After allogeneic hematopoietic stem cell transplantation (allo-HCT), donor-derived B cells develop under selective pressure from alloantigens and play a substantiated role in the autoimmune-like syndrome, chronic graft versus host disease (cGVHD). We performed single-cell RNA-Sequencing (scRNA-Seq) of blood B cells from allo-HCT patients and resolved 10 clusters that were distinguishable by signature genes for maturation, activation and memory. Notably, allo-HCT patient ‘memory’ B cells displayed some striking transcriptional differences when compared to memory B cells from healthy individuals or non-HCT patients, suggesting molecular differences with a propensity for autoreactivity. To inform more specifically about transcriptional programs important for allo-HCT tolerance, we assessed all 10 clusters for differentially expressed genes (DEGs) between patients with Active cGVHD vs. those without disease (No cGVHD). Data reveal insight into DEGs that may influence multiple aspects of B cell function in allo-HCT, leading to chronic B cell activation in Active cGVHD and our observed expansion of potentially pathogenic atypical/age-related memory B cell (ABC) subsets within a B Cell Receptor (BCR)-experienced ‘memory’ cluster. Data also indicate chronic B-cell activation and diversification in allo-HCT is potentially ‘plastic’, and may be manipulated therapeutically. Our findings have implications in understanding how alloreactivity may lead to human autoimmune disease, and identify potentially novel targets for future study and intervention. One Sentence Summary We elucidate B cell subsets and DEGs at the single-cell level in humans receiving allo-HCT, when tolerance is lost or maintained.

Accurate assessment of antigen-specific immune responses is critical in the development of safe and efficacious biotherapeutics and vaccines. Endosomal processing of a protein antigen followed by presentation on major histocompatibility complex (MHC) class II constitute necessary steps in the induction of CD4+ T cell immune responses. Current preclinical methods for assessing immunogenicity risk consist of in vitro cell-based assays and computational prediction tools. Cell-based assays are time and labor-intensive while in silico methodologies have limitations. Here, we propose a novel cell-based assay capable of investigating an antigen's endosomal processing and MHC class II presentation capabilities. This novel assay relies on competition between epitopes for MHC class II binding and employs labeled soluble T cell receptors (sTCRs) as detectors of epitope presentation.