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

Although many T cells carry two in-frame V alpha rearrangements, the products of both V alpha rearrangements have never been shown simultaneously on the surface of normal cells. With the use of monoclonal antibodies to V alpha 2, V alpha 12, and V alpha 24, up to one-third of mature T cells expressed two V alpha chains as part of two functional and independent T cell receptors (TCRs). Thus, the "one cell, one receptor" rule does not apply to a large subset of alpha beta T cells. Cells that belong to this dual TCR subset may be specific for a broader range of antigens than cells with a single receptor, which may be important for autoimmunity and alloreactivity.

SummaryThe T cell receptor (TCR)-c~//~ CD4-8-(double negative, DN) T cell subset is characterized by an oligoclonal repertoire and a restricted V gene usage. By immunizing mice with a DN T cell clone we generated two monodonal antibodies (mAbs) against Vol24 and V311, which have been reported to be preferentially expressed in DN T cells. Using these antibodies, we could investigate the expression and pairing of these Vc~ and V~ gene products among different T cell subsets. Vo~24 is rarely expressed among CD4 + and especially CD8 + T cells. In these cases it is rearranged to different Jcx segments, carries N nucleotides, and pairs with different V3. Remarkably, Voe24 is frequently expressed among DN T cells and is always present as an invariant rearrangement with JolQ, without N region diversity. This invariant Vc~24 chain is always paired to V311. This unique Ve~24-Jc~Q/V311 TCR was found in expanded DN clones from all the individuals tested. These findings suggest that the frequent occurrence of cells carrying this invariant TCR. is due to peripheral expansion of rare clones after recognition of a nonpolymorphic ligand.

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