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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

ABCB5 Identifies Immunoregulatory Dermal Cells.

Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly defined immunomodulatory cell populations poses a barrier to this field. Here, we show that ATP-binding cassette member B5 (ABCB5) identifies dermal immunoregulatory cells (DIRCs) capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1). Purified Abcb5(+) DIRCs suppressed T cell proliferation, evaded immune rejection, homed to recipient immune tissues, and induced Tregs in vivo. In fully major-histocompatibility-complex-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5(+) DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy.

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.

Melanoma Cell Galectin-1 Ligands Functionally Correlate with Malignant Potential.

Galectin-1 (Gal-1)-binding to Gal-1 ligands on immune and endothelial cells can influence melanoma development through dampening antitumor immune responses and promoting angiogenesis. However, whether Gal-1 ligands are functionally expressed on melanoma cells to help control intrinsic malignant features remains poorly understood. Here, we analyzed expression, identity, and function of Gal-1 ligands in melanoma progression. Immunofluorescent analysis of benign and malignant human melanocytic neoplasms revealed that Gal-1 ligands were abundant in severely dysplastic nevi, as well as in primary and metastatic melanomas. Biochemical assessments indicated that melanoma cell adhesion molecule (MCAM) was a major Gal-1 ligand on melanoma cells that was largely dependent on its N-glycans. Other melanoma cell Gal-1 ligand activity conferred by O-glycans was negatively regulated by α2,6 sialyltransferase ST6GalNAc2. In Gal-1-deficient mice, MCAM-silenced (MCAM(KD)) or ST6GalNAc2-overexpressing (ST6(O/E)) melanoma cells exhibited slower growth rates, underscoring a key role for melanoma cell Gal-1 ligands and host Gal-1 in melanoma growth. Further analysis of MCAM(KD) or ST6(O/E) melanoma cells in cell migration assays indicated that Gal-1 ligand-dependent melanoma cell migration was severely inhibited. These findings provide a refined perspective on Gal-1/melanoma cell Gal-1 ligand interactions as contributors to melanoma malignancy.

Isolation and characterization of chimeric human Fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow.

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

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.

Definition of molecular determinants of prostate cancer cell bone extravasation.

Advanced prostate cancer commonly metastasizes to bone, but transit of malignant cells across the bone marrow endothelium (BMEC) remains a poorly understood step in metastasis. Prostate cancer cells roll on E-selectin(+) BMEC through E-selectin ligand-binding interactions under shear flow, and prostate cancer cells exhibit firm adhesion to BMEC via ß1, ß4, and αVß3 integrins in static assays. However, whether these discrete prostate cancer cell-BMEC adhesive contacts culminate in cooperative, step-wise transendothelial migration into bone is not known. Here, we describe how metastatic prostate cancer cells breach BMEC monolayers in a step-wise fashion under physiologic hemodynamic flow. Prostate cancer cells tethered and rolled on BMEC and then firmly adhered to and traversed BMEC via sequential dependence on E-selectin ligands and ß1 and αVß3 integrins. Expression analysis in human metastatic prostate cancer tissue revealed that ß1 was markedly upregulated compared with expression of other ß subunits. Prostate cancer cell breaching was regulated by Rac1 and Rap1 GTPases and, notably, did not require exogenous chemokines as ß1, αVß3, Rac1, and Rap1 were constitutively active. In homing studies, prostate cancer cell trafficking to murine femurs was dependent on E-selectin ligand, ß1 integrin, and Rac1. Moreover, eliminating E-selectin ligand-synthesizing α1,3 fucosyltransferases in transgenic adenoma of mouse prostate mice dramatically reduced prostate cancer incidence. These results unify the requirement for E-selectin ligands, α1,3 fucosyltransferases, ß1 and αVß3 integrins, and Rac/Rap1 GTPases in mediating prostate cancer cell homing and entry into bone and offer new insight into the role of α1,3 fucosylation in prostate cancer development.

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.

Metabolic inhibition of galectin-1-binding carbohydrates accentuates antitumor immunity.

Galectin-1 (Gal-1) has been shown to play a major role in tumor immune escape by inducing apoptosis of effector leukocytes and correlating with tumor aggressiveness and disease progression. Thus, targeting the Gal-1/Gal-1 ligand axis represents a promising cancer therapeutic approach. Here, to test the Gal-1-mediated tumor immune evasion hypothesis and demonstrate the importance of Gal-1-binding N-acetyllactosamines in controlling the fate and function of antitumor immune cells, we treated melanoma- or lymphoma-bearing mice with peracetylated 4-fluoro-glucosamine (4-F-GlcNAc), a metabolic inhibitor of N-acetyllactosamine biosynthesis, and analyzed tumor growth and immune profiles. We found that 4-F-GlcNAc spared Gal-1-mediated apoptosis of T cells and natural killer (NK) cells by decreasing their expression of Gal-1-binding determinants. 4-F-GlcNAc enhanced tumor lymphocytic infiltration and promoted elevations in tumor-specific cytotoxic T cells and IFN-γ levels, while lowering IL-10 production. Collectively, our data suggest that metabolic lowering of Gal-1-binding N-acetyllactosamines may attenuate tumor growth by boosting antitumor immune cell levels, representing a promising approach for cancer immunotherapy.

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.