ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer.

BACKGROUND

Methods to identify and characterize antigen-reactive T cell receptors (TCRs) represent important tools to understand and exploit T cell responses in patients with cancer and beyond. Current methods are hampered by the rarity of individual T cell clones and limited applicability to monitor both major histocompatibility complex (MHC) class I-restricted and MHC class II-restricted responses, hence insufficiently reflecting the entire antigen-reactive repertoire of a patient. To obtain broad and deep insight into polyclonal, antigen-specific TCR repertoires, we developed the 'epitope-specific expansion culture with subsequent identification of TCRs' (ESPEC-SUIT) assay to identify and track antigen-specific TCRs.

METHODS

stimulation of peripheral blood mononuclear cells with (vaccine-targeted) neoantigens was verified in cytokine secretion assays and read-out by TCRβ repertoire sequencing (TCRseq). Candidate antigen-reactive clonotypes were defined by specific expansion in cultures stimulated with relevant antigen, followed by TCR cloning and validation in co-cultures of TCR transgenic effector cells and peptide-presenting targets. Using TCRseq information, candidate and validated clonotypes were traced and characterized in bulk and single-cell repertoire sequencing data of longitudinally collected blood samples and tumor tissue.

RESULTS

In a cohort of 32 patients with cancer, we demonstrate that ESPEC-SUIT supports strong, robust and reproducible expansion of CD4 and CD8 T cells in response to various antigens. TCRseq revealed highly polyclonal neoepitope-specific T cell responses, which can be further characterized with respect to cross-reactivity, affinity or human leukocyte antigen (HLA) restriction. In a subcohort of 10 patients, we selected 341 ESPEC-SUIT-derived TCRs for cloning and functional validation from >2000 candidates and confirmed antigen-reactivity for >75%. We exemplify the usefulness of this TCR discovery method for downstream analysis in neoepitope vaccinated patients with glioma, where we found longitudinal changes in candidate TCR frequencies in blood mirroring antigen-specific Enzyme-Linked ImmunoSpot (ELISpot) responses. Furthermore, up to 67% of candidates could be detected in on-treatment brain tumor tissue and exhibited gene expression signatures overlapping with clonotypes of confirmed specificity to the vaccine antigen.

CONCLUSION

ESPEC-SUIT provides unprecedented insight into highly polyclonal, antigen-specific T cell responses and enables discovery of large numbers of TCRs for a given antigen. It represents an efficient, cost-effective and scalable framework for the interrogation of tumor-reactive T cell responses in patients with cancer.