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AbstractBackgroundPublic neoantigens, including KRAS, TP53, and PIK3CA mutations, which are shared across various tumor types, have demonstrated significant immunogenicity and offer great promise for cancer immunotherapy. Clinical trials targeting these public neoantigens have yielded encouraging results, including tumor regression and prolonged relapse-free survival. This study evaluates the human leukocyte antigen (HLA) binding properties of T-cell epitopes derived from these public neoantigens to identify optimal T-cell target and further develops T-cell receptor (TCR)-based therapeutics.MethodsThe binding properties of public neoantigens to HLA-I molecules were evaluated using peptide-HLA binding affinity and stability assays. Naive T-cell repertoires were used to expand and detect neoantigen-specific TCRs. TCR clones were characterized for functionality using TCR-Jurkat cells and TCR-T cells. Peptide specificity was assessed using an HLA transgenic cell panel and the X-scan assay. In vivo antitumor efficacy of TCR-T cells was tested in xenograft mouse models of solid tumors.ResultsThe analysis of HLA binding properties for public neoantigens revealed that HLA-A*11:01-presented KRASG12V epitopes exhibited the strongest HLA binding stability. Four TCR clones specific to the 9-mer KRASG12V peptide (KRASG12V[9]) were identified. All KRASG12V[9]-specific TCRs, both newly identified by us and previously reported, exhibited varying degrees of cross-recognition of the exogenous self-antigen RAB7B. Among the four TCR clones, one TCR (KT18) exhibited superior functional avidity, effectively recognizing and eliminating KRASG12V mutant tumor cells without off-target activity against endogenous RAB7B or similar peptides. Significantly, KT18 TCR-T cells efficiently mediated tumor regression in multiple xenograft models of solid tumors.ConclusionsThese findings highlight significant differences in peptide-HLA binding affinity and stability across public neoantigen-HLA pairings. The cross-recognition of RAB7B13-21 represents a critical safety consideration when developing HLA-A*11:01-restricted KRASG12V[9]-specific TCRs. KT18 TCR-T cells are highly cytotoxic, exhibiting no off-target recognition and significant potential for clinical applications against KRASG12V-driven solid tumors.

SUMMARY Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in T cells. Promiscuous gene expression by a subpopulation of mTECs regulated by the nuclear protein Aire contributes to the display of self-genomic products to newly generated T cells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations, namely Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21, which attracts developing thymocytes to the medullary region. Here, we report that CCL21+ mTECs and Aire+ mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive T cells and the generation of regulatory T cells. We also detect cooperation for self-tolerance between Aire and Fezf2, the latter of which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance. Graphical Abstract In brief Ushio et al. show that functionally diverse medullary thymic epithelial cell (mTEC) subpopulations cooperate to prevent autoimmune pathology. The results demonstrate the interplay between diverse mTEC subpopulations, namely between CCL21+ mTECs and Aire+ mTECs and between Aire+ mTECs and Fezf2+ mTECs, for optimizing central tolerance.