Endothelial heparan sulfate controls chemokine presentation in recruitment of lymphocytes and dendritic cells to lymph nodes.

Heparan sulfate can bind several adhesion molecules involved in lymphocyte trafficking. However, the in vivo function of endothelial heparan sulfate in lymphocyte homing and stimulation of the immune response has not been elucidated. Here, we generated mutant mice deficient in the enzyme Ext1, which is required for heparan sulfate synthesis, in a Tek-dependent and inducible manner. Chemokine presentation was diminished in the mutant mice, causing the lack of appropriate integrin-mediated adhesion, and resulted in a marked decrease in lymphocyte sticking to high endothelial venules and in recruitment of resident dendritic cells through lymphatic vessels to the lymph nodes. As a consequence, mutant mice displayed a severe impairment in lymphocyte homing and a compromised contact hypersensitivity response. By contrast, lymphocyte rolling was increased because of loss of electrostatic repulsion by heparan sulfate. These results demonstrate critical roles of endothelial heparan sulfate in immune surveillance and immune response generation.

FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc.

O-fucose modulates Notch-controlled blood lineage commitment.

Notch receptors are cell surface molecules essential for cell fate determination. Notch signaling is subject to tight regulation at multiple levels, including the posttranslational modification of Notch receptors by O-linked fucosylation, a reaction that is catalyzed by protein O-fucosyltransferase-1 (Pofut1). Our previous studies identified a myeloproliferative phenotype in mice conditionally deficient in cellular fucosylation that is attributable to a loss of Notch-dependent suppression of myelopoiesis. Here, we report that hematopoietic stem cells deficient in cellular fucosylation display decreased frequency and defective repopulating ability as well as decreased lymphoid but increased myeloid developmental potential. This phenotype may be attributed to suppressed Notch ligand binding and reduced downstream signaling of Notch activity in hematopoietic stem cells. Consistent with this finding, we further demonstrate that mouse embryonic stem cells deficient in Notch1 (Notch1(-/-)) or Pofut1 (Pofut1(-/-)) fail to generate T lymphocytes but differentiate into myeloid cells while coculturing with Notch ligand-expressing bone marrow stromal cells in vitro. Moreover, in vivo hematopoietic reconstitution of CD34(+) progenitor cells derived from either Notch1(-/-) or Pofut1(-/-) embryonic stem cells show enhanced granulopoiesis with depressed lymphoid lineage development. Together, these results indicate that Notch signaling maintains hematopoietic lineage homeostasis by promoting lymphoid development and suppressing overt myelopoiesis, in part through processes controlled by O-linked fucosylation of Notch receptors.

Notch-dependent control of myelopoiesis is regulated by fucosylation.

Cell-cell contact-dependent mechanisms that modulate proliferation and/or differentiation in the context of hematopoiesis include mechanisms characteristic of the interactions between members of the Notch family of signal transduction molecules and their ligands. Whereas Notch family members and their ligands clearly modulate T lymphopoietic decisions, evidence for their participation in modulating myelopoiesis is much less clear, and roles for posttranslational control of Notch-dependent signal transduction in myelopoiesis are unexplored. We report here that a myeloproliferative phenotype in FX(-/-) mice, which are conditionally deficient in cellular fucosylation, is consequent to loss of Notch-dependent signal transduction on myeloid progenitor cells. In the context of a wild-type fucosylation phenotype, we find that the Notch ligands suppress myeloid differentiation of progenitor cells and enhance expression of Notch target genes. By contrast, fucosylation-deficient myeloid progenitors are insensitive to the suppressive effects of Notch ligands on myelopoiesis, do not transcribe Notch1 target genes when cocultured with Notch ligands, and have lost the wild-type Notch ligand-binding phenotype. Considered together, these observations indicate that Notch-dependent signaling controls myelopoiesis in vivo and in vitro and identifies a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in myeloid progenitors.

Differential expression of an alpha-galactosyl-containing trisaccharide on high- and low-malignant murine sarcoma cells: identification and regulation.

Past studies have shown that carbohydrate residues reactive with the Griffonia simplicifolia isolectin B4 (GS I-B4) are present on the surface of highly-malignant murine sarcoma cells but are lacking or expressed in much lower amounts on the surface of low-malignant cells isolated from the same parent tumors (Am J Pathol 111: 27; J Nat Cancer Inst 71: 1281). In the present study it is shown that an antibody which recognizes the trisaccharide Galalpha1-3Galbeta1-4GlcNAc- is reactive with the highly-malignant cells but is non-reactive with the low-malignant cells. Further studies show that the high-malignant cells not only bind GS 1-B4 but also bind Evonymus europaea lectin (which like GS I-B4 recognizes terminal galactose in alpha1-3 linkage) and Erythina crystagalli lectin (which recognizes sub-terminal galactose in the beta1-4 linkage--e.g., Galbeta1-4GlcNAc). In contrast, the low malignant cells bind Erythina crystagalli lectin as efficiently as the high malignant cells but do not bind (or bind much smaller amounts of) either GS I-B4 or Evonymus europaea lectin. The present studies also show that there is no significant difference between high- and low-malignant cells in expression of alpha-galactosidase activity. In contrast, the high-malignant cells express high levels of alpha-galactosyl transferase activity while this enzyme is virtually undetectable in low-malignant cells. Taken together, these studies indicate that differential expression of a single monosaccharide residue distinguishes high- and low-malignant murine sarcoma cells. These studies also identify a mechanism to account for surface carbohydrate differences between the high- and low-malignant cells.

Significant decrease in alpha1,3-linked fucose in association with increase in 6-sulfated N-acetylglucosamine in peripheral lymph node addressin of FucT-VII-deficient mice exhibiting diminished lymphocyte homing.

Lymphocyte homing is mediated by binding of L-selectin on lymphocytes with L-selectin ligands present on high-endothelial venules (HEV) of peripheral and mesenteric lymph nodes. L-selectin ligands are specific O-linked carbohydrates, 6-sulfo sialyl Lewis X, composed of sialylated, fucosylated, and sulfated glycans. Abrogation of fucosyltransferase-VII (FucT-VII) results in almost complete loss of lymphocyte homing, but structural analysis of carbohydrates has not been carried out on FucT-VII null mice. To determine whether functional losses seen in FucT-VII null mice are caused by structural changes in carbohydrates, we elucidated the carbohydrate structure of GlyCAM-1, a major L-selectin counter-receptor. Our results show that most alpha1,3-fucosylated structures in 6-sulfo sialyl Lewis X are absent and 6-sulfo N-acetyllactosamine is increased in the mutant mice. Surprisingly, the amount of 6'-sulfated galactose (Gal) that bound to Sumbucus nigra agglutinin column was also increased. We found that structures of those oligosaccharides containing 6'-sulfated Gal are almost identical to those synthesized by keratan sulfate sulfotransferase (KSST). We then showed that overexpression of KSST suppresses the expression of sialyl Lewis X on Chinese hamster ovary (CHO) cells engineered to express sialyl Lewis X. Moreover, KSST expression in those cells suppressed lymphocyte rolling compared with mock-transfected CHO cells expressing 6-sulfo sialyl Lewis X. 6'-Sulfo sialyl Lewis X can neither be found in GlyCAM-1 from CHO cells expressing both KSST and FucT-VII nor be found in GlyCAM-1 from HEV of mice. These results combined together suggest that KSST competes with FucT-VII for the same acceptor substrate and downregulates the synthesis of L-selectin ligand by inhibiting alpha1,3-fucosylation.

N-acetylglucosamine-6-O-sulfotransferases 1 and 2 cooperatively control lymphocyte homing through L-selectin ligand biosynthesis in high endothelial venules.

Lymphocyte homing is mediated by specific interactions between L-selectin on lymphocytes and sulfated carbohydrates restricted to high endothelial venules in lymph nodes. Here we generated mice deficient in both N-acetylglucosamine-6-O-sulfotransferase 1 (GlcNAc6ST-1) and GlcNAc6ST-2 and found that mutant mice had approximately 75% less homing of lymphocytes to the peripheral lymph nodes than did wild-type mice. Consequently, these mice had lower contact hypersensitivity responses than those of wild-type mice. Carbohydrate structural analysis showed that 6-sulfo sialyl Lewis X, a dominant ligand for L-selectin, was almost completely absent from the high endothelial venules of these mutant mice, whereas the amount of unsulfated sialyl Lewis X was much greater. These results demonstrate the essential function of GlcNAc6ST-1 and GlcNAc6ST-2 in L-selectin ligand biosynthesis in high endothelial venules and their importance in immune surveillance.

Extended core 1 and core 2 branched O-glycans differentially modulate sialyl Lewis X-type L-selectin ligand activity.

It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E- and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAcalpha2-->3Galbeta1-->4(Fucalpha1-->3(sulfo-->6))GlcNAcbeta1--> 3Galbeta1-->3GalNAcalpha1-->Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that beta1,3-N-acetylglucosaminyltransferase-3 (beta3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different beta3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding beta3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 beta1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either beta3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branched O-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells.

Core 2 branching beta1,6-N-acetylglucosaminyltransferase and high endothelial venule-restricted sulfotransferase collaboratively control lymphocyte homing.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to carbohydrate ligands expressed on high endothelial venules (HEV) of the secondary lymphoid organs. Previous studies demonstrated that L-selectin ligand sulfotransferase (LSST) forms 6-sulfo sialyl Lewis x (sLe(x)) on both core 2 branch and MECA-79-positive extended core 1 O-glycans, but the chemical nature and roles of HEV ligands elaborated by LSST and core 2 beta1,6-N-acetylglucosaminyltransferase-1 (Core2GlcNAcT) have been undefined. In the present study, we have generated mutant mice with deficient LSST and show that inactivation of LSST gene alone leads to only partial impairment of lymphocyte homing to peripheral lymph nodes and moderate reduction in lymphocyte counts in the peripheral lymph nodes, despite the fact that L-selectin ligands that contain 6-sulfo sLe(x) are reduced at HEV. By contrast, LSST/Core2GlcNAcT double null mice exhibited a markedly reduced lymphocyte homing and reduced lymphocyte counts as a result of significantly decreased 6-sulfo sLe(x) on HEV L-selectin counterreceptors, relative to LSST- or Core2GlcNAcT-single null mice. Moreover, induction of LSST and Core2GlcNAcT transcripts was observed in HEV-like structure formed in the salivary gland of the non-obese diabetic mouse, which displays chronic inflammation. These results indicate that LSST and Core2GlcNAcT cooperatively synthesize HEV-specific L-selectin ligands required for lymphocyte homing and suggest that LSST and Core2GlcNAcT play a critical role in lymphocyte trafficking during chronic inflammation.