US2-mediated Immune Evasion: A Novel Strategy For Generating Hypoimmunogenic iPSC Lines

Background & Aim

Mismatches in HLA haplotypes between donors and recipients significantly increase the risk of graft failure due to immune rejection. While knocking out beta-2 microglobulin (B2M) is the current standard for preventing HLA class I surface expression and protecting stem cell-derived products from allogeneic rejection, the complete ablation of HLA-I molecules can impair natural killer (NK) cell “self” recognition and disrupt critical immune-regulatory interactions.

Methodology

To address these challenges, we developed a novel hypoimmunogenic induced pluripotent stem cell (iPSC) line by inserting the cytomegalovirus-derived US2 encoding sequence into the AAVS1 safe harbor locus. Transcriptomic analysis of the modified cells, confirmed that US2 expression preserves stemness and differentiation capacity into both endothelial cells (ECs) and kidney organoids.

Results

Flow cytometry (FACS) analysis revealed that US2-expression abrogates HLA-A2 cell surface expression while retaining low levels of non-classical HLA molecules. In co-culture assays, US2 expression abolishes HLA-A2 alloreactive T cell activation and significantly lowers NK cell activation and degranulation compared to B2M KO ECs, demonstrating superior evasion of "missing self"-mediated NK cell responses.

Conclusion

These findings establish US2-mediated "stealth" technology as a refined alternative to B2M knockout, offering selective modulation of HLA expression to mitigate immune rejection while preserving critical immune-regulatory interactions.