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.

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.

Increasing cell permeability of N-acetylglucosamine via 6-acetylation enhances capacity to suppress T-helper 1 (TH1)/TH17 responses and autoimmunity.

N-acetylglucosamine (GlcNAc) branching of Asn (N)-linked glycans inhibits pro-inflammatory T cell responses and models of autoimmune diseases such as Multiple Sclerosis (MS). Metabolism controls N-glycan branching in T cells by regulating de novo hexosamine pathway biosynthesis of UDP-GlcNAc, the donor substrate for the Golgi branching enzymes. Activated T cells switch metabolism from oxidative phosphorylation to aerobic glycolysis and glutaminolysis. This reduces flux of glucose and glutamine into the hexosamine pathway, thereby inhibiting de novo UDP-GlcNAc synthesis and N-glycan branching. Salvage of GlcNAc into the hexosamine pathway overcomes this metabolic suppression to restore UDP-GlcNAc synthesis and N-glycan branching, thereby promoting anti-inflammatory T regulatory (Treg) over pro-inflammatory T helper (TH) 17 and TH1 differentiation to suppress autoimmunity. However, GlcNAc activity is limited by the lack of a cell surface transporter and requires high doses to enter cells via macropinocytosis. Here we report that GlcNAc-6-acetate is a superior pro-drug form of GlcNAc. Acetylation of amino-sugars improves cell membrane permeability, with subsequent de-acetylation by cytoplasmic esterases allowing salvage into the hexosamine pathway. Per- and bi-acetylation of GlcNAc led to toxicity in T cells, whereas mono-acetylation at only the 6 > 3 position raised N-glycan branching greater than GlcNAc without inducing significant toxicity. GlcNAc-6-acetate inhibited T cell activation/proliferation, TH1/TH17 responses and disease progression in Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of MS. Thus, GlcNAc-6-Acetate may provide an improved therapeutic approach to raise N-glycan branching, inhibit pro-inflammatory T cell responses and treat autoimmune diseases such as MS.

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.

Phenotypic and transcriptional fidelity of patient-derived colon cancer xenografts in immune-deficient mice.

Xenografts of human colorectal cancer (CRC) in immune-deficient mice have great potential for accelerating the study of tumor biology and therapy. We evaluated xenografts established in NOD/scid/IL2Rγ-null mice from the primary or metastatic tumors of 27 patients with CRC to estimate their capacity for expanding tumor cells for in vitro studies and to assess how faithfully they recapitulated the transcriptional profile of their parental tumors. RNA-seq analysis of parental human CRC tumors and their derivative xenografts demonstrated that reproducible transcriptional changes characterize the human tumor to murine xenograft transition. In most but not all cases, the human stroma, vasculature, and hematopoietic elements were systematically replaced by murine analogues while the carcinoma component persisted. Once established as xenografts, human CRC cells that could be propagated by serial transplantation remained transcriptionally stable. Three histologically atypical xenografts, established from patients with peritoneal metastases, contained abundant human stromal elements and blood vessels in addition to human tumor cells. The transcriptomes of these mixed tumor/stromal xenografts did not closely resemble those of their parental tumors, and attempts to propagate such xenografts by serial transplantation were unsuccessful. Stable expression of numerous genes previously identified as high priority targets for immunotherapy was observed in most xenograft lineages. Aberrant expression in CRC cells of human genes that are normally only expressed in hematopoietic cells was also observed. Our results suggest that human CRC cells expanded in murine xenografts have great utility for studies of tumor immunobiology and targeted therapies such as immunotherapy but also identify potential limitations.