Abstract
To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals. Using an HLA-A*02-restricted cytomegalovirus (CMV) pp65-derived immunogenic peptide, we found that the more dominant pp65-specific TCR clonotypes in the blood of healthy donors have higher binding affinities for the CMV peptide and arise from clonotypes that are highly shared among individuals. Interestingly, these highly shared HLA-A*02-restricted CMV-specific TCRs were detected in a CMV-seronegative individual as well as in HLA-A*02-negative donors albeit at lower frequency. More intriguingly, these shared TCR clonotypes were abundant in the stem memory T-cell subset, and TCR diversity of the stem memory T-cell repertoire was significantly lower than in the central memory and effector memory T-cell repertoires. These results suggest that the stem memory T-cell subset may serve as a reservoir of highly shared and highly functional memory T-cells.
Highlights
To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals
We evaluated the diversity of the unfractionated entire T-cell repertoire among the donors by calculating Simpson’s Diversity Index (SDI) using the next-generation sequencing (NGS) data
We evaluated the function of identified CMV NLV-specific TCRs by transducing these TCRs into T cells obtained from CMV-seronegative donors, because such knowledge is important for determining details of the mechanisms of cellular immunity in humans as well as for choosing a highly functional TCR suitable for personalized cellular immunotherapy[1, 2]
Summary
To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals. Without this information, it is difficult to evaluate the antigen specificity of the identified TCRs and to conduct functional assays using T cells engineered to express those paired TCRαβ subunits To mitigate this disadvantage, we have developed an integrated methodology that comprehensively and simultaneously analyses paired TCRαβ repertoires and their function by combining semi-quantitative NGS with single-cell multiplex clonotypic analysis to clarify pairs of TCRαβ expressed on T cells at a single-cell level (designated as human T cell efficient cloning within 10 days, hTEC10)[3]. Certain well-screened functional TCRs shared between HLA-matched individuals are designated as public or shared TCRs7
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