Thymic regulatory T cells arise via two distinct developmental programs.

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.

Insulin-Like Growth Factors Are Key Regulators of T Helper 17 Regulatory T Cell Balance in Autoimmunity.

Appropriate balance of T helper 17 (Th17) and regulatory T (Treg) cells maintains immune tolerance and host defense. Disruption of Th17-Treg cell balance is implicated in a number of immune-mediated diseases, many of which display dysregulation of the insulin-like growth factor (IGF) system. Here, we show that, among effector T cell subsets, Th17 and Treg cells selectively expressed multiple components of the IGF system. Signaling through IGF receptor (IGF1R) activated the protein kinase B-mammalian target of rapamycin (AKT-mTOR) pathway, increased aerobic glycolysis, favored Th17 cell differentiation over that of Treg cells, and promoted a heightened pro-inflammatory gene expression signature. Group 3 innate lymphoid cells (ILC3s), but not ILC1s or ILC2s, were similarly responsive to IGF signaling. Mice with deficiency of IGF1R targeted to T cells failed to fully develop disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Thus, the IGF system represents a previously unappreciated pathway by which type 3 immunity is modulated and immune-mediated pathogenesis controlled.

CCR6 Expression on B Cells Is Not Required for Clinical or Pathological Presentation of MOG Protein-Induced Experimental Autoimmune Encephalomyelitis despite an Altered Germinal Center Response.

B cells have been implicated in the pathogenesis of multiple sclerosis, but the mechanisms that guide B cell activation in the periphery and subsequent migration to the CNS remain incompletely understood. We previously showed that systemic inflammation induces an accumulation of B cells in the spleen in a CCR6/CCL20-dependent manner. In this study, we evaluated the role of CCR6/CCL20 in the context of myelin oligodendrocyte glycoprotein (MOG) protein-induced (B cell-dependent) experimental autoimmune encephalomyelitis (EAE). We found that CCR6 is upregulated on murine B cells that migrate into the CNS during neuroinflammation. In addition, human B cells that migrate across CNS endothelium in vitro were found to be CCR6+, and we detected CCL20 production by activated CNS-derived human endothelial cells as well as a systemic increase in CCL20 protein during EAE. Although mice that lack CCR6 expression specifically on B cells exhibited an altered germinal center reaction in response to MOG protein immunization, CCR6-deficient B cells did not exhibit any competitive disadvantage in their migration to the CNS during EAE, and the clinical and pathological presentation of EAE induced by MOG protein was unaffected. These data, to our knowledge, provide new information on the role of B cell-intrinsic CCR6 expression in a B cell-dependent model of neuroinflammation.

Tolerogenic nanoparticles suppress central nervous system inflammation.

Therapeutic approaches for the induction of immune tolerance remain an unmet clinical need for the treatment of autoimmune diseases, including multiple sclerosis (MS). Based on its role in the control of the immune response, the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is a candidate target for novel immunotherapies. Here, we report the development of AhR-activating nanoliposomes (NLPs) to induce antigen-specific tolerance. NLPs loaded with the AhR agonist ITE and a T cell epitope from myelin oligodendrocyte glycoprotein (MOG)35-55 induced tolerogenic dendritic cells and suppressed the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, in preventive and therapeutic setups. EAE suppression was associated with the expansion of MOG35-55-specific FoxP3+ regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells), concomitant with a reduction in central nervous system-infiltrating effector T cells (Teff cells). Notably, NLPs induced bystander suppression in the EAE model established in C57BL/6 × SJL F1 mice. Moreover, NLPs ameliorated chronic progressive EAE in nonobese diabetic mice, a model which resembles some aspects of secondary progressive MS. In summary, these studies describe a platform for the therapeutic induction of antigen-specific tolerance in autoimmune diseases.

Loss of Phosphatidylinositol 3-Kinase Activity in Regulatory T Cells Leads to Neuronal Inflammation.

Class I PI3K enzymes are critical for the maintenance of effective immunity. In T cells, PI3Kα and PI3Kδ are activated by the TCR and costimulatory receptors, whereas PI3Kγ is activated by G protein-coupled chemokine receptors. PI3Kδ is a key regulator of regulatory T (Treg) cell function. PI3K isoform-selective inhibitors are in development for the treatment of diseases associated with immune dysregulation, including chronic inflammatory conditions, cancer, and autoimmune diseases. Idelalisib (PI3Kδ), alpelisib (PI3Kα), duvelisib (PI3Kδ/γ), and copanlisib (pan-PI3K) have recently been approved for use in cancer treatment. Although effective, these therapies often have severe side effects associated with immune dysregulation and, in particular, loss of Treg cells. Therefore, it is important to gain a better understanding of the relative contribution of different PI3K isoforms under homeostatic and inflammatory conditions. Experimental autoimmune encephalitis is a mouse model of T cell-driven CNS inflammation, in which Treg cells play a key protective role. In this study, we show that PI3Kδ is required to maintain normal Treg cell development and phenotype under homeostatic conditions but that loss of PI3Kδ alone in Treg cells does not lead to autoimmunity. However, combined loss of PI3Kα and PI3Kδ signaling resulted in increased experimental autoimmune encephalitis disease severity. Moreover, mice lacking PI3Kα and PI3Kδ in Treg cells developed spontaneous peripheral nerve inflammation. These results show a key role for PI3K signaling in Treg cell-mediated protection against CNS inflammation.

Vascular and immunopathological role of Asymmetric Dimethylarginine (ADMA) in Experimental Autoimmune Encephalomyelitis.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor/uncoupler inducing vascular pathology. Vascular pathology is an important factor for the development and progression of CNS pathology of MS, yet the role of ADMA in MS remains elusive. Patients with multiple sclerosis (MS) are reported to have elevated blood levels of ADMA, and mice with experimental autoimmune encephalomyelitis (EAE, an animal model of MS) generated by auto-immunization of myelin oligodendrocyte glycoprotein (MOG) and blood-brain barrier (BBB) disruption by pertussis toxin also had increased blood ADMA levels in parallel with induction of clinical disease. To explore the role of ADMA in EAE pathogenesis, EAE mice were treated with a daily dose of ADMA. It is of special interest that ADMA treatment enhanced the BBB disruption in EAE mice and exacerbated the clinical and CNS disease of EAE. ADMA treatment also induced the BBB disruption and EAE disease in MOG-immunized mice even without pertussis toxin treatment, suggesting the role of ADMA in BBB dysfunction in EAE. T-cell polarization studies also documented that ADMA treatment promotes TH 1- and TH 17-mediated immune responses but without affecting Treg-mediated immune response in EAE mice as well as in in vitro T-cell culture. Taken together, these data, for the first time, document the vascular and immunopathogenic roles of ADMA in EAE, thus pointing to the potential of ADMA-mediated mechanism as a new target of potential therapy for MS.

Pertussis Toxin Inhibits Encephalitogenic T-Cell Infiltration and Promotes a B-Cell-Driven Disease during Th17-EAE.

Pertussis toxin (PTX) is a required co-adjuvant for experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin antigen. However, PTX's effects on EAE induced by the transfer of myelin-specific T helper cells is not known. Therefore, we investigated how PTX affects the Th17 transfer EAE model (Th17-EAE). We found that PTX significantly reduced Th17-EAE by inhibiting chemokine-receptor-dependent trafficking of Th17 cells. Strikingly, PTX also promoted the accumulation of B cells in the CNS, suggesting that PTX alters the disease toward a B-cell-dependent pathology. To determine the role of B cells, we compared the effects of PTX on Th17-EAE in wild-type (WT) and B-cell-deficient (µMT) mice. Without PTX treatment, disease severity was equivalent between WT and µMT mice. In contrast, with PTX treatment, the µMT mice had significantly less disease and a reduction in pathogenic Th17 cells in the CNS compared to the WT mice. In conclusion, this study shows that PTX inhibits the migration of pathogenic Th17 cells, while promoting the accumulation of pathogenic B cells in the CNS during Th17-EAE. These data provide useful methodological information for adoptive-transfer Th17-EAE and, furthermore, describe another important experimental system to study the pathogenic mechanisms of B cells in multiple sclerosis.

Increase in Autoantibodies-Abzymes with Peroxidase and Oxidoreductase Activities in Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology.

The exact mechanisms of multiple sclerosis (MS) development are still unknown, but the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice is associated with the violation of bone marrow hematopoietic stem cells (HSCs) differentiation profiles associated with the production of harmful for human's autoantibodies hydrolyzing myelin basic protein, myelin oligodendrocyte glycoprotein (MOG35-55), and DNA. It was shown that IgGs from the sera of healthy humans and autoimmune patients oxidize many different compounds due to their H2O2-dependent peroxidase and oxidoreductase activity in the absence of H2O2. Here we first analyzed the change in the relative redox activities of IgGs antibodies from the blood of C57BL/6 mice over time at different stages of the EAE development. It was shown that the peroxidase activity of mice IgGs in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is on average 6.9-fold higher than the oxidoreductase activity. The peroxidase activity of IgGs increased during the spontaneous development of EAE during 40 days, 1.4-fold. After EAE development acceleration due to mice immunization with MOG35-55 (5.3-fold), complexes of bovine DNA with methylated bovine serum albumin (DNA-metBSA; 3.5-fold), or with histones (2.6-fold), the activity was increased much faster. The increase in peroxidase activity after mice immunization with MOG35-55 and DNA-metBSA up to 40 days of experiments was relatively gradual, while for DNA-histones complex was observed its sharp increase at the acute phase of EAE (14-20 days). All data show that IgGs' redox activities can play an important role in the protection of mice from toxic compounds and oxidative stress.

CD49d/CD29-integrin controls the accumulation of plasmacytoid dendritic cells into the CNS during neuroinflammation.

Plasmacytoid dendritic cells (pDCs) are found in the CNS during neuroinflammation and have been reported to exert regulatory functions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of entry of pDCs into the CNS as well as their phenotype and innate functional properties, once recruited into the CNS, have not been thoroughly examined. Herein, we show that pDCs rapidly accumulate into the brain and spinal cord during the acute phase of EAE, and maintain the expression of numerous phenotypic markers typical of peripheral pDCs. Functionally, CNS-pDCs constitutively expressed IRF7 and were able to rapidly produce type I IFNs and IL-12p40 upon ex vivo TLR-9 stimulation. Using adoptive transfer experiments, we provide evidence that CNS-pDC are recruited from the blood and accumulate into the CNS during the acute phase of EAE. Accumulation of pDCs into the CNS was strongly inhibited in the absence of CD29, but not CD18, suggesting a major role for ß1 but not ß2 integrins. Indeed, blocking the CD49d α4-integrins during acute EAE drastically diminished CNS-pDC numbers. Together, our results demonstrate that circulating pDCs are actively recruited into the CNS during acute EAE through a mechanism largely dependent on CD49d/CD29-integrins.

GM-CSF Promotes Chronic Disability in Experimental Autoimmune Encephalomyelitis by Altering the Composition of Central Nervous System-Infiltrating Cells, but Is Dispensable for Disease Induction.

GM-CSF has been portrayed as a critical cytokine in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and, ostensibly, in multiple sclerosis. C57BL/6 mice deficient in GM-CSF are resistant to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 The mechanism of action of GM-CSF in EAE is poorly understood. In this study, we show that GM-CSF augments the accumulation of MOG35-55-specific T cells in the skin draining lymph nodes of primed mice, but it is not required for the development of encephalitogenic T cells. Abrogation of GM-CSF receptor signaling in adoptive transfer recipients of MOG35-55-specific T cells did not alter the incidence of EAE or the trajectory of its initial clinical course, but it limited the extent of chronic CNS tissue damage and neurologic disability. The attenuated clinical course was associated with a relative dearth of MOG35-55-specific T cells, myeloid dendritic cells, and neutrophils, as well as an abundance of B cells, within CNS infiltrates. Our data indicate that GM-CSF drives chronic tissue damage and disability in EAE via pleiotropic pathways, but it is dispensable during early lesion formation and the onset of neurologic deficits.

Clinical and immunological control of experimental autoimmune encephalomyelitis by tolerogenic dendritic cells loaded with MOG-encoding mRNA.

BACKGROUND: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. METHODS: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. RESULTS: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. CONCLUSIONS: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.

A Confocal Reflection Super-Resolution Technique to Image Golgi-Cox Stained Neurons.

Metal-based Golgi-Cox (GC) staining is an established method used to visualise neurons with great morphological detail. Although GC stained samples are imaged routinely under transmitted light microscopy, this method is unable to yield information on the three-dimensional structure of dendrites and neurons and thus help reveal the connective properties of the central nervous system. Although a few studies have attempted simultaneous visualisation of GC staining and antigen-specific fluorescent labelling under a confocal reflection technique, the resolution of both confocal reflection and fluorescence modalities used to acquire GC reflection and fluorescently stained antibody signals are still limited by the diffraction limit of light at about 220 nm. Here, we report a confocal reflection super-resolution technique (CRSR) to break this diffraction barrier, which is achieved by minimising the pinhole size from 1 airy unit (AU) to 0.1 AU. This is achieved by minimising or closing the confocal pinhole size and is possible in this reflection modality, unlike fluorescence, because it is not a photon limited technique. Utilising the lowest wavelength of light available in the system (405 nm), the CRSR technique results in ∼30% lateral and axial resolution improvement. We also show that the CRSR technique can be used in conjunction to visualise both GC and immunofluorescence targets to create precise and improved three-dimensional visualisation and analysis. In addition, using these superresolution confocal reflection data sets from GC in CRSR mode significantly reduced the data overestimation, improving the accuracy of statistical analysis of dendritic spine density and average spine dimensions. Combining the 0.1 AU setting with deconvolution routines, the signal-to-noise ratio and resolution could further be improved an additional ∼20-25%, yielding CRSR images with resolutions up to 2-fold over the diffraction limit both laterally and axially. The improved precision of both visualisation and quantification of subdiffraction limited dendritic spines using the CRSR technique may prove to be critical in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease. LAY DESCRIPTION: For over a century, Golgi-Cox (GC) has been a leading staining technique in the field of neuroscience, used to visualise neurons with great morphological detail. GC stained brain or spinal cord samples are conventionally visualised under transmitted light techniques. This limits the view of Golgi-staining to a two-dimensional image. A recent report showed that Golgi staining can be visualised in three-dimensions using the reflection modality of the confocal microscope. This visualisation also allows for the simultaneous acquisition of immunofluorescence signals. However, the reported resolution of Golgi staining confocal reflection is limited by the diffraction limit of light, which is around 220 nm. Here, we report a superresolution confocal reflection technique (CRSR) that achieves superresolution by minimising the pinhole size used in confocal microscopy. The CRSR technique results in ∼30% lateral and axial resolution improvement. Adding a deconvolution step in the final processing could improve the SNR and resolution even further up to 2-fold improvement in resolution over the diffraction limit both laterally and axially. We hope that this improved visualisation will help in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease.

NF-κB inducing kinase (NIK) is an essential post-transcriptional regulator of T-cell activation affecting F-actin dynamics and TCR signaling.

NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.

Regulatory T Cell Dysfunction Acquiesces to BTLA+ Regulatory B Cells Subsequent to Oral Intervention in Experimental Autoimmune Encephalomyelitis.

Regulatory T cells (Tregs) induced during autoimmunity often become quiescent and unable to resolve disease, suggesting inadequate activation. Resolution of established experimental autoimmune encephalomyelitis (EAE) can be achieved with myelin oligodendrocyte glycoprotein (MOG) fused to reovirus protein σ1 (MOG-pσ1), which activates Tregs, restoring protection, but requiring other regulatory cells to revitalize them. B cells have a dichotomous role in both the pathogenesis and recovery from EAE. Although inflammatory B cells contribute to EAE's pathogenesis, treatment of EAE mice with MOG-pσ1, but not OVA-pσ1, resulted in an influx of IL-10-producing B220(+)CD5(+) B regulatory cells (Bregs) enabling Tregs to recover their inhibitory activity, and in turn, leading to the rapid amelioration of EAE. These findings implicate direct interactions between Bregs and Tregs to facilitate this recovery. Adoptive transfer of B220(+)CD5(-) B cells from MOG-pσ1-treated EAE or Bregs from PBS-treated EAE mice did not resolve disease, whereas the adoptive transfer of MOG-pσ1-induced B220(+)CD5(+) Bregs greatly ameliorated EAE. MOG-pσ1-, but not OVA-pσ1-induced IL-10-producing Bregs, expressed elevated levels of B and T lymphocyte attenuator (BTLA) relative to CD5(-) B cells, as opposed to Tregs or effector T (Teff) cells, whose BTLA expression was not affected. These induced Bregs restored EAE Treg function in a BTLA-dependent manner. BTLA(-/-) mice showed more pronounced EAE with fewer Tregs, but upon adoptive transfer of MOG-pσ1-induced BTLA(+) Bregs, BTLA(-/-) mice were protected against EAE. Hence, this evidence shows the importance of BTLA in activating Tregs to facilitate recovery from EAE.

Accidental Injection of Freund Complete Adjuvant With Mycobacterium Tuberculosis.

Accidental needlestick injuries are common in laboratory and health care workers. Injection of atypical pathogens, such as those encountered in the animal laboratory setting, may pose considerable problems at the site of inoculation. We present the case of an otherwise healthy laboratory worker who accidentally self-injected Freund complete adjuvant with heat-killed Mycobacterium tuberculosis into her hand, requiring multiple debridement operations over a prolonged treatment course.

Changes in haematopoietic progenitor colony differentiation and proliferation and the production of different abzymes in EAE mice treated with DNA.

Immunization of experimental autoimmune encephalomyelitis (EAE)-prone C57BL/6 mice with MOG35-55 (a model used to study aspects of human multiple sclerosis) is known to lead to the production of various abzymes. The production of catalytic IgGs that can efficiently hydrolyse myelin basic protein (MBP), MOG and DNA is associated with changes in the profile of differentiation and level of proliferation of mice bone marrow haematopoietic stem cells (HSCs). As MOG simulates the production of abzymes with high DNase activity, we compared the effects of DNA and MOG immunization on EAE-prone mice. In contrast to MOG, immunization with DNA leads to a suppression of proteinuria, a decrease in the concentrations of antibodies to MOG and DNA and a reduction in abzyme production. Immunization with DNA only resulted in a significant increase in DNase activity over 40 days where it became 122-fold higher than before immunization, and fivefold higher when comparing to the maximal activity obtained after MOG treatment. DNA and MOG immunization had different effects on the differentiation profiles of HSCs, lymphocyte proliferation, and the level of apoptosis in bone marrow and other organs of mice. The data indicate that for C57BL/6 mice, DNA may have antagonistic effects with respect to MOG immunization. The usually fast immune response following MOG injection in C57BL/6 mice is strongly delayed after immunization with DNA, which is probably due to a rearrangement of the immune system following the response to DNA.

Galectin-1 is essential for the induction of MOG35-55 -based intravenous tolerance in experimental autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE), intravenous (i.v.) injection of the antigen, myelin oligodendrocyte glycoprotein-derived peptide, MOG35-55 , suppresses disease development, a phenomenon called i.v. tolerance. Galectin-1, an endogenous glycan-binding protein, is upregulated during autoimmune neuroinflammation and plays immunoregulatory roles by inducing tolerogenic dendritic cells (DCs) and IL-10 producing regulatory type 1 T (Tr1) cells. To examine the role of galectin-1 in i.v. tolerance, we administered MOG35-55 -i.v. to wild-type (WT) and galectin-1 deficient (Lgals1(-/-) ) mice with ongoing EAE. MOG35-55 suppressed disease in the WT, but not in the Lgals1(-/-) mice. The numbers of Tr1 cells and Treg cells were increased in the CNS and periphery of tolerized WT mice. In contrast, Lgals1(-/-) MOG-i.v. mice had reduced numbers of Tr1 cells and Treg cells in the CNS and periphery, and reduced IL-27, IL-10, and TGF-ß1 expression in DCs in the periphery. DCs derived from i.v.-tolerized WT mice suppressed disease when adoptively transferred into mice with ongoing EAE, whereas DCs from Lgals1(-/-) MOG-i.v. mice were not suppressive. These findings demonstrate that galectin-1 is required for i.v. tolerance induction, likely via induction of tolerogenic DCs leading to enhanced development of Tr1 cells, Treg cells, and downregulation of proinflammatory responses.

A20-deficient mast cells exacerbate inflammatory responses in vivo.

Mast cells are implicated in the pathogenesis of inflammatory and autoimmune diseases. However, this notion based on studies in mast cell-deficient mice is controversial. We therefore established an in vivo model for hyperactive mast cells by specifically ablating the NF-κB negative feedback regulator A20. While A20 deficiency did not affect mast cell degranulation, it resulted in amplified pro-inflammatory responses downstream of IgE/FcεRI, TLRs, IL-1R, and IL-33R. As a consequence house dust mite- and IL-33-driven lung inflammation, late phase cutaneous anaphylaxis, and collagen-induced arthritis were aggravated, in contrast to experimental autoimmune encephalomyelitis and immediate anaphylaxis. Our results provide in vivo evidence that hyperactive mast cells can exacerbate inflammatory disorders and define diseases that might benefit from therapeutic intervention with mast cell function.

B cell antigen presentation is sufficient to drive neuroinflammation in an animal model of multiple sclerosis.

B cells are increasingly regarded as integral to the pathogenesis of multiple sclerosis, in part as a result of the success of B cell-depletion therapy. Multiple B cell-dependent mechanisms contributing to inflammatory demyelination of the CNS have been explored using experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-dependent animal model for multiple sclerosis. Although B cell Ag presentation was suggested to regulate CNS inflammation during EAE, direct evidence that B cells can independently support Ag-specific autoimmune responses by CD4 T cells in EAE is lacking. Using a newly developed murine model of in vivo conditional expression of MHC class II, we reported previously that encephalitogenic CD4 T cells are incapable of inducing EAE when B cells are the sole APC. In this study, we find that B cells cooperate with dendritic cells to enhance EAE severity resulting from myelin oligodendrocyte glycoprotein (MOG) immunization. Further, increasing the precursor frequency of MOG-specific B cells, but not the addition of soluble MOG-specific Ab, is sufficient to drive EAE in mice expressing MHCII by B cells alone. These data support a model in which expansion of Ag-specific B cells during CNS autoimmunity amplifies cognate interactions between B and CD4 T cells and have the capacity to independently drive neuroinflammation at later stages of disease.

De novo-induced self-antigen-specific Foxp3+ regulatory T cells impair the accumulation of inflammatory dendritic cells in draining lymph nodes.

Foxp3(+) regulatory T cell (Treg)-based immunotherapy holds promise for autoimmune diseases. However, this effort has been hampered by major caveats, including the low frequency of autoantigen-specific Foxp3(+) Tregs and lack of understanding of their molecular and cellular targets, in an unmanipulated wild-type (WT) immune repertoire. In this study, we demonstrate that infusion of myelin in WT mice results in the de novo induction of myelin-specific Foxp3(+) Tregs in WT mice and amelioration of experimental autoimmune encephalomyelitis. Myelin-specific Foxp3(+) Tregs exerted their effect both by diminishing Ag-bearing inflammatory dendritic cell (iDC) recruitment to lymph nodes and by impairing their function. Transcriptome analysis of ex vivo-isolated Treg-exposed iDCs showed significant enrichment of transcripts involved in functional properties of iDCs, including chemotaxis-related genes. To this end, CCR7 expression by iDCs was significantly downregulated in tolerant mice and this was tightly regulated by the presence of IL-10. Collectively, our data demonstrate a novel model for deciphering the Ag-specific Foxp3(+) Treg-mediated mechanisms of tolerance and delineate iDCs as a Foxp3(+) Treg cellular target in unmanipulated mice.