B Cell With Regulatory Function 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 healthy individuals and their potential role in the pathogenesis of chronic graft-versus-host disease (cGVHD) remains unknown. Here, we examined the functional regulatory properties of peripheral blood (PB)-derived human B cell subsets from healthy individuals. In addition, we carried out studies to explore their role in cGVHD, using B cells from patients following allogeneic stem cell transplantation (HSCT).

We first determined whether human IL-10 producing B cells are enriched within any othe previously described human B cell subsets: CD19⁺IgM⁺CD27⁺ IgM memory, CD19⁺IgM^-CD27⁺ switched memory, CD19⁺IgM⁺CD27^- naive, and and transitional CD19⁺CD24^hiCD38^hi. Following in vitro stimulation with CD40 ligand, the majority of IL-10 producing B cells were found within the CD24^hiCD38^hi transitional and CD19⁺IgM⁺CD27⁺B cell subsets. We next assessed the regulatory properties of the PB-derived B cell subsets, by sort-purifying IgM memory (CD19⁺IgM⁺CD27⁺), switched memory (CD19⁺IgM^-CD27⁺), naïve (CD19⁺IgM⁺CD27^-) and transitional (CD19⁺CD24^hiCD38^hi) B cells from healthy controls, and cultured them 1:1 with autologous magnetic-bead purified CD4⁺ T cells. CD3/CD28 stimulated CD4⁺ T cells cultured with either CD19⁺IgM⁺CD27^- naïve or CD19⁺IgM^-CD27⁺ switched memory B cells proliferated to the same extent and produced equivalent amounts of IFN-γ to cultures containing CD4⁺ T cells alone. In contrast, culture of CD4⁺ T cells with IgM memory and transitional B cells significantly suppressed CD4⁺ T cell proliferation [median percent proliferating CD4⁺ T cells 52.5%; (33%-75%)] and 51% (25%-63%)], respectively when compared with CD3/CD28 stimulated CD4+ T cells (positive control) [89.5% (75%-92%], p=0.0001. The inhibitory effect of IgM memory and transitional B cells on CD4⁺ T cell proliferation was cell dose dependent with the highest suppression observed at a ratio of 1:1. These data suggest that human PB transitional and IgM memory B cells are endowed with regulatory function.

We next examined if the in vitro suppressive effect of transitional and IgM memory B cells is mediated by regulatory T cells (Tregs). For this purpose, CD4+ T cells were depleted of CD127^lo CD25^hi CD4⁺ T cells by magnetic cell purification. B cell subsets were cultured with CD3/CD28 stimulated CD4⁺ CD25^- T cells at a ratio of 1:1. IgM memory and transitional B cells were able to significantly suppress the proliferation and Th1 cytokine response by CD4⁺ CD25^- T cells compared to cultures containing CD4⁺ CD25^-T cells alone, indicating that the suppressive activity of Bregs is independent of Tregs.

To further understand the underlying mechanims though which Bregs exert T-cell suppression, we used antibody blockade experiments and showed that this suppressive effect was mediated partially via the provision of IL-10, but not TGF-ß. Using transwell experiments, we further determined that the suppressive function of Bregs is also partly dependent on direct T cell/B cell contact.

We next assessed whether the activity of Breg cells might be altered in patients with cGVHD. B cells 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, [1.02% (0.22-2.26) vs.1.72% (0.8-5.52) vs. 2.16 (1.3- 5.6), p=0.001]. 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 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 for a defect in the activity of such cells in patients with cGVHD post-SCT. In association with previous reports showing defects in Treg cell activity in GVHD, our results suggest the existence of a broad range of deficiencies in immune regulatory cell function in cGvHD patients.

* Both Anushruti Sarvaria and Ahmad K contributed equally.