These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

Cancer stem cells drive tumor formation and metastasis, but how they acquire metastatic traits is not well understood. Here, we show that all colorectal cancer stem cells (CR-CSCs) express CD44v6, which is required for their migration and generation of metastatic tumors. CD44v6 expression is low in primary tumors but demarcated clonogenic CR-CSC populations. Cytokines hepatocyte growth factor (HGF), osteopontin (OPN), and stromal-derived factor 1α (SDF-1), secreted from tumor associated cells, increase CD44v6 expression in CR-CSCs by activating the Wnt/β-catenin pathway, which promotes migration and metastasis. CD44v6(-) progenitor cells do not give rise to metastatic lesions but, when treated with cytokines, acquire CD44v6 expression and metastatic capacity. Importantly, phosphatidylinositol 3-kinase (PI3K) inhibition selectively killed CD44v6 CR-CSCs and reduced metastatic growth. In patient cohorts, low levels of CD44v6 predict increased probability of survival. Thus, the metastatic process in colorectal cancer is initiated by CSCs through the expression of CD44v6, which is both a functional biomarker and therapeutic target.

Cells of the monocyte-macrophage lineage play a central role in the orchestration and resolution of inflammation. Plasticity is a hallmark of mononuclear phagocytes, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic or M1 and alternative or M2. NF-B is a key regulator of inflammation and resolution, and its activation is subject to multiple levels of regulation, including inhibitory, which finely tune macrophage functions. Here we identify the p50 subunit of NF-B as a key regulator of M2-driven inflammatory reactions in vitro and in vivo. p50 NF-B inhibits NF-B-driven, M1-polarizing, IFN-␤ production. Accordingly, p50-deficient mice show exacerbated M1-driven inflammation and defective capacity to mount allergy and helminth-driven M2-polarized inflammatory reactions. Thus, NF-B p50 is a key component in the orchestration of M2-driven inflammatory reactions.

The increasing evidence that ;D T cells have potent antitumor activity suggests their value in immunotherapy, particularly in areas of unmet need such as metastatic carcinoma. To this end, we initiated a phase I clinical trial in metastatic hormonerefractory prostate cancer to examine the feasibility and consequences of using the ;D T-cell agonist zoledronate, either alone or in combination with low-dose interleukin 2 (IL-2), to activate peripheral blood ;D cells. Nine patients were enlisted to each arm. Neither treatment showed appreciable toxicity. Most patients were treated with zoledronate + IL-2, but conversely only two treated with zoledronate displayed a significant longterm shift of peripheral ;D cells toward an activated effectormemory-like state (T EM ), producing IFN-; and perforin. These patients also maintained serum levels of tumor necrosis factorrelated apoptosis inducing ligand (TRAIL), consistent with a parallel microarray analysis showing that TRAIL is produced by ;D cells activated via the T-cell receptor and IL-2. Moreover, the numbers of T EM ;D cells showed a statistically significant correlation with declining prostate-specific antigen levels and objective clinical outcomes that comprised three instances of partial remission and five of stable disease. By contrast, most patients treated only with zoledronate failed to sustain either ;D cell numbers or serum TRAIL, and showed progressive clinical deterioration. Thus, zoledronate + IL-2 represents a novel, safe, and feasible approach to induce immunologic and clinical responses in patients with metastatic carcinomas, potentially providing a substantially increased window for specific approaches to be administered. Moreover, ;D cell phenotypes and possibly serum TRAIL may constitute novel biomarkers of prognosis upon therapy with zoledronate + IL-2 in metastatic carcinoma. [Cancer Res 2007;67(15):7450-7]

Vδ2 T lymphocytes recognize nonpeptidic antigens without presentation by MHC molecules and mount both immediate effector functions and memory responses after microbial infection. However, how Vδ2 T cells mediate different facets of a memory response remains unknown. Here, we show that the expression of CD45RA and CD27 antigens defines four subsets of human Vδ2 T cells with distinctive compartmentalization routes. Naive CD45RA+CD27+ and memory CD45RA−CD27+ cells express lymph node homing receptors, abound in lymph nodes, and lack immediate effector functions. Conversely, memory CD45RA−CD27− and terminally differentiated CD45RA+CD27− cells, which express receptors for homing to inflamed tissues, are poorly represented in the lymph nodes while abounding at sites of inflammation, and display immediate effector functions. These observations and additional in vitro experiments indicate a lineage differentiation pattern for human Vδ2 T cells that generates naive cells circulating in lymph nodes, effector/memory cells patrolling the blood, and terminally differentiated effector cells residing in inflamed tissues.

BackgroundCD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes.MethodsExosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments.ResultsExosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.ConclusionsOur data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0426-x) contains supplementary material, which is available to authorized users.

IntroductionIL-17 is a cytokine that induces mobilization and activation of neutrophils and triggers the production of proinflammatory cytokines and chemokines by a broad range of cellular targets. 1 It is predominantly produced by ␣␤ T cells, but also by natural killer (NK) T cells, 2 ␥␦ T cells, 3 and other non-T cells, such as macrophages and neutrophils. 4,5 Differentiation of CD4 T cells producing IL-17 (Th17) is initiated in naive Th cells by antigen-specific stimulation in the presence of the polarizing cytokines IL-1␤, TGF-␤, and IL-6 (and autocrine IL-21), which induce the expression of IL-23 receptor (IL-23R), the chemokine receptor CCR6, and the Th17-specifying transcription factor ROR␥t, which is necessary and sufficient for induction of IL-17. 1,6 In mice, ␥␦ T cells represent an innate source of IL-17 and precede the development of the adaptive Th17-cell response. For instance, during Mycobacterium tuberculosis and Escherichia coli infection, ␥␦ T cells are the primary source of 8 and their depletion causes decreased IL-17 production and neutrophil infiltration into the peritoneal cavity. 8 In Listeria monocytogenes infection, ␥␦ T cells producing IL-17 enhance the antibacterial activity of nonphagocytic cells, which correlates with the induction of ␤-defensin gene expression. 9 These results indicate a novel IL-17-dependent protective mechanism of ␥␦ T cells that acts against intracellular bacterial infections in the mouse. The authors of several recent studies have provided data on the differentiation, phenotype, and functions of murine ␥␦ T cells producing IL-17 [10][11][12][13][14][15] and have demonstrated that signals through the ␥␦ TCR are not required for IL-17 production; instead, this process seems to be controlled by innate cytokines produced by accessory cells such as macrophages or dendritic cells (DCs). 11,15,16 Conversely, few groups have investigated IL-17 production by human ␥␦ T cells.Most human peripheral blood ␥␦ T cells express a TCR consisting of the V␥9 and the V␦2 chains (here and thereafter referred to as V␥9V␦2 T cells) and recognize nonpeptidic phosphorylated metabolites of isoprenoid biosynthesis produced by microorganisms and stressed cells. [17][18][19] On activation, V␥9V␦2 T cells promote DC maturation, 20 B-cell activation, 21 and polarize adaptive immunity toward a Th1 immune response. 10 Such a broad plasticity emphasizes the capacity of V␥9V␦2 T cells to influence the nature of immune response to different challenges. Human ␥␦ T cells producing IL-17 have been detected in the peripheral blood of patients with tuberculosis 22 or HIV infection, 23 but in neither of these studies did the authors characterize the IL-17-and IL-22-producing ␥␦ T cells or examine the cytokine requirements for IL-17 production.The authors of a very recent study have demonstrated that IL-17A-and IL-22-producing V␥9V␦2 T cells exist at low but significant frequencies in human and nonhuman primates, 24 and have suggested that V␥9V␦2 T cells can be polarized into Th17 (producing only IL-17),...