Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth.

Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.

IL-17A Orchestrates Reactive Oxygen Species/HIF1α-Mediated Metabolic Reprogramming in Psoriasis.

Immune cell-derived IL-17A is one of the key pathogenic cytokines in psoriasis, an immunometabolic disorder. Although IL-17A is an established regulator of cutaneous immune cell biology, its functional and metabolic effects on nonimmune cells of the skin, particularly keratinocytes, have not been comprehensively explored. Using multiomics profiling and systems biology-based approaches, we systematically uncover significant roles for IL-17A in the metabolic reprogramming of human primary keratinocytes (HPKs). High-throughput liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy revealed IL-17A-dependent regulation of multiple HPK proteins and metabolites of carbohydrate and lipid metabolism. Systems-level MitoCore modeling using flux-balance analysis identified IL-17A-mediated increases in HPK glycolysis, glutaminolysis, and lipid uptake, which were validated using biochemical cell-based assays and stable isotope-resolved metabolomics. IL-17A treatment triggered downstream mitochondrial reactive oxygen species and HIF1α expression and resultant HPK proliferation, consistent with the observed elevation of these downstream effectors in the epidermis of patients with psoriasis. Pharmacological inhibition of HIF1α or reactive oxygen species reversed IL-17A-mediated glycolysis, glutaminolysis, lipid uptake, and HPK hyperproliferation. These results identify keratinocytes as important target cells of IL-17A and reveal its involvement in multiple downstream metabolic reprogramming pathways in human skin.

Homing in on the Sweet Side of Immune Checkpoint Biology.

P-selectin glycoprotein ligand-1 (PSGL-1) and its glycostructural determinants facilitate responses to infection and cancer by promoting immune effector-cell trafficking into inflamed tissue. In this issue of Immunity, Tinoco et al. (2016) report homing-independent functions of PSGL-1 in immune checkpoint regulation and T cell effector activity, in models of chronic viral infection and melanoma.

IL-9 Abrogates the Metastatic Potential of Breast Cancer by Controlling Extracellular Matrix Remodeling and Cellular Contractility.

IL-9 is produced by Th9 cells and is classically known as a growth-promoting cytokine. Although protumorigenic functions of IL-9 are described in T cell lymphoma, recently, we and others have reported anti-tumor activities of IL-9 in melanoma mediated by mast cells and CD8+ T cells. However, involvement of IL-9 in invasive breast and cervical cancer remains unexplored. In this study, we demonstrate IL-9-dependent inhibition of metastasis of both human breast (MDA-MB-231 and MCF-7) and cervical (HeLa) tumor cells in physiological three-dimensional invasion assays. To dissect underlying mechanisms of IL-9-mediated suppression of invasion, we analyzed IL-9-dependent pathways of cancer cell metastasis, including proteolysis, contractility, and focal adhesion dynamics. IL-9 markedly blocked tumor cell-collagen degradation, highlighting the effects of IL-9 on extracellular matrix remodeling. Moreover, IL-9 significantly reduced phosphorylation of myosin L chain and resultant actomyosin contractility and also increased focal adhesion formation. Finally, IL-9 suppressed IL-17- and IFN-γ-induced metastasis of both human breast (MDA-MB-231) and cervical (HeLa) cancer cells. In conclusion, IL-9 inhibits the metastatic potential of breast and cervical cancer cells by controlling extracellular matrix remodeling and cellular contractility.

T-lymphocyte homing: an underappreciated yet critical hurdle for successful cancer immunotherapy.

Advances in cancer immunotherapy have offered new hope for patients with metastatic disease. This unfolding success story has been exemplified by a growing arsenal of novel immunotherapeutics, including blocking antibodies targeting immune checkpoint pathways, cancer vaccines, and adoptive cell therapy (ACT). Nonetheless, clinical benefit remains highly variable and patient-specific, in part, because all immunotherapeutic regimens vitally hinge on the capacity of endogenous and/or adoptively transferred T-effector (Teff) cells, including chimeric antigen receptor (CAR) T cells, to home efficiently into tumor target tissue. Thus, defects intrinsic to the multi-step T-cell homing cascade have become an obvious, though significantly underappreciated contributor to immunotherapy resistance. Conspicuous have been low intralesional frequencies of tumor-infiltrating T-lymphocytes (TILs) below clinically beneficial threshold levels, and peripheral rather than deep lesional TIL infiltration. Therefore, a Teff cell 'homing deficit' may arguably represent a dominant factor responsible for ineffective immunotherapeutic outcomes, as tumors resistant to immune-targeted killing thrive in such permissive, immune-vacuous microenvironments. Fortunately, emerging data is shedding light into the diverse mechanisms of immune escape by which tumors restrict Teff cell trafficking and lesional penetrance. In this review, we scrutinize evolving knowledge on the molecular determinants of Teff cell navigation into tumors. By integrating recently described, though sporadic information of pivotal adhesive and chemokine homing signatures within the tumor microenvironment with better established paradigms of T-cell trafficking under homeostatic or infectious disease scenarios, we seek to refine currently incomplete models of Teff cell entry into tumor tissue. We further summarize how cancers thwart homing to escape immune-mediated destruction and raise awareness of the potential impact of immune checkpoint blockers on Teff cell homing. Finally, we speculate on innovative therapeutic opportunities for augmenting Teff cell homing capabilities to improve immunotherapy-based tumor eradication in cancer patients, with special focus on malignant melanoma.

Melanoma stem cells and metastasis: mimicking hematopoietic cell trafficking?

Malignant melanoma is a highly metastatic cancer that bears responsibility for the majority of skin cancer-related deaths. Amidst the research efforts to better understand melanoma progression, there has been increasing evidence that hints at a role for a subpopulation of virulent cancer cells, termed malignant melanoma stem or initiating cells (MMICs), in metastasis formation. MMICs are characterized by their preferential ability to initiate and propagate tumor growth and their selective capacity for self-renewal and differentiation into less tumorigenic melanoma cells. The frequency of MMICs has been shown to correlate with poor clinical prognosis in melanoma. In addition, MMICs are enriched among circulating tumor cells in the peripheral blood of cancer patients, suggesting that MMICs may be a critical factor in the metastatic cascade. Although these links exist between MMICs and metastatic disease, the mechanisms by which MMICs may advance metastatic progression are only beginning to be elucidated. Recent studies have shown that MMICs express molecules critical for hematopoietic cell maintenance and trafficking, providing a possible explanation for how circulating MMICs could drive melanoma dissemination. We therefore propose that MMICs might fuel melanoma metastasis by exploiting homing mechanisms commonly utilized by hematopoietic cells. Here we review the biological properties of MMICs and the existing literature on their metastatic potential. We will discuss possible mechanisms by which MMICs might initiate metastases in the context of established knowledge of cancer stem cells in other cancers and of hematopoietic homing molecules, with a particular focus on selectins, integrins, chemokines and chemokine receptors known to be expressed by melanoma cells. Biological understanding of how these molecules might be utilized by MMICs to propel the metastatic cascade could critically impact the development of more effective therapies for advanced disease.

Galectin-1 triggers an immunoregulatory signature in Th cells functionally defined by IL-10 expression.

Galectin-1 (Gal-1), a ß-galactoside-binding protein, can alter fate and effector function of Th cells; however, little is known about how Gal-1 induces Th cell differentiation. In this article, we show that both uncommitted and polarized Th cells bound by Gal-1 expressed an immunoregulatory signature defined by IL-10. IL-10 synthesis was stimulated by direct Gal-1 engagement to cell surface glycoproteins, principally CD45, on activated Th cells and enhanced by IL-21 expression through the c-Maf/aryl hydrocarbon receptor pathway, independent of APCs. Gal-1-induced IL-10(+) T cells efficiently suppressed T cell proliferation and T cell-mediated inflammation and promoted the establishment of cancer immune-privileged sites. Collectively, these findings show how Gal-1 functions as a major glycome determinant regulating Th cell development, inflammation, and tumor immunity.

Type I interferon signaling induces melanoma cell-intrinsic PD-1 and its inhibition antagonizes immune checkpoint blockade.

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity, research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast, cancer cell-intrinsic PD-1 functional regulation is not well understood. Here, we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-ß triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling, increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer, and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors.

Tumor cell-intrinsic PD-1 promotes Merkel cell carcinoma growth by activating downstream mTOR-mitochondrial ROS signaling.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer. Inhibitors targeting the programmed cell death 1 (PD-1) immune checkpoint have improved MCC patient outcomes by boosting antitumor T cell immunity. Here, we identify PD-1 as a growth-promoting receptor intrinsic to MCC cells. In human MCC lines and clinical tumors, RT-PCR-based sequencing, immunoblotting, flow cytometry, and immunofluorescence analyses demonstrated PD-1 gene and protein expression by MCC cells. MCC-PD-1 ligation enhanced, and its inhibition or silencing suppressed, in vitro proliferation and in vivo tumor xenograft growth. Consistently, MCC-PD-1 binding to PD-L1 or PD-L2 induced, while antibody-mediated PD-1 blockade inhibited, protumorigenic mTOR signaling, mitochondrial (mt) respiration, and ROS generation. Last, pharmacologic inhibition of mTOR or mtROS reversed MCC-PD-1:PD-L1-dependent proliferation and synergized with PD-1 checkpoint blockade in suppressing tumorigenesis. Our results identify an MCC-PD-1-mTOR-mtROS axis as a tumor growth-accelerating mechanism, the blockade of which might contribute to clinical response in patients with MCC.

PRMT1 Inhibition Selectively Targets BNC1-Dependent Proliferation, but not Migration in Squamous Cell Carcinoma.

Squamous Cell Carcinoma (SCC) develops in stratified epithelial tissues and demonstrates frequent alterations in transcriptional regulators. We sought to discover SCC-specific transcriptional programs and identified the transcription factor Basonuclin 1 (BNC1) as highly expressed in SCC compared to other tumor types. RNA-seq and ChIP-seq analysis identified pro-proliferative genes activated by BNC1 in SCC cells and keratinocytes. Inhibition of BNC1 in SCC cells suppressed proliferation and increased migration via FRA1. In contrast, BNC1 reduction in keratinocytes caused differentiation, which was abrogated by IRF6 knockdown, leading to increased migration. Protein interactome analysis identified PRMT1 as a co-activator of BNC1-dependent proliferative genes. Inhibition of PRMT1 resulted in a dose-dependent reduction in SCC cell proliferation without increasing migration. Importantly, therapeutic inhibition of PRMT1 in SCC xenografts significantly reduced tumor size, resembling functional effects of BNC1 knockdown. Together, we identify BNC1-PRMT1 as an SCC-lineage specific transcriptional axis that promotes cancer growth, which can be therapeutically targeted to inhibit SCC tumorigenesis.

Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-glycans without direct incorporation.

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLe(X)), and related lectin ligands on effector leukocytes. Based on anti-sLe(X) antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLe(X) formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLe(X) (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLe(X) structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLe(X) on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.

Development of a nascent galectin-1 chimeric molecule for studying the role of leukocyte galectin-1 ligands and immune disease modulation.

Galectin-1 (Gal-1), a ß-galactoside-binding lectin, plays a profound role in modulating adaptive immune responses by altering the phenotype and fate of T cells. Experimental data showing recombinant Gal-1 (rGal-1) efficacy on T cell viability and cytokine production, nevertheless, is controversial due to the necessity of using stabilizing chemicals to help retain Gal-1 structure and function. To address this drawback, we developed a mouse Gal-1 human Ig chimera (Gal-1hFc) that did not need chemical stabilization for Gal-1 ligand recognition, apoptosis induction, and cytokine modulation in a variety of leukocyte models. At high concentrations, Gal-1hFc induced apoptosis in Gal-1 ligand(+) Th1 and Th17 cells, leukemic cells, and granulocytes from synovial fluids of patients with rheumatoid arthritis. Importantly, at low, more physiologic concentrations, Gal-1hFc retained its homodimeric form without losing functionality. Not only did Gal-1hFc-binding trigger IL-10 and Th2 cytokine expression in activated T cells, but members of the CD28 family and several other immunomodulatory molecules were upregulated. In a mouse model of contact hypersensitivity, we found that a non-Fc receptor-binding isoform of Gal-1hFc, Gal-1hFc2, alleviated T cell-dependent inflammation by increasing IL-4(+), IL-10(+), TGF-ß(+), and CD25(high)/FoxP3(+) T cells, and by decreasing IFN-γ(+) and IL-17(+) T cells. Moreover, in human skin-resident T cell cultures, Gal-1hFc diminished IL-17(+) T cells and increased IL-4(+) and IL-10(+) T cells. Gal-1hFc will not only be a useful new tool for investigating the role of Gal-1 ligands in leukocyte death and cytokine stimulation, but for studying how Gal-1-Gal-1 ligand binding shapes the intensity of immune responses.

Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking.

How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe(X)) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe(X) and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that alpha1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLe(X) and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that alpha1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism.

Distinct antibody clones detect PD-1 checkpoint expression and block PD-L1 interactions on live murine melanoma cells.

Monoclonal antibodies (abs) targeting the programmed cell death 1 (PD-1) immune checkpoint pathway have revolutionized tumor therapy. Because T-cell-directed PD-1 blockade boosts tumor immunity, anti-PD-1 abs have been developed for examining T-cell-PD-1 functions. More recently, PD-1 expression has also been reported directly on cancer cells of various etiology, including in melanoma. Nevertheless, there is a paucity of studies validating anti-PD-1 ab clone utility in specific assay types for characterizing tumor cell-intrinsic PD-1. Here, we demonstrate reactivity of several anti-murine PD-1 ab clones and recombinant PD-L1 with live B16-F10 melanoma cells and YUMM lines using multiple independent methodologies, positive and negative PD-1-specific controls, including PD-1-overexpressing and PD-1 knockout cells. Flow cytometric analyses with two separate anti-PD-1 ab clones, 29F.1A12 and RMP1-30, revealed PD-1 surface protein expression on live murine melanoma cells, which was corroborated by marked enrichment in PD-1 gene (Pdcd1) expression. Immunoblotting, immunoprecipitation, and mass spectrometric sequencing confirmed PD-1 protein expression by B16-F10 cells. Recombinant PD-L1 also recognized melanoma cell-expressed PD-1, the blockade of which by 29F.1A12 fully abrogated PD-1:PD-L1 binding. Together, our data provides multiple lines of evidence establishing PD-1 expression by live murine melanoma cells and validates ab clones and assay systems for tumor cell-directed PD-1 pathway investigations.

Inhibition of Melanoma Cell-Intrinsic Tim-3 Stimulates MAPK-Dependent Tumorigenesis.

T-cell immunoglobulin mucin family member 3 (Tim-3) is an immune checkpoint receptor that dampens effector functions and causes terminal exhaustion of cytotoxic T cells. Tim-3 inhibitors are under investigation in immuno-oncology (IO) trials, because blockade of T-cell-Tim-3 enhances antitumor immunity. Here, we identify an additional role for Tim-3 as a growth-suppressive receptor intrinsic to melanoma cells. Inhibition of melanoma cell-Tim-3 promoted tumor growth in both immunocompetent and immunocompromised mice, while melanoma-specific Tim-3 overexpression attenuated tumorigenesis. Ab-mediated Tim-3 blockade inhibited growth of immunogenic murine melanomas in T-cell-competent hosts, consistent with established antitumor effects of T-cell-Tim-3 inhibition. In contrast, Tim-3 Ab administration stimulated tumorigenesis of both highly and lesser immunogenic murine and human melanomas in T-cell-deficient mice, confirming growth-promoting effects of melanoma-Tim-3 antagonism. Melanoma-Tim-3 activation suppressed, while its blockade enhanced, phosphorylation of pro-proliferative downstream MAPK signaling mediators. Finally, pharmacologic MAPK inhibition reversed unwanted Tim-3 Ab-mediated tumorigenesis in T-cell-deficient mice and enhanced desired antitumor activity of Tim-3 interference in T-cell-competent hosts. These results identify melanoma-Tim-3 blockade as a mechanism that antagonizes T-cell-Tim-3-directed IO therapeutic efficacy. They further reveal MAPK targeting as a combination strategy for circumventing adverse consequences of unintended melanoma-Tim-3 inhibition. SIGNIFICANCE: Tim-3 is a growth-suppressive receptor intrinsic to melanoma cells, the blockade of which promotes MAPK-dependent tumorigenesis and thus counteracts antitumor activity of T-cell-directed Tim-3 inhibition.

Multiomics Analysis and Systems Biology Integration Identifies the Roles of IL-9 in Keratinocyte Metabolic Reprogramming.

IL-9‒producing T cells are present in healthy skin as well as in the cutaneous lesions of inflammatory diseases and cancers. However, the roles of IL-9 in human skin during homeostasis and in the pathogenesis of inflammatory disorders remain obscure. In this study, we examined the roles of IL-9 in metabolic reprogramming of human primary keratinocytes (KCs). High-throughput quantitative proteomics revealed that IL-9 signaling in human primary KCs disrupts the electron transport chain by downregulating multiple electron transport chain proteins. Nuclear magnetic resonance-based metabolomics showed that IL-9 also reduced the production of tricarboxylic acid cycle intermediates in human primary KCs. An integration of multiomics data with systems-level analysis using the constraint-based MitoCore model predicted marked IL-9-dependent effects on central carbohydrate metabolism, particularly in relation to the glycolytic switch. Stable isotope metabolomics and biochemical assays confirmed increased glucose consumption and redirection of metabolic flux toward lactate by IL-9. Functionally, IL-9 inhibited ROS production by IFN-γ and promoted human primary KC survival by inhibiting apoptosis. In conclusion, our data reveal IL-9 as a master regulator of KC metabolic reprogramming and survival.

Roles of integrin activation in eosinophil function and the eosinophilic inflammation of asthma.

Eosinophilic inflammation is a characteristic feature of asthma. Integrins are highly versatile cellular receptors that regulate extravasation of eosinophils from the postcapillary segment of the bronchial circulation to the airway wall and airspace. Such movement into the asthmatic lung is described as a sequential, multistep paradigm, whereby integrins on circulating eosinophils become activated, eosinophils tether in flow and roll on bronchial endothelial cells, integrins on rolling eosinophils become further activated as a result of exposure to cytokines, eosinophils arrest firmly to adhesive ligands on activated endothelium, and eosinophils transmigrate to the airway in response to chemoattractants. Eosinophils express seven integrin heterodimeric adhesion molecules: alpha 4 beta 1 (CD49d/29), alpha 6 beta 1 (CD49f/29), alpha M beta 2 (CD11b/18), alpha L beta 2 (CD11a/18), alpha X beta 2 (CD11c/18), alpha D beta2 (CD11d/18), and alpha 4 beta 7 (CD49d/beta 7). The role of these integrins in eosinophil recruitment has been elucidated by major advances in the understanding of integrin structure, integrin function, and modulators of integrins. Such findings have been facilitated by cellular experiments of eosinophils in vitro, studies of allergic asthma in humans and animal models in vivo, and crystal structures of integrins. Here, we elaborate on how integrins cooperate to mediate eosinophil movement to the asthmatic airway. Antagonists that target integrins represent potentially promising therapies in the treatment of asthma.

Analysis of glycosyltransferase expression in metastatic prostate cancer cells capable of rolling activity on microvascular endothelial (E)-selectin.

Prostate cancer (PCa) cell tethering and rolling on microvascular endothelium has been proposed to promote the extravasation of PCa cells. We have shown that these adhesive events are mediated through binding interactions between endothelial (E)-selectin and Lewis carbohydrates on PCa cells. Prior data indicate that E-selectin-mediated rolling of bone-metastatic PCa MDA PCa 2b (MDA) cells is dependent on sialyl Lewis X (sLe(X))-bearing glycoproteins. To explore the molecular basis of sLe(X) synthesis and E-selectin ligand (ESL) activity on PCa cells, we compared and contrasted the expression level of glycosyltransferases, characteristically involved in sLe(X) and ESL synthesis, in ESL(+) MDA cells among other ESL(-) metastatic PCa cell lines. We also created and examined ESL(hi) and ESL(lo) variants of MDA cells to provide a direct comparison of the glycosyltransferase expression level. We found that normal prostate tissue and all metastatic PCa cell lines expressed glycosyltransferases required for sialo-lactosamine synthesis, including N-acetylglucosaminyl-, galactosyl-, and sialyltransferases. However, compared with expression in normal prostate tissue, ESL(+) MDA cells expressed a 31- and 10-fold higher level of alpha1,3 fucosyltransferases (FT) 3 and 6, respectively. Moreover, FT3 and FT6 were expressed at 2- to 354-fold lower levels in ESL(-) PCa cell lines. Consistent with these findings, ESL(hi) MDA cells expressed a 131- and 51-fold higher level of FT3 and FT6, respectively, compared with expression in ESL(lo) MDA cells. We also noted that alpha1,3 FT7 was expressed at a 5-fold greater level in ESL(hi) MDA cells. Furthermore, ESL(lo) MDA cells did not display sLe(X) on glycoproteins capable of bearing sLe(X), notably P-selectin glycoprotein ligand-1. These results implicate the importance of alpha1,3 FT3, FT6, and/or FT7 in sLe(X) and ESL synthesis on metastatic PCa cells.

Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans.

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-ß1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.

Cleavage of human 7-domain VCAM-1 (CD106) by thrombin.

Vascular cell adhesion molecule 1 (VCAM-1, CD106) is expressed as a type I transmembrane integrin counter-receptor on activated endothelium and mediates white blood cell attachment. The alternatively spliced 7-domain (7d) form of VCAM-1 contains a potential thrombin cleavage site. Thrombin proteolysis of 7d-VCAM-1 may help regulate adhesive activity ofVCAM-1. We determined whether 7d-VCAM-1 is proteolyzed and rendered inactive by thrombin. Recombinant extracellular domain of 7d-VCAM-1 was cleaved by thrombin to generate 33- and 44-kDa products. Cleavage was in the sequence PGPR/IAAQIG near the N-terminal border of the alternatively spliced fourth immunoglobulin (Ig)-like module. There was no cleavage of 6d-VCAM-1 lacking the fourth module. Expression of full-length 7d-VCAM-1 presented on Chinese hamster ovary (CHO) monolayers, as detected by flow cytometry with an antibody directed to Ig-like modules 1-3, was reduced by thrombin treatment whereas there was no reduction in the expression of full-length 6d-VCAM-1. Adhesion of blood eosinophils to full-length 7d-VCAM-1 was reduced after treatment of CHO cells with thrombin, whereas adhesion to full-length 6d-VCAM-1 was not affected. We conclude that cleavage of 7d-VCAM-1 by thrombin is a potential mechanism for differential regulation of VCAM-1 splice forms in white blood cell adhesion and trafficking.