Occludin Expression in Epidermal δγ T Cells in Response to Epidermal Stress Causes Them To Migrate into Draining Lymph Nodes This information is current as of October 2, 2021. Takahito Saito, Michihiro Yano, Yutaro Ohki, Michio Tomura and Naoko Nakano J Immunol published online 31 May 2017 http://www.jimmunol.org/content/early/2017/05/31/jimmun ol.1600848 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/05/31/jimmunol.160084 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published May 31, 2017, doi:10.4049/jimmunol.1600848 The Journal of Immunology Occludin Expression in Epidermal gd T Cells in Response to Epidermal Stress Causes Them To Migrate into Draining Lymph Nodes Takahito Saito,* Michihiro Yano,* Yutaro Ohki,* Michio Tomura,† and Naoko Nakano* Epidermal gd T cells that reside in the front line of the skin play a pivotal role in stress immune surveillance. However, it is not clear whether these cells are involved in further induction of immune responses after they are activated in dysregulated epidermis. In this study, we found that activated gd T cells expressed occludin and migrated into draining lymph nodes in an occludin- dependent manner. Epidermal gd T cells in occludin-deficient mice exhibited impairments in morphology changes and motility, although they expressed activation markers at levels comparable to those in wild-type cells. Occludin deficiency weakened the induction of allergen-induced contact hypersensitivity, primarily as the result of the impaired migration of epidermal gd T cells. Thus, occludin expression by epidermal gd T cells upon activation in response to epidermal stress allows them to move, which Downloaded from could be important for augmentation of immune responses via collaboration with other cells. The Journal of Immunology, 2017, 199: 000–000. he epidermis provides a barrier against insults from the gdTCRs expressed in DETCs signal constitutively and form the environment, including pathogens, UV light, and chem- immunological synapse at squamous keratinocyte tight junctions T icals. There are two types of immune cells in murine (16). This semiactivated state may be important for interactions http://www.jimmunol.org/ epidermis: Langerhans cells (LCs), which capture invading pathogens between DETCs and keratinocytes. and bring them to the draining lymph nodes, leading to immune re- In stressed epidermis, DETCs are further activated in response to sponses, and dendritic epidermal gd T cells (DETCs), which recog- molecular stress signals expressed in that layer. Several costimu- nize stress- or damage-induced molecules in keratinocytes (1, 2). latory molecules involved in the interaction of DETCs and stressed DETCs have been proposed to eliminate stressed or DNA-damaged keratinocytes have been identified. For example, DETCs express keratinocytes, thereby helping to prevent tumor development (3). In junctional adhesion molecule–like protein (JAML), which inter- addition, in wounded epidermis, DETCs secrete cytokines and play acts with Coxsackie and adenovirus receptor (17). In addition, key roles in wound healing, thereby contributing to epidermal ho- DETCs upregulate CD100, which induces costimulatory signals meostasis (4, 5). Recent work showed that gd T cells promote contact by interacting with plexin B2 on keratinocytes (18). Activated by guest on October 2, 2021 hypersensitivity (CHS) responses (6, 7), skin graft rejection (8), and DETCs undergo morphological changes and detach from tight Ab production (9, 10). However, the molecular mechanisms by which junctions, subsequently reorienting toward basal layers and LCs DETCs activate these immune responses are not well defined. (16). Pathogens and other insults, including chemicals that disrupt DETCs are generated in the first wave of Vg locus rearrange- tight junctions, are also sensed by gdTCRs expressed in DETCs. ments, expressing canonical Vg5Vd1 TCR [nomenclature of Heilig Although there is some evidence that gd T cells function as innate- and Tonegawa (11)], and subsequently migrate to the epidermis. like cells that augment adaptive immune responses, it remains These cells develop in the thymus only during the early embryonic unclear how activated gd T cells interact with other immune cells period and must be selected via mechanisms involving Skint-1, and whether they migrate out of the epidermis. In this study, we which is expressed in thymic epithelial cells and keratinocytes found that DETCs express occludin, a tight junction molecule, in (12–14). Thymocytes expressing this canonical gdTCR engage UVB-irradiated or allergen-treated epidermis. Occludin-deficient Skint-1+ cells and upregulate Egr-3 expression, thus becoming DETCs exhibited impairments in morphological changes and IFN-g–producing gd T cells (15). In the epidermis at steady-state, motility upon activation. Furthermore, allergen-induced CHS re- sponses in occludin-deficient mice were much weaker than those *Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba in wild-type mice. These results suggested that DETCs exert their 278-0022, Japan; and †Department of Pharmaceutical Sciences, Osaka Ohtani Uni- functions by expressing occludin, causing them to become motile versity, Osaka 584-8541, Japan and ultimately leading to the activation of other immune cells. ORCID: 0000-0003-3499-9900 (T.S.). Received for publication May 13, 2016. Accepted for publication May 4, 2017. Materials and Methods This work was supported by a grant from the Center for Technologies against Cancer, Tokyo University of Science. Mice Address correspondence and reprint requests to Dr. Naoko Nakano, Research Insti- C57BL/6J mice were purchased from Sankyo Laboratories (Shizuoka, tute for Biomedical Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Japan). Occludin-deficient mice (Ocln2/2) (19) on the C57BL/6 back- Chiba 278-0022, Japan. E-mail address: [email protected] ground were provided by M. Furuse and were obtained from the Center for The online version of this article contains supplemental material. Animal Resources and Development at Kumamoto University. Littermates +/2 3 +/2 Abbreviations used in this article: CHS, contact hypersensitivity; DETC, dendritic epi- generated from Ocln Ocln crosses were used as occludin-deficient 2/2 +/+ d2/2 dermal gd T cell; DNFB, 2,4-dinitrofluorobenzene; IEL, intraepithelial lymphocyte; (Ocln ) or wild-type control (Ocln ) mice. TCR mice (20) were JAML, junctional adhesion molecule–like protein; LC, Langerhans cell. housed in our mouse facilities. KikGR mice were described previously (21). Six- to ten-week-old male and female mice were used in all exper- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 iments. All mice were maintained under specific pathogen–free conditions in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600848 2 OCCLUDIN-DEPENDENT MIGRATION OF DETCs UPON ACTIVATION the animal facility at Tokyo University of Science, and experimental studies was exposed to violet light (420 nm) for 2 min using an LED hand lamp were approved by the university’s Animal Care and Use Committee. (LED-420P; OptoCode, Tokyo, Japan). Abs and reagents Real-time RT-PCR Abs against TCRgd (UC7-13D5; BioLegend), CD25 (PC61.5; eBioscience), Total RNA was isolated from cells using ISOGEN and reverse transcribed using CD69 (H1.2F3; eBioscience), CD11c (N418; BioLegend), I-A/I-E (M5/ ReverScript III (both from Wako Pure Chemical Industries). PCR was per- 114.15.2; eBioscience), CD100 (BMA12; eBioscience), CD3 (145-2C11; formed in triplicate using GoTaq qPCR Master Mix containing SYBR Green on eBioscience), or Vg5 (7-17; BD Pharmingen), conjugated to FITC, PE, an Applied Biosystems 7500 Fast Real-Time PCR System. Experiments were allophycocyanin, or biotin, were obtained from the indicated suppliers. independently repeated two or three times. Values for each gene were nor- Allophycocyanin-, PE-Cy7–, or Brilliant Violet 421–conjugated streptavidin malized to the corresponding expression levels of b-actin. The specific primers (BioLegend) was used for the second step. CD16/32 (2.4G2) Abs were used used were as follows: claudin-1: 59-ACTCCTTGCTGAATCGAACAGT-39, for Fc blocking. Purified anti-TCRgd (UC7-13D5, functional grade) and 59-GGACACAAAGATTGCGATCAG-39;claudin-3:59-CTCATCGTGGTG- anti-CD3 (145-2C11, functional grade) were purchased from eBioscience. TCCATCC-39,59-ATGGTGATCTTGGCCTTGG-39; claudin-4: 59-GGAA- Rabbit anti-occludin (711500; Invitrogen) and Alexa Fluor 647– or Alexa TCTCCTTGGCAGTCCT-39,59-CACCCACGATGATGCTGAT-39; claudin-6: Fluor 488–conjugated goat anti-rabbit IgG (A21245; Invitrogen) were used 59-TGTGTGGTTCAGAGCACTGG-39,59-AGCAGACAGGAATGAGCGTC-39;