Abstract
Despite the availability of effective vaccines, COVID-19 continues to pose a global health challenge. Severe acute COVID-19 cases are often associated with serious thrombotic complications, including platelet activation, thrombocytopenia, deep vein thrombosis, pulmonary embolism, and endothelial dysfunction, all of which significantly contribute to morbidity and mortality. These complications resemble heparin-induced thrombocytopenia (HIT), which is driven by autoantibodies targeting platelet factor 4 (PF4)/heparin complexes, resulting in platelet activation. We previously demonstrated that a subset of hospitalized COVID-19 patients developed platelet-activating antibodies (PEA) regardless of heparin exposure. However, the B-cell origin of prothrombotic antibodies in COVID-19 remains poorly understood.
To investigate this, we performed single-cell RNA-sequencing (scRNA-seq) and 5' V(D)J sequencing (VDJ-seq) on FACS-sorted CD19+ B cells and CD4+ T cells from PBMCs of 6 COVID-19 patients who tested positive (PEA+) and 5 who tested negative (PEA-) for platelet-activating antibodies in a P-selectin expression assay (PEA). Uniform Manifold Approximation and Projection (UMAP) of transcriptomic data of B cells formed 13 initial Seurat clusters. Subsequently, they consolidated into 6 major populations: naïve, activated naïve, classic memory (cMBC), atypical MBCs B cells (aMBCs), and plasmablast (PB) precursors and PBs. Overall, B cell population distributions were similar between PEA+and PEA- groups.
However, Pseudobulk-corrected differential gene expression (DGE) and gene set enrichment analysis (GSEA) revealed that B cells from PEA+ patients exhibited upregulation of inflammatory and autoimmune-associated gene sets, including the IFNgamma signaling pathway, suggesting an IFNgamma-driven autoantibody response. Additionally, B cell clonal family analysis demonstrated expansion of B cells in different B-cell subsets in PEA+ and PEA- patients, with PEA+patients exhibiting prominent expansion in aMBCs and cMBCs, whereas PEA- patients displayed expansions primarily in PBs. Furthermore, somatic hypermutation analysis at the variable region of immunoglobulin (Ig) heavy (VH) and light (VL) chains of expanded B cell clones showed increased mutation frequencies in aMBCs and reduced frequencies in cMBCs of PEA+ patients relative to PEA-, coupled with reduced replacement to silent (R/S) mutation ratios and reduced mutation frequencies in the Complementarity-Determining Regions (CDRs) relative to framework regions (FWRs) in both aMBCs and cMBCs. The upregulation of IFNgamma signaling in B cells of PEA+ patients, along with the expansion of aMBCs and the unique Ig mutation patterns in their expanded B cells, demonstrates an extrafollicular-biased B cell response with diminished GC involvement, which drives the production of platelet-activating antibodies in COVID-19 patients.
Importantly, compared to PEA- patients, expanded clones within the aMBC clusters of PEA+ patients exhibited significantly greater enrichment for clones possessing R/K/H (RKH) or YYYYY (Y5) motifs in their VHCDR3 region, a feature that was previously associated with platelet-activating antibodies in both HIT and COVID-19, providing support for expansion of clones in aMBCs that are functionally relevant to platelet activation.
Parallel scRNA-seq analysis on CD4+ T cells showed a trend toward increased frequencies of Granzyme B+ Th1 cells (Th1-Gzmb) in PEA+ patients. These cells displayed upregulation of inflammatory and autoimmune-associated signaling pathways, similar to those observed in PEA+ aMBCs. Consistent with the transcriptomic findings, plasma multiplex analysis demonstrated elevated Th1-associated cytokines and chemokines, including IL-2, IL-12p70, CXCL10, and INFgamma. These findings collectively highlight a Th1-skewed inflammatory environment in PEA+ individuals, potentially driving extrafollicular B cell activation and prothrombotic antibody production.Taken together, our study uncovers a mechanistic link between Th1 cytokine-driven response and extrafollicular B cell differentiation in COVID-19 patients who develop platelet-activating antibodies, offering novel insight into the origin of the prothrombotic antibodies and the pathogenesis of COVID-19-associated thrombosis.
