Increasing evidence suggests that ionizing radiation therapy (RT) in combination with checkpoint immunotherapy is highly effective in treating a subset of cancers. To better understand the limited responses to this combination we analysed the genetic, microenvironmental, and immune factors in tumours derived from a transgenic breast cancer model. We identified two tumours with similar growth characteristics but different RT responses primarily due to an antitumour immune response. The combination of RT and checkpoint immunotherapy resulted in cures in the responsive but not the unresponsive tumours. Profiling the tumours revealed that the Axl receptor tyrosine kinase is overexpressed in the unresponsive tumours, and Axl knockout resulted in slower growth and increased radiosensitivity. These changes were associated with a CD8+ T-cell response, which was improved in combination with checkpoint immunotherapy. These results suggest a novel role for Axl in suppressing antigen presentation through MHCI, and enhancing cytokine release, which promotes a suppressive myeloid microenvironment.

Peritoneal metastases are the leading cause of morbidity and mortality in high-grade serous ovarian cancer (HGSOC). Accumulating evidence suggests that mesothelial cells are an important component of the metastatic microenvironment in HGSOC. However, the mechanisms by which mesothelial cells promote metastasis are unclear. Here we report that the HGSOC tumor-mesothelial niche was hypoxic and hypoxic signaling enhanced collagen I deposition by mesothelial cells. Specifically, hypoxic signaling increased expression of lysyl oxidase (LOX) in mesothelial and ovarian cancer cells to promote collagen crosslinking and tumor cell invasion. The mesothelial niche was enriched with fibrillar collagen in human and murine omental metastases. Pharmacologic inhibition of LOX reduced tumor burden and collagen remodeling in murine omental metastases. These findings highlight an important role for hypoxia and mesothelial cells in the modification of the extracellular matrix and tumor invasion in HGSOC.

Tissue hypoxia can occur in physiological and pathological conditions. When O2 availability decreases, the transcription factor hypoxia-inducible factor (HIF)-1α is stabilized and regulates cellular adaptation to hypoxia. The objective of this study was to test whether HIF-1α regulates T cell fate and to define the molecular mechanisms of this control. Our data demonstrate that Th1 cells lose their capacity to produce IFN-γ when cultured under hypoxia. HIF-1α−/− Th1 cells were insensitive to hypoxia, underlining a critical role for HIF-1α. Our results point to a role for IL-10, as suggested by the increased IL-10 expression at low O2 levels and the unchanged IFN-γ production by IL-10–deficient Th1 cells stimulated in hypoxic conditions. Accordingly, STAT3 phosphorylation is increased in Th1 cells under hypoxia, leading to enhanced HIF-1α transcription, which, in turn, may inhibit suppressor of cytokine signaling 3 transcription. This positive-feedback loop reinforces STAT3 activation and downregulates Th1 responses that may cause collateral damage to the host.

The ectonucleotidases CD39 and CD73 sequentially degrade the extracellular ATP pool and release immunosuppressive adenosine, thereby regulating inflammatory responses. This control is likely to be critical in the gastrointestinal tract where high levels of ATP are released in particular by commensal bacteria. The aim of this study was therefore to evaluate the involvement of the adenosinergic regulation in the intestine of mice in steady-state conditions and on acute infection with Toxoplasma gondii. We show that both conventional (Tconv) and regulatory (Treg) CD4(+) T lymphocytes express CD39 and CD73 in the intestine of naive mice. CD73 expression was downregulated during acute infection with T. gondii, leading to impaired capacity to produce adenosine. Interestingly, the expression of adenosine receptors was maintained and treatment with receptor agonists limited immunopathology and dysbiosis, suggesting that the activation of adenosine receptors may constitute an efficient approach to control intestinal inflammation associated with decreased ectonucleotidase expression.

IntroductionThe availability of oxygen in the atmosphere provided protozoans with the ability to meet higher energy demands required for the development of complex multicellular organisms. Consequently, oxygen-dependent cells developed mechanisms to sense and adapt to changes in oxygen levels [1]. In mammals the respiratory, circulatory and nervous system work together to ensure that oxygen levels are precisely maintained, because excess or deficiency of oxygen can lead to cell death and tissue damage [2].The level of oxygen in mammalian tissues is much lower than that in the atmosphere (oxygen partial tension (PO 2 ) = 21.2 kPa). Tissue oxygen can range between 13 kPa in the pulmonary vein down to 2.7 kPa at the interstitial spaces. Intracellular oxygen ranges from 1.3 to 2.7 kPa [3]. In this context, hypoxia is defined as a deficiency in tissue or blood oxygen as compared to physiological levels. HIF-1α has been shown to be stabilized at a PO 2 lower than 2kPa [4].One molecule that plays an important role in mediating adaptive functions to oxygen levels is the hypoxia inducible transcription factor (HIF)-1. HIF-1 is a transcription factor composed of Correspondence: Prof. Muriel Moser e-mail: mmoser@ulb.ac.be two subunits, HIF-1α and HIF-1β (or aryl hydrocarbon receptor nuclear translocator). The beta subunit of HIF is constitutively expressed in mammalian cells, whereas, in the presence of oxygen, the alpha subunit is hydroxylated by proline hydroxylases (PHDs), ubiquitinylated by the von Hippel-Lindau (VHL) protein (E3 ubiquitin ligase), and degraded by the proteasome pathway. Hypoxia leads to stabilization of HIF-1α (through the inhibition of oxygen-dependent PHDs), inducing the transcription of multiple genes unique for each cell type, among them erythropoietin, vascular endothelial growth factor, glycolytic enzymes, etc. (for review see [2]). The transcription function of HIF-1 is also negatively regulated in the presence of oxygen by the dioxygenase factor inhibiting HIF that hydroxylates HIF-1α, thereby preventing the recruitment of the transcriptional machinery [5]. Besides the ubiquitously expressed HIF-1, two paralogs exist in mammals that are also regulated by oxygen: HIF-2 is also widely expressed and plays important roles in erythropoiesis, vascularization, pulmonary development and, more specifically, in iron absorption [6,7]; HIF-3 has been shown to negatively regulates HIF-1 and HIF-2 by competing for the binding on HREs of target genes [8]. A recent report, however, clearly demonstrates that zebrafish Hif-3α has strong transactivation activity and that human HIF-3α isoforms upregulate similar target genes in human cells [9]. Finally, it should be noted that, in addition to hypoxia, other mechanisms [12,13]. Although HIFs appear as major sensors of hypoxia, other transcriptional cascades are triggered at low oxygen tension. In particular, the NF-κB pathway was shown to induce Hif-1α and to be activated by hypoxia through p65 and p50 stabilization and nuclear translocation, linking both pathway...

Monocytes play a major role in the defense against pathogens. They are rapidly mobilized to inflamed sites where they exert both proinflammatory and regulatory effector functions. It is still poorly understood how this dynamic and exceptionally plastic system is controlled at the molecular level. Herein, we evaluated the differentiation process that occurs in Ly6Chi monocytes during oral infection by Toxoplasma gondii. Flow cytometry and single-cell analysis revealed distinct activation status and gene expression profiles in the bone marrow, the spleen and the lamina propria of infected mice. We provide further evidence that acquisition of effector functions, such as the capacity to produce interleukin-27, is accompanied by distinct waves of epigenetic programming, highlighting a role for STAT1/IRF1 in the bone marrow and AP-1/NF-κB in the periphery. This work broadens our understanding of the molecular events that occur in vivo during monocyte differentiation in response to inflammatory cues.

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Purpose: To investigate how induced tumor heterogeneity influences immune responses to radiotherapy with different proportions of mixed immune-responsive and unresponsive tumor cells in a triple-negative breast cancer model. It is hypothesized that studying the immune environment of mixed tumors and responses to radiotherapy could nominate immune active therapies to enhance immune responses after radiotherapy.Experimental Design: Evaluate efficacy and immune responses generated by radiotherapy in tumors with different proportions of immunologically responsive and unresponsive tumor cells. Then study the cellular responses and transcriptomic differences between the tumors to nominate immunotherapy combinations with radiotherapy and evaluate the combination.Results: The addition of the responsive cells to unresponsive tumors led to a greater than expected therapeutic response to radiotherapy with both innate and adaptive immune components.There was a distinct change in myeloid cells, greater inflammatory macrophage activity, and enhanced antigen presentation with responsive cells after radiotherapy. Because differences in matrix components, cell adhesion biology, and innate immune signaling correlated with myeloid cell response and phenotype, we hypothesized that radiotherapy combined with CD40 agonist antibody would sensitize unresponsive tumors. The combination therapy resulted in improved innate and adaptive immune response. Importantly, CD40 treatment increased tumor response to radiotherapy and protected against metastatic spread in a metastatic model.Conclusions: These data combined with transcriptomics from human patients support radiotherapy and myeloid cell targeting for immunologically cold tumors. The established study model presents opportunities to investigate the complex overlapping biologic mechanisms that limit immunotherapy and to implement radiotherapy with different immunotherapy combinations.