IMMUNE REGULATION AND TOLERANCE I

K.L. Pothoven, T. Kheradmand, J.L. Houlihan, X. Luo
Division Of Nephrology, Northwestern University Feinberg School of Medicine, Chicago/UNITED STATES OF AMERICA

Body: Objective – Dendritic cells (DCs) conditioned with the mTOR inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). The present work addresses whether rapamycin conditioned DCs could effectively induce CD4⁺CD25⁺Foxp3⁺ Tregs (iTregs) in cell cultures with antigen specificities to allogeneic antigens, and whether such in vitro differentiated alloantigen-specific CD4⁺CD25⁺Foxp3⁺ iTregs could effectively control acute rejection in allogeneic islet transplantation. Research Design and Methods - C57BL/6 (B6) CD4⁺CD25⁺Foxp3⁺ iTregs were generated from naïve CD4⁺CD25^-Foxp3^- T cells using donor BALB/c bone marrow derived dendritic cells (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin, and tested for their ability to protect BALB/c islet grafts via adoptive transfer in diabetic B6.RAG^-/- mice. Results - CD4⁺CD25⁺Foxp3⁺ iTregs with direct donor antigen-specificity can be effectively generated in vitro using donor BMDCs preconditioned with rapamycin. These in vitro induced CD4⁺CD25⁺Foxp3⁺ iTregs exert donor-specific suppression in vitro, and prolong allogeneic islet graft survival in RAG^-/- hosts upon co-adoptive transfer with T effector cells. This prolongation is donor-specific, as RAG^-/- hosts transplanted with third party islet grafts show no increase of graft survival. To determine the in vivo behavior of adoptively transferred cells, Thy1.2 iTregs were injected with congenic thy1.1 effector T cells. The Thy1.2 iTregs were able to maintain high levels of Foxp3 and CD25 in the draining LN and non-draining LN, however there is a significant loss of the phenotype in the spleen. In the presence of iTregs, Thy1.1 cells also exhibited a decrease in INFγ^hi expression in both CD4 and CD8 populations, suggesting that the iTregs suppress priming of donor-specific CD4 and CD8 effector T cells in vivo. CD4⁺CD25⁺Foxp3⁺ iTregs are further able to potentiate de novo induction of endogenous T cells. Conclusions – Rapamycin conditioned BMDCs can be exploited for efficient in vitro differentiation of donor antigen-specific CD4⁺CD25⁺Foxp3⁺ iTregs. Such in vitro generated CD4⁺CD25⁺Foxp3⁺ iTregs are able to effectively control allogeneic islet graft rejection.

Disclosure: All authors have declared no conflicts of interest.