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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

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.

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.

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.

Accumulation of IL-17–producing Th17 cells is associated with the development of multiple autoimmune diseases; however, the contribution of microRNA (miRNA) pathways to the intrinsic control of Th17 development remains unclear. Here, we demonstrated that miR-21 expression is elevated in Th17 cells and that mice lacking miR-21 have a defect in Th17 differentiation and are resistant to experimental autoimmune encephalomyelitis (EAE). Furthermore, we determined that miR-21 promotes Th17 differentiation by targeting and depleting SMAD-7, a negative regulator of TGF-β signaling. Moreover, the decreases in Th17 differentiation in miR-21–deficient T cells were associated with defects in SMAD-2/3 activation and IL-2 suppression. Finally, we found that treatment of WT mice with an anti–miR-21 oligonucleotide reduced the clinical severity of EAE, which was associated with a decrease in Th17 cells. Thus, we have characterized a T cell–intrinsic miRNA pathway that enhances TGF-β signaling, limits the autocrine inhibitory effects of IL-2, and thereby promotes Th17 differentiation and autoimmunity.

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.

IL-9–producing Th9 cells have been associated with autoimmune diseases, such as experimental autoimmune encephalitis. However, the factors that negatively regulate Th9 cells during autoimmune inflammation are unclear. In this article, we show that IFN-γ inhibits Th9 differentiation both in vitro and in vivo. This suppressive activity was dependent on the transcription factor STAT-1. In addition to its direct inhibitory effect on Th9 differentiation, IFN-γ suppressed Th9 cells through the induction of IL-27 from dendritic cells. In vitro, treatment of naive CD4+ T cells with IL-27 suppressed the development of Th9 cells, which was partially dependent on the transcription factors STAT-1 and T-bet. Moreover, IL-27 treatment completely abrogated the encephalitogenicity of Th9 cells in the experimental autoimmune encephalomyelitis model. Thus, our results identify a previously unknown mechanism by which IFN-γ limits Th9-mediated autoimmune inflammation through dendritic cell modulation of IL-27.

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.

SEE WINGER AND ZAMVIL DOI101093/BRAIN/AWW121 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: The innate immune system plays a central role in the chronic central nervous system inflammation that drives neurological disability in progressive forms of multiple sclerosis, for which there are no effective treatments. The mucosal immune system is a unique tolerogenic organ that provides a physiological approach for the induction of regulatory T cells. Here we report that nasal administration of CD3-specific antibody ameliorates disease in a progressive animal model of multiple sclerosis. This effect is IL-10-dependent and is mediated by the induction of regulatory T cells that share a similar transcriptional profile to Tr1 regulatory cells and that suppress the astrocyte inflammatory transcriptional program. Treatment results in an attenuated inflammatory milieu in the central nervous system, decreased microglia activation, reduced recruitment of peripheral monocytes, stabilization of the blood-brain barrier and less neurodegeneration. These findings suggest a new therapeutic approach for the treatment of progressive forms of multiple sclerosis and potentially other types of chronic central nervous system inflammation.

The gut-associated lymphoid tissue is the largest immune organ in the body and is the primary route by which we are exposed to antigens. Although most immune cells present in the gut display an activated state, in physiological conditions tolerance induction is the default immune pathway at this site. Immunological tolerance to antigens that gain access to the body via the oral route has been termed “oral tolerance” and appears to be physiologically important to prevent intestinal disorders, such as food allergy, celiac disease, and inflammatory bowel disease. Because of its unique immunological properties, oral tolerance is an attractive approach for the development of nontoxic and more effective treatments for different inflammatory and autoimmune diseases. Here we review the basic mechanisms underlying the establishment and maintenance of oral tolerance and discuss the potential to translate it more effectively to the clinic.

In recent years, our knowledge about immunoregulation and autoimmunity has significantly advanced, but nontoxic and more effective treatments for different inflammatory and autoimmune diseases are still lacking. Oral tolerance is of unique immunologic importance because it is a continuous natural immunologic event driven by exogenous antigen and is an attractive approach for treatment of these conditions. Parenteral administration of anti-CD3 monoclonal antibody is an approved therapy for transplantation in humans and is effective in autoimmune diabetes. Orally administered anti-CD3 monoclonal antibody is biologically active in the gut and suppresses experimental models of autoimmune diseases. Orally delivered antibody does not have side effects including cytokine release syndromes, thus oral anti-CD3 antibody is clinically applicable for chronic therapy. Here we review findings that identify a novel and powerful immunologic approach that is widely applicable for the treatment of human autoimmune conditions.

The gut-associated lymphoid tissue is the largest immune organ in the body and is the primary route by which we are exposed to antigens. Tolerance induction is the default immune pathway in the gut, and the type of tolerance induced relates to the dose of antigen fed: anergy/deletion (high dose) or regulatory T-cell (Treg) induction (low dose). Conditioning of gut dendritic cells (DCs) by gut epithelial cells and the gut flora, which itself has a major influence on gut immunity, induces CD103(+) retinoic acid-dependent DC that induces Tregs. A number of Tregs are induced at mucosal surfaces. Th3 type Tregs are transforming growth factor-β dependent and express latency-associated peptide (LAP) on their surface and were discovered in the context of oral tolerance. Tr1 type Tregs (interleukin-10 dependent) are induced by nasal antigen and forkhead box protein 3(+) iTregs are induced by oral antigen and by oral administration of aryl hydrocarbon receptor ligands. Oral or nasal antigen ameliorates autoimmune and inflammatory diseases in animal models by inducing Tregs. Furthermore, anti-CD3 monoclonal antibody is active at mucosal surfaces and oral or nasal anti-CD3 monoclonal antibody induces LAP(+) Tregs that suppresses animal models (experimental autoimmune encephalitis, type 1 and type 2 diabetes, lupus, arthritis, atherosclerosis) and is being tested in humans. Although there is a large literature on treatment of animal models by mucosal tolerance and some positive results in humans, this approach has yet to be translated to the clinic. The successful translation will require defining responsive patient populations, validating biomarkers to measure immunologic effects, and using combination therapy and immune adjuvants to enhance Treg induction. A major avenue being investigated for the treatment of autoimmunity is the induction of Tregs and mucosal tolerance represents a non-toxic, physiologic approach to reach this goal.

A tolerância oral é caracterizada por uma redução da reatividade imunológica específica a uma proteína previamente administrada por via oral. A exposição parenteral a antígenos para os quais a tolerância oral foi previamente induzida afeta (inibe) atividades imunológicas ligadas a antígenos não relacionados, fenômeno que denominamos efeitos indiretos da tolerância oral. Investigamos a possibilidade de transferir a tolerância oral e seus efeitos indiretos para receptores singênicos imunocompetentes por transfusão de células do baço e soro de doadores tolerantes. Investigamos também se os efeitos indiretos da tolerância oral interferem no processo de rejeição de enxertos alogênicos. A transferência adotiva da tolerância oral e de seus efeitos indiretos é mais facilmente observada quando avaliamos a reação de DTH em receptores C57BL/6 e BALB/c. Camundongos BALB/c são mais susceptíveis à transferência adotiva destes fenômenos quando analisamos a produção de anticorpos específicos. Em receptores com o sistema imune intacto, existe uma barreira (barreira singênica) que dificulta a implantação funcional das células transferidas, interferindo em suas atividades imunológicas específicas. Entretanto, a transferência de diferentes componentes do sistema imune é capaz de suplantar essa barreira, demonstrando a importância de interações entre componentes celulares e moleculares para o estabelecimento dos fenômenos aqui estudados. Os efeitos indiretos da tolerância oral parecem retardar a rejeição de enxertos de pele alogênicos, demonstrando seu caráter sistêmico. Fenômenos como a barreira singênica e os efeitos indiretos da tolerância oral são melhor entendidos em modelos que descrevam a interdependência dos componentes do sistema imune. Oral tolerance is characterized by a reduction in specific immunological activities to a protein previously given by oral route. Parenteral exposure to antigens to which oral tolerance had been previously induced affects (inhibits) immunological activities connected to unrelated antigens, a phenomenon we have called indirect effects of oral tolerance. Herein, we investigated the possibility of adoptively transferring oral tolerance and its indirect effects to immunocompetent syngeneic recipients by the transfusion of spleen cells and serum from tolerant donors. We also investigated if the indirect effects of oral tolerance interfere in the rejection of allografts. The adoptive transfer of oral tolerance and its indirect effects is more easily demonstrated when we evaluated DTH reaction in C57BL/6 and BALB/c recipient mice. BALB/c mice are more susceptible to the adoptive transfer of these phenomena when we evaluate specific antibody production. In recipients with an intact immune system, there is a barrier (syngeneic barrier) that impairs the functional implantation of transferred cells, interfering with their specific immunological activities. However, the transfer of different immune system components is able to bypass this barrier, demonstrating the importance of interactions between molecular and cellular components to the establishment of the phenomena studied herein. The indirect effects of oral tolerance seem to delay the rejection of skin allografts, demonstrating its systemic character. Phenomena such as the syngeneic barrier and the indirect effects of oral tolerance will better understood in models that describe the interdependence among the components of the immune system.

A exposição parenteral a antígenos para os quais a tolerância oral foi previamente induzida afeta (inibe) atividades imunológicas ligadas a antígenos não relacionados, fenômeno que denominamos efeitos indiretos da tolerância oral (Carvalho, et al., 1994). Investigamos a possibilidade de transferir a tolerância oral e seus efeitos indiretos para receptores singênicos normais ou previamente irradiados por transfusão de células do baço. Tanto a tolerância oral, quanto seus efeitos indiretos, foram transferidos apenas para receptores irradiados, sendo que os efeitos indiretos requisitaram um maior número de células, o que sugere o envolvimento de um maior número de interações celulares nos efeitos indiretos. Em receptores não-irradiados existe uma barreira (barreira singênica) que impede a implantação funcional das células transferidas, interferindo em suas atividades imunológicas específicas. O padrão de distribuição das células transferidas em diferentes órgãos linfóides foi estudado por marcação com CFSE. Nos receptores irradiados, a porcentagem de células marcadas era maior em todos os órgãos estudados, independentemente se provindas de doadores tolerantes ou não-tolerantes. Entretanto, o número absoluto tende a ser um pouco maior nos receptores não irradiados. Animais receptores de células de doadores não-tolerantes, no terceiro dia após a transferência, apresentaram uma redução, tanto na porcentagem quanto no número absoluto de células marcadas com CFSE, quando comparados com os receptores de células de doadores tolerantes. Isto sugere que há diferenças na dinâmica de decaimento das células transferidas de doadores tolerantes ou não-tolerantes. Fenômenos como a barreira singênica e os efeitos indiretos da tolerância oral talvez sejam melhor entendidos em modelos que descrevam a interdependência dos componentes do sistema imune. Parenteral exposure to antigens to which oral tolerance had been previously induced affects (inhibits) immunological activities connected to unrelated antigens, a phenomenon we have called indirect effects of oral tolerance (Carvalho, et al., 1994). Herein, we investigated the possibility of adoptively transferring oral tolerance and its indirect effects to normal or previously irradiated syngeneic recipients, by spleen cells. Both oral tolerance and its indirect effects were transferred only to irradiated recipients and a larger number of cells was necessary for the transfer of the indirect effects, which suggests the participation of more complex cellular interactions in these effects. In nonirradiated recipients, there is a barrier (syngeneic barrier) that impairs the functional implantation of transferred cells, interfering with their specific immunological activities. We also studied the distribution patterns of transferred cells in different lymphoid organs from recipient mice, by CFSE labeling. In irradiated recipients, independently of the origin of transferred cells, the percentage of labeled cells was higher, in every studied organ. However, the absolute number was a little higher in nonirradiated recipients. Recipients of cells from nontolerant donors displayed a decrease, both in the percentage and in the absolute number of CFSE labeled cells on the third day after cell transference, as compared to recipients of cells from tolerant donors. This suggests different dynamics of decay of adoptively transferred cells from tolerant or non-tolerant donors. Phenomena such as the syngeneic barrier and the indirect effects of oral tolerance will be better understood in models that describe the interdependence among the components of the immune system.

IL-17 producing Th17 cells are the key participants in various autoimmune diseases including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although differentiation of Th17 cells are known to be regulated by specific transcription factors and cytokines, the role of microRNAs that control this inflammatory T cell subset and whether targeting microRNAs can have therapeutic effects are not understood well. We found that microRNA-21 expression is elevated in Th17 cells and Mir-21-/- mice show a defect in Th17 differentiation and are resistant to EAE. Mir-21-/- T cells fail to induce known factors required for Th17 differentiation, such as transcription factor RORgt and the cytokine IL-21. However, Mir-21-/- mice have normal Th1, Th2 and Treg differentiation. The T cell intrinsic function of Mir-21 was clearly demonstrated by the sensitivity to EAE of the Mir-21-/- mice into which WT CD4+ T cells were transferred. Mir-21 promoted Th17 differentiation by targeting Smad-7, a negative regulator of TGF-beta signaling and Th17 differentiation. Finally, we found that anti-Mir-21 treatment reduced clinical severity of EAE associated with decrease in Th17 cells. These findings demonstrate that Mir-21 confers susceptibility to EAE by affecting inflammatory Th17 responses and identify Mir-21 as a new target for therapeutic intervention in MS.