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 < 0.01, Figure 1B), and revealed these B cells were also TACIpos, CD19high, forward scatter high (FSChigh) blast-like cells (Figure 1C). We found that these CD11cpos B cells had mixed expression of CD21, CD27, IgD and CD24 (Figure 1C). Remarkably, other recent studies on bulk patient B cells have suggested that similar CD11cposCD21negCD19highT-BETpos cells are critical drivers of humoral autoimmunity in diseases including systemic lupus erythematosus (SLE; Scharer et al. 2019; Rubtsova et al. 2017; Rubtsov et al. 2011). This subset now identified by single-cell RNA-Seq is consistent with a population of TACIhigh B cells that produced IgG in response to BAFF treatment ex vivo (Sarantopoulos 2009). Data suggest we have identified functionally distinct and potentially targetable B cell subpopulations. We are employing functional assays to determine whether the additional molecular pathways now elucidated account for our previous work showing greater ex vivo B cell survival rates and hyper-responsiveness to surrogate antigen (Allen et al. 2012, 2014), certain TLR agonists (Suthers et al. 2017), and NOTCH ligand (Poe et al. 2017).