Single-cell landscape analysis unravels molecular programming of the human B cell compartment in chronic GVHD.

Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT), chronic graft-versus-host disease (cGVHD), a B cell-associated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B cell receptor (BCR) activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells in allo-HCT, we performed scRNA-Seq analysis on high numbers of purified B cells from patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation, and memory. Within the memory B cell compartment, we found striking transcriptional differences in allo-HCT patients compared with healthy or infected individuals, including potentially pathogenic atypical B cells (ABCs) that were expanded in active cGVHD. To identify intrinsic alterations in potentially pathological B cells, we interrogated all clusters for differentially expressed genes (DEGs) in active cGVHD versus patients who never had signs of immune tolerance loss (no cGVHD). Active cGVHD DEGs occurred in both naive and BCR-activated B cell clusters. Remarkably, some DEGs occurred across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides understanding of altered B cell memory during chronic alloantigen stimulation.

BAFF promotes heightened BCR responsiveness and manifestations of chronic GVHD after allogeneic stem cell transplantation.

Patients with chronic graft-versus-host disease (cGVHD) have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex-mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic µMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell-depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD.

Assaying Spontaneous Network Activity and Cellular Viability Using Multi-well Microelectrode Arrays.

Microelectrode array (MEA) technology is a neurophysiological method that allows for the spontaneous measure of activity in neural cultures and determination of drug and chemical effects thereon. Recent introduction of multi-well MEA (mwMEA) formats have dramatically increased the throughput of this technology, allowing more efficient compound screening. Rapid characterization of compounds for neuroactivity or neurotoxicity hazard evaluation following acute, chronic, or developmental exposures ideally would also consider compound effects on cell health, and to do so in the same well requires a multiplexed approach. Procedures describing the multiplexed method to acute and developmental screening are described in this chapter.