Dendritic epidermal T cells (DETCs) are a well-studied population of γδ T cells that play important roles in wound repair. In this study, we characterize a second major population of γδ T cells in the skin that is present in the dermis. In contrast to DETCs, these Vγ5-negative cells are IL-7RhiCCR6hi retinoic acid-related orphan receptor γt+ and are precommitted to IL-17 production. Dermal γδ T cells fail to reconstitute following irradiation and bone marrow transplantation unless the mice also receive a transfer of neonatal thymocytes. Real-time intravital imaging of CXCR6GFP/+ mouse skin reveals dermal γδ T cells migrate at ∼4 μm/min, whereas DETCs are immobile. Like their counterparts in peripheral lymph nodes, dermal γδ T cells rapidly produce IL-17 following exposure to IL-1β plus IL-23. We have characterized a major population of skin γδ T cells and propose that these cells are a key source of IL-17 in the early hours after skin infection.
The skin has an important barrier function against infection. Part of this function is mediated by resident TCR γδ T cells known as dendritic epidermal T cells (DETCs) because of their morphology and location. Seeded to the epidermis during fetal development, they express Vγ5 (according to Heilig and Tonegawa nomenclature) (1) and produce keratinocyte growth factors following wounding (2). In contrast to this well-recognized subset, the types of T cells resident in the dermis have been less studied.
IL-17 plays an important role in host defense at epithelial surfaces, including the gut, lung, and skin (3). IL-17A is rapidly induced in the skin in response to intradermal Staphylococcus aureus and Candida albicans, and IL-17A–deficient mice fail to optimally resolve infection (4, 5). The cellular source of IL-17 in the first hours to days after skin infection has not been well defined, although it is unlikely to be conventional Th17 cells, as they take ≥3 d to differentiate. Recently, populations of innate IL-17–producing γδ T cells have been described in lymphoid tissues, peritoneum, and intestinal mucosa and appear to arise from IL-17–committed γδ thymocytes (6–10). The presence of IL-17–producing γδ T cells in total skin preparations has also recently been demonstrated (11, 12), but it has been unclear whether these cells correspond to DETCs (4) or to dermal cells (13).
In this study, we characterize a population of IL-17–precommitted Vγ5− γδ T cells in the dermis. We provide evidence that these cells derive from perinatal thymocytes and show by intravital imaging that, in contrast to the DETCs, these cells are motile. We propose that dermal γδ T cells are the major source of skin IL-17 during the early stages of infection.
Materials and Methods
C57BL/6 (CD45.2+), Boy/J (CD45.1+), Cxcr6gfp/+ (14) f05693, B6.129P2-Cxcr6tm1Litt/J), Rorcgfp/+ (15) [007572, B6.129P2(Cg)-Rorctm2Litt/J], and TCRδ−/− (16) (B6.129P2-Tcrdtm1Mom/J) mice were from The Jackson Laboratory or National Cancer Institute. A trend toward fewer dermal and peripheral lymph node (PLN) CCR6+ γδ T cells was noted in mice shipped from the National Cancer Institute compared with animals raised in our internal (The Jackson Laboratory-based) colony. All experiments conformed to ethical principles and guidelines approved by the University of California, San Francisco, Institutional Animal Care and Use Committee.
Tissue preparation and flow cytometry
Lymph nodes (LNs) were digested as described (17) with 67 μg/ml Liberase TM (Roche). Epidermal and dermal sheets were separated with dispase (Life Technologies) and digested with 85 μg/ml Liberase TM (Roche) in DMEM as described (18). Cells were stained (17+IL-7Ra+CCR6+TCRβ−CD11b− cells were sorted (17). To detect IL-17A, cells were stimulated for 2 h with 50 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml ionomycin (I; EMD Biosciences) in brefeldin A (BD Biosciences), stained for surface Ags, treated with BD Cytofix Buffer and Perm/Wash reagent (BD Biosciences), and stained with anti–IL-17A.
In vitro stimulation
Neonatal thymocyte transfer and bone marrow chimeras
A total of 5–10 × 106 thymocytes harvested 0–48 h after birth were transferred i.v. to congenic recipients lethally irradiated with a split dose of 1300 rad. The next day, 3–6 × 106 congenic bone marrow (BM) cells were transferred. Recipient mice were analyzed at least 8 wk later.
Intravital two-photon microscopy
The mouse was anesthetized with ketamine/xylazine, and the dorsal side of the ear was attached to a plastic coverslip mounted on a 37°C heating stage. The ventral side of the ear was imaged with a Zeiss LSM 7MP equipped with a Chameleon laser (Coherent). GFP was excited at 910 nm and detected with a 500–550 nm emission filter. Images were acquired with Zen (Zeiss), and time-lapse images and cell tracks generated with Imaris 5.7.2 (Bitplane). The velocities and turning angles of cells were calculated with MATLAB (The MathWorks). Annotation and final compilation of videos were with After Effects 7.0 software (Adobe Systems).
Results and Discussion
In the course of studies characterizing LN subcapsular and interfollicular regions (17), we identified a population of IL-7Rhi cells enriched in proximity to CD169+ macrophages (Supplemental Fig. 1A). By flow cytometric analysis, the IL-7Rhi cells in PLNs were also high for CXCR6, detected using CXCR6GFP/+ reporter mice (14), mostly CCR6hi, and could be further divided into populations of TCRγδ+, TCRαβ+, and non-T cells (Supplemental Fig. 1B). The γδ T cells represented ∼0.1% of PLN cells but were much less abundant in mesenteric LNs and spleen (Supplemental Fig. 1C). A similar population of PLN-enriched γδ T cells was recently reported by a number of groups and shown to correspond to an innate IL-17–producing cell population (6, 7, 9, 13, 19). However, the basis for the enrichment of these cells in skin-draining LNs compared with mucosal LNs and spleen was not clear and led us to ask whether there was a related cell population in the skin. One study characterizing a novel marker, SCART2, present on PLN γδ T cells noted the presence of SCART2+ Vγ5− γδ T cells in the dermis, providing support for this possibility though the cells were not characterized further (13). Flow cytometric analysis of enzyme-digested epidermis revealed the dominant presence of the expected TCRγδhi Vγ5+ DETCs and few other T cells (Fig. 1A). Strikingly, the dermis also contained a large population of γδ T cells but these cells were TCRγδint, lacked Vγ5 (Fig. 1A), and instead had a surface phenotype similar to the cells in PLNs: they were CCR6hi, IL-7Rαhi, CXCR6hi, and high for αE integrin (CD103; Fig. 1). DETCs differed from dermal γδ T cells in being CCR6lo and IL-7Rαlo (Fig. 1). The DETC-phenotype cells present in dermal sheet preparations (Fig. 1A) were most likely due to contamination by small amounts of epidermis.
The close phenotypic similarity between dermal γδ T cells and PLN γδ T cells, together with the high expression of CCR6, a receptor associated with IL-17–producing cells (20), led us to ask whether dermal γδ T cells were precommitted to IL-17 production. Indeed, when cells prepared from dermal and epidermal sheets were activated by PMA/I, dermal γδ T cells promptly produced large amounts of IL-17, whereas DETCs did not (Fig. 2A). Consistent with this precommitment, dermal γδ T cells had high constitutive expression of IL-17A, IL-17F, and IL-22 transcripts (Fig. 2B). Correspondingly, these transcripts were more abundant in dermis than epidermis (Fig. 2B). Retinoic acid-related orphan receptor γt (RORγt) promotes IL-17 expression and has been detected in γδ T cells isolated from total skin (11). Analysis of separated epidermal and dermal sheets from RORγtGFP/+ mice revealed reporter expression in dermal γδ T cells but no expression in DETCs (Fig. 2A).
Previous work has shown that IL-1β and IL-23 are sufficient to promote IL-17 production by PLN γδ T cells after 3 d of in vitro culture (6). We found that both CCR6+ TCRγδint dermal and PLN cells, but not CCR6− TCRγδhi DETCs, began making IL-17 within 8 h of incubation with IL-1β and IL-23 (Fig. 2C). Our findings contrast with a recent report suggesting IL-17 was made selectively by DETCs following incubation with IL-1β and IL-23 (4). We suspect that this discrepancy reflects contamination of the DETC preparations with dermal γδ T cells.
It is well established that Vγ5+ precursors of DETCs are produced in a wave within the embryonic day 14–17 fetal thymus and are not further produced in the adult thymus (2). Thus, Vγ5+ γδ T cells seed the epidermis in fetal life and give rise to a population of DETCs that are locally maintained (21). Recent studies have demonstrated that IL-17–committed Vγ5− γδ T cells appear in the thymus near the time of birth but remain detectable in significant numbers in the adult thymus (22, 23). Based on these observations, we anticipated that dermal and PLN γδ T cells might be replaced by BM-derived cells following irradiation and reconstitution of adult mice, as recently observed for intestinal γδ T cells (24). However, after 8–26 wk of reconstitution by BM cells, there was little if any reconstitution of dermal and PLN CCR6hi γδ T cells (Fig. 3A, Supplemental Fig. 2A), despite ablation of the majority of the endogenous cells by irradiation. The small number of CCR6hi γδ T cells we did observe were mostly radiation-resistant host CCR6hi γδ T cells. Transferring BM into irradiated TCRδ−/− mice did not improve reconstitution (Supplemental Fig. 2A). We then asked whether cells with reconstituting potential might be most prominent in the thymus during the perinatal window when they first appear (Fig. 3B) (22, 23). When irradiated adult mice were given a combination of BM and neonatal thymocytes, we observed significant reconstitution of CCR6hi dermal and PLN γδ T cells that produced IL-17 when stimulated with PMA/I (Fig. 3C, Supplemental Fig. 2A). These findings suggest that IL-17–producing dermal and PLN γδ T cells can be regenerated by precursors arising in the perinatal thymus, but not adult BM. It will be interesting in future studies to characterize the TCR repertoire of these cells to determine whether perinatal thymic precursors reconstitute the full repertoire of IL-17–producing γδ T cells.
Taking advantage of the abundant expression of CXCR6 in skin γδ T cells (Fig. 1C), we used two-photon laser scanning microscopy to examine their migration dynamics, distinguishing cells in the epidermis and dermis based on their location with respect to the collagen (visible from second harmonic generation emission) that separates these layers (Fig. 4A). In the dermis, 20–35% of CXCR6-GFP+ cells were TCRγδint cells, for which GFP expression was ∼2-fold more abundant than the TCRβ+ and TCR− GFP+ cells (Supplemental Fig. 2B). CXCR6-GFP+ cells in epidermal suspensions were almost exclusively TCRγδhi DETCs and had the same GFP intensity as the dermal γδ T cells (Supplemental Fig. 2B). In the imaging analysis, we compared GFP bright cells in the epidermis (DETC) to GFP bright cells in the dermis, reasoning that the latter would be highly enriched for dermal γδ T cells (Fig. 4A, 4B, Supplemental Videos 1–3). As a second approach to allow imaging-based identification of dermal γδ T cells, we reconstituted wild-type (WT) mice with CXCR6GFP/+ neonatal thymocytes and WT BM. In these chimeric mice, at least 80% of the GFP+ dermal cells were TCRγδint T cells (Supplemental Fig. 2B), whereas the DETCs remained GFP− host-derived cells, as expected (2). Imaging the dermis of these mice revealed similar GFP+ cell behaviors to the GFPhi dermal cells in the CXCR6GFP/+ nonchimeric mice (Fig. 4C, Supplemental Video 4). DETCs and dermal γδ T cells exhibited marked differences in behavior (Fig. 4D–F, Supplemental Videos 1–4). Whereas DETCs had a highly dendritic morphology and were immobile (Fig. 4D, Supplemental Video 5), dermal γδ T cells were more rounded and moved at average speeds of 3–5 μm/min (range 1.4–7.6 μm/min) (Fig. 4F, Supplemental Fig. 2C), pausing occasionally and turning in various directions with a mean angle of ∼85° (Supplemental Fig. 2D). Dermal γδ T cells showed a similar extent of displacement over time whether tracked in CXCR6GFP/+ or chimeric mice, whereas DETCs showed no displacement over time (Fig. 4G, Supplemental Fig. 2E). We suggest that the dermal environment is more permissive for cell motility than the tightly adherent epidermis, and dermal γδ T cells take advantage of this property to achieve surveillance coverage of a larger three-dimensional area.
In summary, although several studies have identified a critical role for IL-17 in the protective response mounted in the early hours to days of cutaneous infections (4, 5, 12), the precise cellular source of this IL-17 was not defined (5, 12) or was suggested to be DETCs (4). Our work demonstrates that dermal γδ T cells are a major subset of IL-17–precommitted cells in the skin. Given the ability of these cells to secrete IL-17 within hours of IL-1β and IL-23 exposure, we propose that they contribute to the rapid IL-17 production following infections. These cells may also contribute to the IL-17–dependent psoriasis-like skin pathology that occurs following repeated intradermal IL-23 injection (25).
The reconstitution of CCR6+ dermal γδ T cells by neonatal thymocytes but not BM suggests that these cells are seeded from the perinatal thymus and subsequently maintained in the periphery. Consistent with this model, BrdU-labeling studies suggest these cells divide at a low rate in situ (data not shown). How they are maintained in the dermis remains to be explored, although given their high IL-7Rα expression (Fig. 1B), IL-7 is a candidate trophic factor. Early studies of transgenic mice overexpressing IL-7 in keratinocytes revealed a marked expansion of Vγ5−TCRγδint T cells in the dermis, and it was suggested that local γδ T cells were responding to the cytokine (26, 27). It is likely that the major population expanded in the skin of these mice was the IL-17–precommitted, CCR6+ γδ T cells; overproduction of IL-17 may well account for the dermatitis suffered by some of the transgenic animals (26, 27). Future studies should examine the possibility that IL-7 production by keratinocytes is important for the local maintenance of dermal γδ T cells. Alteration of IL-7 abundance may be a method to increase or decrease dermal γδ T cell function for therapeutic benefit.
Note added in proof. Following submission of this manuscript, a study from Sumaria et al. (28) was published describing a similar population of Vg5—IL-7Rα+CD103+CXCR6+ T cells in the dermis and demonstrating that these cells are a source of IL-17 following cutaneous Mycobacterium bovis Bacille Calmette–Guérin infection.
The authors have no financial conflicts of interest.
We thank Lisa Kelly for cell sorting and helpful discussion, Irina Grigorova for Matlab code used in data analysis, Andrea Reboldi and Oliver Bannard for helpful discussion, Ying Xu and Jinping An for expert technical assistance, and Jennifer Bando and Richard Locksley for RORγtGFP/+ mice.
This work was supported by National Institutes of Health Grant AI45073. J.G.C. is an Investigator of the Howard Hughes Medical Institute.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- bone marrow
- dendritic epidermal T cell
- lymph node
- peripheral lymph node
- retinoic acid-related orphan receptor γt
- Received February 10, 2011.
- Accepted April 4, 2011.
- Copyright © 2011 by The American Association of Immunologists, Inc.