Volume 4 Supplement 3
Characterization of the TCR V repertoire of piperacillin-specific T-cells
© Al-Attar et al; licensee BioMed Central Ltd. 2014
Published: 18 July 2014
Piperacillin is a -lactam antibiotic that is frequently used to combat bacterial infections in patients with cystic fibrosis. However, its use is associated with a high incidence of delayed-type hypersensitivity reactions. Our previous studies have shown lymphocyte proliferative responses and cytokine secretion from PBMCs in approximately 75% of hypersensitive patients. In contrast, PBMCs from tolerant controls were not activated by these antibiotics. Drug-responsive T-cell clones isolated from piperacillin hypersensitive patients were CD4+ and the drug derived antigen was presented by HLA class II molecules. These data highlight the pivotal role of T cells in mediating the piperacillin hypersensitivity, however, little is known about TCR V usage. Our project aims to explore the phenotype of T cells involved in piperacillin hypersensitivity looking at the TCR V usage, chemokine receptor expression and cytokine release in both hypersensitive patients and in drug naïve healthy donors.
PBMCs from hypersensitive patients were cultured in the presence of piperacillin and drug-responsive T-cell clones were generated by serial dilution. TCR V usage and chemokine receptor expression were analyzed by flow cytometry. Cytokine release was assessed by ELISpot. Naïve T cells from healthy donors were primed to piperacillin using dendritic cells to present the drug antigen.
A total of 20 piperacillin-specific clones were generated from the hypersensitive patients. Most of the clones were CD4+ and drug stimulation was associated with the secretion of IFNγ (85% of the clones), IL5 (65%), IL13 (35%), perforin (60%), granzyme-B (25%) and FasL (65%). Clones expressed high levels of CCR8, CCR4, CCR10, CXCR3, CCR2 and CCR9. TCR V usage was by these clones was diverse. Piperacillin-specific T-cell proliferative responses and cytokine secretion were detected when naïve T-cells were primed with piperacillin for 7 days. TCR V usage was diverse with marked expansion of TCR V9 and V13.2 on memory T-cells post priming. CDR3 spectratyping analysis will be used to characterize whether expansion of the individual TCR Vβs is polyclonal or oligoclonal. In on-going experiments we are cloning T-cells from healthy volunteers to analyse TCR V expression.
These data show that piperacillin-responsive CD4+ T-cells express a diverse repertoire of TCR Vβs.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.