Anti cd3e antibody

T cells from spleen or lymph nodes were enriched by MACS magnetic beads (Miltenyi Biotec) and were sorted by FACS Aria (BD Biosciences) before in vitro stimulation experiments. Purified T cells were cultured in custom ordered Dulbecco’s Modified Eagle Medium (D-MEM, KOHJIN BIO) supplemented with 10% fetal bovine serum (FBS) (Hyclone). For in vitro culture, 2.0 × 10⁵ cells were stimulated in 96-well plates by precoated 2 µg/ml anti-CD3e antibody (553058, BD Biosciences) with 2 µg/ml soluble anti-CD28 antibody (553295, BD Biosciences) for 2 days. Activated T cells were maintained with 40 U/ml rmIL-2 (402-ML, R&D Systems). 2B4 cell line was maintained in RPMI medium (GIBCO) supplemented with 10% FBS and B16-F10 cell line in D-MEM medium (GIBCO) supplemented with 10% FBS. All cell culture medium was supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin.

CD4⁺ naive T cells were isolated from spleen of C57BL/6 (WT) and Arhgef2-/- mice by using CD4⁺CD62L⁺ T Cell Isolation Kit (Miltenyi Biotec, San Diego, CA, USA) and subsequently stained with CellTracer Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the instructions provided. Cells were then incubated with 2 μg/ml of anti-CD28 antibody (BD, New Jersey, USA) in anti-CD3e antibody (BD, New Jersey, USA) pre-coated wells for 4 days at the cell concentration of 1 × 10⁶ cell/ml. 4 days later, cells were collected and stained with APC-conjugated anti-CD3e (Biolegend, San Diego, CA, USA) and BV786-conjugated anti-CD4 (BD, New Jersey, USA). Cells were analyzed by FACS Aria II (BD Bioscience, New Jersey, USA) followed by using FlowJo software (Tree Star). All cells were pre-gated on singlet cells (area & width) and on living cells (7AAD⁻).

Sorted CD4⁺ T cells were labeled with CFSE (ThermoFisher Scientific) and were cultured in custom ordered Dulbecco’s Modified Eagle Medium (D-MEM, KOHJIN BIO) supplemented with 10% heat inactivated FBS (Hyclone). 2.0 × 10⁵ cells were stimulated in 96-well flat-bottomed plate, which was pre-coated with 2 μg ml⁻¹ anti-CD3e antibody (553068, BD Bioscience) and 2 μg ml^-1 soluble anti-CD28 antibody (553295, BD Bioscience), for two days, and were maintained in D-MEM supplemented with 10 ng ml^-1 recombinant mouse IL-2 (402-ML, R&D system) for 2 to 4 days.