Anti cd3 brilliant violet 570

One million T-cells were stained with the following Ab cocktail: anti-CD3 Brilliant Violet 570 (Biolegend, CA, USA), anti-CD4 Brilliant Violet 510 (Biolegend, CA, USA), anti-CD8a APC-Cy7 (BD Biosciences, CA, USA), anti-4-1BB FITC (eBioscience, CA, USA), anti-CD127 APC-AF700 (Beckman Coulter, CA, USA), anti-CD45RA ECD (Beckman Coulter, CA, USA), anti-CCR7 Brilliant Violet 421 (Biolegend, CA, USA), anti-LAG-3 APC (R&D Systems, Minneapolis, MN), anti-CD25 PE-Cy7 (BD Biosciences, CA, USA), anti-CTLA-4 PE-Cy5 (BD Biosciences, CA, USA), anti-TIM3 Percp-eFluor710 (eBioscience, CA, USA) and anti-PD-1 PE (BD Biosciences, CA, USA). After 15 min, T-cells were washed with PBS–0.1% FBS and analyzed using a FACSAria flow cytometer (BD Biosciences, Stockholm, Sweden); data analysis was performed using FlowJo software.

CMV-specific T cells (CMV-CTL) and WT1-specific T cells (WT1-CTL) frequencies were quantified and phenotyped in patients by staining with PE-A^*0201 CMV_NLVPMVATV Dextramer, PE-A^*0201 WT1_RMFPNAPYL Dextramer and APC-A^*0201 WT1_SLGEQQYSV Dextramer (Immudex, Copenhagen, Denmark) (Supplementary Figure 1). Briefly, PBMCs were stained with Dextramers for 30 min at room temperature. Anti-CD3-Brilliant Violet 570, anti-CD4-Brilliant Violet 510, anti-CCR7-Brilliant Violet 421, anti-CD45RA–FITC, anti-PD-1-PE Cy7 (from Biolegend, San Diego, CA, USA), anti-CTLA-4–PE Cy5, anti-CD8-AF700 (from BD Biosciences, San Jose, California, USA) and anti-TIM-3–PerCP eFlour 710 (from eBioscience, USA) were added for the final 20 minutes of incubation. Surface expression of CD45RA and CCR7 was used to characterize naïve (T_Naive, CD45RA+CCR7+), central memory (T_CM, CD45RA-CCR7+), effector memory (T_EM, CD45RA-CCR7-), and terminally differentiated (T_EMRA, CD45RA+CCR7-) phenotypes (Supplementary Figure 2). Appropriate isotype controls or fluorescence minus one control for each fluorochrome were used to assess for nonspecific staining and determine gating strategy, respectively. FACS Aria flow cytometer (BD Biosciences, San Jose, California, USA) and FlowJo software were used for acquisition and data analysis.

TILs were stained with anti-CD3 Brilliant violet 570, anti-CD4 Brilliant violet 510, anti-CXCR3 FITC (all from Biolegend, San Diego, CA) and anti-CD8a APC-Cy7 (BD Biosciences, Franklin Lakes, NJ). After 15 min, cells were washed in PBS-0.1% FBS, and analysed by flow cytometry. Differentiation and maturation marker analysis based on CD45RA and CCR7 expression was performed as described previously.¹⁷