Immunoregulatory B Cells Are Enriched within Transitional and IgM Memory B Cell Subsets in Healthy Donors but Are Reduced and Functionally Impaired in Patients with Chronic Graft-Versus-Host Disease

The immunosuppressive function of IL10 producing regulatory B cells (Bregs) has been shown in several murine models of inflammation and autoimmune disease. However, there is a paucity of data regarding the existence of an equivalent regulatory B cell subset in humans and their relevance in the pathogenesis of chronic graft-versus-host disease (cGVHD) remains unknown. Here, we explored the regulatory properties of peripheral blood (PB)-derived human B cell subsets and their role in cGVHD. Using intracellular cytokine staining following in vitro stimulation with CD40 ligand, we showed that the majority of IL-10 producing B cells in healthy donors are found within the CD24^hiCD38^hi transitional and CD19⁺IgM⁺CD27⁺ memory B cell subsets. Sort-purified IgM memory and transitional B cells suppressed the proliferation, as well as the release of IFN-γ by CD3/CD28 stimulated CD4⁺ T cells. The inhibitory effect of IgM memory and transitional B cells on CD4⁺ T cell proliferation was cell dose dependent with the highest suppression was observed at a ratio of 1:1. These data suggest that human PB transitional and IgM memory B cells are endowed with suppressive function.  This suppression was mediated partially via the provision of IL-10, but not TGF-ß, which we assessed by antibody blockade experiments. Additionally, the suppressive capacity of the B cell subsets was reversed by the addition of CD80 and CD86 mAbs. Using transwell experiments, we further determined that the suppressive function of Bregs is also partly dependent on direct T cell/B cell contact. Although blockade of IL-10 and IL-10R, CD80 and CD86 and separation of B cells and T cells by a transwell membrane individually did not completely reverse the suppressive ability of transitional and IgM memory B cells, a combination of these factors sufficiently reversed the ability of Breg subsets to suppress CD4+ T cell proliferation. Thus, analogous to murine experimental models the suppressive effect of human Breg cells involves both the release of IL-10 and co-receptor interaction. Additionally, Breg cells isolated from patients with cGVHD were refractory to CD40 stimulation and produced less IL-10 when compared to patients without cGVHD post-SCT and healthy controls. Likewise, the absolute number of IL-10 producing B cells was significantly lower in cGvHD patients compared to patients without cGVHD and healthy controls (p=0.007), supporting the existence of both a qualitative and quantitative defect in IL-10 producing B cells in cGvHD.

Our combined studies provide important new data defining the phenotype of B cell populations enriched in regulatory B cells in healthy humans and provide evidence of altered cellular function within such cells that may impact a broad range of deficiencies in immune regulatory cell function in cGvHD post transplant patients.