Extended Data Fig. 3: CD19 trogocytosis in vitro and in vivo.
From: CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape
a, Left, representative FACS profile of CD19 staining on CAR T cells isolated by the time of relapse from bone marrow or extramedullary tumour sites of mice treated with 19-BBζ CAR T cells. Right, MFI of CD19 on CAR T cells (19-BBζ, n = 9 mice; control, n = 4 mice). b, Cd19 mRNA expression in NALM6 cells of relapsed mice treated with 19-BBζ CAR T cells (fold change D0 versus NALM6 in vitro = 0.97; adjusted P = 0.919; fold change D6 vs D0 = 0.625, adjusted P = 0.23; n = 7 independent samples). c, MFI of CD19 on the segregated NALM6 cells in a transwell assay. NALM6 cells alone were plated at the top of the plate and NALM6 cells with CAR T cells at the bottom. Data were collected on days 1, 4, 7 and 14 (n = 3 independent samples). d, Left, diagram illustrating the co-culture of NALM6 cells and CAR T cells. Percentage and MFI of CD19 on NALM6 cell surface (middle) and on CAR T cell surface (right). Data were collected at 0, 1, 2 and 4 h after co-culture gated on live singlet cells (n = 4 donors). e, MFI of CD19 on NALM6 cell surface after 2 h of co-culture with 19-28ζ (left) or 19-BBζ (right) CAR T cells treated with the F-actin inhibitor latrunculin A (n = 5 donors). f, Intensity of heavy amino acid CD19-derived peptides detected in the lysates of trog+ and trog− FACS-sorted cells after 1 h of co-culture with NALM6 cells expressing CD19–mCherry. CD19–mCherry-expressing NALM6 cells are used as a control (n = 2 donors). g, MFI of CD81 on NALM6 cell surface co-cultured with anti-CD19 CAR T cells (n = 3 donors). h, MFI of CD22 on NALM6 cell surface co-cultured with anti-CD19 CAR T cells (data are representative of three donors, n = 3 independent samples). i, MFI of CD22 on cell surface of residual NALM6 retrieved from mouse bone marrow 14 days after 19-BBζ CAR T cell treatment (0.2 × 106 CAR T cell dose, n = 3 mice). ns, non-significant. P values were determined by two-sided Mann–Whitney U-test (a, e and i), binomial false discovery rate-adjusted (b), or two-way ANOVA (d). Data are mean ± s.e.m.
