Lung metastasis is the lethal determinant in many cancers1,2 and a number of lines of evidence point to monocytes and macrophages having key roles in its development3–5. Yet little is known about the immediate fate of incoming tumour cells as they colonize this tissue and even less known about how they make first contact with the immune system. Primary tumours liberate circulating tumour cells (CTCs) into the blood and we have developed a stable intravital two-photon lung imaging model in mice6 for direct observation of the arrival of CTCs and subsequent host interaction. Here we show dynamic generation of tumour microparticles in shear flow in the capillaries within minutes of CTC entry. Rather than dispersing under flow, many of these microparticles remain attached to the lung vasculature or independently migrate along the inner walls of vessels. Using fluorescent lineage reporters and flow cytometry, we observed ‘waves’ of distinct myeloid cell subsets that load differentially and sequentially with this CTC-derived material. Many of these tumour-ingesting myeloid cells collectively accumulated in the lung interstitium along with the successful metastatic cells and, as previously understood, promote the development of successful metastases from surviving tumour cells3. Although the numbers of these cells rise globally in the lung with metastatic exposure and ingesting myeloid cells undergo phenotypic changes associated with microparticle ingestion, a consistently sparse population of resident conventional dendritic cells, among the last cells to interact with CTCs, confer antimetastatic protection. This work reveals that CTC fragmentation generates immune-interacting intermediates, and defines a competitive relationship between phagocyte populations for tumour loading during metastatic cell seeding.

Major histocompatibility complex (MHC) class I molecules associate with a variety of peptide ligands during biosynthesis and present these ligands on the cell surface for recognition by cytotoxic T cells. We have designed conditional MHC ligands that form stable complexes with MHC molecules but degrade on command, by exposure to a defined photostimulus. 'Empty MHC molecules' generated in this manner can be loaded with arrays of peptide ligands to determine MHC binding properties and to monitor antigen-specific T-cell responses in a high-throughput manner. We document the value of this approach by identifying cytotoxic T-cell epitopes within the H5N1 influenza A/Vietnam/1194/04 genome.

• Neutrophil-platelet aggregates are dynamically formed in the lung in response to injury and are regulated by aspirin-triggered lipoxin.• The therapeutic effect of aspirin in acute lung injury is in large part mediated by the production of pro-resolving lipid mediators.Evidence is emerging that platelets are major contributors to innate immune responses in conditions such as acute lung injury (ALI). Platelets form heterotypic aggregates with neutrophils, and we hypothesized that lipoxin mediators regulate formation of neutrophilplatelet aggregates (NPA) and that NPA significantly contribute to ALI. Lipopolysaccharide (LPS)-induced lung injury was accompanied by platelet sequestration, activation, intra-alveolar accumulation, and NPA formation within both blood and alveolar compartments. Using lung intravital microscopy, we observed the dynamic formation of NPA during physiologic conditions, which sharply increased with ALI. Aspirin (ASA) treatment significantly reduced lung platelet sequestration and activation, NPA formation, and lung injury. ASA treatment increased levels of ASA-triggered lipoxin (ATL; 15-epi-lipoxin A 4 ), and blocking the lipoxin A 4 receptor (ALX) with a peptide antagonist (Boc2) or using ALX knockouts (Fpr2/3 2/2 ) reversed this protection. LPS increased NPA formation in vitro, which was reduced by ATL, and engagement of ALX by ATL on both neutrophils and platelets was necessary to prevent aggregation. In a model of transfusion-related acute lung injury (TRALI), Boc2 also reversed ASA protection, and treatment with ATL in both LPS and TRALI models protected from ALI. We conclude that ATL regulates neutrophil-platelet aggregation and that platelet-neutrophil interactions are a therapeutic target in lung injury. (Blood. 2014;124(17):2625-2634

Induction of immunity after DNA vaccination is generally considered a slow process. Here we show that DNA delivery to the skin results in a highly transient pulse of antigen expression. Based on this information, we developed a new rapid and potent intradermal DNA vaccination method. By short-interval intradermal DNA delivery, robust T-cell responses, of a magnitude sufficient to reject established subcutaneous tumors, are generated within 12 d. Moreover, this vaccination strategy confers protecting humoral immunity against influenza A infection within 2 weeks after the start of vaccination. The strength and speed of this newly developed strategy will be beneficial in situations in which immunity is required in the shortest possible time.