Figure 3

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-α, IFN-γ 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.