Cd34 brilliant violet 421

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.

Flow cytometry was performed as described previously (Jeffery et al. 2015 (link)) for BrdU analysis with the following antibodies: CD45 APC-eFluor 780 (eBioscience; 47–0451–80) at 1:1,000, CD31 PE-Cy7 (eBioscience, 25–0311–82), at 1:500, CD29 Alexa Fluor 700 (BioLegend, 102218) at 1:400 and Sca-1 V500 (BD Horizon, 561228) at 1:300. Cells were washed and then fixed and permeabilized using Phosflow lyse/fix and Perm Buffer III (BD Biosciences) according to the manufacturer’s recommendations. Cells were then treated with DNase (deoxyribonuclease I; Worthington; × 200 units/ml) in DPBS (Sigma; with calcium chloride and magnesium chloride) for 2-hrs at 37C and then washed in HBSS with 3% BSA. Cells were then stained with anti-BrdU antibody (Alexa Fluor 647; Phoenix Flow Systems; AX647) at 1:30 in HBSS with 3% BSA overnight in the dark at 4C. Cells were then washed in HBSS with 3% BSA and incubated with CD34 Brilliant Violet 421 (BioLegend 230 119321) at 1:400 and CD24 PerCP-Cyanine 5.5 (eBioscience, 45–0242–80) at 1:250. Following antibody incubation, samples were washed and analyzed on a BD LSRII analyzer. Data analysis was performed using BD FACS Diva software (BD Biosciences).