Extended Data Fig. 2: Characterization of the melanoma–Treg-induced vitiligo mouse model.
From: Anatomically distinct fibroblast subsets determine skin autoimmune patterns
a, Schematic diagram of the melanoma/Treg-induced vitiligo mouse model. First the dorsal skin in the right flank of 8–9-week old C57 mice was inoculated with B16F10 melanoma cells (Day 0), then CD4 depletion antibodies were injected on Days 4 and 10. The tumour was surgically removed on Day 12 to prevent interference of subsequent analysis. b, Representative hair coat images of control and vitiligo induced mice at Day 300 after the vitiligo induction procedure. At 4 weeks after induction, dorsal skin hair follicles close to the B16F10 tumour cell injection and surgical removal sites started to show depigmentation as a result of wounding-induced new hair growth. Then the depigmented hair follicles expanded and eventually rendered the whole dorsal hair coat depigmented at ~Day 300. c, Representative tail skin images of mice at Day 0, 45, 60, 90, 120, 240, 360, 480, 720 post melanoma/Treg-induced vitiligo induction procedure. Melanocytes in mouse dorsal skin are located in hair follicles but not in the epidermis; only in mouse tail skin are melanocytes located in both the hair follicle and epidermis similar to human skin. Skin epidermis depigmentation is the defining feature of vitiligo pathology. Therefore, we only used mouse tail skin for vitiligo analysis. In tail skin, the depigmentation was initially patchy and then progressed to eventually cover the entire epidermal surface, which did not recover even more than a year later. d, Representative whole-mount immunofluorescent staining and density plot images of DCT+ melanocytes and CD8+ T cells in mouse tail skin epidermis at Day 0, 19, 26, 33 after the vitiligo induction procedure. Prior to vitiligo induction, very few if any CD8+ T cells could be detected in epidermis. Starting from Day 19 after the vitiligo induction procedure, CD8+ T cells infiltration and small regions of melanocyte loss could be observed. Note, melanocytes loss only occurred in regions where CD8+ T cells locally aggregated into clusters; as the CD8+ T cells clusters continuously expanded so did the regions of melanocyte loss correspondingly. e, FACS quantifications of CD45+CD3+CD8+ T cells and CD117+ melanocyte in mice with or without vitiligo induction confirmed the loss of melanocytes and enrichment of CD8+ T cells after vitiligo induction. f, Representative whole-mount immunofluorescent staining images and quantifications of DCT+ melanocytes and CD8+ T cells in mouse tail skin epidermis with combined or individual B16F10 inoculation and CD4 depletion antibody treatment, showing the melanoma/Treg-induced vitiligo model requires both B16F10 inoculation and CD4 depletion antibody injection. g, Schematic diagram, representative whole-mount immunofluorescent staining images and quantification of DCT+ melanocytes and CD8+ T cells in the skin epidermis of melanoma/Treg-induced vitiligo model with or without CD8 depletion antibody treatment, showing CD8+ T cells are responsible for melanocyte loss in the melanoma/Treg-induced vitiligo mouse model. h, i, Representative whole-mount images (h) and FACS profiles (i) with corresponding quantification of the percentage of CD8+ T cells that express CD3 or CD11c in mouse epidermis, showing majority of the CD8+ cells are CD3+ T cells, but not CD11c+ dendritic cells. j, Representative whole-mount immunofluorescent staining images and density plot images of CD8+ T cells and DCT+ melanocytes in tail skin epidermis at Day 19, 26, 33 after vitiligo induction procedure. Note the continuously expanding CD8+ T cell clusters in skin epidermis. k, Representative whole-mount immunofluorescent staining images and quantifications of Ki67+, CD3+ T cells, and DCT+ melanocytes in tail skin epidermis at Day 33 after vitiligo induction procedure. Enlarged image on the left represents border of lesion skin, the right one represents lesion region. Quantification showed the proliferation rates of CD8+ T cells at the border versus inside the clusters are equivalent; and percentage of Ki67+ cells in melanocytes, T cells and keratinocytes showed majority of the proliferating cells in skin epidermis are keratinocytes. These data indicated that the continuous expansion of CD8+ T cell clusters mainly results from skin-infiltrated CD8+ T cells being actively recruited into the border regions. Scale bars, 500 µm (c, d, f, g, j), 100 µm (h, k). For exact p values, see Source Data. For statistics, p summary and sample sizes, see Methods.
