Acute HIV-1 and SARS-CoV-2 Infections Share Slan+ Monocyte Depletion-Evidence from an Hyperacute HIV-1 Case Report.

Monocytes are key modulators in acute viral infections, determining both inflammation and development of specific B- and T-cell responses. Recently, these cells were shown to be associated to different SARS-CoV-2 infection outcome. However, their role in acute HIV-1 infection remains unclear. We had the opportunity to evaluate the mononuclear cell compartment in an early hyper-acute HIV-1 patient in comparison with an untreated chronic HIV-1 and a cohort of SARS-CoV-2 infected patients, by high dimensional flow cytometry using an unsupervised approach. A distinct polarization of the monocyte phenotype was observed in the two viral infections, with maintenance of pro-inflammatory M1-like profile in HIV-1, in contrast to the M2-like immunosuppressive shift in SARS-CoV-2. Noticeably, both acute infections had reduced CD14low/-CD16+ non-classical monocytes, with depletion of the population expressing Slan (6-sulfo LacNac), which is thought to contribute to immune surveillance through pro-inflammatory properties. This depletion indicates a potential role of these cells in acute viral infection, which has not previously been explored. The inflammatory state accompanied by the depletion of Slan+ monocytes may provide new insights on the critical events that determine the rate of viral set-point in acute HIV-1 infection and subsequent impact on transmission and reservoir establishment.

An Egg-Derived Sulfated N-Acetyllactosamine Glycan Is an Antigenic Decoy of Influenza Virus Vaccines.

Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess different glycosylation patterns than viruses circulating among humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived glycan that is an antigenic decoy, with egg-binding MAbs reacting with a sulfated N-acetyllactosamine (LacNAc). Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken-derived cells, suggesting that only egg-grown vaccines can induce antiegg antibodies. Notably, antibodies against the egg antigen utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and had a simple heavy-chain complementarity-determining region 3. By analyzing a public data set of influenza virus vaccine-induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.

Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

Current Concepts on 6-sulfo LacNAc Expressing Monocytes (slanMo).

The human mononuclear phagocytes system consists of dendritic cells (DCs), monocytes, and macrophages having different functions in bridging innate and adaptive immunity. Among the heterogeneous population of monocytes the cell surface marker slan (6-sulfo LacNAc) identifies a specific subset of human CD14- CD16+ non-classical monocytes, called slan+ monocytes (slanMo). In this review we discuss the identity and functions of slanMo, their contributions to immune surveillance by pro-inflammatory cytokine production, and cross talk with T cells and NK cells. We also consider the role of slanMo in the regulation of chronic inflammatory diseases and cancer. Finally, we highlight unresolved questions that should be the focus of future research.

Neoadjuvant Radiochemotherapy Significantly Alters the Phenotype of Plasmacytoid Dendritic Cells and 6-Sulfo LacNAc+ Monocytes in Rectal Cancer.

Neoadjuvant radiochemotherapy (nRCT) can significantly influence the tumor immune architecture that plays a pivotal role in regulating tumor growth. Whereas, various studies have investigated the effect of nRCT on tumor-infiltrating T cells, little is known about its impact on the frequency and activation status of human dendritic cells (DCs). Plasmacytoid DCs (pDCs) essentially contribute to the regulation of innate and adaptive immunity and may profoundly influence tumor progression. Recent studies have revealed that higher pDC numbers are associated with poor prognosis in cancer patients. 6-sulfo LacNAc-expressing monocytes (slanMo) represent a particular proinflammatory subset of human non-classical blood monocytes that can differentiate into DCs. Recently, we have reported that activated slanMo produce various proinflammatory cytokines and efficiently stimulate natural killer cells and T lymphocytes. slanMo were also shown to accumulate in clear cell renal cell carcinoma (ccRCC) and in metastatic lymph nodes from cancer patients. Here, we investigated the influence of nRCT on the frequency of rectal cancer-infiltrating pDCs and slanMo. When evaluating rectal cancer tissues obtained from patients after nRCT, a significantly higher density of pDCs in comparison to pre-nRCT tissue samples was found. In contrast, the density of slanMo was not significantly altered by nRCT. Further studies revealed that nRCT significantly enhances the proportion of rectal cancer-infiltrating CD8+ T cells expressing the cytotoxic effector molecule granzyme B. When exploring the impact of nRCT on the phenotype of rectal cancer-infiltrating pDCs and slanMo, we observed that nRCT markedly enhances the percentage of inducible nitric oxide synthase (iNOS)- or tumor necrosis factor (TNF) alpha-producing slanMo. Furthermore, nRCT significantly increased the percentage of mature CD83+ pDCs in rectal cancer tissues. Moreover, the proportion of pDCs locally expressing interferon-alpha, which plays a major role in antitumor immunity, was significantly higher in post-nRCT tissues compared to pre-nRCT tumor specimens. These novel findings indicate that nRCT significantly influences the frequency and/or phenotype of pDCs, slanMo, and CD8+ T cells, which may influence the clinical response of rectal cancer patients to nRCT.

Intracapillary immune complexes recruit and activate slan-expressing CD16+ monocytes in human lupus nephritis.

Lupus nephritis is a major cause of morbidity in patients with systemic lupus erythematosus. Among the different types of lupus nephritis, intracapillary immune complex (IC) deposition and accumulation of monocytes are hallmarks of lupus nephritis class III and IV. The relevance of intracapillary ICs in terms of monocyte recruitment and activation, as well as the nature and function of these monocytes are not well understood. For the early focal form of lupus nephritis (class III) we demonstrate a selective accumulation of the proinflammatory population of 6-sulfo LacNAc+ (slan) monocytes (slanMo), which locally expressed TNF-α. Immobilized ICs induced a direct recruitment of slanMo from the microcirculation via interaction with Fc γ receptor IIIA (CD16). Interestingly, intravenous immunoglobulins blocked CD16 and prevented cell recruitment. Engagement of immobilized ICs by slanMo induced the production of neutrophil-attracting chemokine CXCL2 as well as TNF-α, which in a forward feedback loop stimulated endothelial cells to produce the slanMo-recruiting chemokine CX3CL1 (fractalkine). In conclusion, we observed that expression of CD16 equips slanMo with a unique capacity to orchestrate early IC-induced inflammatory responses in glomeruli and identified slanMo as a pathogenic proinflammatory cell type in lupus nephritis.

Ovarian carcinoma glyco-antigen targeted by human IgM antibody.

Epithelial Ovarian Cancer (EOC) cells expression of a novel carbohydrate antigen was defined using a human VH4-34 encoded IgM monoclonal antibody (mAb216). MAb216 binds to a poly N-acetyllactosamine epitope expressed on B cells and kills normal and malignant B cells in vitro and in vivo. EOC patient ascites and EOC cell lines were used to study the anti tumor effect of mAb216. Various assays were used to characterize the epitope and demonstrate antibody-mediated binding and cytotoxicity in EOC. Drug and antibody combination effects were determined by calculating the combination index values using the Chou and Talalay method. MAb216 displays direct antibody mediated cytotoxicity on a population of human EOC tumor and ascites samples and EOC cell lines, which express high amounts of poly N-acetyllactosamine epitope, carried by CD147/CD98. Eighty four percent of patient samples, including platin resistant, had a tumor population that bound the monoclonal antibody. The binding pattern of mAb216 and mechanism of cytotoxicity was similar to that seen on normal and malignant B cells with unique general membrane disruption and "pore" formation. In vitro incubation with mAb216 and cisplatin enhanced killing of OVCAR3 cell line. In EOC cell lines percent cytotoxicity correlated with percent expression of epitope. Although in vitro data shows specific EOC cytotoxicity, for possible treatment of EOC MAb216 would need to be evaluated in a clinical trial with or without chemotherapy.

Controlling the pro-inflammatory function of 6-sulfo LacNAc (slan) dendritic cells with dimethylfumarate.

BACKROUND: The fumaric acid ester (FAE) dimethylfumarate (DMF) is a small molecule immunomodulator successfully used for the treatment of psoriasis and multiple sclerosis (MS). DMF is thought to inhibit pathogenic immune responses with Th17/Th1T cells, and IL-23/IL-12 producing dendritic cells (DCs). 6-sulfo LacNAc expressing dendritic cells (slanDCs) are a human pro-inflammatory cell type found frequently among the infiltrating leukocytes in skin lesions of psoriasis and brain lesions of MS. OBJECTIVE: To explore the influence of DMF on functional properties and cell signaling pathways of slanDCs. METHODS: In the context of slanDCs we studied the role of DMF in modulating cell migration, phenotypic maturation, cytokine production, cell signaling and T cell stimulation. RESULTS: Initially, we observed the reduction of slanDCs numbers in psoriasis skin lesions of FAE treated patients. Studying whether DMF controls the migratory capacity of slanDCs to chemotactic factors expressed in psoriasis we observed an inhibition of the CX3CL1 and C5a depedent cell migration. DMF also attenuated the rapid spontaneous phenotypic maturation of slanDCs, as judged by a reduced CD80, CD86, CD83 and HLA-DR expression. In addition, we observed a DMF-dependent decrease of IL-23, IL-12, TNF-α and IL-10 secretion, and noticed a reduced capacity to stimulate Th17/Th1 responses. DMF targeted in slanDCs different intracellular cell signaling pathways including NFκB, STAT1 and HO-1. CONCLUSIONS: With this study we identify a frequent pro-inflammatory cell type found in psoriasis and MS as a relevant target for the therapeutic immunomodulatory effects of DMF.

The phosphodiesterase 4 inhibitor apremilast inhibits Th1 but promotes Th17 responses induced by 6-sulfo LacNAc (slan) dendritic cells.

BACKGROUND: The phosphodiesterase 4 (PDE4) inhibitor apremilast increases cellular cAMP levels and has proven effective in the treatment of psoriasis and psoriasis arthritis. We recently described 6-sulfo LacNAc dendritic cells (slanDCs) as immature DCs in blood and as a subset of inflammatory dermal DCs in psoriasis with a pronounced capacity to produce proinflammatory cytokines and to program Th17/Th1 T cell responses. OBJECTIVE: The aim of this study was to investigate possible immune regulatory effects of the PDE4 inhibitor apremilast on slanDCs. METHODS: In vitro studies were performed analyzing the effects of apremilast on the proinflammatory function of slanDCs and their capacity to induce Th1/Th17-biased T cell responses. RESULTS: Increasing cAMP levels in slanDCs by PDE4 inhibition strongly reduced production of IL-12 and TNF-α. In line with these findings, co-culture experiments with apremilast-pulsed slanDCs and allogeneic T cells either from psoriasis patients or healthy controls, revealed a significant reduction of IFN-γ production and expression of the transcription factor T-bet. In parallel, production of IL-23 and IL-1ß by slanDCs was increased and co-cultured T cells revealed a largely augmented IL-17 production and an upregulated RORyt expression. CONCLUSIONS: We here demonstrate anti-inflammatory as well as Th17-promoting effects of apremilast when studying blood precursors of human inflammatory dermal dendritic cells. In the concert of the broad anti-inflammatory effects of apremilast on keratinocytes, fibroblasts and endothelial cells, the dual effect on slan+ inflammatory dermal DCs should be taken into account and may constrain therapeutic responses.

Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4.

Cancer-specific glycans of ovarian cancer are promising epitopes for targeting with monoclonal antibodies (mAb). Despite their potential, structural characterization of these glycan epitopes remains a significant challenge in mAb preclinical development. Our group generated the monoclonal antibody mAb-A4 against human embryonic stem cells (hESC), which also bound specifically to N-glycans present on 11 of 19 ovarian cancer (OC) and 8 of 14 breast cancer cell lines tested. Normal cell lines and tissue were unstained by mAb-A4. To characterize the N-linked glycan epitopes on OC cell lines targeted by mAb-A4, we used glycosidases, glycan microarray, siRNA, and advanced high sensitivity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mAb-A4 epitopes were found to be Fucα1-2Galß1-3GlcNAcß (H type 1) and Galß1-3GlcNAcß (type 1 LacNAc). These structures were found to be present on multiple proteins from hESC and OC. Importantly, endo-ß-galactosidase coupled with MALDI-MS allowed these two epitopes, for the first time, to be directly identified on the polylactosamines of N-glycans of SKOV3, IGROV1, OV90, and OVCA433. Furthermore, siRNA knockdown of B3GALT5 expression in SKOV3 demonstrated that mAb-A4 binding was dependent on B3GALT5, providing orthogonal evidence of the epitopes' structures. The recognition of oncofetal H type 1 and type 1 LacNAc on OC by mAb-A4 is a novel and promising way to target OC and supports the theory that cancer can acquire stem-like phenotypes. We propose that the orthogonal framework used in this work could be the basis for advancing anti-glycan mAb characterization.

Synthesis of di- and tri-saccharide fragments of Salmonella typhi Vi capsular polysaccharide and their zwitterionic analogues.

Zwitterionic polysaccharides (ZPS) behave like traditional T cell-dependent antigens, suggesting the design of new classes of vaccines alternative to currently used glycoconjugates and based on the artificial introduction of a zwitterionic charge motif onto the carbohydrate structure of pathogen antigens. Here we report the new synthesis and antigenic evaluation of di-/tri-saccharide fragments of Salmonella typhi Vi polysaccharide, as well as of their corresponding zwitterionic analogues. Our strategy is based on versatile intermediates enabling chain elongation either by iterative single monomer attachment or by faster and more flexible approach using disaccharide donors. The effect of structural modifications of the synthetic compounds on antigenic properties was evaluated by competitive ELISA. All the oligosaccharides were recognized by specific anti-Vi polyclonal antibodies in a concentration-dependent manner, and the introduction of a zwitterionic motif into the synthetic molecules did not prevent the binding.

Differential accumulation and function of proinflammatory 6-sulfo LacNAc dendritic cells in lymph node and colon of Crohn's versus ulcerative colitis patients.

Human Slan DCs have been studied in patients with psoriasis, rheumatoid arthritis, cancer, and autoimmune diseases. In this study, we investigated the frequency, phenotype, and function of Slan DCs in blood, colon, as well as mLNs of patients with IBD. We first show that the frequency of circulating CD14(dull)Slan DCs was reduced in CD patients refractory to immunosuppressive drugs or TNF-α blockers relative to untreated CD, UC, and healthy subjects. In blood of CD patients, Slan DCs expressed CD172a, as detected by CD47 fusion protein binding, when compared with its lack of expression in control subjects. Next, we demonstrate that CD172a(+)Slan DCs that produced IL-1ß and TNF-α accumulated in mLNs and colons of CD patients. The CD172a(+)Slan DCs up-regulated their expression of CD14 in CD tissues and the proinflammatory cytokines were produced in CD14(bright)CD172a(+)Slan DCs. By contrast, no difference was noted in the frequency of Slan DCs between inflamed, noninflamed colonic mucosa of UC patients and control, non-IBD donors. Finally, the percentage of cytokine-producing Slan DCs also augmented in response to TLR2 and NOD2 in in vitro stimulation in PBMCs of CD, but not UC, patients. In conclusion, we propose that proinflammatory CD14(bright)CD172a(+)Slan DCs are a distinguishing feature between CD and UC, as these cells accumulate uniquely in mLNs and colonic mucosa of CD patients. Thus, Slan DCs may contribute to CD immunopathogenesis.

TNFR2 and IL-12 coactivation enables slanDCs to support NK-cell function via membrane-bound TNF-α.

Human blood NK cells exert strong cytotoxicity against transformed cells and produce different cytokines and chemokines with an important role in modulating immune responses. However, the nature of NK-cell function depends on NK-cell interaction with other immune cells. One type of immune cells that communicate with NK cells are 6-sulfo LacNAc DCs (slanDCs), which comprise a major subpopulation of proinflammatory human blood DCs. In this study, we investigated the molecular mechanisms by which slanDCs interact with NK cells. Our in vitro studies demonstrate that LPS-stimulated slanDCs enhance activation and function of NK cells essentially via membrane-bound TNF-α (mTNF-α). LPS stimulation upregulates expression of mTNF-α in slanDCs, and surface TNF receptor 2 (TNFR2) is upregulated on NK cells after coincubation with slanDCs. IL-12 secreted by slanDCs increases surface expression of TNFR2 in NK cells. TNFR2 signaling in NK cells leads to activation of NF-kB, a transcription factor for cytokines such as GM-CSF. GM-CSF provided by NK cells is responsible for enhancing IL-12 secretion in slanDCs. In conclusion, TNFR2 and IL-12 signaling, which support one another, enables slanDCs to enhance NK-cell function through mTNF-α, thereby regulating immune responses.

T-cell activation by transitory neo-antigens derived from distinct microbial pathways.

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

Immunomodulatory glycan lacto-N-fucopentaose III requires clathrin-mediated endocytosis to induce alternative activation of antigen-presenting cells.

The mechanism of alternative activation of antigen-presenting cells (APCs) is largely unknown. Lacto-N-fucopentaose III (LNFPIII) is a biologically conserved pentasaccharide that contains the Lewis(x) trisaccharide. LNFPIII conjugates and schistosome egg antigens, which contain the Lewis(x) trisaccharide, drive alternative activation of APCs and induce anti-inflammatory responses in vivo, preventing inflammation-based diseases, including psoriasis, transplant organ rejection, and metabolic disease. In this study, we show that LNFPIII conjugates and schistosome egg antigens interact with APCs via a receptor-mediated process, requiring internalization of these molecules through a clathrin/dynamin-dependent but caveolus-independent endocytic pathway. Using inhibitors/small interfering RNA (siRNA) against dynamin and clathrin, we show for the first time that endocytosis of Lewis(x)-containing glycans is required to drive alternative maturation of antigen-presenting cells and Th2 immune responses. We identified mouse SIGNR-1 as a cell surface receptor for LNFPIII conjugates. Elimination of SIGNR-1 showed no effect on uptake of LNFPIII conjugates, suggesting that other receptors bind to and facilitate uptake of LNFPIII conjugates. We demonstrate that disruption of actin filaments partially prevented the entry of LNFPIII conjugates into APCs and that LNFPIII colocalizes with both early and late endosomal markers and follows the classical endosomal pathway leading to lysosome maturation. The results of this study show that the ability of LNFPIII to induce alternative activation utilizes a receptor-mediated process that requires a dynamin-dependent endocytosis. Thus, key steps have been defined in the previously unknown mechanism of alternative activation that ultimately leads to induction of anti-inflammatory responses.

FcγRIII (CD16) equips immature 6-sulfo LacNAc-expressing dendritic cells (slanDCs) with a unique capacity to handle IgG-complexed antigens.

Binding and uptake of immune complexes (ICs) via low-affinity Fcγ receptors (FcγRs) on dendritic cells (DCs) is well known as a booster of immune responses. It can be helpful when stimulating immunity against pathogenic microbes but may be harmful when antibodies form complexes with autologous antigens. To date, no human DC subtype specialized in handling ICs has been identified. By incubating human blood mononuclear cells with ICs and studying their cellular binding, we identified 6-sulfo LacNAc-expressing DCs (slanDCs) as having an outstanding capacity to bind ICs compared with other myeloid DCs, plasmacytoid DCs, or monocytes. Using selective blocking of different (FcγRs), we identified CD16 (FcγRIII) as the major IC-binding structure on slanDCs. In addition, CD16 proved critical for phagocytosis of IgG-coated erythrocytes, and CD16-targeted antigen led to a more efficient proliferation of CD4(+) T cells than CD32 (FcγRII)-targeted antigen. Interestingly, these CD16-mediated functions are short-lived and restricted to the immature stage of slanDCs in blood. We show that CD16 is rapidly shed from the surface of maturing slanDCs, resulting from the combined action of the metalloproteinases ADAM10 and ADAM17. In conclusion, these data provide strong evidence that slanDCs play an important role in IC-driven immune responses.

Human 6-sulfo LacNAc (slan) dendritic cells have molecular and functional features of an important pro-inflammatory cell type in lupus erythematosus.

Lupus erythematosus (LE) is an autoimmune disease with evidence for an IL-23- and IL-17-induced immunopathology. Little is known about the type of dendritic cells supporting this immune response. We recently demonstrated the strong Th1- and Th17-T-cell inducing capacity of human 6-sulfo LacNAc-dendritic cells (slanDCs), and identified slanDCs as inflammatory dermal dendritic cells in psoriasis locally expressing IL-23, TNF-α and inducible nitric oxide synthase (iNOS). In this study, we investigated the role of slanDCs in LE. Using immunohistochemistry, we identified slanDCs at increased frequency in affected skin lesions of cutaneous and systemic LE. slanDCs were found scattered in the dermal compartment and also clustered in lymph follicle-like structures. Here, they colocalized with T cells in the periphery but not with B cells in the center. The positive staining of dermal slanDCs for TNF-α indicated their pro-inflammatory status. In vitro the production of TNF-α was induced when slanDCs were cultured in the presence of serum from patients with LE. Stimulatory components of LE serum were previously identified as autoimmune complexes with ssRNA binding to TLR7 and TLR8. We found that slanDCs express mRNA for TLR7 and TLR8. slanDCs stimulated with ssRNA, selective TLR7 or TLR8 ligands responded with high-level TNF-α and IL-12 production. In contrast to slanDCs, the population of CD1c(+) DCs and plasmacytoid DCs (pDCs) expressed either TLR7 or TLR8, and their production of TNF-α and IL-12 to respective ligands was far less pronounced. We conclude that slanDCs have molecular and functional features of a pro-inflammatory myeloid DC type relevant for the immunopathogenesis of LE.

Immunomodulatory glycan LNFPIII alleviates hepatosteatosis and insulin resistance through direct and indirect control of metabolic pathways.

Parasitic worms express host-like glycans to attenuate the immune response of human hosts. The therapeutic potential of this immunomodulatory mechanism in controlling the metabolic dysfunction that is associated with chronic inflammation remains unexplored. We demonstrate here that administration of lacto-N-fucopentaose III (LNFPIII), a Lewis(X)-containing immunomodulatory glycan found in human milk and on parasitic helminths, improves glucose tolerance and insulin sensitivity in diet-induced obese mice. This effect is mediated partly through increased interleukin-10 (Il-10) production by LNFPIII-activated macrophages and dendritic cells, which reduces white adipose tissue inflammation and sensitizes the insulin response of adipocytes. Concurrently, LNFPIII treatment upregulates nuclear receptor subfamily 1, group H, member 4 (Fxr-α, also known as Nr1h4) to suppress lipogenesis in the liver, conferring protection against hepatosteatosis. At the signaling level, the extracellular signal-regulated kinase (Erk)-activator protein 1 (Ap1) pathway seems to mediate the effects of LNFPIII on both inflammatory and metabolic pathways. Our results suggest that LNFPIII may provide new therapeutic approaches to treat metabolic diseases.

Galectin-1 research in T cell immunity: past, present and future.

Galectin-1 (Gal-1) is one of 15 evolutionarily conserved ß-galactoside-binding proteins that display biologically-diverse activities in pathogenesis of inflammation and cancer. Gal-1 is variably expressed on immune cells and endothelial cells, though is commonly found and secreted at high levels in cancer cells. It induces apoptosis in effector T cells through homodimeric binding of N-acetyllactosamines on membrane glycoproteins (Gal-1 ligands). There is also compelling evidence in models of cancer and autoimmunity that recombinant Gal-1 (rGal-1) can potentiate immunoregulatory function of T cells. Here, we review Gal-1's structural and functional features, while analyzing potential drawbacks and technical difficulties inherent to rGal-1's nature. We also describe new Gal-1 preparations that exhibit dimeric stability and functional activity on T cells, providing renewed excitement for studying Gal-1 efficacy and/or use as anti-inflammatory therapeutics. We lastly summarize strategies targeting the Gal-1-Gal-1 ligand axis to circumvent Gal-1-driven immune escape in cancer and boost anti-tumor immunity.

Identification of mono- and disulfated N-acetyl-lactosaminyl Oligosaccharide structures as epitopes specifically recognized by humanized monoclonal antibody HMOCC-1 raised against ovarian cancer.

A humanized monoclonal antibody raised against human ovarian cancer RMG-I cells and designated as HMOCC-1 (Suzuki, N., Aoki, D., Tamada, Y., Susumu, N., Orikawa, K., Tsukazaki, K., Sakayori, M., Suzuki, A., Fukuchi, T., Mukai, M., Kojima-Aikawa, K., Ishida, I., and Nozawa, S. (2004) Gynecol. Oncol. 95, 290-298) was characterized for its carbohydrate epitope structure. Specifically, a series of co-transfections was performed using mammalian expression vectors encoding specific glycosyltransferases and sulfotransferases. These experiments identified one sulfotransferase, GAL3ST3, and one glycosyltransferase, B3GNT7, as required for HMOCC-1 antigen formation. They also suggested that the sulfotransferase CHST1 regulates the abundance and intensity of HMOCC-1 antigen. When HEK293T cells were co-transfected with GAL3ST3 and B3GNT7 expression vectors, transfected cells weakly expressed HMOCC-1 antigen. When cells were first co-transfected with GAL3ST3 and B3GNT7 and then with CHST1, the resulting cells strongly expressed HMOCC-1 antigen. However, when cells were transfected with a mixture of GAL3ST3 and CHST1 before or after transfection with B3GNT7, the number of antigen-positive cells decreased relative to the number seen with only GAL3ST3 and B3GNT7, suggesting that CHST1 plays a regulatory role in HMOCC-1 antigen formation. Because these results predicted that HMOCC-1 antigens are SO(3) → 3Galß1 → 4GlcNAcß1 → 3(±SO(3) → 6)Galß1 → 4GlcNAc, we chemically synthesized mono- and disulfated and unsulfated oligosaccharides. Immunoassays using these oligosaccharides as inhibitors showed the strongest activity by disulfated tetrasaccharide, weak but positive activity by monosulfated tetrasaccharide at the terminal galactose, and no activity by nonsulfated tetrasaccharides. These results establish the HMOCC-1 epitope, which should serve as a useful reagent to further characterize ovarian cancer.