Patients and clinical samples
Fresh peripheral blood mononuclear cells (PBMCs) were collected from patients with diagnosed with vitiligo at active phase according to clinical manifestations and Wood’s lamp test in the Department of Dermatology of Xijing Hospital and healthy individuals at the Physical Examination Center of Xijing Hospital of the Fourth Military Medical University. 32 patients (3 for the determination of cell IC50 and cell viability of CD8+ T cells, 3 for target prediction, 6 for evaluating of the proliferation and activation of CD8+ T cells, 6 for evaluating the effector function of CD8+ T cells, 6 for the migration of CD8+ T cells, 6 for the detection of chemokine receptors in CD8+ T cells) and sex- and age-matched 15 healthy volunteers (3 for the determination of cell IC50 and cell viability of CD8+ T cells, 6 for evaluating the proliferation and activation of CD8+ T cells, 6 for evaluating the effector function of CD8+ T cells) were recruited in this study. The research protocols for referring to human samples were performed according to the principles of the Declaration of Helsinki. The study was approved by the Ethics Committee of the Fourth Military Medical University, and written informed consent was obtained from all participants.
Induction of vitiligo mice and treatment
All animal studies were reviewed and approved by the Ethics Committee of Animal Care of the Fourth Military Medical University (FMMU). All animal experiments were conducted according to the “Guidelines for Animal. Specific pathogen-free (SPF) female C57BL/6 mice (8-10 weeks old, 18-20 g) were obtained from the Laboratory Animal Center of FMMU. All mice were housed in micro isolator cages in an SPF setting at 24 ± 2˚C and exposed to a 12 h light/12 h dark cycle, with standard feed and water provided ad libitum at the Experimental Animal Center of FMMU.
C57Bl/6 mice were intradermal injected with 2×105 B16F10 cells (B16 melanoma cell line of C57BL/6 mouse origin) in 0.1 ml PBS per mouse (cell viability was more than 96% characterized by harvesting after limited passage in vitro and off-white to white color of cell mass after centrifugation) into the left-back on day 0, and then treated with anti-mouse CD4 antibody (clone, GK1.5, Biolgend) intraperitoneal (i.p.) injection on day 4 and day 10 at 10 ng/kg (only mice with a tumor larger than 1mm in diameter on day 4). CD4+ T cell preferential depletion from mice with advanced melanoma can activate CD8+ T cells against B16 melanoma cells resulting in the destruction of the melanocytes in hair bulbs due to cross-antigenicity between both cell types [24]. On day 12, primary tumors were surgically excised from the skin and the incision was closed (performed on mice with no subcutaneous infiltration). Mice were monitored every other day to ensure no tumor metastases and recurrent primary tumors, and 85% of mice showed vitiligo clinical signs after 40-50 days. The extent of depigmentation was objectively quantified with the percentage of the anatomic site (tail) using Image J software (images converted to 8-bit black on pigmented areas and white on depigmented areas).
Treatments were administrated on day 28 (CD8+ T cells initial infiltration usually began at day 24 to day 28) based on the continuous observation of CD8+ T cell presence in epidermis using whole-mount tail epidermis staining at different periods. To identify the optimal treatment dose of T-96, we used two doses 2.5 mg/kg and 5.0 mg/kg referencing the treatment in a mouse model of breast cancer [22] and the melanoma mouse model [25]. The vitiligo mice were randomly divided into five groups (T-96 at 2.5 mg/kg and 5 mg/kg, Tofa at 5 mg/kg, T-96 combined with Tofa at 5 mg/kg, and vehicle) before treatment. T-96 and Tofa were first dissolved in a small volume of Dimethyl sulfoxide (DMSO, Thermo Fisher Scientific) and later diluted with sterile PBS and then stored according to the manufacturers’ suggestions. T-96 and Tofa treatment were performed by intraperitoneal injection once every other day for five experiments.
Epidermal Whole-mount staining and imaging
The tails were depilated with NairTM, and tail skin was stripped from the tailbone and flattened in a 12-well plate (trimmed into 1 × 1 cm pieces) for 5 min. Then, the plate was incubated with 1ml 20 mM EDTA (pH 8.0) solution at 37°C. After 2 h, carefully obtained the epidermis from the dermis with a fine-tipped tweezer in the anterior-posterior direction under stereomicroscope (also removed the hair follicles and sebaceous gland), flattened and fixed the epidermis in 4% paraformaldehyde for 10 min and further immersed with 0.3% H2O2/methanol for 20 min at -20 °C whereafter washed with PBS for 3 times (45 min per time at room temperature). Blocked in a solution of 2% donkey serum, 1% BSA and 0.3% TritonX-100 for 1 h with shaking, then incubated with primary antibodies (anti-CD8a, and anti-MelanA) (Table S1) in blocking buffer overnight at 4 °C on a shaker. Secondary antibodies (Goat-anti-Rat FITC and Goat-anti-mouse Cy3) (Table S1) with DAPI (0.2ug/ml) in blocking solution were added for 8h at 4 °C on a shaker. Finally, the samples were mounted on to slides using glycerin and DAPI and secured with cover glass. We removed hair follicles and sebaceous glands due to strong non-specific green fluorescence staining with interfering CD8+ T cells for the following experiments. Whole-mount tail epidermis images were acquired in a sequential manner using the sequential scan mode using FV-1000/ES confocal microscope (Olympus, Tokyo, Japan), setting Z-stack to ensure that the z-position covers the entire thickness of samples. Images were exported using ZEN 2012 (Carl Zeiss Microscopy GmbH), optimized globally for color balance, brightness and contrast using Photoshop CC 2018 (Microsoft). Measurements of the numbers of CD8+ T cells and melanocytes were performed using ImageJ software.
CD8+ T cells isolation and proliferation assay
PBMCs were isolated using Ficoll-Hypaque density gradient centrifugation (Dakewei, Shenzhen, China). CD8+ T cells were purified from PBMCs using the CD8+ T Cell Isolation Kit (Miltenyi, USA) according to the manufacturer’s instructions, and the purity of CD8+ T cells was more than 95% monitored by flow cytometry using anti-CD3 and anti-CD8 (Fig. S4A). CD8+ T cells were cultured in complete RMPI medium that contained RPMI 1640 (GlutaMAX™, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), penicillin (1000 U/ml), streptomycin (1 mg/ml) (all from Invitrogen, USA). For proliferation assay, CD8+ T cells were stained using the carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher Scientific, USA). Briefly, purified CD8+ T cells were washed with PBS and incubated with CFSE dye at a final concentration of 5 μM for 15 min at 37°C protected from light. Next, complete RMPI medium was added to the cell suspension and incubated for 5 min before being washed and then resuspended in complete RPMI medium. CFSE-labeled CD8+ T cells were cultured in 96 well plates and stimulated with dynabeads human T-activator CD3/CD28 at 1:200 (Thermo Fisher Scientific, USA) in the presence of hIL-2 (R&D, USA) at 20 ng/ml. Demethylzeylasteral (T-96) (Solarbio Science, CAS No. 107316-88-1, China) or Tofacitinib (Tofa) (MedChem Express, CAS No. 477600-75-2, USA) were stimulated both at 1 µM. After 5 days, the proliferation levels were analyzed using flow cytometry.
CD8+ T cells stimulation and flow cytometry
For the evaluation of activation and effect function, CD8+ T cells were stimulated with plate-bound anti-CD3 (2 μg/ml, BioLegend, USA), plate-bound anti-CD28 (1 μg/ml, BioLegend, USA) and hIL-2 (20 ng/ml) in complete RPMI medium. T-96 and Tofa were treated according to the experimental design. After indicated times, cells were harvested for analysis, and a cocktail of Brefeldin A and Monensin (eBioscience, USA) was added to suppress cytokine release at 6h before intracellular cytokine staining. To investigate the JAK-STAT signaling pathway, CD8+ T cells were pretreated with T-96 and Tofa for 1 h followed by IL-2 stimulation. For surface marker staining, cells were stained with antibodies (anti-CD69 and PE anti-CXCR3) (Table S1) diluted at final concentration 1 μg/ml and human Fc Receptor Blocker (BD, USA) to inhibit nonspecific antibody binding at 1:50 ratio in Facs buffer (PBS, 1%BSA, 0.1% NaN3 sodium azide), then incubated at 4°C in the dark for 30 min. Isotype control antibodies (Table S1) were used to determine the background caused by nonspecific antibodies binding. Next, samples were washed twice using Facs buffer, and suspended with Facs buffer with propidium iodide (1:100), and analyzed with the CytomicsTM FC500 flow cytometer (Beckman Coulter) in 30 min.
For intracellular staining, single-cell suspensions were stained with Fixable Viability Stain 620 (BD, USA) for 15 min at room temperature. The cells were then fixed and permeabilized with Transcription Factor Staining Buffer Set (Invitrogen) according to the manufacturer’s instructions and stained with antibodies (anti-IFN-γ, anti-GzmB, anti-PRF, anti-p-STAT5 (pY694), anti-p-JAK1 (Tyr1022/1023) and p-JAK2 (Tyr1007/1008)) (Table S1) in prepared Facs buffer (supplemented with human Fc Receptor Blocker) for 1 hour at room temperature. For p-JAK3 staining of CD8+ T cells, primary antibody p-JAK3 (Tyr980, Tyr981) at 10 μg/ml was added in Facs buffer incubated for 1 hour at room temperature, washed the cells 3 times with PBS, and then incubated with Goat-anti-Rabbit Cy3 (Table S1) in 1:1000 for 30 min at room temperature. Samples were then analyzed in 48 h using CytomicsTM FC500 flow cytometer (Beckman Coulter, USA) and data was analyzed using FlowJo software.
Cell culture, stimulation, and plasmid transfection
Primary normal human epidermal keratinocytes (NHEKs) were isolated from the epidermis of plastic surgery skin (healthy subjects, 8 to12 years), then cultured in EpiLifeTM medium (Gibco, USA) plus with keratinocyte growth supplement (Gibco, USA). Second to fifth passage NHEKs were used, and each experiment was repeatedly performed with at least three different donors. HaCaT cells (the human keratinocyte cell line) were cultured in RPMI 1640 supplemented with 10% FBS. Mouse melanoma cell line B16F10 was cultured in a complete Dulbecco’s Modified Eagle Medium (DMEM, high glucose, Thermo Fisher Scientific, USA) with 10% FBS. The two cell lines were authenticated by short tandem repeat (STR) fingerprinting by Fourth Military Medical University and showed no mycoplasma contamination. All cell mediums contained penicillin (1000 U/ml), streptomycin (1 mg/ml) (all from Invitrogen, USA). All cells were cultured at 37°C in a humidified incubator with 5% CO2.
Recombinant human IFN-γ at 20 ng/ml (peprotech, USA), T-96 at indicated concentration, and Tofa at 1 µM) was performed in NHEKs and HaCaT cells. Plasmids for overexpression targeting JAK2 (V617F)-pcw107-V5, #64610), were purchased from Addgene, USA. Empty vectors (OE-NC) were used as negative controls. Cells were transfected using the Lipofectamine 3000 transaction Reagent kit (Invitrogen, USA) according to the manufacturer’s instructions.
Cytotoxicity assessment using CCK-8 assay
Cell viability index was measured using the cell counting kit-8 (CCK-8) (Beyotime Biotechnology, China). Briefly, cells were seeded in 96-well plates (3 × 105 for CD8+ T cells or 1 × 105 cells for NHEKs per well) and stimulated with T-96 at a series of expected concentrations for 5 days for the IC50 detection and 48 hours for cell viability. Then CCK-8 reagent (diluted 1:10 in 100 μL fresh medium) was added to each well, and the plates were incubated for another 90 min. The plate was then read with a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA) at an absorbance reading of 450 nm. A logistic regression model for the calculation of the inhibitory concentration 50% (IC50) to determine the cytotoxic activity of T-96.
Pull-down assay
T-96Bio (biotinylated T-96) was synthesized, analyzed by mass spectrometry and HPLC, and stocked at -20°C after a freeze-drying process. CD8+ T cells or NHEKs (stimulated with IFN-γ or H2O2) were lysed with complete lysis buffer that contained NP-40 (Beyotime, China) with protease and phosphatase inhibitor cocktail (Sigma-Aldrich). To obtain the target protein of T-96, we first incubated T-96Bio and the prewashed (with complete lysis buffer) M-280 Streptavidin Dynabeads (Invitrogen, Cat.11205D, USA) at room temperature for 30 min in a shaker and placed the tube into a magnetic stand to collect the T-96Bio-beads mix, then T-96Bio-beads were incubated with the cell protein lysates for each sample overnight at 4°C in a shaker to obtain the T-96Bio-beads-protein compound (washed twice with complete lysis buffer to avoid the non-specific binding protein). All collected T-96Bio-beads-protein complexes were eluted with protein loading buffer at 100°C for 5 min and stored at -80°C for Mass Spectrum analysis.
Mass Spectrum and Gene Ontology analysis
T-96Bio-beads-protein samples were subjected to SDS-PAGE (10% gels), and then gels were stained with Coomassie blue G250 (BIO-RAD, Cat.1610406, USA). The protein bands of interest were excised from Coomassie blue-stained gel, diced into small pieces, and distained by washing twice with 25 mM NH4HCO3/30% acetonitrile. All Samples were prepared according to the standard protocol as described previously [26], and finally dissolved with 2% acetonitrile/98% H2O/0.1% TFA for mass spectrometry analysis using the QExactive system (Thermo Fisher Scientific, USA). The protein identification and quantitation analysis were performed using Protein Pilot software (AB Sciex, USA). Gene Ontology (GO) biological process analysis was performed using Metascape. Dot plots were plotted using http://www.bioinformatics.com.cn, a free online platform for data analysis and visualization.
Molecular docking
A study of in silico docking of T-96 with JAKs was conducted as described previously [27]. In brief, the binding regions and the key amino acids of JAK3, JAK2, and JAK1 were defined according to the published literature and the PDB database. Molecular structures were energy optimized for molecular docking using the Prepare Ligands module present in the Discovery Studio 3.5 (Accelrys Inc.) and converted to the SD file format. The parameters for docking were determined by analysis of poses obtained by docking of JAK1(PDB code: 4EHZ), JAK2 (PDB code: 5UT0), and JAK3 (PDB code: 5TOZ), which were downloaded from Protein Data Bank in PDB format. The number of generated poses was set to 100 for each protein, and default settings were selected for other parameters. Before docking, the original crystal and water molecules were removed from the T-96-protein complexes. Hydrogen atoms were added via the application of CHARMM force field and the Momany-Rone partial charge default settings in Discovery Studio 3.5. Docking analyses of T-96 with JAKs were performed by means of the CDOCKER module, which is accurate when active sites are known. This method meets the requirements of experimental verification.
Quantitative polymerase chain reaction assay
Total RNA was isolated using Trizol reagent (Invitrogen, USA) based on the manufacturer's instruction, then quantified with the Multiskan spectrophotometer (Thermo Scientific, USA). The cDNA synthesis was performed using the PrimeScript RT reagent Kit (Takara, Japan). Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) assays were performed in iQ5 Real-Time PCR Detection System (Bio-Rad) using SYBR Premix Ex Taq II (TaKaRa) to determine the expression of mRNA. The primers used are listed in Supplemental Table S2. Samples were analyzed in triplicate and normalized to ACTIN.
Western blot
After indicated treatment, total protein was obtained from cells with cell lysis buffer RIPA (Beyotime, China) supplemented with protease and Phosphatase Inhibitor (Sigma-Aldrich, USA), and then quantified using BCA protein assay kit (Beyotime, China). Equal amounts of protein were run on 10% SDS-PAGE (Bio-Rad) and blotted into polyvinylidene difluoride membranes (Millipore, Billerica, USA). After blocking in a solution of 5% fat-free dried milk diluted in Tris-buffered saline, the membranes were incubated with primary antibodies (anti-JAK1, anti-p-JAK1 (Tyr1022/1023), anti-JAK2, anti-p-JAK2 (Tyr1007/1008), anti-STAT1, anti-p-STAT1 (Tyr701), anti-β-actin) (Table S1) overnight at 4 °C, and then with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Table S1) at 1:5000 for 1 h at room temperature. Signals were visualized with the ECL western blotting detection system (Millipore, USA) and Image J software (Bio-Rad).
Immunofluorescence staining
NHEKs were grown and treated in single-layer glass slides as previously reported.[28] After washing and fixing, cells were incubated with primary antibodies from Cell Signaling (anti-STAT1) (Table S1) overnight. Then cells were subsequently incubated with the secondary antibodies Goat-anti-Rabbit IgG FITC and goat anti-mouse IgG H&L FITC (Table S1) respectively for 1 h and further the nuclear dye (DAPI) for 15 minutes at room temperature. Fluorescent images were obtained using an FV-1000/ES confocal microscope (Olympus, Tokyo, Japan).
ELISA assay
Cell-free supernatants from stimulated NHEKs (106 cells per mL) were tested. The production of chemokines was detected using ELISA kits CXCL9 (Elabscience, Wuhan, China), and CXCL10 (R&D, USA) according to the corresponding manufacturer’s instructions.
The transwell migration assay
NHEKs were pretreated with T-96, Tofa, T-96 combined with Tofa, and T-96 combined with JAK2 overexpressed following the IFN-γ stimulation. After 48 hours, culture supernatants were collected for the following migration assay. CXCR3+CD8+ T cells (Fig. S4B) sorted from PBMC of patients with vitiligo cultured in the upper chamber (1×105 cells cultured in 100 μl complete RPMI 1640 medium) separated from the cell supernatants in the lower chamber by using a 5.0 μm polycarbonate membrane (Corning, USA). Additionally, neutralization antibodies of CXCL9 (MAB392; R&D, USA) and CXCL10 (MAB266; R&D, USA) were added at 10 ng/mL to block the chemokines CXCL9 and CXCL10 in the culture supernatants. Plates were maintained at 37 °C for 3 h, and then migrated cells in the lower chamber were counted by means of flow cytometric analysis acquisition thereafter. Migration cells were indicated by the percentage of input cells. Each experiment was performed in triplicate.
Statistical analyses
Data analysis was performed using GraphPad Prism version 9.0 software (GraphPad Software, San Diego, CA, USA). All experiments were repeated at least three times unless. Error bars presented as mean ± SEM. Dual comparisons were made with two-tailed Student’s unpaired t test. Groups of three or more were analyzed by one-way analysis of variance (ANOVA) with Dunnett’s test. P values less than 0.05 were considered statistically significant.