Resident memory and recirculating memory T cells cooperate to maintain disease in a mouse model of vitiligo

Abstract

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 IFNγ, 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.

Introduction

Vitiligo is caused by CD8+ T cells that target melanocytes for destruction (van den Boorn et al., 2009), resulting in patchy depigmentation that is disfiguring and distressing to patients (Alikhan et al., 2011; Frisoli and Harris, 2017; Richmond and Harris, 2017; Rodrigues et al., 2017). Depigmentation typically recurs rapidly at the same location after therapy is stopped (Cavalie et al., 2015), 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 et al., 2017; Cheuk et al., 2017; Richmond et al., 2018).

Normally, Trm remain in non-lymphoid tissues to provide tissue surveillance against pathogens (Clark et al., 2012; Gebhardt et al., 2009; Jiang et al., 2012; Schenkel et al., 2013; Watanabe, 2015; Zhu et al., 2013). Upon entering tissues, Trm upregulate CD69 and CD103, downregulate the chemokine receptors S1P₁ and CCR7 to prevent recirculation, and set up residence (Mackay et al., 2013; Skon et al., 2013). We have recently published a strategy for depleting Trm cells in vitiligo by blocking IL-15 signaling (Richmond et al., 2018). 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 et al., 1998), and these cells exhibited skin-homing potential as determined by cutaneous lymphocyte antigen expression (CLA+). 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 largely depends upon signals received in situ (Seidel et al., 2018). 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 et al., 1999). Therefore, the questions that remain are: (1) are Trm sufficient for maintaining disease? and (2) 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 et al., 2010; Overwijk et al., 2003; Overwijk et al., 1998). Our model uses the adoptive transfer of TCR transgenic T cells recognizing the human melanocyte antigen pre-melanosome protein (PMEL) into recipient mice with epidermal melanocytes (Agarwal et al., 2015; Harris et al., 2012; Rashighi et al., 2014; Richmond et al., 2017a; Richmond et al., 2017b; Richmond et al., 2018, Riding et al., 2018). 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 et al., 2011; Frisoli and Harris, 2017; Richmond and Harris, 2017; Rodrigues et al., 2017).

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 et al., 2013), 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 IFNγ 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 (Table S1 & S2). We also assessed expression of other classical T cell memory 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) (Table S1). Epidermal T cells had low CD62L, whereas peripheral T cells expressed it in variable amounts that was highest in spleen and lymph node (Table S1). As we have previously reported, PMEL express high levels of the CD122 chain of the IL-15 receptor (Richmond et al., 2018).

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 OVA (OT-1; (Hogquist et al., 1994)). We induced immune responses with recombinant vaccinia virus (VV) expressing pre-melanosome protein (VV-PMEL) and PMEL, or ovalbumin (VV-OVA; (York et al., 2006)) and OT-1 T cells (Fig 1A). Only PMEL induced disease in mice and established Trm in the epidermis, whereas OT-1 did not despite engrafting in the lymph node (Fig 1B–F). Thus, autoreactive Trm are generated directly in the skin where autoantigen is expressed during vitiligo.

Fig. 1. Melanocyte-specific, but not ovalbumin-specific, T cells, form Trm in the epidermis and secrete IFNγ 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 & CD103 staining used to identify Trm, and (G) IFNγ reporter expression by all GREAT PMEL and by Trm GREAT PMEL. (n=4 mice/group; representative experiment of two shown. Student’s t tests significant as indicated, *p<0.05.) (H) Frequency of epidermal PMEL producing IFNγ and mouse disease scores over time. (n= 2–8 mice per timepoint pooled from 4 separate experiments)

To assess their functional capacity, we bred PMEL donor mice to IFNγ reporter mice (IFN-gamma reporter with IRES poly A tail, or GREAT mouse (Reinhardt et al., 2009)). We first validated that the GREAT reporter accurately represented IFNγ expression by performing co-staining of IFNγ and GFP reporter following in vitro stimulation with anti-CD3/CD28 (Fig S1; method from (Groom et al., 2012)). We then transferred naïve GREAT PMEL in vivo in our vitiligo model and found that PMEL persisted in the epidermis and expressed IFNγ, 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 (Fig 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 (Fig 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 V-beta usage

Melanocyte-specific TCR V-beta usage has successfully been performed on T cells from melanoma patients (Jager et al., 2000); 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 3 stable vitiligo patients, as well as PBMCs, for TCR V-beta sequencing analysis (see Table S3 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 (Fig 2A & B; see Fig S2 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 (Fig 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 (Fig 2D). Thus, TCR V-beta usage is quite heterogeneous, even within a single patient.

Fig. 2. Human Trm from vitiligo patient shave biopsies are polyclonal and have private specificity for TCR Vbeta useage.

(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 3 tissue sites (one-way ANOVA with Tukey’s post tests p<0.0001). (C) TCR Vbeta useage of lesional, nonlesional skin and PBMCs from a vitiligo patient (corresponding Vbetas 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.

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 et al., 2013; Mrass et al., 2008). In order to determine whether these molecules were required on PMEL to initiate or maintain disease, we bred PMEL mice to CD103−/− (Schon et al., 1999) and CD44−/− (Protin et al., 1999) and used these cells to induce vitiligo (confirmation of knockouts in Fig S3). Single transfers of both CD103−/− and CD44−/− PMEL were capable of inducing vitiligo with little effect on epidermal cell numbers or disease score (Fig S4). Numbers of phenotypically different skin memory T cells, namely CD44-CD69+CD103+ and CD69+CD103-, were similar in recipients (Fig S4 D & H). Co-transfers of WT and CD103−/− or CD44−/− PMEL revealed no significant differences in the epidermis, though there was a trend towards more WT cells (Fig S5). In accordance with prior studies in virus and melanoma models (Mackay et al., 2013; Malik et al., 2017), CD44 and CD103 are not required for the 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 which 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 et al., 2011) and we validated the half-life of the reporter in our PMEL T cells (Fig S6). We then used Nur77-GFP PMEL to induce vitiligo, and examined mice 8 weeks post-induction when epidermal Trm are established (Richmond et al., 2018). 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 (Fig 3A & B). 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 (Fig 3C & D), whereas in the dermis CD69+CD103+ PMEL expressed the highest levels of Nur77-GFP reporter (Fig 3E & F). 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.

Fig. 3. Nur77-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 3 separate experiments; one-way ANOVA p= 0.0272, Tukey’s posttests epidermis vs dermis *p=0.0253). (C) Sample flow plots and (D) quantification of frequency of epidermal Nur77-GFP+ cells in the indicated parental Trm phenotyping gates. (repeated measures/matched one-way ANOVA without sphericity p=0.0676, Tukey’s posttests ns) (E) Sample flow plots and (F) quantification of frequency of dermal Nur77-GFP+ cells in the indicated parental Trm phenotyping gates. (repeated measures/matched one-way ANOVA without sphericity p=0.0384, Tukey’s posttests ns, trending towards significance CD69-CD103+ vs CD69+CD103+ p=0.0709).

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 et al., 2012), to determine whether they were capable of secreting chemokines to recruit Tcm to the skin (Fig 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 (Fig 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 (Fig 4C–F).

Fig. 4. REX3 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 ANOVA p=0.0023; repeated measures/matched samples compared for each tissue from every mouse Tukey’s posttests ns for CXCL9; for CXCL10 *p=0.032 epidermis vs dermis, ****p<0.0001 epidermis vs LN, **p=0.001 dermis vs LN; n=10 pooled from 3 separate experiments). (C) Sample flow plots and (D) quantification of frequency of epidermal REX3+ cells in the indicated parental Trm phenotyping gates (repeated measures/matched one-way ANOVA without sphericity p=0.294 for CXCL9, Tukey’s posttests ns; p=0.0382 for CXCL10, Tukey’s posttests CD69-CD103+ vs CD69+CD103- *p=0.0364) (E) Sample flow plots and (F) quantification of frequency of dermal REX3+ cells in the indicated parental Trm phenotyping gates. (repeated measures/matched one-way ANOVA without sphericity p=0.4877 for CXCL9, Tukey’s posttests ns; p= 0.0806 for CXCL10, Tukey’s posttests CD69-CD103+ vs CD69+CD103+ **p=0.0016).

To determine the potential functional role of T cell-derived chemokine production in vitiligo, we bred PMEL mice to CXCL9 (Park et al., 2002) and CXCL10-deficient animals (Dufour et al., 2002) 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 WT PMEL controls (Fig S7). Despite this, CXCL10-deficient PMELs appear to engraft in the lymph node at a higher rate than WT or CXCL9-deficient PMELs (Fig 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 (Fig S8).

Persistence of depigmentation in vitiligo requires Tcm

Since 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, 2005; Murooka et al., 2012; Pham et al., 2007; Schwab et al., 2005) 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 (Fig 5A–E). In accordance with other studies (Pinschewer, 2000), PMEL Trm were still present in the skin after treatment (Fig 5F & G). PMEL numbers in other tissues in FTY720 treated mice were the same as vehicle controls (Fig 5H & I), an expected result based on previous studies demonstrating that FTY720 treatment locks cells in tissues (Murooka et al., 2012; Pham et al., 2007). Therefore, we also used low-dose Thy1.1 depleting antibody to selectively deplete Tcm (Schenkel et al., 2013). These mice also repigmented, supporting the FTY720 data and implicating Tcm as necessary for vitiligo maintenance (Fig 5J–N). Further, Trm were largely intact (Fig 5O & P), whereas lymph node and dermal PMEL populations were significantly reduced (Fig 5Q & R). Thus, Tcm contribute to sustained melanocyte killing during vitiligo maintenance, and Trm are not sufficient effectors for this function.

Fig. 5. Recirculating PMEL 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 PMEL 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+ PMEL 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 PMEL 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+ PMEL normalized to 10,000 live singlet cells in the lymph node and (R) dermis. (student’s t tests significant as indicated; each dot represents one animal pooled from 3 separate experiments).

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 et al., 2012), while others describe Trm as primarily serving an alarm function to efficiently recruit effectors to sites of reinfection (Ariotti, 2014; Schenkel et al., 2013). One study demonstrated Tcm alone are unable to provide efficient responses to reinfection with herpes (Mackay et al., 2012). Cooperation of Trm with other recruited T cell populations has been indicated in cutaneous T cell lymphomas (Watanabe, 2015). 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 et al., 2014; Richmond et al., 2017b). 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 WT controls (Richmond et al., 2017a). In contrast, our studies of human TCR V-beta 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 et al., 2017, which showed high V-beta diversity among vitiligo patients. These findings are also interesting in light of previously reported alarm-related functions of Trm (Ariotti et al., 2014, Schenkel et al., 2013), and they may suggest that breadth of coverage is likely more important rather 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 IFNγ 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 Tcm sense either an undefined melanocyte population in the dermis, or that they sense antigen cross-presented on phagocytes there. This is in contrast to a mouse model of EAE, where a larger proportion (up to 80%) of the cells are exposed to cognate antigen in the brain (Sasaki et al., 2014). However, in EAE 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 IFNγ 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 et al., 2015). 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, 2015), melanoma (Malik et al., 2017) and HSV-1 (Mackay et al., 2013). 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 and Luster, 2011)). Furthermore, the recipient mice were chemokine-sufficient, and could therefore recruit more PMEL 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 et al., 2013).

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 vaccinia virus infection, as FTY720 treatment had no effect on viral clearance in memory hosts (Jiang et al., 2012). 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 et al., 2006).

Materials and Methods

Mice.

All mice were housed in pathogen-free facilities at UMMS, and procedures were approved by the UMMS Institutional Animal Care and Use Committee and in accordance with the National Institutes of Health (NIH) 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 gender bias. Replicate experiments were performed two to five times.

KRT14-Kitl*4XTG2Bjl mice (The Jackson Laboratory, 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-OVA-GFP model, OT-1 mice (stock no. 003831) were used as donors.

Vitiligo induction.

Vitiligo was induced as previously described (Harris et al., 2012). Briefly, PMEL CD8+ T cells from donor mice were negatively selected (Miltenyi Biotec) 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 (pfu) of rVV-hPMEL (N. Restifo, National Cancer Institute, NIH; (Overwijk et al., 1998)). 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 pfu of rVV-OVA (K. Rock, UMass Medical School) in the same manner as the vitiligo model. Vitiligo score was objectively quantified at week 7–10 by an observer blinded to the experimental groups as described previously (Harris et al., 2012). Please see supplemental methods for details.

Repigmentation experiments.

Vitiligo mice with >75% depigmentation and stable disease (week 10–20 post-vitiligo induction) were used for repigmentation studies. FTY720 (Cayman chemical) treatment was performed by i.p. injection of (1) 1mg/kg FTY720 diluted in water or (2) vehicle (water) three times weekly for the duration of the observation period (4 weeks) as previously described (Chiba, 2005; Murooka et al., 2012). For low-dose Thy1.1 depletion, mice received one i.p. injection of (1) 3μg Thy1.1 antibody (BD Biosciences) or (2) PBS as previously described (Schenkel et al., 2013). Repigmentation analysis was performed with ImageJ. 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 Image J software as previously described (Agarwal et al., 2015).

Study subjects.

Patient shave skin biopsies were collected following written informed consent under IRB-approved protocols at UMMS 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 et al., 2015). 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 three months.

Flow cytometry.

Mouse tail skin and draining lymph nodes were harvested at the indicated times as previously described (Richmond et al 2018). All flow data were collected with an LSR II and were analyzed with FlowJo software. Please see supplemental methods for additional information, and Table S4 for antibody clone information.

TCR-V-beta Sequencing.

PBMCs were isolated from heparinized blood via Ficoll density gradient centrifugation and flash-frozen. Epidermis was separated from dermis using 50mg/mL Dispase II for 1h at 37°C. Epidermis was flash-frozen, and all samples were homogenized immediately prior to DNA extraction using a Qiagen DNeasy Blood & Tissue Kit. DNA samples from PBMCs and epidermis were sent to immunoSEQ, and were amplified and sequenced on-site using the hsTCRB kit and an Illumina MiSeq instrument (Carlson et al., 2013; Robins et al., 2009) (Adaptive Biotechnologies, Seattle, WA). 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. Dual comparisons were made with unpaired Student’s t test, and groups of three or more were analyzed by ANOVA with Tukey’s or Dunnett’s post-tests. P values < 0.05 were considered significant.

Supplementary Material

1

Materials & Methods

Fig. S1. Validation of GREAT reporter in PMEL T cells.

Fig. S2. Gating strategy for human flow data.

Fig. S3. Validation of CD44 and CD103 deficient PMEL T cells.

Fig. S4. PMEL do not need CD44 or CD103 to establish vitiligo in mice.

Fig. S5. Cotransfer experiments for WT and CD44 or CD103-deficient PMEL T cells.

Fig. S6. Validation of Nur77-GFP reporter in PMEL T cells.

Fig. S7. CXCL9- and CXCL10-deficient T cells induce vitiligo in recipient mice, but recruit fewer PMEL T cells to the epidermis.

Fig. S8. In situ visualization of reporter PMEL T cells in vitiligo mouse ears.

Table S1. Frequencies of phenotype characteristics of PMEL in different tissues in the vitiligo mouse model.

Table S2. Vitiligo patient characteristics used for V-beta sequencing.

Table S3. Antibodies used for these studies.

References

Abbreviations used:

PMEL

premelanosome protein-specific T cell

Trm

resident memory T cell

Tcm

recirculating memory T cell

OT-1

ovalbumin-specific T cell

GREAT

IFN-gamma reporter with IRES poly A tail

REX3

reporting the expression of CXCR3 ligands