Next Generation Sequencing-Based Characterization of T Cell Receptor Repertoire of Patients with Immune Thrombocytopenia

Background:

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by increased platelet destruction and impaired platelet production. Abnormal T cell immunity plays a critical role in the pathogenesis of ITP. Analysis of T cell receptor (TCR) repertoire can identify specific T cell population proliferations in response to antigenic stimulation. No systematic next-generation sequencing (NGS)-based TCR repertoire characterization of pediatric ITP patients has been reported to date. We herein report our comprehensive analysis of TCR-β repertoires of peripheral blood samples from a cohort of pediatric patients with ITP.

Method:

Pediatric patients from Lucile Packard Children's Hospital (Stanford Children's Health) with newly diagnosed ITP (n=11), chronic ITP (n=8), resolved from ITP (n=3), and healthy individuals (n=4) were consented to be included in the study. Total RNA was extracted from peripheral blood samples of the study subjects and reverse transcribed into cDNA. Completely rearranged TRB gene locus was amplified and used for library preparation, which were subsequently sequenced on the Illumina MiSeq platform. The sequencing reads were analyzed with IMGT (ImMunoGeneTics)/HighV-QUEST tools. Further analysis was performed to evaluate TRB repertoire diversity, V-J gene segment usage, clonotype sharing among patient samples, clonality pattern, predominant clonotype frequency changes in different disease state, and sequence similarity among expanded clonotypes.

Result:

The normalized clonotype counts and the calculated TRB repertoire diversity indices (Shannon and Simpson indices) did not reveal significant difference among newly diagnosed ITP, chronic ITP and healthy control groups. In terms of the V-J gene segment usage among the top 200 clonotypes (as defined by CDR3 amino acid sequences), newly diagnosed ITP group used TRBV12-3 significantly more frequently, and TRBV6-4 significantly less frequently, compared to the healthy control group. The means of overlap coefficient, reflective of TRB clonotype sharing, for newly diagnosed ITP, chronic ITP and healthy control groups are 0.0045 (range 0.005-0.008), 0.0057 (range 0-0.016), 0.0041(range 0.004-0.005), respectively. Chronic ITP patients had significantly more overlap in TRB clonotypes than healthy donors. No common or "public" clonotypes shared within each group were identified. The nucleotide sequence and CDR3 amino acid sequence of dominant TRB clonotypes in ITP patients were compared, and the percent similarity ranged from 30.18% to 88.26% for the former, and from 16.9% to 36.04% for the latter.

Conclusion:

We performed comprehensive analysis of TCR repertoire of peripheral blood samples from pediatric ITP patients by next-generation sequencing. TCR repertoire diversity at different disease states was analyzed. Differences in V-J gene segment usage and clonotype sharing among patients were observed. These findings provide unique insights into the potential pathophysiology of T cell immunity in patients with ITP.