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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., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Well-defined gradients of the lipid mediator sphingosine-1-phosphate (S1P) direct chemotactic egress of mature thymocytes from the thymus into the circulation. Although it is known that these gradients result from low S1P levels in the thymic parenchyma and high S1P concentrations at the exit sites and in the plasma, the biochemical mechanisms that regulate these differential S1P levels remain unclear. Several studies demonstrated that ceramide synthase 2 (Cers2) regulates the levels of the S1P precursor sphingosine. We, therefore, investigated whether Cers2 is involved in the regulation of S1P gradients and S1P-dependent egress into the circulation. By analyzing Cers2-deficient mice, we demonstrate that Cers2 limits the levels of S1P in thymus and blood to maintain functional S1P gradients that mediate thymocyte emigration into the circulation. This function is specific for Cers2, as we also show that Cers4 is not involved in the regulation of thymic egress. Our study identified Cers2 as an important regulator of S1P-dependent thymic egress, and thus contributes to the understanding of how S1P gradients are maintained in vivo., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The DC-derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady-state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α⁻CD11b⁺ LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α-galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α⁻ and CD8α⁺ splenic DC subsets and enhances cross-presentation of exogenous antigens. Based on genome-wide expression profiling, we now show that splenic CD11b⁺ DCs are susceptible to IFN-γ-mediated suppression of CCL17, whereas LN CD11b⁺CCL17⁺ DCs downregulate the IFN-γR and are much less responsive to IFN-γ. Under inflammatory conditions, particularly in the absence of IFN-γ signaling in IFN-γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM-CSF in concert with IL-4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cross-presentation is an important mechanism by which DCs present exogenous antigens on MHC-I molecules, and activate CD8(+) T cells, cells that are crucial for the elimination of tumors. We investigated the feasibility of exploiting the capacity of the mannose receptor (MR) to improve both cross-presentation of tumor antigens and Th polarization, processes that are pivotal for the anti-tumor potency of cytotoxic T cells. To this end, we selected two glycan ligands of the MR, 3-sulfo-Lewis(A) and tri-GlcNAc (N-acetylglucosamine), to conjugate to the model antigen OVA and assessed in vitro the effect on antigen presentation and Th differentiation. Our results demonstrate that conjugation of either 3-sulfo-Lewis(A) or tri-GlcNAc specifically directs antigen to the MR. Both neo-glycoconjugates showed, even at low doses, improved uptake as compared with native OVA, resulting in enhanced cross-presentation. Using MR(-/-) and MyD88-TRIFF(-/-) bone marrow-derived DCs (BMDCs), we show that the cross-presentation of the neo-glycoconjugates is dependent on MR and independent of TLR-mediated signaling. Whereas proliferation of antigen-specific CD4(+) T cells was unchanged, stimulation with neo-glycoconjugate-loaded DCs enhanced the generation of IFN-γ-producing T cells. We conclude that modification of antigen with either 3-sulfo-Lewis(A) or tri-GlcNAc enhances cross-presentation and permits Th1 skewing, through specific targeting of the MR, which may be beneficial for DC-based vaccination strategies to treat cancer., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DC not only activate CD4 T (Th) cell and cytotoxic CD8 T cell (CTL) responses against pathogens, but they also tolerize autoreactive T cells in order to avoid autoimmunity. Previous studies have demonstrated that steady-state DC can tolerize naïve CTL, naïve Th cells and memory CTL. A study in this issue of the European Journal of Immunology demonstrates that DC also tolerize memory Th cells. This is arguably most critical for developing therapies against autoimmune disease; first, because Th cells are the central regulators of all adaptive immune responses, and second because memory, rather than naïve T cells are the clinically relevant cells in established autoimmune diseases. This study fosters hope that DC-based specific immunotherapies for common autoimmune diseases are possible.

DC can present and cross-present self-antigens to autoreactive CD4(+) and CD8(+) T cells, respectively, and incapacitate them by inducing anergy, deletion or converting them into Treg. In this review, we summarize the recent progress in immune tolerance research, which has been achieved by employing antigen- and TCR-transgenic mice. We cover the numerous discoveries that have furthered our knowledge of the DC subsets and maturation pathways involved in tolerance; the signals, such as CD70, TGF-beta, B7-H1/PD-L1, which dictate the decision between immunity and tolerance; and the in vivo role of DC in the maintenance of CD4(+) T-cell tolerance and CD8(+) T-cell cross-tolerance.

To study B-cell tolerance against non-lymphoid tissue autoantigens, we generated transgenic rat insulin promoter (RIP)-OVA/hen egg lysozyme (HEL) mice expressing the model antigens, OVA and HEL, in pancreatic islets. Their vaccination with OVA or HEL induced far less auto-Ab titers compared with non-transgenic controls. Depletion of CD25(+) cells during immunization completely restored auto-Ab production, but did not affect antibodies against a foreign control antigen. Depletion at later time-points was not effective. OVA-specific CD25(+) FoxP3(+) T(reg) were more frequent in the autoantigen-draining pancreatic LN than in other secondary lymphatics of RIP-OVA/HEL mice. Consistently, B cells were suppressed in that LN and also in the spleen, which is known to concentrate circulating antigen, such as the antigens used for vaccination. Suppression involved preventing expansion of autoreactive B cells in response to autoantigen, reducing antibody production per B-cell and isotype changes. These findings demonstrate that CD25(+) T(reg) suppress auto-Ab production against non-lymphoid tissue antigens in an antigen-specific manner.

The induction of robust CD4(+) and CD8(+) T cell responses is a central aim in antiviral and anticancer vaccination. To this end, dendritic cells (DC) need to capture the vaccine, process and present it on MHC class I and II molecules. The mechanisms by which DC acquire antigen predetermine the quantity and quality of the ensuing T cell activation. In this issue of the European Journal of Immunology, it is demonstrated that targeting antigen towards CD11c, an integrin expressed preferentially by murine DC, facilitates efficient CD4(+) and CD8(+) T cell activation. The underlying mechanisms involve efficient antigen delivery into the marginal zone and T/DC zone of the spleen. This commentary discusses these findings in the context of current knowledge on antigen presentation.

The concept that naive CD4+ and CD8+ T cells require co-stimulatory signals for activation and proliferation is well documented. Less clear is the need for co-stimulation during the effector phase of the T cell response. Here we examined the influence of B7-1 (CD80) during the effector phase of an autoimmune response to pancreatic islets using transgenic mouse lines which expressed B7-1 in either all or only some of their beta cells ("confluent" or "patchy" RIP-B7-1 mice). Transgenic expression of B7-1 in normal mouse islets that co-expressed the pro-inflammatory cytokine, IL-2, resulted in early spontaneous autoimmunity. Islets with IL-2 and "confluent" B7-1 expression were destroyed whereas islets with IL-2 and "patchy" B7-1 expression showed selective killing of the B7-1+ beta cells. Islet-reactive T cells, circulating in the RIP-B7-1/IL-2 mice, rejected syngeneic islet grafts, but only if these expressed B7-1. Introduction of the B7-1 transgene into the nonobese diabetic (NOD) genetic background likewise resulted in early spontaneous autoimmunity, but splenocytes from the diabetic animals could only transfer diabetes to NOD scid recipients that expressed B7-1 on their beta cells. In both these transgenic models, therefore, islet destruction required continuous B7-1 expression by target beta cells. Thus, although the normal repertoire contains T cells with potential islet reactivity, these T cells remain harmless because parenchymal cells like the beta cell cannot normally express B7-1. Our results also have implications for tumor immunotherapy in that the ability of T cells to kill poorly immunogenic targets may be dependent upon B7-1 expression by the target cell itself.