Atypical memory B cell clonal expansion and inflammatory programs associate with platelet-activating antibody development in COVID-19
Nathan Witman, Mei Yu, Yuqi Zhang, Kexin Gai, Yuhong Chen, Lu Zhou, Christine Nguyen, Wen Zhu, Yongwei Zheng, Shawn Jobe, Mary Beth Graham, Weiguo Cui, Demin Wang, Renren Wen
Nathan Witman, Mei Yu, Yuqi Zhang, Kexin Gai, Yuhong Chen, Lu Zhou, Christine Nguyen, Wen Zhu, Yongwei Zheng, Shawn Jobe, Mary Beth Graham, Weiguo Cui, Demin Wang, Renren Wen
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Research Article Immunology Vascular biology

Atypical memory B cell clonal expansion and inflammatory programs associate with platelet-activating antibody development in COVID-19

Abstract

Patients with COVID-19 who develop platelet-activating antibodies represent a subset at heightened thrombotic risk, yet the immune features associated with this response remains to be defined. We applied single-cell RNA-seq of B and T cells, single B cell V(D)J-seq, and plasma cytokine and chemokine analysis to define immune signatures distinguishing patients who did (PEA+) or did not (PEA–) develop these antibodies. Patients positive for PEA showed prominent transcriptional enrichment of inflammatory, antigen presentation, and B cell receptor signaling pathways within antigen-experienced B cell subsets. Expanded B cell clones in patients positive for PEA were disproportionately enriched within atypical memory B cells and exhibited upregulated IFN-γ–response signatures, increased proliferative mutational patterns, limited class switching, and a significant overrepresentation of RKH/Y5 heavy-chain motifs associated with platelet-activating antibodies, consistent with an extrafollicular-biased response. Parallel T cell profiling revealed IL-12 pathway enrichment across most T cell subsets, increased IFN-γ transcription, and elevated plasma levels of Th1-associated cytokines in patients positive for PEA. Collectively, these data highlight a coordinated inflammatory environment marked by Th1-skewed T cell activation and selective expansion of atypical memory B cell clones carrying RKH/Y5 motifs, defining immunologic features associated with platelet-activating antibody development in COVID-19.

Authors

Nathan Witman, Mei Yu, Yuqi Zhang, Kexin Gai, Yuhong Chen, Lu Zhou, Christine Nguyen, Wen Zhu, Yongwei Zheng, Shawn Jobe, Mary Beth Graham, Weiguo Cui, Demin Wang, Renren Wen

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Figure 3

Evidence of IFN-γ upregulation and clonal atypical memory B cell expansion in PEA+ patients.

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Evidence of IFN-γ upregulation and clonal atypical memory B cell expansi...
(A) UMAP of 949 clonally expanded B cells, colored by cluster. Expanded clones were defined by shared heavy-chain CDR3 sequences with at least 7 cells per clone and were derived from 5 PEA+ and 3 PEA– patients. (B) UMAP split by PEA status, highlighting expanded clones from PEA+ (n = 350) and PEA– (n = 599) patients. (C) Distribution of expanded clones across B cell subsets. Donut charts show the relative percentage of expanded clones within each patient group, with slice size indicating abundance and color indicating cluster identity. Percentages denote each subset’s contribution to the total expanded pool. Statistical comparisons used a χ2 test. (D) IFN response signature scores for expanded clones across B cell subsets. Scores were calculated from GO:0034341 and HALLMARK_INTERFERON_GAMMA_RESPONSE gene sets, normalized as z scores, and displayed as a heatmap. Differences between PEA groups were assessed with the Wilcoxon rank-sum test. (E) Violin plots showing expression of IFN-γ–associated transcripts and selected control genes in expanded clones from PEA+ versus PEA– patients. Statistics were computed using the Wilcoxon rank-sum test. (F) VH and VL mutation frequencies (total replacement [R] and silent [S]) in atypical memory B cells, cMBCs, and PBs/PCs, colored by PEA status. Data are shown as mean ± SEM. (G) Normalized total mutation frequencies for representative clones collapsed within each clonal family, plotted as average R+S mutations per clone, and colored by PEA status. Data are shown as mean ± SEM. (H) Replacement versus silent mutation frequencies and ratio comparisons in CDR and FWR regions across subsets. Data are shown as mean ± SEM. Statistical significance shown for PEA+ versus PEA– comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.