Fig 2: HLA-I/II-negative human T cells retain functional and phenotypic properties. Primary CD3+ T cells were transfected with a mixture of gRNAs targeting B2M, HLA-DRA, HLA-DQA and HLA-DPA (Quadruple gRNAs) or non-targeting gRNA (control gRNA) together with Cas9 protein and expanded for 14 days ex vivo. (A) A flow cytometry image of HLA ablation in CD3+ T cells transfected with HLA gRNAs. A representative plot is shown from five different donors. Boxes on the plots indicate the HLA-positive control T cells (blue) or HLA-I/II-negative T cells (red) analyzed in (B–E). (B) Distribution of CD4+ and CD8+ T cells in HLA positive control T cells or HLA-I/II-negative T cells gated from (A). Results from three different donors are shown. (C) Histograms depicting expression of activation or exhaustion markers in HLA-positive control T cells (top) or HLA-I/II-negative T cells (bottom) gated from (A). A representative image from five different donors is shown. (D,E) Secretion of TNF-a, IFN-? and CD107a release were measured by intracellular staining in HLA-positive control T cells or HLA-I/II-negative T cells gated from (A). Cells were stimulated with PMA/ionomycin (D) or Dynabeads Human T-Activator CD3/CD28 (E). Results from six (D) or three (E) different PBMC donors are shown. n.s, not significant (p > 0.05) by Wilcoxon matched-pairs signed rank test.

Fig 3: HLA-II ablation in HLA-I-negative T cells further reduces allogeneic T cell activation. (A) A schematic diagram of experimental design for (B,C) Human primary CD3+ T cells isolated from healthy donor PBMC were transfected with non-targeting gRNA (control gRNA), B2M gRNA (B2M-27 gRNA), or a mixture of quadruple gRNAs each targeting B2M, HLA-DRA, HLA-DQA and HLA-DPA (quadruple gRNAs) together with Cas9 protein and expanded ex vivo. On day 14, the expanded T cells were sorted according to their HLA expression and ?-irradiated. Another set of allogeneic human PBMCs stained with CTV was co-cultured with the sorted T cells for 12-h. (B) A representative flow cytometry plot depicting the numbers of cytokine-producing allogeneic CTV+ CD3+ T cells co-cultured with the HLA+ (top), HLA-I- (middle), or HLA-I/II- T cells (bottom), respectively. (C) Quantification of IFN-? (left) or TNF-a-secreting (right) cells among the total allogeneic CD3+ T cells from (B) are presented. Each dot represents different alloreactive donor (n = 13). Pooled results from 4 independently performed experiments are shown. *p < 0.05, by Friedman test followed by Dunn’s multiple comparisons test.