NON-CANONICAL NOTCH SIGNALING REGULATES 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. 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 towards TH1 and iTreg lineages is also Notch dependent but RBP-JΚ independent. Our data provide evidence that non-canonical regulation of these processes likely occurs through NF-ΚB. Additionally, we also provide evidence suggestive of cross-talk between Notch and the mTOR pathway. Notch1, but not RBP-JΚ, is required for phosphorylation of several substrates directly downstream of mTORC2. Collectively, these striking observations demonstrate that many of the cell intrinsic functions of Notch occur independently of RBP-JΚ. This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T-cell responses

Inflammation Triggers Zeb1-Dependent Escape from Tumor Latency

The emergence of metastatic disease in cancer patients many years or decades after initial successful treatment of primary tumors is well documented but poorly understood at the molecular level. Recent studies have begun exploring the cell-intrinsic programs, causing disseminated tumor cells to enter latency and the cellular signals in the surrounding nonpermissive tissue microenvironment that maintain the latent state. However, relatively little is known about the mechanisms that enable disseminated tumor cells to escape cancer dormancy or tumor latency. We describe here an in vivo model of solitary metastatic latency in the lung parenchyma. The induction of a localized inflammation in the lungs, initiated by lipopolysaccharide treatment, triggers the awakening of these cells, which develop into macroscopic metastases. The escape from latency is dependent on the expression of Zeb1, a key regulator of the epithelial-to-mesenchymal transition (EMT). Furthermore, activation of the EMT program on its own, as orchestrated by Zeb1, is sufficient to incite metastatic out-growth by causing carcinoma cells to enter stably into a metastasis-initiating cell state.National Institutes of Health (U.S.) (Grant P01-CA080111)National Institutes of Health (U.S.) (Grant R01-CA078461)National Institutes of Health (U.S.) (Grant U54-CA163109

Epithelial-to-Mesenchymal Transition Contributes to Immunosuppression in Breast Carcinomas

The epithelial-to-mesenchymal transition (EMT) is a cell biological program that confers mesenchymal traits on carcinoma cells and drives their metastatic dissemination. It is unclear, however, whether the activation of EMT in carcinoma cells can change their susceptibility to immune attack. We demonstrate here that mammary tumor cells arising from more epithelial carcinoma cell lines expressed high levels of MHC-I, low levels of PD-L1, and contained within their stroma CD8þT cells and M1 (antitumor) macrophages. In contrast, tumors arising from more mesenchymal carcinoma cell lines exhibiting EMT markers expressed low levels of MHC-I, high levels of PD-L1, and contained within their stroma regulatory T cells, M2 (protumor) macrophages, and exhausted CD8þT cells. Moreover, the more mesenchymal carcinoma cells within a tumor retained the ability to protect their more epithelial counterparts from immune attack. Finally, epithelial tumors were more susceptible to elimination by immunotherapy than corresponding mesenchymal tumors. Our results identify immune cells and immunomodulatory markers that can be potentially targeted to enhance the susceptibility of immunosuppressive tumors to various therapeutic regimens.National Institutes of Health (U.S.) (Grant P01-CA080111

Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells

Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non-small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno-positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89 Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8 + T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89 Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies.National Institutes of Health (U.S.) (Grant R01-AI087879-06)National Institutes of Health (U.S.) (Grant DP1-GM106409-03)National Institutes of Health (U.S.) (Grant R01-GM100518-04)National Institutes of Health (U.S.) (Grant P01 CA080111

Editorial: The Role of the EMT Program in Regulating the Immune Response in Carcinoma

International audienceNo abstract availabl

Low energy electron induced damage to plasmid DNA pQE30

Low energy electrons (LEEs) are produced in copious amounts by the primary radiation used in radiation therapy. The damage caused to the DNA by these secondary electrons in the energy range 5–22 eV has been studied to understand their possible role in radiation induced damage. Electrons are irradiated on dried films of plasmid DNA (pQE30) and analysed using agarose gel electrophoresis. Single strand breaks (SSBs) induced by LEE to supercoiled plasmid DNA show resonance structures at 7, 12, and 15 eV for low doses and 6, 10, and ∼18 eV at saturation doses. The present measurements have an overall agreement with the literature that LEEs resonantly induce SSBs in DNA. Resonant peaks in the SSBs induced by LEEs at 7, 12, and 15 eV with the lowest employed dose in the current study are somewhat different from those reported earlier by two groups. The observed differences are perhaps related to the irradiation dose, conditions and the nature of DNA employed, which is further elaborated

Direct and Indirect Regulators of Epithelial–Mesenchymal Transition–Mediated Immunosuppression in Breast Carcinomas

The epithelial-to-mesenchymal transition, which conveys epithelial (E) carcinoma cells to quasi-mesenchymal (qM) states, enables them to metastasize and acquire resistance to certain treatments. Murine tumors composed of qM mammary carcinoma cells assemble an immunosuppressive tumor microenvironment (TME) and develop resistance to anti-CTLA4 immune-checkpoint blockade (ICB) therapy, unlike their E counterparts. Importantly, minority populations of qM cells within a tumor can cross-protect their more E neighbors from immune attack. The underlying mechanisms of immunosuppression and cross-protection have been unclear. We demonstrate that abrogation of qM carcinoma cell–derived factors (CD73, CSF1, or SPP1) prevents the assembly of an immunosuppressive TME and sensitizes otherwise refractory qM tumors partially or completely to anti-CTLA4 ICB. Most strikingly, mixed tumors in which minority populations of carcinoma cells no longer express CD73 are now sensitized to anti-CTLA4 ICB. Finally, loss of CD73 also enhances the efficacy of anti-CTLA4 ICB during the process of metastatic colonization. Significance: Minority populations of qM carcinoma cells, which likely reside in human breast carcinomas, can cross-protect their E neighbors from immune attack. Understanding the mechanisms by which qM carcinoma cells resist antitumor immune attack can help identify signaling channels that can be interrupted to potentiate the efficacy of checkpoint blockade immunotherapies