B cell responses in heparin-induced thrombocytopenia

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ABSTRACT Heparin-induced thrombocytopenia (HIT) is the most common drug-induced immune thrombocytopenia and can lead to catastrophic thrombosis in an affected patient. Antibodies that recognize the platelet alpha-granule protein, platelet factor 4 (PF4), in a complex with heparin are central to HIT pathogenesis. Human B cells have extensive heterogeneity. Innate-like B cells, such as B1 and marginal zone (MZ) B cells, later germinal center (GC) B cells, different types of memory B cells, and extrafollicular plasmablasts all play an importance and unique role in the development of humoral immunity. Rapid onset of HIT antibody production and apparent lack of immunologic memory suggest T-cell independence whereas IgG antibodies typical of HIT argue for T cell involvement. The profound heterogeneity of human B cells and the ambiguous features of the HIT antibody response have confounded efforts to identify the pathogenic B cells and characterize the atypical immune response in HIT patients. In a mouse model, we have demonstrated that MZ B cells play a critical role in HIT antibody production. In the immediate past grant period, we discovered that in mice, breakdown of immunologic tolerance was involved in HIT antibody production and T helper cells and regulatory T cells could mediate the production of PF4/heparin-specific HIT antibodies. We also identified several novel pathways controlling B cell tolerance that was critical for controlling production of autoantibodies. Importantly, from HIT human patients, we cloned PF4/heparin-specific and platelet-activating antibodies possessing a unique RKH- or Y5-motif in the heavy chain complementarity determining region 3 (HCDR3) region that contributes the most to affinity and specificity of antigen binding. We found that a higher percentage of PF4/heparin-specific B cells, compared to the control B cells, exhibited extrafollicular B-cell features and an atypical memory B cell (atyMB) phenotype. Based on these findings, we hypothesize that PF4/heparin-specific B cells undergo extrafollicular response to follow a distinct differentiation path from activated naïve B cells first into atyMBs and then into plasmablasts in HIT patients. To test this hypothesis, we will (1) study the characteristics of PF4/heparin-specific B cells in HIT patients through integrative analysis of phenotypical, transcriptional and epigenetic responses of these B cells and (2) investigate the origin and developmental trajectory of PF4/heparin-specific B cells that produce platelet- activating antibodies in HIT patients. The proposed studies will identify the key features of PF4/heparin-specific B cells and their correlation with disease progression and severity and will discover the evolution of PF4/heparin- specific B-cell response. Completion of the proposed studies will provide novel insights into the molecular pathogenesis of HIT and guide future diagnosis, prevention and treatment of this potentially devastating disease.