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Replenishing Regulatory T Cells to Halt Depigmentation in Vitiligo

  • I. Caroline Le Poole
    Correspondence
    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.
    Affiliations
    Department of Pathology and Microbiology/Immunology, Oncology Research Institute, Loyola University, Chicago, Illinois, USA
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  • Shikhar Mehrotra
    Affiliations
    Department of Surgery, Medical University of South Carolina, Charleston, South Carolina, USA
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      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.

      Abbreviation:

      Treg (regulatory T cell)

      Vitiligo in Ethnic Skin

      Vitiligo appears in about 0.5% of the world population (
      • Krüger C.
      • Schallreuter K.U.
      A review of the worldwide prevalence of vitiligo in children/adolescents and adults.
      ). No solid data exist on the prevalence of progressive depigmentation among different ethnic groups. Consanguinity can contribute to an elevated prevalence in some communities (
      • Alenizi D.A.
      Consanguinity pattern and heritability of Vitiligo in Arar, Saudi Arabia.
      ). Vitiligo (Figure 1) can be devastating to patients of any skin tone, (
      • Porter J.R.
      • Beuf A.H.
      Racial variation in reaction to physical stigma: a study of degree of disturbance by vitiligo among black and white patients.
      ), yet the particular significance of depigmentation for patients where lesional skin displays such a stark contrast to the unaffected skin is self-evident (
      • Halder R.M.
      • Nootheti P.K.
      Ethnic skin disorders overview.
      ). Blemishes can interfere with social interactions (
      • AlGhamdi K.M.
      Beliefs and perceptions of Arab vitiligo patients regarding their condition.
      ). Thus, it is not surprising that the first prime minister of free India, Nehru, declared vitiligo among the three greatest health problems there (
      • Parsad D.
      • Sunil Dogra S.
      • Kanwar A.J.
      Quality of life in patients with vitiligo.
      ). Disease visibility can leave patients ostracized (
      • Thompson A.R.
      • Clarke S.A.
      • Newell R.J.
      • Gawkrodger D.J.
      Appearance Research Collaboration (ARC)
      Vitiligo linked to stigmatization in British South Asian women: a qualitative study of the experiences of living with vitiligo.
      ), particularly in countries where infectious diseases with a depigmenting component are prevalent (
      • Chaturvedi S.K.
      • Singh G.
      • Gupta N.
      Stigma experience in skin disorders: an Indian perspective.
      ).
      Figure thumbnail gr1
      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.
      Vitiligo can develop at any age but there are two spikes at ages 15 and 45 years that have slightly different causes (
      • Teulings H.E.
      • Ceylan E.
      • Overkamp M.
      • Vrijman C.
      • Bos J.D.
      • Nijsten T.E.
      • et al.
      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 (
      • Phiske M.M.
      “Childhood vitiligo”.
      ). Prior melanoma development likely makes a far greater contribution to vitiligo development in lighter skin (
      • Schild M.
      • Meurer M.
      Vitiligo: clinical presentation and pathogenesis.
      ). In ethnic skin, heredity may thus play a relatively greater role in vitiligo etiology, though there is currently little support for this concept (
      • Boisseau-Garsaud A.M.
      • Garsaud P.
      • Calès-Quist D.
      • Hélénon R.
      • Quénéhervé C.
      • Claire R.C.
      Epidemiology of vitiligo in the French West Indies (Isle of Martinique).
      ). Meanwhile, adolescents are particularly vulnerable to appearance issues (
      • Parsad D.
      • Sunil Dogra S.
      • Kanwar A.J.
      Quality of life in patients with vitiligo.
      ).
      Finally, there are differences in access to treatment. Light treatment is among the most successful treatment modalities (
      • Gawkrodger D.J.
      • Ormerod A.D.
      • Shaw L.
      • Mauri-Sole I.
      • Whitton M.E.
      • Watts M.J.
      • et al.
      Guideline for the diagnosis and management of 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 (
      • Ayanlowo O.
      • Olumide Y.M.
      • Akinkugbe A.
      • Ahamneze N.
      • Otike-Odibi B.I.
      • Ekpudu V.I.
      • et al.
      Characteristics of vitiligo in Lagos, Nigeria.
      ).

      Autoimmune Involvement

      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 (
      • Ganju P.
      • Nagpal S.
      • Mohammed M.H.
      • Nishal Kumar P.
      • Pandey R.
      • Natarajan V.T.
      • et al.
      Microbial community profiling shows dysbiosis in the lesional skin of Vitiligo subjects.
      ).
      In perilesional vitiligo skin, TNF-related apoptosis-inducing ligand (TRAIL)+ dendritic cells (DC) are observed close to TRAIL receptor-expressing melanocytes (
      • Kroll T.M.
      • Bommiasamy H.
      • Boissy R.E.
      • Hernandez C.
      • Nickoloff B.J.
      • Mestril R.
      • et al.
      (2005). 4-Tertiary butyl phenol exposure sensitizes human melanocytes to dendritic cell-mediated killing: relevance to vitiligo.
      ). Circulating TRAIL may also eliminate melanocytes (
      • Edgunlu T.
      • Solak Tekin N.
      • Ozel Turkcu Ü.
      • Karakaş-Çelik S.
      • Urhan-Kucuk M.
      • et al.
      Evaluation of serum trail level and DR4 gene variants as biomarkers for vitiligo patients.
      ). 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 (
      • Gross A.
      • Tapia F.J.
      • Mosca W.
      • Perez R.M.
      • Briceño L.
      • Henriquez J.J.
      • et al.
      Mononuclear cell subpopulations and infiltrating lymphocytes in erythema dyschromicum perstans and vitiligo.
      ,
      • van den Wijngaard R.
      • Wankowicz-Kalinska A.
      • Le Poole C.
      • Tigges B.
      • Westerhof W.
      • Das P.
      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.
      ), accompanied by macrophages to clear cell debris and aid in tissue restoration (
      • Oiso N.
      • Tanemura A.
      • Kotobuki Y.
      • Kimura M.
      • Katayama I.
      • Kawada A.
      Role of macrophage infiltration in successful repigmentation in a new periphery-spreading vitiligo lesion in a male Japanese patient.
      ).
      Recruited T cells are reactive with melanocytes in a T helper type 1 (Th1) cell-dominated response (
      • Wańkowicz-Kalińska A.
      • van den Wijngaard R.M.
      • Tigges B.J.
      • Westerhof W.
      • Ogg G.S.
      • Cerundolo V.
      • et al.
      Immunopolarization of CD4+ and CD8+ T cells to Type-1-like is associated with melanocyte loss in human vitiligo.
      ), as further supported by the apparent IFN-γ dependence of depigmentation in mouse 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.
      ,
      • 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-γ for autoreactive CD8+ T-cell accumulation in the skin.
      ). When incubated with human skin explants, T cells infiltrate the skin, inducing apoptosis (
      • 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.
      ). CD8+ T cells reactive to tyrosinase or MART-1 were readily identified in patient blood (
      • Ogg G.S.
      • Rod Dunbar P.
      • Romero P.
      • Chen J.L.
      • Cerundolo V.
      High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo.
      ). The TCRs from vitiligo T cells expressed enhanced reactivity when compared with those from melanoma patients (
      • Palermo B.
      • Garbelli S.
      • Mantovani S.
      • Scoccia E.
      • Da Prada G.A.
      • Bernabei P.
      • et al.
      Qualitative difference between the cytotoxic T lymphocyte responses to melanocyte antigens in melanoma and vitiligo.
      ). That such T cells contribute to depigmentation is further established by TCR cloning from perilesional skin (
      • Klarquist J.
      • Eby J.M.
      • Henning S.W.
      • Li M.
      • Wainwright D.A.
      • Westerhof W.
      • et al.
      Functional cloning of a gp100-reactive T-cell receptor from vitiligo patient skin.
      ). 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 (
      • Oyarbide-Valencia K.
      • van den Boorn J.G.
      • Denman C.J.
      • Li M.
      • Carlson J.M.
      • Hernandez C.
      • Nishimura M.I.
      • et al.
      Therapeutic implications of autoimmune vitiligo T cells.
      ). Most TCRs identified to date are HLA-A2 restricted (
      • Sensi M.
      • Salvi S.
      • Castelli C.
      • Maccalli C.
      • Mazzocchi A.
      • Mortarini R.
      • et al.
      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 (
      • Klarquist J.
      • Denman C.J.
      • Hernandez C.
      • Wainwright D.A.
      • Strickland F.M.
      • Overbeck A.
      • et al.
      Reduced skin homing by functional Treg in vitiligo.
      ).

      Disease Models

      Several species develop vitiligo, including the Sinclair swine, with skin depigmention after spontaneous regression of melanoma (
      • Misfeldt M.L.
      • Grimm D.R.
      Sinclair miniature swine: an animal model of human melanoma.
      ). Swine skin exhibits similarity to that of humans (
      • Julé S.
      • Bossé P.
      • Egidy G.
      • Panthier J.J.
      Establishment and characterization of a normal melanocyte cell line derived from pig skin.
      ,
      • Summerfield A.
      • Meurens F.
      • Ricklin M.E.
      The immunology of the porcine skin and its value as a model for human skin.
      ). The Smyth line chicken was instrumental in demonstrating humoral reactivity toward melanocytes in vitiligo. This animal develops spontaneous disease in the absence of melanoma (
      • Wang X.
      • Erf G.F.
      Apoptosis in feathers of Smyth line chickens with autoimmune vitiligo.
      ). 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 (
      • Lerner A.B.
      • Shiohara T.
      • Boissy R.E.
      • Jacobson K.A.
      • Lamoreux M.L.
      • Moellmann G.E.
      A mouse model for vitiligo.
      ), but autoimmune disease characteristics are not represented. Vitiligo can be induced by cutaneous overexpression of causative antigens, causing T cell–mediated depigmentation (
      • Denman C.J.
      • McCracken J.
      • Hariharan V.
      • Klarquist J.
      • Oyarbide-Valencia K.
      • Guevara-Patiño J.A.
      • et al.
      HSP70i accelerates depigmentation in a mouse model of autoimmune vitiligo.
      ). Meanwhile, melanoma-responsive TCRs introduced into transgenic strains such as the ‘FH’ mouse were developed as 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.
      ). The Pmel-1 mouse was used to reproduce depigmentation after adoptive T-cell transfer to mice with pigmented skin (
      • 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-γ for autoreactive CD8+ T-cell accumulation in the skin.
      ,
      • 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.
      ), and the Vitesse mouse exhibits the same in a spontaneous fashion, as well as spontaneous repigmentation (
      • Eby J.M.
      • Kang H.K.
      • Klarquist J.
      • Chatterjee S.
      • Mosenson J.A.
      • Nishimura M.I.
      • et al.
      Immune responses in a mouse model of vitiligo with spontaneous epidermal de- and repigmentation.
      ). Two models have been particularly informative regarding the role of Tregs, namely one in which depigmentation develops after Treg depletion and tumor excision (
      • Byrne K.T.
      • Côté A.L.
      • Zhang P.
      • Steinberg S.M.
      • Guo Y.
      • Allie R.
      • et al.
      Autoimmune melanocyte destruction is required for robust CD8+ memory T cell responses to mouse melanoma.
      ) and another (the h3TA2 mouse) serving as a spontaneous model suitable to test Treg-based treatments (
      • Mehrotra S.
      • Al-Khami A.A.
      • Klarquist J.
      • Husain S.
      • Naga O.
      • Eby J.M.
      • et al.
      A coreceptor- independent transgenic human TCR mediates anti-tumor and anti-self immunity in mice.
      ). In h3TA2 mice devoid of IFN-γ, no vitiligo develops (Figure 2), yet vitiligo returns upon depletion of CD25+ Tregs (
      • Chatterjee S.
      • Eby J.M.
      • Al-Khami A.A.
      • Soloshchenko M.
      • Kang H.K.
      • Kaur N.
      • et al.
      A quantitative increase in regulatory T cells controls development of vitiligo.
      ). Once Tregs are removed, IL-17–producing T cells apparently mediate depigmentation (
      • Chatterjee S.
      • Eby J.M.
      • Al-Khami A.A.
      • Soloshchenko M.
      • Kang H.K.
      • Kaur N.
      • et al.
      A quantitative increase in regulatory T cells controls development of vitiligo.
      ). Thus, a Treg barrier can help avoid vitiligo.
      Figure thumbnail gr2
      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).

      Limited Treg Abundance

      Up to 50% of T cells found in healthy skin are Tregs. In vitiligo skin few Tregs are found (
      • Klarquist J.
      • Denman C.J.
      • Hernandez C.
      • Wainwright D.A.
      • Strickland F.M.
      • Overbeck A.
      • et al.
      Reduced skin homing by functional Treg in vitiligo.
      ), 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 (
      • Chattopadhyay S.
      • Chakraborty N.G.
      • Mukherji B.
      Regulatory T cells and tumor immunity.
      ). Until recently, it was difficult to distinguish the two (
      • Lin X.
      • Chen M.
      • Liu Y.
      • Guo Z.
      • He X.
      • Brand D.
      • et al.
      (2013). Advances in distinguishing natural from induced Foxp3(+) regulatory T cells.
      ). Tregs suppress immune cells (
      • Lin X.
      • Chen M.
      • Liu Y.
      • Guo Z.
      • He X.
      • Brand D.
      • et al.
      (2013). Advances in distinguishing natural from induced Foxp3(+) regulatory T cells.
      ) after making contact and secreting inhibitory cytokines (
      • Schmidt A.
      • Oberle N.
      • Krammer P.H.
      Molecular mechanisms of Treg-mediated T cell suppression.
      ). The transcription factor FoxP3 remains the most reliable Treg marker (
      • Chen W.
      • Jin W.
      • Hardegen N.
      • Lei K.J.
      • Li L.
      • Marinos N.
      • et al.
      Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3.
      ), although other cells can sometimes bind FoxP3 antibodies (
      • Schipmann S.
      • Wermker K.
      • Schulze H.J.
      • Kleinheinz J.
      • Brunner G.
      Cutaneous and oral squamous cell carcinoma-dual immunosuppression via recruitment of FOXP3+ regulatory T cells and endogenous tumour FOXP3 expression?.
      ). Other Treg markers include IL-2 receptor CD25, glucocorticoid-induced tumor necrosis factor receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and neuropilin-1 (
      • Venken K.
      • Hellings N.
      • Liblau R.
      • Stinissen P.
      Disturbed regulatory T cell homeostasis in multiple sclerosis.
      ). Tregs have been intensely studied over 15 years, yet little is known about their antigen specificity (
      • Weissler K.A.
      • Caton A.J.
      The role of T-cell receptor recognition of peptide: MHC complexes in the formation and activity of Foxp3+ regulatory T cells.
      ). However, viruses can take advantage of their presumed high-affinity TCRs when viral peptides are recognized by Treg TCRs cross-reactive with self (
      • Weissler K.A.
      • Caton A.J.
      The role of T-cell receptor recognition of peptide: MHC complexes in the formation and activity of Foxp3+ regulatory T cells.
      ), 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).

      ). A paucity of Tregs has been reported in several autoimmune diseases including systemic lupus erythematosus (
      • Ohl K.
      • Tenbrock K.
      Regulatory T cells in systemic lupus erythematosus.
      ), alopecia areata (
      • Han Y.M.
      • Sheng Y.Y.
      • Xu F.
      • Qi S.S.
      • Liu X.J.
      • Hu R.M.
      • et al.
      Imbalance of T-helper 17 and regulatory T cells in patients with alopecia areata.
      ), multiple sclerosis (
      • Kleinewietfeld M.
      • Hafler D.A.
      Regulatory T cells in autoimmune neuroinflammation.
      ) and vitiligo (
      • Dwivedi M.
      • Kemp E.H.
      • Laddha N.C.
      • Mansuri M.S.
      • Weetman A.P.
      • Begum R.
      Regulatory T cells in vitiligo: implications for pathogenesis and therapeutics.
      ). For tissue-specific autoimmunity, reduced Treg infiltration may be limited to the target organ. Alternatively, deranged Treg development can underlie disease (
      • Roychoudhuri R.
      • Hirahara K.
      • Mousavi K.
      • Clever D.
      • Klebanoff C.A.
      • Bonelli M.
      • et al.
      BACH2 represses effector programs to stabilize T(reg)-mediated immune homeostasis.
      ). We reported reduced Treg infiltration of vitiligo skin extending beyond the depigmented lesions (
      • Klarquist J.
      • Denman C.J.
      • Hernandez C.
      • Wainwright D.A.
      • Strickland F.M.
      • Overbeck A.
      • et al.
      Reduced skin homing by functional Treg in vitiligo.
      ). 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 (
      • Dwivedi M.
      • Laddha N.C.
      • Arora P.
      • Marfatia Y.S.
      • Begum R.
      Decreased regulatory T-cells and CD4(+) /CD8(+) ratio correlate with disease onset and progression in patients with generalized vitiligo.
      ), possibly as a consequence of secondary autoimmune responses. Reduced Treg function has been reported by others (
      • Tu C.X.
      • Jin W.W.
      • Lin M.
      • Wang Z.H.
      • Man M.Q.
      Levels of TGF-β(1) in serum and culture supernatants of CD4(+)CD25 (+) T cells from patients with non-segmental 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 (
      • Abbas A.K.
      • Lohr J.
      • Knoechel B.
      • Nagabhushanam V.
      T cell tolerance and autoimmunity.
      ).
      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 (
      • Denman C.J.
      • McCracken J.
      • Hariharan V.
      • Klarquist J.
      • Oyarbide-Valencia K.
      • Guevara-Patiño J.A.
      • et al.
      HSP70i accelerates depigmentation in a mouse model of autoimmune vitiligo.
      ).
      Figure thumbnail gr3
      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.

      Adoptive Treg Transfer

      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 (
      • Gattinoni L.
      Adoptive T cell transfer: imagining the next generation of cancer immunotherapies.
      ). Off-target effects can be addressed by incorporating suicide genes or applying corticosteroids to generically suppress autoimmunity (
      • Griffioen M.
      • van Egmond E.H.
      • Kester M.G.
      • Wikllemze R.
      • Falkenburg J.H.
      • Heemskerk M.H.
      Retroviral transfer of human CD20 as a suicide gene for adoptive T-cell therapy.
      ,
      • Iliopoulou E.G.
      • Koutourakis P.
      • Karamouzis M.V.
      • Doufexis D.
      • Ardavanis A.
      • Baxevanis C.N.
      • et al.
      A phase I trial of adoptive transfer of allogenic natural killer cells in patients with advanced non-small cell lung cancer.
      ).
      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 (
      • Mutis T.
      • van Rijn R.S.
      • Simonetti E.R.
      • Aarts-Riemens T.
      • Emmelot M.E.
      • van Bloois L.
      • et al.
      Human regulatory T cells control xenogeneic graft-versus-host disease induced by autologous T cells in RAG2-/- γc-/-immunodeficient mice.
      ), by preventing the expansion of GVHD–causing T cells. Using an effector-memory enriched population of Tregs may further enhance the suppressive effects (
      • Haase D.
      • Starke M.
      • Puan K.J.
      • Lai T.S.
      • Rotzschke O.
      Immune modulation of inflammatory conditions: regulatory T cells for treatment of GvHD.
      ). We thus introduced traceable Tregs into the h3TA2 model of vitiligo and measured the effects on depigmentation (
      • Chatterjee S.
      • Eby J.M.
      • Al-Khami A.A.
      • Soloshchenko M.
      • Kang H.K.
      • Kaur N.
      • et al.
      A quantitative increase in regulatory T cells controls development of vitiligo.
      ). 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 (
      • Noyan F.
      • Lee Y.S.
      • Zimmermann K.
      • Hardtke-Wolenski M.
      • Taubert R.
      • Warnecke G.
      • et al.
      Isolation of human antigen-specific regulatory T cells with high suppressive function.
      ). Another relevant development is that of HSP70-specific Tregs, recently tested in a model of autoimmune arthritis (
      • van Herwijnen M.J.
      • Wieten L.
      • van der Zee R.
      • van Kooten P.J.
      • Wagenaar-Hilbers J.P.
      • Hoek A.
      • et al.
      Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis.
      ). 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 (
      • Brusko T.M.
      • Koya R.C.
      • Zhu S.
      • Lee M.R.
      • Putnam A.L.
      • McClymont S.A.
      • et al.
      Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.
      ,
      • Eby J.M.
      • Kang H.K.
      • Klarquist J.
      • Chatterjee S.
      • Mosenson J.A.
      • Nishimura M.I.
      • et al.
      Immune responses in a mouse model of vitiligo with spontaneous epidermal de- and repigmentation.
      ;
      • Mehrotra S.
      • Al-Khami A.A.
      • Klarquist J.
      • Husain S.
      • Naga O.
      • Eby J.M.
      • et al.
      A coreceptor- independent transgenic human TCR mediates anti-tumor and anti-self immunity in mice.
      ;). TCR transgenic Tregs specific for collagen II have likewise shown efficacy toward arthritis treatment in mice (
      • Asnagli H.
      • Martire D.
      • Belmonte N.
      • Quentin J.
      • Bastian H.
      • Boucard-Jourdin M.
      • et al.
      Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis.
      ). It can be challenging to maintain a Treg phenotype when introducing TCRs into inducible Tregs (
      • Sarikonda G.
      • Fousteri G.
      • Sachithanantham S.
      • Miller J.F.
      • Dave A.
      • Juntti T.
      • et al.
      BDC12-4.1 T-cell receptor transgenic insulin-specific CD4 T cells are resistant to in vitro differentiation into functional Foxp3+ T regulatory cells.
      ,
      • Yuan X.
      • Malek T.R.
      Cellular and molecular determinants for the development of natural and induced regulatory T cells.
      ), but these studies provide proof of principle for adoptive Treg transfer in vitiligo.

      Supporting Treg Development in Vivo

      Soluble factors supporting Treg development in the thymus include IL-2 (
      • Lio C.W.
      • Hsieh C.S.
      A two-step process for thymic regulatory T cell development.
      ). IL-2 also activates effector T cells, which forms the basis for IL-2 therapy in melanoma (
      • Sim G.C.
      • Wu S.
      • Jin L.
      • Hwu P.
      • Radvanyi L.G.
      Defective STAT1 activation associated with impaired IFN-γ production in NK and T lymphocytes from metastatic melanoma patients treated with IL-2.
      ). This apparent controversy is well recognized, and competition for IL-2 between effector T cells and Tregs likely determines therapeutic outcomes (
      • Jaberi-Douraki M.
      • Pietropaolo M.
      • Khadra A.
      Continuum model of T-cell acidity: understanding autoreactive and regulatory T-cell responses in type 1 diabetes.
      ). IL-2 also maintains the suppressive phenotype (
      • Yates J.
      • Rovis F.
      • Mitchell P.
      • Afzali B.
      • Tsang J.
      • Garin M.
      • et al.
      The maintenance of human regulatory CD4+CD25+ regulatory T cell function: IL-2, IL-4, IL-7 and IL-15 preserve optimal suppressive potency in vitro.
      ). A role for transforming growth factor-β in Treg development is more context dependent (
      • Yuan X.
      • Malek T.R.
      Cellular and molecular determinants for the development of natural and induced regulatory T cells.
      ); 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 (
      • Sauer S.
      • Bruno L.
      • Hertweck A.
      • Finlay D.
      • Leleu M.
      • Spivakov M.
      • et al.
      T cell receptor signaling controls Foxp3 expression via PI3K Akt mTOR.
      ). 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 (
      • Chatterjee S.
      • Eby J.M.
      • Al-Khami A.A.
      • Soloshchenko M.
      • Kang H.K.
      • Kaur N.
      • et al.
      A quantitative increase in regulatory T cells controls development of vitiligo.
      ). Autoimmune myositis (
      • Prevel N.
      • Allenbach Y.
      • Klatzmann D.
      • Salomon B.
      • Benveniste O.
      Beneficial role of rapamycin in experimental autoimmune myositis.
      ) and pancreatitis (
      • Schwaiger T.
      • van den Brandt C.
      • Fitzner B.
      • Zaatreh S.
      • Kraatz F.
      • Dummer A.
      • et al.
      Autoimmune pancreatitis in MRL/Mp mice is a T cell-mediated disease responsive to cyclosporine A and rapamycin treatment.
      ) 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 (
      • Perl A.
      Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases.
      ), psoriasis (
      • Wei K.C.
      • Lai P.C.
      Combination of everolimus and tacrolimus: a potentially effective regimen for recalcitrant psoriasis.
      ), keloid formation (
      • Wong V.W.
      • You F.
      • Januszyk M.
      • Gurtner G.C.
      • Kuang A.A.
      Transcriptional profiling of rapamycin-treated fibroblasts from hypertrophic and keloid scars.
      ), and possibly acne (
      • Monfrecola G.
      • Lembo S.
      • Caiazzo G.
      • De Vita V.
      • Di Caprio R.
      • Balato A.
      • et al.
      Mechanistic target of rapamycin (mTOR) expression is increased in acne patients’ skin.
      ).
      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 (
      • van der Aar A.M.
      • Sibiryak D.S.
      • Bakdash G.
      • van Capel T.M.
      • van der Kleij H.P.
      • Opstelten D.J.
      Vitamin D3 targets epidermal and dermal dendritic cells for induction of distinct regulatory T cells.
      ). In pediatric patients with autoimmune thyroiditis, vitamin D supplementation was helpful in restoring expression of the Treg transcription factor FoxP3 (
      • Şıklar Z.
      • Karataş D.
      • Doğu F.
      • Hacıhamdioğlu B.
      • İkincioğulları A.
      • Berberoğlu M.
      Investigation the effects of functions of regulatory T cells and vitamin D in children with chronic autoimmune thyroiditis.
      ). Its derivatives can augment Treg activity and reduce IL-17–producing T cells (
      • González-Mateo G.T.
      • Fernández-Míllara V.
      • Bellón T.
      • Liappas G.
      • Ruiz-Ortega M.
      • López-Cabrera M.
      • et al.
      Paricalcitol reduces peritoneal fibrosis in mice through the activation of regulatory T cells and reduction in IL-17 production.
      ). Several autoimmune conditions are currently considered for vitamin D3 supplementation (
      • Antico A.
      • Tampoia M.
      • Tozzoli R.
      • Bizzaro N.
      Can supplementation with vitamin D reduce the risk or modify the course of autoimmune diseases? A systematic review of the literature.
      ), including vitiligo (
      • Gorman S.
      • Judge M.A.
      • Hart P.H.
      Immune-modifying properties of topical vitamin D: Focus on dendritic cells and T cells.
      ). 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 (
      • Sehrawat M.
      • Arora T.C.
      • Chauhan A.
      • Kar H.K.
      • Poonia A.
      • Jairath V.
      Correlation of vitamin D levels with pigmentation in vitiligo patients treated with NBUVB therapy.
      ), 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 (
      • Karagün E.
      • Ergin C.
      • Baysak S.
      • Erden G.
      • Aktaş H.
      • Ekiz Ö.
      The role of serum vitamin D levels in vitiligo.
      ,
      • Upala S.
      • Sanquankeo A.
      Low 25-hydroxyvitamin D levels are associated with vitiligo: a systemic review and meta-analysis.
      ). 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 (
      • Sawicki C.M.
      • Van Rompay M.I.
      • Au L.E.
      • Gordon C.M.
      • Sacheck J.M.
      Sun-exposed skin color is associated with changes in serum 25-Hydroxyvitamin D in racially/ethnically diverse children.
      ).

      Driving Tregs to the Skin

      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 (
      • Yi H.
      • Zhao Y.
      Chemokines, chemokine receptors and CD4+CD25+ regulatory T cells.
      ). When observing a paucity of Treg in vitiligo skin tissues compared with control skin, we measured homing receptor expression (
      • Klarquist J.
      • Denman C.J.
      • Hernandez C.
      • Wainwright D.A.
      • Strickland F.M.
      • Overbeck A.
      • et al.
      Reduced skin homing by functional Treg in vitiligo.
      ), including cutaneous lymphocyte antigen (Figure 4), CCR4 (now considered a ubiquitous Treg homing marker), and CCR8 (
      • Colantonio L.
      • Iellem A.
      • Sinigaglia F.
      • D’Ambrosio D.
      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.
      ,
      • Hirahara K.
      • Liu L.
      • Clark R.A.
      • Yamanaka K.
      • Fuhlbrigge R.C.
      • Kupper T.S.
      The majority of human peripheral blood CD4+CD25highFoxp3+ regulatory T cells bear functional skin-homing receptors.
      ), yet found no remarkable differences (
      • Klarquist J.
      • Denman C.J.
      • Hernandez C.
      • Wainwright D.A.
      • Strickland F.M.
      • Overbeck A.
      • et al.
      Reduced skin homing by functional Treg in vitiligo.
      ). However, we observed a marked reduction in the number of CCL22-expressing cells in vitiligo skin (
      • Klarquist J.
      • Denman C.J.
      • Hernandez C.
      • Wainwright D.A.
      • Strickland F.M.
      • Overbeck A.
      • et al.
      Reduced skin homing by functional Treg in vitiligo.
      ). Cutaneous CCL22 is normally expressed by macrophages and dendritic cells (
      • Vulcano M.
      • Albanesi C.
      • Stoppacciaro A.
      • Bagnati R.
      • D’Amico G.
      • Struyf S.
      • et al.
      Dendritic cells as a major source of macrophage-derived chemokine/CCL22 in vitro and in vivo.
      ), the abundance of which is not known to be reduced in vitiligo skin (
      • Le Poole I.C.
      • van den Wijngaard R.M.
      • Westerhof W.
      • Das P.K.
      Presence of T cells and macrophages in inflammatory vitiligo skin parallels melanocyte disappearance.
      ,
      • Kroll T.M.
      • Bommiasamy H.
      • Boissy R.E.
      • Hernandez C.
      • Nickoloff B.J.
      • Mestril R.
      • et al.
      (2005). 4-Tertiary butyl phenol exposure sensitizes human melanocytes to dendritic cell-mediated killing: relevance to vitiligo.
      ). 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 (
      • Ho W.Y.
      • Nguyen H.N.
      • Wolfl M.
      • Kuball J.
      • Greenberg P.D.
      In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naïve repertoire.
      ), thus a paucity of Treg may help determine vitiligo development.
      Figure thumbnail gr4
      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 (
      • Eby J.M.
      • Kang H.K.
      • Tully S.T.
      • Bindeman W.E.
      • Peiffer D.S.
      • Chatterjee S.
      • et al.
      CCL22 to activate Treg migration and suppress depigmentation in 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 (
      • Wang Z.
      • Pratts S.G.
      • Zhang H.
      • Spencer P.J.
      • Yu R.
      • Tonsho M.
      • et al.
      Treg depletion in non-human primates using a novel diphtheria toxin-based anti-human CCR4 immunotoxin.
      ). The impact of CCR4 on Treg homing is further accentuated by attempts to block it in cancer (
      • Sugiyama D.
      • Nishikawa H.
      • Maeda Y.
      • Nishioka M.
      • Tanemura A.
      • Katayama I.
      • et al.
      Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor immune responses in humans.
      ). Promoting Treg homing may augment the inhibition of effector T-cell homing using blocking antibodies to CXCR3 (
      • 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.
      ), 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 (
      • Kakadia P.G.
      • Conway B.R.
      Lipid nanoparticles for dermal drug delivery.
      ,
      • Chaudhary C.
      • Garg T.
      Scaffolds. A novel carrier and potential wound healer.
      ,
      • Mitragotri S.
      Immunization without needles.
      ). 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 (
      • Dwivedi M.
      • Kemp E.H.
      • Laddha N.C.
      • Mansuri M.S.
      • Weetman A.P.
      • Begum R.
      Regulatory T cells in vitiligo: implications for pathogenesis and therapeutics.
      ). Though few laboratory studies have addressed this opportunity in vitiligo, the same strategy is under study for the treatment of other autoimmune diseases (
      • Asnagli H.
      • Jacquin M.
      • Belmonte N.
      • Gertner-Dardenne J.
      • Hubert M.F.
      • Sales A.
      • et al.
      Inhibition of noninfectious uveitis using intravenous administration of collagen II-specific type 1 regulatory T cells.
      ,
      • Boardman D.
      • Maher J.
      • Lechler R.
      • Smyth L.
      • Lombardi G.
      Antigen-specificity using chimeric antigen receptors: the future of regulatory T-cell therapy?.
      ). Antigen-specific Tregs may not maintain their function where effector responses are favored (
      • Sarikonda G.
      • Fousteri G.
      • Sachithanantham S.
      • Miller J.F.
      • Dave A.
      • Juntti T.
      • et al.
      BDC12-4.1 T-cell receptor transgenic insulin-specific CD4 T cells are resistant to in vitro differentiation into functional Foxp3+ T regulatory cells.
      ), and local treatment support by chemokines or immunosuppressive agents may be needed (
      • Auriemma M.
      • Brzoska T.
      • Klenner L.
      • Kupas V.
      • Goerge T.
      • Voskort M.
      • et al.
      α-MSH-stimulated tolerogenic dendritic cells induce functional regulatory T cells and ameliorate ongoing skin inflammation.
      ). With important studies ahead, the future holds great promise for the use of regulatory T cell-based therapeutics in vitiligo.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      The studies described herein have been largely supported by NIH/NIAMS R01AR057643 to CLP and SM.

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