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The tumor microenvironment profoundly influences the behavior of recruited leukocytes and tissue-resident immune cells. These immune cells, which inherently have environmentally driven plasticity necessary for their roles in tissue homeostasis, dynamically interact with tumor cells and the tumor stroma and play critical roles in determining the course of disease. Among these immune cells, neutrophils were once considered much more static within the tumor microenvironment; however, some of these earlier assumptions were the product of the notorious difficulty in manipulating neutrophils in vitro. Technological advances that allow us to study neutrophils in context are now revealing the true roles of neutrophils in the tumor microenvironment. Here we discuss recent data generated by some of these tools and how these data might be synthesized into more elegant ways of targeting these powerful and abundant effector immune cells in the clinic.

Neutrophils are implicated in almost every stage of oncogenesis and paradoxically display anti- and pro-tumor properties. Accumulating evidence indicates that neutrophils display diversity in their phenotype resulting from functional plasticity and/or changes to granulopoiesis. In cancer, neutrophils at a range of maturation stages can be identified in the blood and tissues (i.e., outside of their developmental niche). The functional capacity of neutrophils at different states of maturation is poorly understood resulting from challenges in their isolation, identification, and investigation. Thus, the impact of neutrophil maturity on cancer progression and therapy remains enigmatic. In this review, we discuss the identification, prevalence, and function of immature and mature neutrophils in cancer and the potential impact of this on tumor progression and cancer therapy.

Ly6C – monocytes patrol blood vessels by crawling along uninflamed vasculature. Hedrick and colleagues show that the transcription factor NR4A1 is required for the development and survival of Ly6C – monocytes. The transcription factors that regulate differentiation into the monocyte subset in bone marrow have not yet been identified. Here we found that the orphan nuclear receptor NR4A1 controlled the differentiation of Ly6C − monocytes. Ly6C − monocytes, which function in a surveillance role in circulation, were absent from Nr4a1 −/− mice. Normal numbers of myeloid progenitor cells were present in Nr4a1 −/− mice, which indicated that the defect occurred during later stages of monocyte development. The defect was cell intrinsic, as wild-type mice that received bone marrow from Nr4a1 −/− mice developed fewer patrolling monocytes than did recipients of wild-type bone marrow. The Ly6C − monocytes remaining in the bone marrow of Nr4a1 −/− mice were arrested in S phase of the cell cycle and underwent apoptosis. Thus, NR4A1 functions as a master regulator of the differentiation and survival of 'patrolling' Ly6C − monocytes.

Aberrant expansion of KRT5 + basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5 + cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5 + cells migrate within the fibrotic lung, navigating regional differences in collagen topography. In vitro, KRT5 + cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry- based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5 + cell migration in vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5 + cell behaviour and function contributing to remodelling events in the fibrotic niche. Idiopathic pulmonary fibrosis has been associated with aberrant expansion of KRT5-expressing basal cells. Here the authors show how changes in the ECM glycoprotein SPARC restrict the movement of KRT5+ cells, affecting their retention within fibrotic tissue.

Mutant p53s (mutp53) increase cancer invasiveness by upregulating Rab-coupling protein (RCP) and diacylglycerol kinase-α (DGKα)-dependent endosomal recycling. Here we report that mutp53-expressing tumour cells produce exosomes that mediate intercellular transfer of mutp53’s invasive/migratory gain-of-function by increasing RCP-dependent integrin recycling in other tumour cells. This process depends on mutp53’s ability to control production of the sialomucin, podocalyxin, and activity of the Rab35 GTPase which interacts with podocalyxin to influence its sorting to exosomes. Exosomes from mutp53-expressing tumour cells also influence integrin trafficking in normal fibroblasts to promote deposition of a highly pro-invasive extracellular matrix (ECM), and quantitative second harmonic generation microscopy indicates that this ECM displays a characteristic orthogonal morphology. The lung ECM of mice possessing mutp53-driven pancreatic adenocarcinomas also displays increased orthogonal characteristics which precedes metastasis, indicating that mutp53 can influence the microenvironment in distant organs in a way that can support invasive growth. Some p53 mutants promote invasive migration of cancer cells and metastasis of tumours in vivo. However the key mechanistic details behind these phenomena remain unclear. Here the authors propose a non-cell autonomous mechanism involving fibroblasts, whereby mutant p53-expressing cancer cells activate an exosome-mediated mechanism that influences integrin recycling in fibroblasts, thus influencing extracellular matrix remodelling to favour cancer cell invasion and migration.

In this review, we discuss intravital microscopy of immune cells, starting from its historic origins to current applications in diverse organs. It is clear from a quantitative review of the literature that intravital microscopy is a key tool in both historic and contemporary immunological research, providing unique advances in our understanding of immune responses. We have chosen to focus this review on how intravital microscopy methodologies are used to image specific organs or systems and we present recent descriptions of fundamental immunological processes that could not have been achieved by other methods. The following target organs/systems are discussed in more detail: cremaster muscle, skin (ear and dorsal skin fold chamber), lymph node, liver, lung, mesenteric vessels, carotid artery, bone marrow, brain, spleen, foetus and lastly vessels of the knee joint. Since microscopes were first invented, scientists have constantly been drawn to these marvellous machines. This is because microscopes provide their user with the ability to view first hand minuscule objects and processes they have often dedicated their entire careers to studying. It is this ability to view biology at work in both space and time, from a single molecule to an entire organism that makes imaging such a powerful tool. In this Special Feature, we have compiled a series of articles that discuss the history of microscopes and imaging modalities. We look at how current platforms have influenced basic research of immunology and cell biology as well as their use in the clinic to diagnose and treat disease. We also discuss how future developments in technology will open avenues for an even deeper understanding of fundamental principles in biology and the challenges associated with handling vast amounts of data generated by technology that gives such a high level of deta iled information. Immunology & Cell Biology thanks the coordinator of this Special Feature ‐ Edwin Hawkins ‐ for his planning and input.

BackgroundMetastatic breast cancer is a leading cause of cancer-related death in women worldwide. Infusion of natural killer (NK) cells is an emerging immunotherapy for such malignant tumors, although elimination of the immunosuppressive tumor environment is required to improve its efficacy. The effects of this “metastatic” tumor environment on NK cells, however, remain largely unknown. Previous studies, including our own, have demonstrated that metastasis-associated macrophages (MAMs) are one of the most abundant immune cell types in the metastatic tumor niche in mouse models of metastatic breast cancer. We thus investigated the effects of MAMs on antitumor functions of NK cells in the metastatic tumor microenvironment.MethodsMAMs were isolated from the tumor-bearing lung of C57BL/6 mice intravenously injected with E0771-LG mouse mammary tumor cells. The effects of MAMs on NK cell cytotoxicity towards E0771-LG cells were evaluated in vitro by real-time fluorescence microscopy. The effects of MAM depletion on NK cell activation, maturation, and accumulation in the metastatic lung were evaluated by flow cytometry (CD69, CD11b, CD27) and in situ hybridization (Ncr1) using colony-stimulating factor 1 (CSF-1) receptor conditional knockout (Csf1r-cKO) mice. Finally, metastatic tumor loads in the chest region of mice were determined by bioluminescence imaging in order to evaluate the effect of MAM depletion on therapeutic efficacy of endogenous and adoptively transferred NK cells in suppressing metastatic tumor growth.ResultsMAMs isolated from the metastatic lung suppressed NK cell-induced tumor cell apoptosis in vitro via membrane-bound transforming growth factor β (TGF-β) dependent mechanisms. In the tumor-challenged mice, depletion of MAMs increased the percentage of activated (CD69+) and mature (CD11b+CD27–) NK cells and the number of Ncr1+ NK cells as well as NK cell-mediated tumor rejection in the metastatic site. Moreover, MAM depletion or TGF-β receptor antagonist treatment significantly enhanced the therapeutic efficacy of NK cell infusion in suppressing early metastatic tumor outgrowth.ConclusionThis study demonstrates that MAMs are a main negative regulator of NK cell function within the metastatic tumor niche, and MAM targeting is an attractive strategy to improve NK cell-based immunotherapy for metastatic breast cancer.

The rapid response of neutrophils throughout the body to a systemic challenge is a critical first step in resolution of bacterial infection such as Escherichia coli ( E. coli ). Here we delineated the dynamics of this response, revealing novel insights into the molecular mechanisms using lung and spleen intravital microscopy and 3D ex vivo culture of living precision cut splenic slices in combination with fluorescent labelling of endogenous leukocytes. Within seconds after challenge, intravascular marginated neutrophils and lung endothelial cells (ECs) work cooperatively to capture pathogens. Neutrophils retained on lung ECs slow their velocity and aggregate in clusters that enlarge as circulating neutrophils carrying E. coli stop within the microvasculature. The absolute number of splenic neutrophils does not change following challenge; however, neutrophils increase their velocity, migrate to the marginal zone (MZ) and form clusters. Irrespective of their location all neutrophils capturing heat-inactivated E. coli take on an activated phenotype showing increasing surface CD11b. At a molecular level we show that neutralization of ICAM-1 results in splenic neutrophil redistribution to the MZ under homeostasis. Following challenge, splenic levels of CXCL12 and ICAM-1 are reduced allowing neutrophils to migrate to the MZ in a CD29-integrin dependent manner, where the enlargement of splenic neutrophil clusters is CXCR2-CXCL2 dependent. We show directly molecular mechanisms that allow tissue resident neutrophils to provide the first lines of antimicrobial defense by capturing circulating E. coli and forming clusters both in the microvessels of the lung and in the parenchyma of the spleen.

Lung disease is regularly reported in human filarial infections but the molecular pathogenesis of pulmonary filariasis is poorly understood. We used Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity responsible for pleural inflammation, to model responses to human filarial infections and probe the mechanisms. Wild-type and Th2-deficient mice (ΔdblGata1 and Il-4receptor(r)a-/-/IL-5-/-) were infected with L. sigmodontis. Survival and growth of adult filariae and prevalence and density of microfilariae were evaluated. Cells and cytokines in the pleural cavity and bronchoalveolar space were characterized by imaging, flow cytometry and ELISA. Inflammatory pathways were evaluated by transcriptomic microarrays and lungs were isolated and analyzed for histopathological signatures. 40% of WT mice were amicrofilaremic whereas almost all mutant mice display blood microfilaremia. Microfilariae induced pleural, bronchoalveolar and lung-tissue inflammation associated with an increase in bronchoalveolar eosinophils and perivascular macrophages, production of mucus, visceral pleura alterations and fibrosis. Inflammation and pathology were decreased in Th2-deficient mice. An IL-4R-dependent increase of CD169 was observed on pleural and bronchoalveolar macrophages in microfilaremic mice. CD169+ tissue-resident macrophages were identified in the lungs with specific localizations. Strikingly, CD169+ macrophages increased significantly in the perivascular area in microfilaremic mice. These data describe lung inflammation and pathology in chronic filariasis and emphasize the role of Th2 responses according to the presence of microfilariae. It is also the first report implicating CD169+ lung macrophages in response to a Nematode infection.

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8 ⁺ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c ⁺ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c ⁺ MHCII ʰⁱ CD8α ⁿᵉᵍ epithelial cell adhesion molecule (EpCAM ⁿᵉᵍ) CD11b ⁺ langerin (Lang; CD207) ⁿᵉᵍ DCs, but neither Langerhans cells nor Lang ⁺ DCs were required for CD8 ⁺ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8 ⁺ T-cell priming by live rAdHu5 MAs.