Cutting Edge: Spontaneous Development of IL-17–Producing γδ T Cells in the Thymus Occurs via a TGF-β1–Dependent Mechanism

In naive animals, γδ T cells are innate sources of IL-17, a potent proinflammatory cytokine mediating bacterial clearance as well as autoimmunity. However, mechanisms underlying the generation of these cells in vivo remain unclear. In this study, we show that TGF-β1 plays a key role in the generation of IL-17+ γδ T cells and that it mainly occurs in the thymus particularly during the postnatal period. Interestingly, IL-17+ γδ TCR+ thymocytes were mainly CD44highCD25low cells, which seem to derive from double-negative 4 γδ TCR+ cells that acquired CD44 and IL-17 expression. Our findings identify a novel developmental pathway during which IL-17–competent γδ T cells arise in the thymus by a TGF-β1–dependent mechanism.

In naive animals, gd T cells are innate sources of IL-17, a potent proinflammatory cytokine mediating bacterial clearance as well as autoimmunity. However, mechanisms underlying the generation of these cells in vivo remain unclear. In this study, we show that TGF-b1 plays a key role in the generation of IL-17 + gd T cells and that it mainly occurs in the thymus particularly during the postnatal period. Interestingly, IL-17 + gd TCR + thymocytes were mainly CD44 high CD25 low cells, which seem to derive from double-negative 4 gd TCR + cells that acquired CD44 and IL-17 expression. Our findings identify a novel developmental pathway during which IL-17-competent gd T cells arise in the thymus by a TGF-b1-dependent mechanism. The Journal of Immunology, 2010, 184: 1675-1679.
A lthough they are widely distributed throughout the epithelial cell-rich tissues (1) and are known to be an important source of IL-17 in response to a number of pathogens, recruiting neutrophils to the site of inflammation (2,3), gd T cells constitute a small proportion (,5%) of total peripheral T lymphocytes. gd T cells are also the major IL-17producing cells in naive animals (4)(5)(6). It was reported that Agnaive CD122 2 gd T cells preferentially produce IL-17, whereas Ag-experienced CD122 + gd T cells produce IFN-g (5). It was recently shown that the expression of the TNF family member CD27 better defines these cytokine-producing gd T cell subsets; IL-17 + gd T cells are mostly CD27 2 and IFN-g + gd T cells are mostly CD27 + (7). However, how gd T cells acquire IL-17 expression in vivo remains unclear.
In this study, we report that developing gd T cells acquire IL-17-producing capacity within the thymus via a TGF-b1dependent mechanism. Interestingly, peripheral IL-17 + gd T cells primarily accumulate in the peripheral but not in the mesenteric LN (mLN). An ontogeny study revealed that the highest frequency of IL-17 + gd T cells was found in the postnatal thymus. IL-17 + gd TCR + thymocytes were CD44 high CD25 low (double-negative [DN] 1) phenotype cells, which are in fact the DN4 cells that upregulated CD44 and acquired IL-17 expression. The generation of IL-17 + gd T cells was dramatically abolished by the absence of TGF-b1 but not of other Th17-inducing cytokines. Consistent with this, gd T cells in the thymus expressed the highest levels of TGF-b receptors. Taken together, the current study highlights a unique pathway of thymic gd T cell development during which the differentiation of natural IL-17 + gd T cells takes place, revealing an irreplaceable role for TGF-b1 to promote this process.

Materials and Methods
Mice C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Lymphoid cells from IL-6 2/2 and IL-23 p19 2/2 mice were provided by Drs. Robert Fairchild and Steve Stohlman (Cleveland Clinic Foundation, Cleveland, OH). IL-21 2/2 mice were purchased from the Mutant Mouse Regional Resource Centers (Columbia, MO; www.mmrrc.org). Rag2p-GFP and Tgfb1 2/2 mice were previously described (8,9). All experimental procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee of the Cleveland Clinic Foundation, Case Western Reserve University, and the University of Washington.

Ex vivo stimulation
Spleen, axillary and cervical LN (pLN), and mLN cells were separately harvested and ex vivo stimulated with PMA (10 ng/ml) and ionomycin (1 mM) for 4 h in the presence of 2 mM monensin (Calbiochem, San Diego, CA) during the last 2 h. Cells were immediately fixed with 4% paraformaldehyde, permeabilized, and stained with fluorescence conjugated Abs (see below).

Flow cytometry
The following Abs were used:

Real-time PCR
DN gd TCR 2 , DN gd TCR + , double-positive (DP), and CD4 single-positive (SP) thymocytes were isolated by FACS sorting using a FACSAria cell sorter (Becton Dickinson). RNA was extracted using RNeasy reagent (Qiagen, Valencia, CA), and cDNA was subsequently generated by Superscript III RTase (Invitrogen, Carlsbad, CA). Taqman primers/probes specific for Tgfbr1 (Mm03024015_m1) and Tgfbr2 (Mm03024091_m1) were purchased from Applied Biosystems (Foster City, CA), and their expression was determined using an Applied Biosystems 7500 PCR system. Expression level was normalized based on the 18S rRNA (VIC-TAMRA, purchased from Applied Biosystems) expression.

Data analysis
Statistical significance was determined by the Student t test using the Sig-maPlot 9.0 (SPSS, Chicago, IL). p , 0.05 was considered to indicate a significant difference.

gd T cells are the major IL-17-producing cells in naive animals
In naive animals, very few (∼0.1%) LN CD4 T cells expressed intracellular IL-17 following PMA/ionomycin stimulation, but ∼0.5% non-CD4 T cells expressed IL-17 under the same conditions ( Fig. 1A). IL-17 + non-CD4 T cells were not CD8, B, or NK1.1 + cells (Fig. 1A); instead, ∼75% of the IL-17 + cells expressed gd TCR (Fig. 1B). A significant portion of gd T cells expressed an activated phenotype compared with ab T cells: CD44 high and CD62L low (Fig. 1C). IL-17 production was noticed only from CD44 high gd T cells (Fig. 1D), which differs from a previous study showing that IL-17 is preferentially produced from naive CD122 low gd T cells after TCR cross-linking (5). Our finding agrees with a recent report in that CD27 low gd T cells that mainly produce IL-17 are CD44 high CD62L low cells (7). Indeed, IL-17 + gd T cells in the pLN displayed the same CD27 low phenotype (Fig. 1E, top panel).
Interestingly, the proportion of IL-17 + gd T cells in regional lymphoid tissues displayed a substantial heterogeneity; the highest frequency of IL-17 + gd cells was found in pLN, whereas gd T cells from mLN failed to express IL-17 ( Fig.  1F). IL-17 + gd T cells in the spleen were also present at a low frequency (∼3%). Notably, ,3% of CD27 low mLN gd T cells expressed IL-17, suggesting that CD27 low phenotype does not necessarily define IL-17-producing gd T cells (Fig.  1E). By contrast, IFN-g + gd T cells were found in all lymphoid tissues (Fig. 1F). Consistent with previous reports (5, 7), gd T cells producing IL-17 and IFN-g did not overlap (data not shown). Unlike lymphoid gd T cells, skin-resident gd T cells, gd T cells from Peyer's patches, or intraepithelial gd lymphocytes from the small intestine and the colon expressed very little IL-17 (Fig. 1G). Why peripheral IL-17 + gd FIGURE 1. pLN gd T cells preferentially express IL-17 following activation. A, LN cells were ex vivo stimulated with PMA/ionomycin and subsequently stained for CD4, NK1.1, CD8, B220, and IL-17. B, LN cells stimulated as above were stained for B220, CD4, CD8, IL-17, and gd TCR. Shown is gd T cell IL-17 expression by non-B/non-T cells. C, gd, CD4, and CD8 T cells from the indicated tissues were examined for the surface expression of CD44 and CD62L. D, IL-17 expression of CD44 low and CD44 high gd T cells (spleen and pLN) was examined by intracellular staining. E, gd T cells from the indicated tissues were stimulated and stained for IL-17 and CD27. F, Proportions of IFN-g-and of IL-17-producing gd T cells (and CD4 T cells) in the indicated tissues were examined. Shown is the mean 6 SD (n = 4). G, Cells isolated from the indicated tissues were subsequently stimulated with PMA/ionomycin, and cytokine production was determined by flow cytometric analysis. Shown are cytokine profiles of gd TCR + gated cells. All the experiments were repeated more than twice and similar results were observed. **p , 0.01; ***p , 0.001. DETC, dendritic epidermal gd T cell; IEL, intraepithelial lymphocyte; PP, Peyer's patches; SI, small intestine. T cells display a lymphoid tissue-specific accumulation is unclear. IL-17 + gd T cells may express a chemokine receptor (s) that allows them to preferentially migrate to and accumulate in the pLN. Indeed, Martin et al. (10) recently reported that IL-17 + gd T cells uniformly express CCR6. Whether CCR6 is necessary for IL-17 + gd T cell accumulation in the pLN remains to be determined. Alternatively, a microenvironment within the mLN may suppress IL-17 expression by gd T cells. These results demonstrate that CD44 high gd T cells display IL-17-producing capacity and that these IL-17 + gd T cells are primarily enriched in the pLN but not in the mLN or in other epithelial cell-rich tissues.

Age-dependent generation of IL-17 + gd T cells in the thymus
It was recently demonstrated that the IL-17 + phenotype of gd T cells is established during thymic development (5,7). Analysis of gd TCR + thymocytes from mice of different ages revealed a striking pattern in IL-17 production. Thymus from newborn mice contained gd TCR + thymocytes, 30-40% of which expressed IL-17 following stimulation ( Fig.  2A). The proportions of IL-17 + gd TCR + thymocytes reached a peak around 5 d of age and declined thereafter ( Fig. 2A). Interestingly, IL-17 + gd T cells in the pLN increased as thymic IL-17 + gd TCR + thymocytes declined (between 7 and 14 d of age), suggesting that the IL-17 + cells differentiated within the thymus appear to populate the periphery. Particularly striking is that the total numbers of IL-17 + gd TCR + thymocytes were constant regardless of age (Fig. 2B), strongly suggesting a tight homeostatic mechanism that controls the generation of IL-17 + gd T cells in the thymus. No-tably, thymic gd TCR + thymocytes expressed little IFN-g, whereas peripheral gd T cells uniformly expressed IFN-g in all tested lymphoid tissues (Fig. 2C), suggesting that IFN-g production, unlike IL-17, may be acquired in the periphery at least at the protein level.

DN4 stage gd T cells become CD44 + and express IL-17
To characterize a developmental pathway leading to the generation of IL-17 + gd thymocytes, adult thymocytes were stimulated ex vivo and the phenotypes of IL-17 producing gd TCR + cells were examined. IL-17 + gd TCR + thymocytes were mainly found within CD4 neg CD8 neg CD44 high CD25 low cells, the phenotype of DN1 thymocytes (Fig. 3A). Notably, developing DN thymocytes initiate gd TCR rearrangement following the DN1 stage and start to express surface gd TCR after the DN2/3 stage (11). Therefore, IL-17 + gd T cells found in the DN1 stage might be peripheral gd T cells that have recirculated from the periphery (12). Alternatively, it is possible that the DN1-stage gd T cells are activated DN4 cells that upregulated CD44 and acquired IL-17 expression. To test this, we used Rag2p-GFP transgenic (Tg) mice (13). It was previously shown that Rag2 promoter-driven GFP expression is induced during the late DN2 stage, reaches peak expression in DP thymocytes, and gradually diminishes during the transition from the DP to the SP thymocytes (14). In the periphery, GFP expression is primarily found in recent thymic emigrants, yet the expression is lower than that in DP thymocytes (14). The distribution of gd TCR + thymocytes in Rag2p-GFP Tg adult mice among the DN subsets was comparable to that of non-Tg mice (data not shown). Similarly, most IL-17 + gd T cells were  found within the DN1 subsets (data not shown). DN1 gd TCR + thymocytes showed two populations based on GFP expression (Fig. 3B), and IL-17 + gd TCR + thymocytes were mostly GFP neg cells (Fig. 3C). As expected, only GFP neg gd T cells were found in the pLN (Fig. 3C).
To further examine if GFP neg IL-17 + gd T cells are mature cells recirculated from the periphery, we examined the thymus from 5-d-old Rag2-GFP Tg mice, an age before IL-17 + gd T cells are seen in the periphery (Fig. 2A). Even at this age, the majority of CD44 high gd TCR + thymocytes did not express GFP (data not shown). Moreover, IL-17 + gd TCR + thymocytes from these mice were mostly GFP neg (Fig. 3D), whereas all DN4 gd TCR 2 thymocytes expressed GFP (data not shown). Therefore, it is likely that the GFP neg IL-17 + gd TCR + thymocytes in 5-d-old mice have undergone extensive proliferation and have lost GFP expression (15). A similar pattern was observed in 14-d-old mice (data not shown). Therefore, our data strongly suggest that CD44 high IL-17 + gd TCR + cells in the thymus are DN4 cells that acquire CD44 expression. To test this possibility, sorted DN4 (CD44 low CD25 low ) gd TCR + thymocytes were cocultured with the stromal cell line OP9-DL4 (16). OP9-DL4 cells transduced with the Notch ligands (Delta-like 4) efficiently promoted T lineage development (17). Indeed, ∼10% of cocultured DN4 gd T cells upregulated CD44 (Supplemental Fig. 1). DN4 gd T cells cocultured with the OP9 control cell line failed to survive. These results further support the transition from DN4 to DN1-like cells.

TGF-b is required for the generation of IL-17 + gd T cells
In case of CD4 T cells, multiple factors including IL-23, IL-21, IL-6, and TGF-b play roles in the differentiation of naive CD4 T cells into Th17 cells (18)(19)(20). We thus explored whether these cytokines are required for the endogenous generation of IL-17 + gd T cells. Lymphoid cells from the indicated gene-deficient mice were stimulated and cytokine production was examined. As shown in Supplemental Fig. 2, the lack of an IL-23 p19 subunit and IL-6 did not alter IL-17 production by gd T cells. IL-17 production of gd T cells in the spleen and mLN of IL-21-deficient mice was slightly reduced; yet, pLN gd T cell IL-17 production in these mice was not altered (Supplemental Fig. 2). Notably, the total numbers of gd T cells in the lymphoid tissues of these mice were equivalent, indicating that the generation of gd T cells is independent of these cytokines (data not shown). Endogenous CD4 T cell IL-17 expression was partially reduced by IL-23 p19 deficiency and completely abolished by the lack of IL-6 (data not shown). Therefore, these cytokines, while playing an important role in the generation of endogenous Th17 cells (21), play little or no role in gd T cell acquisition of IL-17 production. Similarly, Lochner et al. (6) also reported that IL-17 + RORgt + gd T cells were unaffected by the absence of IL-6. Whether TGF-b1 plays a role in the generation of IL-17-producing gd T cells was next examined. As all Tgfb1 2/2 mice develop severe lymphoproliferative disease early in life, LN and spleen cells from 2-to 3-wk-old mice were used. TGF-b1 deficiency completely abolished IL-17 expression by gd T cells (Fig. 4A). Of note, gd T cell generation was not impaired in Tgfb1 2/2 mice (Supplemental Fig. 3). Likewise, gd T cells deficient in Smad3, a TGF-b-signaling adaptor molecule (22), expressed significantly lower levels of IL-17 compared with littermate controls (Fig. 4B). In contrast, IFN-g production of gd T cells was not different in Tgfb1 2/2 and in Smad3 2/2 mice (Supplemental Fig. 4).
We then examined if TGF-b1 is needed for the development of IL-17 + gd T cells in the thymus. gd TCR + thymocytes from Tgfb1 2/2 and littermate control mice were analyzed for IL-17 expression. The DN distribution of developing gd TCR + thymocytes was not different (Fig. 4C). However, IL-17producing capacity of the developing gd TCR + thymocytes was greatly impaired in Tgfb1 2/2 mice (Fig. 4D). In support of this finding, gd TCR + thymocytes expressed the highest levels of type I and type II TGF-b receptors (Fig. 4E). Notably, some thymic gd T cells still acquire IL-17 expression in Tgfb1 2/2 mice (Fig. 4D), and this finding might be supported by the expression of either TGF-b2 or TGF-b3 by thymic epithelia. However, these cells disappeared in the periphery (Fig. 4A), suggesting that TGF-b1 may play an important role in maintaining IL-17 + gd T cells in the periphery. It was previously reported that thymic TGF-b is expressed on subcapsular and cortical thymic epithelium, which interacts with developing thymocytes (23). Identifying the source of TGF-b in the thymus as well as in the periphery will be an important subject for future study. Taken together, these results strongly suggest that TGF-b1, although dispensable for the phenotypic maturation (i.e., CD44 upregulation during DN4 to DN1-like transition), plays an irreplaceble role in the acquisition of IL-17-producing capacity in the thymus.
What are the immunologic roles of IL-17-producing gd T cells in vivo? Following Escherichia coli infection, gd T cellderived IL-17 was shown to play critical roles in recruiting neutrophils and in neutrophil-mediated bacterial clearance (2). IL-17 production by gd T cells is also associated with lethal pulmonary aspergillosis in mice with chronic granulomatous disease (24). Moreover, gd T cell IL-17 production is undoubtedly involved in exacerbating collagen-induced arthritis or autoimmunity (25,26). The current study provides an important basis to define the mechanism(s) of how gd T cells acquire IL-17 expression and of how endogenous gd T cell-derived IL-17 influences immunity in vivo.