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CXCR3 Depleting Antibodies Prevent and Reverse Vitiligo in Mice

Open ArchivePublished:January 23, 2017DOI:https://doi.org/10.1016/j.jid.2016.10.048

      Abbreviations:

      Ab (antibody), PMEL (premelanosome protein), WT (wild type)
      To the Editor
      Vitiligo is a disfiguring skin disease in which melanocytes with intrinsic abnormalities are targeted and destroyed by autoreactive CD8+ T cells in the epidermis, resulting in patchy depigmentation (
      • Palermo B.
      • Campanelli R.
      • Garbelli S.
      • Mantovani S.
      • Lantelme E.
      • Brazzelli V.
      • et al.
      Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: the role of cellular immunity in the etiopathogenesis of vitiligo.
      ,
      • 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.
      , and reviewed in
      • Richmond J.M.
      • Frisoli M.L.
      • Harris J.E.
      Innate immune mechanisms in vitiligo: danger from within.
      ). Although it is one of the most common autoimmune diseases, affecting 1% of the population worldwide, there are no Food and Drug Administration-approved treatments. Previous work from our lab has shown that CD8+ T-cell recruitment to the skin in a mouse model of vitiligo is dependent on IFNγ (
      • 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-gamma for autoreactive CD8(+) T-cell accumulation in the skin.
      ) and the downstream CXCR3 chemokine system (
      • 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.
      ). We also demonstrated enrichment of CXCR3 on antigen-specific T cells in the blood of patients with vitiligo compared with healthy controls, and we and others have shown the presence of CXCR3+ cells in skin biopsies from patients with vitiligo (
      • Bertolotti A.
      • Boniface K.
      • Vergier B.
      • Mossalayi D.
      • Taieb A.
      • Ezzedine K.
      • et al.
      Type I interferon signature in the initiation of the immune response in vitiligo.
      ,
      • 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.
      ,
      • Wang X.
      • Wang Q.
      • Wu J.
      • Jiang M.
      • Chen L.
      • Zhang C.
      • et al.
      Increased expression of CXCR3 and its ligands in vitiligo patients and CXCL10 as a potential clinical marker for vitiligo.
      ). Therefore, we sought to determine if targeting CXCR3 could serve as a new treatment for vitiligo.
      We tested different strategies of targeting CXCR3, including blocking and depleting antibodies (Abs), in our mouse model of vitiligo. All mice used for vitiligo studies were on a C57BL/6J background and maintained in pathogen-free facilities at University of Maryland Medical System, and procedures were approved by the University of Maryland Medical System Institutional Animal Care and Use Committee and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (see Supplementary Materials and Methods online for detailed procedures). We first examined whether our candidate molecules could prevent disease in mice by treating animals three times weekly with 100 μg Ab i.p. from weeks 2 to 7 after disease induction. This time period is significant because it occurs after clearance of the virus used to induce disease, but before the onset of autoimmunity. We compared isotype control Ab with a commercially available hamster CXCR3 Ab (depleting; see Supplementary Figure S1 online), a wild-type (WT) mouse CXCR3 Ab (superior depleting), and a mutated mouse CXCR3 Ab called deltaAB (neutralizing) (Figure 1a). Biacore binding data revealed that all Abs had a similar affinity for CXCR3 (Supplementary Figure S2 online). We found that mouse depleting Ab performed the best in preventing clinical disease (Figure 1b). This observation is consistent with data indicating that the WT mouse CXCR3 Ab has better depleting activity than the hamster CXCR3 Ab (Supplementary Figure S1 and data not shown).
      Figure 1
      Figure 1CXCR3 depleting antibodies have the greatest efficacy in prevention of vitiligo in mice. (a) CXCR3 neutralizing (deltaAB [ΔAB]) or depleting (hamster or WT mouse) antibodies were compared in prevention of vitiligo beginning 2 weeks after disease induction. (b) WT mouse CXCR3 depleting Ab performed the best in preventing vitiligo as evidenced by significantly reduced scores 5 weeks after treatment (one-way analysis of variance P = 0.0066, Dunnett’s posttests vs. isotype ns; Tukey’s posttests significant for ΔAB vs. WT and ΔAB vs. hamster). (c) PMEL numbers were significantly reduced in epidermis after treatment with any of the CXCR3 Abs (two-way analysis of variance P < 0.0001 with Bonferroni’s posttests compared with isotype control) and trended toward a reduction in the dermis. Differences in lymph node, spleen, and blood were not significant. (d) Bystander host CD8+ T-cell numbers were significantly reduced in the spleen and LN by hamster or WT mouse CXCR3 Ab treatment, but not in the skin (two-way analysis of variance P < 0.0001 with Bonferroni’s posttests compared with isotype control). (e) Total numbers of CD45+ cells were unchanged after treatment with any of the CXCR3 Abs. (Representative experiment in which all Ab classes were tested at one time; individual treatments vs. isotype control have been repeated two or three times with similar results.) Ab, antibody; LN, lymph node; ns, nonsignificant; PMEL, premelanosome protein; WT, wild type.
      We analyzed our premelanosome protein-specific CD8+ T-cell (called PMEL;
      • 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.
      ) numbers in treated mouse tissues (see Supplementary Figure S3 online for gating strategy). All Abs tested in vitiligo prevention in mice resulted in fewer PMELs in the skin (Figure 1c). However, despite its ability to reduce PMEL number in the skin, neutralizing Ab was less effective than depleting Abs, possibly due to the fact that a low threshold number of PMELs is sufficient for full clinical disease. We observed a similar result in CXCL9-deficient mice (
      • 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.
      ). PMEL numbers were not significantly affected in lymph nodes, whereas treatment with the hamster or WT mouse Ab resulted in fewer PMELs in the spleen and blood, likely due to the fact that depletion is most efficient in these sites (
      • Morelli A.E.
      • Larregina A.T.
      • Shufesky W.J.
      • Zahorchak A.F.
      • Logar A.J.
      • Papworth G.D.
      • et al.
      Internalization of circulating apoptotic cells by splenic marginal zone dendritic cells: dependence on complement receptors and effect on cytokine production.
      ) (Figure 1c). We assessed the effect of CXCR3 depleting Ab on host T cells by measuring the number of host CD8+ T cells and total CD45+ cells in all tissues. The total number of host CD8+ T cells in lymphoid organs and in blood were reduced (Figure 1d); however, total CD45+ cell numbers were not significantly affected during this 5-week treatment period (Figure 1e).
      Because the CXCR3 depleting Abs were the most effective in preventing clinical disease in animals, we evaluated their efficacy in reversal of clinical disease to determine their therapeutic potential. We selected vitiligo mice with >75% depigmentation on their tails and began treating with our candidate Ab 12 weeks after disease induction, when disease was stable. Mice received treatments for a total of 7 to 8 weeks (Figure 2a). WT mouse CXCR3 Ab treatment significantly reversed clinical disease (Figure 2b–e) and reduced PMEL numbers in the epidermis (Figure 2f). The repigmentation pattern was perifollicular, similar to human clinical responses to treatment. Host CD8+ T-cell numbers were slightly reduced in treated mice (Figure 2g), though total CD45+ cell numbers were unchanged (Figure 2h). To determine potential depletion of endogenous immune cells, we performed detailed analysis of spleen populations in WT, unaffected animals. Intravenous administration of one bolus of WT mouse Ab reduced the numbers of host T-cell populations, which is unsurprising considering that these cells may also express CXCR3 (Supplementary Figure S4a–d online). In human vitiligo, multiple T-cell clones likely contribute to disease; therefore, broader depletion of CXCR3+ T-cell pools could be particularly beneficial for disease treatment. We also assessed the effects of CXCR3 Ab treatment on other immune cell populations. Granulocytes and CD4+ natural killer T cells were not significantly affected, indicating that global immunosuppression with WT mouse Ab treatment is unlikely (Supplementary Figure S4e–l). Taken together, these data indicate that CXCR3 depleting Ab can reduce autoreactive T-cell numbers and reverse disease, while having some impact on other compartments of the immune system.
      Figure 2
      Figure 2CXCR3 depleting antibodies reverse vitiligo in mice. (a) WT mouse CXCR3 depleting Ab was tested in reversal of disease. (b) Representative images from tails of vitiligo mice at baseline and after 8 weeks of treatment with isotype or WT mouse Ab. Repigmentation analysis was performed on images for the dorsal and ventral view of each tail. (c) Percent tail pigmentation of isotype or (d) WT mouse CXCR3 Ab before and after treatment. (e) Comparison of the percent change in total pigmentation reveals that WT mouse Ab treatment induces significantly greater repigmentation in all treated animals compared with isotype control. (f) WT mouse Ab treatment reduced PMEL numbers in the skin (two-way analysis of variance P = 0.0402; Bonferroni posttests ns due to differences in skin engraftment levels between both trials; Student’s t-tests were then used to compare treatment for each tissue in each trial, and results were significant as follows: epidermis P = 0.0104 for experiment 1 and P = 0.0166 for experiment 2; dermis P = 0.0414 for experiment 1 and P = 0.008 for experiment 2). (g) There were significantly fewer bystander host CD8+ T cells in the LNs after WT mouse Ab treatment (two-way analysis of variance P = 0.026 with Bonferroni posttests; Student’s t-tests were ns for skin in each trial). (h) There were no significant differences in total CD45+ cells in any tissues (n = 6 mice per group pooled from two separate experiments). Ab, antibody; LN, lymph node; ns, nonsignificant; PMEL, premelanosome protein; WT, wild type.
      Although preliminary, the data provide further rationale for targeting CXCR3 in vitiligo. The quantity of repigmentation induced by CXCR3 depleting Abs outperformed other treatments we have previously explored in this model (
      • Agarwal P.
      • Rashighi M.
      • Essien K.I.
      • Richmond J.M.
      • Randall L.
      • Pazoki-Toroudi H.
      • et al.
      Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo.
      ,
      • 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.
      ). A depleting Ab may have greater clinical efficacy and durability than chemokine neutralizing Abs by removing autoreactive cells rather than blocking their migration, requiring the generation of new effectors to reestablish disease.
      This is especially important in light of controversial evidence surrounding the CXCR3 axis in other autoimmune disease models including type 1 diabetes and multiple sclerosis. Specifically, CXCR3-deficient hosts have been shown to develop accelerated diabetes (
      • Yamada Y.
      • Okubo Y.
      • Shimada A.
      • Oikawa Y.
      • Yamada S.
      • Narumi S.
      • et al.
      Acceleration of diabetes development in CXC chemokine receptor 3 (CXCR3)-deficient NOD mice.
      ), and CXCR3−/− and CXCL10−/− mice develop worse experimental autoimmune encephalomyelitis than WT mice (
      • Klein R.S.
      • Izikson L.
      • Means T.
      • Gibson H.D.
      • Lin E.
      • Sobel R.A.
      • et al.
      IFN-inducible protein 10/CXC chemokine ligand 10-independent induction of experimental autoimmune encephalomyelitis.
      ,
      • Muller M.
      • Carter S.L.
      • Hofer M.J.
      • Manders P.
      • Getts D.R.
      • Getts M.T.
      • et al.
      CXCR3 signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory T cells in the central nervous system.
      ). CXCL10 blockade either had no effect, or reportedly worsened a mouse model of diabetes when administered very early in the disease course, and T-cell migration to the pancreas was normal in the absence of CXCL10 signaling (
      • Coppieters K.T.
      • Amirian N.
      • Pagni P.P.
      • Baca Jones C.
      • Wiberg A.
      • Lasch S.
      • et al.
      Functional redundancy of CXCR3/CXCL10 signaling in the recruitment of diabetogenic cytotoxic T lymphocytes to pancreatic islets in a virally induced autoimmune diabetes model.
      ). However, CXCL10 blockade in experimental autoimmune encephalomyelitis mitigated disease (
      • Fife B.T.
      • Kennedy K.J.
      • Paniagua M.C.
      • Lukacs N.W.
      • Kunkel S.L.
      • Luster A.D.
      • et al.
      CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis.
      ). These data suggest that CXCL10-CXCR3 signaling in autoimmunity is nuanced, and therefore considering CXCL10-CXCR3 signaling as an “all-or-none” phenomenon is too simplistic. However, depleting Abs may remove all CXCR3+ T cells, including pathogenic T effector cells in autoimmunity, thereby potentially “resetting” the disease and possibly influencing tolerance.
      Other examples of autoimmune diseases in which depleting Abs have demonstrated good efficacy are rituximab (anti-CD20) for multiple sclerosis (
      • Hauser S.L.
      • Waubant E.
      • Arnold D.L.
      • Vollmer T.
      • Antel J.
      • Fox R.J.
      • et al.
      B-cell depletion with rituximab in relapsing-remitting multiple sclerosis.
      ) and systemic lupus erythematosus (
      • Anolik J.H.
      • Aringer M.
      New treatments for SLE: cell-depleting and anti-cytokine therapies.
      ), and alemtuzumab (anti-CD52) for multiple sclerosis (
      • Coles A.J.
      • Compston D.A.
      • Selmaj K.W.
      • Lake S.L.
      • Moran S.
      • et al.
      Camms Trial Investigators
      Alemtuzumab vs. interferon beta-1a in early multiple sclerosis.
      ). Vitiligo may be protective against melanoma and nonmelanoma skin cancers (
      • Paradisi A.
      • Tabolli S.
      • Didona B.
      • Sobrino L.
      • Russo N.
      • Abeni D.
      Markedly reduced incidence of melanoma and nonmelanoma skin cancer in a nonconcurrent cohort of 10,040 patients with vitiligo.
      ,
      • Teulings H.E.
      • Overkamp M.
      • Ceylan E.
      • Nieuweboer-Krobotova L.
      • Bos J.D.
      • Nijsten T.
      • et al.
      Decreased risk of melanoma and nonmelanoma skin cancer in patients with vitiligo: a survey among 1307 patients and their partners.
      ). Immunosuppression increases the risk of melanoma and nonmelanoma skin cancers (
      • Long M.D.
      • Martin C.F.
      • Pipkin C.A.
      • Herfarth H.H.
      • Sandler R.S.
      • Kappelman M.D.
      Risk of melanoma and nonmelanoma skin cancer among patients with inflammatory bowel disease.
      ); therefore, this will be important to consider during the development of future treatments for vitiligo. Future studies examining safety and efficacy in patients will need to be conducted to determine the applicability of this treatment strategy for vitiligo.

      Conflict of Interest

      MEY, EM, RC, and JT are employed by Sanofi-Genzyme, and these studies were funded in part through this company.

      Acknowledgments

      We thank Robert Schreiber (Washington University School of Medicine) for the CXCR3-173 cell line, and members of the Harris Lab for technical assistance. This work was funded by Dermatology Foundation Stiefel Award, NIH K08, Kawaja Vitiligo Research Initiative, & Vitiligo Research Foundation (to JEH), and American Skin Association Research Grant and Calder Research Scholar Award (to JMR). These studies were funded in part by Sanofi-Genzyme. Flow cytometry equipment used for this study is maintained by the UMMS Flow Cytometry Core Facility.

      Supplementary Material

      References

        • Agarwal P.
        • Rashighi M.
        • Essien K.I.
        • Richmond J.M.
        • Randall L.
        • Pazoki-Toroudi H.
        • et al.
        Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo.
        J Invest Dermatol. 2015; 135: 1080-1088
        • Anolik J.H.
        • Aringer M.
        New treatments for SLE: cell-depleting and anti-cytokine therapies.
        Best Pract Res Clin Rheumatol. 2005; 19: 859-878
        • Bertolotti A.
        • Boniface K.
        • Vergier B.
        • Mossalayi D.
        • Taieb A.
        • Ezzedine K.
        • et al.
        Type I interferon signature in the initiation of the immune response in vitiligo.
        Pigment Cell Melanoma Res. 2014; 27: 398-407
        • Coles A.J.
        • Compston D.A.
        • Selmaj K.W.
        • Lake S.L.
        • Moran S.
        • et al.
        • Camms Trial Investigators
        Alemtuzumab vs. interferon beta-1a in early multiple sclerosis.
        N Engl J Med. 2008; 359: 1786-1801
        • Coppieters K.T.
        • Amirian N.
        • Pagni P.P.
        • Baca Jones C.
        • Wiberg A.
        • Lasch S.
        • et al.
        Functional redundancy of CXCR3/CXCL10 signaling in the recruitment of diabetogenic cytotoxic T lymphocytes to pancreatic islets in a virally induced autoimmune diabetes model.
        Diabetes. 2013; 62: 2492-2499
        • Fife B.T.
        • Kennedy K.J.
        • Paniagua M.C.
        • Lukacs N.W.
        • Kunkel S.L.
        • Luster A.D.
        • et al.
        CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis.
        J Immunol. 2001; 166: 7617-7624
        • 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-gamma for autoreactive CD8(+) T-cell accumulation in the skin.
        J Invest Dermatol. 2012; 132: 1869-1876
        • Hauser S.L.
        • Waubant E.
        • Arnold D.L.
        • Vollmer T.
        • Antel J.
        • Fox R.J.
        • et al.
        B-cell depletion with rituximab in relapsing-remitting multiple sclerosis.
        N Engl J Med. 2008; 358: 676-688
        • Klein R.S.
        • Izikson L.
        • Means T.
        • Gibson H.D.
        • Lin E.
        • Sobel R.A.
        • et al.
        IFN-inducible protein 10/CXC chemokine ligand 10-independent induction of experimental autoimmune encephalomyelitis.
        J Immunol. 2004; 172: 550-559
        • Long M.D.
        • Martin C.F.
        • Pipkin C.A.
        • Herfarth H.H.
        • Sandler R.S.
        • Kappelman M.D.
        Risk of melanoma and nonmelanoma skin cancer among patients with inflammatory bowel disease.
        Gastroenterology. 2012; 143 (e1): 390-399
        • Morelli A.E.
        • Larregina A.T.
        • Shufesky W.J.
        • Zahorchak A.F.
        • Logar A.J.
        • Papworth G.D.
        • et al.
        Internalization of circulating apoptotic cells by splenic marginal zone dendritic cells: dependence on complement receptors and effect on cytokine production.
        Blood. 2003; 101: 611-620
        • Muller M.
        • Carter S.L.
        • Hofer M.J.
        • Manders P.
        • Getts D.R.
        • Getts M.T.
        • et al.
        CXCR3 signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory T cells in the central nervous system.
        J Immunol. 2007; 179: 2774-2786
        • 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.
        J Exp Med. 2003; 198: 569-580
        • Palermo B.
        • Campanelli R.
        • Garbelli S.
        • Mantovani S.
        • Lantelme E.
        • Brazzelli V.
        • et al.
        Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: the role of cellular immunity in the etiopathogenesis of vitiligo.
        J Invest Dermatol. 2001; 117: 326-332
        • Paradisi A.
        • Tabolli S.
        • Didona B.
        • Sobrino L.
        • Russo N.
        • Abeni D.
        Markedly reduced incidence of melanoma and nonmelanoma skin cancer in a nonconcurrent cohort of 10,040 patients with vitiligo.
        J Am Acad Dermatol. 2014; 71: 1110-1116
        • 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.
        Sci Transl Med. 2014; 6: 223ra23
        • Richmond J.M.
        • Frisoli M.L.
        • Harris J.E.
        Innate immune mechanisms in vitiligo: danger from within.
        Curr Opin Immunol. 2013; 25: 676-682
        • Teulings H.E.
        • Overkamp M.
        • Ceylan E.
        • Nieuweboer-Krobotova L.
        • Bos J.D.
        • Nijsten T.
        • et al.
        Decreased risk of melanoma and nonmelanoma skin cancer in patients with vitiligo: a survey among 1307 patients and their partners.
        Br J Dermatol. 2013; 168: 162-171
        • 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.
        J Invest Dermatol. 2009; 129: 2220-2232
        • Wang X.
        • Wang Q.
        • Wu J.
        • Jiang M.
        • Chen L.
        • Zhang C.
        • et al.
        Increased expression of CXCR3 and its ligands in vitiligo patients and CXCL10 as a potential clinical marker for vitiligo.
        Br J Dermatol. 2016; 174: 1318-1326
        • Yamada Y.
        • Okubo Y.
        • Shimada A.
        • Oikawa Y.
        • Yamada S.
        • Narumi S.
        • et al.
        Acceleration of diabetes development in CXC chemokine receptor 3 (CXCR3)-deficient NOD mice.
        Diabetologia. 2012; 55: 2238-2245