The Characteristics of T Lymphocytic Clones Correlate with the Pathogenesis of Idiopathic Thrombocytopenic Purpura.
Authors: Zhu Ping , Zhu Xia , Guo Xiao-ling , Gu Jiang-ying , Ou Yuan 
Published: April 2020

Abstract
Objective To determine the characteristics of T lymphocytic clones that correlate with the pathogenesis of idiopathic thrombocytopenic purpura (ITP) by investigating complementarity determining region (CDR3) repertoires of T cell receptors (TCRs) β chain variable region (BV).
Methods Twenty-five of patients with idiopathic thrombocytopenic purpura (including 15 patients with acute ITP and 10 patients with chronic ITP), twenty of normal peoples and 20 of normal umbilical cord blood samples were enrolled. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 24 subfamily genes of TCR BV from peripheral blood lymphocytes of ITP patients and normal controls, the PCR products were run on denatured polyacrylamide sequencing gel, establishing spectratyping of TCR BV CDR3 gene expressing repertoire. The bands that dense or disappeared of TCR BV gene repertoire on the electrophoresis gel were used to analyze the variety of T cell clones in cases of ITP. The dense bands in the gel were cut down and sequencing. Compare with those major sequences of TCRBV gene in normal and abnormal T cell clones to understand the relationship among the TCRBV gene and ITP.
Results: In acute ITP (aITP), the spectratyping of TCR BV CDR3 size distribution were similar to normal control (P=0.179), the oligoclonality could be observed with an average value of 2.73±0.88 per person in 15 cases of aITP. TCR BV CDR3 size distribution had little difference compared with aITP and healthy controls. Abnormal spectratyping of TCR BV CDR3 distribution could be observed significantly different between chronic ITP (cITP) patients and healthy controls (P&lt;0.05), with oligoclonality at an average value of 7.2±3.04 per person in 10 cases of cITP. Same T cell clone expansions could be observed in gene subfamilies of TCR BV8,BV13.1,BV14,BV17. In 10 of cITP patients, sequences could be read from 19 out of 20 dense bands on the gene spectratyping of TCR BV CDR3, which suggested some dominant T cell clones expanded in cITPs. In different patients of cITP, the expanded T cell clones shared identical or similar TCR BV gene or CDR3 encoded by the TCR BV gene. Two T cell clones in 2 patients had identical TCR BV8 gene sequences separately. Other 2 T cell clones in another 2 cITP patients had same TCR BV13.1. It showed those expanded T cell clones in the bodies of the cITP patients could recognize common antigen. The similar CDR3 was found in 2/4 expanded TCR BV17 T cell clones, with only 2 amino acids different in framework 4 (FR4). Two T cell clones had almost same sequence of CDR3 in TCR BV17 except for 4 basepairs difference in their full sequences. In analyzing the motifs in CDR3 of different cITPs, we found three common motifs (E/DTQYFGPG;N(K)EQFFGPG;GANVLTFGAG) which were separately used in different TCR BV CDR3 of 19 expanded T cell clones. Among these motifs, TCR BV of 7/19 T cell clones shared common E/DTQYFGPG;TCR BV of 4/19 T cell clones shared common N(K)EQFFGPG; TCR BV17 of 3/4 T cell clones had common GANVLTFGAG. Compared with the various sequence of 10 T cell clones in 6 cases of SLE patients that we had reported (Journal of Chinese Medicine2003;83(10):1648–1652), 5/10 T cell clones of the SLEs shared the motif NEQFFGPG in the CDR3.Three of TCR BV13.1 in these T cell clones showed motif DTQYFGPG.
Conclusion the cITPs have some abnormal expanded T cell clones that correlate with the pathogenesis, while aITPs have no significant expanded clones. All the cITP patients share 3 common motifs in TCR BV CDR3, which is possibly to recognize similar autoantigens.