1. Volume 1, Issue 4, Pages 360-373 (April 2012)
PI3K-Akt-mTORC1-S6K1/2 Axis Controls Th17 Differentiation by Regulating Gfi1 Expression and Nuclear Translocation of RORγ 
Yutaka Kurebayashi, Shigenori Nagai, Ai Ikejiri, Masashi Ohtani, Kenji Ichiyama, Yukiko Baba, Taketo Yamada, Shohei Egami, Takayuki Hoshii, Atsushi Hirao, Satoshi Matsuda, Shigeo Koyasu 
Cell Reports 
Volume 1, Issue 4, Pages (April 2012)
DOI: /j.celrep
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2. Cell Reports 2012 1, 360-373DOI: (10.1016/j.celrep.2012.02.007)
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3. Figure 1
Inhibiting Class IA PI3K Activity Has Little Effect on Th1 and Th2 Differentiation
(A) Immunoblot analysis of Akt phosphorylation and band-shift assay of S6K1. Naive CD4+ T cells were rested on ice or cultured with the indicated combinations of immobilized anti-CD3ε Ab, 1 μg/ml soluble anti-CD28 Ab, and indicated inhibitors for 30 min, and total cell lysates were examined by western blotting with the indicated Abs.
(B and C) Intracellular staining of IFN-γ or IL-4 by p85α+/− or p85α−/− CD4+ T cells (B) or wt CD4+ T cells with or without IC87114 (C) cultured under Th1- or Th2-inducing conditions for 3 days (B) or for 3 and 6 days (C).
(D–F) Concentrations of IFN-γ and IL-4 in the supernatants measured by ELISA after naive p85α+/− or p85α−/− CD4+ T cells (D) or wt CD4+ T cells with or without IC87114 (E and F) were differentiated for 3 days (D and E) or 6 days (F) as described in (B) and (C) and restimulated for overnight with immobilized anti-CD3ε Ab and 1 μg/ml soluble anti-CD28 Ab in fresh media (n = 4). Error bars indicate SEM.
(G) Immunoblot analysis of T-bet, GATA3, and Runx1/3. Naive CD4+ T cells were differentiated to Th1 or Th2 cells for 2, 4, or 6 days (D) with or without IC87114, and nuclear proteins were examined by western blotting with the indicated Abs.
Results are representative of more than three independent experiments.
See also Figure S1.
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4. Figure 2
The Class IA PI3K-Akt-mTORC1 Axis Positively Regulates Th17 Differentiation
(A and B) Intracellular staining of IL-17A and IFN-γ in p85α+/− or p85α−/− CD4+ T cells (A) or wt CD4+ T cells with or without IC87114 (B) cultured under the indicated conditions for 3 days.
(C and D) Concentrations of IL-17A in the supernatants after naive p85α+/− or p85α−/− CD4+ T cells (C) or wt CD4+ T cells with or without IC87114 (D) were cultured as described in (A) and (B) and restimulated for overnight with immobilized anti-CD3ε Ab and 1 μg/ml soluble anti-CD28 Ab in fresh media (n = 4). Error bars indicate SEM.
(E) Intracellular staining of the expression of IL-17A and IL-17F by CD4+ T cells cultured under Th17-inducing conditions with or without IC87114 for 3 days.
(F) Intracellular staining of the expression of IL-17A and IFN-γ by CD4+ T cells expressing Akt-mer cultured under the Th17-inducing conditions with or without 4-HT and indicated inhibitors for 3 days.
(G) Concentrations of IL-17A in the supernatants after Akt-mer tg or wt naive CD4+ T cells were cultured under Th17-inducing conditions with indicated reagents for 3 days and restimulated as described in (C) and (D) (n = 3). Error bars indicate SEM.
Results are representative of more than three independent experiments.
See also Figure S2.
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5. Figure 3 Activation of mTORC1 Is Required for Th17 Differentiation
(A and B) Intracellular staining of the expression of IL-17A and IFN-γ (A) or IL-17A and IL-17F (B) by CD4+ T cells cultured under the indicated conditions with or without rapamycin for 3 days.
(C) Concentrations of IFN-γ and IL-17A in the supernatants after naive CD4+ T cells were cultured under the indicated conditions with or without rapamycin for 3 days and restimulated for overnight with immobilized anti-CD3ε Ab and 1 μg/ml soluble anti-CD28 Ab in fresh media (n = 4). Error bars indicate SEM. n.s., nonsignificant.
(D) Intracellular staining of IL-17A and IFN-γ in Raptorfl/fl or LckCreRaptorfl/fl CD4+ T cells cultured under the indicated conditions for 3 days.
(E) Flow cytometry of the expression of GFP and IL-17A by CD4+ T cells transfected with the indicated vectors and cultured under Th17-inducing conditions for 3 days. The percentage of IL-17Ahigh cells among GFP− and GFP+ populations is shown in bold boxes.
(F) The percentage of IL-17Ahigh cells in (E) is shown (n = 3). Error bars indicate SEM. n.s., nonsignificant.
(G) Representative flow cytometry of intracellular staining for IL-17A and IFN-γ in CD4+ T cells from mesenteric lymph nodes (mLNs) or spleens of the Rag2−/− recipients 6 weeks after the transfer of CD4+CD25−CD45RBhigh T cells with or without rapamycin treatment.
(H) Body weights of Rag2−/− recipients described in (G) (n = 6 each, ∗p < 0.05). Error bars indicate SEM.
(I) Histopathology of hematoxylin and eosin-stained colon tissues from Rag2−/− recipients 6 weeks after the transfer of CD4+CD25−CD45RBhigh T cells with or without rapamycin treatment. Scale bars represent 200 μm. Results are representative of six mice each.
(J) The weight/length ratios of total colons harvested from the Rag2−/− recipients described in (I) (n = 6). Error bars indicate SEM.
Results are representative of more than three independent experiments.
See also Figure S3.
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6. Figure 4
Effects of IC87114 and Rapamycin on the Expression of Transcription Factors
(A and B) Real-time PCR analysis of the indicated mRNA expression in CD4+ T cells cultured under the indicated conditions (Tfh, T follicular helper cells, 30 ng/ml IL-6) with or without indicated inhibitors for 48 hr (n = 3). Error bars indicate SEM.
(C) Gfi1 expression in CD4+ T cells cultured under the indicated conditions with or without indicated inhibitors for 48 hr was examined by real-time PCR (n = 3). Error bars indicate SEM.
(D) Immunoblot analysis of Gfi1 expression. Naive CD4+ T cells were cultured under the indicated conditions with or without indicated inhibitors for 48 hr, and whole-cell extracts were examined by western blotting with the indicated Abs. The numbers indicate the relative band amount of Gfi1 protein in each sample obtained by dividing the Gfi1 band intensity by the GAPDH band intensity in each lane.
Results are representative of more than three independent experiments.
See also Figure S4.
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7. Figure 5
EGR2 and Gfi1 Expressions Are Regulated by S6K1 Downstream of PI3K-Akt-mTORC1 Axis
(A) Immunoblot analysis of EGR1, EGR2, and EGR3. Naive CD4+ T cells were cultured under the indicated conditions with or without indicated inhibitors for 4 or 48 hr. Whole-cell extracts were examined by western blotting with the indicated Abs.
(B) Real-time PCR analysis of the indicated mRNA expression in CD4+ T cells transfected with control or Egr2 lentivirus and cultured under the indicated conditions for following 48 hr (n = 4). Error bars indicate SEM.
(C) Immunoblot analysis of EGR2 and EGR3. Naive CD4+ T cells were transfected with control or S6K1-CA lentivirus and restimulated with immobilized anti-CD3ε Ab and 1 μg/ml soluble anti-CD28 Ab in fresh media for 24 hr. Whole-cell extracts were examined by western blotting with the indicated Abs.
(D) Left panels show immunoblot analysis of S6K1 or S6 phosphorylation with or without lentiviral transfection of S6K1-CA and rapamycin treatment. Naive CD4+ T cells were transfected with control or S6K1-CA lentivirus and cultured under the Th17-inducing conditions with or without rapamycin for 3 days. Whole-cell extracts were examined by western blotting with the indicated Abs. Right panels illustrate flow cytometry of the expression of GFP and IL-17A by CD4+ T cells transfected with the indicated vectors and cultured under Th17-inducing conditions for 3 days. The percentages of IL-17A+ cells among GFP− and GFP+ populations are shown in bold boxes.
(E) Real-time PCR analysis of the indicated mRNA expression in CD4+ T cells transfected with control, Rheb Q64L, or S6K1-CA lentivirus and cultured under the indicated conditions for following 48 hr (n = 4). Error bars indicate SEM. Results are representative of more than three independent experiments. n.s., nonsignificant.
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8. Figure 6
Inhibition of the PI3K-Akt-mTORC1 Axis Impairs the Nuclear Translocation of RORγ during Th17 Differentiation
(A) Immunoblot analysis of RORγ in whole-cell lysates. Naive CD4+ T cells were cultured under the indicated conditions with or without indicated inhibitors for 18 hr. Whole-cell extracts were examined by western blotting with the indicated Abs.
(B) Immunoblot analysis of RORγ. Naive CD4+ T cells were cultured under Th0- or Th17-inducing conditions with or without indicated inhibitors for 18 hr. Left panels show nuclear fractions of naive CD4+ or naive CD8+ cells and cultured Th0 or Th17 cells that were examined by western blotting. Right panels illustrate cytoplasmic and nuclear fractions of cultured Th0 or Th17 cells that were examined for the expression of RORγ.
(C) The ratio of nuclear/cytoplasmic RORγ band amount. Western blotting was performed as described in the right panels of (B), and the ratio was obtained by dividing the nuclear RORγ band intensity normalized with the TBP band intensity by the cytoplasmic RORγ band intensity normalized with the GAPDH band intensity in each lane (n = 3). Error bars indicate SEM.
(D) Immunoblot analysis of T-bet, GATA3, or RORγ expression. Naive CD4+ T cells were cultured under the indicated conditions with or without indicated inhibitors for 48 hr, and nuclear fractions were examined by western blotting.
(E) Immunofluorescence microscopic analyses of RORγ distribution in CD4+ T cells in the absence or presence of IC87114 or rapamycin after cultivation for 18 hr under Th17-inducing conditions.
(F) The frequencies of the cells with RORγ localized in the nucleus or cytoplasm among RORγ+CD4+ T cells in (E) were calculated (n = 3). Error bars indicate SEM.
(G) The frequencies of RORγ+ cells among CD4+ T cells in (E) were calculated (n = 3). Error bars indicate SEM.
(H) Effect of leptomycin B on the localization of RORγ and ERK2 in the cytoplasm and nucleus. Naive CD4+ T cells were cultured under Th0- or Th17-inducing conditions with or without 3 nM rapamycin for 18 hr. Leptomycin B (200 nM) was added for the last 6 hr. Cytoplasmic or nuclear fractions of cultured Th0 or Th17 cells were examined for the expression of RORγ and ERK2.
Results are representative of more than three independent experiments.
See also Figure S5.
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9. Figure 7
S6K2 Interacts with RORγ and Enhances Its Nuclear Translocation
(A and B) Coimmunoprecipitation of RORγ and S6K2. HEK293T cells were transfected with the indicated cDNAs and cultured for 24 hr. Whole-cell lysates were prepared, and immunoprecipitation was performed using anti-Flag Ab (A) or anti-Myc Ab (B). Immunoprecipitates were examined by western blotting.
(C) Immunoblot analysis of Flag-tagged RORγ and Myc-tagged S6K2 in the cytoplasm or nucleus. HEK293T cells were transfected with indicated vectors for 24 hr, and cytoplasmic and nuclear fractions of these cells were examined by western blotting. The numbers indicate the relative band intensities of RORγ-Flag protein in each sample obtained by dividing the cytoplasmic or nuclear RORγ-Flag band intensity by the GAPDH or TBP band intensity, respectively.
(D) Immunoblot analysis of S6K2 in whole-cell lysates. Naive CD4+ T cells were cultured under the indicated conditions with or without indicated inhibitors for 48 hr. Whole-cell extracts were examined by western blotting with the indicated Abs. The relative band intensity of S6K2 protein obtained by dividing the S6K2 band intensity by the GAPDH band intensity in each lane is shown in the right panel (n = 3). Error bars indicate SEM.
(E) Real-time PCR analysis of the Rps6kb2 mRNA expression in CD4+ T cells cultured under the indicated conditions with or without indicated inhibitors for 48 hr (n = 3). Error bars indicate SEM. n.s., nonsignificant.
(F) Immunoblot analysis of S6K2 in the cytoplasm or nucleus. Naive CD4+ T cells were cultured under the indicated conditions with or without indicated inhibitors for 48 hr. Cytoplasmic and nuclear fractions of these cells were examined by western blotting with the indicated Abs.
Results are representative of more than three independent experiments.
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10. Figure S1
Treatment with IC87114 or Loss of p85α Impairs Akt Phosphorylation during In Vitro Differentiation of Th1, Th2, and Th17 Cells, Related to Figure 1
(A and B) Immunoblot analysis of Akt phosphorylation. p85α+/− or p85α−/− naive CD4+ T cells (A) or wt naive CD4+ T cells with or without IC87114 (B) were cultured with 1 μg/ml soluble anti-CD28 Ab alone or a combination of immobilized anti-CD3ε Ab, 1 μg/ml soluble anti-CD28 Ab and Th1-, Th2- or Th17-inducing cytokines (as described in Experimental Procedures) for 30 min and total cell lysates were examined by Western blotting with the indicated Abs.
(C) Immunoblot analysis of p85α and p110δ. B cells or naive CD4+ T cells were purified from p85α+/− or p85α−/− mice and total cell lysates were examined by Western blotting using anti-p110δ and anti-pan-p85α Abs.
(D) Immunoblot analysis of p85α, p110δ and STAT3 phosphorylation. p85α+/− or p85α−/− naive CD4+ T cells were rested or cultured in Th1- or Th17-inducing conditions for 30 min or 24 hr and total cell lysates were examined by Western blotting with the indicated antibodies.
(E) Immunoblot analysis of Akt, Foxo1/3a, PKCθ and S6K1 phosphorylation. Naive CD4+ T cells were rested or cultured with immobilized anti-CD3ε Ab and 1 μg/ml soluble anti-CD28 Ab for 15 min or 24 hr with or without indicated inhibitors and total cell lysates were examined by Western blotting with the indicated antibodies.
Results are representative of more than three independent experiments.
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11. Figure S2
4-HT Treatment Induces the Expression of Tbx21 and IFN-γ during Th17 Differentiation from Akt-mer tg Naive CD4+ T Cells, Related to Figure 2
(A) Immunoblot analysis of Akt and TSC2 phosphorylation. Akt-mer tg naive CD4+ T cells were cultured under Th17-inducing conditions with or without 4-HT for 30 min, 12 hr or 36 hr and total cell lysates were examined by Western blotting with the indicated antibodies.
(B) Intracellular staining of IL-17A and IFN-γ by wt CD4+ T cells cultured under Th17-inducing conditions with or without 4-HT for 3 days.
(C) Intracellular staining of IL-17A and IFN-γ by Akt-mer tg CD4+ T cells cultured under the indicated conditions (3 ng/ml TGF-β, 30 ng/ml IL-6 or the combination of 3 ng/ml TGF-β and 30 ng/ml IL-6) with or without 4-HT for 3 days.
(D) Concentration of IFN-γ in the supernatants after Akt-mer tg naive CD4+ T cells were cultured under Th17-inducing conditions with or without 4-HT for 3 days and re-stimulated for 2 days with immobilized anti-CD3ε Ab and 1 μg/ml soluble anti-CD28 Ab in fresh media (n = 4). Error bars indicate SEM.
(E) RT-PCR analysis for Tbx21 expression in CD4+ T cells expressing Akt-mer cultured under the indicated conditions with or without 4-HT for 48 hr (n = 4). Error bars indicate SEM.
Results are representative of more than three independent experiments.
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12. Figure S3
IL-6 Activates mTORC1 and Rapamycin Treatment Alleviates the Severity of EAE, Related to Figure 3
(A) Three hundred thousand CD4+ T cells were cultured under Th1- or Th17-inducing conditions with the indicated concentrations of rapamycin, and the cell number was determined after 48 hr (n = 3). Error bars indicate SEM.
(B) Immunoblot analysis of S6K1 phosphorylation. Naive CD4+ T cells were cultured with the indicated combinations of cytokines, antibodies and 3 nM rapamycin for 3 days, and total cell lysates were examined by Western blotting with the indicated Abs. Arrowhead indicates the position of a specific band.
(C) Mean clinical scores and disease incidence of EAE. C57BL/6 wt mice were immunized on day 0 and examined for 30 days with or without intraperitoneal rapamycin treatment (n = 9 each, ∗: p < 0.05). Error bars indicate SEM.
(D) (left panels) Intracellular staining of IL-17A and IFN-γ in CD4+ T cells from the draining lymph nodes (LN) or spleens of mice 30 days after the initial immunization. Error bars indicate SEM. Results are representative of 5 mice. (right panels) Mean and SEM values of the percentage of CD4+ T cells expressing IL-17A or IFN-γ are indicated (n = 5).
Results are representative of more than three independent experiments.
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13. Figure S4
STAT3 Phosphorylation and Foxp3 Expression during Th17 Differentiation with or without PI3K-Akt-mTORC1 Inhibition, Related to Figure 4
(A) Immunoblot analyses of Akt and STAT3 phosphorylation. p85α+/− or p85α−/− naive CD4+ T cells were cultured under Th17-inducing conditions for 0, 30 and 90 min and whole cell lysates examined by Western blotting with the indicated Abs.
(B) Immunoblot analysis of Akt or STAT3 phosphorylation. wt naive CD4+ T cells were cultured under Th17-inducing conditions for 0, 30 and 90 min with or without indicated inhibitors and whole cell lysates were examined by Western blotting with the indicated Abs.
(C and D) Immunoblot analysis of STAT3 phosphorylation. Naive CD4+ T cells were cultured under Th17-inducing conditions for 24 hr (C) or 48 hr (D) with or without indicated inhibitors and whole cell lysates were examined by Western blotting with the indicated Abs.
(E) Intracellular staining of Tyr705 pSTAT3 on LckCreRaptorfl/+ or LckCreRaptorfl/fl CD4+ T cells cultured under the indicated conditions for 48 hr.
(F and G) Intracellular staining of Foxp3 expression by wt CD4+ T cells cultured under Th0-, iTreg- or Th17-inducing condition with or without indicated inhibitors for 18 hr (F) or 3 days (G).
(H) Intracellular staining of Foxp3 expression in wt CD4+ T cells cultured under Th0- or iTreg-inducing condition with or without indicated inhibitors for 3 days and further expanded with 10 ng/ml IL-2 and indicated inhibitors for 2 days.
(I) Intracellular staining of IL-17A and IFN-γ in wt or OT-IIxRag2−/−xFoxp3sf CD4+ T cells cultured under Th17-inducing conditions with or without indicated inhibitors for 3 days.
Results are representative of more than three independent experiments.
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14. Figure S5
Nuclear Translocation of RORγ, but Not T-bet or GATA3, Is Impaired by Inhibition of the PI3K-Akt-mTORC1 Axis, Related to Figure 6
(A) Immunofluorescence microscopic analyses of RORγ, T-bet and GATA3 expression in Th17 cells. Naive CD4+ T cells were cultured under Th17-inducing conditions with or without the indicated inhibitors. RORγ expression was examined after 18 hr and the expression of T-bet and GATA3 was examined after 48 hr.
(B and C) Immunofluorescence microscopic analysis of T-bet (B) or GATA-3 (C) expression. Naive CD4+ T cells were cultured under Th1- (B) or Th2-inducing (C) conditions with or without the indicated inhibitors for 48 hr.
(D) Immunoblot analysis of RORγ in the cytoplasm or nucleus. Naive CD4+ T cells were cultured under Th0- or Th17-inducing conditions for 3 days. Indicated inhibitors were added for the last 24 hr. Cytoplasmic or nuclear fractions of cultured Th0 or Th17 cells were extracted and examined for the expression of RORγ.
(E) Intracellular staining of IL-17A and IFN-γ in wt CD4+ T cells cultured under the indicated conditions for 3 days with or without indicated inhibitors from day 0 (upper panel) or from day 2 (lower panel).
Results are representative of more than three independent experiments.
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