Deep immune-phenotyping of HLA-homozygous iPS-cardiomyocytes by spectral flow cytometry

INTRODUCTION: Immunogenicity of allogeneic human induced pluripotent stem cell (hiPSC)-derived transplants limits their applicability in regenerative medicine. Selecting human leukocyte antigen (HLA)-homozygous hiPSC lines could be a mitigation strategy and haplo-matching would profoundly expand thenumberof potential recipients. Here we show deep immune-phenotyping of hiPSC-derived cardiomyocytes(iPS-CM)differentiated from four independentiPSClinesinthree centers under chemically defined conditions. METHODS AND RESULTS: Broad immunophenotyping with 354 antibodies revealed differential expression of 101 immune-related molecules between iPS-CM and the parental hiPSC lines. We selected 54 key immune markers for deep immunephenotyping by spectral flow cytometry at the single-cell level. We found that HLA-homozygousiPSCMsexhibit anoverall stable immune-phenotype across HLAhomozygous and heterozygous hiPSC lines indicating a robust differentiation process. HLA-homozygous iPS-CM displayed significantly reduced HLA-ABC levels compared to heterozygous counterparts with an otherwise conserved immune-phenotype. Upon interferon gamma challenge as a surrogate of immune stress responsiveness, iPS-CM significantly upregulated HLA-ABC,-E,-F, PD-L1, PDL2 and the 'don't eat me' signal CD47. As a proof-of-concept we used this panel to benchmark iPS-CM differentiation across three production sites in this study. DISCUSSION: The data indicate generally stable immune-phenotype of iPS-CM produced at three different sites and support feasibility of monitoring iPS-CM identity by spectral flow cytometry.