*Purpose: TLR-Bregs were defined by our group as B cells generated in vitro. They have demonstrated the ability to inhibit CD4 T cell proliferation. Since CD8 T cells are also major players in rejection of organ transplants, the present report explores the mechanisms by which TLR-Breg suppression may differentially affects CD4 and CD8 T cells in a transplantation setting.

*Methods: B6 TLR-Bregs and OB1 TLR-Bregs were generated from B cells isolated from C57BL/6 and transgenic OB1 mice (B cell receptor specific to ovalbumin, OVA). In vitro MLR was set up in co-culture of CFSE-labeled CD8⁺ T cells (OVA-specific) as responders, irradiated OVA splenic cells as stimulators, and OB1 TLR-Bregs as suppressors. For in vivo suppression experiments, CD8 T cells were adoptively transferred to B6 underwent OVA skin graft, with or without B6 or OB1 TLR-Bregs. Cells from draining lymph nodes (DLN), non-draining lymph nodes (N-DLN), and spleen (SP) were harvested and analyzed by flow cytometry. The combination of anti-CD8 mAb and OB1 TLR-Bregs were administrated to check skin graft survival.

*Results: In vitro assays showed that OB1 TLR-Bregs suppress CD8⁺T cell proliferation and INF-γ production (p<0.05, vs control). In contrast to previous results showing that CD4⁺ T cells were concentrated in DLN, CD8⁺ T cells were not only found in the DLN, but also in the N-DLN and spleen (Figure 1). Suppression assays in vivo confirmed the inability of either B6 or OBI TLR-Bregs to inhibit CD8⁺ T cell proliferation, positive controls of proliferation were at 79.9%±3.8% and had no significant difference with Bregs treatment (64.7%±17.3%, Figure 2). Importantly, the in vivo depletion of CD8⁺T cells in mice treated with OB1 TLR-Bregs (61 days) did not significantly prolong graft survival than OB1 TLR-Bregs alone treatment (38 days).

*Conclusions: The lack of TLR-Breg control over CD8⁺ T cell proliferation may result from a more disperse lymphoid tissue distribution of these cells as compared to that of CD4⁺ T cells. Collectively, these data indicate that ex-vivo generated Bregs promote prolongation of graft survival, mainly by downregulating CD4⁺ helper T cell proliferation, which in turn may curtail graft-specific CD8 reactivity. Additional studies will be required to identify potential therapeutic targets to improve graft survival.

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