Single-Cell RNA-Seq Identifies Potentially Pathogenic B Cell Populations That Uniquely Circulate in Patients with Chronic Gvhd

While B cells are known to contribute to the pathogenesis of chronic graft-versus-host disease (cGVHD) in mice, it has been challenging to elucidate intrinsic mechanisms of tolerance loss in patients. To identify distinct and potentially targetable B-cell subsets in cGVHD, we employed single-cell RNA-Seq along with an unsupervised hierarchical clustering analysis, targeting 10,000 single B cells from each of eight patients who were >12 months post-allogeneic hematopoietic stem cell transplantation (HCT) and either had active cGVHD manifestations (n=4) or never developed cGVHD (n=4). Bioinformatics analysis of pooled cell data (using R with Seurat extension package) identified 6 major B cell clusters common to all patients (Figure 1A). “Intra-cluster” gene comparison (using R package DESeq2, false-discovery rate 0.05) revealed numerous differentially expressed genes between patient groups. The greatest number of differentially-expressed genes occurred in a cluster referred to herein as ‘Cluster 6‘ (Figure 1A, in yellow with asterisk). Within Cluster 6, B cells from active cGVHD patients expressed significantly increased ITGAX (CD11c, Padj =0.007), TNFRSF13B (TACI, a receptor for BAFF, Padj =0.003), IGHG1 (IgG1, Padj =9.3e-06) and IGHG3 (IgG3, Padj =1.7e-12), along with 44 additional genes (to be discussed). Thus, Cluster 6 in cGVHD patients may represent a CD11cpos, BAFF-responsive B cell subset primed to undergo isotype switching in response to alloantigen. Flow cytometry analysis on PBMCs from an independent HCT patient cohort (n=10) confirmed that CD11cpos B cells were indeed significantly expanded in cGVHD (P In addition to more deeply characterizing B-cell subsets in cGVHD, our single-cell RNA-Seq analyses identified several genes significantly altered across multiple B cell clusters in the cGVHD group, implicating more broad alterations of some genes in this disease. Among these is CKS2, a critical cell cycle regulator, which was significantly increased in cGVHD B cells (Padj 1.0e-10 to 0.018, depending on the cluster evaluated). Increased CKS2 expression was validated by qPCR analysis on B cells from a separate HCT patient cohort with or without cGVHD (P In summary, we used an unbiased approach to identify and further characterize an extrafollicular CD11cposTACIposCD19high B cell population in cGVHD patients that appears to be activated and undergoing active IgG isotype switching. This plasmablast-like B cell population is potentially amenable to therapeutic intervention to prevent pathogenic antibody production. Importantly, we also identify gene alterations across the cGVHD peripheral B cell compartment that potentially underpin promotion of hyperactivated B cells in this disease. Therapeutic strategies to target these pathways will also be discussed. This work was supported by a National Institutes of Health grant, R01HL129061. Download : Download high-res image (1MB) Download : Download full-size image Disclosures Ho: Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Horwitz: Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Rizzieri: Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Stemline: Research Funding.