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.

Mutually exclusive expression of T-bet and Eomes drives the development of distinct NK cell lineages with complementary functions.

γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.

The devastating effect of ischemic stroke is attenuated in mice lacking conventional and unconventional T cells, suggesting that inflammation enhances tissue damage in cerebral ischemia. We explored the functional role of ␣␤ and ␥␦ T cells in a murine model of stroke and distinguished 2 different T cell-dependent proinflammatory pathways in ischemiareperfusion injury. IFN-␥ produced by IntroductionIschemic stroke represents a major cause of disability and death in the western world. 1 Although infiltration of inflammatory leukocytes is a well-described feature of human stroke, 2 the perspective that activation of the immune systems is a bystander phenomenon secondary to ischemic tissue damage has changed. Currently, the activation of the immune system is recognized as a major element in all stages of the pathophysiology of stroke, including long lasting regenerative processes. 1,3 Release of danger molecules, local expression of proinflammatory cytokines, the subsequent expression of endothelial adhesion molecules, and breakdown of the blood-brain barrier are among the initial events after arterial occlusion. 3 These events are followed by an amplification of the postischemic inflammation that involves both resident brain cells and infiltrating immune cells. With the use of a mouse model of middle cerebral artery occlusion (MCAO), our group has previously shown a sequentially organized accumulation of immune cells of both the innate and adaptive immune systems in the ischemic brain. 4 The cellular infiltrate is dominated by neutrophils, macrophages, and microglia, but also includes T, natural killer, and dendritic cells.At this early stage, different T-cell subpopulations play important roles even if their absolute abundance in the ischemic brain is low. CD4 ϩ and CD8 ϩ T cells, as well as ␥␦ T cells, promote further tissue damage, 5-8 whereas regulatory T cells and B cells are protective. 9,10 Cytokines involved in the proinflammatory response include IL-1␤, IL-12, and IL-23, as well as interferon ␥ (IFN-␥), IL-17A, and TNF-␣. In contrast IL-4, TGF-␤, and mostly IL-10 are part of protective pathways. 9,11,12 However, the specific integration of each cell type and cytokine in the postischemic inflammatory network still has to be elucidated.In sterile inflammations, including ischemia, IL-17A can be crucial for chemokine induction. 13,14 Importantly, IL-17A can be rapidly released by ␥␦ T cells in response to cytokine activation or engagement of innate receptors, in the absence of TCR activation. 15 Beside IL-17A, IFN-␥ pathways are also implicated in ischemia/reperfusion (I/R) injury. 13,16 In autoimmunity, IFN-␥ production is associated with induction of MHCII expression, production of chemokines, and activation of macrophages. 17 Our analysis of the evolving local and systemic inflammatory responses after stroke has yielded 2 new distinct and crosslinked pathways: First, IFN-␥ produced by ␣␤ T cells induces the expression of TNF-␣ in macrophages. Second, ␥␦ T cells lead to neutrophil infiltration via the IL-1...

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Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C NK cells has remained unclear. Here we found that adaptive NKG2C NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C NK cell populations among HCMV-seropositive people.

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

cd T cells are a potent source of innate IL-17A and IFN-c, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24 low CD44 high effector cd T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6 1 cd T cells produced IL-17A, while NK1.1 1 cd T cells were efficient producers of IFN-c but not of IL-17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1 1 cd T cells. Accordingly, both cd T-cell subsets were rare in gut-associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL-17A and IFN-c in response to TCR-specific and TCR-independent stimuli. IL-12 and IL-18 induced IFN-c and IL-23 induced IL-17A production by NK1.1 1 or CCR6 1 cd T cells, respectively. Importantly, we show that CCR6 1 cd T cells are more responsive to TCR stimulation than their NK1.1 1 counterparts. In conclusion, our findings support the hypothesis that CCR6 1 IL-17A-producing cd T cells derive from less TCR-dependent selection events than IFN-c-producing NK1.1 1 cd T cells.Key words: gd T cells . CCR6 . IFN-g . IL-17A . Innate lymphocytes . NK1.1 Introduction IL-17A and IFN-g are generally regarded as pro-inflammatory effector cytokines that can be produced by Th cells but also by innate lymphocytes such as NK cells, NKT cells and gd T cells. While macrophage activation is supposed to be the main role of IFN-g, the induction of granulopoiesis is ascribed as a key biological function of IL-17A [1]. It is currently emerging that gd T cells are a potent source of IL-17A in the early phases of immune responses (reviewed in [2]). gd T cells constitute a large fraction of all IL-17A-producing cells in healthy mice and humans and are able to secrete IL-17A much more rapidly than CD4 1 Th17 cells [3][4][5]. These observations led to the concept that gd T cells are important players in a transitional response between innate and adaptive immune reactions [6]. The production of innate IL-17A by gd T cells appears to be essential in situations where an effective defence against extra-cellular bacteria or fungi relies on the fast mobilization of neutrophils [4,[6][7][8][9]. Moreover, IL-17A-producing gd T cells have been described to play important roles in immunopathologic diseases such as collageninduced arthritis [10], experimental pulmonary fibrosis [11], and in experimental autoimmune encephalitis [12,13].In contrast to CD4 1 Th cells that can develop into Th17 cells after encounter of specific cognate TCR Ag [14][15][16], it is not clear which stimuli induce IL-17A production by gd T cells in vivo. This essentially results from a lack of information about physiological gd TCR ligands. However, the current literature suggests that the decision whether a gd T cell will produce IL-17A is linked to 3488thymic development. Jensen et al. introduced a concept th...