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Multiple sclerosis is an autoimmune disease that affects the central nervous system. Because of its complexity and the difficulty to treat, searching for immunoregulatory responses that reduce the clinical signs of disease by non-aggressive mechanisms and without adverse effects is a scientific challenge. Herein we propose a protocol of oral tolerance induction that prevented and controlled MOG-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. The genetically modified strain HSP65-producing Lactococcus lactis was orally administered for 5 consecutive days either before or during disease development in mice. Both protocols of feeding HSP65 resulted in significant reduction in the clinical score of EAE. Frequencies of LAP+CD4+Foxp3- regulatory T cells were higher in spleens and inguinal lymph nodes of fed mice. In addition, intravital microscopy showed that adherence of leukocytes to venules in the spinal cord was reduced in orally treated mice. Oral treatment with HSP65-producing L.lactis prevented leukocytes to leave the secondary lymphoid organs, therefore they could not reach the central nervous system. Despite the inhibition of pathological immune response that drive EAE development, activated T cells were at normal frequencies suggesting that oral tolerance did not induce general immunosuppression, but it led to specific control of pathogenic T cells. Our results indicate a novel therapeutic strategy to prevent and control autoimmune diseases such as multiple sclerosis.

Oral tolerance (OT) has classically been defined as the specific suppression of cellular and/or humoral immune responses to an antigen by prior administration of the antigen through the oral route. Multiple mechanisms have been proposed to explain the induction of OT including T cell clonal depletion and anergy when high doses of antigens are fed, and regulatory T (Treg) cell generation following oral administration of low and repeated doses of antigens. Oral antigen administration suppresses the immune response in several animal models of autoimmune disease, including experimental autoimmune encephalomyelitis, uveitis, thyroiditis, myasthenia, arthritis and diabetes, but also non-autoimmune inflammatory conditions such as asthma, atherosclerosis, graft rejection, allergy and stroke. However, human trials have given mixed results and a great deal remains to be learned about the mechanisms of OT before it can be successfully applied to people. One of the possible mechanisms relates to the gut microbiota and in this review, we will explore the cellular components involved in the induction of OT and the role of the gut microbiota in contributing to OT development.

Neutrophils were classically described as powerful effectors of acute inflammation, and their main purpose was assumed to be restricted to pathogen killing through production of oxidants. As consequence, neutrophils also may lead to significant collateral damage to the healthy tissues, and after performing these tasks, these leukocytes are supposed to die within tissues. However, there is a growing body of evidence showing that neutrophils also play a pivotal role in the resolution phases of inflammation, because they can modulate tissue environment due to secretion of different kind of cytokines. Drug-induced liver injury (DILI) is a worldwide concern being one of the most prevalent causes of liver transplantation, and is well established that there is an intense neutrophil recruitment into necrotic liver during DILI. However, information if such abundant granulocyte infiltration is also linked to the tissue repairing phase of hepatic injury is still largely elusive. Here, we investigated the dynamics of neutrophil trafficking within blood, bone marrow, and liver during hepatic inflammation, and how changes in their gene expression profile could drive the resolution events during acetaminophen (APAP)-induced liver injury. We found that neutrophils remained viable during longer periods following liver damage, because they avidly patrolled necrotic areas and up-regulated pro-resolutive genes, including Tgfb, Il1r2, and Fpr2. Adoptive transference of “resolutive neutrophils” harvested from livers at 72 h after injury to mice at the initial phases of injury (6 h after APAP) significantly rescued organ injury. Thus, we provide novel insights on the role of neutrophils not only in the injury amplification, but also in the resolution phases of inflammation.

Oral tolerance is defined as the specific suppression of cellular and/or humoral immune responses to an Ag by prior administration of the Ag through the oral route. Although the investigation of oral tolerance has classically involved Ag feeding, we have found that oral administration of anti-CD3 mAb induced tolerance through regulatory T (Treg) cell generation. However, the mechanisms underlying this effect remain unknown. In this study, we show that conventional but not plasmacytoid dendritic cells (DCs) are required for anti-CD3–induced oral tolerance. Moreover, oral anti-CD3 promotes XCL1 secretion by small intestine lamina propria γδ T cells that, in turn, induces tolerogenic XCR1+ DC migration to the mesenteric lymph node, where Treg cells are induced and oral tolerance is established. Consistent with this, TCRδ−/− mice did not develop oral tolerance upon oral administration of anti-CD3. However, XCL1 was not required for oral tolerance induced by fed Ags, indicating that a different mechanism underlies this effect. Accordingly, oral administration of anti-CD3 enhanced oral tolerance induced by fed MOG35–55 peptide, resulting in less severe experimental autoimmune encephalomyelitis, which was associated with decreased inflammatory immune cell infiltration in the CNS and increased Treg cells in the spleen. Thus, Treg cell induction by oral anti-CD3 is a consequence of the cross-talk between γδ T cells and tolerogenic DCs in the gut. Furthermore, anti-CD3 may serve as an adjuvant to enhance oral tolerance to fed Ags.

BackgroundImmune hyperactivity is an important contributing factor to the morbidity and mortality of COVID-19 infection. Nasal administration of anti-CD3 monoclonal antibody downregulates hyperactive immune responses in animal models of autoimmunity through its immunomodulatory properties. We performed a randomized pilot study of fully-human nasal anti-CD3 (Foralumab) in patients with mild to moderate COVID-19 to determine if its immunomodulatory properties had ameliorating effects on disease.MethodsThirty-nine outpatients with mild to moderate COVID-19 were recruited at Santa Casa de Misericordia de Santos in Sao Paulo State, Brazil. Patients were randomized to three cohorts: 1) Control, no Foralumab (n=16); 2) Nasal Foralumab (100ug/day) given for 10 consecutive days with 6 mg dexamethasone given on days 1-3 (n=11); and 3) Nasal Foralumab alone (100ug/day) given for 10 consecutive days (n=12). Patients continued standard of care medication.ResultsWe observed reduction of serum IL-6 and C-reactive protein in Foralumab alonevs. untreated or Foralumab/Dexa treated patients. More rapid clearance of lung infiltrates as measured by chest CT was observed in Foralumab and Foralumab/Dexa treated subjectsvs. those that did not receive Foralumab. Foralumab treatment was well-tolerated with no severe adverse events.ConclusionsThis pilot study suggests that nasal Foralumab is well tolerated and may be of benefit in treatment of immune hyperactivity and lung involvement in COVID-19 disease and that further studies are warranted.

Conjugated linoleic acid (CLA) has been shown to activate the nuclear receptor PPAR-γ and modulate metabolic and immune functions. Despite the worldwide use of CLA dietary supplementation, strong scientific evidence for its proposed beneficial actions are missing. We found that CLA-supplemented diet reduced mucosal damage and inflammatory infiltrate in the dextran sodium sulfate (DSS)-induced colitis model. Conditional deletion of PPAR-γ in macrophages from mice supplemented with CLA diet resulted in loss of this protective effect of CLA, suggesting a PPAR-γ-dependent mechanism mediated by macrophages. However, CLA supplementation significantly worsened colorectal tumor formation induced by azoxymethane and DSS by inducing macrophage and T-cell-producing TGF-β via PPAR-γ activation. Accordingly, either macrophage-specific deletion of PPAR-γ or in vivo neutralization of latency-associated peptide (LAP, a membrane-bound TGF-β)-expressing cells abrogated the protumorigenic effect of CLA. Thus, the anti-inflammatory properties of CLA are associated with prevention of colitis but also with development of colorectal cancer.

Mammals and microorganisms have evolved a complex and tightly controlled mutual relationship. This interaction grants protection and energy source for the microorganisms, and on the other hand, provides several immunologic, metabolic and physiological advantages for the host. The gastrointestinal tract (GI) harbors the largest bacteria diversity within the body and complex mechanisms control microbiota community under homeostasis. However, once disrupted, microbiota imbalance can lead to overt growth of resident and invasive populations, with potential risk for lethal diseases. In these cases, bacteria might also escape from the intestines and reach different organs through the blood and lymphatic circulation. To control these unwanted conditions, all body tissues are populated with resident macrophages that have the ability to capture and eliminate pathogens, avoiding their dissemination. Here we discuss the different routes for bacterial translocation from the intestinal tract, and how macrophages act in the removal of these microorganisms to prevent systemic infections and restore the homeostasis.

Summary Fecal transfer from healthy donors is being explored as a microbiome modality. MicroRNAs (miRNAs) have been found to affect the microbiome. Multiple sclerosis (MS) patients have been shown to have an altered gut microbiome. Here, we unexpectedly found that transfer of feces harvested at peak disease from the experimental autoimmune encephalomyelitis (EAE) model of MS ameliorates disease in recipients in a miRNA-dependent manner. Specifically, we show that miR-30d is enriched in the feces of peak EAE and untreated MS patients. Synthetic miR-30d given orally ameliorates EAE through expansion of regulatory T cells (Tregs). Mechanistically, miR-30d regulates the expression of a lactase in Akkermansia muciniphila, which increases Akkermansia abundance in the gut. The expanded Akkermansia in turn increases Tregs to suppress EAE symptoms. Our findings report the mechanistic underpinnings of a miRNA-microbiome axis and suggest that the feces of diseased subjects might be enriched with miRNAs with therapeutic properties.

Summary Myeloid suppressor cells promote tumor growth by a variety of mechanisms which are not fully characterized. We identified myeloid cells (MCs) expressing the latency-associated peptide (LAP) of TGF-β on their surface and LAPHi MCs that stimulate Foxp3+ Tregs while inhibiting effector T cell proliferation and function. Blocking TGF-β inhibits the tolerogenic ability of LAPHi MCs. Furthermore, adoptive transfer of LAPHi MCs promotes Treg accumulation and tumor growth in vivo. Conversely, anti-LAP antibody, which reduces LAPHi MCs, slows cancer progression. Single-cell RNA-Seq analysis on tumor-derived immune cells revealed LAPHi dominated cell subsets with distinct immunosuppressive signatures, including those with high levels of MHCII and PD-L1 genes. Analogous to mice, LAP is expressed on myeloid suppressor cells in humans, and these cells are increased in glioma patients. Thus, our results identify a previously unknown function by which LAPHi MCs promote tumor growth and offer therapeutic intervention to target these cells in cancer., Graphical abstract, Highlights • Several myeloid cell subsets express surface LAP • Myeloid cells that express surface LAP possess immunosuppressive properties • LAP expressing myeloid cells induce Tregs and inhibit effector T cell function • LAP expressing myeloid cells promote tumor growth, Immunology; Cancer

International audience; The aim of this study was to investigate the contribution of IL-33/ST2 axis in the onset and progression of acute liver injury using a mice model of drug-induced liver injury (DILI). DILI was induced by overdose administration of acetaminophen (APAP) by oral gavage in wild-type BALB/c, ST2-deficient mice and in different bone marrow chimeras. Neutrophils were depleted by anti-Ly6G and macrophages with clodronate liposomes (CLL). Blood and liver were collected for biochemical, immunologic and genetic analyses. Mice were imaged by confocal intravital microscopy and liver non-parenchymal cells and hepatocytes were isolated for flow cytometry, genetic and immunofluorescence studies. Acetaminophen overdose caused a massive necrosis and accumulation of immune cells within the liver, concomitantly with IL-33 and chemokine release. Liver non-parenchymal cells were the major sensors for IL-33, and amongst them, neutrophils were the major players in amplification of the inflammatory response triggered by IL-33/ST2 signalling pathway. Blockage of IL-33/ST2 axis reduces APAP-mediated organ injury by dampening liver chemokine release and activation of resident and infiltrating liver non-parenchymal cells.

Seizures are due to excessive, synchronous neuronal firing in the brain and are characteristic of epilepsy, the fourth most prevalent neurological disease. We report handling-induced and spontaneous seizures in mice deficient for CD39, a cell-surface ATPase highly expressed on microglial cells. CD39-/- mice with handling-induced seizures had normal input-output curves and paired-pulse ratio measured from hippocampal slices and lacked microgliosis, astrogliosis or overt cell loss in the hippocampus and cortex. As expected, however, the cerebrospinal fluid of CD39-/- mice contained increased levels of ATP and decreased levels of adenosine. To determine if immune activation was involved in seizure progression, we challenged mice with lipopolysaccharide (LPS) and measured the effect on microglia activation and seizure severity. Systemic LPS challenge resulted in increased cortical staining of Iba1/CD68 and gene array data from purified microglia predicted increased expression of interleukin-8, triggering receptor expressed on myeloid cells 1, p38, pattern recognition receptors, death receptor, nuclear factor-κB , complement, acute phase, and interleukin-6 signalling pathways in CD39-/- versus CD39+/+ mice. However, LPS treatment did not affect handling-induced seizures. In addition, microglia-specific CD39 deletion in adult mice was not sufficient to cause seizures, suggesting instead that altered expression of CD39 during development or on non-microglial cells such as vascular endothelial cells may promote the seizure phenotype. In summary, we show a correlation between altered extracellular ATP/adenosine ratio and a previously unreported seizure phenotype in CD39-/- mice. This work provides groundwork for further elucidation of the underlying mechanisms of epilepsy.

Oral administration of Ag induces regulatory T cells that express latent membrane-bound TGF-β (latency-associated peptide [LAP]) and have been shown to play an important role in the induction of oral tolerance. We developed an in vitro model to study modulation of LAP+ on CD4+ T cells. The combination of anti-CD3 mAb, anti-CD28 mAb, and recombinant IL-2 induced expression of LAP on naive CD4+ T cells, independent of Foxp3 or exogenous TGF-β. In vitro generated CD4+LAP+Foxp3− T cells were suppressive in vitro, inhibiting proliferation of naive CD4+ T cells and IL-17A secretion by Th17 cells. Assessing the impact of different cytokines and neutralizing Abs against cytokines, we found that LAP induction was decreased in the presence of IL-6 and IL-21, and to a lesser extent by IL-4 and TNF-α. IL-6 abrogated the in vitro induction of CD4+LAP+ T cells by STAT3-dependent inhibition of Lrrc32 (glycoprotein A repetitions predominant [GARP]), the adapter protein that tethers TGF-β to the membrane. Oral tolerance induction was enhanced in mice lacking expression of IL-6R by CD4+ T cells and by treatment of wild-type mice with neutralizing anti-IL-6 mAb. These results suggest that proinflammatory cytokines interfere with oral tolerance induction and that blocking the IL-6 pathway is a potential strategy for enhancing oral tolerance in the setting of autoimmune and inflammatory diseases.

CD3-specific monoclonal antibody (mAb) treats autoimmune disease in animal models and has shown promise in clinical trials of type 1 diabetes. Whereas intravenous administration of CD3-specific mAb acts primarily by transient depletion of activated effector T cells, oral CD3-specific mAb acts primarily by the induction Tregs. We investigated whether oral CD3-specific mAb inhibits disease in non obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, closely resembling human type 1 diabetes. We found that oral CD3-specific mAb treatment delayed onset and reduced incidence of diabetes in NOD mice, inducing changes in both effector and regulatory T cell compartments. The therapeutic effect was associated with decreased T cell proliferation, decreased IFNγ and IL-17 production, and increased TGF-β and IL-10 production in vitro. In vivo transfer experiments demonstrated that oral CD3-specific mAb decreased diabetogenicity of effector T cells and increased the function of regulatory T cells. Oral OKT3, a monoclonal antibody specific for human CD3 had equivalent effects in transgenic NOD mice expressing the human CD3 epsilon chain which serves as a preclinical model for testing human CD3-specific mAb. These results suggest that oral CD3-specific mAb has the potential for treating autoimmune diabetes in humans.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Experimental Autoimmune Encephalomyelitis (EAE) is the most widely used animal model for the study of MS. The Suppressor of Cytokine Signaling (SOCS) 2 protein plays a critical role in regulating the immune responses. The role of SOCS2 during EAE has not been explored. EAE was induced in WT and SOCS2-/- mice using myelin oligodendrocyte glycoprotein (MOG35-55) peptide. Brain and spinal cord were examined during the peak (day 14) and recovery phase (day 28) of the disease. SOCS2 was upregulated in the brain of WT mice at the peak and recovery phase of EAE. The development of the acute phase was slower in onset in SOCS2-/- mice and was associated with reduced number of Th1 (CD3+CD4+IFN-γ+) cells in the spinal cord and brain. However, while in WT mice, maximal clinical EAE score was followed by a progressive recovery; the SOCS2-/- mice were unable to recover from locomotor impairment that occurred during the acute phase. There was a prolonged inflammatory response (increased Th1 and decreased Th2 and T regulatory cells) in the late phase of EAE in the CNS of SOCS2-/- mice. Transplantation of bone marrow cells from SOCS2-/- into irradiated WT mice resulted in higher lethality at the early phase of EAE. Altogether, these results suggest that SOCS2 plays a dual role in the immune response during EAE. It is necessary for damage during the acute phase damage but plays a beneficial role in the recovery stage of the disease.

Leukocytes are a large population of cells spread within most tissues in the body. These cells may be either sessile (called as resident cells) or circulating leukocytes, which travel long journeys inside the vessels during their lifespan. Although production and maturation of these leukocytes in adults primarily occur in the bone marrow, it is well known that this process—called hematopoiesis—started in the embryonic life in different sites, including the yolk sac, placenta, and the liver. In this review, we will discuss how the liver acts as a pivotal site for leukocyte maturation during the embryo phase, and also how the most frequent liver-resident immune cell populations—namely Kupffer cells, dendritic cells, and lymphocytes—play a vital role in both tolerance and inflammatory responses to antigens from food, microbiota, and pathogens.

Resident and circulating immune cells have been extensively studied due to their almost ubiquitous role in cell biology. Despite their classification under the “immune cell department”, it is becoming increasingly clear that these cells are involved in many different non-immune related phenomena, including fetus development, vascular formation, memory, social behavior and many other phenotypes. There is a huge potential in combining high-throughput assays - including flow cytometry and gene analysis - with in vivo imaging. This can improve our knowledge in both basic and clinical cell biology, and accessing the expression of markers that are relevant in the context of both homeostasis and disease conditions might be instrumental. Here we describe how we generated a novel mouse strain that spontaneously express three different fluorescence markers under control of well-studied receptors (CX3CR1, CCR2 and CD11c) that are involved in a plethora of stages of cell ontogenesis, maturation, migration and behavior. Also, we assess the percentage of the expression and co-expression of each marker under homeostasis conditions, and how these cells behave when a local inflammation is induced in the liver applying a cutting-edge technology to image cells by confocal intravital microscopy.

Since their discovery in the early 90s, microRNAs (miRNAs), small non-coding RNAs, have mainly been associated with posttranscriptional regulation of gene expression on a cell-autonomous level. Recent evidence has extended this role by adding inter-species communication to the manifold functional range. In our latest study [Liu S, et al., 2016, Cell Host & Microbe], we identified miRNAs in gut lumen and feces of both mice and humans. We found that intestinal epithelial cells (IEC) and Hopx+ cells were the two main sources of fecal miRNA. Deficiency of IEC-miRNA resulted in gut dysbiosis and WT fecal miRNA transplantation restored the gut microbiota. We investigated potential mechanisms for this effect and found that miRNAs were able to regulate the gut microbiome. By culturing bacteria with miRNAs, we found that host miRNAs were able to enter bacteria, specifically regulate bacterial gene transcripts and affect bacterial growth. Oral administration of synthetic miRNA mimics affected specific bacteria in the gut. Our findings describe a previously unknown pathway by which the gut microbiome is regulated by the host and raises the possibility that miRNAs may be used therapeutically to manipulate the microbiome for the treatment of disease.

Background & Aims The precise determination of non-alcoholic fatty liver disease (NAFLD) onset is challenging. Thus, the initial hepatic responses to fat accumulation, which may be fundamental to our understanding of NAFLD evolution and clinical outcomes, are largely unknown. Herein, we chronologically mapped the immunologic and metabolic changes in the liver during the early stages of fatty liver disease in mice and compared this with human NAFLD samples. Methods Liver biopsies from patients with NAFLD (NAFLD activity score [NAS] 2–3) were collected for gene expression profiling. Mice received a high-fat diet for short periods to mimic initial steatosis and the hepatic immune response was investigated using a combination of confocal intravital imaging, gene expression, cell isolation, flow cytometry and bone marrow transplantation assays. Results We observed major immunologic changes in patients with NAS 2–3 and in mice in the initial stages of NAFLD. In mice, these changes significantly increased mortality rates upon drug-induced liver injury, as well as predisposing mice to bacterial infections. Moreover, deletion of Toll-like receptor 4 in liver cells dampened tolerogenesis, particularly in Kupffer cells, in the initial stages of dietary insult. Conclusion The hepatic immune system acts as a sentinel for early and minor changes in hepatic lipid content, mounting a biphasic response upon dietary insult. Priming of liver immune cells by gut-derived Toll-like receptor 4 ligands plays an important role in liver tolerance in initial phases, but continuous exposure to insults may lead to damage and reduced ability to control infections. Lay summary Fatty liver is a very common form of hepatic disease, leading to millions of cases of cirrhosis every year. Patients are often asymptomatic until becoming very sick. Therefore, it is important that we expand our knowledge of the early stages of disease pathogenesis, to enable early diagnosis. Herein, we show that even in the early stages of fatty liver disease, there are significant alterations in genes involved in the inflammatory response, suggesting that the hepatic immune system is disturbed even following minor and undetectable changes in liver fat content. This could have implications for the diagnosis and clinical management of fatty liver disease., Graphical abstract, Highlights • Hepatic immune response is already altered in liver biopsies from patients with mild NAFLD. • We designed a novel mouse model to mimic mild NAFLD, enabling the chronological mapping of liver changes. • This revealed an increased mortality rate upon secondary liver damage and a window of increased susceptibility to infection. • NAFLD diagnosis may be significantly improved by a more profound investigation of changes in hepatic immunology. • These data could guide customized nutritional and therapeutic interventions at different stages of NAFLD.

Immunotherapy that acts by counteracting the suppressive environment established by the tumor became a very promising approach for the treatment of cancer. Regulatory immune cells promote cancer by suppressing antitumor immune responses, but there are few cell surface molecules that can specifically be targeted to neutralize their function. We characterized a regulatory T-cell population that expresses surface latency-associated peptide (LAP). LAP is an adaptor and regulator of TGF-beta, a potent immunosuppressive cytokine. An increase in LAP+ CD4 T cells has been reported in human cancer, including head and neck and colorectal cancer. To study the regulatory role of LAP+ cells in cancer, we developed a murine monoclonal anti-LAP antibody. We found that treatment with anti-LAP antibody reduces tumor growth and increases survival in various cancer models, including colon carcinoma, glioblastoma and melanoma. Anti-LAP antibody is able to block TGF-beta release from cells expressing LAP and also reduces both the number and suppressive abilities of tumor-associated LAP+ regulatory cells. Anti-LAP antibody treatment triggers a profound peripheral immune response by acting on both innate and adaptive arms of the immune system. A synergistic antitumor effect was observed by combining anti-LAP antibody with dendritic cell vaccination. To study different LAP+ immune cells on a single-cell level, we developed a 19-panel flow cytometry approach. Based on the TCGA data analysis, the expression of LAP-associated genes correlates inversely with patient survival in a number of cancers. In summary, LAP+ immune cells contribute to cancer progression, and targeting these cells for cancer treatment represents a promising immunotherapeutic approach against cancer. Citation Format: Galina Gabriely, Andre da Cunha, Rafael Rezende, Nathaniel Skillin, Brendan Kenyon, Lena Walton, Murugaiyan Gopal, Howard Weiner. The role of LAP positive immune cells in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A85.

γδ T cells have many known functions, including the regulation of antibody responses. However, how γδ T cells control humoral immunity remains elusive. Here we show that complete Freund’s adjuvant (CFA), but not alum, immunization induces a subpopulation of CXCR5-expressing γδ T cells in the draining lymph nodes. TCRγδ+CXCR5+ cells present antigens to, and induce CXCR5 on, CD4 T cells by releasing Wnt ligands to initiate the T follicular helper (Tfh) cell program. Accordingly, TCRδ−/− mice have impaired germinal center formation, inefficient Tfh cell differentiation, and reduced serum levels of chicken ovalbumin (OVA)-specific antibodies after CFA/OVA immunization. In a mouse model of lupus, TCRδ−/− mice develop milder glomerulonephritis, consistent with decreased serum levels of lupus-related autoantibodies, when compared with wild type mice. Thus, modulation of the γδ T cell-dependent humoral immune response may provide a novel therapy approach for the treatment of antibody-mediated autoimmunity., Many immune functions have been reported for γδ T cells, including the regulation of antibody responses. Here the authors show that CXCR5+ γδ T cells release Wnt ligands to initiate the T follicular helper cell differentiation program and promote antibody production.

Oral tolerance can be defined as an inhibition of specific immune responsiveness to subsequent parenteral injections of proteins to which an individual or animal has been previously exposed via the oral route. Multiple mechanisms of tolerance are induced by oral-fed antigens, but induction of regulatory CD4 T-cells expressing the transcription factor Foxp3 and the membrane-bound TGF-β stands out as the major players in oral tolerance. Oral antigen administration suppresses several animal models of autoimmune disease, including experimental autoimmune encephalomyelitis, uveitis, thyroiditis, myasthenia, arthritis, and diabetes, but also nonautoimmune inflammatory conditions such as asthma, atherosclerosis, graft rejection, allergy, and stroke. However, human trials have produced mixed results, and a great deal remains to be learned about the mechanisms of oral tolerance before it can be successfully applied to people. In this review, we highlight the cellular components involved in oral tolerance induction. A deep knowledge of these intricate cell interactions will pave the way for a successful application of antigen tolerance to treat autoimmune and nonautoimmune inflammatory diseases.

Autoimmune diseases are reversed in mice using nanoparticles that display disease-associated antigens bound to MHC class II molecules.

Regulatory T cells (Tregs) promote cancer by suppressing antitumor immune responses. We found that anti-LAP antibody, which targets the latency-associated peptide (LAP)/transforming growth factor-β (TGF-β) complex on Tregs and other cells, enhances antitumor immune responses and reduces tumor growth in models of melanoma, colorectal carcinoma, and glioblastoma. Anti-LAP decreases LAP+ Tregs, tolerogenic dendritic cells, and TGF-β secretion and is associated with CD8+ T cell activation. Anti-LAP increases infiltration of tumors by cytotoxic CD8+ T cells and reduces CD103+ CD8 T cells in draining lymph nodes and the spleen. We identified a role for CD103+ CD8 T cells in cancer. Tumor-associated CD103+ CD8 T cells have a tolerogenic phenotype with increased expression of CTLA-4 and interleukin-10 and decreased expression of interferon-γ, tumor necrosis factor-α, and granzymes. Adoptive transfer of CD103+ CD8 T cells promotes tumor growth, whereas CD103 blockade limits tumorigenesis. Thus, anti-LAP targets multiple immunoregulatory pathways and represents a potential approach for cancer immunotherapy.

The gastrointestinal immune system plays a pivotal role in the host relationship with food antigens, the homeostatic microbiome and enteric pathogens. Here, we describe how to collect and process liver and intestinal samples to efficiently isolate and analyse resident immune cells. Furthermore, we describe a step-by-step methodology showing how to high-dimensionally immunophenotype resident leucocytes using cytometry by time-of-flight, providing a well-characterized antibody platform that allows the identification of every leucocyte subset simultaneously. This protocol also includes instructions to purify and cultivate primary murine hepatocytes, a powerful tool to assess basic cell biology and toxicology assays. Gut and liver samples from the same mouse can be collected, processed and stained in less than 6 hr. This protocol enables the recovery of several populations of purified and viable immune cells from solid and fibrous organs, preventing unwanted loss of adherent cells during isolation.

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice.

γδ T cells are a subset of lymphocytes specialized in protecting the host against pathogens and tumours. Here we describe a subset of regulatory γδ T cells that express the latency-associated peptide (LAP), a membrane-bound TGF-β1. Thymic CD27+IFN-γ+CCR9+α4β7+TCRγδ+ cells migrate to the periphery, particularly to Peyer's patches and small intestine lamina propria, where they upregulate LAP, downregulate IFN-γ via ATF-3 expression and acquire a regulatory phenotype. TCRγδ+LAP+ cells express antigen presentation molecules and function as antigen presenting cells that induce CD4+Foxp3+ regulatory T cells, although TCRγδ+LAP+ cells do not themselves express Foxp3. Identification of TCRγδ+LAP+ regulatory cells provides an avenue for understanding immune regulation and biologic processes linked to intestinal function and disease., Latency-associated peptide (LAP) is a membrane-bound form of TGF-β1. Here the authors show that LAP marks a subset of regulatory γδ T cells with innate gut-homing properties, which present antigen and induce CD4+ Foxp3+ in Peyer's patches and lamina propria.

Despite accumulating evidence indicating that neurotransmitters released by the sympathetic nervous system can modulate the activity of innate immune cells, we still know very little about how norepinephrine impacts signaling pathways in dendritic cells (DC) and the consequence of that in DC-driven T cell differentiation. In this article, we demonstrate that β2-adrenergic receptor (β2AR) activation in LPS-stimulated DC does not impair their ability to promote T cell proliferation; however, it diminishes IL-12p70 secretion, leading to a shift in the IL-12p70/IL-23 ratio. Although β2AR stimulation in DC induces protein kinase A–dependent cAMP-responsive element–binding protein phosphorylation, the effect of changing the profile of cytokines produced upon LPS challenge occurs in a protein kinase A–independent manner and, rather, is associated with inhibition of the NF-κB and AP-1 signaling pathways. Moreover, as a consequence of the inverted IL-12p70/IL-23 ratio following β2AR stimulation, LPS-stimulated DC promoted the generation of CD4+ T cells that, upon TCR engagement, produced lower amounts of IFN-γ and higher levels of IL-17. These findings provide new insights into molecular and cellular mechanisms by which β2AR stimulation in murine DC can influence the generation of adaptive immune responses and may explain some aspects of how sympathetic nervous system activity can modulate immune function.

Regulatory T cells (Tregs) play a critical role in the maintenance of immunological tolerance. The best-characterized Tregs are those expressing the transcription factor Foxp3 and in vivo modulation of Foxp3 Tregs has been employed to study their role in immune homeostasis. Latency-associated peptide (LAP) is a membrane-bound TGF-β complex that has also been shown to play a role in Treg function and oral tolerance. We developed a novel anti-mouse LAP mAb that allowed us to investigate the effect of targeting LAP in vivo on immune function and on anti-CD3-induced oral tolerance. We found that in vivo anti-LAP mAb administration led to a decrease in the number of CD4+LAP+ Tregs in spleen and lymph nodes without affecting CD4+Foxp3+ Tregs. Spleen cells from anti-LAP-injected mice proliferated more in vitro and produced increased amounts of IL-2, IL-17 and IFN-γ. Moreover, injection of anti-LAP antibody abrogated the protective effect of oral anti-CD3 on experimental autoimmune encephalomyelitis (EAE). Finally, in vivo anti-LAP administration prior to myelin oligodendrocyte glycoprotein immunization resulted in severe EAE in the absence of pertussis toxin, which is used for EAE induction. Our findings demonstrate the importance of CD4+LAP+ T cells in the control of immune homeostasis and autoimmunity and provides a new tool for the in vivo investigation of murine LAP+ Tregs on immune function.