Loss of CD58 in tumor cells gives rise to CAR T-cell dysfunction. (A) Expansion of CD19 CAR T cells after coculturing with WT or CD58^KO Nalm6 or Raji cells at a 1:1 E:T ratio (n = 3). (B) FACS-based measurement of CD107a expression in CD19 CAR T cells stimulated by WT or CD58^KO Nalm6 or Raji cells (n = 4). (C) FACS-based measurement of CD107a expression in CD19 CAR T cells stimulated with Nalm6 or Raji cells pretreated with control or anti-CD58–blocking mAbs (n = 4). (D) FACS-based measurement of CD107a expression in CD19 CAR T cells pretreated with control or anti-CD2–blocking mAbs after stimulation with Nalm6 cells or Raji cells (n = 4). (E) FACS-based quantification of intracellular interleukin-2, IFN-γ, and TNF-α expression in CD19 CAR T cells stimulated with WT or CD58^KO Nalm6 cells (n = 4) or Raji cells (n = 5) at a 1:1 E:T ratio for 8 hours. (F) Schematic showing the functional assessment study. CD19 CAR T cells and WT or CD58^KO Nalm6 cells were initially cocultured at an E:T ratio of 1:1 for 3 days (first coculture). CD19 CAR T cells were sorted by the phycoerythrin magnetic beads method based on CAR staining in first coculture and then cocultured again with WT Nalm6 cells at an E:T ratio of 1:1 for 24 hours and 48 hours (secondary coculture). Expansion (G) and Ki67 expression (H) of CD19 CAR T cells after 24 hours and 48 hours in secondary coculture (n = 3). (I) CD19 CAR T cells were sorted in first coculture and then cocultured again with WT Nalm6 cells at an E:T ratio of 1:1 to test the degranulation (CD107a expression) of CAR T cells for 0.5 hours or 1 hour (n = 4). (J) Survival of WT Nalm6 cells after 24 hours and 48 hours in secondary coculture (n = 3). (K) Representative FACS plots and quantification of annexin V expression of CD19 CAR T cells after 24 hours and 48 hours in secondary coculture (n = 3). (L) Pattern of repeated antigen stimulation in vitro. (M) CD19 CAR T-cell expansion after repeated stimulation with WT or CD58^KO Nalm6 cells. CD25 (N) and CD69 expression (O) in CD19 CAR T cells after repeated stimulation with WT or CD58^KO Nalm6 cells (n = 3). Statistical comparisons were performed using a 2-tailed unpaired t test in panels C-D. Statistical comparisons were performed using a 1-way ANOVA test in panels B and E. Significance was assessed using a 2-way ANOVA test with multiple comparisons in panels A, G-K, and M-O. The values are shown as the means plus or minus SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ↓, the time point when WT or CD58^KO Nalm6 or Raji cells were added; 7-AAD-Percp, 7-Aminoactinomycin D (Peridinin-Chlorophyll-Protein Complex); FACS, fluorescence-activated cell sorter; ns, not significant (P > .05).