*Purpose: Our previous study showed that Bregs-TLR, one type of previously studied Breg expanded in vitro, are capable of suppressing naïve B cell proliferation in the presence of LPS stimulation. Our previous result showed Bregs-TLR rescuing more LPS-stimulated B cells from apoptosis in vitro. However, less is known about how Bregs-TLR influence B cell activation and differentiation when LPS is absent. In the current study, we aim to explore the rescuing role of Bregs-TLR on naive B cells.

*Methods: Bregs-TLR were obtained after purified splenic B6 B cells were given CpG ODN1668 (TLR9 agonist) stimulation for 3 days and LPS (10ug/ml), PMA (50ng/ml), and ionomycin (1ug/ml) stimulation for the last 5 hours. In vitro, Bregs-TLR and naïve B cells w/o LPS stimulation were co-cultured for 3 days. In vivo, B6 recipients were adoptively transferred with Bregs-TLR on D-1 and transplanted with OVA skin grafts on D0. B cell subsets and markers of proliferation were measured by flow cytometry on day 21.

*Results: Flow cytometry found that naive B cells showed low viability and little proliferation in comparison to B cells stimulated for 96h with LPS. In the complete absence of any LPS stimulation, B cells did not survive unless Bregs-TLR or Bregs-CpG were added. Interestingly, while Bregs-CpG induced five generations of B cell proliferation, Bregs-TLR induced a comparable amount of proliferation in fewer generations (5 vs. 2). On the other hand, in vivo data showed that Bregs-TLR could induce B cell proliferation (Figure) and more CD19+IgM^hiIgD^loCD23^loCD21^hi MZ B cells in B6 recipients while Bregs-CpG could not.

*Conclusions: Bregs-TLR regulate the proliferation and differentiation of naïve B cells both in vitro and in vivo. The increase of MZ B cell percentage may indicate that the mechanism for which Bregs-TLR induce the proliferation of other B cells lies in an MZ B cell-relevant pathway.

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