Targeting galectin-1 inhibits pancreatic cancer progression by modulating tumor-stroma crosstalk.

Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal tumor types, with extremely low survival rates due to late diagnosis and resistance to standard therapies. A more comprehensive understanding of the complexity of PDA pathobiology, and especially of the role of the tumor microenvironment in disease progression, should pave the way for therapies to improve patient response rates. In this study, we identify galectin-1 (Gal1), a glycan-binding protein that is highly overexpressed in PDA stroma, as a major driver of pancreatic cancer progression. Genetic deletion of Gal1 in a Kras-driven mouse model of PDA (Ela-KrasG12Vp53-/- ) results in a significant increase in survival through mechanisms involving decreased stroma activation, attenuated vascularization, and enhanced T cell infiltration leading to diminished metastasis rates. In a human setting, human pancreatic stellate cells (HPSCs) promote cancer proliferation, migration, and invasion via Gal1-driven pathways. Moreover, in vivo orthotopic coinjection of pancreatic tumor cells with Gal1-depleted HPSCs leads to impaired tumor formation and metastasis in mice. Gene-expression analyses of pancreatic tumor cells exposed to Gal1 reveal modulation of multiple regulatory pathways involved in tumor progression. Thus, Gal1 hierarchically regulates different events implicated in PDA biology including tumor cell proliferation, invasion, angiogenesis, inflammation, and metastasis, highlighting the broad therapeutic potential of Gal1-specific inhibitors, either alone or in combination with other therapeutic modalities.

Galectin 3 Deficiency Alters Chondrocyte Primary Cilium Formation and Exacerbates Cartilage Destruction via Mitochondrial Apoptosis.

Mechanical overload and aging are the main risk factors of osteoarthritis (OA). Galectin 3 (GAL3) is important in the formation of primary cilia, organelles that are able to sense mechanical stress. The objectives were to evaluate the role of GAL3 in chondrocyte primary cilium formation and in OA in mice. Chondrocyte primary cilium was detected in vitro by confocal microscopy. OA was induced by aging and partial meniscectomy of wild-type (WT) and Gal3-null 129SvEV mice (Gal3-/-). Primary chondrocytes were isolated from joints of new-born mice. Chondrocyte apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), caspase 3 activity and cytochrome c release. Gene expression was assessed by qRT-PCR. GAL3 was localized at the basal body of the chondrocyte primary cilium. Primary cilia of Gal3-/- chondrocytes were frequently abnormal and misshapen. Deletion of Gal3 triggered premature OA during aging and exacerbated joint instability-induced OA. In both aging and surgery-induced OA cartilage, levels of chondrocyte catabolism and hypertrophy markers and apoptosis were more severe in Gal3-/- than WT samples. In vitro, Gal3 knockout favored chondrocyte apoptosis via the mitochondrial pathway. GAL3 is a key regulator of cartilage homeostasis and chondrocyte primary cilium formation in mice. Gal3 deletion promotes OA development.

Lactosamine-Based Derivatives as Tools to Delineate the Biological Functions of Galectins: Application to Skin Tissue Repair.

Galectins have been recognized as potential novel therapeutic targets for the numerous fundamental biological processes in which they are involved. Galectins are key players in homeostasis, and as such their expression and function are finely tuned in vivo. Thus, their modes of action are complex and remain largely unexplored, partly because of the lack of dedicated tools. We thus designed galectin inhibitors from a lactosamine core, functionalized at key C2 and C3' positions by aromatic substituents to ensure both high affinity and selectivity, and equipped with a spacer that can be modified on demand to further modulate their physico-chemical properties. As a proof-of-concept, galectin-3 was selectively targeted. The efficacy of the synthesized di-aromatic lactosamine tools was shown in cellular assays to modulate collective epithelial cell migration and to interfere with actin/cortactin localization.

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin-3 Inhibitor.

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent-field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3'-mono- and 2,3'-diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin-3 for 2'-arylamido derivatives over ureas, thioureas, and amines and that of galectin-7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin-3 versus galectin-1 and galectin-7.

The Parkinsonism-associated protein DJ-1/Park7 prevents glycation damage in human keratinocyte.

Reducing sugars and dicarbonyls form covalent adducts with proteins through a nonenzymatic process known as glycation, which inactivates proteins, is increased in diabetic patients and is associated with diabetic complications, including retinopathy, cataracts, nephropathy, neuropathy, cardiomyopathy and skin defects. We recently characterized DJ-1/Park7 as a protein deglycase that repairs proteins from glycation by glyoxal and methylglyoxal, two major glycating agents which are responsible for up to 65% of glycation events. In this study, we investigated the ability of DJ-1 to prevent protein glycation in keratinocytes. Glycation of collagen and keratinocyte proteins was tested by measuring ultraviolet absorption and fluorescence emission. Protein glycation in HaCaT keratinocytes was investigated by immunodetection with anti-advanced glycation endproduct antibodies, after DJ-1 depletion or overexpression. In vitro, DJ-1 prevented glycation of collagen and keratinocyte protein extracts. In cell culture, DJ-1 depletion by small interfering RNAs resulted in a 3-fold increase in protein glycation levels. Moreover, protein glycation levels were decreased several-fold in cells overexpressing DJ-1 after addition of the Nrf2 inducer sulforaphane or after transfection with a DJ-1 plasmid. Thus, the DJ-1 deglycase plays a major role in preventing protein glycation in eukaryotic cells and might be important for preventing skin glycation.

Astrocyte reactivity after brain injury-: The role of galectins 1 and 3.

Astrocytes react to brain injury in a heterogeneous manner with only a subset resuming proliferation and acquiring stem cell properties in vitro. In order to identify novel regulators of this subset, we performed genomewide expression analysis of reactive astrocytes isolated 5 days after stab wound injury from the gray matter of adult mouse cerebral cortex. The expression pattern was compared with astrocytes from intact cortex and adult neural stem cells (NSCs) isolated from the subependymal zone (SEZ). These comparisons revealed a set of genes expressed at higher levels in both endogenous NSCs and reactive astrocytes, including two lectins-Galectins 1 and 3. These results and the pattern of Galectin expression in the lesioned brain led us to examine the functional significance of these lectins in brains of mice lacking Galectins 1 and 3. Following stab wound injury, astrocyte reactivity including glial fibrillary acidic protein expression, proliferation and neurosphere-forming capacity were found significantly reduced in mutant animals. This phenotype could be recapitulated in vitro and was fully rescued by addition of Galectin 3, but not of Galectin 1. Thus, Galectins 1 and 3 play key roles in regulating the proliferative and NSC potential of a subset of reactive astrocytes.

[Galectins, a class of unconventional lectins]. / Les galectines - Des lectines pas comme les autres.

Galectins constitute a family of soluble animal lectins defined by their evolutionary conserved carbohydrate recognition domain and their affinity for ß-galactosides containing glycoconjugates. Each galectin is characterized by a specific spatio-temporal distribution and a unique set of ligands and molecular partners. Interestingly, galectins are found both extracellularly and intracellularly and modulate various cellular processes. Knock-out mutant mice for galectins-1, 3 or 7 are viable but display a wide range of defects under various stress conditions. Indeed, galectins are multifunctional proteins involved in cell-cell and cell-extracellular matrix interactions, organization of membrane domains, cell signalling and also in intracellular trafficking, apoptosis, regulation of cell cycle. Galectins represent potential therapeutic targets, especially in the context of cancer and inflammatory diseases.

Blocked angiogenesis in Galectin-3 null mice does not alter cellular and behavioral recovery after middle cerebral artery occlusion stroke.

Angiogenesis is thought to decrease stroke size and improve behavioral outcomes and therefore several clinical trials are seeking to augment it. Galectin-3 (Gal-3) expression increases after middle cerebral artery occlusion (MCAO) and has been proposed to limit damage 3days after stroke. We carried out mild MCAO that damages the striatum but spares the cerebral cortex and SVZ. Gal-3 gene deletion prevented vascular endothelial growth factor (VEGF) upregulation after MCAO. This inhibited post-MCAO increases in endothelial proliferation and angiogenesis in the striatum allowing us to uniquely address the function of angiogenesis in this model of stroke. Apoptosis and infarct size were unchanged in Gal-3(-/-) mice 7 and 14 days after MCAO, suggesting that angiogenesis does not affect lesion size. Microglial and astrocyte activation/proliferation after MCAO was similar in wild type and Gal-3(-/-) mice. In addition, openfield activity, motor hemiparesis, proprioception, reflex, tremors and grooming behaviors were essentially identical between WT and Gal-3(-/-) mice at 1, 3, 7, 10 and 14 days after MCAO, suggesting that penumbral angiogenesis has limited impact on behavioral recovery. In addition to angiogenesis, increased adult subventricular zone (SVZ) neurogenesis is thought to provide neuroprotection after stroke in animal models. SVZ neurogenesis and migration to lesion were overall unaffected by the loss of Gal-3, suggesting no compensation for the lack of angiogenesis in Gal-3(-/-) mice. Because angiogenesis and neurogenesis are usually coordinately regulated, identifying their individual effects on stroke has hitherto been difficult. These results show that Gal-3 is necessary for angiogenesis in stroke in a VEGF-dependant manner, but suggest that angiogenesis may be dispensable for post-stroke endogenous repair, therefore drawing into question the clinical utility of augmenting angiogenesis.

Glioma-derived galectin-1 regulates innate and adaptive antitumor immunity.

Galectin-1 is a glycan-binding protein, which is involved in the aggressiveness of glioblastoma (GBM) in part by stimulating angiogenesis. In different cancer models, galectin-1 has also been demonstrated to play a pivotal role in tumor-mediated immune evasion especially by modulating cells of the adaptive immune system. It is yet unknown whether the absence or presence of galectin-1 within the glioma microenvironment also causes qualitative or quantitative differences in innate and/or adaptive antitumor immune responses. All experiments were performed in the orthotopic GL261 mouse high-grade glioma model. Stable galectin-1 knockdown was achieved via transduction of parental GL261 tumor cells with a lentiviral vector encoding a galectin-1-targeting miRNA. We demonstrated that the absence of tumor-derived but not of host-derived galectin-1 significantly prolonged the survival of glioma-bearing mice as such and in combination with dendritic cell (DC)-based immunotherapy. Both flow cytometric and pathological analysis revealed that the silencing of glioma-derived galectin-1 significantly decreased the amount of brain-infiltrating macrophages and myeloid-derived suppressor cells (MDSC) in tumor-bearing mice. Additionally, we revealed a pro-angiogenic role for galectin-1 within the glioma microenvironment. The data provided in this study reveal a pivotal role for glioma-derived galectin-1 in the regulation of myeloid cell accumulation within the glioma microenvironment, the most abundant immune cell population in high-grade gliomas. Furthermore, the prolonged survival observed in untreated and DC-vaccinated glioma-bearing mice upon the silencing of tumor-derived galectin-1 strongly suggest that the in vivo targeting of tumor-derived galectin-1 might offer a promising and realistic adjuvant treatment modality in patients diagnosed with GBM.

Genetic characterization of congenital tufting enteropathy: epcam associated phenotype and involvement of SPINT2 in the syndromic form.

Congenital tufting enteropathy (CTE) is a rare and severe enteropathy recently ascribed to mutations in the epcam gene. Here we establish SPINT2, previously ascribed to congenital sodium diarrhea, as a second gene associated with CTE and report molecular and immunohistochemistry data in 57 CTE patients. Inclusion criteria were early onset diarrhea and intestinal insufficiency with the typical histological CTE abnormalities. The clinical phenotype was registered, the entire coding regions of epcam and SPINT2 sequenced, and immunostaining of EpCAM and SPINT2 performed on intestinal biopsies. An epcam mutation was involved in 41 patients (73 %) who mainly displayed isolated digestive symptoms. Mutations severely affected gene expression since the EpCAM signal on intestinal tissues was either undetectable or low and irregular. Twelve other patients (21 %) carried mutations in SPINT2, and were phenotypically characterized by systematic association with keratitis (p < 10(-4)) and, for half of them, with choanal atresia (p < 10(-4)). Dependency on parenteral nutrition (PN) was comparable in patients with epcam or SPINT2 mutations, but the frequent epcam mutation c.556-14A>G (abnormal splicing) was significantly associated with a better outcome (p = 0.032) with milder PN dependency to weaning in some cases. Finally, four patients (7 %) with isolated digestive symptoms had no detectable epcam or SPINT2 mutation. Two candidate genes, Elf3 and Claudin7, were excluded from this population. Our study allows us to separate CTE patients into at least three genetic classes, each with specific phenotypes. The genetics approach raises the question of the distinction between two congenital enteropathies. Our findings should help improve the diagnosis of CTE, guide toward strategies of long-term PN management, and limit indications for intestinal transplantation to life-threatening PN complications.

Interstrain differences in chronic hepatitis and tumor development in a murine model of inflammation-mediated hepatocarcinogenesis.

UNLABELLED: Chronic inflammation is strongly associated with an increased risk for hepatocellular carcinoma (HCC) development. The multidrug resistance 2 (Mdr2)-knockout (KO) mouse (adenosine triphosphate-binding cassette b4(-/-) ), a model of inflammation-mediated HCC, develops chronic cholestatic hepatitis at an early age and HCC at an adult age. To delineate factors contributing to hepatocarcinogenesis, we compared the severity of early chronic hepatitis and late HCC development in two Mdr2-KO strains: Friend virus B-type/N (FVB) and C57 black 6 (B6). We demonstrated that hepatocarcinogenesis was significantly less efficient in the Mdr2-KO/B6 mice versus the Mdr2-KO/FVB mice; this difference was more prominent in males. Chronic hepatitis in the Mdr2-KO/B6 males was more severe at 1 month of age but was less severe at 3 months of age in comparison with age-matched Mdr2-KO/FVB males. A comparative genome-scale gene expression analysis of male livers of both strains at 3 months of age revealed both common and strain-specific aberrantly expressed genes, including genes associated with the regulation of inflammation, the response to oxidative stress, and lipid metabolism. One of these regulators, galectin-1 (Gal-1), possesses both anti-inflammatory and protumorigenic activities. To study its regulatory role in the liver, we transferred the Gal-1-KO mutation (lectin galactoside-binding soluble 1(-/-) ) from the B6 strain to the FVB strain, and we demonstrated that endogenous Gal-1 protected the liver against concanavalin A-induced hepatitis with the B6 genetic background but not the FVB genetic background. CONCLUSION: Decreased chronic hepatitis in Mdr2-KO/B6 mice at the age of 3 months correlated with a significant retardation of liver tumor development in this strain versus the Mdr2-KO/FVB strain. We found candidate factors that may determine strain-specific differences in the course of chronic hepatitis and HCC development in the Mdr2-KO model, including inefficient anti-inflammatory activity of the endogenous lectin Gal-1 in the FVB strain.

Galectin-3 mediates aldosterone-induced vascular fibrosis.

OBJECTIVE: Aldosterone (Aldo) is involved in arterial stiffness and heart failure, but the mechanisms have remained unclear. Galectin-3 (Gal-3), a ß-galactoside-binding lectin, plays an important role in inflammation, fibrosis, and heart failure. We investigated here whether Gal-3 is involved in Aldo-induced vascular fibrosis. METHODS AND RESULTS: In rat vascular smooth muscle cells Gal-3 overexpression enhanced specifically collagen type I synthesis. Moreover Gal-3 inhibition by modified citrus pectin or small interfering RNA blocked Aldo-induced collagen type I synthesis. Rats were treated with Aldo-salt combined with spironolactone or modified citrus pectin for 3 weeks. Hypertensive Aldo-treated rats presented vascular hypertrophy, inflammation, fibrosis, and increased aortic Gal-3 expression. Spironolactone or modified citrus pectin treatment reversed all the above effects. Wild-type and Gal-3 knock-out mice were treated with Aldo for 6 hours or 3 weeks. Aldo increased aortic Gal-3 expression, inflammation, and collagen type I in wild-type mice at both the short- and the long-term, whereas no changes occurred in Gal-3 knock-out mice. CONCLUSIONS: Our data indicate that Gal-3 is required for inflammatory and fibrotic responses to Aldo in vascular smooth muscle cells in vitro and in vivo, suggesting a key role for Gal-3 in vascular fibrosis.

Seizure-induced neuronal death is suppressed in the absence of the endogenous lectin Galectin-1.

Pilocarpine injection induces epileptic seizures in rodents, an experimental paradigm extensively used to model temporal lobe epilepsy in humans. It includes conspicuous neuronal death in the forebrain and previous work has demonstrated an involvement of the neurotrophin receptor p75(NTR) in this process. Following the identification of Galectin-1 (Gal-1) as a downstream effector of p75(NTR), we examine here the role of this endogenous lectin in pilocarpine-induced cell death in adult mice. We found that most somatostatin-positive neurons also express Gal-1 and that in mice lacking the corresponding gene Lgals1, pilocarpine-induced neuronal death was essentially abolished in the forebrain. We also found that the related lectin Galectin-3 (Gal-3) was strongly upregulated by pilocarpine in microglial cells. This upregulation was absent in Lgals1 mutants and our results with Lgals3-null animals show that Gal-3 is not required for neuronal death in the hippocampus. These findings provide new insights into the roles and regulation of endogenous lectins in the adult CNS and a surprisingly selective proapoptotic role of Gal-1 for a subpopulation of GABAergic interneurons.

Galectin-3 maintains cell motility from the subventricular zone to the olfactory bulb.

The adult brain subventricular zone (SVZ) produces neuroblasts that migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) in a specialized niche. Galectin-3 (Gal-3) regulates proliferation and migration in cancer and is expressed by activated macrophages after brain injury. The function of Gal-3 in the normal brain is unknown, but we serendipitously found that it was expressed by ependymal cells and SVZ astrocytes in uninjured mice. Ependymal cilia establish chemotactic gradients and astrocytes form glial tubes, which combine to aid neuroblast migration. Whole-mount preparations and electron microscopy revealed that both ependymal cilia and SVZ astrocytes were disrupted in Gal3(-/-) mice. Interestingly, far fewer new BrdU(+) neurons were found in the OB of Gal3(-/-) mice, than in wild-type mice 2 weeks after labeling. However, SVZ proliferation and cell death, as well as OB differentiation rates were unaltered. This suggested that decreased migration in vivo was sufficient to decrease the number of new OB neurons. Two-photon time-lapse microscopy in forebrain slices confirmed decreased migration; cells were slower and more exploratory in Gal3(-/-) mice. Gal-3 blocking antibodies decreased migration and dissociated neuroblast cell-cell contacts, whereas recombinant Gal-3 increased migration from explants. Finally, we showed that expression of phosphorylated epidermal growth factor receptor (EGFR) was increased in Gal3(-/-) mice. These results suggest that Gal-3 is important in SVZ neuroblast migration, possibly through an EGFR-based mechanism, and reveals a role for this lectin in the uninjured brain.

Binding of galectin-1 to αIIbß3 integrin triggers "outside-in" signals, stimulates platelet activation, and controls primary hemostasis.

Understanding noncanonical mechanisms of platelet activation represents an important challenge for the identification of novel therapeutic targets in bleeding disorders, thrombosis, and cancer. We previously reported that galectin-1 (Gal-1), a ß-galactoside-binding protein, triggers platelet activation in vitro. Here we investigated the molecular mechanisms underlying this function and the physiological relevance of endogenous Gal-1 in hemostasis. Mass spectrometry analysis, as well as studies using blocking antibodies against the anti-α(IIb) subunit ofα(IIb)ß(3) integrin or platelets from patients with Glanzmann's thrombasthenia syndrome (α(IIb)ß(3) deficiency), identified this integrin as a functional Gal-1 receptor in platelets. Binding of Gal-1 to platelets triggered the phosphorylation of ß(3)-integrin, Syk, MAPKs, PI3K, PLCγ2, thromboxane (TXA(2)) release, and Ca(2+) mobilization. Not only soluble but also immobilized Gal-1 promoted platelet activation. Gal-1-deficient (Lgals1(-/-)) mice showed increased bleeding time (P<0.0002, knockout vs. wild type), which was not associated with an abnormal platelet count. Lgals1(-/-) platelets exhibited normal aggregation to PAR4, ADP, arachidonic acid, or collagen but abnormal ATP release at low collagen concentrations. Impaired spreading on fibrinogen and clot retraction with normal levels of α(IIb)ß(3) was also observed in Lgals1(-/-) platelets, indicating a failure in the "outside-in" signaling through this integrin. This study identifies a noncanonical mechanism, based on galectin-integrin interactions, for regulating platelet activation.

Galectin-7 reprograms skin carcinogenesis by fostering innate immune evasive programs.

Non-melanoma skin cancer (NMSC) has risen dramatically as a result of chronic exposure to sunlight ultraviolet (UV) radiation, climatic changes and clinical conditions associated with immunosuppression. In spite of considerable progress, our understanding of the mechanisms that control NMSC development and their associated molecular and immunological landscapes is still limited. Here we demonstrated a critical role for galectin-7 (Gal-7), a ß-galactoside-binding protein preferentially expressed in skin tissue, during NMSC development. Transgenic mice (Tg46) overexpressing Gal-7 in keratinocytes showed higher number of papillomas compared to WT mice or mice lacking Gal-7 (Lgals7-/-) when subjected to a skin carcinogenesis protocol, in which tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were sequentially administered. RNAseq analysis of Tg46 tumor lesions revealed a unique profile compatible with cells of the myelomonocytic lineage infiltrating these tumors, an effect that was substantiated by a higher number of CD11b+Gr1+ cells in tumor-draining lymph nodes. Heightened c-Met activation and Cxcl-1 expression in Tg46 lesions suggested a contribution of this pathway to the recruitment of these cells. Remarkably, Gal-7 bound to the surface of CD11b+Ly6ChiLy6Glo monocytic myeloid cells and enhanced their immunosuppressive activity, as evidenced by increased IL-10 and TGF-ß1 secretion, and higher T-cell inhibitory activity. In vivo, carcinogen-treated Lgals7-/- animals adoptively transferred with Gal-7-conditioned monocytic myeloid cells developed higher number of papillomas, whereas depletion of these cells in Tg46-treated mice led to reduction in the number of tumors. Finally, human NMSC biopsies showed increased LGALS7 mRNA and Gal-7 protein expression and displayed transcriptional profiles associated with myeloid programs, accompanied by elevated CXCL1 expression and c-Met activation. Thus, Gal-7 emerges as a critical mediator of skin carcinogenesis and a potential therapeutic target in human NMSC.

Binding of Toxoplasma gondii glycosylphosphatidylinositols to galectin-3 is required for their recognition by macrophages.

We showed that the production of tumor necrosis factor (TNF) α by macrophages in response to Toxoplasma gondii glycosylphosphatidylinositols (GPIs) requires the expression of both Toll-like receptors TLR2 and TLR4, but not of their co-receptor CD14. Galectin-3 is a ß-galactoside-binding protein with immune-regulatory effects, which associates with TLR2. We demonstrate here by using the surface plasmon resonance method that the GPIs of T. gondii bind to human galectin-3 with strong affinity and in a dose-dependent manner. The use of a synthetic glycan and of the lipid moiety cleaved from the GPIs shows that both parts are involved in the interaction with galectin-3. GPIs of T. gondii also bind to galectin-1 but with a lower affinity and only through the lipid moiety. At the cellular level, the production of TNF-α induced by T. gondii GPIs in macrophages depends on the expression of galectin-3 but not of galectin-1. This study is the first identification of a galectin-3 ligand of T. gondii origin, and galectin-3 might be a co-receptor presenting the GPIs to the TLRs on macrophages.

Impaired B-cell development at the pre-BII-cell stage in galectin-1-deficient mice due to inefficient pre-BII/stromal cell interactions.

Activation of the pre-B-cell receptor (pre-BCR) in the bone marrow depends on both tonic and ligand-induced signaling and leads to pre-BII-cell proliferation and differentiation. Using normal mouse bone marrow pre-BII cells, we demonstrate that the ligand-induced pre-BCR activation depends on pre-BCR/galectin-1/integrin interactions leading to pre-BCR clustering at the pre-BII/stromal cell synapse. In contrast, heparan sulfates, shown to be pre-BCR ligands in mice, are not implicated in pre-BCR relocalization. Inhibition of pre-BCR/galectin-1/integrin interactions has functional consequences, since pre-BII-cell proliferation and differentiation are impaired in an in vitro B-cell differentiation assay, without affecting cellular apoptosis. Most strikingly, although galectin-1-deficient mice do not show an apparent B-cell phenotype, the kinetics of de novo B-cell reconstitution after hydroxyurea treatment indicates a specific delay in pre-BII-cell recovery due to a decrease in pre-BII-cell differentiation and proliferation. Thus, although it remains possible that the pre-BCR interacts with other ligands, these results highlight the role played by the stromal cell-derived galectin-1 for the efficient development of normal pre-BII cells and suggest the existence of pre-BII-specific stromal cell niches in normal bone marrow.

Galectin-3, a marker for vacuole lysis by invasive pathogens.

Shigella bacteria invade macrophages and epithelial cells and following internalization lyse the phagosome and escape to the cytoplasm. Galectin-3, an abundant protein in macrophages and epithelial cells, belongs to a family of beta-galactoside-binding proteins, the galectins, with many proposed functions in immune response, development, differentiation, cancer and infection. Galectins are synthesized as cytosolic proteins and following non-classical secretion bind extracellular beta-galactosides. Here we analysed the localization of galectin-3 following entry of Shigella into the cytosol and detected a striking phenomenon. Very shortly after bacterial invasion, intracellular galectin-3 accumulated in structures in vicinity to internalized bacteria. By using immuno-electron microscopy analysis we identified galectin-3 in membranes localized in the phagosome and in tubules and vesicles that derive from the endocytic pathway. We also demonstrated that the binding of galectin-3 to host N-acetyllactosamine-containing glycans, was required for forming the structures. Accumulation of the structures was a type three secretion system-dependent process. More specifically, existence of structures was strictly dependent upon lysis of the phagocytic vacuole and could be shown also by Gram-positive Listeria and Salmonella sifA mutant. We suggest that galectin-3-containing structures may serve as a potential novel tool to spot vacuole lysis.

Galectin-1 tunes TCR binding and signal transduction to regulate CD8 burst size.

T cell burst size is regulated by the duration of TCR engagement and balanced control of Ag-induced activation, expansion, and apoptosis. We found that galectin-1-deficient CD8 T cells undergo greater cell division in response to TCR stimulation, with fewer dividing cells undergoing apoptosis. TCR-induced ERK signaling was sustained in activated galectin-1-deficient CD8 T cells and antagonized by recombinant galectin-1, indicating galectin-1 modulates TCR feed-forward/feedback loops involved in signal discrimination and procession. Furthermore, recombinant galectin-1 antagonized binding of agonist tetramers to the TCR on activated OT-1 T cells. Finally, galectin-1 produced by activated Ag-specific CD8 T cells negatively regulated burst size and TCR avidity in vivo. Therefore, galectin-1, inducibly expressed by activated CD8 T cells, functions as an autocrine negative regulator of peripheral CD8 T cell TCR binding, signal transduction, and burst size. Together with recent findings demonstrating that gal-1 promotes binding of agonist tetramers to the TCR of OT-1 thymocytes, these studies identify galectin-1 as a tuner of TCR binding, signaling, and functional fate determination that can differentially specify outcome, depending on the developmental and activation stage of the T cell.