TCR signaling and cellular metabolism regulate the capacity of murine epidermal γδ T cells to rapidly produce IL-13 but not IFN-γ

Resident epidermal T cells of murine skin, called dendritic epidermal T cells (DETCs), express an invariant γδ TCR that recognizes an unidentified self-ligand expressed on epidermal keratinocytes. Although their fetal thymic precursors are preprogrammed to produce IFN-γ, DETCs in the adult epidermis rapidly produce IL-13 but not IFN-γ early after activation. Here, we show that preprogrammed IFN-γ-producing DETC precursors differentiate into rapid IL-13 producers in the perinatal epidermis. The addition of various inhibitors of signaling pathways downstream of TCR to the in vitro differentiation model of neonatal DETCs revealed that TCR signaling through the p38 MAPK pathway is essential for the functional differentiation of neonatal DETCs. Constitutive TCR signaling at steady state was also shown to be needed for the maintenance of the rapid IL-13-producing capacity of adult DETCs because in vivo treatment with the p38 MAPK inhibitor decreased adult DETCs with the rapid IL-13-producing capacity. Adult DETCs under steady-state conditions had lower glycolytic capacity than proliferating neonatal DETCs. TCR stimulation of adult DETCs induced high glycolytic capacity and IFN-γ production during the late phase of activation. Inhibition of glycolysis decreased IFN-γ but not IL-13 production by adult DETCs during the late phase of activation. These results demonstrate that TCR signaling promotes the differentiation of IL-13-producing DETCs in the perinatal epidermis and is needed for maintaining the rapid IL-13-producing capacity of adult DETCs. The low glycolytic capacity of adult DETCs at steady state also regulates the rapid IL-13 response and delayed IFN-γ production after activation.


Introduction
gd T cells represent a minor population of T cells in adult blood and peripheral lymphoid organs but are enriched in epithelial tissues (1,2).In mice, different epithelial tissues are populated by distinct gd T-cell subsets defined by the usage of specific TCR Vg regions (3).These epithelial gd T-cell subsets develop in waves based on ordered Vg gene rearrangement in the fetal and neonatal thymus and are home to specific tissues.Epithelial gd T cells mediate stress surveillance and exert a distinct set of effector functions in a given tissue (4)(5)(6)(7)(8).
Unlike conventional ab T cells, which differentiate into effector subsets during activation in peripheral lymphoid organs (9), most epithelial gd T cells are preprogrammed during thymic development to either IFN-g-or IL-17A-producing effector subsets that exhibit rapid, innate-like responses in the periphery (10)(11)(12).Recent studies have revealed the role of TCR signaling in the differentiation of effector subsets during thymic development.Ligand-induced strong TCR signaling is needed for the differentiation of IFN-g-producing gd T cells, whereas weaker TCR signaling supports the differentiation of IL-17A-producing cells (13)(14)(15)(16)(17)(18).IFN-g-producing gd T-cell development also requires CD27 signaling, and mature IFN-g-producing gd T cells usually express CD27, while IL-17A-producing gd T cells lack CD27 expression (14).
Resident epidermal T cells of murine skin, called dendritic epidermal T cells (DETCs), are prototypic epithelial gd T cells that express an invariant Vg3Vd1 TCR (Garman nomenclature) (19).DETCs contribute to epidermal homeostasis, wound healing, IgE production, and tumor surveillance (7,(20)(21)(22)(23)(24)(25).Although the ligand of the Vg3Vd1 TCR has not been identified, it is expressed on fetal thymic epithelial cells and stressed or transformed epidermal keratinocytes (26)(27)(28)(29).Accumulating evidence suggests that low levels of the TCR ligand are constitutively expressed on keratinocytes at steady state (20,30).DETC precursors develop as the first T cells in the fetal thymus.Thymic maturation of DETC precursors requires ligand-dependent TCR signaling and the thymic stromal determinants Skint1 and Skint2 (15,31,32).TCR signaling in DETC precursors in the fetal thymus promotes the upregulation of skin homing receptors needed for thymic export and skin homing (33)(34)(35), and DETC precursors migrate to the skin before birth.After seeding the epidermis in low numbers, DETCs massively proliferate until 2 to 6 weeks after birth, depending on the mouse strain (36,37), and their numbers become stable by 8 weeks (27).
Ligand-selected mature Vg3 + fetal thymocytes that have received strong TCR signaling express CD27 and Tbx21, which encodes T-bet, the master transcription factor of IFN-g-producing cells, and rapidly produce IFN-g following phorbol 12-myristate 13acetate (PMA) and ionomycin stimulation (15).In contrast, DETCs in the adult epidermis do not express CD27 (38,39) or produce IFN-g within 4 hours after stimulation with PMA/ionomycin (22,40) but do begin to produce IFN-g 12 to 24 hours after stimulation in vitro (39,41).This could be due to the hyporesponsive TCR signaling that occurs during DETC development (42).However, adult DETCs produce IL-13 upon short-term PMA/ionomycin stimulation (22).Therefore, Vg3 + T cells appear to differentiate from preprogrammed IFN-g producers into rapid IL-13 producers after thymic egress.This functional switch in DETCs sets them apart from other tissue-resident gd T cells preprogrammed to produce IFN-g, and the distinct kinetics of IL-13 and IFN-g production by DETCs is important for DETC-mediated stress surveillance in the epidermis (22,24).However, it remains unknown when, where, and how the functional switch of Vg3 + T cells occurs.
We aimed to clarify the timing, location, and underlying mechanisms of the functional switch of Vg3 + T cells.Here, we show that Vg3 + T cells differentiate from preprogrammed IFN-g producers into rapid IL-13 producers in the perinatal epidermis and that this differentiation is dependent on TCR signaling through the p38 mitogen-activated protein kinase (MAPK) pathway.We also show that the rapid IL-13-producing capacity of adult DETCs under steady-state conditions is maintained by continuous TCR signaling and cellular metabolism.

Results
Adult DETCs predominantly produce IL-13 during the early phase of activation Adult DETCs produce IL-13 but not IFN-g upon short-term PMA/ionomycin stimulation (22).To quantify cytokine levels secreted by adult DETCs upon TCR stimulation, we purified DETCs from adult ear epidermal cells without TCR ligation by positive magnetic selection using an anti-integrin b7 mAb.The purified DETCs were >95% pure Vg3 + T cells and contained <1% non-Vg3 + T cells and <1% IA + Langerhans cells (Figure 1A).Although DETCs were reported to constitutively produce IL-13 at steady state (22), purified DETCs cultured without TCR stimulation did not secrete IL-13 or other cytokines (Figure 1B).The purified DETCs predominantly secreted IL-13 during the first 24 hours of TCR stimulation, and IFN-g secretion increased after 24 hours.Consistent with a previous study (39), DETCs secreted a small amount of IL-17A upon TCR stimulation, but IL-4 secretion was not detected (Figure 1B).
RT-PCR analysis revealed that purified naive DETCs constitutively expressed Gata3, the master transcription factor of type 2 cytokine-producing cells, but Tbx21 was expressed only after activation (Figure 1C).Eomes, which encodes eomesodermin that also regulates IFN-g production in type 1 cytokine-producing T cells, was not expressed in DETCs even after activation (Figure 1C).Consistent with the IL-17A-producing capacity of a subpopulation of DETCs, the expression of Rorc, which encodes RORgt, the master transcription factor of IL-17A-producing cells, was detected in naive DETCs (Figure 1C).The lack of IL-4 production by DETCs might be explained by the constitutive expression of Zbtb32 (Figure 1C), which encodes ZBTB32 (Repressor of GATA, ROG) that represses Il4 but not Il13 gene activation in type 2 CD8 + cytotoxic T lymphocytes (43).
These results confirmed that under steady-state conditions, DETCs in the adult epidermis predominantly produce IL-13 during the early phase of activation.However, DETCs can produce IFN-g during the later phase of activation.Therefore, unlike type 2 CD4 + helper T cells (44), the IFN-g-producing capacity of DETCs would not be repressed by stable epigenetic modifications, as is the case for IL-17A-producing CD27 -gd T cells, which can produce IFN-g in a certain inflammatory microenvironment (45).
Purified adult DETCs predominantly produce IL-13 during the early phase of activation.(A) Crude epidermal cells isolated from adult ear skin and DETCs purified from epidermal cells by positive magnetic selection using anti-integrin b7 mAb were stained with the indicated mAbs.Quadrant settings were determined by staining with isotype control mAbs.The numbers denote the percentages of cells in the respective quadrants.Representative profiles from two independent experiments are shown.In each experiment, cells pooled from 3-4 mice were analyzed.(B) Purified DETCs were stimulated with immobilized isotype control or anti-TCR mAb.Culture supernatants were harvested and replaced with fresh culture medium 24 hours after the start of stimulation, and DETCs were stimulated for an additional 24 hours.Cytokine levels in the supernatants were determined by ELISA.Representative data from two independent experiments are shown as the mean and SD of triplicate cultures.(C) mRNA expression of the indicated transcription factors in purified DETCs without stimulation (naive) and purified DETCs stimulated with immobilized anti-TCR mAb for 24 hours (activated) was analyzed in triplicate by real-time RT-PCR.Representative data from two independent experiments are shown as the mean and SD.Significant differences compared with naive DETCs are denoted with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).

Vg3 + T cells lose CD27 expression immediately after migration to the dermis
Similar to embryonic day 17 (E17) mature Vg3 + fetal thymocytes, circulating Vg3 + T cells in the blood at E17 expressed CD27 (Figure 2A).As E17 fetal dermal Vg3 + T cells did not express CD27 (Figure 2A), Vg3 + T cells lost CD27 expression immediately after entering the dermis.A candidate that induces CD27 downregulation on Vg3 + T cells is extracellular ATP, which would be abundant in the perinatal skin, because CD27 on T cells is rapidly shed and downregulated upon treatment with ATP (46).ATP treatment of E17 Vg3 + fetal thymocytes resulted in rapid downregulation of CD27 (Figure 2B).As Vg3 + T cells in day 1 (D1) neonatal epidermis that lacked CD27 expression (Figure 2A) could produce IFN-g (Figure 3), CD27 expression and IFN-g-producing capacity were not perfectly correlated in Vg3 + T cells.

Vg3 + T cells differentiate from preprogrammed IFN-g producers into rapid IL-13 producers in the perinatal epidermis
To clarify when and where Vg3 + T cells functionally switch from preprogrammed IFN-g producers to rapid IL-13 producers, we analyzed cytokines produced by Vg3 + T cells upon short-term stimulation during ontogeny.Vg3 + T cells in the E18 fetal epidermis still predominantly produced IFN-g when stimulated with PMA/ionomycin for 4 hours (Figure 3).While IFN-g-producing cells gradually decreased after birth, IL-13-producing cells gradually increased (Figure 3).As cells producing both IFN-g and IL-13 transiently appeared in the neonatal epidermis (Figure 3), Vg3 + T cells differentiated from preprogrammed IFN-g producers into rapid IL-13 producers via intermediate IFN-g/IL-13 producers during this period in the epidermis.
Epidermal T cells expressing TCRs that recognize the self-ligand on epidermal keratinocytes predominantly produce IL-13 Thymic maturation of Vg3 + T cells to IFN-g-producing cells requires TCR signaling.As the proliferation of Vg3 + T cells in the perinatal epidermis also relies on TCR signaling (47)(48)(49), we hypothesized that TCR signaling also induces the IL-13producing capacity of Vg3 + T cells in the perinatal epidermis.
To determine the role of TCR signaling in the induction of the IL-13-producing capacity of epidermal T cells, we analyzed cytokines produced by resident epidermal T cells of adult TCR dchain-deficient Tcrd -/-mice and TCR Vd1-chain-deficient Tcrd-V1 -/-mice.In Tcrd -/-mice lacking all gd T cells, the epidermal niches of DETCs are replaced by ab T cells, but the ab TCRs expressed on these epidermal T cells cannot recognize the selfligand on epidermal keratinocytes (27,30).In contrast, epidermal T cells of Tcrd-V1 -/-mice express diverse gd TCRs that can recognize the self-ligand on epidermal keratinocytes (50).
Epidermal ab T cells of adult Tcrd -/-mice produced IFN-g but not IL-13 or IL-17A upon PMA/ionomycin stimulation for 4 hours (Figure 4).In contrast, both epidermal Vg3 + and Vg2 + T cells of adult Tcrd-V1 -/-mice, the latter of which are biased to produce IL-17A in the dermis of wild-type mice (38,51,52), predominantly produced IL-13 but not IFN-g or IL-17A (Figure 4).These results suggest that ligand-dependent TCR signaling in the epidermis is needed for the induction and/or maintenance of the rapid IL-13producing capacity of epidermal T cells.

TCR signaling through the p38 MAPK pathway promotes the differentiation of neonatal DETCs into IL-13-producing cells, whereas mammalian target of rapamycin complex 1 (mTORC1) signaling suppresses differentiated IL-13-producing cells
To determine the role of TCR signaling in the functional differentiation of DETCs more directly, we used an in vitro differentiation model of neonatal DETCs.Neonatal epidermal cells were cultured under TCR stimulation in the presence of Vg3 + T cells differentiate from preprogrammed IFN-g producers into rapid IL-13 producers in the perinatal epidermis.(A) Cells isolated from the thymus or epidermis at the indicated time points were stimulated with PMA/ionomycin for 4 hours.Vg3 + T cells were gated, and intracellular IFN-g and IL-13 production was analyzed by flow cytometry.Quadrant settings were determined by staining with isotype control mAbs.The numbers denote the percentages of cells in the respective quadrants.Each profile is representative of three independent experiments.In each experiment, cells pooled from 5-8 fetuses and 1-3 postnatal mice were analyzed.(B) Quantification of (A).Data are expressed as the mean and SEM (n = 3).Significant differences compared with the epidermis at 6 weeks are denoted with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).
various inhibitors of signaling pathways downstream of TCR, and cytokine production by Vg3 + T cells was analyzed after restimulation with PMA/ionomycin for 4 hours.Among the various inhibitors, only the p38 MAPK inhibitor SB203580 blocked the differentiation of IL-13-producing cells from IFN-gproducing cells (Figure 5A).Therefore, it was found that TCR signaling through the p38 MAPK pathway promotes the functional switch of DETCs.

The p38 MAPK inhibitor SB203580 blocks the maintenance of the rapid IL-13producing capacity of adult DETCs in vivo
In the adult epidermis, TCRs on DETCs are triggered at steady state (30).Therefore, TCR-p38 MAPK signaling may also play a role in the maintenance of the IL-13-producing capacity of DETCs in the adult epidermis.Intradermal administration of the p38 MAPK inhibitor SB203580 24 hours before analysis decreased DETCs with the IL-13-producing capacity in the adult epidermis (Figures 5B, C).IL-13 mRNA expression in epidermal cells upon short-term stimulation with PMA/ionomycin in vitro (Figure 5D) and in response to tape-stripping in vivo (Figure 5E) was suppressed by SB203580 pretreatment.As DETCs are the only cells in the epidermis that produce IL-13 (22), these results suggest that continuous TCR signaling through the p38 MAPK pathway is also needed for maintaining the rapid IL-13-producing capacity of adult DETCs under steady-state conditions.
The metabolic switch in DETCs from high glycolytic capacity to higher mitochondrial dependence occurs between 2 and 4 weeks after birth A recent study showed that IFN-g-producing gd T cells use glycolysis for proliferation and to maintain effector functions, but IL-17A-producing gd T cells are dependent on mitochondrial oxidative phosphorylation and fatty acid oxidation (54).As mTORC1, which regulates cellular metabolism, suppressed IL-13-producing cells in our in vitro differentiation model (Figure 5A), the functional switch of DETCs from IFN-g-producing cells to IL-13-producing cells may be associated with the metabolic switch from mTORC1-dependent metabolic pathways to those less dependent on mTORC1.
To analyze the metabolic profiles of DETCs at different ages, we used the recently developed flow cytometry-based method SCENITH ™ (55).D2 neonatal and D14 DETCs displayed higher glucose dependence and higher glycolytic capacity than D21 and D28 adult DETCs (Figure 6A).By D21, DETCs showed low glucose dependence and low glycolytic capacity, with a subsequent high dependence on mitochondrial oxidative phosphorylation and fatty Epidermal T cells expressing TCRs that recognize the self-ligand on epidermal keratinocytes predominantly produce IL-13.Epidermal cells from the indicated adult mice were stimulated with PMA/ionomycin for 4 hours.Vg3 + T cells (wild-type mice), Cb + T cells (Tcrd -/-mice), and Vg3 + or Vg2 + T cells (Tcrd-V1 -/-mice) were gated, and intracellular IFN-g, IL-13, and IL-17A production was analyzed by flow cytometry.Quadrant settings were determined by staining with isotype control mAbs.The numbers denote the percentages of cells in the respective quadrants.Each profile is representative of two to four independent experiments with a single mouse per experiment.acid/amino acid oxidation (Figure 6A).This metabolic reprogramming took place between 2 and 4 weeks after birth (Figure 6A) and thus occurred later than the functional switch observed during the perinatal period (Figure 3).As DETCs have been shown to cease massive proliferation in the epidermis by 2 weeks after birth in C57BL/6 mice (37), mTORC1 activity may be attenuated at this time and maintained at low levels in adult DETCs under steady-state conditions.

B C A
The metabolic switch in DETCs from high glycolytic capacity to higher mitochondrial dependence occurs between 2 and 4 weeks after birth.

Attenuated mTORC1 activity in adult DETCs under steady-state conditions
To determine whether mTORC1 activity is diminished in adult DETCs under steady-state conditions, we compared the levels of phosphorylated 4E-BP1 (p4E-BP1), an mTORC1 downstream target, between neonatal and adult DETCs.Compared with D2 neonatal DETCs, p4E-BP1 levels were lower in adult DETCs (Figures 6B, C).Therefore, mTORC1 activity was found to be attenuated in adult DETCs under steady-state conditions.

Glycolysis inhibition decreases IFN-gproducing cells but not IL-13-producing cells in adult DETCs during the late phase of activation
Finally, we determined whether cellular metabolism regulates cytokine production by adult DETCs.In contrast to adult DETCs under steady-state conditions (Figure 6A), adult DETCs stimulated with anti-TCR mAb for 3 days in vitro showed high glycolytic capacity (Figure 7A).Although adult DETCs predominantly produce IL-13 during the early phase of activation, adult DETCs stimulated for 3 days in vitro produced both IL-13 and IFN-g (Figures 7B, C).Inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) decreased IFN-g-producing cells but not IL-13-producing cells in adult DETCs stimulated with anti-TCR mAb for 3 days in vitro (Figures 7B, C).As TCR-stimulated DETCs cultured in the presence or absence of 2-DG had equivalent levels of p4E-BP1 (Figures 7D,  E), inhibition of glycolysis did not alter mTORC1 activity in adult DETCs.Therefore, it was found that glycolysis acts downstream of mTORC1 in the regulation of DETC cytokine production.

Discussion
We showed that preprogrammed IFN-g-producing Vg3 + T cells differentiate into rapid IL-13 producers in the perinatal epidermis.This functional switch was promoted by TCR signaling through the p38 MAPK pathway.The downstream substrate of p38 MAPK in perinatal DETCs is currently unknown, but a likely candidate is GATA3 because phosphorylation of GATA3 by p38 MAPK is crucial for GATA3 nuclear translocation and IL-13 production in type 2 helper T cells (56) and group 2 innate lymphoid cells (57).As TCR signaling activates p38 MAPK not only through the canonical MAPK cascade but also through direct phosphorylation of p38 MAPK on a non-canonical activating residue by LCK-ZAP70 (58), it is also important to identify the upstream signaling pathway of p38 MAPK in perinatal DETCs in future studies.
Factors present in the perinatal epidermal microenvironment other than TCR signaling may also contribute to the induction of the IL-13-producing capacity of DETCs.Thus far, we have not yet identified such a factor.Treatment of E17 fetal thymocytes with IL-2, IL-4, IL-7, IL-15, TGF-b, or ATP did not induce the IL-13producing capacity of Vg3 + T cells (unpublished data).We also confirmed that adult DETCs of both TSLP receptor-deficient mice and wild-type mice treated with anti-IL-25 and anti-IL-33 mAbs in vivo produced IL-13 but not IFN-g upon short-term PMA/ ionomycin stimulation (unpublished data).Nevertheless, the involvement of cognate signaling through interactions between DETCs and epidermal keratinocytes has not been addressed and warrants further investigation.
We demonstrated that signaling through the p38 MAPK pathway was also needed for the maintenance of the rapid IL-13producing capacity of adult DETCs under steady-state conditions.Although p38 MAPK activation by a receptor other than TCR could be responsible for this finding, our data are consistent with those of a recent study showing that chronic intradermal administration of anti-Skint1 mAb resulted in the loss of IL-13 expression by adult DETCs ( 59) and together support the notion that constitutive TCR signaling at steady state through Skint1-dependent recognition of the self-ligand expressed on healthy keratinocytes ('normality sensing') maintains DETCs in a poised state to rapidly respond to epidermal stress (59).DETCs primarily produce IL-13 when activated in vivo after exposure to a variety of environmental stressors (22) and even after acute upregulation of transgenic NKG2D ligands on epidermal keratinocytes (24).Therefore, IL-13 production can be triggered in steady-state DETCs not only by TCR signaling via stress-induced upregulation of the TCR ligand but also by signaling through other stress-sensing receptors, including NKG2D, to maintain epidermal homeostasis (22).
We identified a role of cellular metabolism in the regulation of cytokine production by adult DETCs.Proliferating neonatal DETCs had high glycolytic capacity, whereas adult DETCs at steady state were more dependent on mitochondrial metabolism.This metabolic switch occurred between 2 and 4 weeks after birth.This is consistent with the fact that DETCs cease massive postnatal proliferation at this time in C57BL/6 mice (37).Accordingly, mTORC1 activity in adult DETCs under steadystate conditions was attenuated compared with that in proliferating neonatal DETCs.Inhibition of glycolysis decreased adult DETCs producing IFN-g during the late phase of activation.As the inhibition of glycolysis resulted in a relative increase in the frequency of IL-13-producing DETCs, IL-13-producing DETCs redifferentiated into IFN-g-producing cells during the late phase of activation.The (re)acquisition of the IFN-g-producing capacity would require higher energy fueled by mTORC1-dependent glycolysis through sustained signaling than IL-13 production.Conversely, the low glycolytic capacity of adult DETCs at steady state contributes to the predominant production of IL-13 over IFNg early after activation as the former is less dependent on glycolysis than the latter.mTORC1 is needed for the proliferation and survival of peripheral gd T cells and is essential for the differentiation of both IFN-g-producing and IL-17A-producing gd T cells (60).In vitro treatment of adult DETCs with a high dose (20 ng/mL) of rapamycin inhibits their proliferation and induces autophagy (61).Therefore, a basal level of mTORC1 activity through low levels of TCR signaling and/or IL-15 receptor signaling is essential for the maintenance of DETCs at steady state.Full activation of DETCs and IFN-g production would require enhanced mTORC1 activity and high glycolytic capacity to meet increased metabolic needs.To avoid complete blocking of mTORC1 activity, we used a low dose (5 ng/mL) of rapamycin for inhibiting mTORC1 activation in our in vitro neonatal DETC differentiation model.Although low-dose rapamycin treatment was reported to activate mTORC2 (62), the involvement of mTORC2 in the increase in IL-13-producing cells by rapamycin treatment was unlikely in our experiments because the addition of Torin 1, which inhibits both mTORC1 and mTORC2, also increased IL-13-producing cells.In our in vitro differentiation model, inhibition of mTORC1dependent glycolysis might prevent differentiated IL-13-producing cells from redifferentiating into IFN-g-producing cells and result in an increase in IL-13-producing cells.
In summary, we demonstrated that TCR signaling through the p38 MAPK pathway promotes the differentiation of IL-13producing Vg3 + T cells in the perinatal epidermis and that constitutive TCR signaling at steady state is also needed for maintaining the rapid IL-13-producing capacity of adult DETCs.In addition, the low mTORC1 activity and low glycolytic capacity of adult DETCs at steady state also regulate the rapid IL-13 response and delayed IFN-g production after activation.A variety of sometimes conflicting effector functions of DETCs have been identified (7,(20)(21)(22)(23)(24)(25).The effector functions of DETCs may be fine-tuned by their metabolic states depending on the epidermal microenvironment.Whether the functions of DETCs other than cytokine production (e.g., growth factor production, cytotoxicity) are regulated by cellular metabolism remains to be determined in future studies.

Mice
C57BL/6J mice were purchased from Japan SLC (Hamamatsu, Japan).Tcrd -/-mice (63) were purchased from the Jackson Laboratory (Bar Harbor, ME).Tcrd-V1 -/-mice (50) were a gift from Yasunobu Yoshikai (Division of Host Defense, Medical Institute of Bioregulation, Kyusyu University, Fukuoka, Japan).TSLP-R -/-mice (64) were a gift from Steven F. Ziegler (Benaroya Research Institute, Seattle, WA).All mice were bred and maintained on a C57BL/6 background in the animal facility of Kagoshima University under specific pathogen-free conditions.Female mice at 4-12 weeks of age were used as adult mice.Fetuses and postnatal mice younger than 4 weeks were used irrespective of sex.Fetuses were obtained from timed pregnant mice.The plug date was defined as embryonic day 0 (E0).

Cells
To isolate epidermal cells, the skin was floated dermal-side down on 1% trypsin (Gibco, Waltham, MA) in PBS for 30 minutes at 37°C.The epidermis was separated and collected in Iscove's modified Dulbecco's medium (IMDM, Gibco) supplemented with 10% FCS (Sigma-Aldrich, St. Louis, MO) and 0.025% DNase I (Sigma-Aldrich).Single-cell suspensions were obtained by mechanical agitation and sequential filtration through 70-and 30mm nylon meshes.
DETCs were purified from epidermal cells by positive magnetic selection using an anti-integrin b7 mAb because DETCs are the only cells in the normal epidermis that express the integrin b7 chain (65).After preincubation with anti-CD16/CD32 mAb (clone 2.4G2; BD Biosciences, Franklin Lakes, NJ), cells were stained with PE-conjugated anti-integrin b7 mAb (clone M293, BD Biosciences), followed by incubation with magnetic particles conjugated with anti-PE mAb (BD IMag ™ Anti-R-PE Magnetic Particles-DM, BD Biosciences).The labeled cells were isolated using the BD IMag ™ Cell Separation Magnet (BD Biosciences) according to the manufacturer's instructions.
To isolate dermal cells, the skin was floated dermal-side down on 1.2 U/mL dispase II (Roche Diagnostics, Basel, Switzerland) in IMDM for 30 minutes at 37°C.After removing the epidermis, small pieces of the dermis were digested in IMDM containing 0.01% DNase I and 250 U/mL collagenase IV (Sigma-Aldrich) for 30 minutes in a shaking water bath at 37°C.The digested dermis was filtered through 70-and 30-mm nylon meshes.
Fetal thymocytes were obtained by teasing the thymic lobes with fine forceps and filtering through a 70-mm nylon mesh.
Blood was collected in 20 mM EDTA in PBS.Lymphocytes were isolated by density gradient centrifugation on Lympholyte ™ -M Cell Separation Media (Cedarlane Laboratories, Burlington, Canada) for 15 minutes at 1000 × g.
After gating on forward and side scatters and viable cells as previously described (29, 65), cells were analyzed on a CytoFLEX flow cytometer with CytExpert software (Beckman Coulter, Brea, CA), and the data were analyzed using FlowJo ™ software (Tree Star, Ashland, OR).

Real-time RT-PCR
Total RNA was extracted from the cells using the RNeasy ™ Plus Mini kit (Qiagen, Venlo, Netherlands) and reverse transcribed using the SuperScript ™ III First-Strand Synthesis System for RT-PCR (Invitrogen, Waltham, MA) with random hexamers.The cDNA was subjected to quantitative real-time PCR in triplicate using Thermal Cycler Dice ™ Real Time System (Takara, Kusatsu, Japan) with FastStart Universal SYBR ™ Green Master (Roche Diagnostics).All primers were purchased from Takara.Primer sequences are available upon request.The cycling conditions were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.Threshold cycle (Ct) values were determined, and mRNA expression relative to that of the Actb mRNA was calculated as 2 -DCt .

ATP treatment of fetal thymocytes
E17 fetal thymocytes were cultured on 24-well plates (2 × 10 6 cells/well) in IMDM supplemented with 10% FCS and 50 mM 2-mercaptoethanol in the presence of PBS or 3 mM ATP (Sigma-Aldrich) for 30 minutes at 37°C.

In vitro differentiation model of neonatal DETCs
Epidermal cells isolated from day 1 neonatal mice were stimulated on 24-well plates (1 × 10 6 cells/well) coated with 10 mg/mL anti-TCR Cd mAb in IMDM supplemented with 10% FCS, 50 mM 2-mercaptoethanol, and 10 ng/mL recombinant mouse IL-2 for 5 days at 37°C.Various inhibitors of signaling pathways downstream of TCR were added during this period.Cells were harvested and rested on uncoated plates in the same culture medium without inhibitors for 2 days at 37°C to allow the recovery of TCR expression before restimulation with PMA/ ionomycin followed by intracellular cytokine staining.
In vivo treatment with the p38 MAPK inhibitor and tape-stripping PBS or 10 mg SB203580 in 25 mL PBS was administered by intradermal injections into the dorsal and ventral sides of the ear pinna using a 29-gauge needle under inhalation anesthesia.Epidermal cells were isolated 24 hours after the injection and stimulated for 4 hours with PMA/ionomycin.To activate DETCs in situ by mild tissue abrasion induced by tape-stripping, the stratum corneum was removed from both sides of the earlobe by application and removal of cellophane tape (Scotch ™ , 3M, St. Paul, MN) seven times (22,24) 24 hours after the injection.Epidermal cells were isolated 4 hours after tape stripping.

Measurement of metabolic dependence and capacity
SCENITH ™ was performed as previously described (55) using the SCENITH ™ kit containing all reagents and protocols (obtained from www.scenith.com/try-it).Briefly, cells were treated on 24-well plates (1 × 10 6 cells/well) with DMSO (control), 100 mM 2-DG, 1 mM oligomycin, or a combination of 2-DG and oligomycin for 40 minutes (for resting cells) or 30 minutes (for activated cells) at 37°C.Puromycin (10 mg/mL) was added for 40 minutes (for resting cells) or for the last 15 minutes (for activated cells) at 37°C.After surface staining, the cells were fixed and permeabilized using Cytofix/ Cytoperm ™ and washed and stained in Perm/Wash ™ buffer with Alexa Fluor 488-conjugated anti-puromycin mAb (clone R4743L-E8) for 30 minutes at 4°C.The impact of the various metabolic inhibitors was quantified as previously described (55).

Statistical analysis
Differences between the two groups were evaluated by t-test.Dunnett's test was used for multiple comparisons to a control.All reported P values are two-tailed, with a P value < 0.05 considered significant.Statistical calculations were performed using JMP ™ software (SAS Institute, Cary, NC).

2
FIGURE 2 Vg3 + T cells lose CD27 expression immediately after migration to the dermis.(A) Cells isolated from the indicated tissues at the indicated embryonic (E) or postnatal day (D) were stained with anti-Vg3 mAb and anti-CD27 or isotype control mAb.Vg3 + T cells were gated, and CD27 expression is shown as open histograms.Shaded histograms indicate cells stained with isotype control mAb.Each profile is representative of three independent experiments.In each experiment, cells pooled from 3-8 fetuses and 1-2 postnatal mice were analyzed.(B) E17 fetal thymocytes were treated with PBS (control) or ATP for 30 minutes and stained with anti-Vg3 mAb and anti-CD27 or isotype control mAb.Vg3 + T cells were gated, and CD27 expression is shown as open histograms.Shaded histograms indicate cells stained with isotype control mAb.Representative profiles from three independent experiments are shown.In each experiment, cells pooled from 9-10 fetuses were analyzed.Relative mean fluorescence intensity (MFI) was determined as (geometric MFI of CD27)/(geometric MFI of isotype control) and is shown in each panel as the mean and SEM (n = 3).Compared with the control, ATP treatment significantly diminished CD27 expression (P < 0.01).

5 TCR
FIGURE 5 TCR signaling through the p38 MAPK pathway promotes the differentiation of neonatal DETCs into IL-13-producing cells in vitro and the maintenance of the rapid IL-13-producing capacity of adult DETCs in vivo, whereas mTORC1 signaling suppresses differentiated IL-13-producing cells in vitro.(A) Day 1 neonatal epidermal cells were stimulated with immobilized anti-TCR mAb in the presence of DMSO (control) or inhibitors of the indicated TCR signaling pathways for 5 days and rested for 2 days.After PMA/ionomycin stimulation for 4 hours, cytokine production by gated Vg3 + T cells was analyzed by flow cytometry.Representative data from three independent experiments are shown as the mean and SEM (n = 3).Significant differences compared with the control are denoted with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).(B-E) PBS (control) and the p38 MAPK inhibitor SB203580 were injected intradermally into each ear of an adult mouse.(B-D) Epidermal cells were isolated from each ear 24 hours after the injection and stimulated with PMA/ionomycin for 4 hours.(B) Cytokine production by gated Vg3 + T cells was analyzed by flow cytometry.Quadrant settings were determined by staining with isotype control mAbs.The numbers denote the percentages of cells in the respective quadrants.Representative profiles from three independent experiments are shown.(C) Quantification of (B).Data are expressed as the mean and SEM (n = 3).Significant difference compared with the control is denoted with asterisks (**P < 0.01).(D) IL-13 mRNA expression in epidermal cells after PMA/ ionomycin stimulation was analyzed in triplicate by real-time RT-PCR.Representative data from two independent experiments are shown as the mean and SD.Significant difference compared with the control is denoted with an asterisk (*P < 0.05).(E) IL-13 mRNA expression in epidermal cells isolated 4 hours after tape-stripping in vivo was analyzed in triplicate by real-time RT-PCR.Representative data from two independent experiments are shown as the mean and SD.Significant difference compared with the control is denoted with asterisks (**P < 0.01).
(A) Epidermal cells isolated at the indicated time points were analyzed without stimulation for the metabolic dependence or capacity of gated Vg3 + T cells by SCENITH ™ .Representative data from two independent experiments are shown.In each experiment, cells pooled from 3-7 mice were analyzed in triplicate.Data are expressed as the mean and SD.Significant differences compared with D2 are denoted with asterisks (**P < 0.01, ***P < 0.001).(B) Epidermal cells were isolated from D2 neonatal mice and 7-week-old adult mice and analyzed without stimulation for intracellular p4E-BP1 expression in gated Vg3 + T cells.Representative profiles from two independent experiments are shown.In each experiment, cells pooled from 3-4 neonatal mice and a single adult mouse were analyzed in triplicate.(C) Quantification of (B).Relative MFI was determined as (geometric MFI of p4E-BP1)/(geometric MFI of isotype control).Data are expressed as the mean and SD.Significant difference compared with D2 is denoted with an asterisk (*P < 0.05).

7
FIGURE 7 Inhibition of glycolysis decreases IFN-g-producing cells but not IL-13-producing cells in adult DETCs during the late phase of activation.(A) Adult epidermal cells stimulated with anti-TCR mAb for 3 days in vitro were analyzed for the metabolic dependence or capacity of gated Vg3 + T cells by SCENITH ™ .Representative data from two independent experiments are shown.In each experiment, cells pooled from 2 mice were analyzed in triplicate.Data are expressed as the mean and SD.(B) Adult epidermal cells were stimulated with anti-TCR mAb in the presence of DMSO (control) or 2-DG for 3 days in vitro.Vg3 + T cells were gated, and intracellular IFN-g and IL-13 production was analyzed by flow cytometry.Quadrant settings were determined by staining with isotype control mAbs.The numbers denote the percentages of cells in the respective quadrants.Representative profiles from two independent experiments are shown.In each experiment, cells pooled from 2 mice were analyzed in triplicate.(C) Quantification of (B).Data are expressed as the mean and SD.Significant differences compared with the control are denoted with asterisks (**P < 0.01, ***P < 0.001).(D) Adult epidermal cells stimulated with anti-TCR mAb in the presence of DMSO (control) or 2-DG for 3 days in vitro were analyzed for intracellular p4E-BP1 expression in gated Vg3 + T cells.Representative profiles from two independent experiments are shown.In each experiment, cells pooled from 2 mice were analyzed in triplicate.(E) Quantification of (D).Relative MFI was determined as (geometric MFI of p4E-BP1)/ (geometric MFI of isotype control).Data are expressed as the mean and SD.Compared with the control, 2-DG treatment did not diminish p4E-BP1 expression in stimulated DETCs (NS, not significant).