Human iPSC-derived neurons stimulated with IFNγ present HLA class I-restricted antigens to cytotoxic CD8⁺ T cells

Multiple sclerosis lesions are dominated by clonally expanded CD8⁺ T cells within an IFNγ-rich inflammatory microenvironment and neurons may be targets of these effector cells. However, the peptide antigens that CD8⁺ T cells recognize on neurons are largely undefined. Neurons constitutively express low levels of HLA class I, and whether inflamed human neurons are competent to present a class I ligandome, what that ligandome contains, and whether presentation has functional consequences for autoreactive CD8⁺ T cells remain open questions. Here we combine human iPSC-derived neural aggregates (HNAs), HLA class I immunoprecipitation coupled to LC-MS/MS immunopeptidomics, and microfluidic co-culture assays to map IFNγ-induced HLA class I presentation by neurons and to test antigen-specific cytotoxicity. IFNγ stimulation induced HLA class I upregulation 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 exogenous 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 antigen-dependent neurite injury. Across four donors, comparative immunopeptidomics identified IFNγ-associated neural peptide repertoires that were distinct from those of matched fibroblasts and enriched for predicted HLA-B binding peptides. β2-microglobulin deletion ablated peptide recovery, and neuron-restricted reconstitution enabled identification of candidate neuron-derived peptides, including recurrent neurofilament light (NEFL)-derived peptides detected across donors. Together, these findings establish a human iPSC-derived platform for studying inflammatory neuronal HLA class I antigen presentation and antigen-dependent CD8⁺ T cell engagement.