Evidence for Expansion of CD21- B Cells with an Exhausted Phenotype in Patients with Active Chronic GvHD

Chronic graft versus host disease (cGvHD) remains a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). At present, the immunopathophysiology of cGVHD is not fully understood. A number of pre-clinical and clinical studies support a role for B cell involvement in the pathogenesis of cGvHD. Increased numbers of CD21^- B cell, believed to be immature/ transitional B cells, were recently reported in the peripheral blood (PB) of cGvHD patients. In this study, we report the expansion of a CD21^- B cell population with an exhausted phenotype in the memory B cell compartment of patients with active cGVHD. Using multicolor flow cytometry we performed an extensive anaylsis of B phenotype and function in 16 patients with active cGvHD compared to 14 age-matched HSCT recipients without clinical evidence of cGVHD and 11 healthy controls. Chronic GvHD patients had significantly higher frequencies of CD21^- B cells in the PB compared with patients without cGvHD and HC (median 12.2% vs. 2.12% vs. 3%; p<0.01). Multi-parameter flow cytometry revealed that the majority of these cells were CD10^- CD27^- CD21^‑ CD20^hi, reminiscent of the recently described exhausted B cells in HIV patients with chronic viremia. We did not observe increased transitional (CD24^hi-CD38^hi- CD19⁺) or immature B cells (CD10⁺CD21^-CD27^-CD19⁺) in cGvHD patients compared with patients with no cGVHD (median transitional 3.4% vs. 6.3%; p=0.08) and (median immature (out of total CD21^- B cells) 3.15 vs.9.34%; p=0.045). As reported in chronic HIV viremia, expanded CD21^- B cells in cGVHD patients also expressed higher inhibitory receptors namely FCRL4, CD22 and CD85J, chemokine receptors CD11c, CXCR3 and lower CD62L and CCR7 compared with naïve (CD27-CD21+CD19+) and classical memory  CD27+CD21+CD19+) B cells. Moreover, B cells in patients with cGVHD had a lower proliferative potential following B-cell receptor crosslinking compared with B cells from patients with no GvHD or HC, a feature of exhaustion associated with high expression of inhibitory receptors. Finally, the frequencies of circulating CD21^- B cells correlated with cGvHD grade (r=0.67; p=0.002) but not with time from SCT (r=0.28; p=0.15). These data suggest that CD21^- B cells may play a role in the pathogenesis of cGVHD and could be used as as a marker of disease severity.