Notch activation, which is associated with basal-like breast cancer (BLBC), normally directs tissue patterning, suggesting that it may shape the tumor microenvironment. Here, we show that Notch in tumor cells regulates the expression of two powerful proinflammatory cytokines, IL1β and CCL2, and the recruitment of tumor-associated macrophages (TAM). Notch also regulates TGFβ-mediated activation of tumor cells by TAMs, closing a Notch-dependent paracrine signaling loop between these two cell types. We use a mouse model in which Notch can be regulated in spontaneous mammary carcinoma to confirm that IL1β and CCL2 production, and macrophage recruitment are Notch-dependent. In human disease, expression array analyses demonstrate a striking association between Notch activation, IL1β and CCL2 production, macrophage infiltration, and BLBC. These findings place Notch at the nexus of a vicious cycle of macrophage infiltration and amplified cytokine secretion and provide immunotherapeutic opportunities in BLBC. BLBC is aggressive and has an unmet need for effective targeted treatment. Our data highlight immunotherapeutic opportunities in Notch-activated BLBC. Effective IL1β and CCL2 antagonists are currently in clinical review to treat benign inflammatory disease, and their transition to the cancer clinic could have a rapid impact. .

The transcription factor AhR modulates immunity at multiple levels. Here we report phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of interleukin 10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in murine systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice and an increased AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

These results provide evidence that MSC-derived MVs can inhibit in vitro a proinflammatory response to an islet antigenic stimulus in type 1 diabetes. The action of MVs involves PGE2 and TGF-β signalling pathways and IL-10 secretion, suggesting a switch to an anti-inflammatory response of T cells.

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General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor–binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

Angiogenesis is considered a hallmark of multiple myeloma (MM) progression. In the present study, we evaluated the morphological and functional features of endothelial cells (ECs) derived from bone marrow (BM) of patients affected by MM (MMECs). We found that MMECs compared with normal BM ECs (BMECs) showed increased expression of syndecan-1. Silencing of syndecan-1 expression by RNA interference technique decreased in vitro EC survival, proliferation and organization in capillary-like structures. In vivo, in severe combined immunodeficient mice, syndecan-1 silencing inhibited MMEC organization into patent vessels. When overexpressed in human umbilical vein ECs and BMECs, syndecan-1 induced in vitro and in vivo angiogenic effects. Flow-cytometric analysis of MMECs silenced for syndecan-1 expression indicated a decreased membrane expression of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2). Immunoprecipitation and confocal analysis showed colocalization of VEGFR-2 with syndecan-1. Absence of nuclear translocation of VEGFR-2 in syndecan-1-knockdown cells together with the shift from perinuclear localization to recycling compartments suggest a role of syndecan-1 in modulation of VEGFR-2 localization. This correlated with an in vitro decreased VEGF-induced invasion and motility. These results suggest that syndecan-1 may contribute to the highly angiogenic phenotype of MMECs by promoting EC proliferation, survival and modulating VEGF–VEGFR-2 signalling.

Disruption of the CD40-CD154 interaction was found to be effective in the prevention and treatment of several immune-mediated diseases. The antibody-based strategy of inhibition was in humans limited by platelet activation leading to thrombotic effects. Other strategies different from antibody technology may be useful to create tools to interfere with CD40-CD154 pathway. In the present study, we selected and characterized from a phage display library, cyclic hepta-peptides specific for human CD154 through biopanning against plate-immobilized recombinant hCD154-muCD8. Nine phage clones were selected for the ability to bind CD154 expressed on the surface of J558L cells transfected with human CD154. From the nine selected phage clones, we obtained seven different amino acidic sequences, and the corresponding hepta-peptides rendered cyclic by two cysteines were synthesized. All the peptides specifically bound CD154 expressed on J558L. However, only the peptide 4.10 (CLPTRHMAC) was found to recognize the active binding site of CD154, as it competed with the blocking anti-CD154 antibody. When changes in the amino acid composition were introduced in the sequence of 4.10 peptide, the binding to CD154 was abrogated, suggesting that the amino acid sequence was critical for its specificity. This peptide was found to inhibit the CD40-CD154 interaction, preventing CD40-dependent activation of B lymphocytes in vitro as it was able, as the blocking anti-human CD154 mAb, to prevent the expression of CD80 and CD86 costimulatory molecules and switching of Ig isotype induced by CD154. Moreover, the peptide 4.10 inhibited the in vitro endothelial cell motility and organization into capillary-like structures, and the in vivo angiogenesis of human umbilical cord-derived endothelial cells implanted in Matrigel in severe combined immunodeficiency mice. In vitro studies on platelet activation demonstrated that the 4.10 peptide, at variance of the anti-CD154 mAb, was unable to prime human platelet activation and aggregation. In conclusion, we identify a cyclic hepta-peptide able to displace the binding of human CD154 to CD40 expressed on cell surface and to abrogate some biological effects related to the CD40 stimulation, such as B cell activation and endothelial triggered angiogenesis.

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