6,7 Next-generation sequencing provides a quantitative readout of V(D)J gene usage and CDR3 sequence-level resolution, assessment of the TCR repertoire, including detection of low-abundance rearrangements from bulk immune cells, or even pairing of the heterodimeric chain sequences with single-cell preparation methods. The simplest method for assessing clonality of a T-cell population employs multiplexed amplification of the TCR α, β, γ, or δ loci using standardized primer sets and qualitative interpretation of fragment size distributions by capillary electrophoresis according to the BIOMED-2 protocol. 4,5Ĭurrent TCR rearrangement profiling assays rely on targeted polymerase chain reaction (PCR) amplification of rearranged TCR genomic loci. 3 T-cell–mediated responses associated with a particular peptide can be detected and monitored by sequencing of the TCR encoding genomic loci derived from a patient sample followed by TCR profiling. 2 Individual T cells contribute to immunologic processes through interactions between the TCR, peptide/major histocompatibility complex, and other costimulatory molecules. 1 The total diversity of a T-cell population is then determined by the structure of the complementarity determining regions (CDRs) of the rearranged and paired α/β or γ/δ chains that interact with the peptide presented in the major histocompatibility complex. The heterodimeric TCR protein includes the variable α and β, or γ and δ chains, which are generated by VDJ recombinase mediated recombination of the variable (V), joining (J), and diversity (D) gene segments at the TCR genomic loci. Mature T-cell lymphomas can be identified by the presence of a clonal population of T cells possessing common T-cell receptor (TCR) rearrangement(s). CapTCR-seq allows for rapid, high-throughput and flexible characterization of dominant clones within TCR repertoire that will facilitate quantitative analysis of patient samples and enhance sensitivity of tumor surveillance over time. Dominant Variable (V) and Joining (J) gene pair rearrangements in cancer cells were confirmed by polymerase chain reaction (PCR) amplification and Sanger sequencing clonality assessment of clinical isolates using BIOMED-2 methods showed agreement for 73% and 77% of samples at the β and γ loci, respectively, whereas β locus V and J allele prevalence in PBMCs were well correlated with results from commercial PCR-based DNA sequencing assays ( r 2 = 0.94 with Adaptive ImmunoSEQ, 0.77-0.83 with Invivoscribe LymphoTrack TRB Assay). We use this method to describe the TCR repertoires of 63 putative lymphoma clinical isolates, 7 peripheral blood mononuclear cell (PBMC) populations, and a collection of tumor infiltrating lymphocytes.
We have developed a hybrid-capture method that enriches DNA sequencing libraries for fragments encoding rearranged TCR genes from all 4 loci in a single reaction. Mature T-cell lymphomas consisting of an expanded clonal population of T cells that possess common rearrangements of the T-cell receptor (TCR) encoding genes can be identified and monitored using molecular methods of T-cell repertoire analysis.
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