Blockade of Notch ligand δ1 promotes allograft survival by inhibiting alloreactive Th1 cells and cytotoxic T cell generation

Abstract

The Notch signaling pathway has been recently shown to contribute to T cell differentiation in vitro. However, the in vivo function of Notch signaling in transplantation remains unknown. In this study, we investigated the importance of Delta1 in regulating the alloimmune response in vivo. Delta1 expression was upregulated on dendritic cells and monocytes/macrophages upon transplantation in a BALB/c into B6 vascularized cardiac transplant model. Whereas administration of anti-Delta1 mAb only slightly delayed survival of cardiac allografts in this fully MHC-mismatched model, it significantly prolonged graft survival in combination with single-dose CTLA4-Ig or in CD28 knockout recipients. The prolongation of allograft survival was associated with Th2 polarization and a decrease in Th1 and granzyme B-producing cytotoxic T cells. The survival benefit of Delta1 blockade was abrogated after IL-4 neutralization and in STAT6KO recipients, but was maintained in STAT4KO recipients, reinforcing the key role of Th2 cell development in its graft-prolonging effects. To our knowledge, these data demonstrate for the first time an important role of Delta1 in alloimmunity, identifying Delta1 ligand as a potential novel target for immunomodulation in transplantation.

Figure 1. Expression of Delta1 on APCs is significantly upregulated upon transplantation

By using a biotinylated anti-Delta1 Ab, we were able to demonstrate a predominant expression of Delta1 on antigen-presenting cells, mainly dendritic cells (CD11c⁺) and monocyte/macrophages (CD11b⁺ CD11c⁻). On the left and middle columns, representative histograms of Delta1 expression on different cell subtypes from splenocytes of naïve B6 mice or B6 recipients of BALB/c allografts 7 days after transplantation (Tx). Isotype control IgG is shown with solid grey line. Right column demonstrates the significant upregulation of Delta1 in the setting of transplantation on APCs (n = 6 each group) (** p<0.001).

Figure 2. Anti-Delta1 mAb delays rejection of BALB/c cardiac allografts in CD28KO recipients

The hearts from BALB/c mice were transplanted into CD28KO recipients on B6 background, which were treated with anti-Delta1 mAb or control hamster IgG. (A) While control-group cardiac grafts had a median survival time (MST) of 12 days (n=8), anti-Delta1-treated group had a delayed rejection with MST of 52 days (n=8, p<0.0001). Days of administration of mAb is depicted on arrow below survival curves. (B) Representative photomicrographs of H&E staining demonstrate more prominent cellular infiltrate on control group compared to anti-Delta1 at 2 weeks after transplantation. (C) At similar time point, scoring of pathology samples from both groups showed significant higher extent of interstitial inflammation and myocyte loss on the control group (n=5 each group). Bars, 200 μm.

Figure 3. Anti-Delta1 mAb prolongs BALB/c cardiac graft survivals in synergy with sCTLA4-Ig in B6 WT recipients

The hearts from BALB/c mice were transplanted into B6 WT recipients, which were treated with single dose of CTLA4-Ig (sCTLA4-Ig) in combination with either anti-Delta1 mAb or control hamster IgG. (A) While control cardiac grafts had a MST of 29 days (n=7), anti-Delta1-cotreated group had a delayed rejection with MST of 58 days (n=7, p=0.0015). (B) Representative photomicrographs of H&E staining demonstrate more prominent cellular infiltrate on control group compared to anti-Delta1-treated group at 2 weeks after transplantation. (C) At similar time point, scoring of pathology samples from both groups showed significant higher extent of interstitial inflammation and myocyte loss on the control group (n=5 each group). (D) Representative photomicrographs of CD4⁺ and CD8⁺ stained sections showed less CD8⁺ and to a lesser extent CD4⁺ infiltration on the combined treated group with significant less T cells/high power field (HPF) as depicted on (E). Bars, 200 μm.

Figure 4. Lower frequency of CD4 eff/ mem and CD8 eff/ mem in the combined sCTLA4-Ig/anti-Delta1-treated group

Flow cytometry analysis of splenocytes from B6 WT recipients of BALB/c hearts 14 days after transplantation showed that anti-Delta1 Ab significantly decreased the percentage of CD4⁺CD44^highCD62L^low and CD8⁺CD44^highCD62L^low cells (CD4 and CD8 eff/ mem, respectively) when compared to sCTLA4-Ig alone. CD4⁺CD25⁺Foxp3⁺ regulatory T cells were no different between groups (p=0.3820). Panels on the left are representative examples of dot plots. Data are representative of three independent experiments.

Figure 5. Anti-Delta1 mAb upregulates Th2 cytokines

(A) Donor specific Th1, Th2 and proinflammatory cytokine production of splenocytes from CD28KO recipients of BALB/c allografts were assessed 14 days after transplantation by Luminex assay. Anti-Delta1 treatment led to a significant increase in the production of Th2 cytokines (IL-4, IL-5), while it decreased the production of IL-2, IL-6 and IL-17. (B) The frequency of GrB-producing cells was also decreased on the Delta1 blockade group by ELISPOT. There was a trend towards lower IFN-γ but it did not reach statistical significance. Data are representative of 3 independent experiments and indicate the mean of triplicate results in each experiment (* p<0.05).

Figure 6. Combination of sCTLA4-Ig and anti-Delta1 lowered GrB and IFN-γ production, while it increased Th2 cytokines

(A) Donor specific cytokine production of splenocytes from B6 WT recipients of BALB/c allografts were assessed 14 days after transplantation by ELISPOT and Luminex assay. GrB (B), IL-2, IFN-γ and IL-6 was significantly decreased in the combined sCTLA4-Ig /anti-Delta1 group, while Th2 cytokines (IL-4, IL-5 and IL-13) were increased when compared to sCTLA4-Ig alone group. Data are representative of 3 independent experiments and indicate the mean of triplicate results in each experiment. (* p<0.05).

Figure 7

Cytotoxicity assay. CD8⁺ T cells from control and anti-Delta1 treated recipients were incubated with allogeneic target cells for 6 hours at effector:target ratios of 1:1 and 5:1 and cytotoxicity activity of these T cells was measured using a live/dead® Viability/Cytotoxicity Kit (Invitrogen). Percentage of specific cytotoxicity with either control or anti-Delta1 CD8⁺ T cells was calculated as described in Materials and Methods (n=3-4, p<0.0001). Data are representative of three independent experiments.

Figure 8. Lower frequency of IFN-γ-producing CD4⁺ and CD8⁺ T cells isolated from heart allografts cotreated with anti-Delta1

Lymphocytes were isolated from heart allografts two weeks after transplantation and were restimulated in vitro for 4 hours. Subsequent intracellular cytokine staining by flow cytometry revealed a lower frequency of IFN-γ-producing T cells in the group treated with both sCTLA4-Ig and anti-Delta1 compared with control (only sCTLA4-Ig). Contour plots are representative examples. Graphs on the right are representative of three independent experiments. (* p<0.01).

Figure 9. Prolongation of allograft survival on anti-Delta1-treated group is dependent on Th2 cytokines

The hearts from B6 mice were transplanted into STAT4KO (A) or STAT6KO (B) recipients on BALB/c background, which were treated with anti-Delta1 mAb or control hamster IgG. While anti-Delta1 delayed allograft rejection in STAT4KO recipients (n=6, p=0.0014), it did not affect graft survival in STAT6KO recipients (n=6, p=0.87). (C) The hearts from BALB/c mice were transplanted into CD28KO recipients on B6 background, which were treated with anti-IL4 mAb in combination with either anti-Delta1 mAb or control hamster IgG. IL-4 neutralization abrogated the prolonging effect of Delta1 blockade (MST=13 vs. 52 in controls, n=5/group, p=0.0002).

Figure 10. The role of Delta1 in T cell differentiation in alloimmunity

Bacteria, virus and TLR ligands are some of the known stimuli that can induce the expression of Delta1 by antigen-presenting cells (APCs), which in turn promote the capacity of APCs to induce cytotoxic CD8⁺ T (CTL) cells and Th1-cell differentiation. When Delta1 interacts with the Notch receptor on T cells, it leads to sequential cleavage of the transmembrane region of Notch, resulting in the release of the Notch intracellular domain that translocates to the nucleus. In combination with co-factors (not shown), it activates the transcription of Notch target genes, including GrB and Eomes on CD8⁺ T cells; and Tbx21 and Ifng on CD4⁺ T cells. Delta1-Notch activation also leads to inhibition of IL-4-signaling on naïve CD4⁺ T cells, suppressing Th2-cell differentiation. As a result, the induction of Th1 cells and CTLs promotes rejection in the transplant setting.