Impact of Notch1 Deletion in Macrophages on Proinflammatory Cytokine Production and the Outcome of Experimental Autoimmune Encephalomyelitis.

Notch signaling is involved in regulating TLR-mediated responses in activated macrophages. In this study, we investigated the impact of Notch signaling in macrophages in an experimental autoimmune encephalomyelitis (EAE) model. To examine the impact of deficiency in Notch signaling in activated macrophages in EAE, an adoptive transfer of activated macrophages derived from Notch1(fl/fl) × Mx1cre(+/-) (Notch1 knockout [N1KO]) or CSL/Rbp-jκ(fl/fl) × Mx1cre(+/-) (CSL/RBP-Jκ KO) mice was performed prior to induction of EAE. Mice receiving activated N1KO macrophages showed decreased severity of EAE compared with mice receiving wild-type or CSL/RBP-Jκ KO macrophages. In vitro restimulation of splenocytes by myelin oligodendrocyte glycoprotein 35-55 peptide from these mice revealed that cells from mice receiving N1KO macrophages produced significantly less IL-17 compared with the control mice, whereas IFN-γ production was similar in both groups. We found that activated N1KO, but not CSL/RBP-Jκ KO, macrophages produced less IL-6 and had lower CD80 expression compared with wild-type and did not exhibit any defect in IL-12p40/70 production, whereas activated macrophages from CSL/RBP-Jκ KO mice phenocopied γ-secretase inhibitor treatment for reduced IL-12p40/70 production. Furthermore, the nuclear translocation of the NF-κB subunit c-Rel was compromised in γ-secretase inhibitor-treated and CSL/RBP-Jκ KO but not N1KO macrophages. These results suggest that Notch1 and CSL/RBP-Jκ in macrophages may affect the severity of EAE differently, possibly through modulating IL-6 and CD80 expression, which is involved in the Th17 but not Th1 response.

Pathways contributing to development of spontaneous mammary tumors in BALB/c-Trp53+/- mice.

Mutation and loss of function in p53 are common features among human breast cancers. Here we use BALB/c-Trp53+/- mice as a model to examine the sequence of events leading to mammary tumors. Mammary gland proliferation rates were similar in both BALB/c-Trp53+/- mice and wild-type controls. In addition, sporadic mammary hyperplasias were rare in BALB/c-Trp53+/- mice and not detectably different from those of wild-type controls. Among the 28 mammary tumors collected from BALB/c-Trp53+/- mice, loss of heterozygosity for Trp53 was detected in more than 90% of invasive mammary tumors. Transplantation of Trp53+/- ductal hyperplasias also indicated an association between loss of the wild-type allele of Trp53 and progression to invasive carcinomas. Therefore, loss of p53 function seems to be a rate-limiting step in progression. Moreover, expression of biomarkers such as estrogen receptor alpha, progesterone receptor, Her2/Neu, and activated Notch1 varied among mammary tumors, suggesting that multiple oncogenic lesions collaborate with loss of p53 function. Expression of biomarkers was retained when tumor fragments were transplanted to syngeneic hosts. Tumors expressing solely luminal or basal keratins were also observed (27 and 11%, respectively), but the largest class of tumors expressed both luminal and basal keratins (62%). Overall, this panel of transplantable tumors provides a resource for detailed evaluation of the cell lineages undergoing transformation and preclinical testing of therapeutic agents targeting a variety of oncogenic pathways including cancer stem cells.

Potent neutralization of staphylococcal enterotoxin B by synergistic action of chimeric antibodies.

Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igkappa and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.

Non-Canonical Notch Signaling Drives Activation and Differentiation of Peripheral CD4(+) T Cells.

Cleavage of the Notch receptor via a γ-secretase, results in the release of the active intra-cellular domain of Notch that migrates to the nucleus and interacts with RBP-Jκ, resulting in the activation of downstream target genes. This canonical Notch signaling pathway has been documented to influence T cell development and function. However, the mechanistic details underlying this process remain obscure. In addition to RBP-Jκ, the intra-cellular domain of Notch also interacts with other proteins in the cytoplasm and nucleus, giving rise to the possibility of an alternate, RBP-Jκ independent Notch pathway. However, the contribution of such RBP-Jκ independent, "non-canonical" Notch signaling in regulating peripheral T cell responses is unknown. In this report, we specifically demonstrate the requirement of Notch1 for regulating signal strength and signaling events distal to the T cell receptor in peripheral CD4(+) T cells. By using mice with a conditional deletion in Notch1 or RBP-Jκ, we show that Notch1 regulates activation and proliferation of CD4(+) T cells independently of RBP-Jκ. Furthermore, differentiation to TH1 and iTreg lineages although Notch dependent, is RBP-Jκ independent. Our striking observations demonstrate that many of the cell-intrinsic functions of Notch occur independently of RBP-Jκ. Such non-canonical regulation of these processes likely occurs through NF-κ B. This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T cell responses.

Inhibitors of gamma-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21.

Notch receptors are processed by gamma-secretase acting in synergy with T cell receptor signaling to sustain peripheral T cell activation. Activated CD4+ T cells differentiate into T helper type 1 (TH1) or TH2 subsets. Molecular cues directing TH1 differentiation include expression of the TH1-specific transcription factor T-bet, encoded by Tbx21. However, the regulation of Tbx21 remains incompletely defined. Here we report that Notch1 can directly regulate Tbx21 through complexes formed on the Tbx21 promoter. In vitro, gamma-secretase inhibitors extinguished expression of Notch, interferon-gamma and Tbx21 in TH1-polarized CD4+ cells, whereas ectopic expression of activated Notch1 restored Tbx21 transcription. In vivo, administration of gamma-secretase inhibitors substantially impeded TH1-mediated disease progression in the mouse experimental autoimmune encephalomyelitis model of multiple sclerosis. Thus, using gamma-secretase inhibitors to modulate Notch signaling may prove beneficial in treating TH1-mediated autoimmunity.