Fig. 2

Trogocytic CAR molecule transfer is related to several factors. a, b U87 cells with different EGFRvIII expression levels were treated with EGFRvIII-targeted CAR-T cells or T cells. Gate on U87 cells (left) and CAR-T cells (right). Flow cytometry results indicate that both CAR molecule acquisition of tumor cells and the loss of CAR-T cells are antigen-density dependent. The mean fluorescence intensity (MFI) of CAR molecules (FLAG-tag labeled) of tumor cells and CAR-T/T cells are presented in b. c, d EGFRvIII-targeted CAR-T cells were successively cocultured with fresh U87 cells of EGFRvIII expression (high antigen density), for a total of three times. CAR molecule transfer was robust in the first stimulation and decreased progressively in subsequent stimulations. Meanwhile, rapid CAR molecule loss was observed in CAR-T cells. Additional replicates are displayed in d. e, f U87 cells of EGFRvIII expression (high antigen density) were cocultured with CAR-T cells of high sensitivity (High-sense, EGFRvIII scFv-CD28-CD3ζ) or low sensitivity (Low-sense, EGFRvIII scFv-CH2CH3-CD28-CD3ζ) overnight. Data reveal that CAR molecule trogocytosis depends on CAR molecule sensitivity. More replicates are shown in f. g, h U87 cells of EGFRvIII expression (high antigen density) were cocultured with CAR-T cells (high sensitivity) at indicated effector-to-target (E: T) ratios. Data reveal that CAR molecule acquisition of tumor cells is positively correlated with the E: T. CAR molecule loss in CAR-T cells was also observed. Independent replicates are presented in h. i, j U87 cells with a high density of EGFRvIII were cocultured with CAR-T cells (high sensitivity) supplemented with (+) or without (−) ZAP70 inhibitor. No significant biological difference was observed between (+) and (−) groups. The result indicates that CD3ζ downstream signaling is not relevant to CAR molecule transfer. Independent replicates are displayed in j. k, l Tyrosine residues of ITAMs were point-mutated to phenylalanine (Y–F) to silence downstream signaling. Mutate (Y-F) or wild type (wt) CAR-T cells (EGFRvIII-targeted, high sensitivity) were cocultured with U87 cells with a high density of EGFRvIII. Again, data demonstrate that trogocytosis is independent of CAR molecule downstream signal. Independent replicates are shown in l. m, n CAR-T cells (EGFRvIII-targeted, high sensitivity) were pre-induced to express PD1 before coculture with U87 cells (high density of EGFRvIII). Data indicate that trogocytic CAR molecule transfer is independent of PD1 expression. Additional replicates are provided in n. Data are represented as mean ± SD, unpaired Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001