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.

We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.

B-cell development in the bone marrow comprises proliferative and resting phases in different niches. We asked whether B-cell metabolism relates to these changes. Compared to pro B and small pre B cells, large pre B cells revealed the highest glucose uptake and ROS but not mitochondrial mass, whereas small pre B cells exhibited the lowest mitochondrial membrane potential. Small pre B cells from Rag1;33.C9 μ heavy chain knock-in mice revealed decreased glycolysis (ECAR) and mitochondrial spare capacity compared to pro B cells from Rag1 mice. We were interested in the step regulating this metabolic switch from pro to pre B cells and uncovered that Swiprosin-2/EFhd1, a Ca-binding protein of the inner mitochondrial membrane involved in Ca-induced mitoflashes, is expressed in pro B cells, but downregulated by surface pre B-cell receptor expression. Knockdown and knockout of EFhd1 in 38B9 pro B cells decreased the oxidative phosphorylation/glycolysis (OCR/ECAR) ratio by increasing glycolysis, glycolytic capacity and reserve. Prolonged expression of EFhd1 in EFhd1 transgenic mice beyond the pro B cell stage increased expression of the mitochondrial co-activator PGC-1α in primary pre B cells, but reduced mitochondrial ATP production at the pro to pre B cell transition in IL-7 cultures. Transgenic EFhd1 expression caused a B-cell intrinsic developmental disadvantage for pro and pre B cells. Hence, coordinated expression of EFhd1 in pro B cells and by the pre BCR regulates metabolic changes and pro/pre B-cell development.

The CC-chemokine receptor 6 (CCR6) can be detected on naive and activated B cells. Counterintuitively, its absence accelerates the appearance of germinal centres (GCs) and increases the production of low-affinity antibodies. The detailed mechanism of CCR6 function during the humoral response has remained elusive, but previously we identified a distinct CCR6 B-cell population in vivo early after antigenic challenge. In this study, we defined this population specifically as early, activated pre-GC B cells. In accordance, we show that CCR6 is upregulated rapidly within hours on the protein or mRNA level after activation in vitro. In addition, only activated B cells migrated specifically towards CCL20, the specific ligand for CCR6. Lack of CCR6 increased the dark zone/light zone ratio of GC and led to decreased antigen-specific IgG1 and IgG2a antibody generation in a B-cell intrinsic manner in mixed bone marrow chimeras. In contrast, antigen-specific IgM responses were normal. Hence, CCR6 negatively regulates entry of activated, antigen-specific pre-GC B cells into the GC reaction.

Activated B cells are selected for in germinal centers by regulation of their apoptosis.The Ca 2+ -binding cytoskeletal adaptor protein Swiprosin-1/EFhd2 (EFhd2) can promote apoptosis in activated B cells. We therefore hypothesized that EFhd2 might limit humoral immunity by repressing both the germinal center reaction and the expected enhancement of immune responses in the absence of EFhd2. Here, we established EFhd2 −/− mice on a C57BL/6 background, which revealed normal B-and T-cell development, basal Ab levels, and T-cell independent type 1, and T-cell independent type 2 responses. However, T cell-dependent immunization with sheep red blood cells and infection with the helminth Nippostrongylus brasiliensis (N.b) increased production of antibodies of multiple isotypes, as well as germinal center formation in EFhd2 −/− mice. In addition, serum IgE levels and numbers of IgE + plasma cells were strongly increased in EFhd2 −/− mice, both after primary as well as after secondary N.b infection. Finally, mixed bone marrow chimeras unraveled an EFhd2-dependent B cell-intrinsic contribution to increased IgE plasma cell numbers in N.b-infected mice. Hence, we established a role for EFhd2 as a negative regulator of germinal center-dependent humoral type 2 immunity, with implications for the generation of IgE. Keywords:Germinal center r IgE r Nippostrongylus brasiliensis r Plasma cell r Swiprosin-1/ EFhd2Additional supporting information may be found in the online version of this article at the publisher's web-site [11]. These short-lived plasma cells provide an initial wave of mostly non class-switched low affinity antibodies. Second, the early activated B cells can form GC where Ab affinity maturation takes place [12,13]. GC B cells upregulate activation induced deaminase (AID), and start to hypermutate the V exons of their Ig genes in the dark zone of the GC [10]. Later, these B cells migrate to the light zone of the GC, where they differentiate into centrocytes. There, they become negatively selected by default through apoptosis but can be rescued by antigen presentation to follicular T helper cells (T FH ) to receive essential survival signals [14]. Those survival signals are usually reserved for high affine, non autoreactive B cells. Centrocytes can leave the GC to develop into long-lived, class-switched and affinity-matured plasma cells that home back to the BM, resulting in sustained production of high-affinity antibodies [15], but to what extent IgE-expressing plasma cells participate in this step is being controversially discussed [16][17][18][19][20].The mechanism of the maturation of IgE-positive B cells is of considerable interest with respect to type I hypersensitivity reactions that can have life-threatening anaphylactic outcomes. Maturation of IgE-positive B cells involves a GC-dependent CSR pathway [16,17] but there is also evidence for indirect CSR to IgE by switching to IgG1 through an initial GC reaction, followed by an extrafollicular maturation phase and switching to IgE [19,20]. It is agreed upon t...