Mangalaparthi, Kiran K.

Tumor-associated antigens (TAAs) are non-mutated antigenic peptides expressed in cancer tissue at abnormally high levels but in normal tissue either at negligible levels, during only particular developmental stages, or in a tissue-restricted manner. The shared nature of TAAs across patients makes them attractive targets for off-the-shelf immunotherapies. However, no studies have comprehensively surveyed the TAA potential of all possible wild-type peptides originating from cancer-associated genes. In this study of 16 cancer tissue types and 39 normal tissue types, we analyzed the expression profiles of protein-coding genes to identify those with aberrantly high RNA levels across multiple solid tumor types and low RNA levels across all normal tissue types examined. We then developed a score to quantify tumor specificity of gene expression. Compared to previously reported TAA genes, those we identified exhibited substantially greater tumor specificity. Seven of these genes demonstrated consistently elevated expression in at least five tumor types and minimal expression across all non-immune-privileged normal tissues. To assess Human Leukocyte Antigen (HLA) class I presentation potential of the multi-cancer-associated genes, we computationally predicted the binding affinities between the most common class I HLA alleles and all possible wild-type TAA epitopes of 8–11 amino acids arising from those genes. We then applied rigorous filtering criteria to prioritize the most promising multi-cancer TAA peptide candidates and evaluated their HLA binding and cell-surface presentation using T2 and immunopeptidome analysis. Our results highlight new potential targets for multi-cancer immunotherapies.

ABSTRACT Interferon-γ (IFNγ) signaling is prominent in inflammatory CNS microenvironments across many neurological disorders, but the neuronal peptides presented on HLA class I under these conditions and their functional consequences for CD8 + T cells remain incompletely defined. Here we combine human iPSC-derived human neural aggregates (HNAs), HLA class I immunoprecipitation coupled to LC-MS/MS immunopeptidomics, and microfluidic co-culture assays to map IFNγ-induced neuronal antigen presentation and test antigen-specific cytotoxicity. IFNγ stimulation induced robust HLA class I expression in HNAs and enabled recovery of a canonical 8-12-mer class I ligandome enriched for 9-mers. Neuron-restricted expression of a synapsin-driven polyepitope cassette yielded presentation of defined neoantigen 9-mer peptides on donor HLA class I molecules and, in the presence of IFNγ, elicited activation of autologous antigen-specific CD8 + T cells and consequent antigen-dependent neurite injury. Across four donors, comparative immunopeptidomics identified large IFNγ-unique neural peptide repertoires distinct from matched fibroblasts and revealed a consistent enrichment of predicted high-affinity binders on HLA-B allotypes. Finally, β2-microglobulin deletion ablated peptide recovery, and neuron-restricted β2-microglobulin reconstitution enabled identification of neuron-derived peptides, including peptides derived from neurofilament light (NEFL) that were shared across donors and presented on multiple HLA allotypes. Together, these data provide an integrated platform for neuronal autoantigen discovery and functional validation and support a model in which IFNγ-driven neuronal HLA class I presentation creates an HLA-B-weighted epitope landscape that can be recognized by autoreactive cytotoxic CD8 + T cells.