The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex - PubMed (original) (raw)

doi: 10.1038/ni.2867. Epub 2014 Apr 6.

Chirag H Patel 1, Sam Collins 2, Adam Waickman 1, Min-Hee Oh 2, Im-Hong Sun 1, Peter Illei 3, Archna Sharma 4, Aniko Naray-Fejes-Toth 5, Geza Fejes-Toth 5, Jyoti Misra-Sen 4, Maureen R Horton 2, Jonathan D Powell 1

Affiliations

The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex

Emily B Heikamp et al. Nat Immunol. 2014 May.

Abstract

SGK1 is an AGC kinase that regulates the expression of membrane sodium channels in renal tubular cells in a manner dependent on the metabolic checkpoint kinase complex mTORC2. We hypothesized that SGK1 might represent an additional mTORC2-dependent regulator of the differentiation and function of T cells. Here we found that after activation by mTORC2, SGK1 promoted T helper type 2 (TH2) differentiation by negatively regulating degradation of the transcription factor JunB mediated by the E3 ligase Nedd4-2. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling expression of the long isoform of the transcription factor TCF-1. Consistent with those findings, mice with selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated substantial IFN-γ in response to viral infection and more readily rejected tumors.

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The authors have no competing financial interests.

Figures

Figure 1

Figure 1. SGK1 is activated downstream of TCR signaling in an mTOR-dependent manner

(a) Immunoblot (IB) of naïve lymphocytes from 5C.C7 mice that were rested for 1 h prior to stimulation with anti-CD3 and anti-CD28. Cells were lysed, and the activity of SGK1 was measured by blotting for phosphorylated NDRG1 (p-T346). The activity of mTORC2 was measured by blotting for phosphorylated Akt (p-S473). Total protein and actin are included as loading controls. (b) IB of CD4+ T cells isolated from 5C.C7 mice, as described in a, and stimulated for 3 h in the presence of polarizing cytokines. Cells were lysed and immunoblotted for SGK1 and Akt activity. (c) IB of naïve 5C.C7 lymphocytes, as described above. Where indicated, the mTOR kinase inhibitor PP242 (1 μM) was added to the cells upon stimulation. Total protein and actin are included as loading controls. Data are representative of 3–5 independent experiments (a–c).

Figure 2

Figure 2. SGK1 is activated downstream of mTORC2 in T cells

(a) IB of wild-type (WT), T-_Rictor_−/−**,** and T-_Sgk1_−/− CD4+ T cells that were stimulated as in Fig. 1a. Cells were lysed and the activity of Akt and SGK1 was measured as above. Total protein and actin are included as loading controls. (b–e) Flow cytometric phenotyping data compiled from multiple mice (n = 6) showing percentages of B220+, CD3+, CD4+ and CD8+ cells from WT and T-_Sgk1_−/− spleen (b–c) and lymph nodes (d–e). CD4+ and CD8+ subsets in (c) and (e) were gated on CD3+. (f) CD4+ T cells were isolated from WT or T-_Sgk1_−/− mice, stained with carboxyfluorescein succinimidyl ester (CFSE), and stimulated with irradiated syngeneic APCs and 1 μg/mL anti-CD3 for 24, 48, 72 or 96 h. Unstimulated (US) cells are shown as a control. (g) Naïve CD4+ T cells were isolated and stimulated overnight with 0.3, 1.0, or 3.0 μg/ml plate-bound anti-CD3 and 2.0 μg/ml soluble anti-CD28. Supernatants were harvested to determine IL-2 production by ELISA. ND = not detectable. Data are representative of 3 independent experiments (a–g) and samples were analyzed in triplicate in each experiment (g). Statistical significance determined by Student’s _t_-test, NS=No Significance, error bars s.e.m.

Figure 2

Figure 2. SGK1 is activated downstream of mTORC2 in T cells

(a) IB of wild-type (WT), T-_Rictor_−/−**,** and T-_Sgk1_−/− CD4+ T cells that were stimulated as in Fig. 1a. Cells were lysed and the activity of Akt and SGK1 was measured as above. Total protein and actin are included as loading controls. (b–e) Flow cytometric phenotyping data compiled from multiple mice (n = 6) showing percentages of B220+, CD3+, CD4+ and CD8+ cells from WT and T-_Sgk1_−/− spleen (b–c) and lymph nodes (d–e). CD4+ and CD8+ subsets in (c) and (e) were gated on CD3+. (f) CD4+ T cells were isolated from WT or T-_Sgk1_−/− mice, stained with carboxyfluorescein succinimidyl ester (CFSE), and stimulated with irradiated syngeneic APCs and 1 μg/mL anti-CD3 for 24, 48, 72 or 96 h. Unstimulated (US) cells are shown as a control. (g) Naïve CD4+ T cells were isolated and stimulated overnight with 0.3, 1.0, or 3.0 μg/ml plate-bound anti-CD3 and 2.0 μg/ml soluble anti-CD28. Supernatants were harvested to determine IL-2 production by ELISA. ND = not detectable. Data are representative of 3 independent experiments (a–g) and samples were analyzed in triplicate in each experiment (g). Statistical significance determined by Student’s _t_-test, NS=No Significance, error bars s.e.m.

Figure 3

Figure 3. SGK1 reciprocally regulates TH1 and TH2 differentiation downstream of mTORC2

(a) IL-4 production of activated CD4+ T cells by ELISA. Naïve CD4+ T cells from WT and T-_Sgk1_−/− mice were isolated based on expression of CD44 and CD62L. Cells were stimulated with irradiated autologous APCs under TH0, TH1 or TH2 polarizing conditions as indicated in the Methods. (b,c) As in a, but supernatants were assayed for IL-5 (b) or IL-13 (c) production by ELISA. (Statistical significance calculated by ANOVA, *P < 0.001, error bars s.e.m.) (d) IFN-γ production of activated CD4+ T cells by intracellular staining. Cells were stimulated and rested as in a prior to activation with overnight plate-bound anti-CD3 and soluble anti-CD28 in the presence of Golgi plug. Numbers represent percentages of cells in each quadrant. (e) IB of activated CD4+ T cells for lineage-specific transcription factors. Cells were stimulated with plate-bound anti-CD3 and anti-CD28 for 48 h then rested for 5 days in IL-2. Live cells were harvested, lysed and immunoblotted for T-bet and GATA-3. Actin is included as a loading control. Right, flow cytometric data showing intracellular staining for the transcription factors T-bet and GATA-3. Cells were fixed and permeabilised to determine expression of T-bet and GATA-3 by flow cytometry. Mean Fluorescence Intensity (MFI) is displayed on each respective flow plot. Data are representative of 4 independent experiments (a–e) and samples were analyzed in triplicate in each experiment (a–c).

Figure 3

Figure 3. SGK1 reciprocally regulates TH1 and TH2 differentiation downstream of mTORC2

(a) IL-4 production of activated CD4+ T cells by ELISA. Naïve CD4+ T cells from WT and T-_Sgk1_−/− mice were isolated based on expression of CD44 and CD62L. Cells were stimulated with irradiated autologous APCs under TH0, TH1 or TH2 polarizing conditions as indicated in the Methods. (b,c) As in a, but supernatants were assayed for IL-5 (b) or IL-13 (c) production by ELISA. (Statistical significance calculated by ANOVA, *P < 0.001, error bars s.e.m.) (d) IFN-γ production of activated CD4+ T cells by intracellular staining. Cells were stimulated and rested as in a prior to activation with overnight plate-bound anti-CD3 and soluble anti-CD28 in the presence of Golgi plug. Numbers represent percentages of cells in each quadrant. (e) IB of activated CD4+ T cells for lineage-specific transcription factors. Cells were stimulated with plate-bound anti-CD3 and anti-CD28 for 48 h then rested for 5 days in IL-2. Live cells were harvested, lysed and immunoblotted for T-bet and GATA-3. Actin is included as a loading control. Right, flow cytometric data showing intracellular staining for the transcription factors T-bet and GATA-3. Cells were fixed and permeabilised to determine expression of T-bet and GATA-3 by flow cytometry. Mean Fluorescence Intensity (MFI) is displayed on each respective flow plot. Data are representative of 4 independent experiments (a–e) and samples were analyzed in triplicate in each experiment (a–c).

Figure 4

Figure 4. SGK1 promotes Th2 differentiation by negatively regulating NEDD4-2

(a) IB of cell extracts from WT and T-_Sgk1_−/− CD4+ T cells that were stimulated in vitro under either TH1 or TH2 polarizing conditions. Cells were lysed and blotted for the expression of JunB and the activity of the E3 ligase NEDD4-2 (by measuring p-S342). Total NEDD4-2 and actin are included as loading controls. ImageJ software was used to calculate band density from 3 independent experiments, and band density was normalized to loading controls and to WT TH1 conditions. (Statistical significance calculated by ANOVA, **P < 0.001, error bars s.e.m.) (b) Intracellular staining of Thy1.1+ adoptively transferred cells for phosphorylated NEDD4-2. OT-II CD4+ T cells bearing the Thy1.1 congenic marker were adoptively transferred into WT Thy1.2+ recipients immunized with OVA adsorbed onto alum to induce a Th2 immune response. Spleens were harvested on day 4, restimulated with OVA Class II peptide, and intracellular staining was performed for phosphorylated NEDD4-2. Plots depict MFI of indicated samples. (Statistical significance calculated by ANOVA, *P =0.0183. (c) IB of cell extracts of WT and T-_Sgk1_−/− treated with MG132. Cells were polarized under TH2 conditions as in a, but with the addition of the proteasome inhibitor MG132 during the final 2 h of stimulation. Lysates were blotted for JunB, and actin is included as a loading control. (d) Immunoprecipitates (IPs) of JunB from WT and T-_Sgk1_−/− CD4+ T cells polarized with IL-4 and treated with MG132. As in c, but lysates were subject to IP with JunB antibody. IPs were blotted for ubiquitin and immunoprecipitated JunB is included as a loading control. (e) IL-4 production of CD4+ T cells transfected with siRNA targeting NEDD4-2 or Ndfip. A non-specific pool of siRNA (scrambled, Scr) was used as a control. Cells were stimulated under TH2 polarizing conditions for 48 h prior to transfection with siRNA then expanded in IL-2 for 5 d. Cells were restimulated overnight with anti-CD3 and anti-CD28 and supernatants were harvested to determine cytokine production. (f) CD4+ T cells from WT or T-_Sgk1_−/− mice were treated as in (e) then lysed for immunoblot to show knockdown of NEDD4-2 and rescue of JunB protein. (Data not shown for Ndfip.) (Statistical significance calculated by ANOVA, **P < 0.001, error bars s.e.m.) Data are representative of 3 independent experiments (a–d), 4 independent experiments (e) or 2 independent experiments (f).

Figure 4

Figure 4. SGK1 promotes Th2 differentiation by negatively regulating NEDD4-2

(a) IB of cell extracts from WT and T-_Sgk1_−/− CD4+ T cells that were stimulated in vitro under either TH1 or TH2 polarizing conditions. Cells were lysed and blotted for the expression of JunB and the activity of the E3 ligase NEDD4-2 (by measuring p-S342). Total NEDD4-2 and actin are included as loading controls. ImageJ software was used to calculate band density from 3 independent experiments, and band density was normalized to loading controls and to WT TH1 conditions. (Statistical significance calculated by ANOVA, **P < 0.001, error bars s.e.m.) (b) Intracellular staining of Thy1.1+ adoptively transferred cells for phosphorylated NEDD4-2. OT-II CD4+ T cells bearing the Thy1.1 congenic marker were adoptively transferred into WT Thy1.2+ recipients immunized with OVA adsorbed onto alum to induce a Th2 immune response. Spleens were harvested on day 4, restimulated with OVA Class II peptide, and intracellular staining was performed for phosphorylated NEDD4-2. Plots depict MFI of indicated samples. (Statistical significance calculated by ANOVA, *P =0.0183. (c) IB of cell extracts of WT and T-_Sgk1_−/− treated with MG132. Cells were polarized under TH2 conditions as in a, but with the addition of the proteasome inhibitor MG132 during the final 2 h of stimulation. Lysates were blotted for JunB, and actin is included as a loading control. (d) Immunoprecipitates (IPs) of JunB from WT and T-_Sgk1_−/− CD4+ T cells polarized with IL-4 and treated with MG132. As in c, but lysates were subject to IP with JunB antibody. IPs were blotted for ubiquitin and immunoprecipitated JunB is included as a loading control. (e) IL-4 production of CD4+ T cells transfected with siRNA targeting NEDD4-2 or Ndfip. A non-specific pool of siRNA (scrambled, Scr) was used as a control. Cells were stimulated under TH2 polarizing conditions for 48 h prior to transfection with siRNA then expanded in IL-2 for 5 d. Cells were restimulated overnight with anti-CD3 and anti-CD28 and supernatants were harvested to determine cytokine production. (f) CD4+ T cells from WT or T-_Sgk1_−/− mice were treated as in (e) then lysed for immunoblot to show knockdown of NEDD4-2 and rescue of JunB protein. (Data not shown for Ndfip.) (Statistical significance calculated by ANOVA, **P < 0.001, error bars s.e.m.) Data are representative of 3 independent experiments (a–d), 4 independent experiments (e) or 2 independent experiments (f).

Figure 5

Figure 5. Loss of SGK1 activity in CD4+ T cells mitigates TH2-mediated disease in an allergen induced asthma model. (a–c)

IL-4, IL-13 and IL-5 production from lung lymphocytes from mice that had been sensitized intraperitoneally and re-challenged intranasally on days 14 and 21 with HDM extract to induce allergic airway inflammation. Lung lymphocytes were stimulated for 3 days ex vivo with HDM extract, and supernatants were harvested and analyzed by ELISA for cytokine production. (d–g) IL-4, IL-13 and IL-5 production from mediastinal lymph nodes from mice that had been immunized as in a–c. Lymphocytes were stimulated ex vivo with PMA and ionomycin for 4 h, then fixed, permeabilised and analyzed by intracellular staining and flow cytometry for cytokine production. (h) Lymphocytes were harvested from lung parenchyma or BAL, stimulated ex vivo with PMA and ionomycin for 4 h, and analyzed for production of IFN-γ by intracellular staining and flow cytometry as described above. (i) As in h, but lymphocytes were harvested from mediastinal lymph nodes. (j–l) Total IgE (j), HDM specific IgE (k), and HDM specific IgG1 (l) in serum from mice that were sensitized and rechallenged with HDM extract as described above. Antibody titers were extrapolated from a standard curve (if available) or absorbance is shown for a dilution of serum for which all samples were in the range of the assay. (m,n) Representative lung sections after periodic acid-Schiff (PAS) (m) or hematoxylin and eosin (H&E) (n) staining for saline control (mock) and HDM-treated mice. Pathologic changes in WT mice include goblet cell hyperplasia (m) and perivascular and peribronchiolar inflammation (n). Images from HDM treated mice are shown at 10× (top, middle rows) and at 20× magnification (bottom row). (o–p) Histologic scoring data of PAS+ cells in large bronchioles (o) and small bronchioles (p) from HDM-treated mice. (q) Histologic scoring data of perivascular and peribronchiolar inflammation as seen in H&E stained lung sections as described above. Data are representative of 3 independent experiments (a–i), 4 independent experiments (j–l) or 2 independent experiments (m–q), n = 5–11 mice per group. (Statistical significance determined by Student’s _t_-test (a-q), **** P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, error bars s.e.m.)

Figure 5

Figure 5. Loss of SGK1 activity in CD4+ T cells mitigates TH2-mediated disease in an allergen induced asthma model. (a–c)

IL-4, IL-13 and IL-5 production from lung lymphocytes from mice that had been sensitized intraperitoneally and re-challenged intranasally on days 14 and 21 with HDM extract to induce allergic airway inflammation. Lung lymphocytes were stimulated for 3 days ex vivo with HDM extract, and supernatants were harvested and analyzed by ELISA for cytokine production. (d–g) IL-4, IL-13 and IL-5 production from mediastinal lymph nodes from mice that had been immunized as in a–c. Lymphocytes were stimulated ex vivo with PMA and ionomycin for 4 h, then fixed, permeabilised and analyzed by intracellular staining and flow cytometry for cytokine production. (h) Lymphocytes were harvested from lung parenchyma or BAL, stimulated ex vivo with PMA and ionomycin for 4 h, and analyzed for production of IFN-γ by intracellular staining and flow cytometry as described above. (i) As in h, but lymphocytes were harvested from mediastinal lymph nodes. (j–l) Total IgE (j), HDM specific IgE (k), and HDM specific IgG1 (l) in serum from mice that were sensitized and rechallenged with HDM extract as described above. Antibody titers were extrapolated from a standard curve (if available) or absorbance is shown for a dilution of serum for which all samples were in the range of the assay. (m,n) Representative lung sections after periodic acid-Schiff (PAS) (m) or hematoxylin and eosin (H&E) (n) staining for saline control (mock) and HDM-treated mice. Pathologic changes in WT mice include goblet cell hyperplasia (m) and perivascular and peribronchiolar inflammation (n). Images from HDM treated mice are shown at 10× (top, middle rows) and at 20× magnification (bottom row). (o–p) Histologic scoring data of PAS+ cells in large bronchioles (o) and small bronchioles (p) from HDM-treated mice. (q) Histologic scoring data of perivascular and peribronchiolar inflammation as seen in H&E stained lung sections as described above. Data are representative of 3 independent experiments (a–i), 4 independent experiments (j–l) or 2 independent experiments (m–q), n = 5–11 mice per group. (Statistical significance determined by Student’s _t_-test (a-q), **** P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, error bars s.e.m.)

Figure 5

Figure 5. Loss of SGK1 activity in CD4+ T cells mitigates TH2-mediated disease in an allergen induced asthma model. (a–c)

IL-4, IL-13 and IL-5 production from lung lymphocytes from mice that had been sensitized intraperitoneally and re-challenged intranasally on days 14 and 21 with HDM extract to induce allergic airway inflammation. Lung lymphocytes were stimulated for 3 days ex vivo with HDM extract, and supernatants were harvested and analyzed by ELISA for cytokine production. (d–g) IL-4, IL-13 and IL-5 production from mediastinal lymph nodes from mice that had been immunized as in a–c. Lymphocytes were stimulated ex vivo with PMA and ionomycin for 4 h, then fixed, permeabilised and analyzed by intracellular staining and flow cytometry for cytokine production. (h) Lymphocytes were harvested from lung parenchyma or BAL, stimulated ex vivo with PMA and ionomycin for 4 h, and analyzed for production of IFN-γ by intracellular staining and flow cytometry as described above. (i) As in h, but lymphocytes were harvested from mediastinal lymph nodes. (j–l) Total IgE (j), HDM specific IgE (k), and HDM specific IgG1 (l) in serum from mice that were sensitized and rechallenged with HDM extract as described above. Antibody titers were extrapolated from a standard curve (if available) or absorbance is shown for a dilution of serum for which all samples were in the range of the assay. (m,n) Representative lung sections after periodic acid-Schiff (PAS) (m) or hematoxylin and eosin (H&E) (n) staining for saline control (mock) and HDM-treated mice. Pathologic changes in WT mice include goblet cell hyperplasia (m) and perivascular and peribronchiolar inflammation (n). Images from HDM treated mice are shown at 10× (top, middle rows) and at 20× magnification (bottom row). (o–p) Histologic scoring data of PAS+ cells in large bronchioles (o) and small bronchioles (p) from HDM-treated mice. (q) Histologic scoring data of perivascular and peribronchiolar inflammation as seen in H&E stained lung sections as described above. Data are representative of 3 independent experiments (a–i), 4 independent experiments (j–l) or 2 independent experiments (m–q), n = 5–11 mice per group. (Statistical significance determined by Student’s _t_-test (a-q), **** P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, error bars s.e.m.)

Figure 5

Figure 5. Loss of SGK1 activity in CD4+ T cells mitigates TH2-mediated disease in an allergen induced asthma model. (a–c)

IL-4, IL-13 and IL-5 production from lung lymphocytes from mice that had been sensitized intraperitoneally and re-challenged intranasally on days 14 and 21 with HDM extract to induce allergic airway inflammation. Lung lymphocytes were stimulated for 3 days ex vivo with HDM extract, and supernatants were harvested and analyzed by ELISA for cytokine production. (d–g) IL-4, IL-13 and IL-5 production from mediastinal lymph nodes from mice that had been immunized as in a–c. Lymphocytes were stimulated ex vivo with PMA and ionomycin for 4 h, then fixed, permeabilised and analyzed by intracellular staining and flow cytometry for cytokine production. (h) Lymphocytes were harvested from lung parenchyma or BAL, stimulated ex vivo with PMA and ionomycin for 4 h, and analyzed for production of IFN-γ by intracellular staining and flow cytometry as described above. (i) As in h, but lymphocytes were harvested from mediastinal lymph nodes. (j–l) Total IgE (j), HDM specific IgE (k), and HDM specific IgG1 (l) in serum from mice that were sensitized and rechallenged with HDM extract as described above. Antibody titers were extrapolated from a standard curve (if available) or absorbance is shown for a dilution of serum for which all samples were in the range of the assay. (m,n) Representative lung sections after periodic acid-Schiff (PAS) (m) or hematoxylin and eosin (H&E) (n) staining for saline control (mock) and HDM-treated mice. Pathologic changes in WT mice include goblet cell hyperplasia (m) and perivascular and peribronchiolar inflammation (n). Images from HDM treated mice are shown at 10× (top, middle rows) and at 20× magnification (bottom row). (o–p) Histologic scoring data of PAS+ cells in large bronchioles (o) and small bronchioles (p) from HDM-treated mice. (q) Histologic scoring data of perivascular and peribronchiolar inflammation as seen in H&E stained lung sections as described above. Data are representative of 3 independent experiments (a–i), 4 independent experiments (j–l) or 2 independent experiments (m–q), n = 5–11 mice per group. (Statistical significance determined by Student’s _t_-test (a-q), **** P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, error bars s.e.m.)

Figure 6

Figure 6. SGK1 negatively regulates TH1 differentiation via the long isoform of TCF-1

(a) IB of cell extracts from WT and T-_Sgk1_−/− CD4+ T cells stimulated under TH2 polarizing conditions and treated overnight with 25 mM LiCl. Lysates were blotted for p-β-catenin (S33/47/T41) and total β-catenin. Total GSK-3β is included as a loading control. (b) IB of cell extracts from WT and T-_Sgk1_−/− CD4+ T cells that were stimulated in vitro under either TH1 or TH2 polarizing conditions. Cells were lysed and blotted for TCF-1. Actin is included as a loading control. Two exposures are shown to appreciate differences in long and short isoforms of TCF-1, short exposure (top), long exposure (bottom). (c) Expression of long isoform of TCF-1 in activated CD4+ T cells at early and late time points during differentiation. CD4+ T cells were purified from WT and T-_Sgk1_−/− mice by magnetic separation, stimulated with anti-CD3 anti-CD28 magnetic beads for 2 d in TH1 or TH2 conditions then rested in IL-2 for 6 d. Fold induction of long isoform of TCF-1 normalized 18S rRNA and to WT TH1 polarizing conditions at 72 h and 8 days post stimulation, as analyzed by quantitative polymerase chain reaction. (d) Flow cytometric analysis of CD4+ T cells polarized under TH2 conditions and transduced to overexpress the long isoform of TCF-1 (FL-TCF-1) with an MSCV retrovirus containing a human CD8 marker. Following transduction, cells were rested then sorted for human CD8 surface expression. Cells were restimulated and analyzed for production of IFN-© by intracellular staining. Cells were gated on low, intermediate, and high expression of human CD8. Histogram overlays of human CD8 high cells are shown to emphasize differences in IFN-© production among cells with similar multiplicity of infection. Data are representative of 3 independent experiments (a–c) or 4 independent experiments (d), and samples were analyzed in triplicate in each experiment (c). (Statistical significance calculated by ANOVA, P<0.001, error bars s.e.m.)

Figure 6

Figure 6. SGK1 negatively regulates TH1 differentiation via the long isoform of TCF-1

(a) IB of cell extracts from WT and T-_Sgk1_−/− CD4+ T cells stimulated under TH2 polarizing conditions and treated overnight with 25 mM LiCl. Lysates were blotted for p-β-catenin (S33/47/T41) and total β-catenin. Total GSK-3β is included as a loading control. (b) IB of cell extracts from WT and T-_Sgk1_−/− CD4+ T cells that were stimulated in vitro under either TH1 or TH2 polarizing conditions. Cells were lysed and blotted for TCF-1. Actin is included as a loading control. Two exposures are shown to appreciate differences in long and short isoforms of TCF-1, short exposure (top), long exposure (bottom). (c) Expression of long isoform of TCF-1 in activated CD4+ T cells at early and late time points during differentiation. CD4+ T cells were purified from WT and T-_Sgk1_−/− mice by magnetic separation, stimulated with anti-CD3 anti-CD28 magnetic beads for 2 d in TH1 or TH2 conditions then rested in IL-2 for 6 d. Fold induction of long isoform of TCF-1 normalized 18S rRNA and to WT TH1 polarizing conditions at 72 h and 8 days post stimulation, as analyzed by quantitative polymerase chain reaction. (d) Flow cytometric analysis of CD4+ T cells polarized under TH2 conditions and transduced to overexpress the long isoform of TCF-1 (FL-TCF-1) with an MSCV retrovirus containing a human CD8 marker. Following transduction, cells were rested then sorted for human CD8 surface expression. Cells were restimulated and analyzed for production of IFN-© by intracellular staining. Cells were gated on low, intermediate, and high expression of human CD8. Histogram overlays of human CD8 high cells are shown to emphasize differences in IFN-© production among cells with similar multiplicity of infection. Data are representative of 3 independent experiments (a–c) or 4 independent experiments (d), and samples were analyzed in triplicate in each experiment (c). (Statistical significance calculated by ANOVA, P<0.001, error bars s.e.m.)

Figure 7

Figure 7. Loss of SGK1 enhances TH1-mediated viral and tumor immunity

(a) IFN-γ production from adoptively transferred OT-II cells that were restimulated ex vivo with PMA and ionomycin. OT-II cells were adoptively transferred into mice that had been simultaneously immunized intravenously with 1 × 106 pfu VAC-OVA. Positive gates for IFN-γ were set using a no stimulation control such that <1.0% of cells were allowed in the positive gate. Plots gated on adoptively transferred cells (left) and bar graph depicts MFI (right). n = 10 mice per group. (b) IFN-γ production from lung lymphocytes of mice infected intranasally with the PR8 strain of influenza. On day 8, lung lymphocytes were harvested and stimulated for 4 h ex vivo with PMA and ionomycin then fixed, permeabilized and analyzed by intracellular staining and flow cytometry for IFN-γ production. (c) Number of B16 melanoma lung metastases. WT and T-_Sgk1_−/− mice were injected intravenously with 2 × 105 B16 melanoma cells, and lungs were harvested 21 days later. The number of lung metastases were counted and expressed as mean + s.e.m. (d) As in b, lung lymphocytes were harvested and stimulated for 4 hours ex vivo with PMA and ionomycin and analyzed by intracellular staining for production of IFN-γ by CD4+ T cells. (e) Kaplan-Meier analysis of WT and T-_Sgk1_−/− mice vaccinated with 1 × 106 pfu VAC-OVA 7 days prior to challenge with 2 × 105 B16 melanoma cells expressing OVA (B16-OVA). T-_Sgk1_−/− mice have prolonged survival (P<0.0001, n = 5–7 mice per group). Data are representative of 2–3 independent experiments, n = 5–15 mice per group. Statistical significance determined by Student’s _t_-test (a–d) or Log-rank (Mantel Cox) survival analysis (e), ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, error bars s.e.m.

Figure 7

Figure 7. Loss of SGK1 enhances TH1-mediated viral and tumor immunity

(a) IFN-γ production from adoptively transferred OT-II cells that were restimulated ex vivo with PMA and ionomycin. OT-II cells were adoptively transferred into mice that had been simultaneously immunized intravenously with 1 × 106 pfu VAC-OVA. Positive gates for IFN-γ were set using a no stimulation control such that <1.0% of cells were allowed in the positive gate. Plots gated on adoptively transferred cells (left) and bar graph depicts MFI (right). n = 10 mice per group. (b) IFN-γ production from lung lymphocytes of mice infected intranasally with the PR8 strain of influenza. On day 8, lung lymphocytes were harvested and stimulated for 4 h ex vivo with PMA and ionomycin then fixed, permeabilized and analyzed by intracellular staining and flow cytometry for IFN-γ production. (c) Number of B16 melanoma lung metastases. WT and T-_Sgk1_−/− mice were injected intravenously with 2 × 105 B16 melanoma cells, and lungs were harvested 21 days later. The number of lung metastases were counted and expressed as mean + s.e.m. (d) As in b, lung lymphocytes were harvested and stimulated for 4 hours ex vivo with PMA and ionomycin and analyzed by intracellular staining for production of IFN-γ by CD4+ T cells. (e) Kaplan-Meier analysis of WT and T-_Sgk1_−/− mice vaccinated with 1 × 106 pfu VAC-OVA 7 days prior to challenge with 2 × 105 B16 melanoma cells expressing OVA (B16-OVA). T-_Sgk1_−/− mice have prolonged survival (P<0.0001, n = 5–7 mice per group). Data are representative of 2–3 independent experiments, n = 5–15 mice per group. Statistical significance determined by Student’s _t_-test (a–d) or Log-rank (Mantel Cox) survival analysis (e), ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, error bars s.e.m.

Comment in

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Publication types

MeSH terms

Substances

Grants and funding

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