γδT17 cells downregulate CCR6 upon activation

We recently reported that Th17 cell development during EAE is coupled with a dynamic, temporally regulated switch from CCR6 to CCR2 expression as Th17 cells propagate their differentiation. Expression patterns of CCR6 and CCR2 define distinct effector phenotypes of Th17 cells, with a CCR6−CCR2+ phenotype marking the encephalitogenic granulocyte–macrophage colony-stimulating factor/interferon-γ-producing population18. Unlike Th17 cells, γδT17 cell effector function is programmed during thymic development and these cells populate barrier tissues prior to inflammation2,21,22. Thus, we initially examined CCR6 and CCR2 expression in sLN and dermis in unimmunized Il17aCre × Rosa26eYFP mice, where Il17a expression drives permanent marking of cells with eYFP23. γδT17 cells in these compartments constitutively co-expressed CCR2 and CCR6 (Fig. 1a and Supplementary Fig. 1a). Expression of CCR6 and CCR2 was restricted to γδ T cells bearing a CD27−CD44hi phenotype, characteristic of γδT17 cells (Supplementary Fig. 2a)24. CCR6/CCR2 co-expression was similar between Vγ4+ and Vγ6+ γδT17 cell subsets as distinguished by both Vγ4 expression and CD3/T-cell receptor (TCR) expression level, as previously reported (‘CD3bright staining’)25 (Supplementary Fig. 1b,c), and both receptors were functional as determined by ex vivo chemotaxis (Fig. 1b). However, examination of γδT17 cells from diverse tissues revealed a heterogeneous pattern of CCR6 expression. While thymic and most lymphoid γδT17 cells uniformly expressed both CCR6 and CCR2, populations of γδT17 cells lacking CCR6 expression (CCR6−CCR2+) were prominent in lung and gut-associated tissues (Fig. 1c). As the gut is tonically immunologically active due to interactions with commensal microbiota, we hypothesized that γδT17 cells downregulate CCR6 expression during inflammation.

Figure 1: γδT17 cells downregulate CCR6 upon activation. (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 Il17aCre × Rosa26eYFP mice (n=3). (b) Ex vivo transwell chemotaxis of Il17aCre × Rosa26eYFPsplenic IL-17A+/− γδ T cells to CCL20 and CCL2 (n=3). (c) Representative flow cytometry of CD45+ γδT17 cells from organs of naïve Il17aCre × Rosa26eYFP 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 Il17aCre × Rosa26eYFP 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 Il17aCre × Rosa26eYFP lymphocytes cultured with indicated stimuli for 72 h (n=5). See also Supplementary Figs 1 and 2. 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. Full size image

In support of this idea, activation of sLN and spleen γδT17 cells in vivo during EAE resulted in downregulation of CCR6 expression compared to unimmunized mice (Fig. 1d). CNS-infiltrating γδT17 cells were also largely CCR6−. BrdU incorporation revealed that CCR6 expression was downregulated in proliferated γδT17 cells, while BrdU− cells remained CCR6+ (Fig. 1e). Other γδ T-cell subsets did not express CCR2 or CCR6 at rest, and did not gain expression of these receptors over the course of EAE (Supplementary Fig. 2a). Unlike Th17 cells, γδT17 cells are predominantly activated by TCR-independent signals including IL-23 and IL-1β1. In vitro stimulation of lymphocytes with a range of known stimuli including IL-23/IL-1β, IL-23/IL-18 (ref. 26), IL-7 (ref. 27) and γδ-TCR signalling28 uniformly repressed CCR6 surface expression in γδT17 cells (Fig. 1f and Supplementary Fig. 2b). IL-12 did not impact CCR6 expression, consistent with a reported absence of IL-12R expression by γδT17 cells29 (Supplementary Fig. 2b). Activation-induced CCR6 downregulation correlated with induction of activation markers CD69 and CD25 and increased CD44 expression, and occurred in both Vγ4+ and Vγ6+ γδT17 cells (Supplementary Fig. 2c,d). In all in vivo and in vitro systems, γδT17 cells maintained high levels of CCR2 following activation, and virtually all γδT17 cells were CCR2+ (Fig. 1a,c,d,f). Therefore, γδT17 cells are programmed to co-express CCR6 and CCR2 during development, but lose CCR6 expression upon activation.

CCR2 drives γδT17 cell recruitment to inflamed tissues

Tissue-infiltrating γδT17 cells are best understood in the context of cancer and autoimmunity. γδT17 cells infiltrate B16 melanomas and promote tumour growth30,31, and infiltrate the CNS at disease onset and exacerbate disease pathogenesis during EAE1,32. How γδT17 cells infiltrate these inflammatory lesions is unknown. We thus used these models to investigate CCR6 and CCR2 function in control of γδT17 cell migration during inflammation. Consistent with the observation that activation induces downmodulation of CCR6 expression, Ccr6-deficiency did not affect γδT17 cell infiltration of B16 melanomas (Fig. 2a,b), nor recruitment to the CNS during EAE onset (Fig. 2c). Thus, γδT17 cell trafficking to inflamed tissues in these settings occurs independently of CCR6.

Figure 2: CCR2 recruits γδT17 cells to inflammatory sites. (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 F 1 (CD45.1+CD45.2+) mice. Ccr2−/−:F 1 total, Vγ4 and Vγ6 γδT17 cell ratios in spleen and tumours were normalized to input ratio. Vγ4 and Vγ6 γδT17 cells were determined by CD3bright 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 F 1 and Ccr2−/− γδT17 cells as in (i). Twenty-four hours later, ratios of Ccr2−/−:F 1 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 Supplementary Figs 3 and 4. 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. Full size image

In contrast, deficiency of Ccr2 abrogated γδT17 cell infiltration of B16 melanomas but did not affect their expansion in draining lymph nodes (LNs) (Fig. 2d). CCR2-driven infiltration of γδT17 cells was consistent with upregulation of its major ligand CCL2 in tumours (Fig. 2e). Similar results were found in EAE, where Ccr2-deficiency inhibited γδT17 cell recruitment to the CNS at both onset and peak disease (Fig. 2f,g), time points at which CCL2 was induced in the CNS as reported33 (Fig. 2h). CCR2 appeared to operate independently of CCR6 in regulation of γδT17 cell trafficking, as compound deficiency of Ccr6 and Ccr2 (Ccr6−/−Ccr2−/−) did not further affect γδT17 cell infiltration in either model (Fig. 2d,f,g).

Ccr2−/−Ccr6−/− mice exhibited enhanced tumour growth, while Ccr2−/− and Ccr2−/−Ccr6−/− mice had decreased EAE severity (Supplementary Fig. 3). Therefore, to examine the cell-intrinsic requirements of CCR2 for γδT17 cell migration, we developed a novel in vitro expansion protocol to generate large numbers of purified activated γδT17 cells, which contained both Vγ4+ and Vγ6+ subsets and maintained functional CCR2 expression (Supplementary Fig. 4). Equal ratios of in vitro-expanded genetically marked wild type (WT) and Ccr2−/− γδT17 cells were co-transferred into B16 melanoma-bearing recipients. While donor γδT17 cells recovered from spleen retained the input ratio, Ccr2−/− γδT17 cells did not migrate efficiently to tumours. This observation was true for both Vγ4+ and Vγ6+ subsets (Fig. 2i). Similar experiments using the EAE model revealed equivalent WT:Ccr2−/− γδT17 cell ratios in spleen and blood but reduced Ccr2−/− γδT17 cell recruitment to the CNS (Fig. 2j). Thus, CCR2, but not CCR6, drives activated γδT17 cell migration to inflammatory sites during B16 melanoma and EAE.

CCR2 is essential for protective γδT17 cell responses

The above models involve γδT17 cell infiltration from circulation, as the CNS and tumours lack resident γδT17 cell populations. However, many inflammatory scenarios implicate tissue-resident γδT17 cells, which survey and rapidly defend against infection at barrier surfaces. The extent to which γδT17 cell migration contributes to host defence during ongoing inflammation is unknown. To investigate whether CCR2 also directs tissue-infiltrating γδT17 cells during infection, we used experimental Streptococcus pneumoniae infection, immunity to which requires γδT17 cells4. Accordingly, Tcrd−/− mice had higher bacterial burden and reduced neutrophils in the nasal wash (NW) than WT at 72 h post-infection (Fig. 3a,b). S. pneumoniae infection induced γδT17 cell expansion in draining LNs and nasal-associated lymphoid tissue (Fig. 3c). CCL2 was induced in the nasal passages (NPs) upon infection (Fig. 3d), and co-transfer of in vitro-expanded WT and Ccr2−/− γδT17 cells into infected mice revealed an intrinsic requirement of CCR2 for γδT17 cell accumulation in NP (Fig. 3e). Thus, CCR2 drives circulating γδT17 cell infiltration of mucosal tissue during S. pneumoniae infection.

Figure 3: CCR2 drives protective γδT17 cell responses. (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 F 1 (CD45.1+CD45.2+) mice. The Ccr2−/−:F 1 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. Full size image

To elucidate the ability of recruited γδT17 cells to control infection, we transferred purified in vitro-expanded γδT17 cells into Tcrd−/− hosts prior to S. pneumoniae infection. In this model, tissue-infiltrating γδT17 cells provide the only source of γδ T-cell-driven protection. Transfer of WT γδT17 cells reduced nasopharyngeal bacterial burden by ∼10-fold, whereas Ccr2−/− γδT17 cells completely failed to control infection (Fig. 3f). Hence, CCR2 drives recruitment of protective γδT17 cells to the nasal mucosa during S. pneumoniae infection. Collectively, we conclude that γδT17 cell trafficking to diverse inflamed tissues is critically dependent on CCR2 signalling.

CCR6 regulates homeostatic positioning of γδT17 cells

Expression of CCR6 during γδT17 cell thymic development followed by rapid downregulation upon activation suggests that CCR6 plays a more prominent function in regulation of γδT17 cell homeostasis. While CCL20 is induced during inflammation, it is constitutively expressed in barrier tissues including skin, Peyer’s patches and large intestine34,35,36,37. Both Ccr6−/− and Ccr2−/−Ccr6−/− mice had markedly reduced number and frequency of γδ T cells expressing intermediate amounts of CD3/TCR in the dermis (γδTlo), a population previously reported to produce IL-17 and distinct from TCRhi dendritic epidermal T cells22 (Fig. 4a). We confirmed that γδTlo cells were entirely marked by eYFP in Il17aCre × Rosa26eYFP mice, despite negligible IL-17A production following ex vivo restimulation (Supplementary Fig. 5a). In contrast to a previous report10, Ccr6-deficiency reduced the number of both Vγ4+ and Vγ4− (Vγ6+) γδTlo cells, although the ratio was skewed slightly towards Vγ6+ cells (Fig. 4b). Examination of other organs revealed that deficiency in Ccr2 had no effect on γδT17 cell homeostasis, while Ccr6-deficiency increased γδT17 cells in the peritoneal cavity (Supplementary Fig. 5b). We conclude that CCR6 regulates dermal γδT17 cell residence.

Figure 4: CCR6 regulates homeostatic γδT17 cell recruitment to dermis. (a) Representative flow cytometry and quantitation of CD45+CD3loTCR-γδlo (γδTlo) 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 γδTlo cells and quantitation of Vγ4+ and Vγ4− γδTlo 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+ γδTlo/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-CAMlo interfollicular epidermis (IE), Sca-1lo/+Ep-CAM+ infundibulum and isthmus (IF & IS), Sca-1loEp-CAMlo double negative (DN)) or CD45− dermal populations (CD31−CD90+CD140α+ fibroblast, gp38+CD31+ lymphatic endothelial cells (LEC), gp38loCD31+ blood endothelial cells (BEC), CD31−CD90−CD140α− double negative (DN)) from naïve WT mice (pooled from 5 mice/experiment). ND, not detected. See also Supplementary Fig. 5. 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. Full size image

To determine whether CCR6 drives recruitment of circulating γδT17 cells into dermis, we transferred unstimulated WT or Ccr6−/− lymphocytes into naïve mice and tracked their accumulation in dermis. Transferred WT γδT17 cells were substantially enriched in dermis, demonstrating that γδT17 cells can constitutively populate the skin from circulation. In contrast, Ccr6−/− γδT17 cells were defective in infiltration of dermis and pooled in the blood (Fig. 4c). In support of earlier results, both Vγ4+ and Vγ4− γδT17 cells were recruited to the dermis, the ratio of which was unaltered by Ccr6 deficiency, suggesting both populations are dependent on CCR6 for circulation-to-dermis trafficking (Fig. 4c). While constitutive expression of CCL20 in epidermis was reported, whether it is expressed in uninflamed dermis is unclear34,38. We found that Ccl20 was constitutively expressed in the dermis by an uncharacterized CD31−CD90−CD140α− stromal population (Fig. 4d). Thus, CCR6 directs homeostatic recruitment of γδT17 cells from circulation into dermis.

IRF4 and BATF regulate CCR6 expression in γδT17 cells

The downregulation of CCR6 upon γδT17 cell activation is surprising, as T cells typically upregulate inflammatory chemokine receptors upon activation. Consequently we investigated the underlying mechanism regulating this process. Ccr6 transcript levels were reduced by approx. fourfold in γδT17 cells within 24 h of stimulation, whereas Ccr2 expression was maintained (Fig. 5a and Supplementary Fig. 6a). This indicated that CCR6 expression is transcriptionally regulated during γδT17 cell activation. We thus examined expression of transcription factors previously implicated directly or indirectly in control of Ccr6 expression, including RORγt17, IRF4 (ref. 39), IRF8 (refs 40, 41), Blimp1 (refs 39, 42), BATF43 and T-bet and Eomes18. Rorc (RORγt) was highly expressed in resting γδT17 cells but was downregulated by 24 h of activation. Batf and Prdm1 (Blimp1) were rapidly upregulated by 24 h, while Irf8 and Irf4 were upregulated by 48 h, although Irf4 was already present in resting γδT17 cells. Expression of Eomes and Tbx21 (T-bet) at rest or following activation was minimal (Fig. 5b and Supplementary Fig. 6b). Therefore, we tested whether RORγt, IRF4, BATF, Blimp1 or IRF8 repressed Ccr6 expression during γδT17 cell activation.

Figure 5: IRF4 and BATF promote CCR6 downregulation in γδT17 cells. (a) Ccr6 and (b) transcription factor mRNA in sorted γδT17 cells from Il17aCre × Rosa26eYFP 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 GFPhi 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 GFPhi 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 Supplementary Figs 6 and 7. (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. Full size image

The similar expression kinetics and known Ccr6 regulatory activity of RORγt presented the possibility that its downregulation may result in loss of CCR6 expression. To test this, we retrovirally forced Rorc expression in in vitro-expanded γδT17 cells (Fig. 5c). However, this failed to alter CCR6 expression even in the highest GFP-expressing cells, suggesting that RORγt downregulation is not required for repression of CCR6 in activated γδT17 cells (Fig. 5d).

To determine whether IRF4, BATF, IRF8 or Blimp1 actively repress Ccr6 expression, we cultured genetically-marked WT and transcription factor-deficient splenocytes with IL-23 and IL-1β. γδT17 cells were present in all strains although at differing frequencies, and homeostatic CCR6 expression was comparable to WT (Supplementary Fig. 6c). IRF4- and BATF-deficient γδT17 cells were intrinsically defective in both proliferation and CCR6 downregulation upon stimulation (Fig. 5e). IRF8 and Blimp1 were not required for these processes, although Blimp1 appeared to moderately promote CCR2 expression in activated γδT17 cells (Supplementary Fig. 6d). Irf4−/− and Batf−/− γδT17 cells exhibited comparable surface expression of IL-23R and IL-1R1 to WT cells, indicating that maintained CCR6 expression was likely due to defective signalling downstream of IL-23 and IL-1β stimulation (Supplementary Fig. 7a). Analysis of our and others’ ChIP-Seq datasets in T cells43,44,45 revealed binding of IRF4 and BATF to a shared site in the Ccr6 promoter (Supplementary Fig. 7b), suggesting that these factors cooperatively and directly repress Ccr6 in γδT17 cells. To assess whether defective proliferation in absence of IRF4 or BATF was the cause of impaired CCR6 downregulation, dye-labelled WT splenocytes were pre-treated with proliferation inhibitor mitomycin C prior to stimulation. Although proliferation was effectively blocked, CCR6 downregulation still occurred upon mitomycin C treatment, suggesting that proliferation and CCR6 downregulation are coincident but independent (Fig. 5f). Together, these data indicate that activation-induced CCR6 downregulation in γδT17 cells is promoted by IRF4 and BATF, and is largely uncoupled from proliferation.

Loss of CCR6 promotes γδT17 cell homing to inflamed tissues

Given the constitutive expression of CCL20 in mucocutaneous sites, we hypothesized that repression of CCR6 during activation enables homing of γδT17 cells toward inflammatory lesions by preventing their accumulation in uninflamed skin. To test this, we first compared the trafficking of in vitro-activated WT γδT17 cells with resting WT and Ccr6−/− γδT17 cells upon transfer into unimmunized hosts. Activated WT γδT17 cells demonstrated the same defect in homing to the dermis as resting Ccr6−/− γδT17 cells, and both pooled in blood compared to resting WT γδT17 cells (Fig. 6a). γδT17 cells lack CD62L and CCR7 expression, and traffic from skin to sLNs in a CCR7-independent manner12. Thus γδT17 cell entry to sLNs following adoptive transfer likely occurs via afferent lymph draining from dermis. In keeping with this idea, resting Ccr6−/− or in vitro-activated WT γδT17 cells, impaired in their ability to home to uninflamed skin, also accumulated less than resting WT γδT17 cells in sLNs (Fig. 6a). These data are consistent with the notion that activation switches off γδT17 cell homeostatic circulation patterns, enabling directed migration toward inflammatory cues.

Figure 6: CCR6 downregulation by γδT17 cells enhances migration to inflamed tissue. (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+ γδTlo/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 F 1 (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 F 1 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 F 1 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 F 1 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. Full size image

To investigate this proposal directly, we studied the migratory patterns of in vitro-activated γδT17 cells retrovirally forced to maintain CCR6 expression. Infection with Ccr6tg virus restored CCR6 expression in activated γδT17 cells, which regained the ability to migrate toward CCL20 (Fig. 6b,c). Genetically marked control- and Ccr6tg-transduced γδT17 cells were mixed 50:50 and transferred into B16 melanoma-bearing recipients. While the input ratio of transferred GFP− cells was maintained in all examined organs as expected, among GFP+ cells, Ccr6tg γδT17 cells were enriched in the dermis but deficient in tumours (Fig. 6d). Similar results were observed during S. pneumoniae infection: Ccr6tg γδT17 cells were selectively deficient at homing to NP, but accumulated to a greater extent than control-transduced cells in uninflamed dermis (Fig. 6e). Ccr6tg γδT17 cells also homed less efficiently to the CNS during EAE, although subcutaneous complete Freund’s adjuvant immunization precluded analysis of homing to uninflamed skin in this model (Fig. 6f). Together, these experiments demonstrated that activated γδT17 cells with forced CCR6 expression were recruited to uninflamed dermis at the expense of homing to inflamed tissue. Thus, CCR6 downregulation promotes γδT17 cell migration to inflammatory sites.