Chimeric Ag receptor T cells derived from universal donors are susceptible to recipient immunologic rejection, which may limit their in vivo persistence and compromise treatment efficacy. In this study, we generated HLA class I–deficient T cells by disrupting β₂-microglobulin to evade recognition by HLA-mismatched CD8⁺ T cells, and then restored NK cell tolerance by forced expression of an HLA-E single-chain receptor. We specifically report on an optimized hypoimmunogenic disulfide trap HLA-E4 (dtHLA-E⁴) molecule that exhibited increased surface expression, enhanced NK cell inhibitory potential, and abrogated CD8-dependent T cell recognition. Our dtHLA-E⁴ molecule comprised the CD4 (4) transmembrane domain and truncated cytoplasmic region, as well as disulfide trap mutations to anchor an HLA class I signal sequence-derived peptide. Functional comparison of dtHLA-E⁴ molecules fused to different VL9 epitopes showed that peptides derived from HLA-A and HLA-C allotypes maximized NK cell inhibition and minimized NKG2C⁺ NK cell activation. Furthermore, incorporation of mutations into the α3 domain of HLA-E diminished the immunogenicity of dtHLA-E⁴ by reducing CD8⁺ T cell recognition, but crucially, these mutations left NK cell inhibitory function intact. These findings demonstrate the systematic construction of a hypoimmunogenic dtHLA-E⁴ molecule, which promises to facilitate persistence of allogeneic HLA class I–deficient chimeric Ag receptor T cells by overcoming NK cell missing-self recognition.



Summary

This study engineered hypoimmunogenic T cells for universal donor CAR-T cell therapy. Researchers created HLA class I-deficient T cells to avoid recipient CD8+ T cell rejection. To prevent NK cell-mediated “missing-self” rejection, they introduced a disulfide trap HLA-E4 (dtHLA-E4) molecule. Optimization included increased surface expression, strong NK cell inhibition, and reduced CD8+ T cell recognition. Different VL9 peptide epitopes and α3 domain mutations further minimized T cell immunogenicity while maintaining NK cell inhibition. This dtHLA-E4 molecule facilitates the persistence of allogeneic HLA class I-deficient CAR-T cells by avoiding both T and NK cell rejection.

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