Notch receptor signaling plays a key role in T cell activation and differentiation, though limited data exist on its importance in immune regulation. Herein, we explored the role of Notch-1 in alloimmunity using both a genetic and antibody approach.

We first examined the expression of Notch-1 on lymphocyte subsets: Notch-1 was predominantly expressed by T cells and was upregulated in the setting of in vitro stimulation with aCD3/CD28 or irradiated allo-APCs. We then investigated the effect of a novel, selective blocking Notch-1 antibody (aNotch-1) on graft survival in a full MHC-mismatch (Balb/c⇒B6) transplant model. Mice treated with aNotch-1 for 5 days had significantly prolonged graft survival compared to IgG-treated controls (MST 13 vs 7 days;p<0.0001), while the addition of aNotch-1 to a single dose of CTLA4-Ig (sCTLA4-Ig) synergistically induced long-term graft survival (MST >200 vs 42 days in sCTLA4-Ig alone).

We determined that aNotch-1 significantly inhibited CD4+ and CD8+ effector memory T cells, with a corresponding decrease in the frequency of cells secreting IFN-g (250±4 vs 1014±8;p<0.0001) and Granzyme B (114±2 vs 390±1;p<0.0001). Use of aNotch-1 was also found to decrease T follicular helper cells and alloantibody production (IgG2>IgG1;p<0.01). In contrast, aNotch-1 led to a 2-fold expansion of peripheral Tregs (CD4+CD25+FoxP3+;p<0.001). Furthermore, the enhanced graft survival seen with aNotch-1 was abrogated by Treg depletion (achieved by use of aCD25 on days -6, -1 pre-transplant), demonstrating a crucial contribution of Tregs to control the alloimmune response.

To isolate and clarify the role of Notch-1 in Tregs, we crossed Notch-1fl/fl mice with GFP.Foxp3Cre mice: selective genetic deletion of Notch-1 (N1cKO) on Tregs increased their proportion, proliferation and in vitro suppressive function. To assess their function in vivo, we utilized a cardiac transplant model, in which RAG-/- mice receive a BALB/c cardiac allograft followed by infusion of CD4+GFP.Foxp3- cells and either control (GFP.Foxp3Cre) Tregs or N1cKO Tregs. Graft survival in recipients of N1cKO Tregs far exceeded that in recipients of control Tregs (MST >100 vs 47 days; p=0.0006).

Our data reveal a promising novel approach for immune modulation in transplantation by selectively targeting Notch-1.

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