Journal
IMMUNOLOGY AND CELL BIOLOGY
Volume 94, Issue 6, Pages 604-611Publisher
WILEY
DOI: 10.1038/icb.2016.16
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Funding
- NHMRC
- ACH2
- University International Postgraduate Award UNSW Australia
- NHMRC Practitioner Fellowship [1043067]
- NHMRC Career Development Fellowship [1060443, 1067590]
- NHMRC SRFB fellowship [1102792]
- National Health and Medical Research Council of Australia [1067590, 1060443] Funding Source: NHMRC
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Heterogeneity of T cells is a hallmark of a successful adaptive immune response, harnessing the vast diversity of antigen-specific T cells into a coordinated evolution of effector and memory outcomes. The T cell receptor (TCR) repertoire is highly diverse to account for the highly heterogeneous antigenic world. During the response to a virus multiple individual clones of antigen specific CD8+ (Ag-specific) T cells can be identified against a single epitope and multiple epitopes are recognised. Advances in single-cell technologies have provided the potential to study Ag-specific T cell heterogeneity at both surface phenotype and transcriptome levels, thereby allowing investigation of the diversity within the same apparent sub-population. We propose a new method (VDJPuzzle) to reconstruct the native TCR alpha beta from single cell RNA-seq data of Ag-specific T cells and then to link these with the gene expression profile of individual cells. We applied this method using rare Ag-specific T cells isolated from peripheral blood of a subject who cleared hepatitis C virus infection. We successfully reconstructed productive TCR alpha beta in 56 of a total of 63 cells (89%), with double a and double beta in 18, and 7% respectively, and double TCR alpha beta in 2 cells. The method was validated via standard single cell PCR sequencing of the TCR. We demonstrate that single-cell transcriptome analysis can successfully distinguish Ag-specific T cell populations sorted directly from resting memory cells in peripheral blood and sorted after ex vivo stimulation. This approach allows a detailed analysis of the TCR diversity and its relationship with the transcriptional profile of different clones.
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