T-Cell Transcriptomic Signatures in Adults with Primary Untreated Immune Thrombocytopenia Segregate By Age, Revealing Distinct Druggable Pathways
Authors: Shao Lipei , Boothby Aaron , Bailey S. Lawrence , Chauhan Aastha , Boechenek Martin , Gernsheimer Terry B. , Semple John W. , Zehnder James L. , Rao V. Koneti , Ping Jin , Stroncek David F. , Stolla Moritz , Panch Sandya R. 
Published: December 2024

Background:
Immune thrombocytopenia (ITP) is a common autoimmune manifestation of inborn errors of immunity (IEI). As monogenic disorders, some IEIs can now be treated with mutation specific drugs which also treat the co-existing ITP. In adults, 80% of ITP is thought to be “primary” or “idiopathic” and is characterized by significant heterogeneity in phenotype, treatment choices and response. A recent study at our institution suggested a high rate of heterozygous IEI-associated pathogenic gene variants in adult patients (age ≥18 years) with chronic refractory ITP, although no cases of IEI were identified. We hypothesized that genomic, transcriptomic, and cytokine profiles vary among adults with “primary” ITP, providing a way to refine this diagnostic category and optimize treatments.
Methods:
Following IRB approval, we enrolled patients (age ≥18 years) with primary ITP (platelet count ≤100x103/µL) not on active treatment and age/sex matched healthy controls. Targeted gene sequencing for IEIs, peripheral blood mononuclear cell transcriptomics on the nCounter T-cell (780 genes) and autoimmune (770 genes) panels (NanoString Technologies), single cell RNA sequencing (CITE-seq) and cytokine assays (Bio-rad Inc.) were performed.
Results:
Between 9/2023 and 7/2024 we enrolled 23 adult patients and 23 healthy controls. Of the 3 males and 20 females, 3 were excluded due to possible congenital macrothrombocytopenia, H. pylori, and immunosuppression at sample collection. Mean age at sample collection and at ITP diagnosis were 46 and 36, respectively. 55% identified as white, 80% had chronic ITP, 20% reported a family history of autoimmunity, and 10% had prior splenectomy. Mean platelet count at sample collection was 47x103/µL with a mean immature platelet fraction of 18%. T, B, NK cell subset abnormalities were noted in 55%. Quantitative Immunoglobulins were normal in all cases.
On unsupervised hierarchical clustering of transcriptomic data, young adult ITP patients (age 18-40 years) formed a unique cluster, while older patients (≥40 years) and 82% controls cluster separately (p= &lt;0.0001). When analyzed by age at disease onset, these clusters segregated similarly into 2 distinct groups (p=0.009), particularly evident on T-cell transcriptomics. No significant differences were noted in other demographic or cellular characteristics among the clusters (E.g. platelet count at sample collection). Pathway analyses revealed a relative overexpression of MTOR, NFKB, and MAPK-Pi3K in younger patients and increased autophagy and JAK-STAT signaling in older patients. Single cell RNA seq in a subgroup of old vs. young patients suggested differences in certain T cell subsets; more T-effector memory cells in older vs. a higher proportion of CD4-CD8- (double) negative T cells in younger patients. Cytokine studies revealed higher CCL-2 among older compared to younger ITP (p&lt;0.05).
IEI gene panel found a mean of 3.5 (SD 1.2) gene variants per patient. All patients carried at least 1 variant. One patient with relatively poor increments on eltrombopag had 2 variants in LRBA (orientation pending family testing). No significant differences were noted by patient age groups. Control subject results are pending. Additional immunophenotyping and functional studies are underway.
Discussion:
In this prospective controlled study using a comprehensive multi-omic approach to profile adult patients with primary untreated ITP, we identified age-specific differences in T-cell transcriptomic pathways. These data corroborate the previously recognized role of T-cells in ITP pathogenesis. Increased mTOR expression in younger adults with ITP is intriguing given studies suggesting efficacy of sirolimus (mTOR inhibitor) in this group. Similarly, PI3K inhibitors and co-stimulatory blockade have been used in certain IEIs, may counter overexpression of this pathway notable in younger ITP patients. Successful JAK inhibitor treatment in refractory ITP may also be explained by the pathway overexpression in elderly with ITP. While genomic analysis suggested possible immune-dysregulating variants in this cohort, more work is needed to determine the role of early omic testing in clinical practice.
Conclusion:
Age-associated T-cell transcriptomic clusters among adult patients with “primary” ITP are a novel finding and merit further study for tailored therapeutics and precision medicine initiatives.