Phenotype of anti-CD19-CAR–expressing T cells. (A) Infusion cells of patient 5 are shown after staining with a CAR-specific antibody. The first plot is gated on CD3⁺CD8⁺ cells. The phenotype of the CD3⁺CD8⁺CAR⁺ cells is shown on the subsequent plots of CD45RA vs CCR7, programmed cell death protein 1 (PD-1) vs CD57, and CD27 vs CD28. (B) CAR expression was assessed on pretreatment lymphocytes of patient 5 by flow cytometry. The plot shows minimal background staining when the cells were stained with a CAR-specific monoclonal antibody. The plot is gated on CD3⁺CD8⁺ lymphocytes. (C) The first plot is gated on CD3⁺CD8⁺ lymphocytes that were obtained from patient 5 12 days after CAR T-cell infusion. CAR⁺ cells made up 2.2% of the CD3⁺CD8⁺ lymphocytes. The subsequent CD45RA vs CCR7, PD-1 vs CD57, and CD27 vs CD28 plots are gated on CD3⁺CD8⁺ lymphocytes that are either CAR⁺ or CAR^–. (D) Infusion cells of patient 5 are shown. The first plot is gated on CD3⁺CD4⁺ cells. Subsequent plots show the phenotype of CD3⁺CD4⁺CAR⁺ cells. (E) The plot shows minimal background staining when pretreatment lymphocytes from patient 5 were stained with a CAR-specific antibody. The plot is gated on CD3⁺CD4⁺ lymphocytes. (F) The first plot is gated on CD3⁺CD4⁺ lymphocytes that were obtained from patient 5 12 days after CAR T-cell infusion. CAR⁺ cells made up 7.4% of the CD3⁺CD4⁺ lymphocytes. Subsequent plots show the phenotype of CD3⁺CD4⁺ cells that were either CAR⁺ or CAR^–. Increased expression of the T-cell inhibitory protein PD-1 was present on CD4⁺CAR⁺ T cells (G) and CD8⁺CAR⁺ T cells (H) relative to CAR^– T cells from the same patient at the time of the peak in the level of CAR⁺ blood T cells in each patient. The percentage of PD-1⁺ T cells is shown for the CAR⁺ and CAR^– T cells of the indicated patients. Note that an insufficient number of CAR⁺CD8⁺ T cells were present in the blood of patient 10 to allow accurate determination of PD-1–expressing CD8⁺ cells in this patient. PD-1 expression was determined as shown in panels C and F. For CD4⁺ T cells, the difference in PD-1 expression between CAR⁺ cells and CAR^– cells was statistically significant (P = .03, Mann-Whitney U test). PD-1 levels are only shown for 4 patients because some patients had very low or undetectable levels of CAR⁺ T cells in their blood and because we lacked sufficient PBMCs from relevant time points to conduct the experiment in some other patients.