Anti cd4 clone rpa t4

Naïve CD4⁺ T cells were isolated from fresh human blood samples with a negative selection isolation kit from Miltenyi Biotec as per protocol. The purity of naïve CD4⁺ T cells was evaluated with staining of anti-CD3 (Clone UCHT1, BioLegend, San Diego, USA), anti-CD4 (clone RPA-T4, BioLegend, San Diego, USA), and anti-CD45RA (HI100, BioLegend, San Diego, USA). Isolated cell purities were over 95% (Supplementary Figure S1). Cells were cultured in RPMI 1640 media (GE Health Care Life Sciences, Marlborough, USA) supplemented with 10% FBS (Life Technologies, Carlsbad, USA). Cells were stimulated overnight with anti-CD3 (10 μg/mL, pre-coated on plate, Clone UCHT1, BD Biosciences, San Jose, USA) and anti-CD28 (2.5 μg/mL, Clone CD28.2, BD Biosciences, San Jose, USA), with and without glycolysis inhibitor 2-deoxy-d-glucose (2-DG, 2 mg/mL, Acros Organics, New Jersey, USA), mTOR inhibitor rapamycin (100 Nm, Alfa Aesar, Ward Hill, USA) or H₂O₂ (50 μM, Sigma-Aldrich, St. Louis, USA). The next day the antibodies were removed and fresh media (RPMI and 10% FBS) were added with and without 2-DG, rapamycin, or H₂O₂. The cells were cultured for a total of 3 days before harvesting.

Fresh tumor tissue was obtained for CITE-seq. Tumor cells were dissociated, washed, and resuspended. Antibodies for CITE-seq were anti-CD34 (clone 581), anti-CD31 (clone WM59), anti-CD45RA (clone HI100), anti-CD3 (clone UCHT1), anti-CD8A (clone RPAT8), anti-CD4 (clone RPAT4), anti-CD14 (clone 63D3), and anti-CD19 (clone HIB19) TotalSeq-A antibodies (BioLegend, San Diego, CA). Cells from one tumor were used to optimize preparation by adding variable antibody concentrations to 1 million cells in 50 mcl, washing with 200 mcl FACS buffer, resuspending in 200 mcl FACS buffer, and analyzing by flow cytometry. Otherwise, cells were used for CITE-seq using 3′ v3 Single Cell Immune Profiling technology according to the manufacturer’s protocol (10× Genomics, San Diego, CA).

CD4-positive and CD8-positive populations were negative sorted with CD8 and CD4 MicroBeads (Miltenyi Biotec), respectively, and preincubated with anti-CD4 (clone RPA-T4, 300501, BioLegend), anti-CD8 (clone SK1, 344702, BioLegend), or isotype control (Mouse Igg1,κ, 400101, BioLegend) antibodies for 30 min. T cells (10,000 per well) were then cultured with mesothelin and MAGE-A4–positive (A375 transfected with MSLN; S1 Methods, 10,000 cells per well) target cells at an effector-to-target ratio of 1:1. IFN-γ secretion was assessed in the supernatant by ELISA after overnight incubation at 37°C and 5% CO₂, according to the manufacturer’s instructions (R&D Systems). Data were analyzed using a two-way analysis of variance with Tukey correction for multiple comparisons in GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA),