Pd 1 percp cy5.5 eh12 2h7

Each set of patient samples was stained with the following antibodies: CD3-Brilliant Violet 785 (OKT3, Biolegend), CD4-APC/Cy7 (OKT4, Biolegend), CD8-Alexafluor 700 (SK1, Biolegend), CD11b-Brilliant Violet 570 (M1/70, Biolegend), CD19-Brilliant Violet 570 (HIB19, Biolegend), CD34-Brilliant Violet 421 (561, Biolegend), TCR vβ12-PE (VER2.32.1, Beckman Coulter), CD25-Brilliant Violet 711 (BC96, Biolegend), CD69-PE/Cy7 (FN50, Biolegend), OX40-FITC (Ber-ACT35, Biolegend), PD-1-PerCP/Cy5.5 (EH12.2H7, Biolegend), T cell immunoglobulin and mucin-domain containing-3 (TIM-3)-APC (F38-2E2, Biolegend), CTLA-4-PE-CF594 (BNI3, BD Biosciences), C-C motif chemokine receptor 7(CCR7, CD197)-Brilliant Violet 650 (G043H7, Biolegend), and a viability dye (Live/Dead Aqua, Invitrogen). Representative stains are shown in Supplementary Figure 3. All samples were analyzed on the LSRFortessa in the Loyola University Chicago Flow Cytometry Core. The absolute number of TCR-transduced T cells per milliliter (mL) of study blood was estimated by combining percentages of TCR-transduced T cells with counts of total white blood cells from known volumes of whole blood.

Fresh tumor samples were manually disaggregated between frosted-glass slides to obtain a single-cell suspension for analysis. Both the disaggregated tissue samples and expanded TIL were stained on ice in FACS Wash Buffer (Dulbecco’s Phosphate Buffered Saline 1× with 1% Bovine Serum Albumin) for 30 min with fluorochrome-conjugated monoclonal antibodies for CD3 FITC (SK7), CD4 PerCP-Cy5.5 (RPA-T4), CD8 PB (RPA-T8), CD16 PE (B159), CD28 PE-Cy7 (CD28.2), CD56 PE-Cy7 (B159), TCR y8 APC (B1), BTLA PE (J168 & J168–540), PD-1 BV650 (EH12) HLA-ABC PE (G46–2.6) (all BD Bioscience, San Jose, CA, USA), and PD-1 PerCP-Cy5.5 (EH12.2H7) (Biolegend, San Diego, CA, USA). Dead cells were excluded using the Aqua or Yellow LIVE/DEAD viability stain (ThermoFisher). Stained cells were fixed in 1% paraformaldehyde solution for 20 min at RT. Samples were acquired using the BD FACSCanto™ II or BD LSRFortessa X-20 and analyzed using FlowJo Software v10.5 (Tree Star). Subpopulations were excluded from analysis if less than 100 events.