High-throughput identification of antigen-specific TCRs by TCR gene capture

Abstract

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

“…Therefore, to determine the population dynamics of a given emulsion RT-PCRderived pair precisely, this technique must be paired with more established TCR profiling methods, such as single-cell RT-PCR from sorted cells, cloning of tumor T cells, and/or RNA-or DNAbased bulk sequencing (11,(22)(23)(24)(25). Additionally, our observation that differences in TCR transcript abundance in laboratorygenerated T-cell hybridomas results in differential recovery of TCR sequences has implications in profiling diverse T-cell pools.…”

“…The alpha-and beta-chains of the TCR undergo V(D)J recombination and heterodimerize in the thymus, resulting in a diverse T-cell repertoire that specifically recognizes peptide-MHC complexes. Many studies have analyzed the alpha-and beta-chains of TIL TCRs separately using highthroughput sequencing to describe the diversity of TILs (10)(11)(12)(13). Pairs are readily identified after expansion of T-cell clones, although culture of T cells can lead to substantial skewing of the repertoire (14), which may select for T cells of varied affinity or avidity (15).…”

“…Single-cell sequencing identifies alpha-beta pairs, but is often laborious and has relatively low throughput. Tumor-reactive TCR pairs can be generated from activated T cells after culture with an autologous tumor using gene capture followed by frequency-based matching (11). Recently, a high-throughput statistical method to determine the matched alpha-beta pairs was published, demonstrating high-fidelity pairing by this combinatorial analysis (16).…”

Functional TCR Retrieval from Single Antigen-Specific Human T Cells Reveals Multiple Novel Epitopes

“…Only recently three different approaches addressing the latter limitation were reported. Linnemann and colleagues (20) exploited the quantitative nature of TCR gene capture for frequency-based matching of TCR-a/b chains from oligoclonal T-cell populations. Turchaninova and colleagues (21) introduced an interesting cell-based emulsion real-time PCR (RT-PCR) technique for the identification of TCR-a/b chain pairing.…”

Immunotherapy for opportunistic infections: Current status and future perspectives

“…61 Techniques to isolate each peptide-specific T cells might be useful to facilitate the identification and isolation of antigen-specific T cells. 62 T cells with genetically engineered TCR would contribute to break a major obstacle in a patient or donor with low frequency of specific T cells.…”