Intrinsic in vitro functionality of HLA class I and II–deficient primary human T cells. (A) Representative flow-cytometric intracellular cytokine staining of interferon gamma (IFN-γ) and tumor necrosis factor α (TNFα) in response to no antigen, increasing amounts of antigen or phorbol 12-myristate 13-acetate (PMA) and ionomycin; human PBMCs as effector cells underwent orthotopic TCR replacement with CMV NLV–specific TCR 6-2 (TCR KI) and were simultaneously edited with B2M gRNA (B2M KO) or CIITA gRNA (CIITA KO); K562 cells loaded with NLV-peptide pp65_495-503 as antigen were used for stimulation. (B) Quantification of data from panel A (n = 2-3 technical replicates, mean ± standard deviation [SD]), x-axis showing logarithmic molar peptide concentration; neg, negative control. (C) Killing of target cells (HepG2 cells pulsed with NLV-peptide pp65_495-503) by HLA class II KO T cells as measured through changes in cell index over time (left panel) in an xCELLigence assay and quantified as the area under the curve (right panel) calculated over the entire time period shown; addition of effector cells indicated by dashed line; positive control of target-cell lysis achieved through addition of detergent Triton-X; negative control (neg. ctrl.) shows uninhibited target-cell growth through absence of effector cells; mock-edited effector cells were used as control to show nonspecific effect on target-cell growth through superseding; CD8⁺ T cells from panels A and B were used as effector cells and sorted for successful editing (CD8⁺ hTCR⁻ mTRBC⁺ β2m⁻/CIITA⁻); statistical testing by ordinary 1-way analysis of variance (ANOVA) and Tukey multiple comparisons test), n = 3 technical replicates, mean ± SD; ns, not significant; ∗∗∗∗P < .0001. (D) As in panel C, with HLA class I KO T cells as investigated effector cells.