Notch receptor signaling is crucial to cell development and has been found to play a key role in T cell activation and differentiation. Using a novel antibody approach, we sought to investigate the role of Notch-1 in both the cellular and antibody-mediated aspects of the alloimmune response.

We first investigated the effect of a novel, selective, blocking antibody against Notch-1 (aNotch-1; Wu et al, Nature 2010) on graft survival in a fully MHC mismatched (Balb/c⇒B6) transplant model. Mice treated with aNotch-1 for 6 days had significantly prolonged graft survival compared to IgG-treated controls (MST 13 vs 7 days; p=0.0019), while the addition of aNotch-1 to a single dose of CTLA4-Ig synergistically prolonged graft survival from 30 to 66 days (p=0.002).

Using this model, we determined that aNotch-1 significantly inhibited CD4⁺ and CD8⁺ effector memory T cells, with a corresponding decrease in the frequency of cells secreting IFNΓ (250±40 vs 1014±7.9; p<0.0001) and Granzyme B (114±22 vs 390±10; p<0.0001). Use of aNotch-1 was also found to decrease T follicular helper cells and alloantibody production (IgG2>IgG1; p<0.01).

Characterization of the thymus of aNotch-1-treated mice revealed marked interruption of the maturation of CD4^–CD8^– double negative (DN) to CD4⁺CD8⁺ double positive (DP) thymocytes (6.44±3.7 vs 81.2±0.38%; p<0.0001) at DN stage I (CD44⁺CD25^–). Strikingly, however, these mice were found to have a 10-fold increase in the thymic CD4⁺CD25⁺FoxP3⁺ Treg population compared to controls (2.83±0.34 vs 0.27±0.03%; p=0.0016).

To determine the importance of Tregs in the prolonged graft survival seen with aNotch-1, we administered anti-CD25 pre-transplant to B6 mice then treated with either aNotch-1 or IgG. Indeed, the contribution of the Treg population was shown to be crucial, as depletion of Tregs near-completely abrogated the improved survival seen with aNotch-1 alone (MST 9 vs 13 days; p=0.005), but had no effect on the survival of IgG-treated mice (MST 7.5 vs 7 days; p=0.34).

These data reveal an important role of Notch-1 in both cellular and antibody-mediated effector responses in transplantation, and suggest that the regulatory response may be Notch-1-independent, thereby identifying a promising novel approach for immune modulation in transplantation.

Siebel, C.: Employee, Genentech, Inc.

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