Figure 2.

HLA-I retention by allogeneic T cells broadly inhibits NK cell reactivity

(A) Schematic of allogeneic HLA-I-deficient (B2M^KO) T cells expressing an HLA-I single-chain (HLA^SC) molecule that inhibits NK cells by engaging a cognate HLA-specific inhibitory receptor. (B) Bubble plot indicates frequency of CD56⁺ NK cells expressing the indicated HLA-specific inhibitory receptor from 14 independent human donors. (C and D) NK cells were stimulated with allogeneic HLA-I⁺ T cells, B2M^KO T cells, or B2M^KO T cells engineered to express one HLA^SC: HLA-Bw4^SC (HLA-B∗57), HLA-C1^SC (HLA-C∗01:02 or C∗07:02), HLA-C2^SC (HLA-C∗04:01, C∗05:01, C∗06:02, or C∗18:01), or HLA-E^SC (HLA-E∗01:03). (C) Summarized data indicate frequency of NK cell subsets expressing the indicated HLA-specific inhibitory receptor that were CD107a⁺ after stimulation with allogeneic HLA-I⁺ T cells, B2M^KO T cells, or B2M^KO T cells expressing the HLA^SC inhibitory ligand for the corresponding NK cell subset (i.e., KIR3DL1-HLA-Bw4^SC, KIR2DL2/L3-HLA-C1^SC, KIR2DL1-HLA-C2^SC, NKG2A-HLA-E^SC). Horizontal dashed line indicates average frequency of CD107a⁺ NK cells after stimulation with allogeneic HLA-I⁺ T cells. (D) Frequency of total CD107a⁺ NK cells after stimulation with the indicated target T cell population. Vertical dashed line indicates average frequency of CD107a⁺ NK cells in the absence of stimulation. (E) NK cell cytotoxicity assay as described in materials and methods. Percentage specific lysis of allogeneic HLA-I⁺ T cells, B2M^KO T cells, or B2M^KO T cells engineered to express the indicated HLA^SC molecule 48 h post-culture at different E/T ratios. (C–E) Symbols represent aggregated data from three to five independent NK cell donors in duplicate. Bars indicate mean and error bars show ±SEM. Statistical significance was calculated by Wilcoxon matched pairs signed rank test (C) and Kruskall-Wallace test with Dunn’s test for multiple comparisons (D and E).