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.

N-Glycan Branching Regulates BTLA Opposite to PD-1 to Limit T Cell Hyperactivity Induced by Branching Deficiency.

N-glycan branching is a potent and multifaceted negative regulator of proinflammatory T cell and B cell function. By promoting multivalent galectin-glycoprotein lattice formation at the cell surface, branching regulates clustering and/or endocytosis of the TCR complex (TCR+CD4/CD8), CD45, CD25, BCR, TLR2 and TLR4 to inhibit T cell and B cell activation/proliferation and proinflammatory TH1 and TH17 over TH2 and induced T regulatory cell responses. In addition, branching promotes cell surface retention of the growth inhibitory receptor CTLA-4. However, the role of N-glycan branching in regulating cell surface levels of other checkpoint receptors such as BTLA (B and T lymphocyte attenuator) and PD-1 (programmed cell death protein 1) is unknown. In this study, we report that whereas branching significantly enhances PD-1 cell surface expression by reducing loss from endocytosis, the opposite occurs with BTLA in both T cells and B cells. T cell hyperactivity induced by branching deficiency was opposed by BTLA ligation proportional to increased BTLA expression. Other members of the BTLA/HVEM (herpesvirus entry mediator) signaling axis in T cells, including HVEM, LIGHT, and CD160, are largely unaltered by branching. Thus, branching-mediated endocytosis of BTLA is opposite of branching-induced inhibition of PD-1 endocytosis. In this manner, branching deficiency-induced upregulation of BTLA appears to serve as a checkpoint to limit extreme T cell hyperactivity and proinflammatory outcomes in T cells with low branching.

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.

Targeting neural precursors in the adult brain rescues injured dopamine neurons.

In Parkinson's disease, multiple cell types in many brain regions are afflicted. As a consequence, a therapeutic strategy that activates a general neuroprotective response may be valuable. We have previously shown that Notch ligands support neural precursor cells in vitro and in vivo. Here we show that neural precursors express the angiopoietin receptor Tie2 and that injections of angiopoietin2 activate precursors in the adult brain. Signaling downstream of Tie2 and the Notch receptor regulate blood vessel formation. In the adult brain, angiopoietin2 and the Notch ligand Dll4 activate neural precursors with opposing effects on the density of blood vessels. A model of Parkinson's disease was used to show that angiopoietin2 and Dll4 rescue injured dopamine neurons with motor behavioral improvement. A combination of growth factors with little impact on the vasculature retains the ability to stimulate neural precursors and protect dopamine neurons. The cellular and pharmacological basis of the neuroprotective effects achieved by these single treatments merits further analysis.

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.

Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation.

Rapidly proliferating cells switch from oxidative phosphorylation to aerobic glycolysis plus glutaminolysis, markedly increasing glucose and glutamine catabolism. Although Otto Warburg first described aerobic glycolysis in cancer cells >90 years ago, the primary purpose of this metabolic switch remains controversial. The hexosamine biosynthetic pathway requires glucose and glutamine for de novo synthesis of UDP-GlcNAc, a sugar-nucleotide that inhibits receptor endocytosis and signaling by promoting N-acetylglucosamine branching of Asn (N)-linked glycans. Here, we report that aerobic glycolysis and glutaminolysis co-operatively reduce UDP-GlcNAc biosynthesis and N-glycan branching in mouse T cell blasts by starving the hexosamine pathway of glucose and glutamine. This drives growth and pro-inflammatory TH17 over anti-inflammatory-induced T regulatory (iTreg) differentiation, the latter by promoting endocytic loss of IL-2 receptor-α (CD25). Thus, a primary function of aerobic glycolysis and glutaminolysis is to co-operatively limit metabolite supply to N-glycan biosynthesis, an activity with widespread implications for autoimmunity and cancer.

Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis.

Essential biological systems employ self-correcting mechanisms to maintain cellular homeostasis. Mammalian cell function is dynamically regulated by the interaction of cell surface galectins with branched N-glycans. Here we report that N-glycan branching deficiency triggers the Golgi to generate bioequivalent N-glycans that preserve galectin-glycoprotein interactions and cellular homeostasis. Galectins bind N-acetyllactosamine (LacNAc) units within N-glycans initiated from UDP-GlcNAc by the medial-Golgi branching enzymes as well as the trans-Golgi poly-LacNAc extension enzyme ß1,3-N-acetylglucosaminyltransferase (B3GNT). Marginally reducing LacNAc content by limiting N-glycans to three branches results in T-cell hyperactivity and autoimmunity; yet further restricting branching does not produce a more hyperactive state. Rather, new poly-LacNAc extension by B3GNT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by redistribution of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules. These data demonstrate the functional equivalency of structurally dissimilar N-glycans and suggest a self-correcting feature of the Golgi that sustains cellular homeostasis.

Hypomorphic MGAT5 polymorphisms promote multiple sclerosis cooperatively with MGAT1 and interleukin-2 and 7 receptor variants.

Deficiency of the Golgi N-glycan branching enzyme Mgat5 in mice promotes T cell hyperactivity, endocytosis of CTLA-4 and autoimmunity, including a spontaneous multiple sclerosis (MS)-like disease. Multiple genetic and environmental MS risk factors lower N-glycan branching in T cells. These include variants in interleukin-2 receptor-α (IL2RA), interleukin-7 receptor-α (IL7RA), and MGAT1, a Golgi branching enzyme upstream of MGAT5, as well as vitamin D3 deficiency and Golgi substrate metabolism. Here we describe linked intronic variants of MGAT5 that are associated with reduced N-glycan branching, CTLA-4 surface expression and MS (p=5.79×10(-9), n=7,741), the latter additive with the MGAT1, IL2RA and IL7RA MS risk variants (p=1.76×10(-9), OR=0.67-1.83, n=3,518).

Interleukin-2, Interleukin-7, T cell-mediated autoimmunity, and N-glycosylation.

T cell activation and self-tolerance are tightly regulated to provide effective host defense against foreign pathogens while deflecting inappropriate autoimmune responses. Golgi Asn (N)-linked protein glycosylation coregulates homeostatic set points for T cell growth, differentiation, and self-tolerance to influence risk of autoimmune disorders such as multiple sclerosis (MS). Human autoimmunity is a complex trait that develops from intricate and poorly understood interactions between an individual's genetics and their environmental exposures. Recent evidence from our group suggests that in MS, additive and/or epistatic interactions between multiple genetic and environmental risk factors combine to dysregulate a common biochemical pathway, namely Golgi N-glycosylation. Here, we review the multiple regulatory mechanisms controlling N-glycan branching in T cells and autoimmunity, focusing on recent data implicating a critical role for interleukin-2 (IL-2) and IL-7 signaling.

Pathogenesis of multiple sclerosis via environmental and genetic dysregulation of N-glycosylation.

Autoimmune diseases such as multiple sclerosis (MS) result from complex and poorly understood interactions of genetic and environmental factors. A central role for T cells in MS is supported by mouse models, association of the major histocompatibility complex region, and association of critical T cell growth regulator genes such as interleukin-2 receptor (IL-2RA) and interleukin-7 receptor (IL-7RA). Multiple environmental factors (vitamin D(3) deficiency and metabolism) converge with multiple genetic variants (IL-7RA, IL-2RA, MGAT1, and CTLA-4) to dysregulate Golgi N-glycosylation in MS, resulting in T cell hyperactivity, loss of self-tolerance and in mice, a spontaneous MS-like disease with neurodegeneration. Here, we review the genetic and biological interactions that regulate MS pathogenesis through dysregulation of N-glycosylation and how this may enable individualized therapeutic approaches.

Genetics and the environment converge to dysregulate N-glycosylation in multiple sclerosis.

How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice, N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity, cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis, spontaneous inflammatory demyelination and neurodegeneration, the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3), including genetic variants in interleukin-7 receptor-α (IL7RA*C), interleukin-2 receptor-α (IL2RA*T), MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3), optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway, namely N-glycosylation.