We showed that regulatory B cells (Bregs) promote long-term islet allograft survival. Using mice with a novel inducible B cell-specific deletion of IL-10 (IL-10fl/flXhCD20TAM-Cre) we now confirm that IL-10 is essential for Breg activity. However, many aspects of Breg biology remain unclear, including how and where Bregs regulate immune responses within lymphoid tissues. Unfortunately, Bregs lack a specific marker and IL-10 protein can generally only be detected after ex vivo stimulation. Thus, the actual B cell subsets that express IL-10 in vivo remain unknown. Moreover, Breg localization and cellular interactions required for their suppressive function in vivo have never been examined. Using IL-10eGFP reporter mice we could detect GFP+ B cells without ex vivo stimulation. B cell IL-10 increased 2.5x after mice were immunized (2.0% ±0.09 vs. 0.8% ±0.1%; p = 0.0001). Analysis of IL-10+ B cells in spleen by FACs revealed that marginal zone B cells, follicular B cells and plasmablasts made similar large contributions to total B cell lineage IL-10 (~25% each). To examine Breg inter-cellular interactions in vivo, GFP+ (IL-10+) Bregs and GFP- Control B cells were sorted from mice immunized with NP-CGG (to boost NP-reactive B cells). Sorted Bregs or Control B cells were pulsed in vitro with NP-Ova (specific Ag) or NP-HEL (non-specific Ag) and then transferred into WT B6 mice along with fluorescently labeled Ova-pulsed DCs and Ova-specific OT-I CD8+ T cells. 2-Photon intra-vital microscopy revealed that Bregs made more frequent contacts with T cells at the T:B border (Bregs; 1.0 ± 0.14 vs. Control; 0.091 ± 0.037 contacts/cell, p = 0.0001) and 4.5x more prolonged interactions with T cells, compared to Control B cells. Breg:T cell interactions were Ag-dependent. Similar results were found with OT-II CD4 T cells. While, Bregs made almost no contacts with DC's, T cells made 25% fewer contacts with DCs in the presence of Bregs (1.4 ±0.17 vs. 1.9 ±0.18 contacts/cell, p = 0.01) and T cell:DC contact duration was ~40% shorter in the presence of Bregs (10 ±11 vs. 16 ±12 min, p = 0.0001). Bregs expressed higher levels of CCR-7 than Control B cells, which may explain their capacity to localize more frequently to the T-B border and interact with T cells. Our results are the first to show a comprehensive examination of the B cell subsets that make up Breg in-vivo and Breg in-vivo dynamic interaction with T cells and DCs.

CITATION INFORMATION: Mohib K., Cherukuri A., Zhou Y., David R. In-Vivo Visualization of Regulatory B Cells Reveals Ag-Dependent T Cell Interactions That Suppress Subsequent T Cell:DC Interactions Am J Transplant. 2017;17 (suppl 3).

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