Figure 2.
Phenotypic and functional properties of CAR T cells. (A) Serum interferon gamma (IFN-γ) fold change from baseline is displayed. Levels increase rapidly following CAR T-cell infusion and peak at D+7 after infusion at 2576-fold increase. Baseline value was 1.29 pg/mL, increasing to 3323 pg/mL on D+7. (B) Flow cytometry assessment of T-cell receptor Vβ repertoire gated on CAR+ T cells from a D+90 peripheral blood mononuclear cell (PBMC) sample. This assay detected Vβ repertoire of 62.8% of CAR+ T cells, which was a similar level of coverage to healthy donor T cells run in parallel (data not shown). (C) The relative clonal abundance of lentiviral integration sites is displayed. Most are of low abundance (gray shading), consistent with a polyclonal population of transduced cells. At D+45, a small clone (12%) with an integration site in VARS1 emerged. Numbers above the columns indicate the numbers of cells sampled. Genomic locations of the most abundant clones (all found at D+45) are shown in the key at right (numbers refer to locations on the hg38 draft of the human genome sequence). (D) Immunophenotyping of CD4 CAR+ and CD8 CAR+ T cells from D+21 and D+90 PBMCs is shown. Naïve-like (CD45RA+CCR7+CD95−), stem cell memory (Scm)–like (CD45RA+CCR7+CD95+), central memory (CM)–like (CD45RA−CCR7+), effector memory (EM)–like (CD45RA−CCR7−), and EMRA-like (CD45RA+CCR7−) populations are displayed. Most CAR T cells are EM-like, with a small population of EMRA-like CAR T cells emerging at D+90. (E) PD1, TIM3, and LAG3 expression levels were assessed by immunophenotyping. The percentages of CAR+ T cells at D+21 and D+90 expressing these inhibitory receptors are displayed. (F) A real-time cytotoxicity assay of PBMCs from D+21 and D+90 was performed against the BCMA/green fluorescent protein (GFP)–positive target cell line RPMI-8226 (in a 1:1 effector–to–target cell ratio). The green area corresponds to viable GFP+ cells and inversely relates to cytotoxicity. Triplicates were performed, and mean and standard error of the mean are plotted. Both D+21 and D+90 PBMCs rapidly eliminated tumor cells, compared with tumor cells cultured alone or with baseline (D-5) PBMCs.

Phenotypic and functional properties of CAR T cells. (A) Serum interferon gamma (IFN-γ) fold change from baseline is displayed. Levels increase rapidly following CAR T-cell infusion and peak at D+7 after infusion at 2576-fold increase. Baseline value was 1.29 pg/mL, increasing to 3323 pg/mL on D+7. (B) Flow cytometry assessment of T-cell receptor Vβ repertoire gated on CAR+ T cells from a D+90 peripheral blood mononuclear cell (PBMC) sample. This assay detected Vβ repertoire of 62.8% of CAR+ T cells, which was a similar level of coverage to healthy donor T cells run in parallel (data not shown). (C) The relative clonal abundance of lentiviral integration sites is displayed. Most are of low abundance (gray shading), consistent with a polyclonal population of transduced cells. At D+45, a small clone (12%) with an integration site in VARS1 emerged. Numbers above the columns indicate the numbers of cells sampled. Genomic locations of the most abundant clones (all found at D+45) are shown in the key at right (numbers refer to locations on the hg38 draft of the human genome sequence). (D) Immunophenotyping of CD4 CAR+ and CD8 CAR+ T cells from D+21 and D+90 PBMCs is shown. Naïve-like (CD45RA+CCR7+CD95−), stem cell memory (Scm)–like (CD45RA+CCR7+CD95+), central memory (CM)–like (CD45RA−CCR7+), effector memory (EM)–like (CD45RA−CCR7−), and EMRA-like (CD45RA+CCR7−) populations are displayed. Most CAR T cells are EM-like, with a small population of EMRA-like CAR T cells emerging at D+90. (E) PD1, TIM3, and LAG3 expression levels were assessed by immunophenotyping. The percentages of CAR+ T cells at D+21 and D+90 expressing these inhibitory receptors are displayed. (F) A real-time cytotoxicity assay of PBMCs from D+21 and D+90 was performed against the BCMA/green fluorescent protein (GFP)–positive target cell line RPMI-8226 (in a 1:1 effector–to–target cell ratio). The green area corresponds to viable GFP+ cells and inversely relates to cytotoxicity. Triplicates were performed, and mean and standard error of the mean are plotted. Both D+21 and D+90 PBMCs rapidly eliminated tumor cells, compared with tumor cells cultured alone or with baseline (D-5) PBMCs.

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