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Epithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy.We evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55).We uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3Our results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

This file contains Supplementary Figures 1-8 and Supplementary Tables #1-2

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.This article is highlighted in the In This Issue feature, p. 995

Supplementary Figure 3 shows immunosuppressive cells in tumors arising from Snail+ cells relative to epithelial Slug+ cells

Supplementary Figure legends shows legends for each figure

In vivo and in vitro growth curves of D2A1-d and parental D2A1 cells

Supplementary Figure 7 shows Epithelial and Mesenchymal cells in mixed tumors before and after immunotherapy

Supplementary Figure 1 shows enrichment of immunosuppressive cells in tumors arising from mesenchymal cell lines

Summary of tumor incidences at limiting dilutions for D2A1-d + Zeb1 knockdown cells

Lung metastases can form after two weeks of continuous induction of the EMT program.

Supplementary Figure 4 shows differential expression of molecules associated with immune-evasion and immune-suppression by epithelial and mesenchymal cells

Summary of tumor incidences at limiting dilutions for CD24negative and CD24positive cell populations

Supplementary Figure 5 shows activation of an EMT leads to an immunosuppressive phenotype.

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 CD8+ T 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 CD8+ T 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. Cancer Res; 77(15); 3982–9. ©2017 AACR.

Detailed description of supplementary figures

LPS-induced inflammation involves the native but not the adaptive immune system.

Summary of incidence of metastasis formation at limiting dilutions in D2A1-d and parental D2A1 cells

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 outgrowth by causing carcinoma cells to enter stably into a metastasis-initiating cell state. Cancer Res; 76(23); 6778–84. ©2016 AACR.

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. This article is highlighted in the In This Issue feature, p. 995

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs), and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs, and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

Epithelial–mesenchymal transition (EMT) is a cellular programme that is known to be crucial for embryogenesis, wound healing and malignant progression. During EMT, cell–cell and cell–extracellular matrix interactions are remodelled, which leads to the detachment of epithelial cells from each other and the underlying basement membrane, and a new transcriptional programme is activated to promote the mesenchymal fate. In the context of neoplasias, EMT confers on cancer cells increased tumour-initiating and metastatic potential and a greater resistance to elimination by several therapeutic regimens. In this Review, we discuss recent findings on the mechanisms and roles of EMT in normal and neoplastic tissues, and the cell-intrinsic signals that sustain expression of this programme. We also highlight how EMT gives rise to a variety of intermediate cell states between the epithelial and the mesenchymal state, which could function as cancer stem cells. In addition, we describe the contributions of the tumour microenvironment in inducing EMT and the effects of EMT on the immunobiology of carcinomas. Epithelial–mesenchymal transition (EMT) is crucial for embryogenesis, wound healing and cancer development, and confers greater resistance to cancer therapies. This Review discusses the mechanisms of EMT and its roles in normal and neoplastic tissues, the contribution of cell-intrinsic signals and the microenvironment to inducing EMT, and its effects on the immunobiology of carcinomas.

Background Immune checkpoint blockade (ICB) has generated some dramatic responses in certain types of human tumors, most notably, melanomas. However, the response of breast tumors has been largely limited. We have previously demonstrated that the residence of breast cancer cells in the epithelial or mesenchymal phenotypic states can itself be used as an important determinant of the success or failure of ICB. Specifically, we have shown that while epithelial tumors are sensitive to anti-CTLA4, mesenchymal tumors are highly resistant. Most strikingly, in tumors arising from a mixture of both cell types, a minority population (10%) of mesenchymal cells can cross-protect the vast majority (90%) of their epithelial neighbors from immune attack.1 However, the mechanisms underlying such cross-protection remain elusive. This is particularly important as most human breast cancers contain minority populations of such mesenchymal cells which can protect the tumor as a whole from immune attack. Methods Using a combination of transcriptomic and CRISPR/Cas9 approaches, we first identified that mesenchymal carcinoma cells express high levels of CD73, an ecto-enzyme that catalyzes the production of adenosine. Additionally, we used digital spatial profiling to determine whether CD73 expression differs across distinct epithelial and mesenchymal sectors in mixed tumors. Results Abrogation of CD73 from mesenchymal carcinoma cells prevented the assembly of an immunosuppressive tumor microenvironment and resulted instead in increased numbers of tumor-infiltrating lymphocytes and cross-presenting dendritic cells. Most strikingly, abrogation of CD73 sensitized previously refractory mesenchymal tumors completely to ICB. In the context of mixed tumors comprised of both epithelial and mesenchymal carcinoma cells, gradients in expression of CD73 were observed corresponding to the epithelial or mesenchymal sectors of these mixed tumors. Importantly, mixed tumors in which the minority population of mesenchymal carcinoma cells lacked the expression of CD73, were also sensitized partially to ICB. Thus, these mesenchymal carcinoma cells knocked out for CD73 could no longer protect their epithelial neighbors from immune attack. Conclusions Taken together, our work suggests that mesenchymal carcinoma cells exert immune-suppressive effects which are also prominent in heterogeneous tumors. Furthermore, targeting the adenosinergic signaling pathway in mesenchymal carcinoma cells can potentiate the efficacy of ICB. Reference Dongre A, Rashidian M, Reinhardt F, Bagnato A, Keckesova Z, Ploegh HL, Weinberg RA, Epithelial-to-mesenchymal transition contributes to immunosuppression in breast carcinomas. Cancer Res 2017 Aug 1;77(15):3982–3989.

Rashidian et al. show that 89Zr-PEGylated single-domain antibodies that target CD8+ T cells can be used to monitor and evaluate the response to immunotherapy as a predictive tool., 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 89Zr-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 89Zr-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.

Immunotherapy using checkpoint-blocking antibodies against PD-1 has produced impressive results in a wide range of cancers. However, the response remains heterogeneous among patients. We used noninvasive immuno-positron emission tomography (PET), using (89)Zr-labeled PEGylated single-domain antibody fragments (nanobodies or VHHs), to explore the dynamics and distribution of intratumoral CD8(+) T cells and CD11b(+) myeloid cells in response to anti–PD-1 treatment in the MC38 colorectal mouse adenocarcinoma model. Responding and nonresponding tumors showed consistent differences in the distribution of CD8(+) and CD11b(+) cells. Anti–PD-1 treatment mobilized CD8(+) T cells from the tumor periphery to a more central location. Only those tumors fully infiltrated by CD8(+) T cells went on to complete resolution. All tumors contained CD11b(+) myeloid cells from the outset of treatment, with later recruitment of additional CD11b(+) cells. As tumors grew, the distribution of intratumoral CD11b(+) cells became more heterogeneous. Shrinkage of tumors in responders correlated with an increase in the CD11b(+) population in the center of the tumors. The changes in distribution of CD8(+) and CD11b(+) cells, as assessed by PET, served as biomarkers to gauge the efficacy of anti–PD-1 treatment. Single-cell RNA sequencing of RNA from intratumoral CD45(+) cells showed that CD11b(+) cells in responders and nonresponders were markedly different. The responders exhibited a dominant population of macrophages with an M1-like signature, while the CD45(+) population in the nonresponders displayed an M2-like transcriptional signature. Thus, by using immuno-PET and single-cell RNA sequencing, we show that anti–PD-1 treatment not only affects interactions of CD8(+) T cells with the tumor but also impacts the intratumoral myeloid compartment.

Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.

Epithelial-mesenchymal transition (EMT) is a cellular programme that is known to be crucial for embryogenesis, wound healing and malignant progression. During EMT, cell-cell and cell-extracellular matrix interactions are remodelled, which leads to the detachment of epithelial cells from each other and the underlying basement membrane, and a new transcriptional programme is activated to promote the mesenchymal fate. In the context of neoplasias, EMT confers on cancer cells increased tumour-initiating and metastatic potential and a greater resistance to elimination by several therapeutic regimens. In this Review, we discuss recent findings on the mechanisms and roles of EMT in normal and neoplastic tissues, and the cell-intrinsic signals that sustain expression of this programme. We also highlight how EMT gives rise to a variety of intermediate cell states between the epithelial and the mesenchymal state, which could function as cancer stem cells. In addition, we describe the contributions of the tumour microenvironment in inducing EMT and the effects of EMT on the immunobiology of carcinomas.

The epithelial-to-mesenchymal transition (EMT), which conveys epithelial (E) carcinoma cells to quasi-mesenchymal (qM) states, enables these cells to gain tumor-initiating stem like abilities, metastasize and acquire resistance to several drug and chemotherapeutic regimens. In addition to these aforementioned features, we have recently demonstrated that the EMT program also contributes to the generation of an immunosuppressive tumor microenvironment (TME) in breast carcinomas and confers resistance to immune checkpoint blockade therapies. Specifically, qM tumors recruit immunosuppressive cells to their TME and are refractory to checkpoint blockade, while E tumors recruit CD8+ T-cells instead and are sensitive to checkpoint blockade. Importantly, minority populations of qM cells within a tumor can cross-protect their more E neighbors from immune attack. The underlying mechanisms by which this immunosuppression and cross-protection are achieved have been unclear. Using a combination of novel E and qM tumor models arising in syngeneic immunocompetent hosts combined with CRISPR/Cas9 and transcriptomic approaches, we demonstrate that qM but not E tumors are specifically associated with an immunosuppressive gene signature. Furthermore, abrogation of carcinoma cell-derived factors (CD73, Csf1 or Spp1) specifically associated with the qM state, prevents the assembly of an immunosuppressive TME and sensitizes otherwise refractory qM tumors partially or completely to checkpoint blockade immunotherapy. Taken together, our work indicates that carcinoma cell-intrinsic factors specifically associated with residence in the qM state can directly influence their response to anti-tumor immunity and checkpoint blockade. As a result, this work brings to the forefront the possibility of using the epithelial-mesenchymal state of carcinoma cells as an important surrogate marker that can be used to predict responses to immune checkpoint blockade therapies. This abstract is also being presented as PO038. Citation Format: Anushka Dongre, Mohammad Rashidian, Elinor Ng Eaton, Ferenc Reinhardt, Prat Thiru, Maria Zagorulya, Sunita Nepal, Tuba Banaz, Anna Martner, Stefani Spranger, Robert A. Weinberg. Potentiating the efficacy of immune checkpoint blockade by targeting the epithelial-to-mesenchymal transition (EMT) in breast carcinomas [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR010.

The epithelial-to-mesenchymal transition (EMT) is a cell-biologic program that confers mesenchymal traits on carcinoma cells and drives their metastatic dissemination. We recently demonstrated that epithelial and mesenchymal carcinomas recruit distinct immune cells to their tumor microenvironments (TME) and differ in their susceptibility to checkpoint blockade. Specifically, mesenchymal tumors recruit immunosuppressive cells to their TME and are refractory to checkpoint blockade, while epithelial tumors recruit CD8+ T cells instead and are sensitive to checkpoint blockade. The mechanism(s) underlying such EMT-mediated immunosuppression remains elusive. Furthermore, the biologic relevance of various immune cells infiltrating epithelial versus mesenchymal tumors requires further understanding. Using a combination of novel epithelial and mesenchymal tumor models arising in syngeneic immunocompetent hosts combined with transcriptomic approaches, we demonstrate that mesenchymal but not epithelial tumors are specifically associated with an immunosuppressive gene signature. Furthermore, while CD8+ T cells play a critical role in regulating tumor progression and responses to checkpoint blockade in epithelial breast tumors, they do not do so in mesenchymal breast tumors. On the contrary, mesenchymal tumors recruit and rely on M2-macrophages to generate an immunosuppressive TME and mount refractory responses to checkpoint blockade. Finally, we demonstrate that mesenchymal tumors utilize a combination of carcinoma cell-intrinsic direct and indirect mechanism(s) to assemble an immunosuppressive TME. This work suggests that epithelial and mesenchymal breast carcinomas have dramatically different immunologic profiles. Thus, the residence of carcinoma cells in the epithelial or mesenchymal states can dictate their corresponding responses to checkpoint blockade immunotherapies. This in turn can therefore have a direct translational impact on breast cancer patients. Citation Format: Anushka Dongre, Mohammad Rashidian, Elinor Ng Eaton, Ferenc Reinhardt, Sunita Nepal, Hidde Ploegh, Robert Weinberg. Direct and indirect regulators of epithelial-to-mesenchymal transition mediated immunosuppression in breast carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A82.

Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1β (IL-1β)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1β maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1β expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival.

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)

Notch1 signaling sustains the proinflammatory behavior of Th1 cells, implicated in the development of aplastic anemia in humans and mice., Severe aplastic anemia (AA) is a bone marrow (BM) failure (BMF) disease frequently caused by aberrant immune destruction of blood progenitors. Although a Th1-mediated pathology is well described for AA, molecular mechanisms driving disease progression remain ill defined. The NOTCH signaling pathway mediates Th1 cell differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by γ-secretase. Using a mouse model of AA, we demonstrate that expression of both intracellular NOTCH1IC and T-BET, a key transcription factor regulating Th1 cell differentiation, was increased in spleen and BM-infiltrating T cells during active disease. Conditionally deleting Notch1 or administering γ-secretase inhibitors (GSIs) in vivo attenuated disease and rescued mice from lethal BMF. In peripheral T cells from patients with untreated AA, NOTCH1IC was significantly elevated and bound to the TBX21 promoter, showing NOTCH1 directly regulates the gene encoding T-BET. Treating patient cells with GSIs in vitro lowered NOTCH1IC levels, decreased NOTCH1 detectable at the TBX21 promoter, and decreased T-BET expression, indicating that NOTCH1 signaling is responsive to GSIs during active disease. Collectively, these results identify NOTCH signaling as a primary driver of Th1-mediated pathogenesis in AA and may represent a novel target for therapeutic intervention.

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.

The Notch receptor is activated by ligands that belong to the Delta or Jagged family. Ligand binding is followed by proteolytic cleavages resulting in the release of intra-cellular Notch which translocates to the nucleus and functions as a transcriptional activator. While the role of Notch in thymocyte maturation has been studied, the precise role of Notch in T-cell activation and cellular metabolism remains incompletely understood. Evidence from our lab has suggested that TCR mediated activation leads to the generation of active Notch and inhibition via Gamma Secretase Inhibitors (GSI) decreases T cell activation and proliferation. However, since GSI’s are known to have multiple substrates, whether such a decrease in activation is indeed dependent on Notch or is simply a GSI mediated effect needs to be examined. To determine the role of Notch in T cell activation we conditionally deleted Notch1 in peripheral T cells. Our data demonstrate that CD4 cells have reduced expression of activation markers accompanied by a decrease in IL-2 and IFN-Gamma secretion as well as impaired proliferation in the absence of Notch1. Notch1 was also required for polarization towards Th1. Furthermore, absence of Notch1 decreased pAkt-S473 implying reduced mTORC2 activity. Our data implicate the importance of Notch1 in T cell function. Future studies are aimed to delineate the role of non-canonical Notch1 signaling in T cells and how it may influence molecular pathways downstream of the TCR.