Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition

Abstract

Alopecia areata (AA) is a common autoimmune disease resulting from damage of the hair follicle by T cells. The immune pathways required for autoreactive T cell activation in AA are not defined limiting clinical development of rational targeted therapies. Genome-wide association studies (GWAS) implicated ligands for the NKG2D receptor (product of the KLRK1 gene) in disease pathogenesis. Here, we show that cytotoxic CD8(+)NKG2D(+) T cells are both necessary and sufficient for the induction of AA in mouse models of disease. Global transcriptional profiling of mouse and human AA skin revealed gene expression signatures indicative of cytotoxic T cell infiltration, an interferon-γ (IFN-γ) response and upregulation of several γ-chain (γc) cytokines known to promote the activation and survival of IFN-γ-producing CD8(+)NKG2D(+) effector T cells. Therapeutically, antibody-mediated blockade of IFN-γ, interleukin-2 (IL-2) or interleukin-15 receptor β (IL-15Rβ) prevented disease development, reducing the accumulation of CD8(+)NKG2D(+) T cells in the skin and the dermal IFN response in a mouse model of AA. Systemically administered pharmacological inhibitors of Janus kinase (JAK) family protein tyrosine kinases, downstream effectors of the IFN-γ and γc cytokine receptors, eliminated the IFN signature and prevented the development of AA, while topical administration promoted hair regrowth and reversed established disease. Notably, three patients treated with oral ruxolitinib, an inhibitor of JAK1 and JAK2, achieved near-complete hair regrowth within 5 months of treatment, suggesting the potential clinical utility of JAK inhibition in human AA.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare competing financial interests: details are available in the online version of the paper.

Figures

Figure 1

CD8+NKG2D+ cytotoxic T lymphocytes accumulate in the skin and are necessary and sufficient to induce disease in AA mice. (a) Immunofluorescence staining of NKG2D ligand (H60) in the hair follicle inner root sheath (marked by K71). Scale bar, 100 μm. (b) CD8+NKG2D+ cells in hair follicles of C57BL/6, healthy C3H/HeJ and C3H/HeJ AA mice. Top scale bar, 100 μm; bottom scale bar, 50 μm. (c) Cutaneous lymphadenopathy and hypercellularity in C3H/HeJ AA mice. (d) Frequency (number shown above boxed area) of CD8+NKG2D+ T cells in the skin and skin-draining lymph nodes in alopecic mice versus ungrafted mice. (e) Immunophenotype of CD8+NKG2D+ T cells in cutaneous lymph nodes of C3H/HeJ alopecic mice. (f) Left, Rae-1t–expressing dermal sheath cells grown from C3H/HeJ hair follicles. Right, dose-dependent specific cell lysis induced by CD8+NKG2D+ T cells isolated from AA mice cutaneous lymph nodes in the presence of blocking anti-NKG2D antibody or isotype control. Effector to target ratio given as indicated. Data are expressed as means ± s.d. (g) Hair loss in C3H/HeJ mice injected subcutaneously with total lymph node (LN) cells, CD8+NKG2D+ T cells alone, CD8+NKG2D− T cells or lymph node cells depleted of NKG2D+ (5 mice per group). Mice are representative of two experiments. ***P < 0.001 (Fisher’s exact test). For c,d,f, n and number of repeats are detailed in the Supplementary Methods.

Figure 2

Prevention of AA by blocking antibodies to IFN-γ, IL-2 or IL-15Rβ. C3H/HeJ grafted mice were treated systemically from the time of grafting. (a–h) AA development in C3H/HeJ grafted mice treated systemically from the time of grafting with antibodies to IFN-γ (a,b), IL-2 (d,e) and IL-15Rβ (g,h). Frequency (number shown above boxed area) of CD8+NKG2D+ T cells in the skin of mice treated with antibodies to IFN-γ (b), IL-2 (e) and IL-15Rβ (h) compared to PBS-treated mice. (*P < 0.05, **P < 0.01, ***P < 0.001, statistical methods described in the Supplementary Methods. Immunohistochemica staining of skin biopsies showing CD8 and MHC class I and II expression in skin of mice treated with isotype control antibody or with antibodies to IFN-γ (c), IL-2 (f) or IL-15Rβ (i). Scale bars, 100 μm. For each experiment, n and number of repeats are detailed in the Supplementary Methods.

Figure 3

Systemic JAK1/2 or JAK3 inhibition prevents the onset of AA in grafted C3H/HeJ mice. (a–j) AA development in C3H/HeJ grafted mice treated systemically from the time of grafting with ruxolitinib (JAK1/2i) (a,b) or tofacitinib (JAK3i) (f,g) (**P < 0.01). Frequency (number shown above boxed area) of CD8+NKG2D+ T cells in skin and cutaneous lymph nodes of mice treated with PBS or with JAK1/2i (c) or JAK3i (h) (***P < 0.001, statistical methods described in Supplementary Methods). Immunohistochemical staining of skin biopsies showing CD8 and MHC class I and II expression in skin of mice treated with PBS or with JAK1/2i (d) or JAK3i (i). ALADIN score of transcriptional analysis from mice treated with PBS or with JAK1/2i (e) or JAK3i (j), given as log2 mean expression Z-scores as indicated in the Supplementary Methods. Hair regrowth after an additional 12 weeks after treatment withdrawal is also shown. (a,f). Scale bars, 100 μm. For each experiment, n and number of repeats are detailed in the Supplementary Methods.

Figure 4

Reversal of established AA with topical small-molecule inhibitors of the downstream effector kinases JAK1/2 or JAK3, and clinical results of patients with AA. (a) Three mice per group with long-standing AA (at least 12 weeks after grafting) treated topically on the dorsal back with 0.5% JAK1/2i (center), 0.5% JAK3i (bottom) or vehicle alone (Aquaphor, top) by daily application for 12 weeks. This experiment was repeated three times. Hair regrowth at an additional 8 weeks after treatment withdrawal is also shown. (b) Time course of hair regrowth index shown as weeks after treatment. (c) The frequency (number shown above boxed area) of CD8+NKG2D+ T cells in the skin of mice treated with JAK1/2i or JAK3i compared to vehicle control mice (mean ± s.e.m., n = 3 per group, *P < 0.05, **P < 0.01, statistical methods described in the Supplementary Methods). NS, not significant. (d) The ALADIN score shows treatment-related loss of CTL and IFN signatures, given as log2 mean expression Z-scores as indicated in the Supplementary Methods. (e) Immunohistochemical staining of mouse skin biopsies shows treatment-related loss of expression of CD8 and MHC class I and II markers. Scale bar, 100 μm. (f) Treatment of patient 3 with AA, who had hair loss involving >80% of his scalp at baseline, with ruxolitinib and hair regrowth after 12 weeks of oral treatment. (g) Clinical correlative studies of biopsies obtained before treatment (baseline) and after 12 weeks of treatment of patient 2, including immunostains for CD4, CD8 and human leukocyte antigen (HLA) class I (A, B, C) and class II (DP, DQ, DR). Scale bar, 200 μm. (h,i) RNA microarray analysis from treated patients 1 and 2 with AA (before treatment versus after treatment versus 3 normal subjects) presented as a heatmap (h) and as a cumulative ALADIN index (i). KRT, hair follicle keratins.