JAK inhibitors reverse vitiligo in mice but do not deplete skin resident memory T cells

To the Editor,

Vitiligo is an autoimmune skin disease presenting with distinct, patchy areas of depigmentation that can appear anywhere on the body. These white patches occur when autoreactive, cytotoxic CD8+ T cells promote elimination of melanocytes, the pigment-producing cells of the epidermis (Frisoli et al. 2020). Vitiligo affects approximately 1% of the global population equally across races and sexes, and frequently presents before the age of 20 (Taieb and Picardo 2009).

Currently, there are no U.S Food and Drug Administration (FDA)-approved medical treatments to promote repigmentation in vitiligo, while topical immunosuppressants and phototherapy are moderately effective when used off-label (Frisoli et al. 2020). Recent studies report efficacy of the Janus kinase inhibitors (JAKi) tofacitinib and ruxolitinib in vitiligo patients through case reports and case studies, as well as ongoing clinical trials (Craiglow and King 2015; Harris et al. 2016; Kim et al. 2018; Liu et al. 2017). However, studies also reported rapid relapse of disease after stopping both conventional treatments (Cavalié et al. 2015) as well as JAKi (Harris et al. 2016; Liu et al. 2017).

We and others recently reported that autoreactive resident memory T cells (Trm) form in the lesions of vitiligo patients, and that these cells are responsible for disease relapse (Boniface et al. 2018; Cheuk et al. 2017; Richmond et al. 2018b). Trm function to recruit autoreactive T cells from the circulation, which participate in killing repigmenting melanocytes (Richmond et al. 2018a). Treatments that inhibit Trm function, but do not remove them from the skin, are not durable (Riding and Harris 2019). Therefore, we hypothesized that Trm numbers are largely unaffected by JAKi treatment based on the lack of durable responses in patients.

To test our hypothesis, we used JAKi in our mouse model of vitiligo, which is induced by adoptive transfer of melanocyte-specific CD8+ T cells, also known as premelanosome protein-specific T cells (PMEL), into recipient hosts with pigmented skin (Harris et al. 2012). We administered tofacitinib or ruxolitinib orally once daily to hosts prior to disease onset (prevention) or after stabilization (reversal) and measured their effect on extent of disease, as well as T cell populations within the epidermis and dermis by flow cytometry.

In the prevention model, hosts were treated daily with vehicle or JAKi from week two to seven post-induction of vitiligo (Fig 1A). We observed a mild, but statistically insignificant, decrease in the vitiligo score in hosts treated with ruxolitinib (p=0.4255), and a larger, statistically significant decrease in vitiligo score with tofacitinib treatment (p= 0.0037, n=36 vehicle, 21 tofacitinib, & 13 ruxolitinib mice; Fig 1B-C). Whether this indicates that ruxolitinib will be less effective at controlling active disease in humans is not clear, but should be monitored in clinical studies.

Figure 1. JAKi reduce vitiligo and prevent epidermal accumulation of autoreactive T cells in mice.

(a) Prevention experiments were completed according to the schedule above. (b) Sample photographs from mouse tails after 5 weeks of treatment. (c) Clinical scores of vitiligo in vehicle, tofacitinib (tofa), and ruxolitinib (rux)-treated mice. Total number of premelanosome protein specific T cells (PMEL) in the epidermis (d) and dermis (e) by flow cytometry (each dot represents one animal; n=36 vehicle, 21 tofacitinib and 13 ruxolitinib pooled from at least 3 separate experiments).

To understand how the JAKi were impacting disease, we examined the number of immune cells in the skin using flow cytometry. We found that hosts treated with either JAKi had fewer, though not statistically significant, PMELs in the epidermis (Fig 1D, tofacitinib vs vehicle p=0.1076, ruxolitinib vs vehicle p=0.2779). Ruxolitinib, but not tofacitinib, appeared to reduce PMEL in the dermis as well (Fig 1E, tofacitinib vs vehicle p=0.5861, ruxolitinib vs vehicle p=0.0655).

In the reversal model, we treated hosts twelve weeks post-induction with JAKi for eight more weeks (Fig 2A). Both tofacitinib and ruxolitinib significantly reversed disease as compared to vehicle controls (Fig 2B-C, p =0.0010 and 0.0071 respectively, n=13 vehicle, 14 tofacitinib and 8 ruxolitinib). Interestingly, in contrast to the prevention model, JAKi did not decrease the overall number of PMEL in the epidermis (Fig 2D, p=0.2032 and p=0.3958), dermis (Fig 2E, p= 0.1539 and p=0.5399), or specifically the Trm subpopulation, marked by expression of CD103 and CD69 (Fig 2F, p=0.1189; p=0.228). Thus, Trm populations remained in the skin during reversal, even after significant repigmentation was observed.

Figure 2. JAKi reverse depigmentation in vitiligo but do not clear Trm in the skin.

(a) Reversal experiments were completed according to the schedule above. (b) Photographs of mouse tails at the beginning and end of treatment after 8 weeks. (c) Change in tail pigment was calculated using before and after photos of each mouse. PMEL count within the epidermis (d) and dermis (e) was assessed at the end of treatment (each dot represents one animal; n=13 vehicle, 14 tofacitinib, and 8 ruxolitinib). (f) Resident memory T cells (Trm) were stained in the epidermis and quantified after treatment (n=6 vehicle, 8 tofacitinib, and 8 ruxolitinib pooled from 2 separate experiments).

Taken together, our data suggest that JAKi may prevent accumulation of T cells in the skin during disease progression, but do not affect T cell numbers once established. The observed efficacy of treatments to promote reversal of disease may instead be through inhibition of T cell function within the skin, rather than by decreasing their numbers. Both tofacitinib and ruxolitinib inhibit signaling through IFN-g (Damsky and King 2017), a cytokine produced by Trms and required for recruitment of cytotoxic cells from the circulation (Richmond et al. 2018a). While IFN-y signaling through JAKs appears to be necessary for depigmentation, it is dispensable for Trm maintenance in the skin, which supports clinical observations that JAK inhibition does not result in durable treatment responses.