Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (T1) activity and reduced immune cytotoxicity or increased TGFβ levels predict adverse outcomes in patients with colorectal cancer. Here we analyse the interplay between genetic alterations and the tumour microenvironment by crossing mice bearing conditional alleles of four main colorectal cancer mutations in intestinal stem cells. Quadruple-mutant mice developed metastatic intestinal tumours that display key hallmarks of human microsatellite-stable colorectal cancers, including low mutational burden, T-cell exclusion and TGFβ-activated stroma. Inhibition of the PD-1-PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFβ unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFβ signalling rendered tumours susceptible to anti-PD-1-PD-L1 therapy. Our data show that increased TGFβ in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the T1-effector phenotype. Immunotherapies directed against TGFβ signalling may therefore have broad applications in treating patients with advanced colorectal cancer.

The impact of the nutritional status during foetal life in the overall health of adults has been recognised1. However dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs (SLOs) occurs during embryogenesis and is considered to be developmentally programmed2,3. SLO formation dependents on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells2,3,4,5. Here we show that foetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero pre-setting the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi differentiation was controlled by maternal retinoid intake and foetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, while RA receptors directly regulated the Rorc locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and infection at mucosal barriers1. ILC3 development has been considered to be programmed1. Nevertheless, how ILC3 perceive, integrate and respond to local environmental signals remains unclear. Here we show that ILC3 sense their environment and control gut defence as part of a novel glial-ILC3-epithelial cell unit orchestrated by neurotrophic factors. We found that enteric ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led to decreased innate interleukin-22 (IL-22), impaired epithelial reactivity, dysbiosis and increased susceptibility to bowel inflammation and infection. Neurotrophic factors directly controlled innate II22, downstream of p38 MAPK/ERK-AKT cascade and STAT3 activation. Strikingly, ILC3 were adjacent to neurotrophic factor expressing glial cells that exhibited stellate-shaped projections into ILC3 aggregates. Glial cells sensed microenvironmental cues in a MYD88 dependent manner to control neurotrophic factors and innate IL-22. Accordingly, glial-intrinsic Myd88 deletion led to impaired ILC3-derived IL-22 and pronounced propensity to gut inflammation and infection. Our work sheds light into a novel multi-tissue defence unit, revealing glial cells as central hubs of neuron and innate immune regulation via neurotrophic factor signals.

SummaryAntigen-specific cognate interaction of T lymphocytes with antigen-presenting cells (APCs) drives major morphological and functional changes in T cells, including actin rearrangements at the immune synapse (IS) formed at the cell-cell contact area. Here we show, using cell lines as well as primary cells, that clathrin, a protein involved in endocytic processes, drives actin accumulation at the IS. Clathrin is recruited towards the IS with parallel kinetics to that of actin. Knockdown of clathrin prevents accumulation of actin and proteins involved in actin polymerization, such as dynamin-2, the Arp2/3 complex and CD2AP at the IS. The clathrin pool involved in actin accumulation at the IS is linked to multivesicular bodies that polarize to the cell-cell contact zone, but not to plasma membrane or Golgi complex. These data underscore the role of clathrin as a platform for the recruitment of proteins that promote actin polymerization at the interface of T cells and APCs.

The role of nitric oxide (NO) in T cells remains controversial, and the origin and localization of endogenous NO and whether it regulates lymphocyte activation are unclear. We show here that, within minutes of binding to antigen, T cells produce NO via endothelial nitric oxide synthase (eNOS). This process required increased intracellular Ca2+ and phosphoinositide3-kinase activity. By using an eNOS-green fluorescent fusion protein and fluorescent probes to detect NO, we show that eNOS translocates with the Golgi apparatus to the immune synapse of T helper cells engaged with antigen-presenting cells (APC), where it was fully activated. Overexpression of eNOS prevented the central coalescence of CD3 at the T cell-APC contact site, which was accompanied by increased phosphorylation of CD3zeta chain, ZAP-70, and extracellular signal-regulated kinases and increased IFN-gamma synthesis, but reduced production of IL-2. Therefore, eNOS-derived NO selectively potentiates T cell receptor signaling to antigen at the immunological synapse.