PS1493 NEXT‐GENERATION SEQUENCING REVEALS DISTINCT EXPANDED T‐CELL RECEPTOR (TCR) B‐CHAIN CLONOTYPES IN PATIENTS WITH CHRONIC IMMUNE THROMBOCYTOPENIA (ITP)
Authors: Malik A. , Watt E. , Sayed A.A. , Tsiamita O. , Adams S. , Cooper N. 
Published: June 2019

Background:

Immune thrombocytopenia (ITP) is an autoimmune disease characterised by isolated low platelet count. The pathophysiology of ITP is multifactorial and the role of CD8+ T‐cells remains largely unexplored. Our recent studies show that ITP patients have an increase in effector CD8+ T‐cell population. In order to further explore the nature of these expanded cells, we analysed the TCR β‐chain repertoires in PBMC and sorted CD8+ T‐cells of patients with ITP.

Aims:

To fully characterise the TCRβ repertoire of patients with chronic ITP.

Methods:

The study population consisted of 36 samples from 22 patients diagnosed with chronic ITP with a median age of 46yrs (IQR 33–58) and median platelet count of 36 × 109/L (IQR 21–142) at the time of sampling. Patients were either on no treatment (n = 10) or thrombopoietin‐receptor agonists (n = 12) and were compared to nine age and gender‐matched healthy controls (HC). DNA was extracted from 31 PBMC and five sorted CD8+ T‐cell samples. Next generation sequencing of TCRβ was carried out using the Illumina MiSeq platform. A multiplex of primers was used for PCR amplification of variable‐diversity‐joining (VDJ) gene segments of rearranged TCRβ gene. Analysis of the raw TCR sequences was performed using MiXCR, and data is presented as median values.

Results:

In total, 28,000,000 sequence reads were generated. When compared to HC, the number of unique TCRβ clonotypes in PBMC of ITP patients were significantly less (1556 vs 5757, p < 0.0001; Fig1a). Principal component analysis of V and/or J gene usage demonstrated a difference in usage pattern between HC and patients as they clustered separately. When analysed by the differences in individual genes, Vβ 20.1 was significantly over‐represented in ITP patients (9.01% vs 5.37%; p < 0.001) and Jβ 2.1 was significantly under‐represented (2.63% vs 12.63%; P < 0.0001), compared to HC.

In PBMC repertoires of ITP patients, there was a significantly higher prevalence of expanded clonotypes (defined as ≥1% frequency), compared to HC (81% vs 18%; p < 0.0001; Fig 1b). Longitudinally, most expanded clones persisted over multiple years. The expanded clones in ITP patients were not present in HC. We also explored whether these expanded clonotypes were shared in ITP patients, i.e. present in more than one individual. We found two shared sequences present in two ITP patients in both sorted CD8+ T‐cell and PBMC repertoires at multiple timepoints (both absent in HC). For ITP patients, where repertoires of both PBMC and sorted CD8+ T‐cell samples were available, the top five expanded clones in PBMC were also the top five expanded clones in sorted CD8+ T‐cell population.

Summary/Conclusion:

Our findings reveal that the TCR repertoires of ITP patients display a significantly decreased diversity compared to HC and display a preferential use of unique V and J genes. The presence of the same expanded clonotypes in PBMC and CD8 T‐cells in ITP patients suggest that these expansions arise from CD8+ T‐cells rather than CD4+ T‐cells. The restricted TCR repertoire and stability of expanded clonotypes over time in ITP patients might be attributed to continuous CD8+ T‐cell responses to platelet antigens. Further exploration of these clonotypes, including isolation of shared clonotypes may allow us to determine disease causing cells in ITP, with potential prognostic and therapeutic value.

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