Abstract

Chronic idiopathic/immune thrombocytopenic purpura (ITP) is an autoimmune disorder in which antiplatelet antibodies induce destruction of platelets. T cells may play a critical role in controlling the synthesis of autoantibodies, and may relate to initiation of ITP. In order to identify the feature of T cells immune state in patients with ITP, TCR Vβ repertoire which can identify T cell proliferation in response to antigenic stimulation; signal joint T-cell receptor excision DNA circles (TRECs) which can use to estimate the thymic recent naïve T cells output function and the expression level of TCR ζ chain gene which defined as an important factor in T cells signaling. Specific Vβ family primers, RT-PCR and genescan technique were use to analyze the expression of TCR Vβ subfamily and the clonality of TCR Vβ T cells in cDNA form peripheral blood mononuclear cells (PBMCs) of 5 ITP patients; quantitative analysis of TRECs in DNA of PBMCs from 15 cases were preformed by real-time PCR (TaqMan); real-time PCR with SYBR Green I technique was used for detecting TCR ζ gene expression level in cDNA of PBMCs from of 18 patients, β2 -microglobulin gene was used as an endogenous reference, relative changes in TCR ζ chain gene expression level were used by the 2 ΔCt method. 25 normal individuals served as control. The results showed that

  1. only 4–11 Vβ subfamily T cells could be identified in ITP cases. The frequent expression Vβ subfamilies were Vβ2 (100%), Vβ3 (100%), Vβ19 (80%) and Vβ21 (80%), while it could not detected the expression of Vβ4, Vβ6–12, Vβ17, Vβ20 and Vβ24. However, all 24 Vβ subfamilies could be detected in samples from normal individuals and all of them displayed polyclonality, clonal expansion Vβ T cells could be found in some subfamilies in every patient. The frequent clonal expansion T cells were Vβ21 subfamily which could be identified in 4 out of 5 cases.

  2. The mean value of TRECs was 2.60±2.99 copies/1000 PBMCs in ITP patients, it was not significantly difference (p>0.05) in comparison with normal group (3.76±3.42 copies/1000 PBMCs), although the TRECs level displayed changefully in different patients with ITP. In four out of fifteen ITP cases no TRECs copies could be detected in more than 10000 PBMCs. The higher frequency of TRECs was found in female group, however it was not statistical significance in comparison with male group.

  3. The expression of TCR ζ chain gene could be detected in all of 25 normal samples, and TCR ζ gene was found in 17 out of 18 cases with ITP.

However, the expression level of TCR ζ gene was lower in ITP samples than those from normal individuals (P=0.017). The expression level of TCR ζ gene is nonsignificat age-associated or gender-link in ITP patients. In conclusions, the restricted expression of TCR Vb subfamilies and clonal expanded Vβ T cells are the T-cell immune feature in ITP, and the frequent presentation of clonal expanded Vβ21 T cells may relate to the disorder of cellular immune function in ITP. At least, in most cases with ITP, the normal level of naïve T cells and thymic recent output function were detected. It is, to our knowledge, the first description in the expression feature of TCR ζ gene in ITP patients, which displayed down expression, it might be a feature in autoimmune disease.

Author notes

Disclosure:Research Funding: The project was sponsored by grants from National Natural Science Foundation of China (No. 30424003) and the Medical scientic research funding of Guangdong Province (No. A2003396).