Abstract
BackgroundT cell receptor (TCR) reflects the status and function of T cells. We previously developed a gene melting spectral pattern (GMSP) assay, which rapidly detects clonal expansion of the T cell receptor β variable gene (TCRBV) in patients with HBV by using quantitative real-time reverse transcription PCR (qRT-PCR) with DNA melting curve analysis. However, the molecular profiles of TCRBV in peripheral blood mononuclear cells (PBMCs) and CD8+, CD8- cell subsets from chronic severe hepatitis B (CSHB) patients have not been well described.MethodsHuman PBMCs were separated and sorted into CD8+ and CD8- cell subsets using density gradient centrifugation and magnetic activated cell sorting (MACS). The molecular features of the TCRBV CDR3 motif were determined using GMSP analysis; the TCRBV families were cloned and sequenced when the GMSP profile showed a single-peak, indicative of a monoclonal population.ResultsThe number of skewed TCRBV in the CD8+ cell subset was significantly higher than that of the CD8- cell subset as assessed by GMSP analysis. The TCRBV11 and BV7 were expressed more frequently than other members of TCRBV family in PBMCs and CD8+, CD8- subsets. Also the relatively conserved amino acid motifs were detected in the TCRBV22, BV18 and BV11 CDR3 in PBMCs among patients with CSHB.ConclusionsThe molecular features of the TCRBV CDR3 were markedly different among PBMCs and CD8+, CD8- cell subsets derived from CSHB patients. Analysis of the TCRBV expression in the CD8+ subset was more accurate in assessing the status and function of circulating T cells. The expression of TCRBV11, BV7 and the relatively conserved CDR3 amino acid motifs could also help to predict and treat patients with CSHB.
Highlights
T cell receptor (TCR) reflects the status and function of T cells
Skewed T cell receptor b variable gene (TCRBV) repertoire within total peripheral blood mononuclear cells (PBMCs) and CD8+, CD8- subsets The CD8+ and CD8- PBMC, which were mostly CD4+, were obtained using magnetic sorting
The TCRBV complementarity determining region 3 (CDR3) profiles were compared between two sorted cell populations (15 samples sorted) and the 27 PBMCs populations isolated from patients with chronic severe hepatitis B (CSHB)
Summary
T cell receptor (TCR) reflects the status and function of T cells. We previously developed a gene melting spectral pattern (GMSP) assay, which rapidly detects clonal expansion of the T cell receptor b variable gene (TCRBV) in patients with HBV by using quantitative real-time reverse transcription PCR (qRT-PCR) with DNA melting curve analysis. The molecular profiles of TCRBV in peripheral blood mononuclear cells (PBMCs) and CD8+, CD8- cell subsets from chronic severe hepatitis B (CSHB) patients have not been well described. Chronic severe hepatitis B (CSHB) is associated with a high mortality rate; its pathogenesis is not well understood. Measuring the frequency of specific CDR3 sequences can reflect the degree of T cell clonal expansion and provide some understanding of T cell function
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