative of two independent experiments (n 5 per genotype). (b) Expression of IL-6R in, and + T cells compared by mean fluorescence intensity (MFI). Graph shows pooled data from n 5 mice per genotype and bars and error bars represent mean ± SEM. ns, not-significant (one-way ANOVA followed by Dunnett s post-test with as control group). (c) Immunoblot analysis of Akt activation, assessed by phosphorylation of Akt (p-akt) and Foxo (p-foxo1/3a) proteins, in, and + T cells stimulated with IL-6 for 3 min. Phosphorylation of STAT3 (p-stat3) serves as positive control. Data are representative of two independent experiments. (d) Phospho-flow analysis of Akt activation in, and + T cells stimulated with IL-6 for 3 min. Shaded histogram represents unstimulated cells. Data are representative of two independent experiments with n 5 per genotype.
a 4.±1. 4.6±.8 IFNγ CFSE b 38.1±2.7 35.8±1.1 IL-4 CFSE c Akt2 ΔT IL-17 44.1 25. Supplementary Figure 9. T H 1 and T H 2 cell differentiation is intact in and + T cells. (a) CFSE dilution and IFN-γ production is shown for and + T cells that were cultured with plate-bound anti-cd3 plus anti-cd28 in T H 1-polarizing condition for 4 days. (b) CFSE dilution and IL-4 production is shown for and + T cells that were cultured with plate-bound anti-cd3 plus anti-cd28 in T H 2-polarizing condition for 4 days. (c) it H 17 differentiation is defective in Akt ΔT mice. IL-17 production in (Akt fl/fl cre ) and Akt2 ΔT + T cells cultured with plate-bound anti-cd3 plus anti-cd28 in T H 17- polarizing condition for 3 days. Data are representative of at least two independent experiments (a-c).
a HIF1 fl/fl HIF2 fl/fl Vav-cre 5.2.8 Th17 Treg HIF1α".871.1 b Th Th1 Th17.55 12.5 Treg.629 HIF1α Ab HIF1α" see Fig. 6c.813.61.216 Isotype Ab Akt2 ΔT HIF1α Ab HIF1α" Isotype".15 1.7 see Fig. 6c.926.89.58.913.211 Akt2 ΔT Isotype Ab Isotype" Supplementary Figure 1. Detection of intracellular HIF1α protein expression using flow cytometry. (a) HIF1α expression in and Hif1a fl/fl Hif2 fl/fl Vav-cre + T cells cultured with plate-bound anti-cd3 plus anti-cd28 in T H 17- or T reg -polarizing condition; n = 1. (b) Intracellular staining using HIF1α or isotype antibody to assess HIF1α expression in and Akt2 fl/fl Vav-cre (Akt2 ΔT ) + T cells cultured under T H, T H 1, T H 17 or T reg -polarizing conditions. Representative plots from 2 independent experiments are shown.