The lipid globotriaosylceramide promotes germinal center B cell responses and antiviral immunity.

Influenza viruses escape immunity owing to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. We found that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 bound and disengaged CD19 from its chaperone CD81, permitting CD19 to translocate to the B cell receptor complex to trigger signaling. Moreover, Gb3 regulated major histocompatibility complex class II expression to increase diversity of T follicular helper and GC B cells reactive with subdominant epitopes. In influenza infection, elevating Gb3, either endogenously or exogenously, promoted broadly reactive antibody responses and cross-protection. These data demonstrate that Gb3 determines the affinity and breadth of B cell immunity and has potential as a vaccine adjuvant.

Hyperglycosylation of prosaposin in tumor dendritic cells drives immune escape.

Tumors develop strategies to evade immunity by suppressing antigen presentation. In this work, we show that prosaposin (pSAP) drives CD8 T cell-mediated tumor immunity and that its hyperglycosylation in tumor dendritic cells (DCs) leads to cancer immune escape. We found that lysosomal pSAP and its single-saposin cognates mediated disintegration of tumor cell-derived apoptotic bodies to facilitate presentation of membrane-associated antigen and T cell activation. In the tumor microenvironment, transforming growth factor-ß (TGF-ß) induced hyperglycosylation of pSAP and its subsequent secretion, which ultimately caused depletion of lysosomal saposins. pSAP hyperglycosylation was also observed in tumor-associated DCs from melanoma patients, and reconstitution with pSAP rescued activation of tumor-infiltrating T cells. Targeting DCs with recombinant pSAP triggered tumor protection and enhanced immune checkpoint therapy. Our studies demonstrate a critical function of pSAP in tumor immunity and may support its role in immunotherapy.

The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity.

Influenza viruses escape immunity due to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. Here, we demonstrate that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 binds and disengages CD19 from its chaperone CD81 for subsequent translocation to the B cell receptor (BCR) complex to trigger signaling. Abundance of Gb3 amplifies the PI3-kinase/Akt/Foxo1 pathway to drive affinity maturation. Moreover, this lipid regulates MHC-II expression to increase diversity of T follicular helper (Tfh) and GC B cells reactive with subdominant epitopes. In influenza infection, Gb3 promotes broadly reactive antibody responses and cross-protection. Thus, we show that Gb3 determines affinity as well as breadth in B cell immunity and propose this lipid as novel vaccine adjuvant against viral infection. One Sentence Summary: Gb3 abundance on GC B cells selects antibodies with high affinity and broad epitope reactivities, which are cross-protective against heterologous influenza infection.

Hyperglycosylation of prosaposin in tumor DCs promotes immune escape in cancer.

Tumors develop strategies to evade immunity by suppressing antigen presentation. Here, we show that prosaposin drives CD8 T cell-mediated tumor immunity and that its hyperglycosylation in tumor DCs leads to cancer immune escape. We found that lysosomal prosaposin and its single saposin cognates mediated disintegration of tumor cell-derived apoptotic bodies to facilitate presentation of membrane-associated antigen and T cell activation. In the tumor microenvironment, TGF-ß induced hyperglycosylation of prosaposin and its subsequent secretion, which ultimately caused depletion of lysosomal saposins. In melanoma patients, we found similar prosaposin hyperglycosylation in tumor-associated DCs, and reconstitution with prosaposin rescued activation of tumor-infiltrating T cells. Targeting tumor DCs with recombinant prosaposin triggered cancer protection and enhanced immune checkpoint therapy. Our studies demonstrate a critical function of prosaposin in tumor immunity and escape and introduce a novel principle of prosaposin-based cancer immunotherapy. One Sentence Summary: Prosaposin facilitates antigen cross-presentation and tumor immunity and its hyperglycosylation leads to immune evasion.

GARP on hepatic stellate cells is essential for the development of liver fibrosis.

BACKGROUND & AIMS: Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-ß docking molecule. While the immune regulatory properties of GARP on blood cells have been studied, the function of GARP on tissue stromal cells remains unclear. Here, we investigate the role of GARP expressed on hepatic stellate cells (HSCs) in the development of liver fibrosis. METHODS: The function of GARP on HSCs was explored in toxin-induced and metabolic liver fibrosis models, using conditional GARP-deficient mice or a newly generated inducible system for HSC-specific gene ablation. Primary mouse and human HSCs were isolated to evaluate the contribution of GARP to the activation of latent TGF-ß. Moreover, cell contraction of HSCs in the context of TGF-ß activation was tested in a GARP-dependent fashion. RESULTS: Mice lacking GARP in HSCs were protected from developing liver fibrosis. Therapeutically deleting GARP on HSCs alleviated the fibrotic process in established disease. Furthermore, natural killer T cells exacerbated hepatic fibrosis by inducing GARP expression on HSCs through IL-4 production. Mechanistically, GARP facilitated fibrogenesis by activating TGF-ß and enhancing endothelin-1-mediated HSC contraction. Functional GARP was expressed on human HSCs and significantly upregulated in the livers of patients with fibrosis. Lastly, deletion of GARP on HSCs did not augment inflammation or liver damage. CONCLUSIONS: GARP expressed on HSCs drives the development of liver fibrosis via cell contraction-mediated activation of latent TGF-ß. Considering that systemic blockade of TGF-ß has major side effects, we highlight a therapeutic niche provided by GARP and surface-mediated TGF-ß activation. Thus, our findings suggest an important role of GARP on HSCs as a promising target for the treatment of liver fibrosis. IMPACT AND IMPLICATIONS: Liver fibrosis represents a substantial and increasing public health burden globally, for which specific treatments are not available. Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-ß docking molecule. Here, we show that GARP expressed on hepatic stellate cells drives the development of liver fibrosis. Our findings suggest GARP as a novel target for the treatment of fibrotic disease.

Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation.

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired.

The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells.

Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms.

Scramblase TMEM16F terminates T cell receptor signaling to restrict T cell exhaustion.

In chronic infection, T cells become hyporesponsive to antigenic stimulation to prevent immunopathology. Here, we show that TMEM16F is required to curb excessive T cell responses in chronic infection with virus. TMEM16F-deficient T cells are hyperactivated during the early phase of infection, exhibiting increased proliferation and cytokine production. Interestingly, this overactivation ultimately leads to severe T cell exhaustion and the inability of the host to control viral burden. Mechanistically, we identify TMEM16F as the dominant lipid scramblase in T lymphocytes that transports phospholipids across membranes. TMEM16F is located in late endosomes, where it facilitates the generation of multivesicular bodies for TCR degradation and signal termination. Consequently, TMEM16F deficiency results in sustained signaling and augmented T cell activation. Our results demonstrate that scramblase restricts TCR responses to avoid overactivation, ensuring a well-balanced immune response in chronic infectious disease.

CD1a on Langerhans cells controls inflammatory skin disease.

CD1a is a lipid-presenting molecule that is abundantly expressed on Langerhans cells. However, the in vivo role of CD1a has remained unclear, principally because CD1a is lacking in mice. Through the use of mice with transgenic expression of CD1a, we found that the plant-derived lipid urushiol triggered CD1a-dependent skin inflammation driven by CD4(+) helper T cells that produced the cytokines IL-17 and IL-22 (TH17 cells). Human subjects with poison-ivy dermatitis had a similar cytokine signature following CD1a-mediated recognition of urushiol. Among various urushiol congeners, we identified diunsaturated pentadecylcatechol (C15:2) as the dominant antigen for CD1a-restricted T cells. We determined the crystal structure of the CD1a-urushiol (C15:2) complex, demonstrating the molecular basis of urushiol interaction with the antigen-binding cleft of CD1a. In a mouse model and in patients with psoriasis, CD1a amplified inflammatory responses that were mediated by TH17 cells that reacted to self lipid antigens. Treatment with blocking antibodies to CD1a alleviated skin inflammation. Thus, we propose CD1a as a potential therapeutic target in inflammatory skin diseases.

Thinking inside the box: endogenous α-anomeric lipid antigens.

The most powerful iNKT cell antigen is α-galactosylceramide (α-GalCer), derived from the marine sponge. However, α-anomeric glycolipids are thought to be absent in mammals. In this issue of Immunity, Kain et al., (2014) demonstrate the presence of mammalian α-linked glycosylceramides, such as α-GalCer.

Seven steps to stellate cells.

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes(1,2). Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A(1, 2). Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis(3). Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension(4). Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes(5). Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Lysosomal alpha-galactosidase controls the generation of self lipid antigens for natural killer T cells.

Natural Killer T (NKT) cells are lipid-reactive, CD1d-restricted T lymphocytes important in infection, cancer, and autoimmunity. In addition to foreign antigens, NKT cells react with endogenous self lipids. However, in the face of stimulating self antigen, it remains unclear how overstimulation of NKT cells is avoided. We hypothesized that constantly degraded endogenous antigen only accumulates upon inhibition of alpha-galactosidase A (alpha-Gal-A) in lysosomes. Here, we show that alpha-Gal-A deficiency caused vigorous activation of NKT cells. Moreover, microbes induced inhibition of alpha-Gal-A activity in antigen-presenting cells. This temporary enzyme block depended on Toll-like receptor (TLR) signaling and ultimately triggered lysosomal lipid accumulation. Thus, we present TLR-dependent negative regulation of alpha-Gal-A as a mechanistic link between pathogen recognition and self lipid antigen induction for NKT cells.

The immunological functions of saposins.

Saposins or sphingolipid activator proteins (SAPs) are small, nonenzymatic glycoproteins that are ubiquitously present in lysosomes. SAPs comprise the five molecules saposins A-D and the GM2 activator protein. Saposins are essential for sphingolipid degradation and membrane digestion. On the one hand, they bind the respective hydrolases required to catabolize sphingolipid molecules; on the other hand, saposins can interact with intralysosomal membrane structures to render lipids accessible to their degrading enzymes. Thus, saposins bridge the physicochemical gap between lipid substrate and hydrophilic hydrolases. Accordingly, defects in saposin function can lead to lysosomal lipid accumulation. In addition to their specific functions in sphingolipid metabolism, saposins have membrane-perturbing properties. At the low pH of lysosomes, saposins get protonated and exhibit a high binding affinity for anionic phospholipids. Based on their universal principle to interact with membrane bilayers, we present the immunological functions of saposins with regard to lipid antigen presentation to CD1-restricted T cells, processing of apoptotic bodies for antigen delivery and cross-priming, as well as their potential antimicrobial impact.

Natural killer T cells activated by a lipopeptidophosphoglycan from Entamoeba histolytica are critically important to control amebic liver abscess.

The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jalpha18(-/-) mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using alpha-galactosylceramide (alpha-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d(-/-) mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-gamma but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28:0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-gamma production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to alpha-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess.

Viral danger signals control CD1d de novo synthesis and NKT cell activation.

The nonpolymorphic CD1 molecules present lipid antigens to T cells. In myeloid DC humans express five different CD1 proteins (CD1a-e; the corresponding CD1 genes are designated CD1A-E). A role for CD1d-restricted NKT cells in the control of virus infections has been delineated from clinical observations, mouse models and viral evasion mechanisms targeting CD1d. How NKT cells are activated by virus infections is unclear. We found that human myeloid DC differentially regulate CD1 antigen presentation in response to viral danger signals. Stimulation with type I IFN, viral TLR ligands or viruses strongly enhanced the number of CD1D transcripts in human myeloid DC but diminished the abundance of CD1A, CD1B and CD1E mRNA. These changes on the transcriptional level were mirrored by altered cellular distribution and increased surface expression of CD1d. As a consequence NKT cells were activated and showed a Th1-like response. Moreover, NKT cell activation in PBMC exposed to viral danger signals was dependent on human plasmacytoid DC which produce large amounts of IFN-alpha. In conclusion, our data indicate that viral danger signals trigger NKT cell activation by enhancing CD1d de novo synthesis through increasing the abundance of CD1D mRNA in human myeloid DC.

Inhibition of CD1 antigen presentation by human cytomegalovirus.

The betaherpesvirus human cytomegalovirus (HCMV) encodes several molecules that block antigen presentation by the major histocompatibility complex (MHC) proteins. Humans also possess one other family of antigen-presenting molecules, the CD1 family; however, the effect of HCMV on CD1 expression is unknown. The majority of CD1 molecules are classified on the basis of homology as group 1 CD1 and are present almost exclusively on professional antigen-presenting cells such as dendritic cells, which are a major target for HCMV infection and latency. We have determined that HCMV encodes multiple blocking strategies targeting group 1 CD1 molecules. CD1 transcription is strongly inhibited by the HCMV interleukin-10 homologue cmvIL-10. HCMV also blocks CD1 antigen presentation posttranscriptionally by the inhibition of CD1 localization to the cell surface. This function is not performed by a known HCMV MHC class I-blocking molecule and is substantially stronger than the blockage induced by herpes simplex virus type 1. Antigen presentation by CD1 is important for the development of the antiviral immune response and the generation of mature antigen-presenting cells. HCMV present in antigen-presenting cells thus blunts the immune response by the blockage of CD1 molecules.

Starring stellate cells in liver immunology.

Stellate cells are star-shaped cells located in the liver and mediate a multitude of primarily non-immunological functions. They play a pivotal role in the metabolism of vitamin A and store 80% of total body retinol. Upon activation, stellate cells differentiate to myofibroblasts for production of extracellular matrix, leading to liver fibrosis. Moreover, activated stellate cells regulate liver blood flow through vasoconstriction implicated in portal hypertension. Earlier work demonstrated stellate cell derived secretion of chemokines and cytokines such as transforming growth factor beta (TGF-beta), suggesting an association with immunological processes. Indeed, recent evidence indicated that hepatic stellate cells perform potent APC function for stimulation of NKT cells as well as CD8 and CD4 T cells. Additionally, stellate cell mediated antigen presentation induced protective immunity against bacterial infection. Current experiments reveal that the presenting ability of stellate cells is the key to antigen-dependent T cell instruction by vitamin A derived retinoic acid. Finally, future studies will show whether in the firmament of immunology stellate cells will represent fixed or falling stars.

Ito cells are liver-resident antigen-presenting cells for activating T cell responses.

Here we identified Ito cells (hepatic stellate cells, HSC), known for storage of vitamin A and participation in hepatic fibrosis, as professional liver-resident antigen-presenting cells (APC). Ito cells efficiently presented antigens to CD1-, major histocompatibility complex (MHC)-I-, and MHC-II-restricted T cells. Ito cells presented lipid antigens to CD1-restricted T lymphocytes such as natural killer T (NKT) cells and promoted homeostatic proliferation of liver NKT cells through interleukin-15. Moreover, Ito cells presented antigenic peptides to CD8(+) and CD4(+) T cells and mediated crosspriming of CD8(+) T cells. Peptide-specific T cells were activated by transgenic Ito cells presenting endogenous neoantigen. Upon bacterial infection, Ito cells elicited antigen-specific T cells and mediated protection. In contrast to other liver cell types that have been implicated in induction of immunological tolerance, our data identify Ito cells as professional intrahepatic APCs activating T cells and eliciting a multitude of T cell responses specific for protein and lipid antigens.

CD1 antigen presentation by human dendritic cells as a target for herpes simplex virus immune evasion.

In contrast to MHC molecules, which present peptides, the CD1 molecules have been discovered to present lipid Ags to T cells. CD1-restricted T lymphocytes have been recently associated with resistance to virus infection. The mechanisms underlying activation of CD1-restricted T cells in the course of virus infection are not defined. In this study, we wanted to investigate the interaction of HSV with the antiviral CD1 Ag presentation system in human dendritic cells (DC). In response to low titers of HSV, the surface expression of CD1b and CD1d on human DC was up-regulated. These phenotypic changes enhanced the capacity of infected DC to stimulate proliferation of CD1-restricted T lymphocytes. High titers of HSV, however, lead to strong down-regulation of all surface CD1 molecules. This modulation of surface expression was associated with intracellular accumulation, colocalization with viral proteins, and disruption of the CD1 recycling machinery. Finally, even at low titers HSV interfered with the capacity of infected DC to stimulate the release of important cytokines by CD1d-restricted NKT cells. Thus, we demonstrate both the existence of a CD1 pathway allowing human DC to react to viral infection, as well as its blockage by a human herpesvirus.

Cholesterol glucosylation promotes immune evasion by Helicobacter pylori.

Helicobacter pylori infection causes gastric pathology such as ulcer and carcinoma. Because H. pylori is auxotrophic for cholesterol, we have explored the assimilation of cholesterol by H. pylori in infection. Here we show that H. pylori follows a cholesterol gradient and extracts the lipid from plasma membranes of epithelial cells for subsequent glucosylation. Excessive cholesterol promotes phagocytosis of H. pylori by antigen-presenting cells, such as macrophages and dendritic cells, and enhances antigen-specific T cell responses. A cholesterol-rich diet during bacterial challenge leads to T cell-dependent reduction of the H. pylori burden in the stomach. Intrinsic alpha-glucosylation of cholesterol abrogates phagocytosis of H. pylori and subsequent T cell activation. We identify the gene hp0421 as encoding the enzyme cholesterol-alpha-glucosyltransferase responsible for cholesterol glucosylation. Generation of knockout mutants lacking hp0421 corroborates the importance of cholesteryl glucosides for escaping phagocytosis, T cell activation and bacterial clearance in vivo. Thus, we propose a mechanism regulating the host-pathogen interaction whereby glucosylation of a lipid tips the scales towards immune evasion or response.