Ctla4 bv786

Human peripheral blood samples were collected in 4 heparin tubes at baseline, following nivolumab infusion and multiple timepoints following TIL infusion. Peripheral blood mononuclear cells (PBMC) were collected using a Ficoll gradient and cryopreserved in 10% DMSO and FBS. Cells were thawed in media, and subsequently stained in PBS containing 5% FBS (vol/vol, FACS buffer) with: CD3 BUV496, CD56 BUV563, CD4 BUV737, CD197 BV421, CD28 BV480, CD14 BV605, CD19 BV605, CD95 BV711, CD195 BV786, CD127 PE, CD194 PE-Cy7, CD45RA Alexa488, CD25 PerCP-Cy5.5, NKG2D APC, Tim3 BV421, PD1 BV480, CD226 BV711, CTLA4 BV786, Lag3 PE, TIGIT PE-Cy7, CD244 Alexa488, CD27 PerCP-Cy5.5, BTLA APC from BD Biosciences. Dead cells were excluded using the Zombie NIR Fixable Viability Kit from Biolegend, incubated at 4 °C for 1 hr, then washed twice with FACS buffer, and finally fixed in PBS containing 1% paraformaldehyde before running flow cytometry. Cells were acquired on a BD FACSymphony™ A5, and data were analyzed with FlowJo Version 10.0 software. All cell gates were drawn uniformly for analysis across patients and time points, with gating strategy provided in Supplementary Appendix 2. Plots of t-SNE were generated by Flowjo Version 10.6.1 according to the expression of CD45RA, CCR7, CD28 and CD95. Different memory T cell subsets were shown using separate colors.