Identification of mITGB2 as an MHC-II neoantigen using hepatocellular carcinoma organoids

The immune microenvironment in hepatocellular carcinoma (HCC) is highly complex and immunosuppressive, limiting the efficacy of immunotherapy. Insufficient antigen presentation and T cell dysfunction remain major barriers. Neoantigen-based vaccines offer a targeted strategy to elicit tumor-specific immune responses. In this study, we applied a tumor organoid–based platform for neoantigen screening and immunogenicity evaluation in HCC. An organoid–immune cell co-culture model was first established to assess antigen-specific cytotoxicity. Proteomic profiling of tumor and matched peritumoral organoids identified differentially expressed peptides, from which candidate MHC class II neoantigens were predicted using NetMHCIIpan and IEDB. Among these, mITGB2—a peptide encoded by an ITGB2 splice variant—was selected based on its high HLA-DRB1 binding affinity and consistent overexpression in tumor organoids. The expression profile and immune relevance of ITGB2 were analyzed using TCGA and clinical samples. Functional validation included in vitro antigen presentation and CD4⁺ Th1 polarization assays, followed by in vivo testing in humanized HCC mouse models. Co-culture assays showed that dendritic cells (DCs) loaded with tumor organoid lysates activated PBMCs and induced specific cytotoxicity against HCC organoids. Proteomic analysis revealed upregulation of HLA-DRB1, indicating enhanced antigen presentation. ITGB2 was prioritized for its predicted immunogenicity and overexpression. High ITGB2 expression was associated with increased CD4⁺ T cell infiltration and favorable prognosis. mITGB2-loaded DCs promoted CD4⁺ Th1 polarization in vitro and suppressed tumor growth in vivo. We identified mITGB2 as a functionally immunogenic MHC class II–restricted neoantigen in HCC, supporting its potential as a promising candidate for neoantigen-based immunotherapy.