Resident Memory T Cells in Autoimmune Skin Diseases

1Tissue resident memory T cells (TRM) are long-lived lymphocytes that reside in tissues and develop after a T cell-mediated immune response is initiated. Subpopulations of memory T cells can recirculate through the blood and lymphoid organs T recirculating memory cells (TRCM), though evidence suggests that true TRM have a reduced capacity for recirculation. Both CD4+ and CD8+ TRM have been described in skin and other tissues including the mucosa, lung, brain, GI tract and pancreas. Because of the very different locations that TRM are found, they are an extremely diverse population, showing specializations for their resident tissue. Mouse models reveal that TRMs function as sentinel-alarm cells that secrete cytokines and chemokines upon encountering their cognate antigen, with some infections such as vaccinia virus requiring TRM for optimal viral clearance. There is mounting evidence that TRM work together with other effector and memory T cell populations to provide tissue surveillance and clear infections.

2Compared to TRM in other tissues, skin TRM persist for years and express skin-specific homing antigens, such as cutaneous lymphocyte antigen (CLA) and CCR8. In addition, certain skin retention markers, including CD103 and CD69, are upregulated. Mouse models have demonstrated that TRM require IL-15 and TGFβ for their differentiation in the skin, and human TRM express their receptors. The tissue-residency program that ensues involves downregulation of chemokine receptors involved in recirculation, including CCR7 and S1P1, as well as upregulation of key integrins, cytokine/growth factor receptors, and signaling molecules including CD69, CD103, CD49a, CD122 and PD-1, a pattern that is conserved in both mice and humans. Skin TRM survival is dependent upon IL-15 and fatty acid metabolism ( Figure 1 ).

3In addition to anti-viral responses, TRM also develop in the context of xenobiotic and/or toxin exposure, allergies and autoimmune diseases. For example, fixed drug eruption is thought to be mediated by TRM. Type IV hypersensitivity to poison ivy, other contact dermatitis irritants or allergens induces TRM development. Healthy skin donors exhibit autoreactive T cell clones, which are thought to provide immune surveillance against tumors. However, in autoimmune skin conditions, environmental and genetic factors precipitate inappropriate activation of autoreactive T cells and promote onset of autoimmunity. Further, a reduced ratio of Tregs to TRM and T effectors and/or reduced functionality of Tregs in skin tissue tips the balance towards development of autoimmunity, which may become recalcitrant or relapse following treatment cessation due to the persistence of autoreactive TRM. In this minireview, we will focus on TRM in autoimmune skin diseases, including discussions of how they may be targeted for durable treatment options.

4Vitiligo is characterized by distinct patchy white spots present on patients’ skin, which are caused by CD8+ T cells inappropriately targeting melanocytes for destruction. Many studies have implicated both genetic and environmental factors are important in the pathogenesis of vitiligo. Familial clustering of vitiligo suggested that there was a genetic component before genetic studies were available. Alkhateeb et al. reported in 2003 that approximately 20% of people with vitiligo reported at least one affected first-degree relative. Since then, genome-wide association studies (GWAS) have identified multiple genes associated with a higher risk for developing vitiligo, including genes involved in both innate and adaptive immunity. It is now recognized that melanocytes in vitiligo patients have a reduced ability to manage cellular stress, making them more vulnerable to environmental stressors such as UV radiation and certain chemicals. Cytokines and danger associated molecules released in response to states of high stress in the skin attract and activate immune cells. In addition, the IFNγ-CXCR3 pathway has been determined to be critical to the migration of autoreactive CD8+ T cells in vitiligo in mice and humans, and correlates with disease progression and severity. While treatments are available for vitiligo patients, the white spots rapidly reappear in the same location if therapy is stopped, demonstrating that memory persists in the skin even after treatment. For example, JAK/STAT signaling is recognized as a key mediator in many of the effector functions of TRM cells, including the production of cytokines and inflammation inducers. JAK inhibitors interrupt the IFNγ signaling pathway and cause skin repigmentation; however, depigmentation recurs when treatment is stopped. Azzolino et al. recently reported that established TRM numbers in skin in a mouse model of vitiligo are not affected by Jak inhibitors, thus providing an explanation for why JAKi do not provide durable treatment responses.

5T cells in vitiligo patients react to melanocyte self-antigens including gp100/Pmel-17, melan-A/MART-1, tyrosinase and tyrosinase-related proteins 1 & 2. Recent studies have characterized melanocyte-specific CD8+ TRMs from patients with active and stable vitiligo using blister biopsies and punch biopsies. Approximately 80% of the melanocyte-specific CD8+ T cells express TRM markers, including CD69 single positive cells (which may be actively differentiating, or recirculating cells) and CD69/CD103 double positive cells. CD49a, a marker that distinguishes a subset of CD8+ CD103+ T cells with the cytotoxic potential to produce IFNγ, is expressed in half of epidermal and less than a quarter of dermal CD8+ CD103+ T cells in patients with vitiligo. Comparatively, in healthy skin, it is expressed by one third of epidermal and less than 5% of dermal CD8+ CD103+ T cells. In all patients with vitiligo, these cells were enriched in the skin; moreover, in patients with active disease, there was a significant increase in the number of T cells as compared to those with stable disease, particularly CD69+. Boniface et al. found co-expression of CXCR3 on CD69+CD103+ melanocyte-specific T cells in vitiligo skin at a higher frequency than psoriasis or healthy skin. CD8+ TRM are also present in depigmenting lesions in post-melanoma-immunotherapy-associated vitiligo.

6We examined factors required for maintenance of these autoreactive TRM in the skin during vitiligo, with the hope of identifying new durable treatments targeting autoreactive TRM. Based on Mackay et al, which demonstrated TRM require IL-15 for their differentiation, we tested whether blocking this cytokine pathway could deplete TRM. Treating mice with anti-CD122 antibodies, which block IL-15 signaling and thus TRM proliferation and survival, provided long-lasting repigmentation ( Figure 2A ). Other labs have mapped additional roles IL-15 plays in vitiligo. Chen et al. demonstrated that oxidative stress can result in increased IL-15 transpresentation on CD215 by keratinocytes. Jacquemin et al. demonstrated that IL-15 can induce NKG2D expression on effector memory CD8+ T cells, which, based on our FTY720 blockade data demonstrating that TRM are not sufficient for maintaining depigmentation in mice, supports a role for effector memory T cells as the melanocyte killers. IL-15 blocking antibodies are now being explored as a potential durable therapy for vitiligo patients (NCT04338581; Table S1 ).

7Cutaneous lupus erythematosus (CLE) is a spectrum of autoimmune skin diseases encompassing several clinical subtypes, all of which are characterized by interface dermatitis, or inflammation at the dermal-epidermal junction. The most well-known manifestation of CLE is the characteristic erythematous malar rash, which is associated with systemic lupus erythematosus (SLE). Other CLE subtypes can present without any systemic involvement, with different entities conferring different levels of risk of developing SLE. Genetic factors and environmental stressors both contribute to the etiology of CLE ( Figure 2B ).

8The T cell specificities in CLE skin are largely unknown, but SLE studies identified T cells reactive to nucleosomes/histones, which can induce anti-dsDNA antibody production. A recent study characterized antigen specificities of T cells in the kidney in lupus nephritis patients, and found that they recognize SmD1, RNP70, histone, Ro, La, and nuclear antigen. A population of CD8+ CD103+ tissue-resident memory T cells was identified and correlated positively with disease severity in patients. Interestingly, tofacitinib, a JAK3>1>>2 inhibitor, was effective in preventing disease progression in mouse models of lupus nephritis and suppressed the development of CD8+ TRM cells. Thus, the microenvironment that the TRM cells create in lupus nephritis appears to be crucial for disease progression.

9Many studies have shown aberrant T cell signaling contributes to the pathogenesis of CLE, including expression of cytotoxic markers characteristic of T cell function. However, it is only recently that TRM cells have been linked to disease progression: In the skin, increased TRM were found in CLE patients refractory to antimalarials. JAK/STAT inhibitors have also demonstrated efficacy in treating CLE: In a case study of a patient with chilblain lupus, a subtype of CLE, the JAK 1/2-kinase inhibitor ruxolitinib provided a rapid response. When ruxolitinib was discontinued, the skin lesions re-appeared, but upon re-starting the drug they resolved completely. Thus, similar to vitiligo studies, TRM in CLE are not affected permanently by Jak inhibitors, and disease recurs once treatment is stopped. Examination of skin biopsies taken from the patient revealed that ruxolitinib inhibited the production of CLE-typical chemokines, specifically CXCL10, supporting the idea that TRM cytokine production and downstream signaling drives recruitment of recirculating cytotoxic cells to the skin, as was demonstrated in vitiligo mouse models described above. Another study identified IL-17+ cells by immunohistochemistry; a trial assessing IL-17 blockade for discoid lupus erythematosus is recruiting ( Table S1 ).

10Psoriasis is a chronic and relapsing inflammatory skin disease mediated by T cells. As in other autoimmune skin diseases, the relapsing nature of psoriasis suggested the presence of immune memory in the skin. Using a humanized mouse model of psoriasis, transplantation of unaffected skin from a patient with psoriasis was grafted onto mice deficient in type I and type II interferons and the recombination activating gene 2, (IFNG-/- IFNAR-/- RAG2-/-) and the skin graft spontaneously developed psoriasis. Thus, the immune cells already resident in the grafted skin were sufficient for inducing disease. Moreover, E-selectin inhibitors are not efficacious in preventing psoriasis, further supporting that nonmigratory immune cells can mediate disease. However, the question of whether TRM are sufficient, or whether they also require TRCM, has not yet definitively been addressed, because the resident immune cells in skin graft experiments may have given rise to TRCM, and there may be redundant pathways in addition to E-selectin that mediate skin recruitment in psoriasis.

11Cheuk et al. demonstrated that the marker CD49a identifies two distinct populations of CD8+ CD103+ TRM cells. While vitiligo disease is driven by CD49a+ TRM cells producing IFNγ, CD49a- TRM cells in psoriasis mediate disease through the production of interleukin-17 (IL-17), ( Figure 2C ). The identification of CD49a- IL-17+ T cells as critical mediators of disease fits with the previous success of anti-IL-17 drugs that mediate successful amelioration of disease (UptoDate). The FDA has approved several monoclonal antibodies to IL-17 for the treatment of psoriasis: tildrakizumab, guselkumab, risankizumab, secukinumab, ixekizumab, and brodalamub ( Table S1 ). Scientists have also developed drugs to target IL-23, a cytokine produced by myeloid dendritic cells to activate IL-17-producing CD49a- TRM cells as well as non-TRM IL-17-producing cells. Nevertheless, IL-17 blockade may require long-term administration, as it is blocking a molecule made by the TRM rather than targeting TRM themselves. In a clinical study of ten patients with psoriasis receiving secukinumab for 24 weeks, Fujiyama et al. demonstrated that while there was a significant decrease in CD8+ CD103+ cells in lesional skin, there was only a slight decrease in CD8+ CD103+ CD49- cells, suggesting that TRM cells are preserved.

12While CD8+ CD103+ TRM cells make up the majority of the T cell infiltrate in psoriatic lesions, CD4+ TRM cells are also present in low numbers in the epidermis and dermis. Like CD8+ TRM, CD4+ TRM are identified by the marker CD69+. However, unlike CD8+ TRM, CD4+ TRM cells show variability in the expression of CD103, and the majority of CD4+ TRM do not express it. CD4+ TRM were further distinguished from CD8+ TRM cells by Cheuk et al, who demonstrated that epidermal CD4+ TRM cells in resolved psoriatic lesions produced IL-22 while CD8+ T cells mainly produced IL-17A. IL-22 is a pro-inflammatory cytokine that promotes the expression of multiple chemokines in the skin. In addition, through the STAT3 pathway, IL-22 promotes the proliferation and decreases the differentiation of keratinocytes, a hallmark of psoriatic skin. Using immunohistochemistry, Tohyama et al. reported that the production of IL-17A by CD8+ TRM cells and IL-22 by CD4+ TRM cells correlated with Bcl-3 production, IL-22-induced gene expression, and expression of other genes associated with psoriasis in lesional skin as compared to healthy control skin. By targeting Bcl-3, both CD4+ and CD8+ TRM disease pathways in psoriasis could be interrupted. It is now understood that expression of Bcl-3 promotes CD4+ T cell survival and is necessary for the proper development of Th1, Th2, and Th17 cells. While Bcl-3 inhibitors have been extensively studied as potential therapeutics in cancer, their role as a therapeutic in psoriasis or other skin diseases has not yet been explored.

13Although blocking the psoriatic microenvironment produced by TRM cells has proven effective in treating disease, scientists are also looking at how to directly target TRM cells. Pan et al. examined the metabolic pathways of mouse CD8+ TRM cells in order to elucidate how TRM cells survive in the nutrient-poor conditions of the skin. In both mouse models and skin biopsies from patients with psoriatic lesions, it was found that TRM T cells required exogenous free fatty acids to survive in the skin. This characteristic is unique to TRM T cells and could be a potential target for treatment for skin diseases mediated by TRM T cells.

14The hair follicle has long been recognized as an immune privileged site, as it protects the hair from potential autoreactive immune attacks through a system of complex regulation and suppression of the immune system. When this system breaks down, hair loss can result. Alopecia areata (AA) and cicatricial alopecia (CA) are common, inflammatory hair loss conditions. While CA is scarring and has a distinct clinical presentation of hair loss along the frontotemporal hairline and eyebrows, AA can present variably, ranging from a single patch of hair loss to total body hair loss and is non-scarring. A combination of genetics and environmental triggers have been implicated in the pathogenesis of both alopecias.

15CA is a diverse group of scarring hair loss encompassing several clinical variants including frontal fibrosing alopecia (FFA), lichen planopilaris (LPP) and central centrifugal cicatricial alopecia (CCCA). FFA and LPP most commonly occur in postmenopausal Caucasian women, however there have been a few cases in younger women. Autoimmune thyroid disease is the most common comorbid autoimmune disorder with FFA and LPP. In contrast, CCCA almost exclusively occurs in African American women, and Type 2 diabetes is the most common comorbidity.

16AA affects people of all different ages, sexes, and ethnic backgrounds. To date, the immunopathogenesis of AA is the most well-characterized of the alopecias: AA is driven by CD8+ T cells and natural killer (NK) cells as evidenced by both mouse models and studies of ex vivo human tissues. NKG2D+ cells are central to pathogenesis, as NKG2D activates both CD8+ T cells and NK cells to promote destruction of the hair follicle. A clinical trial targeting NK cell lectin-like-receptor subfamily K is currently enrolling ( Table S1 ).

17While anagen hair follicle epithelium is the target of CD8+ T cells in AA, FFA is mediated by destruction of stem cells in the hair follicle. Targets in CCCA have not been well-characterized, and future studies of alopecia in patients with skin of color are warranted to understand nuances in immunopathogenesis and potential novel treatment options.

18Del Duca et al. recently determined that lesional skin from AA and FFA patients has significantly more CD8+ cytotoxic T cells, CD11c+ dendritic cells, and CD103+ CD69+ TRM cells when compared to nonlesional skin. In particular, FFA was shown to have a significant upregulation of the IFNγ/CXCL9/CXCL10 pathway and the JAK-STAT pathway, ( Figure 2D ). In a recent case series, Yang et al. reported significant clinical improvement as measured using LPPAI scoring in a patient treated with the JAK inhibitor (JAKi) tofacitinib. This patient had previously failed monthly intralesional triamcinolone, topical steroids, doxycycline, and hydroxychloroquine. Similarly, AA patients have successfully been treated with JAKi. However, it is important to note that, like vitiligo, alopecia recurs upon cessation of JAKi. The known recurrence of AA and FFA, the confirmed upregulation of CD103+CD69+ TRM cells, and the reported clinical significance of JAKi support the crucial role of TRM cells in disease pathogenesis. A new clinical trial for a selective and simultaneous inhibitor of cytokines IL-2, IL-9, and IL-15 is enrolling for alopecia, which may have an impact on TRM ( Table S1 ).

19The complex roles of TRM cells in autoimmune skin diseases are just beginning to be understood. However, it is clear that TRM play significant roles in disease reactivation and flares, and must be targeted if we hope to achieve long-lasting and durable treatment options for patients. The tremendous strides in the last ten years in understanding the pathophysiology and treatment of autoimmune skin disease is incredibly exciting, emphasizing not only how much we still have to learn about these diseases, but also the enormous impact it will have on patient survival and quality of life.

20GR: original draft and figure drafts. JR and GR: literature search. GR and JR: digital artwork generation. JR and JH: editing. All authors contributed to the article and approved the submitted version.

21GR was supported by the UMass Research & Curriculum Exploration program. JR is supported by a Career Development Award from the Dermatology Foundation, a Target Identification in Lupus Award from the Lupus Research Alliance and a Concept Award from the US Department of Defense Lupus Research Program #LR190030. JH is supported by NIH R01 AR069114, R61 AR070302, the Hartford Foundation, and a Calder Research Scholar Award from the American Skin Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

22The views expressed in this article are those of the authors and may not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.

23JH is the founder of Villaris Therapeutics and NIRA Biosciences. JH and JR are inventors on patent application #62489191, “Diagnosis and Treatment of Vitiligo” which covers targeting IL-15 and TRM for the treatment of vitiligo; and on patent application #15/851,651, “Anti-human CXCR3 antibodies for the Treatment of Vitiligo” which covers targeting CXCR3 for the treatment of vitiligo. JR is a reviewer for Frontiers Immunology journal.