Role of CD58 in resistance to CAR T-cell–mediated killing. (A-C) CD58^KO/WT Nalm6 cell ratio in growth competition assays. CD58^KO Nalm6 cells referred to sgCD58-2 Nalm6 cells. (A) A mixture of Nalm6 cells were cocultured with control T cells or CD20 CAR T cells at a 1:1 E:T ratio (n = 3). (B) A mixture of Nalm6 cells were cocultured with control T cells or tandem CD19/20 CAR T cells at a 1:20 E:T ratio (n = 3). (C) A mixture of Nalm6 cells were cocultured with control T cells or CD19.28z CAR T cells at a 1:20 E:T ratio (n = 4). (D-G) CD58^KO/WT Raji cell ratio in a growth competition assay. CD58^KO Raji cells referred to sgCD58-2 Raji cells. (D) A mixture of Raji cells were cocultured with control T cells or CD19 CAR T cells at a 1:20 E:T ratio (n = 3). (E) A mixture of Raji cells were cocultured with control T cells or CD20 CAR T cells at a 1:20 E:T ratio (n = 3). (F) A mixture of Raji cells were cocultured with control T cells or CD19/20 CAR T cells at a 1:20 E:T ratio (n = 4). (G) A mixture of Raji cells were cocultured with control T cells or CD19.28z CAR T cells at a 1:20 E:T ratio (n = 4). (H) Cytotoxic analysis of WT and CD58^KO Nalm6 cells cocultured with CD19 CAR T cells at indicated E:T ratios for 24 hours (n = 3). (I) Cytotoxic analysis of WT and CD58^KO Nalm6 cells cocultured with CD19/20 CAR T cells at indicated E:T ratios for 24 hours (n = 4). (J) Cytotoxic analysis of WT and CD58^KO Nalm6 cells cocultured with CD19.28z CAR T cells at indicated E:T ratios for 24 hours (n = 4). (K) Cytotoxic analysis of WT and CD58^KO Raji cells cocultured with CD19 CAR T cells at indicated E:T ratios for 24 hours (n = 4). (L) Cytotoxic analysis of WT and CD58^KO Raji cells cocultured with CD19/20 CAR T cells at indicated E:T ratios for 24 hours (n = 4). (M) FACS histogram showing the CD58 level in Nalm6 or Raji cell lines pretreated with control or blocking anti-CD58 mAbs for 30 minutes. (N) Cytotoxic analysis of Nalm6 or Raji cells pretreated with control or anti-CD58–blocking mAbs and cocultured with CD19 CAR T cells for 24 hours at a 1:20 E:T ratio (n = 3). (O) Representative FACS histogram showing the CD2 level in CD19 CAR T cells pretreated with control or anti-CD2–blocking mAbs for 30 minutes. (P) Cytotoxicity analysis of Nalm6 or Raji cells cocultured with control or anti-CD2 mAb-blocked CD19 CAR T cells at a 1:20 E:T ratio for 24 hours (n = 3). (Q) Representative FACS plot showing the level of CD19 expression in WT and CD58^KO cell lines. (R) Representative FACS plot showing the level of CD20 expression in WT and CD58^KO cell lines. Significance was assessed using a 2-way ANOVA test with multiple comparisons in panels A-L. Statistical comparisons were performed using a 2-tailed unpaired t test in panels N and P. The values are shown as the means plus or minus SD. ∗P < .05; ∗∗P < .01; ∗∗∗ P < .001. FACS, fluorescence-activated cell sorter; ns, not significant (P > .05).