Presentation of graft-derived antigens with correct co-stimulation drives the allo-immune response. Conversely, in the absence of these factors graft-specific tolerance may result. For DC’s, the A20 gene is an antigen presentation attenuator; increased A20 levels negatively regulate DC maturation and cytokine production. We examined whether A20’s inhibitory capacity could be harnessed to promote graft-specific tolerance. For our transplant model, islets from BALB/c H-2d donors were transplanted under the renal capsule of full MHC-mismatched diabetic C57BL/6 H-2b recipients. A20 was over-expressed using a rAd.A20 construct. All control grafts (n=10) were rapidly rejected whereas 45 % (n=27) of A20-expressing grafts were permanently accepted (> 200 days) in the absence of exogenous immunosuppression. Long-term surviving A20-transduced grafts showed strong insulin-labeling, with increased Foxp3+ mononuclear cells at the islet/kidney parenchyma border. A number of experiments were conducted to assess the importance of these Foxp3+ cells. In the case of A20 long-term surviving grafts, purified T cells were adoptively transferred into immune deficient RAG-/- mice pre-transplanted with either a BALB/c allograft or a third party CBA (H-2k) allograft. ∼ 70% of BALB/c grafts were accepted, whereas, all third party CBA grafts were rejected. Next, only effector T cells (CD25 depleted) were adoptively transferred into RAG-/- mice pre-transplanted with either a BALB/c or CBA allograft. In this case all grafts were rejected. Thus A20-expression induced graft-specific tolerance dependent upon CD25+ T cells. To determine if CD25+ T cells were necessary early, C57BL/6 recipients were administered mAb PC61 prior to the transplantation of A20-overexpressing BALB/c islet grafts. In this case all A20 transduced grafts were rejected. Thus, A20 expression allows Treg control of the allo-immune response. We examined whether A20 attenuated graft-derived inflammation. RTqPCR analysis at POD10 revealed that A20 transduced grafts exhibited reduced mRNA levels of CXCL10, INFΓ, IL6 and IL17, but increased mRNA levels for IL10, CTLA4 and TGFΒ. In vitro experiments demonstrated that A20 suppressed cytokine-induced NF-ΚB and JNK/AP1 activation, whereas no change was detected in ERK and p38 signaling. We interpret these findings to indicate that A20 re-programmed intra-graft inflammatory circuits, generating a local milieu supporting tolerance versus rejection.

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