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Due to the rise in overnutrition, the incidence of obesity-induced hepatocellular carcinoma (HCC) will continue to escalate; however, our understanding of the obesity to HCC developmental axis is limited. We constructed a single-cell atlas to interrogate the dynamic transcriptomic changes during hepatocarcinogenesis in mice. Here we identify fatty acid binding protein 5 (FABP5) as a driver of obesity-induced HCC. Analysis of transformed cells reveals that FABP5 inhibition and silencing predispose cancer cells to lipid peroxidation and ferroptosis-induced cell death. Pharmacological inhibition and genetic ablation of FABP5 ameliorates the HCC burden in male mice, corresponding to enhanced ferroptosis in the tumour. Moreover, FABP5 inhibition induces a pro-inflammatory tumour microenvironment characterized by tumour-associated macrophages with increased expression of the co-stimulatory molecules CD80 and CD86 and increased CD8 + T cell activation. Our work unravels the dual functional role of FABP5 in diet-induced HCC, inducing the transformation of hepatocytes and an immunosuppressive phenotype of tumour-associated macrophages and illustrates FABP5 inhibition as a potential therapeutic approach., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Blood vessels are continually exposed to circulating lipids, and elevation of ApoB-containing lipoproteins causes atherosclerosis. Lipoprotein metabolism is highly regulated by lipolysis, largely at the level of the capillary endothelium lining metabolically active tissues. How large blood vessels, the site of atherosclerotic vascular disease, regulate the flux of fatty acids (FAs) into triglyceride-rich (TG-rich) lipid droplets (LDs) is not known. In this study, we showed that deletion of the enzyme adipose TG lipase (ATGL) in the endothelium led to neutral lipid accumulation in vessels and impaired endothelial-dependent vascular tone and nitric oxide synthesis to promote endothelial dysfunction. Mechanistically, the loss of ATGL led to endoplasmic reticulum stress-induced inflammation in the endothelium. Consistent with this mechanism, deletion of endothelial ATGL markedly increased lesion size in a model of atherosclerosis. Together, these data demonstrate that the dynamics of FA flux through LD affects endothelial cell homeostasis and consequently large vessel function during normal physiology and in a chronic disease state.

Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3'-untranslated region (3'-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3'UTR of SERINC5 mRNA, reducing SERINC5 expression in vitro . Moreover, we found that an anti-svRNA treatment to Vero E6 cells before SARS-CoV-2 infection recovered the levels of SERINC5 and reduced the levels of N and S viral proteins. Finally, we showed that SERINC5 positively controls the levels of Mitochondrial Antiviral Signalling (MAVS) protein in Vero E6. These results highlight the therapeutic potential of targeting svRNAs based on their action on key proteins of the innate immune response during SARS-CoV-2 viral infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Meseguer, Rubio, Lainez, Pérez-Benavente, Pérez-Moraga, Romera-Giner, García-García, Martinez-Macias, Cremades, Iborra, Candelas-Rivera, Almazan and Esplugues.)

Adaptive immunity provides life-long protection by generating central and effector memory T cells and the most recently described tissue resident memory T (T RM ) cells. However, the cellular origin of CD4 T RM cells and their contribution to host defense remain elusive. Using IL-17A tracking-fate mouse models, we found that a significant fraction of lung CD4 T RM cells derive from IL-17A-producing effector (T H 17) cells following immunization with heat-killed Klebsiella pneumonia (Kp). These exT H 17 T RM cells are maintained in the lung by IL-7, produced by lymphatic endothelial cells. During a memory response, neither antibodies, γδ T cells, nor circulatory T cells are sufficient for the rapid host defense required to eliminate Kp. Conversely, using parabiosis and depletion studies, we demonstrated that exT H 17 T RM cells play an important role in bacterial clearance. Thus, we delineate the origin and function of airway CD4 T RM cells during bacterial infection, offering novel strategies for targeted vaccine design., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The gut microbiota has been causally linked to cancer, yet how intestinal microbes influence progression of extramucosal tumors is poorly understood. Here we provide evidence implying that Prevotella heparinolytica promotes the differentiation of Th17 cells colonizing the gut and migrating to the bone marrow (BM) of transgenic Vk*MYC mice, where they favor progression of multiple myeloma (MM). Lack of IL-17 in Vk*MYC mice, or disturbance of their microbiome delayed MM appearance. Similarly, in smoldering MM patients, higher levels of BM IL-17 predicted faster disease progression. IL-17 induced STAT3 phosphorylation in murine plasma cells, and activated eosinophils. Treatment of Vk*MYC mice with antibodies blocking IL-17, IL-17RA, and IL-5 reduced BM accumulation of Th17 cells and eosinophils and delayed disease progression. Thus, in Vk*MYC mice, commensal bacteria appear to unleash a paracrine signaling network between adaptive and innate immunity that accelerates progression to MM, and can be targeted by already available therapies.

T H 17 cells are major drivers of inflammation and involved in several autoimmune diseases. Tissue inflammation is a beneficial host response to infection, but it can also contribute to autoimmunity. The crosstalk between a tissue and the immune system during an inflammatory response is key for preserving tissue integrity and restoring physiological processes. However, how the inflamed tissue regulates the magnitude of an immune response by controlling pro-inflammatory T cells is not well characterized so far. Here we show that T H 17 cells accumulating in the small intestine upon inflammation express the IL-33 receptor (ST2) and intestinal epithelial cells (IEC) are the main source of the alarmin interleukin-33 (IL-33). We show that pro-inflammatory T H 17 cells acquire a regulatory phenotype with immunosuppressive properties in response to IL-33. Absence of ST2 signaling promotes the secretion of pro-inflammatory cytokines by T H 17 cells and dampens the secretion of IL-10. Our results provide new insights into the mechanisms by which IEC, via IL-33/ST2 axis, may control pro-inflammatory T H 17 cells in the small intestine to sustain homeostasis.

Growing insight into the pathogenesis of autoimmune diseases and numerous studies in preclinical models highlights the potential of regulatory T cells to restore tolerance. By using non-obese diabetic (NOD) BDC2.5 TCR-transgenic (Tg), and IL-10 and Foxp3 double-reporter mice, we demonstrate that alteration of gut microbiota during cohousing experiments or treatment with anti-CD3 mAb significantly increase intestinal IL-10-producing type 1 regulatory T (Tr1) cells and decrease diabetes incidence. These intestinal antigen-specific Tr1 cells have the ability to migrate to the periphery via a variety of chemokine receptors such as CCR4, CCR5, and CCR7 and to suppress proliferation of Th1 cells in the pancreas. The ability of Tr1 cells to cure diabetes in NOD mice required IL-10 signaling, as Tr1 cells could not suppress CD4 + T cells with a dominant-negative IL-10R. Taken together, our data show a key role of intestinal Tr1 cells in the control of effector T cells and development of diabetes. Therefore, modulating gut-associated lymphoid tissue to boost Tr1 cells may be important in type 1 diabetes management., Competing Interests: The authors declare no conflict of interest.

IL-10 is essential to maintain intestinal homeostasis. CD4 + T regulatory type 1 (T R 1) cells produce large amounts of this cytokine and are therefore currently being examined in clinical trials as T cell therapy in patients with inflammatory bowel disease. However, factors and molecular signals sustaining T R 1 cell regulatory activity still need to be identified to optimize the efficiency and ensure the safety of these trials. We investigated the role of IL-10 signaling in mature T R 1 cells in vivo. Double IL-10 eGFP Foxp3 mRFP reporter mice and transgenic mice with impairment in IL-10 receptor signaling were used to test the activity of T R 1 cells in a murine inflammatory bowel disease model, a model that resembles the trials performed in humans. The molecular signaling was elucidated in vitro. Finally, we used human T R 1 cells, currently employed for cell therapy, to confirm our results. We found that murine T R 1 cells expressed functional IL-10Rα. T R 1 cells with impaired IL-10 receptor signaling lost their regulatory activity in vivo. T R 1 cells required IL-10 receptor signaling to activate p38 MAPK, thereby sustaining IL-10 production, which ultimately mediated their suppressive activity. Finally, we confirmed these data using human T R 1 cells. In conclusion, T R 1 cell regulatory activity is dependent on IL-10 receptor signaling. These data suggest that to optimize T R 1 cell-based therapy, IL-10 receptor expression has to be taken into consideration., Competing Interests: The authors have declared that there is no conflict of interest., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Germinal center (GC) B cells undergo affinity selection, which depends on interactions with CD4(+) follicular helper T cells (TFH cells). We found that TFH cells progressed through transcriptionally and functionally distinct stages and provided differential signals for GC regulation. They initially localized proximally to mutating B cells, secreted interleukin 21 (IL-21), induced expression of the transcription factor Bcl-6 and selected high-affinity B cell clones. As the GC response evolved, TFH cells extinguished IL-21 production and switched to IL-4 production, showed robust expression of the co-stimulatory molecule CD40L, and promoted the development of antibody-secreting B cells via upregulation of the transcription factor Blimp-1. Thus, TFH cells in the B cell follicle progressively differentiate through stages of localization, cytokine production and surface ligand expression to 'fine tune' the GC reaction.

Microbial infections often precede the onset of autoimmunity. How infections trigger autoimmunity remains poorly understood. We investigated the possibility that infection might create conditions that allow the stimulatory presentation of self peptides themselves and that this might suffice to elicit autoreactive T cell responses that lead to autoimmunity. Self-reactive CD4(+) T cells are major drivers of autoimmune disease, but their activation is normally prevented through regulatory mechanisms that limit the immunostimulatory presentation of self antigens. Here we found that the apoptosis of infected host cells enabled the presentation of self antigens by major histocompatibility complex class II molecules in an inflammatory context. This was sufficient for the generation of an autoreactive TH17 subset of helper T cells, prominently associated with autoimmune disease. Once induced, the self-reactive TH17 cells promoted auto-inflammation and autoantibody generation. Our findings have implications for how infections precipitate autoimmunity.

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c(+) cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c(+) cells. These CD11c(+) cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c(+) cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c(+) cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS., (© 2015 The Authors.)

Control of chronic viral infections by CD8 T cells is critically dependent on CD4 help. In particular, helper-derived IL-21 plays a key role in sustaining the CD8 T cell response; however, the molecular pathways by which IL-21 sustains CD8 T cell immunity remain unclear. We demonstrate that IL-21 causes a phenotypic switch of transcription factor expression in CD8 T cells during chronic viral infection characterized by sustained BATF expression. Importantly, BATF expression during chronic infection is both required for optimal CD8 T cell persistence and anti-viral effector function and sufficient to rescue "unhelped" CD8 T cells. Mechanistically, BATF sustains the response by cooperating with IRF4, an antigen-induced transcription factor that is also critically required for CD8 T cell maintenance, to preserve Blimp-1 expression and thereby sustain CD8 T cell effector function. Collectively, these data suggest that CD4 T cells "help" the CD8 response during chronic infection via IL-21-induced BATF expression., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Inflammation is a beneficial host response to infection but can contribute to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (they can cease to express their signature cytokine, IL-17A) and plasticity (they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However, technical limitations have prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as transdifferentiation. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two new fate-mapping mouse models to track Th17 cells during immune responses to show that CD4(+) T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a change in their signature transcriptional profile and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF-β signalling and consequently for the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 cells transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest that Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.

T follicular helper (T(FH)) cells select high-affinity, antibody-producing B cells for clonal expansion in germinal centers (GCs), but the nature of their interaction is not well defined. Using intravital imaging, we found that selection is mediated by large but transient contacts between T(FH) and GC B cells presenting the highest levels of cognate peptide bound to major histocompatibility complex II. These interactions elicited transient and sustained increases in T(FH) intracellular free calcium (Ca(2+)) that were associated with T(FH) cell coexpression of the cytokines interleukin-4 and -21. However, increased intracellular Ca(2+) did not arrest TFH cell migration. Instead, T(FH) cells remained motile and continually scanned the surface of many GC B cells, forming short-lived contacts that induced selection through further repeated transient elevations in intracellular Ca(2+)., (Copyright © 2014, American Association for the Advancement of Science.)

Interchromosomal associations can regulate gene expression, but little is known about the molecular basis of such associations. In response to antigen stimulation, naive T cells can differentiate into Th1, Th2, and Th17 cells expressing IFN-γ, IL-4, and IL-17, respectively. We previously reported that in naive T cells, the IFN-γ locus is associated with the Th2 cytokine locus. Here we show that the Th2 locus additionally associates with the IL-17 locus. This association requires a DNase I hypersensitive region (RHS6) at the Th2 locus. RHS6 and the IL-17 promoter both bear Oct-1 binding sites. Deletion of either of these sites or Oct-1 gene impairs the association. Oct-1 and CTCF bind their cognate sites cooperatively, and CTCF deficiency similarly impairs the association. Finally, defects in the association lead to enhanced IL-17 induction. Collectively, our data indicate Th17 lineage differentiation is restrained by the Th2 locus via interchromosomal associations organized by Oct-1 and CTCF., (Copyright © 2014 Elsevier Inc. All rights reserved.)

T follicular helper cells (Tfh cells) play a pivotal role in germinal center reactions, which require B cell lymphoma 6 (Bcl6) transcription factor. To analyze their relationships with other effector T cell lineages and their stability in vivo, we developed and analyzed a new Bcl6 reporter mouse alone or together with other lineage reporter systems. Assisted with genome-wide transcriptome analysis, we show substantial plasticity of T cell differentiation in the early phase of immune response. At this stage, CXCR5 appears to be expressed in a Bcl6-independent manner. Once Bcl6 is highly expressed, Tfh cells can persist in vivo and some of them develop into memory cells. Together, our results indicate Bcl6 as a bona fide marker for Tfh polarized program.

Intracellular (clade B) OVA-serpin protease inhibitors play an important role in tissue homeostasis by protecting cells from death in response to hypo-osmotic stress, heat shock, and other stimuli. It is not known whether these serpins influence immunological tolerance and the risk for autoimmune diseases. We found that a fraction of young autoimmune diabetes-prone NOD mice had elevated levels of autoantibodies against a member of clade B family known as serpinB13. High levels of anti-serpinB13 Abs were accompanied by low levels of anti-insulin autoantibodies, reduced numbers of islet-associated T cells, and delayed onset of diabetes. Exposure to anti-serpinB13 mAb alone also decreased islet inflammation, and coadministration of this reagent and a suboptimal dose of anti-CD3 mAb accelerated recovery from diabetes. In a fashion similar to that discovered in the NOD model, a deficiency in humoral activity against serpinB13 was associated with early onset of human type 1 diabetes. These findings suggest that, in addition to limiting exposure to proteases within the cell, clade B serpins help to maintain homeostasis by inducing protective humoral immunity.

Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. microRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene expression, acting predominantly at posttranscriptional level. Recent work from our group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the Srebp genes, regulate cholesterol and fatty acid metabolism in concert with their host genes. Here, we show that hsa-miR-33 family members modulate the expression of genes involved in cell cycle regulation and cell proliferation. MiR-33 inhibits the expression of the cyclin-dependent kinase 6 (CDK6) and cyclin D1 (CCND1), thereby reducing cell proliferation and cell cycle progression. Overexpression of miR-33 induces a significant G 1 cell cycle arrest in Huh7 and A549 cell lines. Most importantly, inhibition of miR-33 expression using 2'fluoro/methoxyethyl-modified (2'F/MOE-modified) phosphorothioate backbone antisense oligonucleotides improves liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these results suggest that Srebp/miR-33 locus may cooperate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration.

Genome-wide association studies (GWAS) in immune-mediated diseases have identified over 150 associated genomic loci. Many of these loci play a role in T cell responses, and regulation of T cell differentiation plays a critical role in immune-mediated diseases; however, the relationship between implicated disease loci and T cell differentiation is incompletely understood. To further address this relationship, we examined differential gene expression in naïve human CD4+ T cells, as well as in in vitro differentiated Th1, memory Th17-negative and Th17-enriched CD4+ T cells subsets using microarray and RNASeq. We observed a marked enrichment for increased expression in memory CD4+ compared to naïve CD4+ T cells of genes contained among immune-mediated disease loci. Within memory T cells, expression of disease-associated genes was typically increased in Th17-enriched compared to Th17-negative cells. Utilizing RNASeq and promoter methylation studies, we identified a differential regulation pattern for genes solely expressed in Th17 cells (IL17A and CCL20) compared to genes expressed in both Th17 and Th1 cells (IL23R and IL12RB2), where high levels of promoter methylation are correlated to near zero RNASeq levels for IL17A and CCL20. These findings have implications for human Th17 celI plasticity and for the regulation of Th17-Th1 expression signatures. Importantly, utilizing RNASeq we found an abundant isoform of IL23R terminating before the transmembrane domain that was enriched in Th17 cells. In addition to molecular resolution, we find that RNASeq provides significantly improved power to define differential gene expression and identify alternative gene variants relative to microarray analysis. The comprehensive integration of differential gene expression between cell subsets with disease-association signals, and functional pathways provides insight into disease pathogenesis.

Recent studies have shown that IL-17 can contribute beneficially to pathogen defense but also that excessive IL-17 levels are associated with chronic inflammation and autoimmune disorders. To date, the role of IL-17 in viral infections and type 1 diabetes is ambiguous. In this study, we used IL-17A enhanced green fluorescent protein bicistronic reporter mouse strains to analyze in situ production of IL-17A. Upon Klebsiella pneumoniae bacterial infection, CD4(+) and γδ T cells produce IL-17A. In contrast, CD4(+) or CD8(+) T cells do not produce IL-17A in response to acute or protracted viral infection with lymphocytic choriomeningitis virus or during autoimmune diabetes development in the CD8-driven lymphocytic choriomeningitis virus-induced model of type 1 diabetes. We conclude that viral elimination and type 1 diabetes can occur in the absence of detectable IL-17A production, suggesting IL-17A is not essential in these settings.

Interleukin (IL)-17-producing T helper cells (T(H)17) are a recently identified CD4(+) T cell subset distinct from T helper type 1 (T(H)1) and T helper type 2 (T(H)2) cells. T(H)17 cells can drive antigen-specific autoimmune diseases and are considered the main population of pathogenic T cells driving experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis. The factors that are needed for the generation of T(H)17 cells have been well characterized. However, where and how the immune system controls T(H)17 cells in vivo remains unclear. Here, by using a model of tolerance induced by CD3-specific antibody, a model of sepsis and influenza A viral infection (H1N1), we show that pro-inflammatory T(H)17 cells can be redirected to and controlled in the small intestine. T(H)17-specific IL-17A secretion induced expression of the chemokine CCL20 in the small intestine, facilitating the migration of these cells specifically to the small intestine via the CCR6/CCL20 axis. Moreover, we found that T(H)17 cells are controlled by two different mechanisms in the small intestine: first, they are eliminated via the intestinal lumen; second, pro-inflammatory T(H)17 cells simultaneously acquire a regulatory phenotype with in vitro and in vivo immune-suppressive properties (rT(H)17). These results identify mechanisms limiting T(H)17 cell pathogenicity and implicate the gastrointestinal tract as a site for control of T(H)17 cells.

Cellular imbalances of cholesterol and fatty acid metabolism result in pathological processes, including atherosclerosis and metabolic syndrome. Recent work from our group and others has shown that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are located within the sterol regulatory element-binding protein-2 and -1 genes, respectively, and regulate cholesterol homeostasis in concert with their host genes. Here, we show that miR-33a and -b also regulate genes involved in fatty acid metabolism and insulin signaling. miR-33a and -b target key enzymes involved in the regulation of fatty acid oxidation, including carnitine O-octaniltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA-dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. Moreover, miR-33a and -b also target the insulin receptor substrate 2, an essential component of the insulin-signaling pathway in the liver. Overexpression of miR-33a and -b reduces both fatty acid oxidation and insulin signaling in hepatic cell lines, whereas inhibition of endogenous miR-33a and -b increases these two metabolic pathways. Together, these data establish that miR-33a and -b regulate pathways controlling three of the risk factors of metabolic syndrome, namely levels of HDL, triglycerides, and insulin signaling, and suggest that inhibitors of miR-33a and -b may be useful in the treatment of this growing health concern.

T helper 17 (Th17) cells are important for host defense against extracellular microorganisms. However, they are also implicated in autoimmune and chronic inflammatory diseases, and as such need to be tightly regulated. The mechanisms that directly control committed pathogenic Th17 cells in vivo remain unclear. We showed here that IL-17A-producing CD4+ T cells expressed interleukin-10 receptor α (IL-10Rα) in vivo. Importantly, T cell-specific blockade of IL-10 signaling led to a selective increase of IL-17A+IFN-γ⁻ (Th17) and IL-17A+IFN-γ+ (Th17+Th1) CD4+ T cells during intestinal inflammation in the small intestine. CD4+Foxp3⁻ IL-10-producing (Tr1) cells and CD4+Foxp3+ regulatory (Treg) cells were able to control Th17 and Th17+Th1 cells in an IL-10-dependent manner in vivo. Lastly, IL-10 treatment of mice with established colitis decreased Th17 and Th17+Th1 cell frequencies via direct signaling in T cells. Thus, IL-10 signaling directly suppresses Th17 and Th17+Th1 cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mouse infection with intracellular bacteria induces a potent inflammatory response that requires protective mechanisms to avoid infection-induced immune pathology. CD69 is expressed in all leukocytes during activation after infection with a wide range of microbial pathogens. This study explores the way in which CD69 affects cell activation after Listeria monocytogenes (Lm) infection and its effects on host protection. We show that infectivity and bacterial clearance capability are unaltered in CD69(-/-) peritoneal macrophages, bone marrow-derived macrophages and dendritic cells. We found no major altered cell populations in splenocytes of Lm-infected CD69(-/-) mice. However, an increase in the expression of Th1 cytokines was observed after infection, with increased production of type I and II interferon (IFN). In addition, CD69(-/-) splenocytes showed increased apoptosis, consistent with IFN enhancement of lymphocyte apoptosis in response to Lm infection. CD69(-/-) mice showed liver and spleen damage, and greatly increased susceptibility to Lm infection, compared with wild-type controls. Lm-specific T cells were decreased in CD69(-/-) mice even if T-cell cross-presentation and T-cell intrinsic priming response were not compromised. As listeriosis was increased as early as day 1 post-infection but CD69(-/-)RAG2(-/-) mice were more efficient at controlling Listeria, we propose that CD69 controls the cross-talk between innate components and lymphocytes. These results highlight a role for CD69 in preventing infection-induced immunopathology.

CD69 expression is induced following activation of leukocytes at inflammatory sites and plays a negative regulatory role in the development of collagen-induced arthritis (CIA). To evaluate potential strategies of CD69 targeting in chronic inflammatory diseases, two different anti-CD69 mAbs were generated and their effects on CIA were studied. Administration of the IgG1 anti-CD69 mAb 2.2 to DBA/1 mice with CIA led to an exacerbation of the disease, correlated with down-modulation of CD69 from the cell surface, and reproduced the phenotype of the CD69(-/-) mouse in wild-type animals. In contrast, treatment with the IgG2a anti-CD69 mAb 2.3 was effective in ameliorating CIA when administered in the early or intermediate phases of the disease, causing a decreased production of proinflammatory cytokines in inflammatory foci. Monoclonal antibody 2.3 induces partial depletion of CD69+ cells in vivo. Moreover, adoptive transfer of type-II collagen (CII)-sensitized cells treated with mAb 2.3 to deplete CD69+ cells did not result in arthritis. The attenuation of inflammation correlates with reduced lymphocyte proliferative response in response to CII and with a reduction in the frequency of CII-specific T cells producing IFN-gamma. We thus conclude that CD69 targeting by mAbs can either enhance or dampen the immune response.

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-gamma secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-gamma release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-gamma release involve distinct NK pathways.

The leukocyte activation marker CD69 is a novel regulator of the immune response, modulating the production of cytokines including transforming growth factor-beta (TGF-beta). We have generated an antimurine CD69 monoclonal antibody (mAb), CD69.2.2, which down-regulates CD69 expression in vivo but does not deplete CD69-expressing cells. Therapeutic administration of CD69.2.2 to wild-type mice induces significant natural killer (NK) cell-dependent antitumor responses to major histocompatibility complex (MHC) class I low RMA-S lymphomas and to RM-1 prostatic carcinoma lung metastases. These in vivo antitumor responses are comparable to those seen in CD69(-/-) mice. Enhanced host NK cytotoxic activity correlates with a reduction in NK-cell TGF-beta production and is independent of tumor priming. In vitro studies demonstrate the novel ability of anti-CD69 mAbs to activate resting NK cells in an Fc receptor-independent manner, resulting in a substantial increase in both NK-cell cytolytic activity and interferon gamma (IFNgamma) production. Modulation of the innate immune system with monoclonal antibodies to host CD69 thus provides a novel means to antagonize tumor growth and metastasis.

Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation and cell death induction in several cell types. The biological effects of TNF-alpha are mediated via the cell-surface TNF receptors TNFR1 and TNFR2. Soluble forms of these two receptors, which contain the extracellular ectodomains, are proteolytically cleaved from the membrane. High levels of soluble (s) TNFR2 in serum have been documented in multiple inflammatory pathologies. We describe here a new differential spliced isoform of human TNFR2 missing exons 7 and 8, DS-TNFR2(Delta7,8). This novel isoform lacks the transmembrane and cytoplasmic domains. Expression studies with DS-TNFR2(Delta7,8) cDNA transiently transfected COS cells showed that it encodes a sTNFR2 receptor of approximately 42 kDa. Soluble DS-TNFR2(Delta7,8) blocked TNF-alpha-induced apoptosis, which suggests that it regulates TNF-alpha function by antagonizing its biological activity. An ELISA was developed that quantifies sTNFR2 generated by alternative splicing. Our data show that sTNFR2 generated by alternative splicing can be found in sera of healthy individuals, at increased levels in patients with sepsis and at high concentrations in rheumatoid arthritis patients.

CD69 is induced after activation of leukocytes at inflammatory sites, but its physiological role during inflammation remains unknown. We explored the role of CD69 in autoimmune reactivity by analyzing a model of collagen-induced arthritis (CIA) in WT and CD69-deficient mice. CD69-/- mice showed higher incidence and severity of CIA, with exacerbated T and B cell immune responses to type II collagen. Levels of TGF-beta1 and TGF-beta2, which act as protective agents in CIA, were reduced in CD69-/- mice inflammatory foci, correlating with the increase in the proinflammatory cytokines IL-1beta and RANTES. Local injection of blocking anti-TGF-beta antibodies increased CIA severity and proinflammatory cytokine mRNA levels in CD69+/+ but not in CD69-/- mice. Moreover, in vitro engagement of CD69 induced total and active TGF-beta1 production in Concanavalin A-activated splenocyte subsets, mouse and human synovial leukocytes, and Jurkat stable transfectants of human CD69 but not in the parental CD69 negative cell line. Our results show that CD69 is a negative modulator of autoimmune reactivity and inflammation through the synthesis of TGF-beta, a cytokine that in turn downregulates the production of various proinflammatory mediators.

We investigated the in vivo role of CD69 by analyzing the susceptibility of CD69-/- mice to tumors. CD69-/- mice challenged with MHC class I- tumors (RMA-S and RM-1) showed greatly reduced tumor growth and prolonged survival compared with wild-type (WT) mice. The enhanced anti-tumor response was NK cell and T lymphocyte-mediated, and was due, at least in part, to an increase in local lymphocytes. Resistance of CD69-/- mice to MHC class I- tumor growth was also associated with increased production of the chemokine MCP-1, diminished TGF-beta production, and decreased lymphocyte apoptosis. Moreover, the in vivo blockade of TGF-beta in WT mice resulted in enhanced anti-tumor response. In addition, CD69 engagement induced NK and T cell production of TGF-beta, directly linking CD69 signaling to TGF-beta regulation. Furthermore, anti-CD69 antibody treatment in WT mice induced a specific down-regulation in CD69 expression that resulted in augmented anti-tumor response. These data unmask a novel role for CD69 as a negative regulator of anti-tumor responses and show the possibility of a novel approach for the therapy of tumors.

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4-CD8- thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-gamma secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.

To determine the prevalence and significance of the systolic compression of the anterior descending coronary artery in hypertrophic cardiomyopathy, we studied 54 consecutive patients out of a catheterization laboratory population of 1619. This angiographic finding was found to be more prevalent (P less than 0.001) and severe in myopathic than in secondary hypertrophy. Complete systolic occlusion occurred in 5 of the 6 patients with nonobstructive cardiomyopathy showing the systolic narrowing. Severe septal squeezing was also present in these cases and the diastolic time lag to refill the distal branches reached 20-33% of the diastolic period. This subset of patients showed the least dynamic anterior wall contraction (P less than 0.001) and the highest incidence of thallium-201 perfusion defects (P less than 0.05) and of recurrent cardiac arrest (P less than 0.05). We conclude that severe systolic compression of the descending coronary artery in hypertrophic cardiomyopathy may be an angiographic marker of the myopathic hypertrophy extending to the anterior wall and might contribute to ischemia when the time to restore the distal perfusion is greatly delayed.