Targeting Sag in Donor T Cells As a Novel Strategy for Reducing Gvhd

Neddylation is crucial for the degradation of certain proteins. However its role in regulating T cells is unknown. Neddylation is mediated by cullin-RING ligase (CRL) protein complex, an E3 ubiquitin ligase and its critical adapter element, SAG protein (sensitive to apoptosis gene protein). We explored the role of SAG and thus neddylation in T cells by utilizing two different, but complementary approaches, namely, genetic knock-out and chemical inhibition with small molecule. The T cell specific SAG KO animals were generated by crossing B6 SAG^fl/fl mice with B6 LCK-Cre mice. The KO animals were viable. The splenic and thymic analyses showed no significant differences in the numbers of conventional T cells (Tcons) and Tregs between the KO and WT animals. In vitro functional analysis of Tcons, however, revealed that stimulation with either allogeneic splenocytes or by α-CD3 and α-CD28 antibody, SAG^-/- T cells showed significantly decreased proliferation (P<0.002). Phenotypic analysis following stimulation demonstrated that SAG^-/- T cells showed reduced expression of CD69, CD44 and greater expression of CD62L when compared to WT-T cells (P<0.04). The KO-T cells also demonstrated reduced expression of T effector signature cytokines, IL-17, IFN-γ and IL-4. Similar reduction in proliferation, activation marker expression and release of cytokines was observed when the WT-T cells were treated with small molecule inhibitor of neddylation, MLN4924.

We next determined the in vivo relevance of SAG and neddylation in Tcons by utilizing the MHC disparate (B6→BALB/c) model of allogeneic BMT. The BALB/c animals were lethally irradiated and transplanted with TCD BM from either syngeneic or allogeneic WT-B6 animals along with 5x10⁵ splenic T cells from either the WT B6 or SAG^-/- B6 animals. The allogeneic animals that received SAG^-/- T cells demonstrated markedly reduced clinical GVHD and significantly increased survival when compared to those that received WT-B6 T cells (P<0.001). Similar results were observed in B6→B6D2F1 model. To further confirm our results and to determine potential translational application, we utilized the small molecule MLN4924, once again in the B6→BALB/c system. The recipient mice were lethally irradiated and received 5 doses of MLN4924 (20mg/kg, day-1 to day +3 of BMT) along with WT-B6 T cells. Mice receiving MLN4924 demonstrated significantly decreased clinical GVHD and improved survival. Our studies thus demonstrate that SAG is a novel molecular target for regulating T cell responses and mitigating GVHD. Furthermore, the clinical availability of the small molecule, MLN4924, suggests that this strategy could be tested in carefully designed human clinical trial for attenuating GVHD.