Serum O-Glycan Antigenicity in Pediatric Immune Thrombocytopenia Predicts Disease Persistence

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count, leading to symptoms of bleeding and fatigue. While many cases are mild with limited mucocutaneous bleeding, severe and life-threatening bleeding can occur. ITP is a diagnosis of exclusion. It may be idiopathic or attributed to another underlying immune disorder. The heterogeneity of clinical behavior and treatment responses in ITP reflects its complex underlying pathophysiology, underscoring the need for further research and innovation. Predicting the disease outcome, whether a patient will have spontaneous disease resolution in <1 year (acute) or chronic disease lasting longer than a year, anticipating bleeding severity and determining the best therapeutic approaches remain challenging.

Reduced platelet count in ITP is caused by premature platelet destruction and insufficient production. Mechanisms include classically described antibody-mediated destruction, deficient regulatory T cell activity, T-cell-mediated apoptosis of platelets and megakaryocytes, as well as inhibition of platelet production. Glycosylation significantly affects platelet count. Platelets are marked as aged when their terminal carbohydrate moiety, sialic acid (Sia), is lost and they are subsequently removed from circulation. Data indicate that changes in platelet glycosylation, particularly the loss of Sia, may contribute to thrombocytopenia in ITP. Our recent data showed that antibodies against the exposed Thomsen-Friedenreich (TF) antigen, which is typically sialylated and cryptic, in plasma from pediatric patients with ITP could distinguish healthy individuals from those with ITP. This data indicates the role of immune cells in recognizing altered sialylation of aberrantly exposed TF antigens in ITP.

Here, we hypothesize that a specific anti-glycan antibody signature could prove valuable in distinguishing spontaneously resolving and chronic ITP from healthy based on glycan alterations, specifically Sia residues. We have analyzed O-glycan binding by antibodies in pediatric ITP patient plasma to identify antibody-glycan motifs predictive of healthy vs. ITP and spontaneously resolving vs. chronic ITP cases. In healthy controls, Sia antibody generation is preferential towards α2,3-Sia epitopes, whereas α2,6-Sia appears more immunogenic in ITP. Specifically, α2,6-Sia biosynthetic enzyme ST6GAL1 and its products have been implicated in malignancy and inflammatory conditions. Additionally, we discovered that IgG antibodies in healthy and pediatric ITP patients can distinguish between glycan epitopes and amino acid presentation, with IgG antibodies showing increased binding of serine O-glycans relative to threonine O-glycans. In order to understand the global transcriptional landscape, we also conducted RNA sequencing of peripheral blood mononuclear cells (PBMCs) in pediatric ITP patients. Sialyltransferase and glycogene expression for generating the H antigen, the precursor to ABO blood groups antigens, in PBMCs correlate with O-glycan antibody signatures observed in healthy vs. ITP children. Studies have shown that ITP patients exhibit aberrant expression levels of different epigenetic modifiers. Gene Set Enrichment Analysis (GSEA) between chronic and acute ITP patients demonstrates downregulation of pathways associated with histone demethylation, regulation of histone modifications, and histone H3 lysine 9 demethylations (FDR <0.05). Conversely, critical cellular metabolic-associated processes were enriched (FDR<0.05).

Due to diagnostic challenges of ITP, including prediction of disease persistence, we show that high-throughput detection of the plasma O-glycome by microarrays can aid in the stratification of spontaneously resolving vs. chronic patients as a therapeutic tool.