A mouse model of vitiligo with focused epidermal depigmentation requires IFN-γ for autoreactive CD8⁺ T-cell accumulation in the skin

Abstract

Vitiligo is an autoimmune disease of the skin causing disfiguring patchy depigmentation of the epidermis and, less commonly, hair. Therapeutic options for vitiligo are limited, reflecting in part limited knowledge of disease pathogenesis. Existing mouse models of vitiligo consist of hair depigmentation but lack prominent epidermal involvement, which is the hallmark of human disease. They are thus unable to provide a platform to fully investigate disease mechanisms and treatment. CD8(+) T cells have been implicated in the pathogenesis of vitiligo, and expression of IFN-γ is increased in the lesional skin of patients, however, it is currently unknown what role IFN-γ has in disease. Here, we have developed an adoptive transfer mouse model of vitiligo using melanocyte-specific CD8(+) T cells, which recapitulates the human condition by inducing epidermal depigmentation while sparing the hair. Like active lesions in human vitiligo, histology of depigmenting skin reveals a patchy mononuclear infiltrate and single-cell infiltration of the epidermis. Depigmentation is accompanied by accumulation of autoreactive CD8(+) T cells in the skin, quantifiable loss of tyrosinase transcript, and local IFN-γ production. Neutralization of IFN-γ with antibody prevents CD8(+) T-cell accumulation and depigmentation, suggesting a therapeutic potential for this approach.

Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1. Depigmentation targets the epidermis and spares the hair

(a) Within 4–5 wks after treatment, hosts developed patchy depigmentation (arrows) at visible epidermal surfaces, including the ears, nose, feet, tail, and occasionally on the trunk under the hair (photographed at 7 wks). (b) Chronic vitiligo resulted in widespread depigmentation of the epidermis but spared the hair (photographed at 18 wks). (c) Mice with vitiligo were assigned a Vitiligo Score based on the extent of involvement, and a representative result at 5 weeks is shown (Error bars = mean +/− SEM, n=3 control, n=5 vitiligo).

Figure 2. T cells accumulate in the skin during vitiligo

(a) GFP-labeled CD8+ T cells were quantified by flow cytometry of ear skin at the indicated times (error bars = mean +/− SEM, n=3 per time point). Example flow plots of the CD45+ gate are shown. (b) Tail skin from affected mice harvested 5 wks after transfer (mouse) is shown with affected skin from a vitiligo patient (human), revealing a patchy mononuclear infiltrate at the dermal-epidermal junction and epidermal infiltration (arrows). Normal mouse skin is shown at inset, scale bars=50μm. (c) Ears were harvested from mice with vitiligo at 5 wks after induction and GFP+ T cells were visualized with fluorescent microscopy (arrow). Scale bars=100μm. Hairs are visible as short, linear yellow structures due to autofluorescence.

Figure 3. Depigmentation correlates with loss of melanocytes

Ear skin from affected mice was harvested at the indicated times. qRT-PCR revealed a quantifiable decrease in tyrosinase, reflecting the progressive loss of melanocytes during disease (error bars = mean +/− SEM, n=3 per time point).

Figure 4. IFN-γ expression is induced in the skin during vitiligo and is produced by autoreactive CD8+ T cells

(a) qRT-PCR for IFN-γ was performed on ear skin of mice at the indicated times (error bars = mean +/− SEM, n=3 mice per time point). (b) Single-cell suspensions were isolated from skin-draining lymph nodes 7 weeks after vitiligo induction and either left unstimulated, or stimulated with PMEL peptide or PMA and ionomycin as indicated. Following stimulation, cells were stained for IFN-γ, and flow cytometry revealed IFN-γ expression in transferred melanocyte-specific CD8+ T cells (GFP+) and endogenous CD8+ T cells (GFP−). Cells shown are gated on the total CD8+ population, and the percentage shown is of either the total GFP− or GFP+ gate. A representative result is shown.

Figure 5. IFN-γ neutralization prevents depigmentation and melanocyte-specific CD8+ T cell accumulation in the skin

IFN-γ was neutralized through intraperitoneal injection of IFN-γ neutralizing antibody (500 μg) beginning 2 weeks after induction and twice weekly until scored at 5 weeks (error bars = mean +/− SEM, n=4 control, n=5 vitiligo, n=4 vitiligo + IFN-γ blockade). (a) Vitiligo Score is decreased by IFN-γ neutralization. (b–c) The effects of IFN-γ neutralizing antibody on the total numbers of melanocyte-specific CD8+ T cells in the ear (b) and in skin-draining lymph nodes (c).