Anti cd3 clone hit3a

To activate and expand T cells from PBMCs, thawed PBMCs from patient, patient’s father, or healthy donor resuspended in 10% complete RPMI 1640 medium [18 (link)] were stimulated with 1 μg/ml of anti-CD3 (clone HIT3a, BD Pharmingen 555336) and anti-CD28 (clone CD28.2, BD Pharmingen 555725) antibodies, and plated at 2 × 10⁶ cells per well of 48-well plate. PMBCs were incubated at 37 °C and under 5% CO₂ for overnight and then continue cultured in the presence of 30 U/ml hIL-2 (PeproTech) in medium for 7 days. Fresh hIL-2 containing medium was added every 2 days. Orai1-KO HEK293 [19 (link)] cells were in DMEM media (Invitrogen) supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin. Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The transfection efficiency was routinely checked by low magnification microscopy on an EVOS FL Cell Imaging System (ThermoFisher Scientific). For 4-PBA treatment, cells were first transfected with plasmids for 6 h and then treated with 10 μM of 4-PBA for additional 12 h.

PBLs from patients with SSc and healthy donors were cultured for 5 days on plates previously coated with anti-CD3 (clone HIT3a, 2 μg/ml; BD Pharmingen, San Diego, CA, USA) antibody at the concentration of 0.5 × 10⁶ cells/ml in complete RPMI 1640 medium supplemented with soluble anti-CD28 (clone CD28.2, 1 μg/ml; BD Pharmingen) antibody and IL-2 (20 U/ml; PeproTech, Rocky Hill, NJ, USA). At the sixth day, cells were stimulated for the last 4 h with a combination of phorbol 12-myristate 13-acetate (10 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) and ionomycin (1 μM; Sigma-Aldrich) in the presence or absence of the optimal concentration of 5 ng/ml TGF-β1 (PeproTech), named TGF-β hereafter, in 0.5 % FCS-containing RPMI 1640 medium with or without a 1-h preincubation with specific inhibitors (SB431542 from Sigma-Aldrich; SB203580 or SIS3 from Calbiochem, San Diego, CA, USA). Jurkat T cells were cultured in 0.5 % FCS-containing medium for 16 h before addition of 5 ng/ml TGF-β for 30 min or 4 h.

Mouse antibodies for γ-adaptin and GFP were obtained from Sigma; rat anti-HA clone 3E10 was from Roche; anti-human CD4 mAbs clone 4B12 and clone OKT4 were from Santacruz and Leica; purified anti-MCHI mAb (WG-32) was kindly donated by A. Corbí (CIB-CSIC, Madrid); anti-Tf-R mAb was from Zymed and Alexa-594 Transferrin conjugate was from Molecular Probes. Anti-p56^Lck mAb was from BD Transduction Laboratories and pTyr505-Lck mAb was from Millipore. PE-, PC7-conjugated anti-human CD4 mAbs, PE-conjugated anti-CD3 mAb, and PE- and PC7-conjugated anti-mouse immunoglobulin G were purchased by BD Pharmingen. Secondary antibodies Alexa-555- or Alexa-647- conjugated anti-murine, and Alexa-555-conjugated anti-rat were from Molecular Probes. For T cell spreading, the monoclonal antibody (mAb) anti-CD3 (clone HIT3a) was used (BD Pharmingen). For F-actin stain, tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Sigma) was used. For Western blotting, secondary horseradish peroxidase-conjugated anti-IgGs were from Dako.