Extending the residence period of allogeneic iPSC-derived invariant natural killer T cells in humanized mice by editing HLA class I expression

Abstract

Induced pluripotent stem cell (iPSC)-derived immune cells show promise for allogeneic immunotherapies. However, their clinical efficacy can be limited by early rejection of grafted iPSC-derived cells by host T cells and natural killer (NK) cells due to HLA mismatching. In theory, disrupting HLA class I expression can prevent T cell-mediated rejection, but, in its place, NK cells can eliminate “missing-self” targets due to the absence of HLA-C and HLA-E. Therefore, we devised a strategy to extend the residence time of iPSC-derived invariant natural killer T (iPSC-derived iNKT) cells in humanized mice harboring allogeneic peripheral blood mononuclear cells (PBMCs) by genetically modifying HLA class I expression. To this end, we disrupted HLA class I expression by deleting the β2-microglobulin (B2M) gene, and then recapitulated HLA-C and/or HLA-E expression in the iPSC-derived iNKT cells. Parental and modified iPSC-derived iNKT cells were assessed for their susceptibility to T- or NK cell-mediated cytotoxicity in vitro and for their residence time in allogeneic humanized mice. In vitro, dual co-expression of HLA-C and HLA-E facilitated escape from NK cell-mediated cytotoxicity, whereas single expression of either HLA-C or HLA-E provided inconsistent protection across NK cell donors. Since allogeneic HLA-C alleles can be recognized by T cells, we examined the impact of HLA-C mismatching on T cell-mediated cytotoxicity. T cell-mediated cytotoxicity against B2M-deficient iPSC-derived iNKT cells was restored when mismatched HLA-C was re-expressed. In vivo, B2M deficiency extended the residence time of iPSC-derived iNKT cells in allogeneic humanized mice. This effect was further enhanced by expression of HLA-C alleles matched to donor PBMCs, whereas expression of incompatible HLA-C reduced residence time. Taking into account the limitations of the humanized mouse model in recapitulating human NK cell function, these observations highlight the dominant role of T cell-mediated mechanisms in the rejection of allogeneic iPSC-derived iNKT cells in vivo. This study presents a promising approach for generating iPSC-derived iNKT cells tailored for a limited master cell bank, with the potential to develop universal off-the-shelf immunotherapy.