We also isolated cells from Foxp3EGFPCreNotch1fl/fl mice (N1cKO), wherein Notch-1 is specifically deleted within the Treg population (Supplemental Figure S5)

We also isolated cells from Foxp3EGFPCreNotch1fl/fl mice (N1cKO), wherein Notch-1 is specifically deleted within the Treg population (Supplemental Figure S5). lung transplant models, and in a humanized skin transplant model. Based on our findings, we further employed a genetic approach to investigate the effect of selective Notch-1 inhibition in Tregs. Results We observed an increased proportion of Tregs expressing surface and intracellular (activated) Notch-1 compared to conventional T cells (Tconv), both in transplanted mice and in the peripheral blood of transplanted patients. In the murine cardiac transplant model, peri-transplant administration of aNotch-1 (days 0, 2, 4, 6, 8, 10) significantly prolonged allograft survival compared to IgG-treated controls. Similarly, aNotch-1 treatment improved both histological and functional outcomes in the murine lung transplant model. The use of aNotch-1 resulted in a reduced proportion of both splenic and intra-graft Tconv, while increasing the proportion of Tregs. Furthermore, Tregs isolated from aNotch-1 treated mice showed enhanced suppressive function on a per-cell basis, confirmed with selective Notch-1 deletion in Tregs (Foxp3EGFPCreNotch1fl/fl). Notch-1 blockade inhibited the mTOR pathway and increased the phosphorylation of STAT5 in murine Tregs. Notch-1low Tregs isolated from human peripheral blood exhibited more potent Tmem34 suppressive capacity than Notch-1high Tregs. Lastly, the combination of aNotch-1 with costimulation blockade induced long-term tolerance in a cardiac transplant model, and this tolerance was dependent on CTLA-4 signaling. Conclusion Our GLPG0187 data reveal a promising, clinically relevant approach for immune modulation in transplantation by selectively targeting Notch-1. Il2rg 0.05. Prism software was used for data analysis and drawing graphs (GraphPad Software, Inc., San-Diego, CA). Data represent mean SD with at least 3 samples per studied group for all experiments. Morpheus matrix visualization and analysis tool (Broad Institute; https://software.broadinstitute.org/morpheus) was used to create a heatmap of the mean fluorescence intensity (MFI) of the markers analyzed; MFI values in the heat map were represented using the minimum and maximum of each independent row. RESULTS Notch-1 was highly expressed in Tregs compared to conventional T cells in mice and kidney transplant patients As Notch-1 is critical in T helper cell differentiation27, GLPG0187 we first examined the differential Notch-1 expression in Tconv and Tregs in the transplant setting. In the full MHC-mismatch murine cardiac transplant model, in which hearts retrieved from BALB/c mice (I-Ad/H-2d) were transplanted into C57BL/6J (B6; I-Ab/H-2b) recipients, a greater proportion of Tregs (CD4+Foxp3+) expressed surface Notch-1 than Tconv (CD4+FoxP3?, GLPG0187 Figure 1A). This pattern of expression was also seen in kidney transplant patients (Figure 1B), while the proportion of Notch-1+Tregs was significantly higher in kidney transplant patients than non-transplanted healthy control subjects (Figure 1C). Upon ligand-receptor interaction and consequent receptor activation, the intracellular domain of Notch-1 is cleaved and translocates from the cellular surface into the nucleus, where it forms a nuclear transcription complex and promotes target gene transcription7, 28. We found that both mouse and human Tregs have a higher expression of activated intracellular Notch-1 compared to Tconv cells (Figure 1D and ?andE),E), indicating that Notch-1 signaling pathway is more activated in Tregs. These results suggest that Notch-1 signaling within the Treg population might play a role in immune regulation in the transplant setting. Open in a separate window Figure 1. Regulatory T cells (Tregs) express higher levels of Notch-1 than conventional T cells (Tconv).A, Mouse splenic Tregs (CD4+Foxp3+) expressed higher levels of Notch-1 than Tconv (CD4+Foxp3?) on day 7 post full MHC-mismatch cardiac transplantation. Representative flow cytometry plots illustrating the MFI of Notch-1 in Tconv and Tregs are shown (meanSD, n=4, paired t-test, *aNotch-1 therapy blocked Notch signaling in graft-infiltrating and splenic T cells by evaluating.