Improved functionality of HLA–reduced primary human T cells in the presence of HLA mismatched T and NK cells in vivo. (A) Experimental setup for in vivo transfer of HLA–reduced aCD19-CAR T cells; effector cells from HLA-A∗02⁺ donor A (same donor as in Figures 4 and 5) were administered IV 1 day after assessment of humanization of hu-CD34 NSG-SGM3 mice; humanization was performed with cord blood derived hCD34⁺ cells from donor C, which were different from cells of donor A for every HLA class I allele except for HLA-A∗02; every 3 days, blood samples were drawn and analyzed via flow cytometry; CAR T cells used as effector cells all received an aCD19-CAR knock-in into the endogenous TRAC gene locus and were simultaneously edited for all HLA class I alleles except for HLA∗02, and were administered directly after electroporation without prior sorting. (B) CD19⁺ B cells (percentage of living hCD45⁺ lymphocytes) on day 11 after administration of no cells or aCD19 CAR T cells with unedited or reduced HLA alleles into humanized mice. (C) CD19⁺ B cells shown as change in percentage relative to 1 day prior to administration of no cells or aCD19 CAR T cells with unedited or reduced HLA alleles into humanized mice; statistical testing by ordinary 2-way ANOVA and Tukey multiple comparisons test; n = 2-3 mice. (C) CD19⁺ B cells (percentage of hCD45⁺ living lymphocytes) recovered in indicated organs on day 14 after aCD19 CAR T-cell administration with unedited or reduced HLA alleles into humanized mice; statistical testing was done using an unpaired t test; n = 2-3 mice. (E) Numbers of CD19⁺ B cells recovered in blood of humanized mice on day 7 after aCD19 CAR T-cell administration with unedited or reduced HLA alleles, pooled data from 2 independent experiments (dot or diamond symbol barcode), normalized to mean of allogeneic CAR T cells in both experiments; statistical testing was done using an unpaired t test; n = 8 mice; ∗P < .05. Bar height indicates mean, error bars indicate SD for panels C-E. ns, not significant.