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Correspondence: I. Caroline Le Poole, Department of Pathology, Microbiology and Immunology, Oncology Research Institute Room 203, Loyola University Chicago, 2160 South First Avenue, Maywood, Illinois 60153, USA.
Vitiligo is a cutaneous autoimmune disease, especially devastating to patients with darker skin tones because of the contrast between unaffected and lesional skin. We studied immune cells infiltrating vitiligo skin and found very few regulatory T cells (Tregs). Vitiligo was not associated with a reduced frequency or function of circulating Tregs. To manipulate Treg function, we used mouse models expressing melanocyte-reactive TCRs, following changes in pelage color. We also isolated splenocytes to measure Treg function and evaluated cutaneous Treg abundance. Even small numbers of Tregs transferred into depigmenting mice could effectively interfere with depigmentation. The same holds true for treatment with rapamycin, readily translatable for use in human patients; such treatment may be well tolerated. Because vitiligo skin is relatively devoid of cells that produce the chemokine CCL22, whereas circulating Tregs express normal levels of its receptor CCR4, we overexpressed Ccl22 in the skin of vitiligo-prone mice to assess the resulting levels of depigmentation. Markedly reduced depigmentation was accompanied by Treg infiltration to the skin. With several options available to support a healthy balance between Tregs and effector T cells, the next challenge will be to render such treatment antigen specific and avoid general immunosuppression.
). No solid data exist on the prevalence of progressive depigmentation among different ethnic groups. Consanguinity can contribute to an elevated prevalence in some communities (
), yet the particular significance of depigmentation for patients where lesional skin displays such a stark contrast to the unaffected skin is self-evident (
Appearance Research Collaboration (ARC) Vitiligo linked to stigmatization in British South Asian women: a qualitative study of the experiences of living with vitiligo.
Figure 1Examples of vitiligo in skin of different origins. Vitiligo is a skin disease that can occur in patients of any ethnicity, yet its presence is most readily noticeable in people with darker skin tones. Examples with country of origin are shown.
Nonsegmental vitiligo disease duration and female sex are associated with comorbidity and disease extent; A retrospective analysis in 1307 patients older than 50 years.
). In the latter group environmental factors can play a greater role, whereas a greater percentage of pediatric patients have family members with vitiligo (
), yet access to costly equipment may be limited in less affluent environments. Dedicated treatment centers can then offer a reliable base for disease management and for meeting community members with vitiligo (
Antigen-presenting cells are activated in part via toll-like receptors in response to common toll-like receptor ligands. Antigen-presenting cell activation is associated with triggers of disease, such as a cell damage, entry of skin microbes and release of CpG nucleotides and LPS (
In perilesional vitiligo skin, TNF-related apoptosis-inducing ligand (TRAIL)+ dendritic cells (DC) are observed close to TRAIL receptor-expressing melanocytes (
). Dying melanocytes then become a source of antigen for processing by DCs and presentation to T cells. Resulting infiltrates of CD4+ and CD8+ T cells are found in vitiligo skin (
Local immune response in skin of generalized vitiligo patients. Destruction of melanocytes is associated with the prominent presence of CLA+ T cells at the perilesional site.
). Such TCRs can be incorporated into melanoma treatments. Ethnic skin is generally less susceptible to melanoma. However, the disease is generally further advanced when diagnosed in ethnic skin. Patients could then benefit from therapy that uses TCR-transduced T cells (
T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone.
). With limited HLA-A2 expression among ethnic patients, compatible vitiligo skin is a more beneficial source of T-receptors for such treatments.
Because regulatory T cells (Tregs) are relatively less abundant in vitiligo skin, this would suggest that Treg/T effector (Teff) cell ratios are unfavorable and that uninhibited cytotoxic T cells can contribute to depigmentation (
). The Smyth line chicken was instrumental in demonstrating humoral reactivity toward melanocytes in vitiligo. This animal develops spontaneous disease in the absence of melanoma (
). Genetically modified strains of mice have also offered insight into vitiligo development. The VIT mouse exhibits gradual depigmentation through gradual loss of Mitf-deficient melanocytes (
), but autoimmune disease characteristics are not represented. Vitiligo can be induced by cutaneous overexpression of causative antigens, causing T cell–mediated depigmentation (
). Two models have been particularly informative regarding the role of Tregs, namely one in which depigmentation develops after Treg depletion and tumor excision (
Figure 2IFN-γ drives depigmentation in vitiligo-prone h3TA2 mice. On an IFN-γ–knockout background, no depigmentation is observed, whereas a lack of pigment in the skin and hair of h3TA2 mice is readily apparent. Depigmentation in the IFN-γ–knockout mice was restored by depleting Tregs (not shown).
), and melanocyte-reactive cytotoxic T cells can act unopposed. Tregs serve to terminate ongoing immune responses. Thymic T cells that exhibit high affinity for self-antigen can develop into natural Tregs, whereas inducible Tregs develop in the periphery (
Cutaneous and oral squamous cell carcinoma-dual immunosuppression via recruitment of FOXP3+ regulatory T cells and endogenous tumour FOXP3 expression?.
), which can provide an explanation for virus-induced autoimmunity.
Without Tregs, autoimmune responses can do persistent damage, as observed in patients with FoxP3 mutations exhibiting immune dysregulation, polyendocrinopathy, enteropathy, X-linked (i.e., IPEX) syndrome (
Bacchetta R, Barzaghi F, Roncarolo MG (2016). From IPEX syndrome to FOXP3 mutation: a lesson on immune dysregulation [e-pub ahead of print]. Ann N Y Acad Sci 2016; http://dx.doi.org/10.1111/nyas.13011 (accessed 21 October 2016).
). For tissue-specific autoimmunity, reduced Treg infiltration may be limited to the target organ. Alternatively, deranged Treg development can underlie disease (
). Reduced Treg abundance appears to result from reduced expression of CCL22. Although we found that the abundance of circulating Tregs trends toward an increase, some have reported systemically reduced Tregs in vitiligo (
), measurable as changes in immunosuppressive cytokine secretion or T-cell proliferation. Effector responses are then reduced through cell cycle arrest, apoptosis, and anergy of responder T cells (
Different methods can enhance the abundance, activity, proliferation, homing, and cytokine secretion by Tregs. For such studies we rely on the mouse models of disease, and TCR transgenic models of disease will also exhibit an underdeveloped regulatory component (Figure 3). Thus, restored pigmentation can readily serve as a readout for therapeutic activity (
Figure 3Limited abundance of Tregs in the skin of vitiligo-prone h3TA2 mice. FoxP3+ CD3+ Tregs compared in skin homogenates from A2 and h3TA2 mice. Abundance of Tregs identified as FoxP3+ CD3+ lymphocytes among cells infiltrating the skin shows markedly (∼20-fold) reduced Tregs in h3TA2 mice. SSC, side scatter; FSC, forward scatter.
To replenish Tregs, autologous cells can be amplified in vitro and returned to patients when the disease flares up. Such adoptive cell transfer has been put to the test for antitumor responses. Genetically modified T cells have been tested in clinical settings with some remarkable responses (
Safety concerns are less pronounced when transferring regulatory T cells that interfere with ongoing immunity. Preclinical studies have been performed to demonstrate the benefit of Treg transfer for reducing the expression of graft-versus-host disease (GVHD) in mice (
), by preventing the expansion of GVHD–causing T cells. Using an effector-memory enriched population of Tregs may further enhance the suppressive effects (
). A single dose of 200,000 cells proved sufficient to maintain elevated Treg/Teff ratios for 6 weeks, when significantly reduced depigmentation was found among treated mice, where Tregs were more abundant.
The specificity of adoptive Treg treatment can be improved using antigen-specific Tregs as purified on the basis of latency-associated peptide (LAP) or glycoprotein A repetitions predominant (GARP) surface expression (
). Antigen-specific Tregs are now under development for several autoimmune diseases with known target antigens. With vitiligo as a prime example, tyrosinase-reactive Tregs were generated using the same TCR used to generate the h3TA2 and Vitesse mouse models of vitiligo (
); it is added when expanding Tregs in vitro in the presence of IL-2 but can also support T helper 17 cell development when accompanied by IL-6. Also, protein kinase B activation and subsequent mTOR activity impairs Treg development, whereas rapamycin treatment can promote Tregs (
). In our h3TA2 model, rapamycin was administered on alternate days for 2 weeks at 5 mg/kg of body weight, resulting in remarkable vitiligo inhibition and prolonged Treg skin infiltration (
) can also benefit from rapamycin in animal disease models, accompanied by reduced effector T cell and increased Treg abundance. The same is being considered for rheumatic disease (
Another option of interest is cholecalciferol (vitamin D) treatment. Applied topically, induction of different subsets of regulatory T cells depends on the origin of the dendritic cells being primed (
). In pediatric patients with autoimmune thyroiditis, vitamin D supplementation was helpful in restoring expression of the Treg transcription factor FoxP3 (
). Because light treatment is among the most successful therapeutics available for vitiligo today and narrow-band UVB can enhance vitamin D levels and stimulate pigmentation (
), improved Treg induction may explain the observed correlation between these parameters. Although contested, some studies suggest that patients with vitiligo have reduced serum vitamin D levels (
). Because ethnic skin is considerably more resilient to sunlight and people with darker skin tones can be at risk for vitamin D deficiency, in some places this is a risk factor to consider (
Circulating T cells are attracted to tissues through the expression of homing receptors. Chemokines and their receptors will likewise incentivize Tregs to extravasate in tissues where an ongoing immune response has run its course (
Skin-homing CLA+ T cells and regulatory CD25+ T cells represent major subsets of human peripheral blood memory T cells migrating in response to CCL1/I-309.
). The responsible cell type is thus still at large. We do know that reduced CCL22 expression extends to unaffected skin, suggesting that a paucity of Tregs can set the stage for cytotoxic T cells to attack. The presence of melanocyte-reactive CD8+ T cells is not unique to patients (
), thus a paucity of Treg may help determine vitiligo development.
Figure 4Skin-homing Tregs in healthy skin express cutaneous lymphocyte antigen (CLA). In adult human skin (left), intense red staining for FoxP3 to locate Tregs in the skin is accompanied by membrane expression of CLA as detected by antibody Heca452 in blue. By contrast, CLA+ T cells in nonlesional vitiligo skin (right) are not Tregs. Scale bars = 100 μm.
Treg paucity in affected mouse skin is shown in Figure 3, where skin tissue from h3TA2 and wild-type mice was dissociated and Tregs were identified by CD3/FoxP3 coexpression. Cutaneous Ccl22 overexpression can replenish the resident Treg population and prevent vitiligo (
). Other chemokines may support skin homing by Tregs, but CCL22 and possibly CCL17 are expected to be superior chemoattractants given the high percentage of Tregs that express CCR4 (
), and repopulating vitiligo skin with a healthy population of Tregs may be beneficial for normalizing off-balance immune responses in patient skin overall.
Toward Treg Therapy for Vitiligo
When testing therapeutics in patients, safety must be established. Suppressing unwanted immune responses is safe only if necessary immunity remains intact. This may be accomplished by a cutaneous application to the border of expanding lesions. Different modes of application besides subcutaneous injection include needleless injectors, electroporation, microneedle applications, tattooing, nanoparticles, and scaffolds (
). The depth and longevity of the application will help determine its efficacy and safety. Vitiligo is among the first of autoimmune diseases where target antigens have been identified, thus enabling the generation of antigen-specific Tregs (
). Though few laboratory studies have addressed this opportunity in vitiligo, the same strategy is under study for the treatment of other autoimmune diseases (
Bacchetta R, Barzaghi F, Roncarolo MG (2016). From IPEX syndrome to FOXP3 mutation: a lesson on immune dysregulation [e-pub ahead of print]. Ann N Y Acad Sci 2016; http://dx.doi.org/10.1111/nyas.13011 (accessed 21 October 2016).
Skin-homing CLA+ T cells and regulatory CD25+ T cells represent major subsets of human peripheral blood memory T cells migrating in response to CCL1/I-309.
Cutaneous and oral squamous cell carcinoma-dual immunosuppression via recruitment of FOXP3+ regulatory T cells and endogenous tumour FOXP3 expression?.
T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone.
Nonsegmental vitiligo disease duration and female sex are associated with comorbidity and disease extent; A retrospective analysis in 1307 patients older than 50 years.
Local immune response in skin of generalized vitiligo patients. Destruction of melanocytes is associated with the prominent presence of CLA+ T cells at the perilesional site.
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