Several platforms exist for single-cell-combined TCR repertoire and transcriptomics analysis, including 10× Genomics and more recently the ICELL8 single-cell system. Transcript sequencing provides gene expression and TCR repertoire information at the single-cell level. The combination of single-cell transcriptomics with TR Over the last 5 years, single-cell transcriptomics has become a popular approach, as it allows to detect the heterogeneity in gene expression among individual cells and the discovery of small subpopulations. Most importantly perhaps, with any of these approaches, it remains difficult to examine changes in TCRαβ repertoire diversity within a heterogeneous pool of T cells or low-abundant population like antigen-specific T cells without purifying them first and/or acquiring large enough numbers of cells. Moreover, neither of these approaches allows to evaluate the actual composition of the total TCRαβ receptor, as no information on TCRα (TRA) profiles is obtained. A major disadvantage of bulk sequencing approaches is the large number of cells required, whereas flow cytometry-based TCRVβ assays suffer from the limitation that the 24 different TCRVβ antibodies collectively cover only 70% of the normal human TCRVβ repertoire. Varying from DNA- or RNA-based TRBīulk sequencing assays to flow cytometry-based single-cell TCRVβ approaches, all suffer from drawbacks. Historically, TCR repertoire diversity assays have mostly focused on TCRβ (TRB) chain profiling. Importantly, whereas antigen-inexperienced or naïve T cells have a broad, unselected TCR repertoire, antigen-experienced or memory T cells generally contain more narrow TCR repertoires, mostly consisting of particular antigen-selected specificities. Estimates of the number of possible different TCRαβ receptors amount to 10 12 molecules. ) of the variable domain, which collectively mediate the specific recognition of antigens. The V(D)J junction is one of the complementarity-determining regions (i.e., CDR3 Repertoire diversity, which is especially apparent in the V(D)J junction. In this way the V(D)J recombination process generates a huge TR Diversity in these variable domains arises from complex recombination processes involving V, D, and J genes in the TCR chain-encoding loci. All four TCR chains are highly diverse in their variable domains. Approximately 95% of T cells express a TCRαβ receptor, consisting of a TCRα and a TCRβ chain, whereas the remaining 5% possess a TCRγδ receptor, consisting of a TCRγ and a TCRδ chain. T cells recognize antigens via unique T-cell receptor (TCR) molecules. Here we present the ICELL8 single-cell method for the parallel analysis of the TR repertoire and transcriptome, which is especially useful in samples that contain relatively few cells. An additional advantage of such single-cell technologies is that the combination of both IG and both TR chains can be studied on a per cell basis, which better reflects the antigen receptor reactivity of cells. By combining the IG/TR repertoire with transcriptome data, the reactivity of the B or T cell can be associated with activation or maturation stages. This is especially relevant in cell samples in which much heterogeneity of the cell population is expected. Single-cell technologies do allow to study the IG/TR repertoire at the individual cell level. Even though many of these strategies do provide in-depth and high-resolution information of the immunoglobulin (IG) and/or T-cell receptor (TR) repertoire, one clear disadvantage is that the IG/TR profiles cannot be connected to individual cells. With the advent of next-generation sequencing (NGS) methodologies, the total repertoires of B and T cells can be disclosed in much more detail than ever before.
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