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The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., (© 2021 Wiley-VCH GmbH.)

Inflammatory bowel diseases (IBD) are chronic conditions that result from uncontrolled intestinal inflammation. Pathogenic Th17 cells, characterized by production of IL-17A in the absence of IL-10, are thought to contribute to this inflammation, but in humans, antibody-mediated blockade of IL-17A is an ineffective IBD therapy whereas IL-23 blockade is effective. Here, we investigated the effects of pharmacological inhibition of RORC2, the Th17 cell lineage-defining transcription factor, on in vivo-differentiated human Th17 cells and Th17-like Tregs (Th17-Tregs). BMS-336, a small molecule RORC2 inverse agonist, inhibited expression of RORC2-regulated genes in peripheral Th17 cells (CD4 + CD25 - CD127 + CXCR3 - CCR4 + CCR6 + ) in a dose-dependent manner, with similar inhibitory effects on laminar propria mononuclear cells from IBD and non-IBD subjects. Exposure of peripheral Th17-Tregs (CD4 + CD25 hi CD127 lo CXCR3 - CCR4 + CCR6 + ) to BMS-336 also inhibited IL-17A production and prevented inflammatory cytokine-induced destabilization, as evidenced by preserved FOXP3 expression and epigenetic status of the Treg-specific demethylation region. In parallel, RORC2 inhibition increased the production of IL-10 in Th17-Tregs, resulting in enhanced suppression of inflammatory cytokines from myeloid cells. Thus, via its ability to simultaneously inhibit Th17 cells and enhance the stability and function of Th17-Tregs, pharmacological inhibition of RORC2 is a promising approach to suppress inflammation and promote immune regulation in IBD., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.)

Y-box-binding protein 1 (YB-1) is a regulatory protein that is associated with drug resistance and relapse in solid tumors. As YB-1 mediates some of its activity through growth factor receptor signaling dysregulation, the present study compared the expression of YB-1 and interleukin 7 (IL-7) receptor α (IL-7Rα) in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) and normal BCP cells. The expression levels of IL-7Rα and YB-1 were higher in relapsed vs. diagnostic samples of primary BCP ALL; however, co-expression was also observed in a minor BCP cell population in samples from healthy donors. Functional crosstalk between YB-1 and IL-7R was detected: Overexpression of YB-1 increased surface levels of IL-7R in B cells, and the stimulation of BCP ALL cell lines and primary samples by IL-7 activated YB-1 by phosphorylation at S102 in a phosphatidylinositol 3-kinase-independent and MEK1/2-dependent manner. Targeted knockdown of YB-1 reduced IL-7-mediated protection against rapamycin, and an inhibitor of MEK1/2 potentiated rapamycin-mediated killing in the presence of IL-7. These data establish a novel link between two well-characterized pro-survival factors in acute leukemia, and suggest that YB-1 inhibition may represent a novel therapeutic strategy for increasing sensitivity to chemotherapy in patients with refractory acute B-cell leukemia.

Cathepsin (Cathepsin) S, L, and B proteases mediate antigen presentation on major histocompatibility complex (MHC) class II by degrading the invariant chain Ii, which blocks peptide loading. The ability of the Cathepsin S inhibitor LHVS (morpholinurea-leucine-homophenylalanine-vinylsulfone phenyl) to impede antigen presentation has led its development as a therapy for autoimmune diseases. There is substantial evidence that donor T cell recognition of host minor histocompatibility antigens (miHA) and subsequent destruction of host tissue mediates graft-versus-host disease (GVHD). We hypothesized that enzymes involved in antigen presentation may play a role in the development of GVHD. Using the C57BL/6 → BALB.B minor mismatch acute GVHD (aGVHD) model, we found that the cathepsin S activity of spleens from allogenetically transplanted mice were significantly increased 1 week after transplantation compared with syngeneic mice. Although LHVS decreased T cell priming responses against both single OVA antigen and miHA in vitro, LHVS did not reduce the severity of aGVHD. In fact, LHVS exacerbated a CD4(+)-T cell-dependent model of GVHD similar to chronic GVHD. This suggests that cytokines rather than T cells may mediate much of the damage in the aGVHD model and that therapeutics based on inhibition of antigen presentation for GVHD must be approached with caution., (Copyright © 2012 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Although intestinal epithelial cells (IECs) are continually exposed to commensal microbes, under healthy conditions they contribute to intestinal homeostasis while keeping inflammatory responses in check. In response to invading pathogens, however, IECs respond vigorously by producing inflammatory mediators. To better understand the signals that regulate the inflammatory responses of IECs, we investigated whether the danger signal ATP (which is released from injured cells) could alter responses to bacterial products., Methods: We measured chemokine production from Caco-2 cells stimulated with the Toll-like receptor 5 agonist flagellin with or without ATP. ATP increased flagellin-induced IL-8 secretion but reduced CCL20 secretion via distinct signaling pathways., Results: ATP-enhanced IL-8 production was only partly blocked by the P(2) receptor antagonist suramin and required activation of NF-κB while ATP-mediated reduction of CCL20 was completely blocked by suramin and required activation of ERK1/2. The effects of ATP on both chemokines required extracellular calcium but not phospholipase C, implicating P(2) X receptor involvement. To investigate how ATP alters IEC responses to bacterial products in vivo, mice receiving dextran sodium sulfate were given intrarectal flagellin with or without ATP. Addition of ATP to flagellin caused greater weight loss and increased antiflagellin antibody titers, as well as decreased colonic interferon gamma (IFN-γ) and higher antiflagellin IgG1/IgG2 ratios, which indicate decreased Th1 polarization., Conclusions: Together, these data indicate that stress, in the form of extracellular ATP, reshapes both the inflammatory response of flagellin-stimulated IECs and downstream adaptive immunity, representing a possible strategy by which these cells differentiate between commensal and pathogenic bacteria., (Copyright © 2010 Crohn's & Crohn's & Colitis Foundation of America, Inc.)

Intestinal epithelial cells act as innate immune sentinels, as the first cells that encounter diarrheal pathogens. They use pattern recognition molecules such as the Toll-like receptors (TLRs) to identify molecular signals found on microbes but not host cells or food components. TLRs cannot generally distinguish the molecular signals on pathogenic bacteria from those found in commensals, yet under healthy conditions epithelial immune responses are kept in check. We hypothesized that, in the setting of tissue damage or stress, intestinal epithelial cells would upregulate their responses to TLR ligands to reflect the greater need for immediate protection against pathogens. We treated Caco-2 cells with the TLR5 agonist flagellin in the presence or absence of H(2)O(2) and measured chemokine production and intracellular signaling pathways. H(2)O(2) increased flagellin-induced IL-8 (CXCL8) production in a dose-dependent manner. This was associated with synergistic phosphorylation of p38 MAP kinase and with prolonged I-kappaB degradation and NF-kappaB activation. The H(2)O(2)-mediated potentiation of IL-8 production required the activity of p38, tyrosine kinases, phospholipase Cgamma, and intracellular calcium, but not protein kinase C or protein kinase D. H(2)O(2) prolonged and augmented NF-kappaB activation by flagellin. In contrast to IL-8, CCL20 (MIP3alpha) production by flagellin was reduced by H(2)O(2), and this effect was not calcium dependent. Oxidative stress biases intestinal epithelial responses to flagellin, leading to increased production of IL-8 and decreased production of CCL20. This suggests that epithelial cells are capable of sensing the extracellular environment and adjusting their antimicrobial responses accordingly.

Enteroaggregative Escherichia coli (EAEC) causes diarrhea in diverse populations worldwide. The AraC-like regulator AggR is a key virulence regulator in EAEC. AggR-regulated genes include those encoding the Aggregative Adherence Fimbria, the dispersin protein, and a type VI secretion system. This study characterizes the regulation of the aggR promoter (P(aggR)). Using primer extension analysis, the transcriptional start site of the aggR promoter was located 40 nucleotides upstream of the translational start. P(aggR) was found to be autoregulated and DNA footprinting revealed the presence of two AggR-binding sites: one upstream of the transcriptional start site and one downstream. Additionally, P(aggR) was found to be positively regulated by the DNA-binding protein FIS and negatively regulated by the global regulator H-NS. To further understand this complex regulation scheme, a bacterial luciferase reporter system was used with a mouse model of EAEC colonization. This allowed for the in vivo measurement of P(aggR), P(fis), and P(hns) activity. EAEC present in the mouse intestine possessed relatively high levels of P(fis) and P(aggR) activity and a low level of P(hns) when compared with in vitro experiments. The data provide significant insights into the regulation cascade leading to aggR expression in the mammalian intestine during EAEC infection.

Background: The two forms of human inflammatory bowel disease, Crohn's disease (CD) and ulcerative colitis (UC), are both associated with loss of tolerance to gut microbial antigens. The dominant antigen recognized by antibody and T-cell responses in patients with CD is bacterial flagellin. Flagellin is also the only known ligand for Toll-like receptor 5 (TLR5), a key protein in innate immunity. Although flagellin activates TLR5 to produce inflammatory responses in many cell types in the gut, there is conflicting evidence as to whether TLR5 is harmful or protective in CD and murine colitis models. A recent study found that administration of flagellin enemas to mice along with dextran sodium sulfate (DSS) made their colitis worse., Methods: We sought to determine whether this exacerbation was due to TLR5 ligation, or to TLR5-independent adaptive immune responses to flagellin as an antigen, by using a transposon insertional mutant of the Escherichia coli H18 flagellin, 2H3, which lacks TLR5 stimulatory activity., Results: We found that flagellin enemas produced only a mild exacerbation of DSS colitis, and that 2H3 was equivalent to or worse than wildtype flagellin. Moreover, we found that DSS colitis was more severe in TLR5(-/-) mice than wildtype C57BL/6 mice., Conclusions: Together, these results suggest that flagellin-mediated exacerbation of colitis is independent of TLR5.

The protein kinase D (PKD) family consists of three serine-threonine kinases involved in cellular proliferation, motility, and apoptosis. We previously reported that human toll-like receptor 5 (TLR5) contains a consensus PKD phosphorylation site. Flagellin stimulation of cells activated PKD1, and inhibition of PKD1 reduced flagellin-induced interleukin-8 (IL-8) production in epithelial cells. In the current work, we examined PKD1 and PKD2 involvement downstream of TLR5, TLR4 and TLR2. We found that inhibition of either kinase with shRNA reduced IL-8 and CCL20 release due to TLR4 and TLR2 agonists to a similar extent as previously reported for TLR5. PKD1 and PKD2 inhibition reduced NF-kappaB activity but not MAPK activation. These results demonstrate that both PKD1 and PKD2 are required for inflammatory responses following TLR2, TLR4, or TLR5 activation, although PKD1 is more strongly involved. These kinases likely act downstream of the TLRs themselves to facilitate NF-kappaB activation but not MAP kinase phosphorylation., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Bacterial flagellin triggers inflammation in mammalian cells via Toll-like receptor (TLR) 5. Release of the chemokine IL-8 in response to flagellin involves NF-κB, p38 MAP kinase, and phosphatidylinositol 3-kinase (PI3K). However, PI3K has been reported to be either pro- or anti-inflammatory in different model systems. We hypothesized that this could be due to different activities of the p110α and β isoforms of PI3K., Results: PI3K and Akt were rapidly activated in Caco-2 colon carcinoma cells by flagellin. Using a plasmid-based shRNA delivery system and novel p110 isoform-specific inhibitors, we found that flagellin-induced IL-8 production was dependent on both p110α and p110β. However in the mouse, inhibition of p110β but not p110α reduced the increase of serum IL-6 levels induced by intraperitoneal injection of flagellin., Conclusions: These data demonstrate that the p110α and β isoforms of class IA PI3K are both required for the proinflammatory response to flagellin.

Protein kinase D (PKD), also called protein kinase C (PKC)mu, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein secretion. After identifying a putative PKD phosphorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative Escherichia coli flagellin in human epithelial cell lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target peptide in vitro, and phosphorylation of the putative target serine 805 in HEK 293T cell-derived TLR5 was identified by mass spectrometry. Furthermore, mutation of serine 805 to alanine abrogated responses of transfected HEK 293T cells to flagellin. Second, TLR5 interacted with PKD in coimmunoprecipitation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with Gö6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor Gö6983 had no significant effects on these phenotypes. Finally, involvement of PKD in the p38-mediated IL-8 response to flagellin was confirmed by small hairpin RNA-mediated gene silencing. Together, these results suggest that phosphorylation of TLR5 by PKD may be one of the proximal elements in the cellular response to flagellin, and that this event contributes to p38 MAPK activation and production of inflammatory cytokines in epithelial cells.

Flagellin, the major structural subunit of bacterial flagella, potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) has been reported to play a role in early TLR signalling. We identified a putative binding site for PI3K at tyrosine 798 in the TLR5 TIR domain, at a site analogous to the PI3K recruitment domain in the interleukin-1 receptor. Mutation of this residue did not affect homodimerization, but prevented inflammatory responses to flagellin. While we did not detect direct interaction of PI3K with TLR5, we demonstrated by mass spectrometry that Y798 is phosphorylated in flagellin-treated HEK 293T cells. Together, these results suggest that phosphorylation of Y798 in TLR5 is required for signalling, but not for TLR5 dimerization.