Volume 31, Issue 2, Pages 331-341 (August 2009) Interleukin-1 and IL-23 Induce Innate IL-17 Production from γδ T Cells, Amplifying Th17 Responses and Autoimmunity  Caroline E. Sutton, Stephen J. Lalor, Cheryl M. Sweeney, Corinna F. Brereton, Ed C. Lavelle, Kingston H.G. Mills  Immunity  Volume 31, Issue 2, Pages 331-341 (August 2009) DOI: 10.1016/j.immuni.2009.08.001 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 IL-1β and IL-23 Promote IL-17 Production by CD3+ but Not by CD4+ or CD8+ T Cells in the Absence of TCR Engagement (A) Spleen cells from C57BL/6 or Il1r1−/− mice were stimulated with IL-1β and/or IL-23 with and without anti-CD3 for 3 days. IL-17 concentrations in supernatants were quantified by ELISA. ∗∗p < 0.01 and ∗∗∗p < 0.001, versus C57BL/6. (B) Spleen cells from C57BL/6 mice were stimulated as indicated without anti-CD3 for 3 days and stained for intracellular IL-17 and for surface CD3. Numbers refer to percentages of CD3+IL-17+ cells. (C) Spleen cells, CD4+ T cells, or CD4+CD62LhiCD44lo T cells from C57BL/6 mice were stimulated for 3 days as indicated. IL-17 concentrations were quantified by ELISA. ∗∗p < 0.01 and ∗∗∗p < 0.001, versus medium control. (D) Spleen cells from C57BL/6 mice were cultured for 3 days with medium only or IL-1β and IL-23, and intracellular IL-17 was determined in CD3, CD4, or CD8 cells. Numbers refer to percentages of IL-17+ cells. Results for ELISA data are means ± SE of triplicate culture and representative of three experiments. All error bars represent mean ± SD. Immunity 2009 31, 331-341DOI: (10.1016/j.immuni.2009.08.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Killed M. tuberculosis or Purified TLR and NLR Agonists Induce Innate IL-17 Production by T Cells by Stimulating DC IL-23 and IL-1β Production (A and B) Murine bone marrow-derived DCs were incubated with LPS, MDP, PGN, LPS + PGN, MDP + PGN or with medium alone (A), or with killed MTB (B). Supernatants were removed after 24 hr and IL-23, IL-1β, IL-12p70, and IL-6 concentrations quantified by ELISA. (C) DCs were stimulated with MTB or medium only for 24 hr, washed, and added to CD3+ T cells with IL-1Ra, anti-IL-23R, both, or a control antibody (not shown). After 3 days, IL-17 was quantified in the supernatants by ELISA. ∗p < 0.05 and ∗∗p < 0.01, versus MTB alone. Data are means for triplicate cultures and are representative of two experiments. All error bars represent mean ± SD. Immunity 2009 31, 331-341DOI: (10.1016/j.immuni.2009.08.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 IL-1β and IL-23 Stimulate IL-17A, IL-17F, IL-21, IL-22, and IL-23R Expression by γδ T Cells (A–C) CD3+ T cells and CD3+ T cells depleted of γδ T cells (A), spleen cells from C57BL/6 and Tcrd−/− mice (B), purified γδ T cells, and γδ-depleted spleen cells from C57BL/6 mice (C) were stimulated for 3 days as indicated. IL-17 concentrations in supernatants were quantified by ELISA. ∗∗∗p < 0.001 versus WT or nondepleted. (D) Spleen cells from C57BL/6 mice were cultured with IL-1β and IL-23 or medium alone for 3 days, and intracellular staining was performed for IL-17 and surface γδ TCR. Numbers refer to the percentage of positive cells. (E) γδ-depleted CD3+ T cells or purified γδ T cells were stimulated with medium only, IL-1, IL-23, or IL-1 and IL-23 both. IL-17A, IL-17F, IL-21, IL-22, RORγt, and IL-23R mRNA expression was evaluated by real-time PCR normalized to 18 s rRNA and relative to γδ-depleted T cells stimulated with medium only. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus medium control; +p < 0.05, 2+p < 0.01, and +++p < 0.001, versus γδ-depleted T cells. (F) Spleen cells were stimulated with IL-23 or medium for 24 hr and were then analyzed for IL-23R expression. Dot plots are shown for staining with anti-IL-23R and an isotype control antibody. Numbers on the dot plots indicate the percentage of gated γδ T cells. All data are representative of at least three independent experiments. All error bars represent mean ± SD. Immunity 2009 31, 331-341DOI: (10.1016/j.immuni.2009.08.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 A High Frequency of IL-17-Secreting γδ T Cells Infiltrate the CNS of Mice at the Onset of EAE, which Correlates with IL-1β and IL-23p19 mRNA Expression EAE was induced in C57BL/6 mice. Brain mononuclear cells from naive mice and mice 7, 10, 14, and 21 days after induction of EAE, were stained for intracellular IL-17 or IFN-γ and surface CD45, CD3, and γδ TCR or CD4. Representative dot plots of IL-17 or IFN-γ-secreting γδ T cells (A) or IL-17 or IFN-γ-secreting CD4+ T cells (B) on day 10 are shown. (C) shows the mean percentage and absolute number of IL-17 or IFN-γ-secreting γδ T cells for five control (Cont.) mice and five mice at 7, 10, 14, and 21 days after induction of EAE; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus control. As shown in (D), brain mononuclear cells from mice 10 days after induction of EAE were stained for intracellular IL-17 and for surface γδ TCR, Vγ4, Vγ5, and Vδ6.3/2. Numbers refer to percentages of total γδ T cells. As shown in (E), IL-17A, IL-1β, and IL-23p19 mRNA expression was evaluated in the brain (cortex) by real-time PCR normalized to 18 s rRNA and relative to naive control mice. ∗∗p < 0.01, versus control. Dot plots are representative of five individual mice per group. Data are representative of three experiments. All error bars represent mean ± SD. Immunity 2009 31, 331-341DOI: (10.1016/j.immuni.2009.08.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 γδ T Cells Cooperate with Conventional T Cells in the Development of EAE (A and B) EAE was induced in C57BL/6 or Tcrd−/− mice. (A) Mice were observed daily for clinical symptoms of EAE. ∗p < 0.05 versus Tcrd−/−. (B) Mice were sacrificed and lymph node cells stimulated for 3 days with MOG peptide, medium, or PMA and anti-CD3. IL-17 concentrations in supernatants were determined by ELISA. ∗∗p < 0.01 and ∗∗∗p < 0.001 versus C57BL/6. (C) IL-17A and IL-17F mRNA expression was evaluated in spleen tissue by real-time PCR normalized to 18 s rRNA and relative to naive control mice. ∗p < 0.05 versus WT with EAE. (D) EAE was induced in C57BL/6 or Il1r1−/− mice. In addition, CD3+ T cells or CD3+ T cells depleted of γδ T cells from WT mice were transferred into Il1r1−/− mice 24 hr before induction of EAE. Mice were observed daily for clinical signs of EAE; n = 5. (E and F) Spleen and lymph node cells from mice with EAE were stimulated in vitro with MOG or MOG + IL-1 + IL-23. After 3 days, unseparated cells, CD4+ T cells, or γδ T cells purified from the cultures (E) or CD4+ and CD8+ or CD4+ and CD8+ plus γδ T cells (F) were transferred to naive recipient mice. Mice were observed for symptoms of EAE. Data are representative of two to three independent experiments. All error bars represent mean ± SD. Immunity 2009 31, 331-341DOI: (10.1016/j.immuni.2009.08.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 γδ T Cells Activated with IL-1β and IL-23 Promote IL-17 Production from CD4+ T Cells (A) Spleen cells from C57BL/6 and Tcrd−/− mice were stimulated as indicated for 3 days and IL-17 concentrations in supernatants quantified by ELISA. ∗∗∗p < 0.001 versus WT. (B and C) γδ T cells were incubated with IL-1β and IL-23 or medium alone. After 3 days, cells were washed and added to purified CD4+ T cells with and without anti-CD3 and anti-CD28 (B) or anti-CD3 and DCs (C). After an additional 3 days, supernatants were removed and IL-17 concentrations quantified by ELISA. ∗∗∗p < 0.001 versus no anti-CD3, +++p < 0.001 γδ (medium) versus γδ (IL-1β + IL-23), ###p < 0.001, versus without DCs. (D) Purified CD4+ T cells were stimulated with anti-CD3 and anti-CD28 and medium only or with supernatants from γδ T cells stimulated with IL-1β + IL-23 (washed on day 1), with or without anti-IL-17, anti-IL-21, or both antibodies. IL-17A and IL-17F mRNA expression was evaluated by real-time PCR normalized to 18 s rRNA and relative to CD4+ T cells stimulated with medium only. ∗p < 0.05 and ∗∗p < 0.01, with versus without antibody. Data are representative of three independent experiments. All error bars represent mean ± SD. Immunity 2009 31, 331-341DOI: (10.1016/j.immuni.2009.08.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 IL-17A Induces IL-17A and IL-17F Production from CD4+ T Cells and Enhances TLR-Induced IL-1β and IL-23 Production from DCs (A) IL-17R mRNA expression was evaluated in CD4+ T cells, lung, spleen, and control tissue (Cont; liver) from naive mice by real-time PCR normalized to 18 s rRNA and relative to control tissue. (B) Purified CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 in the presence of IL-17 or medium only (Med), and IL-17A and IL-17F mRNA expression was evaluated by real-time PCR normalized to 18 s rRNA. (C) Purified CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 and IL-21, IL-23, or both cytokines or with medium only (Med). IL-17A and IL-17F mRNA expression was evaluated by real-time PCR normalized to 18 s rRNA and expressed relative to medium only. (D) BMDC were stimulated with medium (Med) only, LPS, or CpG, and IL-17R mRNA expression was evaluated by real-time PCR normalized to 18 s rRNA and relative to a control (Cont.; liver). (E) BMDC were stimulated with IL-17 or medium only. After 24 hr, IL-23 production in supernatants was quantified by ELISA. (F) DCs were stimulated with LPS, IL-17, LPS + IL-17, or medium alone. After 24 hr, supernatants were removed and IL-23, IL-1β, IL-12p40, and IL-6 concentrations were quantified by ELISA. (G) DCs were stimulated with LPS alone or with IL-17 in the presence or absence of IL-17RFc. After 24 hr, TGF-β, IL-1β, IL-6, CCL2, and CXCL10 mRNA was evaluated by real-time PCR normalized to 18 s rRNA and relative to DC stimulated with medium only. Data are representative of at least two independent experiments. All error bars represent mean ± SD. Immunity 2009 31, 331-341DOI: (10.1016/j.immuni.2009.08.001) Copyright © 2009 Elsevier Inc. Terms and Conditions