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Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic condition in childhood. Disease etiology remains largely unknown, however a key role in JIA pathogenesis is surely mediated by T cells. T lymphocytes activity is controlled via signals, known as immune-checkpoints (IC). Delivering an inhibitory signal or blocking a stimulatory signal to achieve immune suppression is critical in autoimmune diseases. However, the role of IC in chronic inflammation and autoimmunity must still be deciphered. In this study, we investigated at single cell level the feature of T cells in JIA chronic inflammation both at transcriptome level via single-cell RNA sequencing and at protein level by flow cytometry. We found that despite the heterogeneity in the composition of synovial CD4+ and CD8+ T cells, those characterized by PD-1 expression were clonally expanded Trm-like cells and displayed the highest pro-inflammatory capacity, suggesting their active contribution in sustaining chronic inflammation in situ. Our data support the concept that novel therapeutic strategies targeting PD-1 may be effective in the treatment of JIA. With this approach, it may become possible to target overactive T regardless of their cytokine production profile.

Cells experience intrinsic and extrinsic pressures that affect their proclivity to expand and persistin vivo. In congenital disorders caused by loss-of-function mutations in mitochondrial DNA (mtDNA), metabolic vulnerabilities may result in cell-type specific phenotypes and depletion of pathogenic alleles, contributing to purifying selection. However, the impact of pathogenic mtDNA mutations on the cellular hematopoietic landscape is not well understood. Here, we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from Pearson syndrome patients. We resolve the interdependence between pathogenic mtDNA and lineage, including purifying selection against deletions in effector/memory CD8 T-cell populations and recent thymic emigrants and dynamics in other hematopoietic populations. Our mapping of lineage-specific purifying selection dynamics in primary cells from patients carrying pathogenic heteroplasmy provides a new perspective on recurrent clinical phenotypes in mitochondrial disorders, including cancer and infection, with potential broader relevance to age-related immune dysfunction.

T lymphocytes accumulate in inflamed tissues of patients with chronic inflammatory diseases (CIDs) and express pro-inflammatory cytokines upon re-stimulation in vitro. Further, a significant genetic linkage to MHC genes suggests that T lymphocytes play an important role in the pathogenesis of CIDs including juvenile idiopathic arthritis (JIA). However, the functions of T lymphocytes in established disease remain elusive. Here we dissect the transcriptional and the clonal heterogeneity of synovial T lymphocytes in JIA patients by single-cell RNA sequencing combined with T cell receptor profiling on the same cells. We identify clonally expanded subpopulations of T lymphocytes expressing genes reflecting recent activation by antigen in situ. A PD-1+ TOX+ EOMES+ population of CD4+ T lymphocytes expressed immune regulatory genes and chemoattractant genes for myeloid cells. A PD-1+ TOX+ BHLHE40+ population of CD4+ , and a mirror population of CD8+ T lymphocytes expressed genes driving inflammation, and genes supporting B lymphocyte activation in situ. This analysis points out that multiple types of T lymphocytes have to be targeted for therapeutic regeneration of tolerance in arthritis.

Fiskin et al. (2021) developed a "multi-omics" approach that integrates phage-displayed single-domain antibodies ("nanobodies") with the assay for transposase-accessible chromatin (PHAGE-ATAC) to simultaneously determine protein expression, chromatin accessibility, and mitochondrial DNA mutations (for clonal tracing) in single cells.

Foxp3+ regulatory T cells (Treg cells) are crucial for the maintenance of immune homeostasis both in lymphoid tissues and in non-lymphoid tissues. Here we demonstrate that the ability of intestinal Treg cells to constrain microbiota-dependent interleukin (IL)-17-producing helper T cell (TH17 cell) and immunoglobulin A responses critically required expression of the transcription factor c-Maf. The terminal differentiation and function of several intestinal Treg cell populations, including RORγt+ Treg cells and follicular regulatory T cells, were c-Maf dependent. c-Maf controlled Treg cell-derived IL-10 production and prevented excessive signaling via the kinases PI(3)K (phosphatidylinositol-3-OH kinase) and Akt and the metabolic checkpoint kinase complex mTORC1 (mammalian target of rapamycin) and expression of inflammatory cytokines in intestinal Treg cells. c-Maf deficiency in Treg cells led to profound dysbiosis of the intestinal microbiota, which when transferred to germ-free mice was sufficient to induce exacerbated intestinal TH17 responses, even in a c-Maf-competent environment. Thus, c-Maf acts to preserve the identity and function of intestinal Treg cells, which is essential for the establishment of host-microbe symbiosis.

Inflammatory bowel disease (IBD) is a group of severe chronic inflammatory conditions of the human gastrointestinal tract. Murine models of colitis have been invaluable tools to improve the understanding of IBD development and pathogenesis. While the disease etiology of IBD is complex and multifactorial, CD4

Why do we still have no cure for chronic inflammatory diseases? One reason could be that current therapies are based on the assumption that chronic inflammation is driven by persistent 'acute' immune reactions. Here we discuss a paradigm shift by suggesting that beyond these reactions, chronic inflammation is driven by imprinted, pathogenic 'memory' cells of the immune system. This rationale is based on the observation that in patients with chronic inflammatory rheumatic diseases refractory to conventional immunosuppressive therapies, therapy-free remission can be achieved by resetting the immune system; that is, by ablating immune cells and regenerating the immune system from stem cells. The success of this approach identifies antigen-experienced and imprinted immune cells as essential and sufficient drivers of inflammation. The 'dark side' of immunological memory primarily involves memory plasma cells secreting pathogenic antibodies and memory T lymphocytes secreting pathogenic cytokines and chemokines, but can also involve cells of innate immunity. New therapeutic strategies should address the persistence of these memory cells. Selective targeting of pathogenic immune memory cells could be based on their specificity, which is challenging, or on their lifestyle, which differs from that of protective immune memory cells, in particular for pathogenic T lymphocytes. The adaptations of such pathogenic memory cells to chronic inflammation offers entirely new therapeutic options for their selective ablation and the regeneration of immunological tolerance.

Inflammatory bowel disease (IBD) is a group of severe chronic inflammatory conditions of the human gastrointestinal tract. Murine models of colitis have been invaluable tools to improve the understanding of IBD development and pathogenesis. While the disease etiology of IBD is complex and multifactorial, CD4+ T helper cells have been shown to strongly contribute to the disease pathogenesis of IBD. Here, we present a detailed protocol of the preclinical model of T-cell transfer colitis, which can easily be utilized in the laboratory to study T helper cell functions in intestinal inflammation.

T lymphocytes accumulate in inflamed tissues of patients with chronic inflammatory diseases (CIDs) and express pro-inflammatory cytokines upon re-stimulation in vitro. Further, a significant genetic linkage to MHC genes suggests that T lymphocytes play an important role in the pathogenesis of CIDs including juvenile idiopathic arthritis (JIA). However, the functions of T lymphocytes in established disease remain elusive. Here we dissect the transcriptional and the clonal heterogeneity of synovial T lymphocytes in JIA patients by single-cell RNA sequencing combined with T cell receptor profiling on the same cells. We identify clonally expanded subpopulations of T lymphocytes expressing genes reflecting recent activation by antigen in situ. A PD-1

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility., Nature Communications, 11 (1), ISSN:2041-1723

Conflicting evidence has been provided as to whether induction of intestinal inflammation by adoptive transfer of naïve T cells into Rag-/- mice requires expression of the transcription factor T-bet by the T cells. Here, we formally show that the intestinal microbiota composition of the Rag-/- recipient determines whether or not T-bet-deficient Th cells can induce colitis and we have resolved the differences of the two microbiomes, permissive or non-permissive to T-bet-independent colitis. Our data highlight the dominance of the microbiota over particular T cell differentiation programs in the pathogenesis of chronic intestinal inflammation. © 2017 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim.

Introduction/AbstractT lymphocytes accumulate in inflamed tissues of patients with chronic inflammatory diseases (CIDs) and express pro-inflammatory cytokines upon re-stimulation in vitro1–29. Further, a significant genetic linkage to MHC genes suggests that T lymphocytes play an important role in the pathogenesis of CIDs including juvenile idiopathic arthritis (JIA)30–33. However, the functions of T lymphocytes in established disease remain elusive. Here we dissect the heterogeneity of synovial T lymphocytes in JIA patients by single cell RNA-sequencing. We identify subpopulations of T lymphocytes expressing genes reflecting recent activation by antigen in situ. A PD-1+TOX+EOMES+ population of CD4+ T lymphocytes expressed immune regulatory genes and chemoattractant genes for myeloid cells. A PD-1+TOX+BHLHE40+ population of CD4+, and a mirror population of CD8+ T lymphocytes expressed genes driving inflammation, and genes supporting B lymphocyte activation. This analysis points out that multiple types of T lymphocytes have to be targeted for therapeutic regeneration of tolerance in arthritis.

Career situation of first and presenting author Post-doctoral fellow. Introduction Juvenile idiopathic arthritis (JIA) is a chronic inflammatory disease (CID) of unknown origin and is characterized by joint inflammation in children and young adults.1 Evidence suggests a strong contribution of memory T cells to disease pathogenicity in JIA. While few markers for T cells adapted to chronic inflammation have been identified.2 3 a comprehensive analysis of what distinguishes pathogenic memory T cells in inflamed tissues of chronic inflammation from protective, circulating memory T cells is still lacking. Objectives To characterize the transcriptional profiles of memory T cells that putatively maintain chronic inflammation in JIA patients. To identify biomarkers that are associated with autoantigen-specific clonotypes among memory T cells in JIA. Methods Memory T cells were isolated from the synovial fluid (SF) and the peripheral blood (PB) of oligoarticular JIA patients and purified by fluorescence-activated cell sorting (FACS). Subsequently, single cell sequencing including T cell receptor (TCR) sequencing was performed on ∼18.000 memory T cells of each JIA patient. Results Memory T cell populations both from the blood and from the SF are heterogenous populations according to their transcriptional expression patterns. The SF harbored a larger population of enriched T memory cell clonotypes than the blood. In addition, enriched memory T helper cell clones in the SF showed a transcriptional pattern of activation compared to non-enriched clonotypes. Finally, small subpopulations of enriched memory T helper cell clones in the SF show a transcriptional signature that resembles transcriptomes obtained by bulk sequencing. Thus, a rather small subpopulation of antigen-specific cells might be responsible for the overall transcriptional character of T cells found at the inflamed sites of CIDs. Conclusions Single cell sequencing combined with TCR sequencing is a powerful tool to identify and characterize subsets of T memory cells in chronic inflammation. The obtained data might be useful to better understand how T cell subsets contribute to disease pathogenicity in CIDs and reveals putative targets that could be therapeutically exploited in order to selectively deplete pathogenic memory T cells. References Prakken B, et al. Juvenile idiopathic arthritis. Lancet 2011. Niesner U, et al. Autoregulation of Th1-mediated inflammation by twist1. J Exp Med 2008. Maschmeyer P, et al. Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo. J Autoimmun 2018. Acknowledgements This work is supported by the European Regional Development Fund (ERDF 2014–2020 and EFRE 1.8/11). Disclosure of Interest None declared.

Many health problems facing society are multifactorial and often require social and political input as well as interventions from medical and technological experts. For example, the treatment of chronic pain requires expertise from multiple disciplines: imaging technology, cellular electrophysiology, neurochemistry, genetics, social, psychological, and cultural studies (1). While these activities are coordinated by the treating physician, they usually remain parallel and are never fully integrated to create an innovative therapy for the patient. From a research standpoint, we argue that for these new solutions to emerge, there needs to be a concerted effort to move from multidisciplinarity to interdisciplinarity. Multidisciplinary research is defined as work involving researchers from different fields who “remain conceptually and methodologically anchored in their respective fields” (2). In contrast, interdisciplinary research is defined as “a mode of research by teams or individuals that integrates information, data, techniques, tools, perspectives, concepts, and/or theories from two or more disciplines or bodies of specialized knowledge, to advance fundamental understanding or to solve problems whose solutions are beyond the scope of a single discipline or area of research practice” (3). It may lead to the creation of a new scientific field, such as environmental humanities (4–6). The major difference between the two types of research is that while interdisciplinarity involves deep and robust integration of distinct disciplines, multidisciplinarity implicates juxtaposition of a variety of expertises (5). By these definitions, both research types are clearly valuable, but interdisciplinary research should drive more impactful results for complicated problems. These advances come at a cost for researchers because interdisciplinarity has its own set of unique challenges, ranging from communication issues to allocation of credits among a team. In this article, we discuss these hurdles and potential solutions to raise awareness amongst researchers keen to lead a successful interdisciplinary project.

Proinflammatory type 1 T helper (Th1) cells are enriched in inflamed tissues and contribute to the maintenance of chronic inflammation in rheumatic diseases. Here we show that the microRNA- (miR-) 31 is upregulated in murine Th1 cells with a history of repeated reactivation and in memory Th cells isolated from the synovial fluid of patients with rheumatic joint disease. Knock-down of miR-31 resulted in the upregulation of genes associated with cytoskeletal rearrangement and motility and induced the expression of target genes involved in T cell activation, chemokine receptor- and integrin-signaling. Accordingly, inhibition of miR-31 resulted in increased migratory activity of repeatedly activated Th1 cells. The transcription factors T-bet and FOXO1 act as positive and negative regulators of T cell receptor (TCR)-mediated miR-31 expression, respectively. Taken together, our data show that a gene regulatory network involving miR-31, T-bet, and FOXO1 controls the migratory behavior of proinflammatory Th1 cells.

OBJECTIVE Inflamed tissue is characterized by low availability of oxygen and nutrients. Yet CD4+ T helper lymphocytes persist over time in such tissue and probably contribute to the chronicity of inflammation. This study was undertaken to analyze the metabolic adaptation of these cells to the inflamed environment. METHODS Synovial and blood CD4+ T cells isolated ex vivo from patients with juvenile idiopathic arthritis (JIA) and murine CD4+ T cells were either stimulated once or stimulated repeatedly. Their dependency on particular metabolic pathways for survival was then analyzed using pharmacologic inhibitors. The role of the transcription factor Twist 1 was investigated by determining lactate production and oxygen consumption in Twist1-sufficient and Twist1-deficient murine T cells. The dependency of these murine cells on particular metabolic pathways was analyzed using pharmacologic inhibitors. RESULTS Programmed death 1 (PD-1)+ T helper cells in synovial fluid samples from patients with JIA survived via fatty acid oxidation (mean ± SEM survival of 3.4 ± 2.85% in the presence of etomoxir versus 60 ± 7.08% in the absence of etomoxir on day 4 of culture) (P < 0.0002; n = 6) and expressed the E-box-binding transcription factor TWIST1 (2-14-fold increased expression) (P = 0.0156 versus PD-1- T helper cells; n = 6). Repeatedly restimulated murine T helper cells, which expressed Twist1 as well, needed Twist1 to survive via fatty acid oxidation. In addition, Twist1 protected the cells against reactive oxygen species. CONCLUSION Our findings indicate that TWIST1 is a master regulator of metabolic adaptation of T helper cells to chronic inflammation and a target for their selective therapeutic elimination.

Foxp3

It is current belief that numbers of CD8+ memory T lymphocytes in the memory phase of an immune response are maintained by homeostatic proliferation. Here, we compare the proliferation of CD8+ memory T lymphocytes, generated by natural infections and by intentional immunization, in spleen and bone marrow (BM). Fifty percent of CD8+ memory T lymphocytes in the spleen are eliminated by cyclophosphamide within 14 days, indicating that numbers of at least 50% of splenic CD8+ memory T lymphocytes are maintained by proliferation. The numbers of CD8+ memory T lymphocytes in the BM, however, were not affected by cyclophosphamide. This stability was independent of circulating CD8+ memory T cells, blocked by FTY720, showing that BM is a privileged site for the maintenance of memory T lymphocytes, as resident cells, resting in terms of proliferation.

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory., Highlights • Th1 cells expressing miR-148a mediate colitis in a murine model of IBD. • Antagomir-148a inhibits colitis by selectively depleting Th1 cells from the colon. • Antagomir-148a does not affect the protective immunological memory.

The transcription factor T-bet is highly expressed by Th cells isolated from the inflamed intestine of Crohn's disease patients, and has been regarded a critical driver of murine T cell-induced colitis. However, we show here that T-bet expression by Th cells is not required for the manifestation of T-cell-induced colitis in the presence of segmented filamentous bacteria and Helicobacter hepaticus. T-bet expression by Th cells controls their survival and localization, their repertoire of chemokine and chemokine receptor expression, the accumulation of monocytes and macrophages in the inflamed colon, and their differentiation to the M1 type, i.e., type 1 inflammation. Nevertheless, T-bet-deficient Th cells efficiently induce colitis, as reflected by weight loss, diarrhea, and colon histopathology. T-bet-deficient Th cells differentiate into Th1/17 cells, able to express IFN-γ and IL-17A upon restimulation. While neutralization of IL-17A exacerbated colitis induced by wild-type or T-bet-deficient Th cells, neutralization of IFN-γ completely abolished colitis.

Exercise at regular intervals is assumed to have a positive effect on immune functions. Conversely, after spaceflight and under simulated weightlessness (e.g., bed rest), immune functions can be suppressed. We aimed to assess the effects of simulated weightlessness (Second Berlin BedRest Study; BBR2-2) on immunological parameters and to investigate the effect of exercise (resistive exercise with and without vibration) on these changes. Twenty-four physically and mentally healthy male volunteers (20-45 years) performed resistive vibration exercise (n=7), resistance exercise without vibration (n=8) or no exercise (n=9) within 60 days of bed rest. Blood samples were taken 2 days before bed rest, on days 19 and 60 of bed rest. Composition of immune cells was analyzed by flow cytometry. Cytokines and neuroendocrine parameters were analyzed by Luminex technology and ELISA/RIA in plasma. General changes over time were identified by paired t-test, and exercise-dependent effects by pairwise repeated measurements (analysis of variance (ANOVA)). With all subjects pooled, the number of granulocytes, natural killer T cells, hematopoietic stem cells and CD45RA and CD25 co-expressing T cells increased and the number of monocytes decreased significantly during the study; the concentration of eotaxin decreased significantly. Different impacts of exercise were seen for lymphocytes, B cells, especially the IgD(+) subpopulation of B cells and the concentrations of IP-10, RANTES and DHEA-S. We conclude that prolonged bed rest significantly impacts immune cell populations and cytokine concentrations. Exercise was able to specifically influence different immunological parameters. In summary, our data fit the hypothesis of immunoprotection by exercise and may point toward even superior effects by resistive vibration exercise.

The initial inflammatory phase of bone fracture healing represents a critical step for the outcome of the healing process. However, both the mechanisms initiating this inflammatory phase and the function of immune cells present at the fracture site are poorly understood. In order to study the early events within a fracture hematoma, we established an in vitro fracture hematoma model: we cultured hematomas forming during an osteotomy (artificial bone fracture) of the femur during total hip arthroplasty (THA) in vitro under bioenergetically controlled conditions. This model allowed us to monitor immune cell populations, cell survival and cytokine expression during the early phase following a fracture. Moreover, this model enabled us to change the bioenergetical conditions in order to mimic the in vivo situation, which is assumed to be characterized by hypoxia and restricted amounts of nutrients. Using this model, we found that immune cells adapt to hypoxia via the expression of angiogenic factors, chemoattractants and pro-inflammatory molecules. In addition, combined restriction of oxygen and nutrient supply enhanced the selective survival of lymphocytes in comparison with that of myeloid derived cells (i.e., neutrophils). Of note, non-restricted bioenergetical conditions did not show any similar effects regarding cytokine expression and/or different survival rates of immune cell subsets. In conclusion, we found that the bioenergetical conditions are among the crucial factors inducing the initial inflammatory phase of fracture healing and are thus a critical step for influencing survival and function of immune cells in the early fracture hematoma.

Background and objectives MiR-31 is upregulated in repeatedly activated Th1 and in effector/memory Th lymphocytes isolated from the synovial fluid of patients suffering from rheumatic joint diseases. Several respective miR-31 targets are downregulated and essential for the cytoskeletal rearrangement of repeatedly activated Th1 cells. This suggests that miR-31 regulates the motility and immobilises proinflammatory Th1 cells in inflamed tissues. Materials and methods Assuming that Th cells involved in chronic inflammation have a history of repeated stimulation by autoantigens, we have in vitro generated four times activated Th1 cells. We have determined the transcriptomes and the migratory behaviour of these cells after Antagomir mediated miR-31 knockdown by using microarrays and in vitro transwell migration assays. For the identification of potential direct targets we have applied the target prediction algorithm TargetScan. In order to determine the putative function of miR-31 in proinflammatory Th1 cells a gene set enrichment (GSE) analysis of the obtained transcriptome data after miR-31 knockdown was performed. Results Specific inhibition of miR-31 in repeatedly activated Th1 cells promoted their migration up to 30% in a transwell migration assay. In sum, we have identified 283 differentially expressed genes in repeatedly activated Th1 cells after treatment with a miR-31 specific or an unspecific Antagomir for 36, 48 and 72 h. The GSE analysis revealed an enrichment of genes, which regulate cell-intrinsic and extrinsic pathways involved in motility and tissue localization. Among the putative direct targets we found actin binding LIM Protein 1 (Ablim1), Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Epsilon (Ywhae), actin gamma 1 (Actg1), actin beta (Actb), Lamellipodin (Raph1), T-cell lymphoma invasion and metastasis-inducing protein 1 (TIAM1) and Ras homolog gene family, member A (RhoA). From these genes, we could verify the upregulation of Actg1, Actb, Raph1 and RhoA after 48 to 72 h of Antagomir-31 treatment from 1.2 to 3 fold using quantitative RT-PCR. Conclusion We show that miR-31 restricts the motility of repeatedly activated Th1 cells in vitro most likely by repressing genes involved in cytoskeletal rearrangement. MiR-31 might represent a molecular switch important for the persistence of proinflammatory Th cells in inflamed tissues.

Background and objectives T helper type 1 (Th1) cells are involved in rheumatic diseases such as Crohn´s disease or rheumatoid arthritis and have a history of chronic autoantigenic stimulation. We have shown that Th1 cells adapt to chronic inflammation by upregulating the expression of the microRNA (miR)-148a. MiR-148a promotes the survival of chronically activated Th1 cells by regulating the expression of the proapoptotic protein Bim. Thus, we tested the suitability of miR-148a as a therapeutic target for the selective elimination of proinflammatory Th1 cells in a preclinical model of colitis. Methods Chronically activated Th1 cells were transferred into Rag1-deficient mice to induce colitis. Then, mice were intravenously injected with antagomirs that specifically target the microRNA-148a (antagomir-148a) or with control antagomirs (antagomir-scr). For assessing the function of T cell-intrinsic expression of miR-148a in colitis, we transduced Th1 cells with inducible microRNA sponges prior to transfer into Rag1-deficient mice. Colitic inflammation was determined by the weight-to-length ratios of colons. Colonic Th cells were sorted by FACS to measure miR-148a expression by qRT-PCR. The expression of Bim and Bcl-2 as well as numbers of viable Th1 cells, were determined by flow cytometry. Results Systemic inhibition of miR-148a by antagomirs alleviated colitis in mice as measured by reduced weight-to-length ratios of their colons. The numbers of viable Th1 cells were reduced up to 50% in mice that were treated with antagomir-148a when compared to mice that were injected with antagomir-scr. Antagomir-148a injections were efficient and resulted in a 30% reduction of miR-148a in colonic Th1 cells. The expression of the proapoptotic protein Bim was increased up to 30% in Th cells from antagomir-148a treated mice, while the anti-apoptotic protein Bcl-2 was unchanged. Th cell specific inhibition of miR-148a by miR-sponges during colitis led to a 30% reduction of Th cells in the colons of colitic mice and reduced the weight-to-length ratio. Conclusions We suggest that miR-148a controls the survival of proinflammatory Th1 cells in chronic inflammation by inhibiting Bim expression in a Th cell intrinsic fashion. Thus, miR-148a might represent a suitable target for the selective depletion of proinflammatory Th1 cells in chronic inflammation.

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes(1,2). Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A(1, 2). Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis(3). Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension(4). Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes(5). Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Hypoxia, a feature of inflammation and tumors, is a potent inducer of the proinflammatory cytokine macrophage migration inhibitory factor (MIF). In transformed cells, MIF was shown to modulate and to be modulated via the oxygen-sensitive transcription factor hypoxia-inducible factor (HIF)-1. Furthermore, anti-inflammatory glucocorticoids (GCs) were described to regulate MIF action. However, in-depth studies of the interaction between MIF and HIF-1 and GC action in nontransformed primary human CD4+ T cells under hypoxia are missing. Therefore, we investigated the functional relationship between MIF and HIF and the impact of the GC dexamethasone (DEX) on these key players of inflammation in human CD4+ T cells. In this article, we show that hypoxia, and specifically HIF-1, is a potent and rapid inducer of MIF expression in primary human CD4+ T cells, as well as in Jurkat T cells. MIF signaling via CD74, in turn, is essential for hypoxia-mediated HIF-1α expression and HIF-1 target gene induction involving ERK/mammalian target of rapamycin activity complemented by PI3K activation upon mitogen stimulation. Furthermore, MIF signaling enhances T cell proliferation under normoxia but not hypoxia. MIF also counterregulates DEX-mediated suppression of MIF and HIF-1α expression. Based on these data, we suggest that hypoxia significantly affects the expression of HIF-1α in a MIF-dependent manner leading to a positive-feedback loop in primary human CD4+ T cells, thus influencing the lymphoproliferative response and DEX action via the GC receptor. Therefore, we suggest that HIF and/or MIF could be useful targets to optimize GC therapy when treating inflammation.

Saposins or sphingolipid activator proteins (SAPs) are small, nonenzymatic glycoproteins that are ubiquitously present in lysosomes. SAPs comprise the five molecules saposins A-D and the GM2 activator protein. Saposins are essential for sphingolipid degradation and membrane digestion. On the one hand, they bind the respective hydrolases required to catabolize sphingolipid molecules; on the other hand, saposins can interact with intralysosomal membrane structures to render lipids accessible to their degrading enzymes. Thus, saposins bridge the physicochemical gap between lipid substrate and hydrophilic hydrolases. Accordingly, defects in saposin function can lead to lysosomal lipid accumulation. In addition to their specific functions in sphingolipid metabolism, saposins have membrane-perturbing properties. At the low pH of lysosomes, saposins get protonated and exhibit a high binding affinity for anionic phospholipids. Based on their universal principle to interact with membrane bilayers, we present the immunological functions of saposins with regard to lipid antigen presentation to CD1-restricted T cells, processing of apoptotic bodies for antigen delivery and cross-priming, as well as their potential antimicrobial impact.

Background Proinflammatory T helper (Th) cells are critically involved in the initiation and perpetuation of chronic inflammatory diseases (CID). In these diseases, proinflammatory Th cells persist in inflamed tissues and are refractory to current immunosuppressive therapies. The molecular factors regulating the survival of proinflammatory Th cells are not fully understood. Recently, we have shown that proinflammatory Th1 cells adapt to chronic inflammation by upregulating the expression of the microRNA (miRNA, miR)-148a 1 . MiR-148a promotes the survival of proinflammatory Th1 cells by regulating the expression of the pro-apoptotic protein Bim. Memory Th cells isolated from inflamed synovia of patients with rheumatoid arthritis induce the expression of miR-148a, presumably enhancing their survival. Therefore we hypothesize, that miR-148a could represent a therapeutic target for the selective elimination of proinflammatory Th1 cells in CID. Objectives To test the suitability of miR-148a as a therapeutic target in a preclinical murine model of transfer colitis. Methods We have generated a retroviral based inducible miR-148a inhibitor, containing complementary binding sites for miR-148a, termed “miR-148a sponge”. We transduced transgenic Th cells with the miR-148a sponge or a scrambled (scr) control sponge. Induction of sponge expression was initiated by tamoxifen treatment. Expression of miRNA sponges was detected by GFP reporter gene expression and the functionality of miR148a inhibition was determined by real-time PCR for the miR-148a target Bim. MiR-148a sponge-transduced CD4 + Th1 cells were FACS-sorted and transferred into Rag1 -/- mice. After manifestation of colitis, mice were sacrificed and T cells were re-isolated from the spleens and colons for determining their phenotype and number by FACS analysis. Results Expression of miR-148a sponges resulted in a 2 fold increase of Bim expression in activated Th1 cells in vitro . In addition, we found reduced numbers of miR-148a-sponge expressing Th cells as compared to scr-sponge expressing Th cells in the spleens and colons of colitic mice. Finally, miR-148a sponge-expressing Th cells from spleens and colons of colitic mice showed higher levels of Bim expression per cell than scr-sponge expressing cells. Conclusions Our data suggest that miR-148a controls the survival of pro-inflammatory Th1 cells in chronic inflammation by inhibiting Bim expression. Thus, therapeutic targeting of miR-148a probably is suitable for the selective depletion of proinflammatory Th1 cells in chronic inflammation. References Haftmann, C. et al. miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim. European journal of immunology, doi:10.1002/eji.201444633 (2014). Acknowledgements Patrick Maschmeyer is supported by EUTRAIN, a FP7 Marie Curie Initial Training Network for Early Stage Researchers funded by the European Union. Disclosure of Interest None declared

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. 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Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., 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., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).