An efficient single‐cell based method for linking human T cell phenotype to T cell receptor sequence and specificity

Abstract

Single-cell antigen-receptor gene amplification and sequencing platforms have been used to characterize T cell receptor (TCR) repertoires but typically fail to generate paired fulllength gene products for direct expression cloning and do not enable linking this data to cell phenotype information. To overcome these limitations, we established a highthroughput platform for the quantitative and qualitative analysis of human TCR repertoires that provides insights into the clonal and functional composition of human CD4 + and CD8 + αβ T cells at the molecular and cellular level. The strategy is a powerful tool to qualitatively assess differences between antigen receptors of phenotypically defined αβ T cell subsets, e.g. in immune responses to cancer, vaccination, or infection, and in autoimmune diseases.

Clonal evolution and TCR specificity of the human T FH cell response to Plasmodium falciparum CSP

“…To enable the unbiased integration of cell phenotype data with TCR gene information, we used indexed flow cytometric cell sorting to isolate single PD-1 high ICOS + cT FH cells from all sampling time points, and we amplified and sequenced the paired TRB and TRA genes from more than 3000 cells without antigen-mediated enrichment or additional in vitro antigenic stimulation ( 19 ). Upon immunization, cT FH cells expanded clonally, resulting in a reduction in repertoire diversity quantified by normalized Shannon-Wiener index (Fig.…”

“…Through gene cloning of paired TRA and TRB genes and subsequent retroviral transduction, we stably expressed the selected TCRs in CD3 + CD4 + , endogenous TCR-negative Jurkat76 cells (fig. S5) ( 19 ). To screen for PfCSP reactivity, the cell lines were cocultured with Epstein-Barr virus (EBV)–immortalized autologous B cells pulsed with pools of overlapping 15-mer peptides covering the complete N terminus and central repeat region or the C terminus (hereafter referred to as N-CSP or C-CSP, respectively; table S2).…”

“…Complementary DNA (cDNA) synthesis and TCR gene amplification were performed as described previously ( 19 ). Briefly, cDNA was prepared in a total volume of 4 μl in the original sort plates using random hexamer primers.…”

“…The cloning and stable expression of TCRs in TCR neg CD3 + CD4 + Jurkat76 cells (J76-CD4) was performed as described previously ( 19 ). Briefly, full-length TRA and TRB genes were cloned into retroviral pMSCV-PImC expression vectors.…”

“…TCR reactivity tests were performed as described ( 19 ). Briefly, 2.13 × 10 5 EBV immortalized B cells were seeded in 96-well tissue culture plates (U-bottom) in AIM V medium and loaded with peptide (2.5 μg/ml) for 18 hours at 37°C and 5% CO 2 before the addition of 4.27 × 10 5 TCR-transgenic Jurkat76 cells.…”