*Purpose: The development and maintenance of peripheral tolerance is required for long term success of solid organ transplantation without immuno-suppressive drugs. Using a novel, post-transplant, mixed chimerism-tolerance induction regimen in a Rhesus Macaque (RM) model of kidney transplantation, using total lymphoid irradiation (TLI) and bone marrow transplantation (BMT), we observed a correlation between the generation of donor and recipient cell-chimerism with graft survival. Furthermore, an Interleukin 35 (IL35) expressing regulatory B cell (Breg) is over expressed in long term surviving animals on this regimen. We set out to explore whether IL35 expressing Bregs are part of a potential mechanism associated with immune tolerance.

*Methods: Isolated peripheral blood mononuclear cells (PBMCs) from blood draws of 5 transplant recipient animals on the kidney+TLI+BMT regimen following recovery of lymphocytes (40 days post-transplant), were stained with various fluorochrome conjugated antibodies and used in multi-parameter flow cytometry.

*Results: We found that Breg lymphocyte populations (CD3^neg,CD20⁺, CD24⁺, CD27⁺) are dramatically over expressed (42% of CD20⁺ B cells) in long term graft survival animals vs healthy controls (0.8% of CD20⁺ B cells) or transplant animals that never attained chimerism (0.6% of CD20⁺B cells). Furthermore, an increase in IL35⁺ Bregs (CD3^neg,CD20⁺, CD24⁺, CD27⁺,Ebi3⁺, p35⁺), was associated with chimerism and increased long term graft survival (50% of CD20⁺ Bregs in chimeric animals vs 0.3% of Bregs in non-chimeric animals).

*Conclusions: These preliminary findings support further investigation into the potential role that IL35⁺ Bregs play in chimerism associated immune tolerance. As a potential component of a mechanism for establishing an anti-inflammatory milieu promoting tolerance over inflammation, the knowledge gained from continued investigations of IL35⁺ Bregs will provide tremendous insight into not just transplantation immunology, but also to auto-immune and chronic inflammatory diseases.

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