This article and associated images are based on a poster originally authored by Alex Chenchik, Lester Kobzik, Tianbing Liu, Dongfang Hu, Kitt Paraiso, Khadija Ghias, and Paul Diehl and presented at ELRIG Drug Discovery 2025 in affiliation with Cellecta, Inc.
This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.
Adaptive immune receptor (AIR) sequencing of T- and B-cell receptors (TCRs, BCRs) has broad applications in infectious disease, autoimmunity, and cancer immunotherapy, but connecting receptor discovery to antigen specificity remains a major bottleneck. The researchers present a comprehensive workflow that integrates bulk and single-cell repertoire sequencing, paired-chain reconstruction, and functional assays to bridge this gap. By sequencing DNA and RNA from the same sample, the study distinguishes clonal expansion from transcriptional activation.
In the work, paired TCR sequences are resolved using a plate-based, single-cell assay, cloned, and expressed in GFP-reporter Jurkat cells. These engineered cells enable sensitive antigen-screening assays, including dextramer binding and co-culture with K562 antigen-presenting cells, yielding fluorescent readouts of antigen recognition.
This platform unifies discovery and validation, providing a direct route from repertoire profiling to antigen identification. It can be applied to map disease-relevant clonotypes, interrogate peptide and small-molecule libraries, validate immunotherapies, and dissect receptor signaling pathways that underlie adaptive immune responses.
Figure 1. Schematic representation of TCR repertoire diversity and VDJ recombination. Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
We demonstrate an integrated workflow that focuses on three analytical approaches, which together provide a comprehensive approach to identify and characterize therapeutically important TCR/BCR-antigen interactions.
Repertoire diversity was analyzed using the DriverMap™ Targeted Multiplex RT-PCR technology, which enables simultaneous amplification and sequencing of 100,000 TCR and BCR receptors in a single assay. Empirically tested optimized primers enable the following:
Figure 2. Schematic for multiplex RT-PCR primers for RNA and DNA assays. Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
Figure 3. Schematic shows how the level of receptor RNA and DNA before and after antigen activation can be used to differentiate clone proliferation from increased receptor expression. Parallel profiling and normalization of RNA and DNA can elucidate differences between induced expression (i.e., gene activation by RNA assay) from proliferating cells clones (by DNA assay). Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
Figure 4. Peripheral blood was collected for controls (pre-vaccination and multiple weeks after vaccination) and treatment (few days/weeks post-vaccination). Longitudinal analysis of the adaptive immune repertoire by DriverMap™ AIR RNA and DNA shows differences in sensitivity in identifying clonotype activation pre- and post-vaccination. Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
Figure 5A. DriverMap™ Single-Cell AIR Immunophenotyping Kit which, in addition to profiling TCRs, includes multiplex primers in the same reaction to profile expression of 36 T-cell marker genes, to identify the most abundant single TCR α/β clonotype pair in leukemic T-cells (35 “Normal” blue wells). Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
Figure 5B. Gene expression analysis of the primers for the 36 T-cell marker genes included in the assay revealed that the NKG7 and CCL5 are highly expressed in the most abundant clone. Co-expression of these genes has been associated with neutropenia. Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
By expressing a TCR-αβ receptor in Cellecta’s Jurkat TCR KO GFP Reporter cells, it is possible to test and screen TCRs with multiple antigens to identify which ones they react with.
Figure 6A. The schematic shows the Jurkat TCR K/O NFAT-GFP reporter line transduced with a control TCR construct targeting EBV, FLU, or CMV peptides. Fluorescently labeled dextramers specific for each peptide bind to the corresponding TCRs expressed on the surface of the Jurkat reporter cells and detected via FACS analysis. Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
Figure 6B. Jurkat Reporter Clone staining with peptide-specific dextramer (CMV, FLU and EBV). FACS results show positive staining of Jurkat clone expressing peptide-specific TCR. Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
Figure 7A. Schematic shows the interaction of a Jurkat GFP reporter cell line expressing a specific TCR with K562 APC cells expressing the complementary antigen. TCR-Epitope-MHC interactions activate expression of GFP, which labels APC-interacting peptides. Amplification and NGS analysis from APCs allow the identification of peptide epitope candidates. Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
Figure 7B. Shows that the Jurkat NFAT GFP reporter cells expressing CMV, MART1, or P53 peptide-specific TCR and K562_HLA cells expressing CMV, MART1, or P53 short peptide sequence were co-cultured with each peptide-specific TCR reporter cell (Jc/Jm/Jp). FACS results show activation of the Jurkat reporter cells after incubation as compared to the negative control. Image Credit: Image courtesy of Alex Chenchik et al., in partnership with ELRIG (UK) Ltd.
