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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.

Most eukaryotic cells have a primary cilium which acts as a sensory organelle1. Cilia are assembled by intraflagellar transport (IFT), a process mediated by multimeric IFT particles and molecular motors2. Here we show that lymphoid and myeloid cells, which lack primary cilia, express IFT proteins. IFT20, an IFT component essential for ciliary assembly3 , 4, was found to colocalize with both the MTOC and Golgi and post-Golgi compartments in T-lymphocytes. In antigen-specific conjugates, IFT20 translocated to the immune synapse (IS). IFT20 knockdown resulted in impaired TCR/CD3 clustering and signaling at the IS due to defective polarized recycling. Moreover, IFT20 was required for the inducible assembly of a complex with other IFT components (IFT57, IFT88) and the TCR. The results identify IFT20 as a novel regulator of IS assembly in T-cells and provide the first evidence that IFT is implicated in membrane trafficking in cells lacking primary cilia, thereby opening a new perspective on IFT function beyond its role in ciliogenesis.When naive T-cells encounter antigen presenting cells (APC) bearing cognate MHC-bound peptide antigen, a dynamic rearrangement of membrane and cytosolic molecules occurs at the T-cell:APC contact area. This results in the formation of a highly organized interface known as immune synapse (IS), which acts as a platform for signal integration, fine-tuning and extinction5 , 6. A hallmark of the nascent IS is reorientation of the microtubule organizing center (MTOC) towards the APC7, which ensures targeted delivery of signaling molecules from intracellular pools to the IS8. This includes the TCR/CD3 complex itself, which is carried to the IS through polarized recycling9. Directional movement of structural and regulatory molecules, orchestrated by the MTOC, is a characteristic feature of primary cilia. These structures, present on most non-dividing cells, act as sensory organelles, relaying information from the external environment into the cell through intraflagellar transport (IFT), a process carried out by multimeric IFT particles and molecular motors2 , 10.At variance with most other eukaryotic cell types, hematopoietic cells lack primary cilia10. Surprisingly, IFT20, an IFT component essential for ciliary assembly3 ,4 , was found to be Correspondence should be addressed to CTB. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 November 1. Published in final edited form as:Nat Cell Biol. 2009 November ; 11(11): 1332-1339. doi:10.1038/ncb1977. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript expressed in human cells of both lymphoid (peripheral blood lymphocytes, Jurkat T-lymphoma cells) and myeloid (monocytes, monocyte-derived DC) lineages (Fig.1a). Similar results were obtained in the mouse, where IFT20 was detectable in central (thymus, bone marrow) and peripheral (lymph node, spleen) lymphoid organs (Fig.1a). Immunofluorescence analysis of Jurkat cells and human peripheral blood lymphocytes (PBL) showed that IFT20...

Helicobacter pylori toxin, VacA, damages the gastric epithelium by erosion and loosening of tight junctions. Here we report that VacA also interferes with T cell activation by two different mechanisms. Formation of anion-specific channels by VacA prevents calcium influx from the extracellular milieu. The transcription factor NF-AT thus fails to translocate to the nucleus and activate key cytokine genes. A second, channel-independent mechanism involves activation of intracellular signaling through the mitogen-activated protein kinases MKK3/6 and p38 and the Rac-specific nucleotide exchange factor, Vav. As a consequence of aberrant Rac activation, disordered actin polymerization is stimulated. The resulting defects in T cell activation may help H. pylori to prevent an effective immune response leading to chronic colonization of its gastric niche.

Anthrax is an infection caused by pathogenic strains of Bacillus anthracis, which secretes a three-component toxic complex consisting of protective antigen (PA), edema factor (EF), and lethal factor (LF). PA forms binary complexes with either LF or EF and mediates their entry into host cells. Although the initial phases of bacterial growth occur in the lymph node, the host fails to mount an effective immune response. Here, we show that LT and ET are potent suppressors of human T cell activation and proliferation triggered through the antigen receptor. Both LT and ET inhibit the mitogen-activated protein and stress kinase pathways, and both toxins inhibit activation of NFAT and AP-1, two transcription factors essential for cytokine gene expression. These data identify a novel strategy of immune evasion by B. anthracis, based on both effector subunits of the toxic complex, and targeted to a key cellular component of adaptive immunity.