Anti cd3 clone hit3a

Anti-CD3 (clone: HIT3a) and anti-CD28 (clone: CD28.2) antibodies for T cell stimulation were purchased from Biolegend. Anti-TRAF3IP2 antibody was purchased from Ebioscience (Cat #14-4040-80), anti-THEMIS was from Thermo Fisher (Cat #PA5-56740), anti-β-tubulin was from Millipore Sigma (Cat #05-661), and anti-GAPDH was from Cell Signaling Technology (Cat #2118S). AlexaFluor-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Cat #32735 and Cat #32729).

Human PBMCs were isolated by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation, washed twice in phosphate-buffered saline (PBS), and resuspended at 10⁶/mL in complete RPMI 1640 (cRPMI) medium containing 10% fetal bovine serum, 2 mM glutamine, and 100 U/mL each of penicillin and streptomycin (Invitrogen). For T cell activation, human primary T cells were isolated by MACS beads (MACS, 130–096-535) and activated with 1 μg/mL anti-CD3 (clone HIT3a, BioLegend) and 1 μg/mL anti-CD28 (clone CD28.2, BioLegend) or with Dynabeads® Human T-Activator CD3/CD28 (Thermo Fisher Scientific).

Patient or control T cell blasts, between 2 and 3 weeks after initial activation, were collected and resuspended in fresh IL-2-containing media. Cells were then added to 96-well plates in triplicate and stimulated with the indicated dose of anti-CD3 (clone: HIT3a, BioLegend) in the presence of 1 ug/mL Protein A for 18 hours, as previously described.[15 (link)] Cells were then collected by repeat pipetting and stained with AnnexinV-APC and propidium iodide and analyzed by flow cytometry. The percentage of live cells was determined by AnnexinV and propidium iodide staining and expressed as a percentage of cells lost in the control condition due to TCR restimulation.

For murine cytotoxicity analysis, B16-SIY organoids were treated with IgG/α-PD-L1/α-PD-1 as indicated. Organoids were dissociated to single cells and pelleted as described above. Cells were incubated with anti-Melanoma antibody (Abcam, 1:80) for 30 minutes on ice. For human tumor cell cytotoxicity assay, PDOs were cultured for 1 week in the presence of anti-CD3 (clone HIT3a, 2 mg/ml) (cat no: 300332, BioLegend) and anti-CD28 (clone CD28.2, 2 μg/ml) (cat no: 302923, BioLegend) with either 10 μg/ml anti-PD-1 (nivolumab) or IgG4 control. For both mouse and human analysis, cells were washed twice with cold Cell Staining Buffer, and resuspended in Annexin V Binding Buffer (Biolegend, FITC Annexin V Apoptosis Detection Kit) at a concentration of 0.25−1.0 × 10⁷ cells/ml. 100 μl of cell suspension were transferred in a 5 ml test tube and incubated with 5 μl of Annexin V-FITC and 5 μl of 7-AAD Viability Staining Solution for 15 min at room temperature (25°C) in the dark and analyzed by FACS for Annexin V and 7-AAD, yielding Annexin-V(+)/7-AAD(−) early apoptotic and Annexin-V(+)/7-AAD(+) late apoptotic/necrotic cells. The Annexin-V, 7-AAD double positive tumor cells were pre-gated based on forward and side scatter properties to enrich for tumor epithelium, eliminate hematopoietic and debris populations and further pre-gated for single cells.