62 Screening of peptide epitopes for T-cell receptors using a Jurkat-NFAT-GFP reporter system and K562 antigen-presenting cells
Year: 2024
Type: conference-abstract
Abstract: Background The activation of T-cell receptors (TCRs) is a critical component of the adaptive immune response, driven by the interaction between TCRs and peptide-major histocompatibility complex (MHC) molecules on antigen-presenting cells. Understanding this interaction is pivotal for the development of improved checkpoint and cell-based immunotherapies. Methods To characterize antigen specificity and discover peptide epitopes recognized by specific TCRs, we developed a technological platform based on using Jurkat-NFAT-GFP reporter cells in combination with artificial antigen-presenting K562 cells (APC) expressing any class I HLAs. We developed an effective protocol for cloning full-length candidate TCRs or a library of TCRs in Jurkat reporter cells and predicted the minigene peptide epitopes library in the APC-HLA. Co-culture experiments of Jurkat-TCR reporter cells expressing specific TCRs with APC cells expressing the library of peptide-MHC complexes allow us to identify specific peptide epitopes recognized by candidate TCRs. Specific TCR-Epitope-MHC interactions activate expression of GFP and secretion of IL2 which specifically label APC expressing interacting peptide. Amplification and NGS analysis of minigenes from IL2-labelled APCs allow the identification of peptide epitope candidates which are used for follow-up validation and characterization of TCR candidate antigen specificity using synthetic peptides. Results To validate the performance of the TCR-Epitope screening platform, we expressed TCRs specific for CMV-MHC, EBV-MHC, or FLU-MHC in Jurkat reporter cells and co-cultured these cells with a K562 APC-HLA-A 02:01 expressing library of 100 minigenes comprising candidate peptide epitopes for matched control viral antigens. Activation of Jurkat-TCR reporter cells was detected in 24 hours. IL2-labelled APCs were sorted and matched viral antigens were successfully identified for all three control TCRs. TCR epitope specificity was additionally validated by activation of Jurkat-TCR clones with APC labeled with synthetic identified viral peptides. Conclusions Our study demonstrates the efficacy of the developed TCR-epitope screening platform in the discovery and characterization of novel TCR-peptide-MHC interactions. The developed off-the-shelf universal Jurkat reporter and panel of artificial APCs designed with any class I HLA could be effectively used for screening novel shared and patient-specific epitopes using several pre-made or custom neo-antigen libraries. Furthermore, Jurkat reporter and APC-HLA cells can be used to clone any specific/candidate TCR and study its activation using different predicted/candidate synthetic peptides.
Institution: Cellecta (United States)
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W4404064147