Group 3 innate lymphoid cells (ILC3s) are key sensors of the intestinal environment, integrating dietary and microbial cues to maintain intestinal immunity. We found that intestinal ILC3s were reduced in overweight and obese humans and in high-fat diet (HFD)-fed mice. ILC3 loss occurred independently of caloric excess, weight gain, or glucose intolerance. Instead, impairment arose within hours of HFD consumption and was initiated by microbiota-driven intestinal barrier permeability and concomitant activation of inflammatory mononuclear phagocytes (MNPs). This response to inflammation impaired fatty acid oxidation in lipid-loaded ILC3s, resulting in mitochondrial damage and cell death. Intestinal ILC3 cell death was rescued by removal of excess fats from the diet. ILC3s from individuals with obesity also exhibited impaired fatty acid oxidation. Together, our findings define a malleable mechanism whereby dietary fats and microbial cues drive ILC3 maladaptation and death, with consequences for intestinal homeostasis.

Gamma-delta (γδ) T cells are a major cell population in the intestinal mucosa and are key mediators of mucosal tolerance and microbiota composition. Little is known about the mechanisms by which intestinal γδ T cells interact with the gut microbiota to maintain tolerance. We found that antibiotic treatment impaired oral tolerance and depleted intestinal γδ T cells, suggesting that the gut microbiota is necessary to maintain γδ T cells. We also found that mice deficient for γδ T cells (γδ−/−) had an altered microbiota composition that led to small intestine (SI) immune dysregulation and impaired tolerance. Accordingly, colonizing WT mice with γδ−/− microbiota resulted in SI immune dysregulation and loss of tolerance whereas colonizing γδ−/− mice with WT microbiota normalized mucosal immune responses and restored mucosal tolerance. Moreover, we found that SI γδ T cells shaped the gut microbiota and regulated intestinal homeostasis by secreting the fecal micro-RNA let-7f. Importantly, oral administration of let-7f to γδ−/− mice rescued mucosal tolerance by promoting the growth of the γδ−/−-microbiota-depleted microbe Ruminococcus gnavus. Taken together, we demonstrate that γδ T cell-selected microbiota is necessary and sufficient to promote mucosal tolerance, is mediated in part by γδ T cell secretion of fecal micro-RNAs, and is mechanistically linked to restoration of mucosal immune responses.

Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public-private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.

Group 3 innate lymphoid cells (ILC3s) are critical sensory hubs to maintain intestinal immunity while adapting to environmental changes, including food intake and microbiota composition. Nutritional stress such as feeding on a high-fat diet (HFD) impairs ILC3s homeostasis, but the drivers underlying this phenotype remain elusive. Here, we found that intestinal ILC3s are depleted in overweight and obese humans, similarly to HFD-induced obese mice. However, we identified that ILC3s loss is not dependent on excessive calorie intake, weight gain, or glucose intolerance. Instead, we found that ILC3s impairment starts upon hours of HFD consumption, inducing microbial efflux. As a result, tissue-resident mononuclear phagocytes (MNPs) become proinflammatory, driving ILC3 cell death. We determined that intracellular lipid uptake hyperactivates ILC3s but triggers lipotoxicity in the context of inflammation. Collectively, our findings define the mechanism driving ILC3s maladaptation to fat-microbiota crosstalk and raise new considerations for understanding intestinal homeostasis and inflammation.Funding Information: We thank funding from Ann Romney Center for Neurologic Diseases (ARCND).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: Animal experiments followed ethical regulations for animal testing and research according to the animal protocol guidelines of Brigham and Women’s Hospital Institutional Animal Care and Use Committee (IACUC). After obtaining written informed consent, terminal Ileal tissue was collected from adult non-smoker male and female subjects at Boston Children’s Hospital or Brigham and Women’s Hospital under Institutional Review Board protocol IRB-P00000529. All animal procedures were approved by IACUC under the protocol number: 2016000230.

Chronic inflammation can drive tumor development. Here, we have identified microRNA-146a (miR-146a) as a major negative regulator of colonic inflammation and associated tumorigenesis by modulating IL-17 responses. MiR-146a-deficient mice are susceptible to both colitis-associated and sporadic colorectal cancer (CRC), presenting with enhanced tumorigenic IL-17 signaling. Within myeloid cells, miR-146a targets RIPK2, a NOD2 signaling intermediate, to limit myeloid cell-derived IL-17-inducing cytokines and restrict colonic IL-17. Accordingly, myeloid-specific miR-146a deletion promotes CRC. Moreover, within intestinal epithelial cells (IECs), miR-146a targets TRAF6, an IL-17R signaling intermediate, to restrict IEC responsiveness to IL-17. MiR-146a within IECs further suppresses CRC by targeting PTGES2, a PGE2 synthesis enzyme. IEC-specific miR-146a deletion therefore promotes CRC. Importantly, preclinical administration of miR-146a mimic, or small molecule inhibition of the miR-146a targets, TRAF6 and RIPK2, ameliorates colonic inflammation and CRC. MiR-146a overexpression or miR-146a target inhibition represent therapeutic approaches that limit pathways converging on tumorigenic IL-17 signaling in CRC.

Oral tolerance (OT) plays a critical role in maintaining the gut homeostatic environment. OT is defective in mice lacking microbiota or gamma-delta T cells (γδ-/-). We found that γδ-/- mice have an altered microbiota, including a decrease in Rumincoccus gnavus. Transfer of microbiota from γδ-/- mice to WT mice impaired OT, whereas transfer of WT microbiota or R. gnavus to γδ-/- mice restored OT. Restoration of OT was associated with increased IL-10 production by CX3CR1+ mononuclear phagocytes and Treg cells, and decreased Th17 cells. Fecal micro-RNAs can regulate the microbiota and we found that intestinal γδ T cells produced the miRNA let-7f and that oral administration of let-7f to γδ-/- mice increased R. gnavus and restored OT. Taken together, we demonstrate that the γδ T cell-selected microbiota is necessary and sufficient to promote OT, which is mechanistically linked to γδ T cell secretion of a microbiota-modulating miRNA.

Smad7, a negative regulator of TGF-β signaling, has been implicated in the pathogenesis and treatment of inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC). Here, we found that Smad7 mediates intestinal inflammation by limiting the PDL2/1-PD1 axis in dendritic cells (DCs) and CD4+T cells. Smad7 deficiency in DCs promotes TGF-β responsiveness and the co-inhibitory molecules PDL2/1 on DCs, and it further imprints T cell-PD1 signaling to promote Treg differentiation. DC-specific Smad7 deletion mitigates DSS-induced colitis by inducing CD103+PDL2/1+DCs and Tregs. In addition, Smad7 deficiency in CD4+T cells promotes PD1 and PD1-induced Tregs in vitro. The transfer of Smad7-deficient CD4+T cells enhances Tregs in vivo and protects against T cell-mediated colitis. Furthermore, Smad7 antisense ameliorates DSS-induced UC, increasing TGF-β and PDL2/1-PD1 signaling. Enhancing PD1 signaling directly via Fc-fused PDL2/1 is also beneficial. Our results identify how Smad7 mediates intestinal inflammation and leverages these pathways therapeutically, providing additional strategies for IBD intervention.

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.

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.

Oral administration of biologics may be a feasible approach for immune therapy that improves drug safety and potentiates mechanisms of tolerance at mucosal barriers. We tested the ability of a fully human non-FcR binding anti-CD3 mAb, foralumab, to prevent skin xenograft rejection in mice with human immune systems. At an intragastric dose of 15μg, the drug could transit through the small bowel. Serum absorption and binding of lymphoid cells was seen and proliferative responses of splenic CD8+ T cells to mitogen were reduced. Five consecutive daily doses, then weekly dosing led to indefinite graft acceptance without depletion of peripheral T cells. Proliferative and cytokine responses to activation of splenocytes with PHA were reduced. The serum levels of IL-10 but not TNF were increased 6days after application of the skin graft. Oral treatment with anti-CD3 mAb may represent a feasible approach for immune modulation.

Bortezomib (BTZ) is a first-in-class proteasome inhibitor approved for the therapy of multiple myeloma that also displays unique regulatory activities on immune cells. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme exerting potent immunoregulatory effects when expressed in dendritic cells (DCs), the most potent antigen-presenting cells capable of promoting either immunity or tolerance. We previously demonstrated that, in inflammatory conditions, IDO1 is subjected to proteasomal degradation in DCs, turning these cells from immunoregulatory to immunostimulatory. In non-obese diabetic (NOD) mice, an experimental model of autoimmune diabetes, we also identified an IDO1 defect such that the DCs do not develop tolerance toward pancreatic islet autoantigens. We found that BTZ rescues IDO1 protein expression in vitro in a particular subset of DCs, i.e., plasmacytoid DCs (pDCs) from NOD mice. When administered in vivo to prediabetic mice, the drug prevented diabetes onset through IDO1- and pDC-dependent mechanisms. Although the drug showed no therapeutic activity when administered alone to overtly diabetic mice, its combination with otherwise suboptimal dosages of autoimmune-preventive anti-CD3 antibody resulted in disease reversal in 70% diabetic mice, a therapeutic effect similar to that afforded by full-dosage anti-CD3. Thus, our data indicate a potential for BTZ in the immunotherapy of autoimmune diabetes and further underline the importance of IDO1-mediated immune regulation in such disease.

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.

Mentor circles combine the advantages of both mentoring networks and peer-to-peer mentoring.

Intestinal T cells balance the immune response to microbes and food [Also see Report by Kim et al. ]

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.

OBJECTIVE: To investigate tolerogenic mechanisms in IL-27-primed myeloid dendritic cells (mDCs) in relapsing multiple sclerosis (RRMS). BACKGROUND: Relapsing MS is related to encephalitogenic T-cells that invade the CNS. DCs contribute significantly to both effector immunity and tolerance by their effects on T-cell differentiation. Interleukin-27 increases expression of indoleamine 2,3 dioxygenase (IDO) and programmed death ligand 2 (PD-L2) on mDCs which in turn induces IL-10 secreting regulatory T-cells (Tr1 cells) and thus acts as a negative feedback mechanism against proinflammatory immune responses. DESIGN/METHODS: FACS sorted peripheral blood natural mDCs were isolated from 13 RRMS patients and 13 controls and activated during 24h with LPS or LPS+IL-27. RNA was extracted and their profile was analyzed for gene expression (Nanostring) and microRNAs (Exiqon) assays. Genes that were found differentially expressed (PD-L2, IDO, SOCS1, IRF1, IL6R) were confirmed by RT-qPCR and their proteins studied by FACS and confocal microscopy. Activated DCs were also co-cultured with naive T-cells for 5 days from the same donors at which time T-cell proliferation was measured and supernatants assayed for cytokines measurements using LUMINEX. RESULTS: We found decreased IL-27-induced PD-L2 expression in mDCs stimulated with LPS+IL27 in MS vs. HCs (p<0.01). PD-L2 induction in IL-27-stimulated DCs correlated with the expression of IL-10 as neutralization of IL-10 abrogated IL-27-induced PD-L2 expression (p<0.01). In HCs, T-cells cultured with mDCs treated with IL-27 proliferated less and secreted increased amounts of IL-10 and less pro-inflammatory cytokines (IL17F, IL23, TNF-alpha). This tolerogenic effect of IL-27 was not observed in DCs isolated from MS patients. CONCLUSION: We found that the induction of tolerogenic mDCs by IL-27 was defective in RRMS and that this was related to the PD-L2/PD1 pathway. Disclosure: Dr. Von Glehn Silva has nothing to disclose. Dr. Murugaiyan has nothing to disclose. Dr. Regev has nothing to disclose. Dr. Kuhn has nothing to disclose. Dr. Raheja has nothing to disclose. Dr. Mazzola has nothing to disclose. Dr. Paul has nothing to disclose. Dr. Jangi has nothing to disclose. Dr. Kivisakk received research support from Merck-Serono. Dr. Gandhi has received personal compensation for activities with Biogen and Novartis. Dr. Weiner has nothing to disclose.

The induction of tolerance is a major goal of immunotherapy. Investigations over the last 20 years have shown that anti-CD3 monoclonal antibodies (mAbs) effectively treat autoimmune disease in animal models and have also shown promise in clinical trials. Tolerance induction by anti-CD3 mAbs is related to the induction of Tregs that control pathogenic autoimmune responses. Here, we review preclinical and clinical studies in which intravenous or mucosal administration of anti-CD3 mAbs has been employed and provide an outlook on future developments to enhance the efficacy of this promising therapeutic approach.

In this brief review we propose to discuss salient data showing the importance of immune regulatory mechanisms, and in particular of Treg, for the control of pathogenic anti-β-cell response in autoimmune diabetes. Disease progression that culminates with the massive destruction of insulin-secreting β-cells and advent of hyperglycemia and glycosuria tightly correlates with a functional deficit in immune regulation. Better dissection of the cellular and molecular mechanisms through which the immune system normally sustains tolerance to "self", and which become defective when autoimmune aggression is overt, is the only direct and robust way to learn how to harness these effectively, so as to restore immune tolerance in patients with insulin-dependent type 1 diabetes. No doubt that regulatory T cells are a privileged mechanism underlying this self-tolerance in the periphery. The discovery of the key role of the transcription factor FoxP3, represented the cornerstone leading to the great advances in the field we are witnessing today. Type 1 diabetes is certainly one of the prototypic T cell-mediated autoimmune diseases where immune regulatory mechanisms relying on specialized subsets of T cells have been the most thoroughly analyzed from the fundamental point of view and also largely exploited in a translational therapeutic perspective.

Oral antigen administration results in systemic hyporesponsiveness to a subsequent challenge with the fed antigen (oral tolerance) and has been shown to inhibit autoimmune disease in multiple animal models. However, transfer of this strategy to the clinics has not yet been achieved. Our aim is to better understand mechanisms implicated in the generation of Th3 cells and oral tolerance in order to enhance oral tolerance induction for a use in the clinics. Low dose oral tolerance depends on the presence of Th3 type Tregs, characterized by surface expression of latent TGF-beta (LAP and TGF-beta) and its adapter protein GARP (LRRC32). We established an in vitro system to induce Th3 like cells that express LAP and GARP on their surface. In vitro induced CD4+LAP+ T cells suppress proliferation of naïve T cells and production of IL-17A by Th17 cells. RT- PCR showed that membrane bound TGF-beta correlated with mRNA transcription levels of GARP but not TGF-beta, suggesting that the availability of GARP directly limits the expression of membrane bound TGF-beta. We used this in vitro system to screen for molecules that modulate generation of Th3 like cells and identified IL-6 as a potent inhibitor of membrane TGF-beta. IL-6 inhibited transcription of GARP in a STAT3 dependent manner. To test the therapeutic potential of targeting IL-6R signaling additionally to oral tolerance induction we assessed the DTH (delayed-type hypersensitivity) response in wild-type or CD4CreIL6Rafl/fl mice after feeding with ovalbumin for 5 consecutive days. Importantly, oral tolerance was significantly improved in mice that lack IL-6R signaling in CD4+ T cells. This demonstrates that blocking of IL-6R signaling is a promising approach to enhance oral tolerance induction.