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Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.Multispectral large-scale single-cell resolution 3D imaging allows up to eight fluorophores to be captured in a single acquisition. This protocol enables the visualization and exploration of large intact tissue volumes.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning–based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor’s spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors. An imaging software for large-scale microscopy data shows how cells are organized in tissues.

Our understanding of the cellular composition and architecture of cancer has primarily advanced using 2D models and thin slice samples. This has granted spatial information on fundamental cancer biology and treatment response. However, tissues contain a variety of interconnected cells with different functional states and shapes, and this complex organization is impossible to capture in a single plane. Furthermore, tumours have been shown to be highly heterogenous, requiring large‐scale spatial analysis to reliably profile their cellular and structural composition. Volumetric imaging permits the visualization of intact biological samples, thereby revealing the spatio‐phenotypic and dynamic traits of cancer. This review focuses on new insights into cancer biology uniquely brought to light by 3D imaging and concomitant progress in cancer modelling and quantitative analysis. 3D imaging has the potential to generate broad knowledge advance from major mechanisms of tumour progression to new strategies for cancer treatment and patient diagnosis. We discuss the expected future contributions of the newest imaging trends towards these goals and the challenges faced for reaching their full application in cancer research.

Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor's spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.

I was cynical at first, but it’s proved a powerful tool, says Ellen J. Wehrens. I was cynical at first, but it’s proved a powerful tool, says Ellen J. Wehrens.

During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA-sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants that controlled the fate specification of CD8+ T lymphocytes. Our findings suggest a model for the differentiation of terminal effector cells initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power and necessity of single-cell approaches.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

The role of IL-27 in antiviral immunity is still incompletely understood, especially in the context of chronic viruses that induce a unique environment in their infected host. Cytomegalovirus (CMV) establishes a persistent, tissue localized infection followed by lifelong latency. CMV infects the majority of people and although asymptomatic in healthy individuals, can cause serious disease or death in those with naïve or compromised immune systems. Therefore, there is an urgent need to develop a protective CMV vaccine for people at-risk and identifying key regulators of the protective immune response towards CMV will be crucial. Here we studied mouse CMV (MCMV) in IL-27 receptor deficient animals (Il27ra-/-) to assess the role of IL-27 in regulating CMV immunity. We found that IL-27 enhanced the number of antiviral CD4 T cells upon infection. However, in contrast to a well-established role for CD4 T cells in controlling persistent replication and a positive effect of IL-27 on their numbers, IL-27 promoted MCMV persistence in the salivary gland. This coincided with IL-27 mediated induction of IL-10 production in CD4 T cells. Moreover, IL-27 reduced expression of the transcription factor T-bet and restricted a cytotoxic phenotype in antiviral CD4 T cells. This is a highly intriguing result given the profound cytotoxic phenotype of CMV-specific CD4 T cells seen in humans and we established that dendritic cell derived IL-27 was responsible for this effect. Together, these data show that IL-27 regulates the number and effector functions of MCMV-specific CD4 T cells and could be targeted to enhance control of persistent/latent infection.

OBJECTIVE: Resistance of effector T cells (Teff) to regulatory T cell (Treg)-mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of Juvenile Idiopathic Arthritis (JIA) patients. However, an unanswered question is whether this resistant phenotype is self-sustained and whether CD8(+) and CD4(+) Teff share the same mechanism of resistance to suppression. Here, we investigated CD8(+) Teff intrinsic resistance to suppression and how this can be targeted therapeutically. METHODS: CD8(+) or CD4(+) Teff were cultured with or without antigen presenting cells (APC) in Treg-dependent and -independent suppression assays. Synovial fluid (SF)-derived Teff were cross-cultured with peripheral blood (PB) Treg from JIA patients or healthy controls. TNF-α or IFN-γ blocking agents were used to restore Teff responsiveness to suppression. RESULTS: Suppression of cell proliferation and cytokine production by CD8(+) Teff from the SF of JIA patients was severely impaired compared to PB of JIA patients, regardless of APC and CD4(+) Teff cell presence. Similarly to CD4(+) Teff, impaired suppression of CD8(+) Teff was shown to be an intrinsic feature of this cell population. Whereas TNF-α blockade rescued both CD8(+) and CD4(+) Teff resistance, autocrine release of IFN-γ selectively sustained CD8(+) Teff resistance, which could be relieved by IFN-γ blockade. CONCLUSION: Unlike CD4(+) Teff, resistance of CD8(+) Teff to suppression at the site of autoimmune inflammation is maintained by autocrine release of IFN-γ and blockade of IFN-γ restores CD8(+) Teff responsiveness to suppression. These findings indicate a potential therapeutic value of blocking IFN-γ to restore immune regulation in JIA. This article is protected by copyright. All rights reserved.

Chronic viral infections represent a major challenge to the host immune response, and a unique network of immunological elements, including cytokines, are required for their containment. By using a model persistent infection with the natural murine pathogen lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) we investigated the role of one such cytokine, interleukin-27 (IL-27), in the control of chronic infection. We found that IL-27 receptor (IL-27R) signaling promoted control of LCMV Cl13 as early as days 1 and 5 after infection and that il27p28 transcripts were rapidly elevated in multiple subsets of dendritic cells (DCs) and myeloid cells. In particular, plasmacytoid DCs (pDCs), the most potent type 1 interferon (IFN-I)-producing cells, significantly increased il27p28 in a Toll-like receptor 7 (TLR7)-dependent fashion. Notably, mice deficient in an IL-27-specific receptor, WSX-1, exhibited a pleiotropy of innate and adaptive immune alterations after chronic lymphocytic choriomeningitis virus (LCMV) infection, including compromised NK cell cytotoxicity and antibody responses. While, the majority of these immune alterations appeared to be cell extrinsic, cell-intrinsic IL-27R was necessary to maintain early pDC numbers, which, alongside lower IFN-I transcription in CD11b+ DCs and myeloid cells, may explain the compromised IFN-I elevation that we observed early after LCMV Cl13 infection in IL-27R-deficient mice. Together, these data highlight the critical role of IL-27 in enabling optimal antiviral immunity early and late after infection with a systemic persistent virus and suggest that a previously unrecognized positive-feedback loop mediated by IL-27 in pDCs might be involved in this process. IMPORTANCE Persistently replicating pathogens, such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, represent major health problems worldwide. These infections impose a long-term challenge on the host immune system, which must be heavily and continuously regulated to keep pathogen replication in check without causing fatal immunopathology. Using a persistently replicating rodent pathogen, LCMV, in its natural host, we identified the cellular sources and effects of one important regulatory pathway, interleukin-27 receptor WSX-1 signaling, that is required for both very early and late restriction of chronic (but not acute) infection. We found that WSX-1 was necessary to promote innate immunity and the development of aberrant adaptive immune responses. This not only highlights the role of IL-27 receptor signaling in regulating distinct host responses that are known to be necessary to control chronic infections, but also positions IL-27 as a potential therapeutic target for their modulation.

Although characterization of Tcell exhaustion has unlocked powerful immunotherapies, the mechanisms sustaining adaptations of short-lived innate cells to chronic inflammatory settings remain unknown. During murine chronic viral infection, we found that concerted events in bone marrow and spleen mediated by type I interferon (IFN-I) and Toll-like receptor 7 (TLR7) maintained a pool of functionally exhausted plasmacytoid dendritic cells (pDCs). In the bone marrow, IFN-I compromised the number and the developmental capacity of pDC progenitors, which generated dysfunctional pDCs. Concurrently, exhausted pDCs in the periphery were maintained by self-renewal via IFN-I- and TLR7-induced proliferation of CD4- subsets. On the other hand, pDC functional loss was mediated by TLR7, leading to compromised IFN-I production and resistance to secondary infection. These findings unveil the mechanisms sustaining a self-perpetuating pool of functionally exhausted pDCs and provide a framework for deciphering long-term exhaustion of other short-lived innate cells during chronic inflammation.

Objective To determine whether therapeutic strategies that block interleukin-6 (IL-6) or tumor necrosis factor α (TNFα) can improve the responsiveness of Teff cells to suppression in patients with juvenile idiopathic arthritis (JIA). Methods Synovial fluid mononuclear cells (SFMCs) from the inflamed joints of patients with JIA were cultured in the presence of etanercept or anti–IL-6 in vitro, and protein kinase B (PKB)/c-Akt activation and responsiveness to suppression were measured. In addition, the in vivo effects of TNFα blockade were investigated using peripheral blood mononuclear cells obtained from patients before and after the start of etanercept therapy. Results In vitro treatment of SFMCs with anti–IL-6 led to improved Treg cell–mediated suppression of cell proliferation in some but not all patients. Blocking TNFα with etanercept, however, clearly enhanced suppression, especially that of CD8+ T cells. In the presence of etanercept, PKB/c-Akt activation of Teff cells was reduced, and cells became more susceptible to transforming growth factor β–mediated suppression, indicating that anti-TNFα directly targets resistant Teff cells. Conclusion This study is the first to show that anti-TNFα targets the resistance of Teff cells to suppression, resulting in improved regulation of inflammatory effector cells.

Autologous hematopoietic stem cell transplantation (HSCT) is increasingly considered for patients with severe autoimmune diseases whose prognosis is poor with standard treatments. Regulatory T cells (Tregs) are thought to be important for disease remission after HSCT. However, eliciting the role of donor and host Tregs in autologous HSCT is not possible in humans due to the autologous nature of the intervention. Therefore, we investigated their role during immune reconstitution and re-establishment of immune tolerance and their therapeutic potential following congenic bone marrow transplantation (BMT) in a proteoglycan-induced arthritis (PGIA) mouse model. In addition, we determined Treg T-cell receptor (TCR) CDR3 diversity before and after HSCT in patients with juvenile idiopathic arthritis and juvenile dermatomyositis. In the PGIA BMT model, after an initial predominance of host Tregs, graft-derived Tregs started dominating and displayed a more stable phenotype with better suppressive capacity. Patient samples revealed a striking lack of diversity of the Treg repertoire before HSCT. This ameliorated after HSCT, confirming reset of the Treg compartment following HSCT. In the mouse model, a therapeutic approach was initiated by infusing extra Foxp3(GFP+) Tregs during BMT. Infusion of Foxp3(GFP+) Tregs did not elicit additional clinical improvement but conversely delayed reconstitution of the graft-derived T-cell compartment. These data indicate that HSCT-mediated amelioration of autoimmune disease involves renewal of the Treg pool. In addition, infusion of extra Tregs during BMT results in a delayed reconstitution of T-cell compartments. Therefore, Treg therapy may hamper development of long-term tolerance and should be approached with caution in the clinical autologous setting.

Suppression of CD8 and CD4 T cells is a hallmark in chronic viral infections, including hepatitis C and HIV. While multiple pathways are known to inhibit CD8 T cells, the host molecules that restrict CD4 T cell responses are less understood. Here, we used inducible and CD4 T cell–specific deletion of the gene encoding the TGF-β receptor during chronic lymphocytic choriomeningitis virus infection in mice, and determined that TGF-β signaling restricted proliferation and terminal differentiation of antiviral CD4 T cells. TGF-β signaling also inhibited a cytotoxic program that includes granzymes and perforin expression at both early and late stages of infection in vivo and repressed the transcription factor eomesodermin. Overexpression of eomesodermin was sufficient to recapitulate in great part the phenotype of TGF-β receptor–deficient CD4 T cells, while SMAD4 was necessary for CD4 T cell accumulation and differentiation. TGF-β signaling also restricted accumulation and differentiation of CD4 T cells and reduced the expression of cytotoxic molecules in mice and humans infected with other persistent viruses. These data uncovered an eomesodermin-driven CD4 T cell program that is continuously suppressed by TGF-β signaling. During chronic viral infection, this program limits CD4 T cell responses while maintaining CD4 T helper cell identity.

Since the discovery of FOXP3+ regulatory T (T(REG)) cells over 15 years ago, intensive research has focused on their presence, phenotype and function in autoimmune disease. Whether deficiencies in T(REG) cells underlie autoimmune pathology and whether, or how, therapeutic approaches based on these cells might be successful is still the subject of debate. The potential role of T(REG)-cell extrinsic factors, such as proinflammatory cytokines and resistance of effector T cells to suppression, as the cause of regulatory defects in chronic autoimmune inflammation is an intensive area of research. It is now clear that, at the site of inflammation, antigen presenting cells (APCs) and proinflammatory cytokines drive effector T cell skewing and plasticity, and that these T cells can become unresponsive to regulation. In addition, expansion and function of T(REG) cells is affected by the inflammatory environment; indeed, new data suggest that, in certain conditions, T(REG) cells promote inflammation. This Review summarizes the latest findings on changes in effector T cell homeostasis in autoimmune disease and focuses on how mechanisms that normally regulate these cells are affected in the inflamed joints of patients with arthritis. These findings have important clinical implications and will affect the development of new therapeutic strategies for autoimmune arthritis.

During the last decade research has focused on the application of FOXP3+ regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well.

The discovery of regulatory T cells almost 15 years ago initiated a new and exciting research area. The growing evidence for a critical role of these cells in controlling autoimmune responses has raised expectations for therapeutic application of regulatory T cells in patients with autoimmune arthritis. Here, we review recent studies investigating the presence, phenotype and function of these cells in patients with RA and juvenile idiopathic arthritis (JIA) and consider their therapeutic potential. Both direct and indirect methods to target these cells will be discussed. Arguably, a therapeutic approach that combines multiple regulatory T-cell-enhancing strategies could be most successful for clinical application.

Contains fulltext : 51645.pdf (Publisher’s version ) (Closed access) Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.

Resistance of Teff cells to Treg cell-mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). However, unanswered questions are whether this resistant phenotype is self-sustained and whether CD8+ and CD4+ Teff cells share the same mechanism of resistance to suppression. We undertook this study to investigate intrinsic resistance of CD8+ Teff cells to suppression and to determine how this can be targeted therapeutically.CD8+ or CD4+ Teff cells were cultured with or without antigen-presenting cells (APCs) in Treg cell-dependent and -independent suppression assays. Synovial fluid (SF)-derived Teff cells were crosscultured with peripheral blood (PB) Treg cells from JIA patients or healthy controls. Tumor necrosis factor (TNF) or interferon-γ (IFNγ) blocking agents were used to restore Teff cell responsiveness to suppression.Suppression of cell proliferation and cytokine production in CD8+ Teff cells from the SF of JIA patients was severely impaired compared to that in CD8+ Teff cells from the PB of JIA patients, regardless of the presence of APCs and CD4+ Teff cells. Similar to CD4+ Teff cells, impaired suppression of CD8+ Teff cells was shown to be an intrinsic feature of this cell population. While TNF blockade restored both CD8+ and CD4+ Teff cell susceptibility to suppression, autocrine release of IFNγ selectively sustained CD8+ Teff cell resistance, which could be relieved by IFNγ blockade.Unlike CD4+ Teff cells, resistance of CD8+ Teff cells to suppression at the site of autoimmune inflammation is maintained by autocrine release of IFNγ, and blockade of IFNγ restores CD8+ Teff cell responsiveness to suppression. These findings indicate a potential therapeutic value of blocking IFNγ to restore immune regulation in JIA.

Autologous bone marrow transplantation (aBMT) is a last resort treatment for patients with refractory juvenile idiopathic arthritis (JIA). It can induce disease remission for 80 months after transplantation. Unfortunately, relapses also occur. The underlying working mechanisms remain largely unknown.

Cytomegalovirus (CMV) is a large DNA β-herpes virus that infects the majority of humans and is endemic in wild mice. CMV establishes life-long latency with sporadic reactivation, and both primary infection and reactivation can cause serious disease in patients with compromised or naïve immunity. Horizontal CMV transmission commonly occurs through prolonged replication in the salivary gland (SG). In mice, CD4 T cells are absolutely required to curtail SG persistence, whereas CD8 T cells control acute systemic infection. Both CD4 and CD8 CMV-specific T cells from infected individuals display a strong cytotoxic phenotype and are able to kill target cells ex vivo. However, the immune factors that promote or regulate differentiation of these cytotoxic T cells remain largely unclear. Here we show that the pleiotropic cytokine IL-27 plays a key role in regulating CMV-specific T cell responses. Infection of mice deficient for the IL-27 receptor a subunit (Il27ra−/−) with mouse CMV (MCMV), resulted in enhanced expression of KLRG1 by both CD4 and CD8 T cells, a marker associated with terminal differentiation and cytotoxicity. Consistently, MCMV-specific CD4 and CD8 T cells from Il27ra−/− mice showed increased expression of both granzyme A and FasL. Importantly, this enhanced cytotoxic phenotype correlated with improved control of MCMV persistence in the SG. Taken together, our data reveal an unexpected and novel role for IL-27 in restricting T cell cytotoxicity and control of MCMV persistence, and suggest that targeting IL-27 has the potential to limit disease caused by persistent viral infection.

To determine whether therapeutic strategies that block interleukin-6 (IL-6) or tumor necrosis factor α (TNFα) can improve the responsiveness of Teff cells to suppression in patients with juvenile idiopathic arthritis (JIA).Synovial fluid mononuclear cells (SFMCs) from the inflamed joints of patients with JIA were cultured in the presence of etanercept or anti-IL-6 in vitro, and protein kinase B (PKB)/c-Akt activation and responsiveness to suppression were measured. In addition, the in vivo effects of TNFα blockade were investigated using peripheral blood mononuclear cells obtained from patients before and after the start of etanercept therapy.In vitro treatment of SFMCs with anti-IL-6 led to improved Treg cell-mediated suppression of cell proliferation in some but not all patients. Blocking TNFα with etanercept, however, clearly enhanced suppression, especially that of CD8+ T cells. In the presence of etanercept, PKB/c-Akt activation of Teff cells was reduced, and cells became more susceptible to transforming growth factor β-mediated suppression, indicating that anti-TNFα directly targets resistant Teff cells.This study is the first to show that anti-TNFα targets the resistance of Teff cells to suppression, resulting in improved regulation of inflammatory effector cells.

Background FOXP3+ regulatory T cells (Treg) are critical in maintaining self tolerance and are therefore considered important targets for the treatment of autoimmune disease. However, environmental factors at the site of autoimmune inflammation, such as enhanced costimulatory potential of antigen presenting cells (APC) and increased proinflammatory cytokine production, can negatively affect Treg function, thereby limiting effectiveness of these Treg targeted approaches. Aim Here we studied the phenotype of APC present at the site of inflammation in patients with Juvenile Idiopathic Arthritis (JIA) and investigated whether these cells can interfere with Treg mediated suppression. Methods Mononuclear cells were isolated from peripheral blood (PB) of healthy controls (HC) and from paired PB and synovial fluid (SF) of JIA patients. The phenotype of APC was analysed using flow cytometry. In vitro suppression assays were performed to study T cell activation and Treg mediated suppression in the presence of SF and PB derived APC. Results Monocytes from the site of inflammation displayed a more pro-inflammatory phenotype, with significantly increased costimulatory molecule expression, compared to monocytes from PB. In line with this pro-inflammatory phenotype, SF APC induced enhanced proliferation of effector cells and decreased suppression of effector cell proliferation in the presence of Treg. Conclusions APC from the site of inflammation have an enhanced stimulatory capacity that interferes with Treg mediated suppression. Therefore, this increased stimulatory potential should be targeted as well, in order for a Treg enhancing approach to be fully effective in the treatment of autoimmune inflammation.

Contains fulltext : 52899.pdf (Publisher’s version ) (Closed access) BACKGROUND: Naturally occurring CD4+CD25+ regulatory T cells (Tregs) play a critical role in the maintenance of self-tolerance and it has been suggested that these Tregs may also be involved in preventing allergic disease. OBJECTIVE: The precise role of CD4+CD25+ T cells in the regulation of allergic responses to mucosal antigens remains to be elucidated. In the present study, it was investigated whether CD4+CD25+ T cells are involved in the induction of oral tolerance and whether they play a role in controlling hypersensitivity responses to food proteins. METHODS: CD4+CD25+ T cells were depleted with PC61 mAb before the induction of low dose oral tolerance to peanut extract (PE). In addition, CD4+CD25+ T cell depletion was performed during sensitization or before oral challenge, using a C3H/HeOuJ mouse model of allergic sensitization to peanut. RESULTS: Oral tolerance to PE could not be induced in CD4+CD25+ T cell-depleted mice. However, CD4+CD25+ T cell depletion during long-term exposure to PE alone did not result in allergic sensitization. In sensitized mice, anti-CD25 treatment during oral exposure resulted in higher levels of PE-specific IgE and increased mast cell degranulation upon an oral challenge. In contrast, anti-CD25 treatment of PE-sensitized mice before oral challenges did not affect the level of mast cell degranulation. CONCLUSION: These results indicate that CD4+CD25+ Tregs are involved in maintaining tolerance to oral antigens and regulate the intensity of an IgE-mediated food hypersensitivity response, but are not crucial in preventing sensitization. Accordingly, CD4+CD25+ Tregs may represent a potential tool for the treatment of food allergic disorders.

Ever since their discovery research has focused on whether deficiencies in FOXP3+ regulatory T cells (Treg) underlie human autoimmune pathology. Very recently however, the topic of Treg extrinsic factors as the cause of regulatory defects in chronic autoimmune inflammation has become more prominent in the discussion. It has become clear that resistance of effector cells (Teff) to suppression contributes to disturbed immune regulation in autoimmune inflammation, especially at the site of inflammation. Therefore, targeting this unresponsiveness to suppression could be a promising treatment option for patients with autoimmune disease. It remains unknown how resistance of T cells to suppression is induced.

Background Resistance of effector T cells (Teff) to suppression contributes to disturbed immune regulation in autoimmune disease. Targeting this unresponsiveness to suppression might therefore have beneficial effects in autoimmune inflammation1. In juvenile idiopathic arthritis (JIA) we have recently shown that Teff from inflamed joints are refractory to suppression, which was associated with enhanced PKB/c-akt activation in these cells2. Objectives To investigate whether anti-IL-6 and anti-TNFa influence unresponsiveness of Teff to suppression in patients with JIA. Methods Resistant Teff from the inflamed joints of JIA patients were cultured in the presence of etanercept or anti-IL-6 in vitro and PKB/c-akt activation and responsiveness to suppression was measured. In addition, in vivo effects of TNFa blockade were investigated using peripheral blood samples of patient before and after start of etanercept therapy. Results In vitro treatment of synovial fluid Teff with anti-IL-6 led to improved Treg mediated suppression of cell proliferation in some, but not all patients. Blocking TNFa with etanercept however clearly enhanced suppression in all samples analyzed. In the presence of etanercept PKB/c-akt activation of Teff was reduced and Teff became more susceptible to TGFb mediated suppression, indicating that anti-TNFa directly targets resistant Teff. This was confirmed by ex vivo data from patients treated with etanercept demonstrating enhanced responsiveness of effector cells to suppression after therapy. Conclusions This study is the first to show resistance of Teff to suppression as a target of anti-TNFatherapy in arthritis, resulting in improved regulation of inflammatory effector cells. References Wehrens EJ, Prakken BJ, van WF. T cells out of control-impaired immune regulation in the inflamed joint. Nat.Rev.Rheumatol. Nat Rev Rheumatol. 2012 Sep 18;9(1):34-42. Wehrens EJ, Mijnheer G, Duurland CL et al. Functional human regulatory T cells fail to control autoimmune inflammation due to PKB/c-akt hyperactivation in effector cells. Blood 2011;118:3538-3548. Disclosure of Interest None Declared

Background Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists. Results Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre‐tubular aggregates, renal vesicles, and in segments of S‐shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single‐cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long‐term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons. Conclusions Given the profound early mesenchymal expression and MET signature of LGR6+ cells, together with the lineage tracing of mesenchymal LGR6+ cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability., Key Findings Lgr6 is expressed in the earliest cap mesenchyme pool, a niche where nephrogenic progenitor cells (NPCs) are found.Lgr6 marks NPCs undergoing mesenchymal to epithelial transition, following the main process of nephron development.Using ex vivo and vivo lineage tracing, we show that mesenchymal Lgr6 expressing cells give rise to multiple types of mesenchymal derived nephron segments, including specialized glomerular epithelium, such as podocytes.

Resistance of effector T cells (Teff) to suppression contributes to disturbed immune regulation in autoimmune disease. Targeting this unresponsiveness to suppression might therefore have beneficial effects in autoimmune inflammation. In juvenile idiopathic arthritis (JIA) we have recently shown that Teff from inflamed joints are refractory to suppression, which was associated with enhanced PKB/c-akt activation in these cells.

Autologous stem cell transplantation (ASCT) is a last resort treatment for refractory juvenile idiopathic arthritis (JIA). It has been shown to induce dramatic and long-term improvements, but the underlying mechanisms remain to be elucidated. We investigated the potential role of regulatory T cells (Treg) in the processes of immune reconstitution and re-establishment of immune tolerance following ASCT. In a mouse model of proteoglycan-induced arthritis, (pseudo)ASCT following lethal irradiation reduced arthritis scores and restored the immune balance of pro-inflammatory effector T cells and Treg. Directly following the ASCT the majority of Treg consisted of Treg that survived the conditioning and had expanded vigorously. After a few weeks the infused stem cell-derived Treg started dominating the Treg pool and these “new” thymus-derived Treg showed more suppressive capacity than the remaining host Treg including a more stable expression of Foxp3. A therapeutic approach was initiated by infusing extra Foxp3GFPTreg together with the stem cells graft. These Treg expanded vigorously and were still present two months after the ASCT. However, by infusion of Treg the induction of stem cell-derived Treg was delayed and also the restoration of immune tolerance was impaired. These data indicate that restoration of the immune balance following ASCT depends on renewal of the natural Treg pool. Furthermore, infusion of Treg during ASCT seems to have a long-term negative effect on T cell reconstitution and re-establishment of immune tolerance.

Autoimmune diseases are characterized by an imbalance between regulatory T cells (Treg) and effector T cells (Teff) both in terms of number and function. Our group previously showed that Teff from the site of inflammation (i.e. synovial fluid, SF) of patients affected by Juvenile Idiopathic Arthritis (JIA) are resistant to autologous Treg-mediated suppression irrespective of the source of Tregs (SF or peripheral blood(PB)-derived). However, it is still unclear whether resistance to suppression is an intrinsic characteristic of SF-derived Teff or it is induced/maintained by local pro-inflammatory antigen presenting cells (APC).