T Cell Clonality in Paediatric Immune Thrombocytopenia
Authors: Tan Michelle MH , Malik Amna , Wang Jiawen , Lo Clara , Hart Alice , Joshi Nehal , Adams Stuart , Zhang Patrick , Rezvani Katayoun , Zhang Bing , Cooper Nichola 
Published: December 2024

Introduction

Immune thrombocytopenia (ITP) is a complex autoimmune disorder characterised by persistent low platelet counts. The cause of ITP is poorly understood and given the heterogeneity of clinical outcome, may involve different pathogenic mechanisms in individual patients. Despite novel therapies, some patients are unresponsive to any treatment and often have severe symptoms. Understanding why some patients progress from newly diagnosed ITP to chronic disease, and why some develop refractory disease will help us to better direct therapy for these patients.

We have previously described different TCRVβ families in children with newly diagnosed ITP vs chronic ITP. In this extended cohort of children, we utilised next-generation sequencing of T-cell receptor (TCR) genes, combined single-cell RNA and TCR sequencing to identify the differences between disease states and explore potential disease outcome predictors.

Methods

50 children with primary ITP and 10 healthy controls (HC) were recruited from the Imperial College National Health Service Trust and Lucile Packard Children's Hospital (Stanford Children's Health). Next-generation sequencing of the TCR β gene was carried out using in-house methods and sequenced on the Illumina MiSeq platform. Analysis of raw TCR sequences was performed using MiXCR. Combined single-cell and RNA sequencing was carried out using the 10x Genomics platform (Chromium Single Cell VDJ, and 5′ Library kits) on two refractory patients. Cell Ranger was used to perform sample demultiplexing, barcode processing, and single-cell 5′ unique molecular identifier counting. Seurat was used for gene expression analysis.

Patients were defined based on the stage of ITP disease at time of patient sampling; newly diagnosed (within 3 months of diagnosis, n=24); chronic (&gt; 12 months, n=25); refractory if they were unresponsive to &gt; 3 prior therapies including thrombopoietin receptor agonists (TPO-RAs), mycophenolate mofetil and rituximab (n=5).

Results

Children with newly diagnosed ITP had more restricted TCR repertoire and expanded T cell clones compared to those with chronic ITP. Patients who had expanded T cell clones, occupying &gt; 1% of the repertoire, within three months of diagnosis were less likely to go into long term remission (p=0.03). We also found preferential use of certain TCRVβ gene in patients who developed refractory disease. In one patient, followed over multiple years, T cell clones persisted over many years, and correlated with the platelet count.

Clustering analysis of scRNA sequencing data showed 15 distinct clusters and subclusters of immune cells. Compared to HC, children with refractory ITP showed higher proportions of CD14 and CD16 monocytes and B memory cells, and a lower proportion of CD8 TCM. Chronic refractory ITP patients had CD8 TEM subsets that expressed high levels of GZMA, NKG7, GZMB and KLRD1 genes.

scTCR-seq revealed that children with refractory ITP have clonally expanded T cells with the top 10 expanded clones taking up 25-50% of the TCR repertoire space. Mapping TCR sequencing to gene expression, we found that clonally expanded T cells all comprised of CD8 TEM subset. The expanded clones expressed cytotoxic genes, including GNLY, NKG7, GZMB, and GZMA, and interferon genes (IFITM3 and ISG15) without evidence of exhaustion (no expression of CTLA4, HAVCR2, and TIGIT).

Conclusion

Using next-generation sequencing to study the TCR repertoire in paediatric ITP patients we demonstrate, for the first time, that expanded T cell clones are present within the first three months of diagnosis and predict whether patients will go into a long-term response. T cell clones persist overtime, and correlate with platelet count. Certain V-J gene families were more common in children who develop refractory disease and using single-cell gene expression profiling combined with TCR sequencing, we show that the expanded clones are enriched in patients with refractory disease and exhibit a cytotoxic phenotype.

Our findings provide critical insight into the role of T-cell-mediated disease in children with refractory ITP.