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ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with
Katharina Lindner1,2,3,4, Saskia R Stange1,2,3,4,5, Julian Benedikt Hlawatsch1,2,3,4,6
1Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany.
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:
In vitro 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 in vitro 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 ex vivo 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.
Insights
The novel ESPEC-SUIT assay effectively identifies and tracks numerous antigen-specific T cell receptors (TCRs) from polyclonal T cell responses. This method offers deep insights into cancer immunity and aids in developing targeted immunotherapies.
Area of Science:
- Immunology
- Oncology
- Molecular Biology
Background:
- Current methods for identifying antigen-reactive T cell receptors (TCRs) are limited by T cell clone rarity and restricted applicability to MHC class I and II responses.
- A comprehensive understanding of the entire antigen-reactive repertoire is crucial for exploiting T cell responses in cancer and other diseases.
Purpose of the Study:
- To develop a novel assay, Epitope-Specific Expansion Culture with Subsequent Identification of TCRs (ESPEC-SUIT), for broad and deep insight into polyclonal, antigen-specific TCR repertoires.
- To identify and track antigen-specific TCRs for a better understanding of T cell responses in cancer patients.
Main Methods:
- Peripheral blood mononuclear cells were stimulated with neoantigens, and TCRβ repertoire sequencing (TCRseq) was used for readout.
- Candidate antigen-reactive clonotypes were identified by expansion, followed by TCR cloning and validation in co-cultures.
- TCRseq data was used to trace and characterize clonotypes in longitudinal blood and tumor tissue samples.
Main Results:
- The ESPEC-SUIT assay demonstrated robust expansion of CD4+ and CD8+ T cells in response to various antigens in 32 cancer patients.
- TCRseq revealed highly polyclonal neoepitope-specific T cell responses, with >75% of validated TCRs confirming antigen-reactivity.
- Longitudinal analysis in glioma patients showed changes in TCR frequencies mirroring ex vivo responses, with detected TCRs in tumor tissue exhibiting relevant gene expression signatures.
Conclusions:
- ESPEC-SUIT provides unprecedented insight into polyclonal, antigen-specific T cell responses and enables the discovery of numerous TCRs for specific antigens.
- The assay offers an efficient, cost-effective, and scalable framework for interrogating tumor-reactive T cell responses in cancer patients.
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