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

Journal name:
Nature Medicine
Volume:
20,
Pages:
1043–1049
Year published:
DOI:
doi:10.1038/nm.3645
Received
Accepted
Published online

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 therapies1. Genome-wide association studies (GWAS)2 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.

At a glance

Figures

  1. CD8+NKG2D+ cytotoxic T lymphocytes accumulate in the skin and are necessary and sufficient to induce disease in AA mice.
    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-1–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.

  2. Prevention of AA by blocking antibodies to IFN-[gamma], IL-2 or IL-15R[beta].
    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. (ah) 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. Immunohistochemical 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.

  3. Systemic JAK1/2 or JAK3 inhibition prevents the onset of AA in grafted C3H/HeJ mice.
    Figure 3: Systemic JAK1/2 or JAK3 inhibition prevents the onset of AA in grafted C3H/HeJ mice.

    (aj) 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.

  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.
    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.

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References

  1. Gilhar, A., Etzioni, A. & Paus, R. Alopecia areata. N. Engl. J. Med. 366, 15151525 (2012).
  2. Petukhova, L. et al. Genome-wide association study in alopecia areata implicates both innate and adaptive immunity. Nature 466, 113117 (2010).
  3. Gilhar, A., Ullmann, Y., Berkutzki, T., Assy, B. & Kalish, R.S. Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice. J. Clin. Invest. 101, 6267 (1998).
  4. McElwee, K.J. et al. Transfer of CD8+ cells induces localized hair loss whereas CD4+/CD25 cells promote systemic alopecia areata and CD4+/CD25+cells blockade disease onset in the C3H/HeJ mouse model. J. Invest. Dermatol. 124, 947957 (2005).
  5. Ito, T. et al. Maintenance of hair follicle immune privilege is linked to prevention of NK cell attack. J. Invest. Dermatol. 128, 11961206 (2008).
  6. Sundberg, J.P., Cordy, W.R. & King, L.E. Jr. Alopecia areata in aging C3H/HeJ mice. J. Invest. Dermatol. 102, 847856 (1994).
  7. McElwee, K.J., Boggess, D., King, L.E. Jr. & Sundberg, J.P. Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts. J. Invest. Dermatol. 111, 797803 (1998).
  8. Bertolini, M. et al. Abnormal interactions between perifollicular mast cells and CD8+ T cells may contribute to the pathogenesis of alopecia areata. PLoS ONE 9, e94260 (2014).
  9. Best, J.A. et al. Transcriptional insights into the CD8+ T cell response to infection and memory T cell formation. Nat. Immunol. 14, 404412 (2013).
  10. Bezman, N.A. et al. Molecular definition of the identity and activation of natural killer cells. Nat. Immunol. 13, 10001009 (2012).
  11. Brajac, I., Gruber, F., Petrovecki, M. & Malnar-Dragojevic, D. Interleukin-2 receptor α-chain expression in patients with alopecia areata. Acta Dermatovenerol. Croat. ADC 12, 154156 (2004).
  12. Fehniger, T.A. & Caligiuri, M.A. Interleukin 15: biology and relevance to human disease. Blood 97, 1432 (2001).
  13. Ye, W., Young, J.D. & Liu, C.C. Interleukin-15 induces the expression of mRNAs of cytolytic mediators and augments cytotoxic activities in primary murine lymphocytes. Cell. Immunol. 174, 5462 (1996).
  14. Meresse, B. et al. Reprogramming of CTLs into natural killer-like cells in celiac disease. J. Exp. Med. 203, 13431355 (2006).
  15. Saikali, P., Antel, J.P., Pittet, C.L., Newcombe, J. & Arbour, N. Contribution of astrocyte-derived IL-15 to CD8 T cell effector functions in multiple sclerosis. J. Immunol. 185, 56935703 (2010).
  16. Meresse, B. et al. Coordinated induction by IL15 of a TCR-independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells in celiac disease. Immunity 21, 357366 (2004).
  17. Freyschmidt-Paul, P. et al. Interferon-γ-deficient mice are resistant to the development of alopecia areata. Br. J. Dermatol. 155, 515521 (2006).
  18. Gilhar, A., Kam, Y., Assy, B. & Kalish, R.S. Alopecia areata induced in C3H/HeJ mice by interferon-γ: evidence for loss of immune privilege. J. Invest. Dermatol. 124, 288289 (2005).
  19. Freyschmidt-Paul, P. et al. Reduced expression of interleukin-2 decreases the frequency of alopecia areata onset in C3H/HeJ mice. J. Invest. Dermatol. 125, 945951 (2005).
  20. O'Shea, J.J., Kontzias, A., Yamaoka, K., Tanaka, Y. & Laurence, A. Janus kinase inhibitors in autoimmune diseases. Ann. Rheum. Dis. 72 (suppl. 2), ii111ii115 (2013).
  21. Quintás-Cardama, A. et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood 115, 31093117 (2010).
  22. Ghoreschi, K. et al. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J. Immunol. 186, 42344243 (2011).
  23. Eichler, G.S., Huang, S. & Ingber, D.E. Gene Expression Dynamics Inspector (GEDI): for integrative analysis of expression profiles. Bioinformatics 19, 23212322 (2003).
  24. Verstovsek, S. et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N. Engl. J. Med. 363, 11171127 (2010).
  25. Harrison, C. et al. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N. Engl. J. Med. 366, 787798 (2012).
  26. Punwani, N. et al. Preliminary clinical activity of a topical JAK1/2 inhibitor in the treatment of psoriasis. J. Am. Acad. Dermatol. 67, 658664 (2012).
  27. Paus, R., Nickoloff, B.J. & Ito, T.A. A 'hairy' privilege. Trends Immunol. 26, 3240 (2005).
  28. Waldmann, T.A. The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat. Rev. Immunol. 6, 595601 (2006).
  29. Dolgin, E. Companies hope for kinase inhibitor JAKpot. Nat. Rev. Drug Discov. 10, 717718 (2011).

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Author information

  1. These authors contributed equally to this work.

    • Luzhou Xing,
    • Zhenpeng Dai &
    • Ali Jabbari
  2. These authors jointly directed this work.

    • Angela M Christiano &
    • Raphael Clynes

Affiliations

  1. Department of Pathology, Columbia University, New York, New York, USA.

    • Luzhou Xing &
    • Raphael Clynes
  2. Department of Dermatology, Columbia University, New York, New York, USA.

    • Zhenpeng Dai,
    • Ali Jabbari,
    • Jane E Cerise,
    • Claire A Higgins,
    • Weijuan Gong,
    • Annemieke de Jong,
    • Sivan Harel,
    • Gina M DeStefano,
    • Lisa Rothman,
    • Pallavi Singh,
    • Lynn Petukhova,
    • Julian Mackay-Wiggan,
    • Angela M Christiano &
    • Raphael Clynes
  3. Department of Psychiatry, Columbia University, New York, New York, USA.

    • Jane E Cerise
  4. Department of Epidemiology, Columbia University, New York, New York, USA.

    • Gina M DeStefano
  5. Department of Genetics and Development, Columbia University, New York, New York, USA.

    • Angela M Christiano
  6. Department of Medicine, Columbia University, New York, New York, USA.

    • Raphael Clynes

Contributions

L.X., Z.D. and A.J. were responsible in large part for performing the studies reported herein and participated in the design, execution and interpretation of the data. C.A.H. was responsible for establishing the C3H/HeJ graft model. A.d.J., S.H., G.M.D., L.R. and P.S. were involved in additional molecular and cell biological experiments. W.G. performed immunofluorescence and morphometric studies. L.P. and J.E.C. performed biostatistical analysis of all data sets. J.M.-W. was instrumental in human sample acquisition and analysis. A.M.C. and R.C. were responsible for conception, design, oversight, execution and interpretation of data for this study. All authors contributed to drafts, writing, figure preparation and editing of the final manuscript.

Competing financial interests

Columbia University has filed for intellectual property protection on the treatment of AA with small-molecule JAK inhibitors (PCT/US2011/059029 and PCT/US2013/034688).

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    Supplementary Figures 1–17, Supplementary Tables 1–5 and Supplementary Methods

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