Key postinfusion PD-1⁺ CAR⁺ CD8⁺ T-cell populations correlate with clinical outcomes. 2D flow plot analysis was performed on spectral flow cytometry data from day-14 post-CAR-T samples. (A) Representative 2D flow cytometry plots showing individual patient PD-1 and TCF1 expression in CAR⁺ CD8⁺ T cells and corresponding box plot quantification in CR vs PD cohorts. (B) Representative 2D flow cytometry plots showing individual patient EOMES and CD45RO expression in CAR⁺ CD8⁺ PD-1⁺ TOX⁻ T cells and corresponding box plot quantification in CR vs PD cohorts. (C) Representative 2D flow cytometry plots showing individual patient PD-1 and TIM3 expression in CAR⁺ CD8⁺ T-bet⁺ GZMB⁺ T cells and corresponding box plot quantification in CR vs PD cohorts. (D, E, F) Kaplan-Meier (KM) analysis was used to generate PFS curves stratified by high vs low percent of CD8⁺ CAR-T populations. (D) KM analysis of PFS for patients with high (>4%) or low (<4%) percent of this cell type; high group, n = 6; low group, n = 20. (E) KM analysis of PFS for patients with high (>4.7%) or low (<4.7%) percent of this cell type; high group, n = 13; low group, n = 13. (F) KM analysis of PFS for patients with high (>12%) or low (<12%) percent of this cell type; high group, n = 17; low group, n = 9. (G) Box plot of the combination of PD-1⁺ TCF1⁺ cells and PD-1⁺ TIM3⁺ T-bet⁺ GZMB⁺ cells in CR vs PD (left). KM analysis of PFS for patients with high (>24%) or low (<24%) percent of this combination of cell types (right); high group, n = 13; low group, n = 13. (H) Box plot of the combination of PD-1⁺ TCF1⁺ cells, PD-1⁺ TOX⁻ EOMES⁺ CD45RO⁺ cells, and PD-1⁺ TIM3⁺ T-bet⁺ GZMB⁺ cells in CR vs PD (left). KM analysis of PFS for patients with high (>25%) or low (<25%) percent of this combination of cell types (right); high group, n = 15; low group, n = 11. For all KM curves, the x-axis was time in days from date of CAR-T infusion. Dotted lines on box plots indicate separation lines between high and low percentages of CAR-Ts in each population and were selected based on optimal response separation between cohorts. Because clinical outcomes were known during patient stratification, P values need to be interpreted with caution. Box plots in panels A, B, and C show quartiles with bands at the median; whiskers indicate 1.5 interquartile range; all observations overlaid as dots. P values are from linear regression analysis (cell type % changes) and log-rank tests (PFS); ∗P < .05, ∗∗P < .01; ∗∗∗P < .001. Pt, patient.