1. 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 (August 2009)
DOI: /j.immuni
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. 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  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. 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  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. 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 < 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 < 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  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. 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 < 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  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. 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 < 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  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. 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 < versus no anti-CD3, +++p < 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  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. 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  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions