Anti cd8 af700

Cryopreserved PBMNCs were thawed, incubated with an Fc receptor blocking reagent (ChromPure Mouse IgG, Jackson ImmunoResearch Suffolk, UK) to inhibit non-specific binding before incubated with the following, pre-titrated antibodies (all from BD Biosciences, San Jose, CA): anti-CD3 V450, anti-CD45RO FITC, anti-PD-1 (CD279) PE, and anti-CD8 AF700. Flow cytometry was acquired by a LSRFortessa (BD Biosciences, San Diego, CA) and data analyzed using FlowJo version 10.2 (Tree Star Inc., Ashland, OR). Compensation was performed using BD CompBeads (BD Biosciences). Applying gating strategies based on unstained controls and for PD-1 the fluorescence minus one (FMO), the expression of PD-1 was measured on CD3⁺/CD45RO⁺/CD8⁺ singlets and given by the median fluorescense intensity (MFI) (Supplementary Figure 4).

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.