Hypoimmunogenic HLA-E Single Chain Inhibits Alloreactive Immune Responses

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.