Supplementary Materials1. cells acquired a hyporesponsive phenotype that blocked cytokine production but maintained their growth in irradiated allo-BMT recipients, as well as their and cytotoxic potential. Our results reveal parallel functions for Notch signaling in alloreactive CD4+ and CD8+ T cells that differ from past reports of Notch action and spotlight the therapeutic potential of Notch inhibition in GVHD. Introduction Notch signaling is usually a highly conserved cell-to-cell communication pathway with multiple functions in health and disease (1). Notch receptors interact with Jagged or Delta-like ligands, leading to proteolytic release of intracellular Notch (ICN). ICN translocates into the nucleus to interact with CSL/RBP-Jk Avanafil (encoded by locus. Upon expression, pan-Notch inhibition was achieved in mature CD4+ and CD8+ T cells without interference with early stages of T cell development (7, 9, 10). DNMAML blocks the Notch transcriptional activation complex downstream of all Notch receptors, with comparable effects to those observed in the absence Avanafil of CSL/RBP-Jk (the DNA-binding transcription factor that mediates all the effects of canonical Notch signaling). In mouse allo-BMT models, pan-Notch blockade in donor CD4+ T cells led to markedly reduced GVHD severity and improved survival (7). Notch-deprived alloreactive CD4+ T cells had decreased production of inflammatory cytokines, including IFN, TNF, IL-17A, IL-4 and IL-2. Concomitantly, Notch inhibition Avanafil led to increased accumulation of regulatory T cells (Tregs). However, Notch-deprived CD4+ alloreactive T cells were capable of extensive proliferation, allowing for their enhanced accumulation in lymphoid tissues. Despite reduced cytokine production, Notch-deprived CD4+ T cells retained potent cytotoxic potential and expression during Th2 differentiation (9C12). In Th1 cells, pharmacological inhibitors and a antisense strategy suggested that Notch controlled expression of transcription (13). Notch signaling was also shown to influence Th17 and Treg differentiation, as well as CD4+ T cell longevity, at least (14C17). In CD8+ Igf1 T cells, Notch was suggested to act directly at the and loci, with an impact on differentiation and function (18C20). However, these findings originate from heterogeneous experimental systems, different immune contexts and variable strategies to manipulate Notch signaling, including gain-of-function approaches and pharmacological inhibitors. These results can be confounded by off-target effects and may not reflect the physiological functions of Notch in T cells. Here, we investigated the cellular and molecular mechanisms underlying the effects of Notch signaling in alloreactive CD4+ and CD8+ T cells during GVHD. Our strategy relied on priming of donor T cells in the presence or absence of all canonical CSL/RBP-Jk and MAML-dependent Notch signals specifically in T cells, ensuring that T cells were exposed to relevant Notch ligands in the post-transplantation environment. Notch-deprived alloreactive CD4+ and CD8+ T cells shared a profound defect in IFN production, suggesting parallel effects of Notch in both T cell subsets. Decreased IFN was observed despite preserved or enhanced expression of the transcription factors T-bet and Eomesodermin, consistent with the absence of a classical Th1 or effector CD8+ T cell differentiation defect. Notch-deprived alloreactive CD4+ and CD8+ T cells acquired a hyporesponsive phenotype with decreased Ras/MAPK and NF-kB signaling. Notch inhibition led to increased expression of selected unfavorable regulators of T cell activation. Some of these characteristics have been observed in anergic T cells, suggesting that Notch-inhibited CD4+ and CD8+ T cells acquire an anergy-like phenotype after allo-BMT, resulting in decreased production of inflammatory cytokines. Despite these changes, Notch inhibition preserved alloreactive T cell growth and only had modest effects on Avanafil their proliferative potential, while increasing growth of preexisting natural Tregs and preserving high cytotoxic potential. Altogether, our data demonstrate a novel, shared mechanism of Notch action in alloreactive CD4+ and CD8+ T cells during allo-BMT which differs from all previous reports of Notch activity in T cells. Understanding these effects is essential to harness the therapeutic benefits of Notch blockade to control GVHD after allo-BMT. Methods Mice BALB/c (H-2d) and C57BL/6 (B6, H-2b, CD45.2+) mice were from Harlan (Indianapolis, IN); C57BL/6.Ptprca (B6-SJL, H-2b, CD45.1+) from the NCI (Frederick, MD); BALB/b (H-2b) and Foxp3-IRES-RFP (FIR) from Jackson Laboratories (Bar Harbor, ME) (21). NF-kB reporter mice (NGL) were described previously (22). B6.129S6-mice by Dr. Reiner (Columbia University) (24); mice by Dr. Honjo (Kyoto, Japan) (4). mice (DNMAML) contain a Cre-inducible cassette encoding the.