T Cell Repertoires Correlate With Pathogenesis Of Idiopathic Thrombocytopenic Purpura

Objective: To determine the 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( β V).Methods: Twenty-five patients with idiopathic thrombocytopenic purpura (including 15 patients with acute ITP and 10 patients with chronic ITP) were enrolled. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 24 subfamily genes of TCRβV from peripheral blood lymphocytes (PBLs) of FTP patients and the amplications run on denatured polyacrylamide sequencing gel, establishing a map of TCR β VCDR3 gene expressing repertoire. The bands that widened or disappeared of TCR β V gene repertoire on the electrophoresis gel were used to analyze the variety of T cell clones in cases of ITP. Comparing sequences of TCR β gene repertoire in normal and abnormal T cell clones made it possible to understand the relationship between TCR β V gene and ITP. Statistical analysis was performed with SPSS 11.0 software. Statistical significance was accepted with a β value less than 0.05.Results: (1)TCR β V CDR3 size distribution: Normally each lane of TCR β V displayed eight to ten bands that generally formed a Gaussian distribution. There were 8 β V subfamilies in which oligoclonality could be observed in all 6 controls, with an average value of 1.5±0.54 per person.In aITP, TCRβV CDR3 size distribution wassimilar to that of normal controls and oligoclonality could be observed in 15 cases with an average value of 2.73±0.88 per person. Abnormal CDR3 size distribution in alTP had little difference compared with healthy controls(P>0.05). There was no common expanded T cell clones in alTP, while abnormal CDR3 distribution could be observed significantly different between clTP patients and healthy controls (P<0.001), with oligoclonality at an average value of 7.2±3.04 per person in 10 cases. Common clonal T cell expansions could be observed in V^ V￡13.1> VP14, V017 subfamily. ?Normally a band consisted of multiple CDR3 clones with the same length, therefore sequence of the band is not readable. In 10 clTP patients, sequence could be read from 19 out of 21 dense and strong T cell clone bands on the map of TCR P V CDR3, which suggested that a dominant monoclonal T cell expanded in clTP. Different patients shared a common or similar CDR3 encoded by the TCR P V gene of the expanded T cell clones. The same CDR3 was found in 2/4 expanded 3 V 17 T cell clones, with only 2 amino acids different in framework four (FR4). Two T cell clones had almost the same sequence of CDR3 ofPV17 except for only four basepair difference in their full sequences. The full TCR 3 V gene sequences remained the same in 2 T cell clones for TCR 3 V8 and in other 2 T cell clones for TCR 3 V13.1 in clTP group. It showed that an expanded T cell clones with common function existed and could recognize common antigen in the body of patients with clTP. In analyzing the common motif in CDR3 of different clTP, we found three common mitifs (GANVLTFGAG; E/DTQYFGPG; N(K)EQFFGPG) which were separately used in different TCR6VCDR3 of 19 expanded T cell clones. Among these motifs, TCRPV17 of 3/4 T cell clones had common GANVLTFGAG; TCR 3 V of 4/17 T cell clones had common N(K)EQFFGPG; TCR 3 V of 7/17 T cell clones had common E/DTQYFGPG Not only was the amino acid of the latter very similar to the sequence of experimental autoimmune encephalomyelitis(EAE)-specific Vbeta8.2 TCR CDR3 (CASDSSYEQYEGPG ) in the rat's models, but also the amino acid sequence of TCRBV8 CDR3 in case 8 (CASSYQGSEQYFGPG) was almost the same, compared with the various sequence of 10 T cell clones of 6 SLE cases we reported a few years ago. NEQFFGPG sequence wasshown in the CDR3 region of 5/10 T cell clones, while 3 TCR P V13.1 showed DTQYFGPG Conclusion1. alTP has no significant T cell clones expanded, while cITP usually demonstrates some expanded abnormal oligoclones. This indicate that alTP and cITP have different relations with T cells;2. cITP patients share a common motif for TCR P V CDR3, which is possibly related to recognization of the autoantigens;3. Abnormal T cell clones could be found by TCR P V CDR3 size distribution, which will be contribute to the diagnosis and discrimination of alTP and cITP . It could develop a new therapeutic method for cITP by the preparation of corresponding monoclonal antibody or DNA bacterin.