N-glycosylation bidirectionally extends the boundaries of thymocyte positive selection by decoupling Lck from Ca²âº signaling.

Positive selection of diverse yet self-tolerant thymocytes is vital to immunity and requires a limited degree of T cell antigen receptor (TCR) signaling in response to self peptide-major histocompatibility complexes (self peptide-MHCs). Affinity of newly generated TCR for peptide-MHC primarily sets the boundaries for positive selection. We report that N-glycan branching of TCR and the CD4 and CD8 coreceptors separately altered the upper and lower affinity boundaries from which interactions between peptide-MHC and TCR positively select T cells. During thymocyte development, N-glycan branching varied approximately 15-fold. N-glycan branching was required for positive selection and decoupled Lck signaling from TCR-driven Ca(2+) flux to simultaneously promote low-affinity peptide-MHC responses while inhibiting high-affinity ones. Therefore, N-glycan branching imposes a sliding scale on interactions between peptide-MHC and TCR that bidirectionally expands the affinity range for positive selection.

Metabolism, cell surface organization, and disease.

Genetic information flows from DNA to macromolecular structures-the dominant force in the molecular organization of life. However, recent work suggests that metabolite availability to the hexosamine and Golgi N-glycosylation pathways exerts control over the assembly of macromolecular complexes on the cell surface and, in this capacity, acts upstream of signaling and gene expression. The structure and number of N-glycans per protein molecule cooperate to regulate lectin binding and thereby the distribution of glycoproteins at the cell surface. Congenital disorders of glycosylation provide insight as extreme hypomorphisms, whereas milder deficiencies may encompass many common chronic conditions, including autoimmunity, metabolic syndrome, and aging.

N-acetylglucosamine inhibits inflammation and neurodegeneration markers in multiple sclerosis: a mechanistic trial.

BACKGROUND: In the demyelinating disease multiple sclerosis (MS), chronic-active brain inflammation, remyelination failure and neurodegeneration remain major issues despite immunotherapy. While B cell depletion and blockade/sequestration of T and B cells potently reduces episodic relapses, they act peripherally to allow persistence of chronic-active brain inflammation and progressive neurological dysfunction. N-acetyglucosamine (GlcNAc) is a triple modulator of inflammation, myelination and neurodegeneration. GlcNAc promotes biosynthesis of Asn (N)-linked-glycans, which interact with galectins to co-regulate the clustering/signaling/endocytosis of multiple glycoproteins simultaneously. In mice, GlcNAc crosses the blood brain barrier to raise N-glycan branching, suppress inflammatory demyelination by T and B cells and trigger stem/progenitor cell mediated myelin repair. MS clinical severity, demyelination lesion size and neurodegeneration inversely associate with a marker of endogenous GlcNAc, while in healthy humans, age-associated increases in endogenous GlcNAc promote T cell senescence. OBJECTIVES AND METHODS: An open label dose-escalation mechanistic trial of oral GlcNAc at 6 g (n = 18) and 12 g (n = 16) for 4 weeks was performed in MS patients on glatiramer acetate and not in relapse from March 2016 to December 2019 to assess changes in serum GlcNAc, lymphocyte N-glycosylation and inflammatory markers. Post-hoc analysis examined changes in serum neurofilament light chain (sNfL) as well as neurological disability via the Expanded Disability Status Scale (EDSS). RESULTS: Prior to GlcNAc therapy, high serum levels of the inflammatory cytokines IFNγ, IL-17 and IL-6 associated with reduced baseline levels of a marker of endogenous serum GlcNAc. Oral GlcNAc therapy was safe, raised serum levels and modulated N-glycan branching in lymphocytes. Glatiramer acetate reduces TH1, TH17 and B cell activity as well as sNfL, yet the addition of oral GlcNAc dose-dependently lowered serum IFNγ, IL-17, IL-6 and NfL. Oral GlcANc also dose-dependently reduced serum levels of the anti-inflammatory cytokine IL-10, which is increased in the brain of MS patients. 30% of treated patients displayed confirmed improvement in neurological disability, with an average EDSS score decrease of 0.52 points. CONCLUSIONS: Oral GlcNAc inhibits inflammation and neurodegeneration markers in MS patients despite concurrent immunomodulation by glatiramer acetate. Blinded studies are required to investigate GlcNAc's potential to control residual brain inflammation, myelin repair and neurodegeneration in MS.

N-Glycan Branching Is Required for Development of Mature B Cells.

Galectins have been implicated in inhibiting BCR signaling in mature B cells but promoting pre-BCR signaling during early development. Galectins bind to branched N-glycans attached to cell surface glycoproteins to control the distribution, clustering, endocytosis, and signaling of surface glycoproteins. During T cell development, N-glycan branching is required for positive selection of thymocytes, inhibiting both death by neglect and negative selection via enhanced surface retention of the CD4/CD8 coreceptors and limiting TCR clustering/signaling, respectively. The role of N-glycan branching in B cell development is unknown. In this study, we report that N-glycan branching is absolutely required for development of mature B cells in mice. Elimination of branched N-glycans in developing B cells via targeted deletion of N-acetylglucosaminyl transferase I (Mgat1) markedly reduced cellularity in the bone marrow and/or spleen and inhibited maturation of pre-, immature, and transitional stage 2 B cells. Branching deficiency markedly reduced surface expression of the pre-BCR/BCR coreceptor CD19 and promoted spontaneous death of pre-B cells and immature B cells in vitro. Death was rescued by low-dose pre-BCR/BCR stimulation but exacerbated by high-dose pre-BCR/BCR stimulation as well as antiapoptotic BclxL overexpression in pre-B cells. Branching deficiency also enhanced Nur77 induction, a marker of negative selection. Together, these data suggest that, as in T cells, N-glycan branching promotes positive selection of B cells by augmenting pre-BCR/BCR signaling via CD19 surface retention, whereas limiting negative selection from excessive BCR engagement.

N-acetylglucosamine drives myelination by triggering oligodendrocyte precursor cell differentiation.

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.

Long-term plasma exchange as maintenance therapy for cerebellar-type Hashimoto's encephalopathy, a case report.

Hashimoto's encephalopathy (HE) is a presumed autoimmune disorder associated with anti-thyroid autoantibodies and signs and symptoms of encephalopathy. A sub-type of HE is associated with cerebellar dysfunction and ataxia. Immunosuppressive therapy, particularly corticosteroid treatment, is utilized in the majority of cases. Short-term apheresis has been reported with variable patient responses. Here we report the case of a 72 year-old female with an ∼15 year history of cerebellar type HE that had profound improvement in symptoms after long-term apheresis treatment over an ∼2 year period. Following an induction phase, twice-weekly maintenance apheresis of 1 plasma volume reversed long-standing severe gait ataxia that had required a walker, as well as mild cognitive symptoms. This paralleled reductions in anti-thyroid antibody levels. Holidays from apheresis lasting several weeks and/or reductions in maintenance apheresis frequency to once per-week resulted in re-expression of ataxia and cognitive impairments along with a rise in anti-thyroid antibody levels. An apheresis dose-effect was observed whereby parallel rise and fall in both symptomatology and antibody levels would mirror duration between apheresis intervals. To our knowledge, this is the first report of profound therapeutic benefit and a dose-response relationship to long-term apheresis in cerebellar-type HE. This case suggests that maintenance apheresis be considered in responsive patients, particularly in those with contraindications to medical immunosuppression.

Genome-Wide Analysis of Gene-Gene and Gene-Environment Interactions Using Closed-Form Wald Tests.

Despite the successful discovery of hundreds of variants for complex human traits using genome-wide association studies, the degree to which genes and environmental risk factors jointly affect disease risk is largely unknown. One obstacle toward this goal is that the computational effort required for testing gene-gene and gene-environment interactions is enormous. As a result, numerous computationally efficient tests were recently proposed. However, the validity of these methods often relies on unrealistic assumptions such as additive main effects, main effects at only one variable, no linkage disequilibrium between the two single-nucleotide polymorphisms (SNPs) in a pair or gene-environment independence. Here, we derive closed-form and consistent estimates for interaction parameters and propose to use Wald tests for testing interactions. The Wald tests are asymptotically equivalent to the likelihood ratio tests (LRTs), largely considered to be the gold standard tests but generally too computationally demanding for genome-wide interaction analysis. Simulation studies show that the proposed Wald tests have very similar performances with the LRTs but are much more computationally efficient. Applying the proposed tests to a genome-wide study of multiple sclerosis, we identify interactions within the major histocompatibility complex region. In this application, we find that (1) focusing on pairs where both SNPs are marginally significant leads to more significant interactions when compared to focusing on pairs where at least one SNP is marginally significant; and (2) parsimonious parameterization of interaction effects might decrease, rather than increase, statistical power.

Incorporating parental information into family-based association tests.

Assumptions regarding the true underlying genetic model, or mode of inheritance, are necessary when quantifying genetic associations with disease phenotypes. Here we propose new methods to ascertain the underlying genetic model from parental data in family-based association studies. Specifically, for parental mating-type data, we propose a novel statistic to test whether the underlying genetic model is additive, dominant, or recessive; for parental genotype-phenotype data, we propose three strategies to determine the true mode of inheritance. We illustrate how to incorporate the information gleaned from these strategies into family-based association tests. Because family-based association tests are conducted conditional on parental genotypes, the type I error rate of these procedures is not inflated by the information learned from parental data. This result holds even if such information is weak or when the assumption of Hardy-Weinberg equilibrium is violated. Our simulations demonstrate that incorporating parental data into family-based association tests can improve power under common inheritance models. The application of our proposed methods to a candidate-gene study of type 1 diabetes successfully detects a recessive effect in MGAT5 that would otherwise be missed by conventional family-based association tests.

T-cell growth, cell surface organization, and the galectin-glycoprotein lattice.

Basal, activation, and arrest signaling in T cells determines survival, coordinates responses to pathogens, and, when dysregulated, leads to loss of self-tolerance and autoimmunity. At the T-cell surface, transmembrane glycoproteins interact with galectins via their N-glycans, forming a molecular lattice that regulates membrane localization, clustering, and endocytosis of surface receptors. Galectin-T-cell receptor (TCR) binding prevents ligand-independent TCR signaling via Lck by blocking spontaneous clustering and CD4-Lck recruitment to TCR, and in turn F-actin transfer of TCR/CD4-Lck complexes to membrane microdomains. Peptide-major histocompatibility complexes overcome galectin-TCR binding to promote TCR clustering and signaling by Lck at the immune synapse. Galectin also localizes the tyrosine phosphatase CD45 to microdomains and the immune synapse, suppressing basal and activation signaling by Lck. Following activation, membrane turnover increases and galectin binding to cytotoxic T-lymphocyte antigen-4 (CTLA-4) enhances surface expression by inhibiting endocytosis, thereby promoting growth arrest. Galectins bind surface glycoproteins in proportion to the branching and number of N-glycans per protein, the latter an encoded feature of protein sequence. N-glycan branching is conditional to the activity of Golgi N-acetylglucosaminyl transferases I, II, IV and V (Mgat1, 2, 4, and 5) and metabolic supply of their donor substrate UDP-GlcNAc. Genetic and metabolic control of N-glycan branching co-regulate homeostatic set-points for basal, activation, and arrest signaling in T cells and, when disturbed, result in T-cell hyperactivity and autoimmunity.

The Cancer Moonshot Immuno-Oncology Translational Network at 5: accelerating cancer immunotherapies.

The Immuno-Oncology Translational Network (IOTN) was established in 2018 as part of the Cancer Moonshot. In 2022, President Joe Biden set new goals to reduce the cancer death rate by half within 25 years and improve the lives of people with cancer and cancer survivors. The IOTN is focused on accelerating translation of cancer immunology research, from bench to bedside, and improving immunotherapy outcomes across a wide array of cancers in the adult population. The unique structure and team science approach of the IOTN is designed to accelerate discovery and evaluation of novel immune-based therapeutic and prevention strategies. In this article, we describe IOTN progress to date, including new initiatives and the development of a robust set of resources to advance cancer immunology research. We summarize new insights by IOTN researchers, some of which are ripe for translation for several types of cancers. Looking to the future, we identify barriers to the translation of immuno-oncology concepts into clinical trials and key areas for action and improvements that are suitable for high-yield investments. Based on these experiences, we recommend novel National Institutes of Health funding mechanisms and development of new resources to address these barriers.

N-acetylglucosamine inhibits T-helper 1 (Th1)/T-helper 17 (Th17) cell responses and treats experimental autoimmune encephalomyelitis.

Current treatments and emerging oral therapies for multiple sclerosis (MS) are limited by effectiveness, cost, and/or toxicity. Genetic and environmental factors that alter the branching of Asn (N)-linked glycans result in T cell hyperactivity, promote spontaneous inflammatory demyelination and neurodegeneration in mice, and converge to regulate the risk of MS. The sugar N-acetylglucosamine (GlcNAc) enhances N-glycan branching and inhibits T cell activity and adoptive transfer experimental autoimmune encephalomyelitis (EAE). Here, we report that oral GlcNAc inhibits T-helper 1 (Th1) and T-helper 17 (Th17) responses and attenuates the clinical severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE when administered after disease onset. Oral GlcNAc increased expression of branched N-glycans in T cells in vivo as shown by high pH anion exchange chromatography, MALDI-TOF mass spectroscopy and FACS analysis with the plant lectin l-phytohemagglutinin. Initiating oral GlcNAc treatment on the second day of clinical disease inhibited MOG-induced EAE as well as secretion of interferon-γ, tumor necrosis factor-α, interleukin-17, and interleukin-22. In the more severe 2D2 T cell receptor transgenic EAE model, oral GlcNAc initiated after disease onset also inhibits clinical disease, except for those with rapid lethal progression. These data suggest that oral GlcNAc may provide an inexpensive and nontoxic oral therapeutic agent for MS that directly targets an underlying molecular mechanism causal of disease.

N-Glycans differentially regulate eosinophil and neutrophil recruitment during allergic airway inflammation.

Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-D-mannoside ß1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.

Inhibition of glyceraldehyde-3-phosphate dehydrogenase activity by antibodies present in the cerebrospinal fluid of patients with multiple sclerosis.

We have previously shown that B cells and Abs reactive with GAPDH and antitriosephosphate isomerase (TPI) are present in lesions and cerebrospinal fluid (CSF) in multiple sclerosis (MS). In the current study, we studied the effect of anti-GAPDH and anti-TPI CSF IgG on the glycolytic enzyme activity of GAPDH and TPI after exposure to intrathecal IgG from 10 patients with MS and 34 patients with other neurologic diseases. The degree of inhibition of GAPDH activity by CSF anti-GAPDH IgG in the seven MS samples tested varied from 13 to 98%, which seemed to correlate with the percentage of anti-GAPDH IgG in the CSF IgG (1-45%). Inhibition of GAPDH activity (18 and 23%) by CSF IgG was seen in two of the 34 patients with other neurologic diseases, corresponding to the low percentage of CSF anti-GAPDH IgG (1 and 8%). In addition, depletion of anti-GAPDH IgG from CSF IgG, using immobilized GAPDH, removed the inhibitory effect of the IgG on GAPDH. No inhibition of GAPDH activity was seen with CSF samples not containing anti-GAPDH IgG. No inhibition of TPI activity was seen with any purified CSF IgG sample. These findings demonstrate an increased percentage of anti-GAPDH Abs in the CSF of patients with MS that can inhibit GAPDH glycolytic enzyme activity and may contribute to neuroaxonal degeneration.

Age-associated impairment of T cell immunity is linked to sex-dimorphic elevation of N-glycan branching.

Impaired T cell immunity with aging increases mortality from infectious disease. The branching of Asparagine-linked glycans is a critical negative regulator of T cell immunity. Here we show that branching increases with age in females more than males, in naïve more than memory T cells, and in CD4+ more than CD8+ T cells. Female sex hormones and thymic output of naïve T cells (TN) decrease with age, however neither thymectomy nor ovariectomy altered branching. Interleukin-7 (IL-7) signaling was increased in old female more than male mouse TN cells, and triggered increased branching. N-acetylglucosamine, a rate-limiting metabolite for branching, increased with age in humans and synergized with IL-7 to raise branching. Reversing elevated branching rejuvenated T cell function and reduced severity of Salmonella infection in old female mice. These data suggest sex-dimorphic antagonistic pleiotropy, where IL-7 initially benefits immunity through TN maintenance but inhibits TN function by raising branching synergistically with age-dependent increases in N-acetylglucosamine.

Adaptive regulation at the cell surface by N-glycosylation.

The association of receptors and solute transporters with components of the endocytic machinery regulates their surface levels, and thereby cellular sensitivity to cytokines, ligands and nutrients in the extracellular environment. Most transmembrane receptors and solute transporters are glycoproteins, and the Asn (N)-linked oligosaccharides (N-glycans) can bind animal lectins, forming multivalent lattices or microdomains that regulate glycoprotein mobility in the plane of membrane. The N-glycan number (sequence-encoded NXS/T) and context-dependent Golgi N-glycan branching cooperate to regulate glycoprotein affinities for the galectin family of lectins. Galectin-3 binding reduces EGF receptor trafficking into clathrin-coated pits and caveolae lipid rafts, decreases ligand-independent receptor activation and promotes alpha5beta1 integrin remodelling in focal adhesions. N-glycan branching in the medial Golgi increases glycan affinity for galectins, and the Golgi pathway is sensitive to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) supply, in turn hexosamine pathway metabolites (fructose-6-P, glutamine and acetyl-CoA). Thus, lattice avidity and cellular responsiveness to extracellular cues are regulated in an adaptive manner by metabolism and Golgi modification to glycoproteins. Computational modelling of the hexosamine/Golgi/lattice has provided new insight on cell surface adaptation in cancer and autoimmune disease.

Association of a Marker of N-Acetylglucosamine With Progressive Multiple Sclerosis and Neurodegeneration.

Importance: N-glycan branching modulates cell surface receptor availability, and its deficiency in mice promotes inflammatory demyelination, reduced myelination, and neurodegeneration. N-acetylglucosamine (GlcNAc) is a rate-limiting substrate for N-glycan branching, but, to our knowledge, endogenous serum levels in patients with multiple sclerosis (MS) are unknown. Objective: To investigate a marker of endogenous serum GlcNAc levels in patients with MS. Design, Setting, and Participants: A cross-sectional discovery study and cross-sectional confirmatory study were conducted at 2 academic MS centers in the US and Germany. The discovery study recruited 54 patients with MS from an outpatient clinic as well as 66 healthy controls between April 20, 2010, and June 21, 2013. The confirmatory study recruited 180 patients with MS from screening visits at an academic MS study center between April 9, 2007, and February 29, 2016. Serum samples were analyzed from December 2, 2013, to March 2, 2015. Statistical analysis was performed from February 23, 2020, to March 18, 2021. Main Outcomes and Measures: Serum levels of GlcNAc plus its stereoisomers, termed N-acetylhexosamine (HexNAc), were assessed using targeted tandem mass spectroscopy. Secondary outcomes (confirmatory study) comprised imaging and clinical disease markers. Results: The discovery cohort included 66 healthy controls (38 women; mean [SD] age, 42 [20] years), 33 patients with relapsing-remitting MS (RRMS; 25 women; mean [SD] age, 50 [11] years), and 21 patients with progressive MS (PMS; 14 women; mean [SD] age, 55 [7] years). The confirmatory cohort included 125 patients with RRMS (83 women; mean [SD] age, 40 [9] years) and 55 patients with PMS (22 women; mean [SD] age, 49 [80] years). In the discovery cohort, the mean (SD) serum level of GlcNAc plus its stereoisomers (HexNAc) was 710 (174) nM in healthy controls and marginally reduced in patients with RRMS (mean [SD] level, 682 [173] nM; P = .04), whereas patients with PMS displayed markedly reduced levels compared with healthy controls (mean [SD] level, 548 [101] nM; P = 9.55 × 10-9) and patients with RRMS (P = 1.83 × 10-4). The difference between patients with RRMS (mean [SD] level, 709 [193] nM) and those with PMS (mean [SD] level, 405 [161] nM; P = 7.6 × 10-18) was confirmed in the independent confirmatory cohort. Lower HexNAc serum levels correlated with worse expanded disability status scale scores (ρ = -0.485; P = 4.73 × 10-12), lower thalamic volume (t = 1.7; P = .04), and thinner retinal nerve fiber layer (B = 0.012 [SE = 7.5 × 10-11]; P = .008). Low baseline serum HexNAc levels correlated with a greater percentage of brain volume loss at 18 months (t = 1.8; P = .04). Conclusions and Relevance: This study suggests that deficiency of GlcNAc plus its stereoisomers (HexNAc) may be a biomarker for PMS. Previous preclinical, human genetic, and ex vivo human mechanistic studies revealed that N-glycan branching and/or GlcNAc may reduce proinflammatory responses, promote myelin repair, and decrease neurodegeneration. Combined, the data suggest that GlcNAc deficiency may be associated with progressive disease and neurodegeneration in patients with MS.

The Crossroads of Glycoscience, Infection, and Immunology.

Advances in experimental capabilities in the glycosciences offer expanding opportunities for discovery in the broad areas of immunology and microbiology. These two disciplines overlap when microbial infection stimulates host immune responses and glycan structures are central in the processes that occur during all such encounters. Microbial glycans mediate host-pathogen interactions by acting as surface receptors or ligands, functioning as virulence factors, impeding host immune responses, or playing other roles in the struggle between host and microbe. In the context of the host, glycosylation drives cell-cell interactions that initiate and regulate the host response and modulates the effects of antibodies and soluble immune mediators. This perspective reports on a workshop organized jointly by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research in May 2020. The conference addressed the use of emerging glycoscience tools and resources to advance investigation of glycans and their roles in microbe-host interactions, immune-mediated diseases, and immune cell recognition and function. Future discoveries in these areas will increase fundamental scientific understanding and have the potential to improve diagnosis and treatment of infections and immune dysregulation.

T cell receptor signaling co-regulates multiple Golgi genes to enhance N-glycan branching.

T cell receptor (TCR) signaling enhances beta1,6GlcNAc-branching in N-glycans, a phenotype that promotes growth arrest and inhibits autoimmunity by increasing surface retention of cytotoxic T lymphocyte antigen-4 (CTLA-4) via interactions with galectins. N-Acetylglucosaminyltransferase V (MGAT5) mediates beta1,6GlcNAc-branching by transferring N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to N-glycan substrates produced by the sequential action of Golgi alpha1,2-mannosidase I (MIa,b,c), MGAT1, alpha1,2-mannosidase II (MII, IIx), and MGAT2. Here we report that TCR signaling enhances mRNA levels of MIa,b,c and MII,IIx in parallel with MGAT5, whereas limiting levels of MGAT1 and MGAT2. Blocking the increase in MI or MII enzyme activity induced by TCR signaling with deoxymannojirimycin or swainsonine, respectively, limits beta1,6GlcNAc-branching, suggesting that enhanced MI and MII activity are both required for this phenotype. MGAT1 and MGAT2 have an approximately 250- and approximately 20-fold higher affinity for UDP-GlcNAc than MGAT5, respectively, and increasing MGAT1 expression paradoxically inhibits beta1,6GlcNAc branching by limiting UDP-GlcNAc supply to MGAT5, suggesting that restricted changes in MGAT1 and MGAT2 mRNA levels in TCR-stimulated cells serves to enhance availability of UDP-GlcNAc to MGAT5. Together, these data suggest that TCR signaling differentially regulates multiple N-glycan-processing enzymes at the mRNA level to cooperatively promote beta1,6GlcNAc branching, and by extension, CTLA-4 surface expression, T cell growth arrest, and self-tolerance.

N-Glycan Branching Decouples B Cell Innate and Adaptive Immunity to Control Inflammatory Demyelination.

B cell depletion potently reduces episodes of inflammatory demyelination in multiple sclerosis (MS), predominantly through loss of innate rather than adaptive immunity. However, molecular mechanisms controlling innate versus adaptive B cell function are poorly understood. N-glycan branching, via interactions with galectins, controls endocytosis and signaling of cell surface receptors to control cell function. Here we report that N-glycan branching in B cells dose dependently reduces pro-inflammatory innate responses by titrating decreases in Toll-like receptor-4 (TLR4) and TLR2 surface expression via endocytosis. In contrast, a minimal level of N-glycan branching maximizes surface retention of the B cell receptor (BCR) and the CD19 co-receptor to promote adaptive immunity. Branched N-glycans inhibit antigen presentation by B cells to reduce T helper cell-17 (TH17)/TH1 differentiation and inflammatory demyelination in mice. Thus, N-glycan branching negatively regulates B cell innate function while promoting/maintaining adaptive immunity via BCR, providing an attractive therapeutic target for MS.

Effect of vitamin D supplementation on N-glycan branching and cellular immunophenotypes in MS.

OBJECTIVE: To investigate the effect of cholecalciferol (vitamin D3) supplementation on peripheral immune cell frequency and N-glycan branching in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: Exploratory analysis of high-dose (20 400 IU) and low-dose (400 IU) vitamin D3 supplementation taken every other day of an 18-month randomized controlled clinical trial including 38 RRMS patients on stable immunomodulatory therapy (NCT01440062). We investigated cholecalciferol treatment effects on N-glycan branching using L-PHA stain (phaseolus vulgaris leukoagglutinin) at 6 months and frequencies of T-, B-, and NK-cell subpopulations at 12 months with flow cytometry. RESULTS: High-dose supplementation did not change CD3+ T cell subsets, CD19+ B cells subsets, and NK cells frequencies, except for CD8+ T regulatory cells, which were reduced in the low-dose arm compared to the high-dose arm at 12 months. High-dose supplementation decreased N-glycan branching on T and NK cells, measured as L-PHA mean fluorescence intensity (MFI). A reduction of N-glycan branching in B cells was not significant. In contrast, low-dose supplementation did not affect N-glycan branching. Changes in N-glycan branching did not correlate with cell frequencies. INTERPRETATION: Immunomodulatory effect of vitamin D may involve regulation of N-glycan branching in vivo. Vitamin D3 supplementation did at large not affect the frequencies of peripheral immune cells.