Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity.

T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR specific for a tumor epitope, is a form of adoptive cell therapy (ACT) that has demonstrated promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, but also attractive targets for TCR-T cell therapy. However, MHC class I-restricted TCRs specifically targeting G12-mutant KRAS epitopes in the context of tumors expressing HLA-A2, the most common human HLA-A allele, have remained elusive despite evidence that an epitope encompassing the mutation can bind HLA-A2 and induce T cell responses. We report that post-translational modifications of the protein on this epitope may allow tumor cells to evade immunologic pressure from TCR-T cells. A lysine side chain-methylated KRAS G12V peptide, rather than unmodified epitope, may be presented in HLA-A2 by tumor cells and impact recognition by TCRs. Using a novel computationally guided approach to design TCRs, we developed by mutagenesis TCRs that recognize this methylated peptide, enhancing tumor recognition and destruction. Additionally, we identified TCRs with similar functional activity in normal repertoires from rare primary T cells by stimulation with modified peptide, clonal expansion, and selection. Mechanistically, a gene knockout screen to identify mechanism(s) by which tumor cells methylate or demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these TCRs. These findings highlight the role of post-translational modifications in immune evasion and suggest that identifying and targeting such modifications should facilitate development of more effective TCR-T cell therapies.

Large libraries of single-chain trimer peptide-MHCs enable antigen-specific CD8+ T cell discovery and analysis.

The discovery and characterization of antigen-specific CD8+ T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapt single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We use this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then construct SCT libraries to capture SARS-CoV-2 specific CD8+ T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes is validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease.

The effects of low-dose irradiation on inflammatory response proteins in a 3D reconstituted human skin tissue model.

Skin responses to moderate and high doses of ionizing radiation include the induction of DNA repair, apoptosis and stress response pathways. Additionally, numerous studies indicate that radiation exposure leads to inflammatory responses in skin cells and tissue. However, the inflammatory response of skin tissue to low-dose radiation (≤10 cGy) is poorly understood. To address this, we have utilized a reconstituted human skin tissue model (MatTek EpiDermFT™) and assessed changes in 23 cytokines, 24 and 48 h after treatment of skin with either 3 or 10 cGy low dose of radiation. Three cytokines, IFN-γ, IL-2, MIP-1α, were significantly altered in response to low-dose radiation. In contrast, seven cytokines were significantly altered in response to a high radiation dose of 200 cGy (IL-2, IL-10, IL-13, IFN-γ, MIP-1α, TNFα and VEGF) or the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (G-CSF, GM-CSF, IL-1α, IL-8, MIP-1α, MIP-1ß and RANTES). Additionally, radiation induced inflammation appears to have a distinct cytokine response relative to the nonradiation induced stressor, TPA. Overall, these results indicate that there are subtle changes in the inflammatory protein levels after exposure to low-dose radiation and this response is a subset of what is seen after a high dose in a human skin tissue model.