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Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the β-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic β-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of β-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4+T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause β-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed β-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or β-cell toxicity in NOD mice, the use of gal-1 to control β-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D. Copyright © 2009 by The American Association of Immunologists, Inc.

Type 1 diabetes (T1D) is a disease caused by the destruction of the β cells of the pancreas by activated T cells. Dendritic cells (DC) are the APC that initiate the T cell response that triggers T1D. However, DC also participate in T cell tolerance, and genetic engineering of DC to modulate T cell immunity is an area of active research. Galectin-1 (gal-1) is an endogenous lectin with regulatory effects on activated T cells including induction of apoptosis and down-regulation of the Th1 response, characteristics that make gal-1 an ideal transgene to transduce DC to treat T1D. We engineered bone marrow-derived DC to synthesize transgenic gal-1 (gal-1-DC) and tested their potential to prevent T1D through their regulatory effects on activated T cells. NOD-derived gal-1-DC triggered rapid apoptosis of diabetogenic BDC2.5 TCR-transgenic CD4+ T cells by TCR-dependent and -independent mechanisms. Intravenously administered gal-1-DC trafficked to pancreatic lymph nodes and spleen and delayed onset of diabetes and insulitis in the NODrag1−/− lymphocyte adoptive transfer model. The therapeutic effect of gal-1-DC was accompanied by increased percentage of apoptotic T cells and reduced number of IFN-γ-secreting CD4+ T cells in pancreatic lymph nodes. Treatment with gal-1-DC inhibited proliferation and secretion of IFN-γ of T cells in response to β cell Ag. Unlike other DC-based approaches to modulate T cell immunity, the use of the regulatory properties of gal-1-DC on activated T cells might help to delete β cell-reactive T cells at early stages of the disease when the diabetogenic T cells are already activated.

Galectin-1 kills immature thymocytes and activated peripheral T cells by binding to glycans on T cell glycoproteins including CD7, CD45, and CD43. Although roles for CD7 and CD45 in regulating galectin-1-induced death have been described, the requirement for CD43 remains unknown. We describe a novel role for CD43 in galectin-1-induced death, and the effects of O-glycan modification on galectin-1 binding to CD43. Loss of CD43 expression reduced galectin-1 death of murine thymocytes and human T lymphoblastoid cells, indicating that CD43 is required for maximal T cell susceptibility to galectin-1. CD43, which is heavily O-glycosylated, contributes a significant fraction of galectin-1 binding sites on T cells, as T cells lacking CD43 bound ∼50% less galectin-1 than T cells expressing CD43. Although core 2 modification of O-glycans on other glycoprotein receptors is critical for galectin-1-induced cross-linking and T cell death, galectin-1 bound to CD43 fusion proteins modified with either unbranched core 1 or branched core 2 O-glycans and expression of core 2 O-glycans did not enhance galectin-1 binding to CD43 on T cells. Moreover, galectin-1 binding clustered CD43 modified with either core 1 or core 2 O-glycans on the T cell surface. Thus, CD43 bearing either core 1 or core 2 O-glycans can positively regulate T cell susceptibility to galectin-1, identifying a novel function for CD43 in controlling cell death. In addition, these studies demonstrate that different T cell glycoproteins on the same cell have distinct requirements for glycan modifications that allow recognition and cross-linking by galectin-1.

Lectins play a critical role in host protection against infection. The galectin family of lectins recognizes saccharide ligands on a variety of microbial pathogens, including viruses, bacteria, and parasites. Galectin-3, a galectin expressed by macrophages, dendritic cells, and epithelial cells, binds bacterial and parasitic pathogens including Leishmania major, Trypanosoma cruzi, and Neisseria gonorrhoeae. However, there have been no reports of galectins having direct effects on microbial viability. We found that galectin-3 bound only to Candida albicans species that bear β-1,2-linked oligomannans on the cell surface, but did not bind Saccharomyces cerevisiae that lacks β-1,2-linked oligomannans. Surprisingly, binding directly induced death of Candida species containing specific β-1,2-linked oligomannosides. Thus, galectin-3 can act as a pattern recognition receptor that recognizes a unique pathogen-specific oligosaccharide sequence. This is the first description of antimicrobial activity for a member of the galectin family of mammalian lectins; unlike other lectins of the innate immune system that promote opsonization and phagocytosis, galectin-3 has direct fungicidal activity against opportunistic fungal pathogens.

Dendritic cells (DCs) are potent mediators of the immune response, and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved β-galactoside-binding lectin family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However, its effects on human DCs are unknown. In this study, we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli, LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40, CD83, CD86, and HLA-DR), secreted high levels of IL-6 and TNF-α, stimulated T cell proliferation, and showed reduced endocytic capacity, similar to LPS-matured MDDCs. However, unlike LPS-matured DCs, galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS, galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed, compared with LPS, galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel, an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM, suggesting that galectin-1 may be an important component in initiating an immune response.

Galectins are a family of mammalian β-galactoside-binding proteins that positively and negatively regulate T cell death. Extracellular galectin-1 directly induces death of T cells and thymocytes, while intracellular galectin-3 blocks T cell death. In contrast to the antiapoptotic function of intracellular galectin-3, we demonstrate that extracellular galectin-3 directly induces death of human thymocytes and T cells. However, events in galectin-3- and galectin-1-induced cell death differ in a number of ways. Thymocyte subsets demonstrate different susceptibility to the two galectins: whereas galectin-1 kills double-negative and double-positive human thymocytes with equal efficiency, galectin-3 preferentially kills double-negative thymocytes. Galectin-3 binds to a complement of T cell surface glycoprotein receptors distinct from that recognized by galectin-1. Of these glycoprotein receptors, CD45 and CD71, but not CD29 and CD43, appear to be involved in galectin-3-induced T cell death. In addition, CD7 that is required for galectin-1-induced death is not required for death triggered by galectin-3. Following galectin-3 binding, CD45 remains uniformly distributed on the cell surface, in contrast to the CD45 clustering induced by galectin-1. Thus, extracellular galectin-3 and galectin-1 induce death of T cells through distinct cell surface events. However, as galectin-3 and galectin-1 cell death are neither additive nor synergistic, the two death pathways may converge inside the cell.

During a viral response, Ag-specific effector T cells show dramatically increased binding by the mAb 1B11 and the lectin peanut agglutinin (PNA). We investigated the contribution of CD43 expression to 1B11 and PNA binding as well as its role in generation and maintenance of a CD8 T cell response. Analysis of CD43−/− mice revealed no increased 1B11 binding and reduced PNA binding on virus-specific CD8 T cells from −/− mice compared with +/+ mice. Furthermore, we examined the role of CD43 in the kinetics of an immune response. We show that CD43 expression modestly effects generation of a primary virus-specific CD8 T cell response in vivo but plays a more significant role in trafficking of CD8 T cells to tissues such as the brain. More interestingly, CD43 plays a role in the contraction of the immune response, with CD43−/− mice showing increased numbers of Ag-specific CD8 T cells following initial expansion. Following the peak of expansion, Ag-specific CD8 T cells from −/− mice show similar proliferation but demonstrate increased Bcl-2 levels and decreased apoptosis of Ag-specific effector CD8 T cells in vitro. Consistent with a delay in the down-modulation of the immune response, following chronic viral infection CD43−/− mice show increased morbidity. These data suggest a dynamic role of CD43 during an immune response: a positive regulatory role in costimulation and trafficking of T cells to the CNS and a negative regulatory role in the down-modulation of an immune response.

Galectin-1 induces death of immature thymocytes and activated T cells. Galectin-1 binds to T cell-surface glycoproteins CD45, CD43, and CD7, although the precise roles of each receptor in cell death are unknown. We have determined that CD45 can positively and negatively regulate galectin-1-induced T cell death, depending on the glycosylation status of the cells. CD45+ BW5147 T cells lacking the core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) were resistant to galectin-1 death. The inhibitory effect of CD45 in C2GnT− cells appeared to require the CD45 cytoplasmic domain, because Rev1.1 cells expressing only CD45 transmembrane and extracellular domains were susceptible to galectin-1 death. Moreover, treatment with the phosphotyrosine-phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate(V) enhanced galectin-1 susceptibility of CD45+ T cell lines, but had no effect on the death of CD45− T cells, indicating that the CD45 inhibitory effect involved the phosphatase domain. Expression of the C2GnT in CD45+ T cell lines rendered the cells susceptible to galectin-1, while expression of the C2GnT in CD45− cells had no effect on galectin-1 susceptibility. When CD45+ T cells bound to galectin-1 on murine thymic stromal cells, only C2GnT+ T cells underwent death. On C2GnT+ cells, CD45 and galectin-1 co-localized in patches on membrane blebs while no segregation of CD45 was seen on C2GnT− T cells, suggesting that oligosaccharide-mediated clustering of CD45 facilitated galectin-1-induced cell death.

Galectin-1, an endogenous lectin expressed in lymphoid organs and immune-privileged sites, induces death of human and murine thymocytes and T cells. Galectin-1 binds to several glycoproteins on the T cell surface, including CD7. However, the T cell surface glycoprotein receptors responsible for delivering the galectin-1 death signal have not been identified. We show that CD7 is required for galectin-1-mediated death. This demonstrates a novel function for CD7 as a death trigger and identifies galectin-1/CD7 as a new biologic death signaling pair.

Galectin-1 induces apoptosis of human thymocytes and activated T cells by an unknown mechanism. Apoptosis is a novel function for a mammalian lectin; moreover, given the ubiquitous distribution of the oligosaccharide ligand recognized by galectin-1, it is not clear how susceptibility to and signaling by galectin-1 is regulated. We have determined that galectin-1 binds to a restricted set of T cell surface glycoproteins, and that only CD45, CD43, and CD7 appear to directly participate in galectin-1-induced apoptosis. To determine whether these specific glycoproteins interact cooperatively or independently to deliver the galectin-1 death signal, we examined the cell surface localization of CD45, CD43, CD7, and CD3 after galectin-1 binding to human T cell lines and human thymocytes. We found that galectin-1 binding resulted in a dramatic redistribution of these glycoproteins into segregated membrane microdomains on the cell surface. CD45 and CD3 colocalized on large islands on apoptotic blebs protruding from the cell surface. These islands also included externalized phosphatidylserine. In addition, the exposure of phosphatidylserine on the surface of galectin-1-treated cells occurred very rapidly. CD7 and CD43 colocalized in small patches away from the membrane blebs, which excluded externalized phosphatidylserine. Receptor segregation was not seen on cells that did not die in response to galectin-1, including mature thymocytes, suggesting that spatial redistribution of receptors into specific microdomains is required for triggering apoptosis.

Galectin-1 is an endogenous lectin expressed by thymic and lymph node stromal cells at sites of Ag presentation and T cell death during normal development. It is known to have immunomodulatory activity in vivo and can induce apoptosis in thymocytes and activated T cells (1-3). Here we demonstrate that galectin-1 stimulation cooperates with TCR engagement to induce apoptosis, but antagonizes TCR-induced IL-2 production and proliferation in a murine T cell hybridoma and freshly isolated mouse thymocytes, respectively. Although CD4+ CD8+ double positive cells are the primary thymic subpopulation susceptible to galectin-1 treatment alone, concomitant CD3 engagement and galectin-1 stimulation broaden susceptible thymocyte subpopulations to include a subset of each CD4- CD8-, CD4+ CD8+, CD4- CD8+, and CD4+ CD8- subpopulations. Furthermore, CD3 engagement cooperates with suboptimal galectin-1 stimulation to enhance cell death in the CD4+ CD8+ subpopulation. Galectin-1 stimulation is shown to synergize with TCR engagement to dramatically and specifically enhance extracellular signal-regulated kinase-2 (ERK-2) activation, though it does not uniformly enhance TCR-induced tyrosine phosphorylation. Unlike TCR-induced IL-2 production, TCR/galectin-1-induced apoptosis is not modulated by the expression of kinase inactive or constitutively activated Lck. These data support a role for galectin-1 as a potent modulator of TCR signals and functions and indicate that individual TCR-induced signals can be independently modulated to specifically affect distinct TCR functions.

The polyphenol group rutin (R) appears to influence isotype expression, because R-BSA conjugates induce anti-BSA responses in mice that show a significant decrease in hemagglutinating antibodies (HA) to BSA, as compared to mice immunized with BSA. However, the level of IgE antibodies to BSA is unaltered. To determine if suppressor cells for isotypes other than IgE are induced by R-BSA, cell transfers were performed. The results were consistent with the view that the decrease in HA titer to BSA in R-BSA immunized mice is not due to the activation of suppressor cells for isotypes other than IgE. Inasmuch as the IgE response in mice is associated with the production of IL-4 by Th2 cells, we analyzed the factors produced by spleen cells cultured with R-BSA. We found that supernatant from spleen cells cultured with R-BSA contained IL-4 as determined by the enhanced expression of Fc epsilon R (CD23) on B cells. This enhancement was inhibited by 11B11, the anti-IL-4 mAb. IL-2, a product of Th1 cells, was not detected in these supernatants. Moreover, IL-4 mRNA, but not IL-2 mRNA, was detected by Northern blot analysis of RNA from spleen cells cultured with R-BSA. Taken together the data suggest that the polyphenol containing compounds preferentially activate Th2 cells, thereby favoring IgE production.