Generation of Human Cells with Monoallelic Expression of HLA‑A in HLA Class I Knockout Cells for Evaluation of Antigen Presentation.

Cells expressing monoallelic HLA class I molecules represent a convenient in vitro model for studying the immunogenicity of tumor protein neoepitopes. Such models can be generated using endogenous HLA class I-deficient cells. Here, we describe an effective method for eliminating HLA class I expression in HEK293T cells via simultaneous CRISPR/Cas9 editing of the HLA‑A, HLA‑B, and HLA‑C genes using a single guide RNA. An important advantage of this method is the possibility to maintain the unedited HLA‑E gene. The antigen-presenting function of the resulting cells was successfully restored by transduction of a single HLA class I allele. Cells expressing monoallelic HLA‑A, after processing SARS-CoV‑2 epitopes, induced the allele-specific immune response in leukocytes isolated from patients who had recovered from COVID-19. Thus, the obtained HLA class I-knockout cells can be used to evaluate allele-specific neoantigen presentation.