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

METHODS
Methods and any associated references are available in the online version of the paper.

ONLINE METHODS

Mice
7- to 10-week-old female C57BL/6 and C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME) were used and maintained under specific pathogen-free conditions. Experiments were performed in compliance with institutional guidelines as approved by the Institutional Animal Care and Use Committee of Columbia University Medical Center.

Transfer of alopecia areata using grafted C3H/HeJ skin
Normal-haired C3H/HeJ mice were grafted at 8 weeks of age (during the second telogen) with skin from a C3H/HeJ mouse that developed AA spontaneously, as described previously7. In brief, mice spontaneously affected with AA were euthanized, and full thickness skin grafts of approximately 2 cm in diameter were removed and grafted to normal-haired C3H/HeJ mice. All experiments included 10 grafted mice (5 treated and 5 untreated) and 3 sham grafted mice (mice grafted with autologous grafts). Hair loss typically began at around 4–6 weeks after grafting. No sham grafted mouse developed hair loss.

Flow cytometric analysis of skin and cutaneous lymph nodes
To make a single-cell suspension of mouse skin, fat was removed from the overlying skin in cold PBS and then incubated in collagenase type I (2 mg/ml in PBS) at 32 °C for 75 min. After digestion, the skin was minced in RPMI/10% FBS, filtered through a 70-μM cell strainer and centrifuged at 1100g for 5 min. The pellet was resuspended in RPMI/10% FBS, filtered through a 40-μM cell strainer and spun at 400g for 5 min. The pellet was resuspended in FACS buffer (PBS/5% BSA), DAPI to gate on live cells and staining antibodies (listed in Supplementary Methods). Cutaneous lymph nodes were pooled, minced in RPMI, filtered through a 40-μM cell strainer, centrifuged at 400g for 5 min, stained and analyzed on a BD LSR II flow cytometer.

Transfer of T cell populations into recipient C3H/HeJ mice
For positive selection of T cell populations, lymph node cells were obtained from 5 C3H/HeJ alopecic mice, stained with anti-CD4, anti-CD8 and anti-NKG2D antibodies) and then sorted (BD Influx) to obtain two fractions: CD8+NKG2D+ T cells and CD8+NKG2D− T cells. Antibody dilutions are given in the Supplementary Methods. Three to five 7-week-old C3H/HeJ mice per group were injected subcutaneously with two million sorted cells of each population. For negatively selected populations, NKG2D+ cells were depleted by incubating total lymph node cells from 3 alopecic C3H/HeJ mice with biotinylated anti-NKG2D (CX5) and then with streptavidin-conjugated beads (Miltenyi) before removal on a Miltenyi magnetic column. Five million cells (either CD8/NKG2D-depleted or total lymph node cells) were suspended in 100 ul PBS and transferred into each of 5 mice by subcutaneous injection.

Prevention and treatment studies in mice
For prevention studies, mice were treated beginning on the day of grafting (n = 5–10 mice per group). For anti-IFN-γ experiments, i.p. injections of hamster isotype control IgG or hamster polyclonal anti-IFN-γ IgG (BioXCell) 300 μg in 100 μl PBS were administered ten times weekly for 12 weeks. For anti-IL-2 experiments, i.p. injections of control rat isotype control IgG or simultaneous administration of two anti-IL-2 rat monoclonal antibodies (BioXCell) (250 μg clone S4B6 and 250 μg JES6-1 together in 100 μl PBS) were administered three times weekly for 12 weeks. For anti-IL-15-Rβ experiments, i.p. injections of rat isotype control IgG or anti-IL-15Rβ antibody (Biolegend, clone TM-β1) (200 μg in 100 μl PBS) were administered two times weekly for 12 weeks. For JAK1/2i experiments, mice were administered vehicle (0.5% methylcellulose; Sigma-Aldrich) or vehicle-containing 50 mg/kg of JAK1/2i ruxolitinib (ChemieTek) daily by oral gavage for 12 weeks. For JAK3i experiments, mice were implanted subcutaneously with Alzet osmotic mini-pumps (pumps, model 2004, Durect Corporation) on the back of each mouse to deliver vehicle (poly(ethylene glycol) (PEG)300) or vehicle containing the JAK3i tofacitinib (Abmole) at 15 mg/kg/day for 12 weeks.
For topical treatment studies, grafted mice with long-standing AA (more than 8 weeks) were treated once daily for 12 weeks to affected skin on the dorsal back with vehicle (10% DMSO in Aquaphor) or vehicle containing JAK inhibitors, initially dissolved in DMSO and then diluted 1:10 in Aquaphor, to achieve 0.5% JAKi ointment). Full-thickness skin biopsies were excised from the dorsal surface of each mouse at interim time points, and skin samples were either snap frozen in liquid nitrogen for RNA extraction or snap frozen in OCT for immunostaining. Hair status was examined twice weekly.

Human clinical studies
We initiated a single center, proof-of-concept clinical trial in the Clinical Trials Unit in the Department of Dermatology at the Columbia University Medical Center entitled “An Open-Label Pilot Study to Evaluate the Efficacy of ruxolitinib in Moderate to Severe Alopecia Areata,” ClinicalTrials.gov identifier NCT01950780. The protocol for this intervention trial was reviewed and approved by the Institutional Review Board at Columbia University and conducted under the Declaration of Helsinki principles. Informed consent was received before inclusion in the study. Eligibility criteria included >30% hair loss for at least 3 months in duration with no evidence for hair regrowth at the time of enrollment. The first three treated patients are described here. The ruxolitinib dose was 20 mg orally twice daily for 3–6 months. Skin punch biopsies were obtained at baseline and after 12 weeks of treatment. Consent for photography was obtained for the patients shown in Figure 4 and in the Supplementary Figures 15 and 16.

Immunohistochemistry and immunofluorescence
8 μM acetone-fixed frozen mouse skin or human skin sections were air-dried and stained overnight with anti-mouse or anti-human antibodies (see Supplementary Methods) at 4 °C in a moist chamber. Human hair follicles were microdissected and embedded in OCT compound before sectioning and staining (see Supplementary Methods).

Primary dermal sheath and lymphokine-activated killer (LAK) cell culture
Dermal sheath (DS) cells were isolated from microdissected mouse vibrissa follicles and cultured in 20% FBS DMEM with 5 ng/ml murine FGF (Pepro Tech). LAK cells were generated from bulk splenocytes plated at 4 × 106 in 6-well plates with 50 ng/ml murine IL-15 (Pepro Tech), 50 nM JAK3i (tofacitinib) or 50 ng/ml murine IL-15 plus 50 nM JAK3i and incubated at 37 °C in a 5% CO2 incubator for 96 h.

In vitro cytotoxicity assays
Determination of specific killing of target cells was performed using CFSE-labeled DS cells as targets mixed with different ratios of effector cells incubated for 5 h at 37 °C 5% CO2 with or without neutralizing rat anti-mouse NKG2D antibody (20 ug/ml) (Biolegend, CX5). Specific lysis of DS cells was determined flow cytometrically by measuring cell death of CFSE + DS cells using Annexin V/7-AAD.

Gene expression sample preparation in human and mouse skin and T cells
Total RNA was isolated using the miRNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) with on-column DNA digestion using the RNase-free DNase set (Qiagen, Inc.). For RNA-seq analysis CD3+CD8+CD44+NKG2D+ and CD3+CD8+CD44+NKG2D− cells were flow-sorted from lymph nodes of alopecic C3H/HeJ mice. RNA was extracted as above and prepared for RNA-seq using the TruSeq RNA Sample Prep Kit v2. Samples were sequenced on the HiSeq 2000 sequencer (Illumina, San Diego, CA) for 50 cycles. RNA-seq files were demultiplexed by the Rockefeller University Genomics Core Facility.
For global transcriptional profiling in mouse skin, total extracted RNA was processed using the 3′ IVT Express Kit from Affymetrix. Resulting biotinylated cDNA samples were hybridized to the Mouse Genome 430 2.0 gene chips and subsequently washed, stained with streptavidin-phycoerythrin and scanned on an HP GeneArray Scanner (Hewlett-Packard Company, Palo Alto, CA). For gene expression studies, mice grafted with autologous healthy skin were included as sham-operated controls.
For human AA samples, perilesional punch biopsies from 5 patients with patchy alopecia areata who were not undergoing local or systemic treatments were collected and compared to scalp biopsies from 5 unrelated unaffected individuals. All procedures were performed under Institutional Review Board–approved protocols at Columbia University and conducted under the Declaration of Helsinki principles. Informed consent was received before inclusion in the study. Extracted total RNA was reverse transcribed and amplified using the Ovation RNA Amplification V2 kit (NuGEN Technologies, Inc., San Carlos, CA). Amplified cDNA was biotinylated with the Encore Biotin Module (NuGEN Technologies) and then hybridized to the U133 Plus 2.0 gene chips. RT-PCR confirmations done as described in the Supplementary Methods. Primer sequences are given in Supplementary Tables 4 and 5.

Statistical analyses
Statistical methods for each figure are given in the Supplementary Methods. No statistical method was used to predetermine sample size. The investigators were not blinded to allocation during experiments and outcome assessment. The experiments were not randomized.

Supplementary Material
Supplementary Note