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Resident Memory and Recirculating Memory T Cells Cooperate to Maintain Disease in a Mouse Model of Vitiligo

Open ArchivePublished:November 10, 2018DOI:https://doi.org/10.1016/j.jid.2018.10.032
      Tissue resident memory T cells (Trm) form in the skin in vitiligo and persist to maintain disease, as white spots often recur rapidly after discontinuing therapy. We and others have recently described melanocyte-specific autoreactive Trm in vitiligo lesions. Here, we characterize the functional relationship between Trm and recirculating memory T cells (Tcm) in our vitiligo mouse model. We found that both Trm and Tcm sensed autoantigen in the skin long after stabilization of disease, producing IFN-γ, CXCL9, and CXCL10. Blockade of Tcm recruitment to the skin with FTY720 or depletion of Tcm with low-dose Thy1.1 antibody reversed disease, indicating that Trm cooperate with Tcm to maintain disease. Taken together, our data provide characterization of skin memory T cells in vitiligo, demonstrate that Trm and Tcm work together during disease, and indicate that targeting their survival or function may provide novel, durable treatment options for patients.

      Abbreviations:

      GREAT (IFN-γ reporter with IRES poly A tail), OT-1 (ovalbumin-specific T cell), PMEL (premelanosome protein-specific T cell), Tcm (recirculating memory T cell), Trm (resident memory T cell), VV (vaccinia virus)

      Introduction

      Vitiligo is caused by CD8+ T cells that target melanocytes for destruction (
      • van den Boorn J.G.
      • Konijnenberg D.
      • Dellemijn T.A.
      • van der Veen J.P.
      • Bos J.D.
      • Melief C.J.
      • et al.
      Autoimmune destruction of skin melanocytes by perilesional T cells from vitiligo patients.
      ), resulting in patchy depigmentation that is disfiguring and distressing to patients (
      • Alikhan A.
      • Felsten L.M.
      • Daly M.
      • Petronic-Rosic V.
      Vitiligo: a comprehensive overview Part I. Introduction, epidemiology, quality of life, diagnosis, differential diagnosis, associations, histopathology, etiology, and work-up.
      ,
      • Frisoli M.L.
      • Harris J.E.
      Vitiligo: mechanistic insights lead to novel treatments.
      ,
      • Richmond J.M.
      • Harris J.E.
      Vitiligo.
      ,
      • Rodrigues M.
      • Ezzedine K.
      • Hamzavi I.
      • Pandya A.G.
      • Harris J.E.
      Vitiligo Working Group
      Current and emerging treatments for vitiligo.
      ). Depigmentation typically recurs rapidly at the same location after therapy is stopped (
      • Cavalie M.
      • Ezzedine K.
      • Fontas E.
      • Montaudie H.
      • Castela E.
      • Bahadoran P.
      • et al.
      Maintenance therapy of adult vitiligo with 0.1% tacrolimus ointment: a randomized, double blind, placebo-controlled study.
      ), indicating that autoimmune memory persists in the skin and permits disease reactivation after cessation of treatment. We and others have shown that lesional skin biopsies from patients contain antigen-specific CD8+ resident memory T cells (Trm), supporting a role for these cells in human vitiligo (
      • Boniface K.
      • Jacquemin C.
      • Darrigade A.S.
      • Dessarthe B.
      • Martins C.
      • Boukhedouni N.
      • et al.
      Vitiligo skin is imprinted with resident memory CD8 T cells expressing CXCR3.
      ,
      • Cheuk S.
      • Schlums H.
      • Gallais Serezal I.
      • Martini E.
      • Chiang S.C.
      • Marquardt N.
      • et al.
      CD49a Expression defines tissue-resident CD8+ T cells poised for cytotoxic function in human skin.
      ,
      • Richmond J.M.
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      • Zapata Jr., L.
      • Garg M.
      • Riding R.L.
      • Refat M.A.
      • et al.
      Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo.
      ).
      Normally, Trm remain in non-lymphoid tissues to provide tissue surveillance against pathogens (
      • Clark R.A.
      • Watanabe R.
      • Teague J.E.
      • Schlapbach C.
      • Tawa M.C.
      • Adams N.
      • et al.
      Skin effector memory T cells do not recirculate and provide immune protection in alemtuzumab-treated CTCL patients.
      ,
      • Gebhardt T.
      • Wakim L.M.
      • Eidsmo L.
      • Reading P.C.
      • Heath W.R.
      • Carbone F.R.
      Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus.
      ,
      • Jiang X.
      • Clark R.A.
      • Liu L.
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      • Fuhlbrigge R.C.
      • Kupper T.S.
      Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.
      ,
      • Schenkel J.M.
      • Fraser K.A.
      • Vezys V.
      • Masopust D.
      Sensing and alarm function of resident memory CD8(+) T cells.
      ,
      • Watanabe R.
      • Gehad A.
      • Yang C.
      • Scott L.L.
      • Teague J.E.
      • Schlapbach C.
      • et al.
      Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.
      ,
      • Zhu J.
      • Peng T.
      • Johnston C.
      • Phasouk K.
      • Kask A.S.
      • Klock A.
      • et al.
      Immune surveillance by CD8alphaalpha+ skin-resident T cells in human herpes virus infection.
      ). Upon entering tissues, Trm upregulate CD69 and CD103, downregulate the chemokine receptors S1P1 and CCR7 to prevent recirculation, and set up residence (
      • Mackay L.K.
      • Rahimpour A.
      • Ma J.Z.
      • Collins N.
      • Stock A.T.
      • Hafon M.L.
      • et al.
      The developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of skin.
      ,
      • Skon C.N.
      • Lee J.Y.
      • Anderson K.G.
      • Masopust D.
      • Hogquist K.A.
      • Jameson S.C.
      Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells.
      ). We have recently published a strategy for depleting Trm cells in vitiligo by blocking IL-15 signaling (
      • Richmond J.M.
      • Strassner J.P.
      • Zapata Jr., L.
      • Garg M.
      • Riding R.L.
      • Refat M.A.
      • et al.
      Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo.
      ). In contrast to Trm, recirculating memory T cells (Tcm) are able to migrate back and forth through the blood and lymph to tissues such as the skin. Antigen-specific Tcm have previously been identified in the blood of vitiligo patients (
      • Ogg G.S.
      • Dunbar P.R.
      • Romero P.
      • Chen J.L.
      • Cerundolo J.
      High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo.
      ), and these cells exhibited skin-homing potential, as determined by cutaneous lymphocyte antigen expression. However, less is known about the functional capacity of Tcm versus Trm in vitiligo. Recent studies have begun to address this issue by looking at cytokine production of Tcm pools in the skin, and have concluded that the effector function of Tcm depends largely upon signals received in situ (
      • Seidel J.A.
      • Vukmanovic-Stejic M.
      • Muller-Durovic B.
      • Patel N.
      • Fuentes-Duculan J.
      • Henson S.M.
      • et al.
      Skin resident memory CD8+ T cells are phenotypically and functionally distinct from circulating populations and lack immediate cytotoxic function.
      ). In agreement with these studies, melanocyte-specific Tcm have been identified in healthy individuals, but appear to lack effector functions seen in vitiligo patients (
      • Pittet M.J.
      • Valmori D.
      • Dunbar P.R.
      • Speiser D.E.
      • Lienard D.
      • Lejeune F.
      • et al.
      High frequencies of naive Melan-a/Mart-1–specific Cd8+ T cells in a large proportion of human histocompatibility leukocyte antigen (Hla)-A2 individuals.
      ). Therefore, the following questions remain: (i) are Trm sufficient for maintaining disease? and (ii) what is the role of Tcm in vitiligo?
      To begin to answer these questions, we sought to define autoreactive Tcm in our vitiligo mouse model, which was adapted from previous studies of melanoma-associated vitiligo models (
      • Gregg R.K.
      • Nichols L.
      • Chen Y.
      • Lu B.
      • Engelhard V.H.
      Mechanisms of spatial and temporal development of autoimmune vitiligo in tyrosinase-specific TCR transgenic mice.
      ,
      • Overwijk W.W.
      • Tsung A.
      • Irvine K.R.
      • Parkhurst M.R.
      • Goletz T.J.
      • Tsung K.
      • et al.
      gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand.
      ,
      • Overwijk W.W.
      • Theoret M.R.
      • Finkelstein S.E.
      • Surman D.R.
      • de Jong L.A.
      • Vyth-Dreese F.A.
      • et al.
      Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells.
      ). Our model uses the adoptive transfer of TCR transgenic T cells recognizing the human melanocyte antigen premelanosome protein (PMEL) into recipient mice with epidermal melanocytes (
      • Agarwal P.
      • Rashighi M.
      • Essien K.I.
      • Richmond J.M.
      • Randall L.
      • Pazoki-Toroudi H.
      • et al.
      Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo.
      ,
      • Harris J.E.
      • Harris T.H.
      • Weninger W.
      • Wherry E.J.
      • Hunter C.A.
      • Turka L.A.
      A mouse model of vitiligo with focused epidermal depigmentation requires IFN-gamma for autoreactive CD8(+) T-cell accumulation in the skin.
      ,
      • Rashighi M.
      • Agarwal P.
      • Richmond J.M.
      • Harris T.H.
      • Dresser K.
      • Su M.W.
      • et al.
      CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.
      ,
      • Richmond J.M.
      • Bangari D.S.
      • Essien K.I.
      • Currimbhoy S.D.
      • Groom J.R.
      • Pandya A.G.
      • et al.
      Keratinocyte-derived chemokines orchestrate T-cell positioning in the epidermis during vitiligo and may serve as biomarkers of disease.
      ,
      • Richmond J.M.
      • Masterjohn E.
      • Chu R.
      • Tedstone J.
      • Youd M.E.
      • Harris J.E.
      CXCR3 depleting antibodies prevent and reverse vitiligo in mice.
      ,
      • Richmond J.M.
      • Strassner J.P.
      • Zapata Jr., L.
      • Garg M.
      • Riding R.L.
      • Refat M.A.
      • et al.
      Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo.
      ,
      • Riding R.L.
      • Richmond J.M.
      • Harris J.E.
      Mouse model for human vitiligo.
      ). These T cells (also called PMEL) like their antigenic target accumulate in the epidermis, kill mouse melanocytes, and induce patchy epidermal depigmentation that mirrors human disease (
      • Alikhan A.
      • Felsten L.M.
      • Daly M.
      • Petronic-Rosic V.
      Vitiligo: a comprehensive overview Part I. Introduction, epidemiology, quality of life, diagnosis, differential diagnosis, associations, histopathology, etiology, and work-up.
      ,
      • Frisoli M.L.
      • Harris J.E.
      Vitiligo: mechanistic insights lead to novel treatments.
      ,
      • Richmond J.M.
      • Harris J.E.
      Vitiligo.
      ,
      • Rodrigues M.
      • Ezzedine K.
      • Hamzavi I.
      • Pandya A.G.
      • Harris J.E.
      Vitiligo Working Group
      Current and emerging treatments for vitiligo.
      ).
      Here, we show that Tcm sense antigen, secrete cytokines and chemokines, and cooperate with Trm to maintain disease in mice. Importantly, inhibiting T cell recruitment to the skin with FTY720, or depleting Tcm with low-dose Thy1.1 antibody (
      • Schenkel J.M.
      • Fraser K.A.
      • Vezys V.
      • Masopust D.
      Sensing and alarm function of resident memory CD8(+) T cells.
      ), reversed disease. Thus, our data indicate that Trm must cooperate with Tcm cells to maintain lesions, and targeting their survival or function may provide novel treatment options for patients.

      Results

      Autoreactive Trm cells require self-antigen and make IFN-γ in the epidermis

      We employed our vitiligo mouse model to address the functional roles of Trm and Tcm in vitiligo. We first performed phenotypic characterization of PMEL in tissues in mice with established vitiligo. We found that a large fraction of epidermal PMEL expressed the Trm makers CD69 and CD103 (Supplementary Tables S1 and S2 online). We also assessed expression of other classical Tcm markers and found that the majority of PMEL expressed CD127, PD-1, CD44, KLRG1 (all tissues), CCR5 (all tissues except blood), and CXCR3 (all tissues except directly in the skin, possibly due to internalization in sites of high ligand production) (Supplementary Table S1). Epidermal T cells had low CD62L, whereas peripheral T cells expressed it in variable amounts, which was highest in spleen and lymph node (Supplementary Table S1). As we have reported previously, PMEL express high levels of the CD122 chain of the IL-15 receptor (
      • Richmond J.M.
      • Strassner J.P.
      • Zapata Jr., L.
      • Garg M.
      • Riding R.L.
      • Refat M.A.
      • et al.
      Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo.
      ).
      To determine the role of self-antigen in the recruitment and retention of Trm, we compared the generation of epidermal Trm that recognize PMEL physiologically expressed in melanocytes to T cells that recognize the irrelevant foreign antigen ovalbumin (OT-1) (
      • Hogquist K.A.
      • Jameson S.C.
      • Heath W.R.
      • Howard J.L.
      • Bevan M.J.
      • Carbone F.R.
      T cell receptor antagonist peptides induce positive selection.
      ). We induced immune responses with recombinant vaccinia virus expressing premelanosome protein (VV-PMEL) and PMEL, or ovalbumin (
      • York I.A.
      • Brehm M.A.
      • Zendzian S.
      • Towne C.F.
      • Rock K.L.
      Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an important role in immunodominance.
      ) and OT-1 T cells (Figure 1a). Only PMEL induced disease in mice and established Trm in the epidermis, whereas OT-1 did not, despite engrafting in the lymph node (Figure 1b–1f). Thus, autoreactive Trm are generated directly in the skin where autoantigen is expressed during vitiligo.
      Figure thumbnail gr1
      Figure 1Melanocyte-specific, but not ovalbumin-specific, T cells, form Trm in the epidermis and secrete IFN-γ in the mouse model of vitiligo. (a) Schematic of the mouse model. (b) Example photos and (c) vitiligo scores from mice that received PMEL+VV-PMEL versus OT-1+VV-OVA. Sample flow plots and quantification of transferred T cells in the (d) lymph node and (e) epidermis. Sample flow plots demonstrating (f) CD69 and CD103 staining used to identify Trm, and (g) IFN-γ reporter expression by all GREAT PMEL and by Trm GREAT PMEL. (n = 4 mice/group; representative experiment of two shown. Student t tests significant as indicated, P < 0.05.) (h) Frequency of epidermal PMEL producing IFN-γ and mouse disease scores over time. (n = 2–8 mice per time point pooled from 4 separate experiments). GREAT, IFN-γ reporter with IRES poly A tail; OVA, ovalbumin; PMEL, premelanosome protein-specific T cell; Trm, resident memory T cell; VV, virus vaccinia.
      To assess their functional capacity, we bred PMEL donor mice to IFN-γ reporter mice (IFN-gamma reporter with IRES poly A tail [GREAT] mouse) (
      • Reinhardt R.L.
      • Liang H.E.
      • Locksley R.M.
      Cytokine-secreting follicular T cells shape the antibody repertoire.
      ). We first validated that the GREAT reporter accurately represented IFN-γ expression by performing co-staining of IFN-γ and GFP reporter following in vitro stimulation with anti-CD3/CD28 (Supplementary Figure S1 online) (method from
      • Groom J.R.
      • Richmond J.
      • Murooka T.T.
      • Sorensen E.W.
      • Sung J.H.
      • Bankert K.
      • et al.
      CXCR3 chemokine receptor-ligand interactions in the lymph node optimize CD4+ T helper 1 cell differentiation.
      ). We then transferred naïve GREAT PMEL in vivo in our vitiligo model and found that PMEL persisted in the epidermis and expressed IFN-γ, but this was not limited to Trm PMEL: rather, similar frequencies of total PMEL and Trm PMEL expressed the GREAT reporter at the peak of disease week 8 (Figure 1g). We therefore quantified GREAT reporter expression over time and found all epidermal PMEL express GREAT reporter at least 27 weeks following disease induction, and by 62 weeks the expression was reduced (Figure 1h). These data indicate that all autoreactive PMEL, not just Trm, have functional capacity for long periods of time.

      Autoreactive Trm within lesions of vitiligo patients are polyclonal as defined by private specificity for TCR Vβ usage

      Melanocyte-specific TCR Vβ usage has been performed successfully on T cells from melanoma patients (
      • Jager E.
      • Maeurer M.
      • Hohn H.
      • Karbach J.
      • Jager D.
      • Zidianakis Z.
      • et al.
      Clonal expansion of Melan A-specific cytotoxic T lymphocytes in a melanoma patient responding to continued immunization with melanoma-associated peptides.
      ); therefore, we hypothesized that this method would allow us to assess T cell clonality in human vitiligo, and to determine whether specific clones could be identified in lesional vitiligo skin that differ from nonlesional skin and blood (as opposed to our single clone-mediated mouse model). We obtained epidermis from shave biopsies from two lesional and two nonlesional sites in three stable vitiligo patients, as well as peripheral blood mononuclear cells, for TCR Vβ sequencing analysis (see Supplementary Table S3 online for patient characteristics). We identified Trm in these patients using a portion of the tissue for flow cytometry, and found that 80% of epidermal T cells were Trm, as defined by CD3+CD8+CD69+CD103+ in both lesional and nonlesional skin, whereas only 20% were found in PBMCs (Figure 2a, 2b; see Supplementary Figure S2 online for flow gating strategy). The presence of the Trm in nonlesional skin could be due to subclinical involvement or due to memory to different antigens, such as pathogens. We did not detect a unique dominant clone, and clonality varied among lesions (Figure 2c). Our data revealed non-conserved sequences at each biopsy site and among patients, suggesting that multiple different T cell clones infiltrate different lesions, a phenomenon described as private specificity (Figure 2d). Thus, TCR Vβ usage is quite heterogeneous, even within a single patient.
      Figure thumbnail gr2
      Figure 2Human Trm from vitiligo patient shave biopsies are polyclonal and have private specificity for TCR Vβ usage. (a) Sample flow cytometry of CD8+ T cells and Trm in human lesional and nonlesional shave biopsies and PBMCs from vitiligo patients. Cells were gated on live, single, CD45+, CD3+CD8+ T cells then CD69+CD103+ for Trm identification. (b) Quantification of CD69+CD103+CD8+CD3+ cells in all three tissue sites (one-way analysis of variance with Tukey’s post tests P < 0.0001). (c) TCR Vβ usage of lesional, nonlesional skin, and PBMCs from a vitiligo patient (corresponding Vβs indicated by symbols in the key). (d) Overlap and correlation values for each lesion from each patient reveals no significant overlap except with each lesion to itself (dark purple squares in bold boxes), indicating private specificity. PBMC, peripheral blood mononuclear cells; Trm, resident memory T cell.

      CD44 and CD103 are dispensable for vitiligo in mice

      Previous studies reported that CD103 and CD44 are upregulated on Trm as part of their developmental program (
      • Mackay L.K.
      • Rahimpour A.
      • Ma J.Z.
      • Collins N.
      • Stock A.T.
      • Hafon M.L.
      • et al.
      The developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of skin.
      ,
      • Mrass P.
      • Kinjyo I.
      • Ng L.G.
      • Reiner S.L.
      • Pure E.
      • Weninger W.
      CD44 mediates successful interstitial navigation by killer T cells and enables efficient antitumor immunity.
      ). In order to determine whether these molecules were required on PMEL to initiate or maintain disease, we bred PMEL mice to CD103–/– (
      • Schon M.P.
      • Arya A.
      • Murphy E.A.
      • Adams C.M.
      • Strauch U.G.
      • Agace W.W.
      • et al.
      Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice.
      ) and CD44–/– (
      • Protin U.
      • Schweighoffer T.
      • Jochum W.
      • Hilberg F.
      CD44-deficient mice develop normally with changes in subpopulations and recirculation of lymphocyte subsets.
      ) and used these cells to induce vitiligo (confirmation of knockouts in Supplementary Figure S3 online). Single transfers of both CD103–/– and CD44–/– PMEL were capable of inducing vitiligo with little effect on epidermal cell numbers or disease score (Supplementary Figure S4 online). Numbers of phenotypically different skin memory T cells, namely CD44CD69+CD103+ and CD69+CD103, were similar in recipients (Supplementary Figure S4d, S4h). Co-transfers of wild-type and CD103–/– or CD44–/– PMEL revealed no significant differences in the epidermis, though there was a trend towards more wild-type cells (Supplementary Figure S5 online). In accordance with prior studies in virus and melanoma models (
      • Mackay L.K.
      • Rahimpour A.
      • Ma J.Z.
      • Collins N.
      • Stock A.T.
      • Hafon M.L.
      • et al.
      The developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of skin.
      ,
      • Malik B.T.
      • Byrne K.T.
      • Vella J.L.
      • Zhang P.
      • Shabaneh T.B.
      • Steinberg S.M.
      • et al.
      Resident memory T cells in the skin mediate durable immunity to melanoma.
      ), CD44 and CD103 are not required for generation or function of PMEL memory during vitiligo.

      PMEL in both the epidermis and dermis encounter self-antigen

      Self-reactive Trm differ from viral-reactive Trm in that they can be re-exposed to autoantigens, in contrast to viral antigens that are cleared. Further, melanocytes are capable of regenerating and, therefore, Trm retained within the skin are likely to re-encounter self-antigen expressed in repopulating cells. To evaluate the frequency with which these encounters result in TCR stimulation, we bred PMEL mice to Nur77-GFP mice (
      • Moran A.E.
      • Holzapfel K.L.
      • Xing Y.
      • Cunningham N.R.
      • Maltzman J.S.
      • Punt J.
      • et al.
      T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse.
      ) and we validated the half-life of the reporter in our PMEL T cells (Supplementary Figure S6 online). We then used Nur77-GFP PMEL to induce vitiligo, and examined mice 8 weeks post-induction when epidermal Trm are established (
      • Richmond J.M.
      • Strassner J.P.
      • Zapata Jr., L.
      • Garg M.
      • Riding R.L.
      • Refat M.A.
      • et al.
      Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo.
      ). In these mice, approximately 10% of epidermal and lymph node PMEL were GFP-positive in mice with established disease, whereas 30% of dermal PMEL were GFP-positive (Figure 3a, 3b). We further characterized PMEL activation based on CD69 and CD103 expression, and found that epidermal CD69+CD103 PMEL expressed the highest levels of Nur77-GFP (Figure 3c, 3d), whereas in the dermis, CD69+CD103+ PMEL expressed the highest levels of Nur77-GFP reporter (Figure 3e, 3f). Thus, new immigrants in the epidermis are most likely to detect self-antigen, whereas long-lived Trm in the epidermis sense self-antigen at a lower but consistent level.
      Figure thumbnail gr3
      Figure 3Nur77-GFP TCR activation reporter reveals epidermal and dermal PMEL sense antigen. (a) Sample flow plots and (b) quantification of frequency of PMEL expressing the Nur77-GFP reporter in the indicated tissues (pre-gated on live single PMEL, n = 10 pooled from three separate experiments; one-way analysis of variance; P = 0.0272, Tukey’s post-tests epidermis vs. dermis; P = 0.0253). (c) Sample flow plots and (d) quantification of frequency of epidermal Nur77-GFP+ cells in the indicated parental resident memory T cell phenotyping gates (repeated measures/matched one-way analysis of variance without sphericity; P = 0.0676, Tukey’s post-tests NS). (e) Sample flow plots and (f) quantification of frequency of dermal Nur77-GFP+ cells in the indicated parental resident memory T cell phenotyping gates. (repeated measures/matched one-way analysis of variance without sphericity P = 0.0384, Tukey’s post-tests, NS, trending towards significance CD69CD103+ versus CD69+CD103+; P = 0.0709). LN, lymph node; PMEL, premelanosome protein-specific T cell.

      PMEL Trm produce CXCR3 chemokines for the potential recruitment of Tcm

      We bred PMEL mice to REX3 mice, which report expression of CXCL9 and CXCL10 (
      • Groom J.R.
      • Richmond J.
      • Murooka T.T.
      • Sorensen E.W.
      • Sung J.H.
      • Bankert K.
      • et al.
      CXCR3 chemokine receptor-ligand interactions in the lymph node optimize CD4+ T helper 1 cell differentiation.
      ), to determine whether they were capable of secreting chemokines to recruit Tcm to the skin (Figure 4a). The highest frequency of chemokine expression was in the epidermis, followed by dermis and lymph node, potentially providing a gradient for Tcm to follow in order to find their melanocyte targets (Figure 4b). We further characterized the PMEL based on CD69 and CD103 expression and found that both epidermal and dermal CD69+CD103+ PMEL expressed the highest levels of CXCL9 and CXCL10, though other phenotypes were also capable of producing these chemokines at lower levels (Figure 4c–4f).
      Figure thumbnail gr4
      Figure 4REX3 reporter reveals PMEL express the alarm/recruitment chemokines CXCL9 and CXCL10. (a) Sample flow plots and (b) quantification of frequency of PMEL expressing the CXCL9-RFP and CXCL10-BFP reporters in the indicated tissues (pre-gated on live single PMEL for REX3 reporting, two-way analysis of variance P = 0.0023; repeated measures/matched samples compared for each tissue from every mouse Tukey’s post-tests, NS for CXCL9; for CXCL10 P = 0.032 epidermis versus dermis, ∗∗∗∗P < 0.0001 epidermis versus lymph node, ∗∗P = 0.001 dermis versus lymph node; n = 10 pooled from three separate experiments). (c) Sample flow plots and (d) quantification of frequency of epidermal REX3+ cells in the indicated parental resident memory T cell phenotyping gates (repeated measures/matched one-way analysis of variance without sphericity P = 0.294 for CXCL9, Tukey’s post-tests, NS; P = 0.0382 for CXCL10, Tukey’s post-tests CD69CD103+ versus CD69+CD103 P = 0.0364) (e) Sample flow plots and (f) quantification of frequency of dermal REX3+ cells in the indicated parental resident memory T cell phenotyping gates (repeated measures/matched one-way analysis of variance without sphericity P = 0.4877 for CXCL9, Tukey’s post-tests, NS; P = 0.0806 for CXCL10, Tukey’s post-tests CD69CD103+ versus CD69+CD103+ ∗∗P = 0.0016). PMEL, premelanosome protein-specific T cell.
      To determine the potential functional role of T cell-derived chemokine production in vitiligo, we bred PMEL mice to CXCL9- (
      • Park M.K.
      • Amichay D.
      • Love P.
      • Wick E.
      • Liao F.
      • Grinberg A.
      • et al.
      The CXC chemokine murine monokine induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs, targets lymphocytes including activated B cells, and supports antibody responses to a bacterial pathogen in vivo.
      ) and CXCL10-deficient animals (
      • Dufour J.H.
      • Dziejman M.
      • Liu M.T.
      • Leung J.H.
      • Lane T.E.
      • Luster A.D.
      IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking.
      ) and used these as T cell donors. Mice that received CXCL9- and CXCL10-deficient PMELs had significantly fewer PMELs in the epidermis and a trend towards fewer PMELs in the dermis, though clinical disease scores were similar and the frequency of epidermal Trm was also similar to wild-type PMEL controls (Supplementary Figure S7 online). Despite this, CXCL10-deficient PMELs appear to engraft in the lymph node at a higher rate than wild-type or CXCL9-deficient PMELs (Supplementary Figure S7e). Thus, autoreactive Trm in our model serve a sensing/alarm function to recruit Tcm that target regenerating melanocytes and maintain white patches in vitiligo, but likely use multiple or redundant chemokine signals to do so.
      We next performed en face microscopy of whole ear tissue from vitiligo mice that had received any of the reporter PMEL T cells (GREAT, Nur77-GFP, or REX3) to visualize their location within the skin tissue. We found that all PMEL reporter T cells were often sparsely populated, but sometimes clustered near hair follicles, as determined by CD200 staining (Supplementary Figure S8 online).

      Persistence of depigmentation in vitiligo requires Tcm

      Because dermal PMEL sense self-antigen as measured by Nur77-GFP, and antigen-specific T cells secreted chemokine as measured by REX3, we sought to determine whether Trm were sufficient to maintain depigmentation, or if Tcm help maintain vitiligo. We used the S1P1 inhibitor FTY720 (
      • Chiba K.
      FTY720, a new class of immunomodulator, inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptors.
      ,
      • Murooka T.T.
      • Deruaz M.
      • Marangoni F.
      • Vrbanac V.D.
      • Seung E.
      • von Andrian U.H.
      • et al.
      HIV-infected T cells are migratory vehicles for viral dissemination.
      ,
      • Pham T.H.M.
      • Okada T.
      • Matloubian M.
      • Lo C.G.
      • Cyster J.G.
      S1P1 receptor signaling overrides retention mediated by Gai-coupled receptors to promote T cell egress.
      ,
      • Schwab S.R.
      • Pereira J.P.
      • Matloubian M.
      • Xu Y.
      • Huang Y.
      • Cyster J.G.
      Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients.
      ) to inhibit the recirculation of T cells from the lymph nodes and evaluated repigmentation. We found that treatment with FTY720 resulted in rapid reversal of disease (Figure 5a–5e). In accordance with other studies (
      • Pinschewer D.
      • Ochsenbein A.F.
      • Odermatt B.
      • Brinkmann V.
      • Hengartner H.
      • Zinkernagel R.M.
      FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion, and memory.
      ), PMEL Trm were still present in the skin after treatment (Figure 5f, 5g). PMEL numbers in other tissues in FTY720-treated mice were the same as vehicle controls (Figure 5h, 5i), an expected result based on previous studies demonstrating that FTY720 treatment locks cells in tissues (
      • Murooka T.T.
      • Deruaz M.
      • Marangoni F.
      • Vrbanac V.D.
      • Seung E.
      • von Andrian U.H.
      • et al.
      HIV-infected T cells are migratory vehicles for viral dissemination.
      ,
      • Pham T.H.M.
      • Okada T.
      • Matloubian M.
      • Lo C.G.
      • Cyster J.G.
      S1P1 receptor signaling overrides retention mediated by Gai-coupled receptors to promote T cell egress.
      ). Therefore, we also used low-dose Thy1.1 depleting antibody to selectively deplete Tcm (
      • Schenkel J.M.
      • Fraser K.A.
      • Vezys V.
      • Masopust D.
      Sensing and alarm function of resident memory CD8(+) T cells.
      ). These mice also repigmented, supporting the FTY720 data and implicating Tcm as necessary for vitiligo maintenance (Figure 5j–5n). Further, Trm were largely intact (Figure 5o, 5p), whereas lymph node and dermal PMEL populations were significantly reduced (Figure 5l, 5r). Thus, Tcm contribute to sustained melanocyte killing during vitiligo maintenance, and Trm are not sufficient effectors for this function.
      Figure thumbnail gr5
      Figure 5Recirculating PMELs are required for maintenance of depigmentation in vitiligo mice. (a) Timing of treatments for the FTY720 repigmentation model. (b) Sample photographs of tails from vehicle- or FTY720-treated animals. (c) Quantification of pigment before/after in PBS or (d) FTY720-treated animals, and (e) total change in pigment. (f) Number of live, single CD45+CD8+Thy1.1+ epidermal PMELs normalized to 10,000 live singlet cells and (g) frequency of CD69+CD103+ PMEL Trm in the repigmentation mice. (h) Number of live, single CD45+CD8+Thy1.1+ PMELs normalized to 10,000 live singlet cells in the lymph node and (i) dermis. (j) Timing of Thy1.1 depletion for the repigmentation model. (k) Sample photographs of tails from control or Thy1.1-depleted animals. (l) Quantification of pigment before/after in PBS or (m) Thy1.1 depleted animals, and (n) total change in pigment. (o) Number of live, single CD45+CD8+Thy1.1+ epidermal PMELs normalized to 10,000 live singlet cells and (p) frequency of CD69+CD103+ PMEL Trm in the repigmentation mice. (q) Number of live, single CD45+CD8+Thy1.1+ PMELs normalized to 10,000 live singlet cells in the lymph node and (r) dermis (Student t tests significant as indicated; each dot represents one animal pooled from three separate experiments). PBS, phosphate buffered saline; PMEL, premelanosome protein-specific T cell; Trm, resident memory T.

      Discussion

      Previous studies in virus models are conflicted as to the function of Trm and Tcm within tissues. Some studies report enhanced effector function of Trm compared to Tcm (
      • Jiang X.
      • Clark R.A.
      • Liu L.
      • Wagers A.J.
      • Fuhlbrigge R.C.
      • Kupper T.S.
      Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.
      ), while others describe Trm as primarily serving an alarm function to efficiently recruit effectors to sites of reinfection (
      • Ariotti S.
      • Hogenbirk M.A.
      • Dijkgraaf F.E.
      • Visser L.L.
      • Hoekstra M.E.
      • JY Song, H
      • et al.
      Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert.
      ,
      • Schenkel J.M.
      • Fraser K.A.
      • Vezys V.
      • Masopust D.
      Sensing and alarm function of resident memory CD8(+) T cells.
      ). One study demonstrated Tcm alone are unable to provide efficient responses to reinfection with herpes (
      • Mackay L.K.
      • Stock A.T.
      • Ma J.Z.
      • Jones C.M.
      • Kent S.J.
      • Mueller S.N.
      • et al.
      Long-lived epithelial immunity by tissue-resident memory T (TRM) cells in the absence of persisting local antigen presentation.
      ). Cooperation of Trm with other recruited T cell populations has been indicated in cutaneous T cell lymphomas (
      • Watanabe R.
      • Gehad A.
      • Yang C.
      • Scott L.L.
      • Teague J.E.
      • Schlapbach C.
      • et al.
      Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.
      ). Our data support the role of autoreactive Trm as sentinel/alarm cells that work together with Tcm populations to maintain depigmentation during vitiligo. Further, we previously reported that blocking CXCL10 or CXCR3 not only prevented the progression of vitiligo, but also reversed stable disease after melanocytes were destroyed and Trm became established (
      • Rashighi M.
      • Agarwal P.
      • Richmond J.M.
      • Harris T.H.
      • Dresser K.
      • Su M.W.
      • et al.
      CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.
      ,
      • Richmond J.M.
      • Masterjohn E.
      • Chu R.
      • Tedstone J.
      • Youd M.E.
      • Harris J.E.
      CXCR3 depleting antibodies prevent and reverse vitiligo in mice.
      ). This suggested that Trm may not be sufficient for the memory observed in lesions.
      Our mouse model of vitiligo is driven predominantly by the PMEL T cell clone, and we previously reported that CD8–/– host mice develop vitiligo comparable to wild-type controls (
      • Richmond J.M.
      • Bangari D.S.
      • Essien K.I.
      • Currimbhoy S.D.
      • Groom J.R.
      • Pandya A.G.
      • et al.
      Keratinocyte-derived chemokines orchestrate T-cell positioning in the epidermis during vitiligo and may serve as biomarkers of disease.
      ). In contrast, our studies of human TCR Vβ usage revealed a highly polyclonal response, with private specificity across patients and even different clones in different lesions within the same patient. These data corroborate
      • Cheuk S.
      • Schlums H.
      • Gallais Serezal I.
      • Martini E.
      • Chiang S.C.
      • Marquardt N.
      • et al.
      CD49a Expression defines tissue-resident CD8+ T cells poised for cytotoxic function in human skin.
      , who reported high Vβ diversity among vitiligo patients. These findings are also interesting in light of previously reported alarm-related functions of Trm (
      • Ariotti S.
      • Hogenbirk M.A.
      • Dijkgraaf F.E.
      • Visser L.L.
      • Hoekstra M.E.
      • JY Song, H
      • et al.
      Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert.
      ,
      • Schenkel J.M.
      • Fraser K.A.
      • Vezys V.
      • Masopust D.
      Sensing and alarm function of resident memory CD8(+) T cells.
      ), and they may suggest that breadth of coverage is likely more important than clonal expansion. Future mechanistic studies evaluating the relative contributions of these clones, as well as human Trm versus Tcm pools, will need to be conducted.
      Our analysis of reporter PMEL indicated that they are able to sense antigen and produce IFN-γ and alarm chemokines in situ. Interestingly, 30% of dermal PMEL sensed self-antigen, despite the fact that melanocytes predominantly reside in the epidermis. This suggests that either Tcm sense an undefined melanocyte population in the dermis or they sense antigen cross-presented on phagocytes there. This is in contrast to a mouse model of experimental autoimmune encephalomyelitis, where a larger proportion (up to 80%) of the cells are exposed to cognate antigen in the brain (
      • Sasaki K.
      • Bean A.
      • Shah S.
      • Schutten E.
      • Huseby P.G.
      • Peters B.
      • et al.
      Relapsing-remitting central nervous system autoimmunity mediated by GFAP-specific CD8 T cells.
      ). However, in experimental autoimmune encephalomyelitis, autoantigens are present throughout brain tissue, whereas melanocyte stem cell reservoirs are confined to hair follicles. We suspect that Trm are exposed to regenerating melanocytes as they exit the follicle due to their proximity, as observed in confocal microscopy. Thus, only a small subset of Trm may be activated at a particular time. Furthermore, we hypothesize that in chronically depigmented mice, the melanocyte stem cell reservoirs become exhausted and therefore no longer emerge from hair follicles to stimulate Trm. This is based on our observations that PMELs in aged mice produce less IFN-γ and Nur77-GFP reporter compared to those with recent disease activity.
      All phenotypes of PMEL were capable of producing CXCL9 and CXCL10, but at different frequencies in different tissues. It is possible that these signals provide a chemokine network for Tcm to follow, similar to other studies that have shown small foci of CXCR3 ligands directing T cell migration to virus-infected tissues (
      • Ariotti S.
      • Beltman J.B.
      • Borsje R.
      • Hoekstra M.E.
      • Halford W.P.
      • Haanen J.B.
      • et al.
      Subtle CXCR3-dependent chemotaxis of CTLs within infected tissue allows efficient target localization.
      ). In the epidermis, all PMEL phenotypes were capable of producing chemokines, indicating the presence of memory populations that do not express the classical CD69 and CD103 markers, similar to observations in patients with cutaneous T cell lymphomas (
      • Watanabe R.
      • Gehad A.
      • Yang C.
      • Scott L.L.
      • Teague J.E.
      • Schlapbach C.
      • et al.
      Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.
      ), melanoma (
      • Malik B.T.
      • Byrne K.T.
      • Vella J.L.
      • Zhang P.
      • Shabaneh T.B.
      • Steinberg S.M.
      • et al.
      Resident memory T cells in the skin mediate durable immunity to melanoma.
      ), and HSV-1 (
      • Mackay L.K.
      • Rahimpour A.
      • Ma J.Z.
      • Collins N.
      • Stock A.T.
      • Hafon M.L.
      • et al.
      The developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of skin.
      ). Indeed, our experiments using CXCL9-, CXCL10-, CD44-, or CD103-deficient PMEL indicate that alternative pathways may compensate for epidermal recruitment, residence, and effector function, as recipients still developed vitiligo. In the case of chemokines, a caveat to our studies is that many serve redundant functions (reviewed in
      • Groom J.R.
      • Luster A.D.
      CXCR3 ligands: redundant, collaborative and antagonistic functions.
      ). Furthermore, the recipient mice were chemokine-sufficient, and could therefore recruit more PMELs through endogenous chemokine production. Taken together, our data support the model of Trm in autoimmunity as acting as sentinel/alarm cells that work together with Tcm to mediate disease (
      • Schenkel J.M.
      • Fraser K.A.
      • Vezys V.
      • Masopust D.
      Sensing and alarm function of resident memory CD8(+) T cells.
      ).
      FTY720 treatment and low-dose Thy1.1 depletion resulted in repigmentation in our model, indicating that the Trm coordinate Tcm responses to efficiently kill melanocytes. This is in contrast to what was observed in a model of cutaneous VV infection, as FTY720 treatment had no effect on viral clearance in memory hosts (
      • Jiang X.
      • Clark R.A.
      • Liu L.
      • Wagers A.J.
      • Fuhlbrigge R.C.
      • Kupper T.S.
      Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.
      ). A possible explanation for this is specific tuning of Trm responses in the context of different inflammatory environments. A viral infection induces a highly inflammatory environment, whereas regeneration of target cells occurs without inflammation. Nevertheless, targeting Tcm through S1P1 inhibitors or other similar drugs may be effective treatments for vitiligo and other autoimmune diseases, consistent with clinical efficacy of fingolimod in multiple sclerosis (
      • Kappos L.
      • Antel J.
      • Comi G.
      • Montalban X.
      • O'Connor P.
      • Polman C.H.
      • et al.
      Oral fingolimod (FTY720) for relapsing multiple sclerosis.
      ).

      Materials and Methods

      Mice

      All mice were housed in pathogen-free facilities at University of Massachusetts Medical School, and procedures were approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Age and sex-matched mice were used, and both male and female mice of all strains were tested to avoid sex bias. Replicate experiments were performed two to five times.
      KRT14-Kitl*4XTG2Bjl mice (The Jackson Laboratory, Bar Harbor, ME; stock no. 009687) were bred as heterozygotes and used as hosts for the vitiligo model. Thy1.1+ PMEL TCR transgenic mice (stock no. 005023) were used as donors. The following strains were bred to PMEL mice for use as T cell donors in these studies: GREAT (stock no. 017580), Nur77-GFP (stock no. 016617), CD103–/– (stock no. 006144), CD44–/– (stock no. 005085), CXCL9–/– (stock no. 030285), CXCL10–/– (stock no. 006087), and REX3 (provided by A. Luster, Massachusetts General Hospital). For the OT-1 VV-ovalbumin-GFP model, OT-1 mice (stock no. 003831) were used as donors.

      Vitiligo induction

      Vitiligo was induced as described previously (
      • Harris J.E.
      • Harris T.H.
      • Weninger W.
      • Wherry E.J.
      • Hunter C.A.
      • Turka L.A.
      A mouse model of vitiligo with focused epidermal depigmentation requires IFN-gamma for autoreactive CD8(+) T-cell accumulation in the skin.
      ). Briefly, PMEL CD8+ T cells from donor mice were negatively selected (Miltenyi Biotec, Bergisch Gladbach, Germany) from spleens according to the manufacturer’s instructions. One million T cells were injected intravenously into sublethally irradiated (500 rads) Krt14-Kitl* hosts, and were activated in vivo using intraperitoneal injection of 1 × 106 plaque-forming units of rVV-hPMEL (N. Restifo, National Cancer Institute, National Institutes of Health) (
      • Overwijk W.W.
      • Tsung A.
      • Irvine K.R.
      • Parkhurst M.R.
      • Goletz T.J.
      • Tsung K.
      • et al.
      gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand.
      ). For comparison to an irrelevant antigen, 1 × 106 purified CD8+ OT-1 T cells were injected intravenously into sublethally irradiated Krt14-Kitl* hosts, along with 1 × 106 plaque-forming units of rVV-ovalbumin (K. Rock, University of Massachusetts Medical School) in the same manner as the vitiligo model. Vitiligo score was objectively quantified at weeks 7–10 by an observer blinded to the experimental groups, as described previously (
      • Harris J.E.
      • Harris T.H.
      • Weninger W.
      • Wherry E.J.
      • Hunter C.A.
      • Turka L.A.
      A mouse model of vitiligo with focused epidermal depigmentation requires IFN-gamma for autoreactive CD8(+) T-cell accumulation in the skin.
      ). See Supplementary Methods online for details.

      Repigmentation experiments

      Vitiligo mice with >75% depigmentation and stable disease (weeks 10–20 post-vitiligo induction) were used for repigmentation studies. FTY720 (Cayman Chemical, Ann Arbor, MI) treatment was performed by intraperitoneal injection of 1 mg/kg FTY720 diluted in water or vehicle (water) three times weekly for the duration of the observation period (4 weeks), as described previously (
      • Chiba K.
      FTY720, a new class of immunomodulator, inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptors.
      ,
      • Murooka T.T.
      • Deruaz M.
      • Marangoni F.
      • Vrbanac V.D.
      • Seung E.
      • von Andrian U.H.
      • et al.
      HIV-infected T cells are migratory vehicles for viral dissemination.
      ). For low-dose Thy1.1 depletion, mice received one intraperitoneal injection of 3 μg Thy1.1 antibody (BD Biosciences, San Jose, CA) or phosphate buffered saline, as described previously (
      • Schenkel J.M.
      • Fraser K.A.
      • Vezys V.
      • Masopust D.
      Sensing and alarm function of resident memory CD8(+) T cells.
      ). Repigmentation analysis was performed with ImageJ software (National Institutes of Health, Bethesda, MD). Photos were taken of each individual mouse before treatment and again after treatment was completed. The images were converted into black and white and the change in pigment was quantified with ImageJ software, as described previously (
      • Agarwal P.
      • Rashighi M.
      • Essien K.I.
      • Richmond J.M.
      • Randall L.
      • Pazoki-Toroudi H.
      • et al.
      Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo.
      ).

      Study subjects

      Patient shave skin biopsies were collected following written informed consent under Institutional Review Board−approved protocols at University of Massachusetts Medical School by board-certified dermatologists. All samples were de-identified before use in experiments. Stable patients were defined as having no changes in their lesions over the previous 6 months, as well as the absence of confetti depigmentation, a recently described clinical sign of active vitiligo (
      • Sosa J.J.
      • Currimbhoy S.D.
      • Ukoha U.
      • Sirignano S.
      • O'Leary R.
      • Vandergriff T.
      • et al.
      Confetti-like depigmentation: a potential sign of rapidly progressing vitiligo.
      ). Non-lesional sites were selected as normal-appearing, non-depigmented skin when examined by Wood’s lamp at least 2 cm from the nearest depigmented macule. Patients were excluded from the study if they had received treatment within the previous 3 months.

      Flow cytometry

      Mouse tail skin and draining lymph nodes were harvested at the indicated times, as described previously (
      • Richmond J.M.
      • Strassner J.P.
      • Zapata Jr., L.
      • Garg M.
      • Riding R.L.
      • Refat M.A.
      • et al.
      Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo.
      ). All flow data were collected with an LSR II and were analyzed with FlowJo software (FlowJo LLC, Ashland, OR). Please see Supplementary Methods for additional information, and Supplementary Table S4 online for antibody clone information.

      TCR–Vβ Sequencing

      Peripheral blood mononuclear cells were isolated from heparinized blood via Ficoll density gradient centrifugation and flash-frozen. Epidermis was separated from dermis using 50 mg/ml Dispase II for 1 hour at 37°C. Epidermis was flash-frozen, and all samples were homogenized immediately prior to DNA extraction using a Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). DNA samples from peripheral blood mononuclear cells and epidermis were sent to immunoSEQ (Adaptive Biotechnologies, Seattle, WA), and were amplified and sequenced on-site using the hsTCRB kit and an Illumina MiSeq instrument (
      • Carlson C.S.
      • Emerson R.O.
      • Sherwood A.M.
      • Desmarais C.
      • Chung M.W.
      • Parsons J.M.
      • et al.
      Using synthetic templates to design an unbiased multiplex PCR assay.
      ,
      • Robins H.S.
      • Campregher P.V.
      • Srivastava S.K.
      • Wacher A.
      • Turtle C.J.
      • Kahsai O.
      • et al.
      Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells.
      ) (Adaptive Biotechnologies). Data were analyzed with the immunoSEQ Analyzer, and data have been submitted to the immunoSEQ public database (http://doi.org/10.21417/B7V884).

      Statistics

      All statistical analyses were performed with GraphPad Prism software (La Jolla, CA). Dual comparisons were made with unpaired Student t test, and groups of three or more were analyzed by analysis of variance with Tukey’s or Dunnett’s post-tests. A P value < 0.05 was considered significant.

      Conflict of Interest

      JMR, JPS, and JEH are inventors on patent application #62489191, "Diagnosis and Treatment of Vitiligo," which covers targeting IL-15 and Trm for the treatment of vitiligo. JMR and JEH are inventors on patent application #15/851,651, "Anti-Human CXCR3 Antibodies for the Treatment of Vitiligo," which covers targeting CXCR3 for the treatment of vitiligo. The remaining authors state no conflict of interest.

      Author Contributions

      JMR and JEH designed the study. JMR, JPS, MR, PA, MG, KIE, LSP performed the experiments, JMR, and JEH drafted the manuscript, and all authors critically revised the manuscript.

      Acknowledgments

      We thank clinic patients of JEH for donating tissue, and C. Hartigan for patient management. We thank B. J. Longley for Krt14-Kitl* mice; A.D. Luster for REX3 mice; J.M. Farber for CXCL9–/– mice; S. Swain for OT-1 mice; K. Rock for rVV-ovalbumin; and N. Restifo for rVV-hPMEL. We thank M. Damiani, V. Azzolino, and M. Frisoli of the Harris Lab and A. Leporati of the UMass Infectious Disease Department for technical assistance. Supported by a research grant and a Calder Research Scholar Award from the American Skin Association, a Career Development Award from the Dermatology Foundation, and an immunoSEQ Young Investigator Award (to JMR), the National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the National Institutes of Health , under award numbers AR061437 and AR069114 , and research grants from the Kawaja Vitiligo Research Initiative, Vitiligo Research Foundation, and Dermatology Foundation Stiefel Scholar Award (to JEH), and the National Institutes of Health Training Grant AI095213 (to JPS and KIE). Flow cytometry and confocal microscopy equipment used for this study is maintained by the University of Massachusetts Medical School Flow Cytometry Core Facility and Morphology Core Facility. The University of Massachusetts Center for Clinical Research was responsible for blood and biopsy collection and is supported by National Institutes of Health Clinical and Translational Sciences Award UL1TR000161 . Mice were obtained through respective institutions under a material transfer agreement and Vβ sequencing data have been submitted to the immunoSEQ public database (http://doi.org/10.21417/B7V884).

      Supplementary Material

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