(a) Representative flow cytometry of CCR6 and CCR2 expression in skin-draining lymph nodes (sLN) and dermal CD3⁺TCR-γδ⁺IL-17A-YFP⁺ γδT17 cells from Il17a^Cre × Rosa26^eYFP mice (n=3). (b) Ex vivo transwell chemotaxis of Il17a^Cre × Rosa26^eYFPsplenic IL-17A^+/− γδ T cells to CCL20 and CCL2 (n=3). (c) Representative flow cytometry of CD45⁺ γδT17 cells from organs of naïve Il17a^Cre × Rosa26^eYFP mice (n=3). mLN, mesenteric lymph node; PP, Peyer's patches; siLPL, small intestinal lamina propria lymphocytes. (d) Representative flow cytometry and quantitation of CCR6 and CCR2 expression by γδT17 cells from organs of Il17a^Cre × Rosa26^eYFP mice either naïve (n=6) or at experimental autoimmune encephalomyelitis (EAE) onset (n=7) or peak (n=5). CNS, central nervous system; iLN, inguinal lymph node; ND, not detected. (e) Representative flow cytometry and quantitation of CCR6 expression by γδT17 cells from wild type (WT) mice given BrdU at d3 post-immunization for EAE, and analysed at d8 (n=4). (f) Representative flow cytometry and frequency of CCR6 and CCR2 expression by γδT17 cells from Il17a^Cre × Rosa26^eYFP lymphocytes cultured with indicated stimuli for 72 h (n=5). See also . Mean±s.e.m. (a–c) Representative of two experiments. (d,f) Pooled from two experiments. (e) Paired two-tailed Student's t-test, (f) one-way paired ANOVA with Dunnett's multiple comparisons test relative to unstimulated control. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

(a) CD45⁺CD3⁺TCRγδ⁺IL-17A⁺ γδT17 cell numbers in tumour-infiltrating lymphocytes (TIL) following B16 melanoma challenge (n=5/time point). (b) γδT17 cell numbers in TIL d7 post-challenge with B16 melanoma in wild type (WT) (n=12) and Ccr6^−/− mice (n=13). (c) γδT17 cell numbers in central nervous system (CNS) at experimental autoimmune encephalomyelitis (EAE) onset in WT (n=7) and Ccr6^−/− mice (n=6). (d) γδT17 cell numbers in TIL and inguinal lymph nodes (iLN) d7 post-challenge with B16 melanoma in WT (n=15 (TIL), 9 (iLN)), Ccr2^−/− (n=13 (TIL), 10 (iLN)) and Ccr2^−/−Ccr6^−/− mice (n=9 (TIL), 5 (iLN)). (e) ELISA for CCL2 in tumour supernatant from WT mice challenged with B16 melanoma (n=5/time point). (f) γδT17 cell numbers in CNS and iLN at EAE onset in WT (n=14), Ccr2^−/− (n=13) and Ccr2^−/−Ccr6^−/− mice (n=12). (g) γδT17 cell numbers in CNS at peak disease in WT (n=6), Ccr2^−/− (n=5) and Ccr2^−/−Ccr6^−/− mice (n=6). (h) ELISA for CCL2 in CNS of WT mice with EAE (n=4/time point). (i) Ly5.1 mice (n=4) at d5 post-challenge with B16 melanoma were transferred i.v. with in vitro-expanded γδT17 cells from Ccr2^−/− (CD45.2⁺) and F₁ (CD45.1⁺CD45.2⁺) mice. Ccr2^−/−:F₁ total, Vγ4 and Vγ6 γδT17 cell ratios in spleen and tumours were normalized to input ratio. Vγ4 and Vγ6 γδT17 cells were determined by CD3^bright gating. Representative flow cytometry of CD45.2⁺ γδT17 cells at d7 or input. (j) Ly5.1 mice (n=7) at EAE onset were transferred with F₁ and Ccr2^−/− γδT17 cells as in (i). Twenty-four hours later, ratios of Ccr2^−/−:F₁ γδT17 cells in spleen, blood and CNS were normalized to input. Representative flow cytometry of CD45.2⁺ γδT17 cells 24 h later or input. See also . Mean±s.e.m. (a,c,e,i) Representative of two experiments. (b,d,f,j) Pooled from two experiments. (b,c) Unpaired two-tailed Student's t-test, (d,f–g,j) one-way ANOVA with Bonferroni's multiple comparisons test (paired in j)), (i) paired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

(a) Colony-forming units (c.f.u.) and (b) CD45⁺CD11b⁺Ly6G⁺ neutrophils recovered from nasal wash (NW) of wild type (WT) (n=9) and Tcrd^−/− mice (n=10) 72 h post-infection with S. pneumoniae. (c) γδT17 cell numbers in cervical lymph node (cLN) and nasal-associated lymphoid tissue (NALT) and (d) ELISA for CCL2 in digested nasal passage (NP) supernatant in unimmunized mice (n=7) and at 72 h post-S. pneumoniae infection (n=13). (e) Ly5.1 mice (n=5) 24 h post-S. pneumoniae infection were transferred i.v. with expanded γδT17 cells from Ccr2^−/− (CD45.2⁺) and F₁ (CD45.1⁺CD45.2⁺) mice. The Ccr2^−/−:F₁ γδT17 cell ratio in spleen and NP was normalized to input ratio. (f) Twenty-four hours prior to S. pneumoniae infection, Tcrd^−/− hosts received PBS (n=8) or expanded and purified γδT17 cells from WT (n=9) or Ccr2^−/− (n=7) mice. c.f.u. recovered from NW 72 h post-infection. Mean±s.e.m. (a–d) Pooled from two experiments. (a,b) Mann–Whitney test, (c,d) unpaired two-tailed Student's t-test, (e) paired two-tailed Student's t-test, (f) Kruskal–Wallis test with Dunn's multiple comparisons test. *P<0.05, **P<0.01.

(a) Representative flow cytometry and quantitation of CD45⁺CD3^loTCR-γδ^lo (γδT^lo) cells from ear skin dermis of naïve wild type (WT) (n=13), Ccr6^−/− (n=11), Ccr2^−/− (n=10) and Ccr2^−/−Ccr6^−/− mice (n=5). (b) Representative flow cytometry of Vγ4 expression by dermal γδT^lo cells and quantitation of Vγ4⁺ and Vγ4⁻ γδT^lo cells in dermis of WT and Ccr6^−/− mice (n=7/group). (c) WT or Ccr6^−/− lymphocytes were transferred i.v. into naïve Ly5.1 mice (n=4/group). After 36 h, number of CD45.2⁺ γδT^lo/γδT17 cells recovered was expressed as % of number transferred. Representative flow cytometry of dermal CD45.2⁺ cells and quantitation of γδT17 cell recovery and Vγ4⁺:Vγ4⁻ ratio (normalized to input). (d) Ccl20 mRNA from whole tissues or sorted CD45⁻ epidermal keratinoctyes (Sca-1⁺Ep-CAM^lo interfollicular epidermis (IE), Sca-1^lo/+Ep-CAM⁺ infundibulum and isthmus (IF & IS), Sca-1^loEp-CAM^lo double negative (DN)) or CD45⁻ dermal populations (CD31⁻CD90⁺CD140α⁺ fibroblast, gp38⁺CD31⁺ lymphatic endothelial cells (LEC), gp38^loCD31⁺ blood endothelial cells (BEC), CD31⁻CD90⁻CD140α⁻ double negative (DN)) from naïve WT mice (pooled from 5 mice/experiment). ND, not detected. See also . Mean±s.e.m. (a,d) Pooled from three experiments, (c) representative of two similar experiments. (a) One-way ANOVA with Bonferroni's multiple comparisons test, (b,c) unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

(a) Ccr6 and (b) transcription factor mRNA in sorted γδT17 cells from Il17a^Cre × Rosa26^eYFP lymphocytes ex vivo or cultured with IL-23/IL-1β for indicated times (pooled from 5 to 7 mice). ND, not detected. (c,d) Expanded γδT17 cells (n=3) were transduced with empty pMIG or pMIG-Rorc retrovirus. (c) Representative flow cytometry of RORγt expression in GFP^hi γδT17 cells (gated as in d), relative to isotype (grey) and geometric mean fluorescence intensity (gMFI) relative to GFP fluorescence intensity (FI). (d) Representative flow cytometry of CCR6 expression and quantitation in GFP^hi γδT17 cells. (e) Splenocytes from Ly5.1 and either wild type (WT), Irf4^−/− or Batf^−/− mice were 670 dye-labelled, mixed 50:50 and stimulated with IL-23/IL-1β for 72 h. Representative flow cytometry and quantitation of CCR6 expression and proliferation in CD45.1⁺ or CD45.2⁺ γδT17 cells (n=3/group). (f) Representative flow cytometry and quantitation of CCR6 expression by 670 dye-labelled γδT17 cells from WT splenocytes cultured with IL-23/IL-1β for 72 h with/without mitomycin C pre-treatment (n=3). See also . (a,b) Mean±s.d., (c–f) Mean±s.e.m. (a–f) Representative of two similar experiments. (d,e) Paired two-tailed Student's t-test, (f) one-way paired ANOVA with Bonferroni's multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001.

(a) Resting lymphocytes from wild type (WT) (n=3) or Ccr6^−/− (n=4) mice, or WT lymphocytes stimulated with IL-23/IL-1β for 72 h (n=4) were transferred i.v. into separate naïve Ly5.1 hosts. After 36 h, number of CD45.2⁺ γδT^lo/γδT17 cells recovered was expressed as % of number transferred. sLN, skin-draining lymph node. (b) Representative flow cytometry for CCR6 expression by GFP⁺ in vitro-expanded γδT17 cells transduced with empty pMIG or pMIG-Ccr6, relative to isotype (grey) (n=3). (c) Chemotaxis of GFP⁺ γδT17 cells transduced as in (b) to CCL20 (n=3). (d) In vitro-expanded γδT17 cells from F₁ (CD45.1⁺CD45.2⁺) or WT (CD45.2⁺) mice were transduced with empty pMIG or pMIG-Ccr6, respectively. Equal numbers of mixed GFP⁺ cells were transferred i.v. into Ly5.1 mice challenged with B16 melanoma 5 days prior and analysed at d7 (n=5). Representative flow cytometry and ratio of recovered F₁ to WT γδT17 cells within transduced (GFP⁺) and untransduced (GFP⁻) populations. Recovered values were normalized to input values. TIL, tumour-infiltrating lymphocytes. (e,f) In vitro-expanded γδT17 cells from WT or F₁ mice were transduced with empty pMIG or pMIG-Ccr6, respectively. Equal numbers of mixed GFP⁺ cells were transferred i.v. into Ly5.1 mice either (e) 24 h post-infection with S. pneumoniae (n=4) or (f) at experimental autoimmune encephalomyelitis (EAE) onset (n=3) and organs were analysed 48 h later. Ratio of recovered WT to F₁ γδT17 cells within transduced (GFP⁺) and untransduced (GFP⁻) populations, normalized to input values. CNS, central nervous system; NP, nasal passage. Mean±s.e.m. (a) Representative of three similar experiments, (b,d) representative of two experiments. (a) One-way ANOVA with Dunnett's multiple comparisons test relative to resting WT γδT17 cells, (d–f) paired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.