Galectin-1 binds different CD43 glycoforms to cluster CD43 and regulate T cell death

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 approximately 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.     

CD7 delivers a pro-apoptotic signal during galectin-1-induced T 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.     

CD45 modulates galectin-1-induced T cell death: regulation by expression of core 2 O-glycans.

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 beta-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.     

The ST6Gal I sialyltransferase selectively modifies N-glycans on CD45 to negatively regulate galectin-1-induced CD45 clustering,phosphatase modulation,and T cell death

The addition of sialic acid to T cell surface glycoproteins influences essential T cell functions such as selection in the thymus and homing in the peripheral circulation. Sialylation of glycoproteins can be regulated by expression of specific sialyltransferases that transfer sialic acid in a specific linkage to defined saccharide acceptor substrates and by expression of particular glycoproteins bearing saccharide acceptors preferentially recognized by different sialyltransferases. Addition of alpha2,6-linked sialic acid to the Galbeta1,4GlcNAc sequence, the preferred ligand for galectin-1, inhibits recognition of this saccharide ligand by galectin-1. SAalpha2,6Gal sequences, created by the ST6Gal I enzyme, are present on medullary thymocytes resistant to galectin-1-induced death but not on galectin-1-susceptible cortical thymocytes. To determine whether addition of alpha2,6-linked sialic acid to lactosamine sequences on T cell glycoproteins inhibits galectin-1 death, we expressed the ST6Gal I enzyme in a galectin-1-sensitive murine T cell line. ST6Gal I expression reduced galectin-1 binding to the cells and reduced susceptibility of the cells to galectin-1-induced cell death. Because the ST6Gal I preferentially utilizes N-glycans as acceptor substrates, we determined that N-glycans are essential for galectin-1-induced T cell death. Expression of the ST6Gal I specifically resulted in increased sialylation of N-glycans on CD45, a receptor tyrosine phosphatase that is a T cell receptor for galectin-1. ST6Gal I expression abrogated the reduction in CD45 tyrosine phosphatase activity that results from galectin-1 binding. Sialylation of CD45 by the ST6Gal I also prevented galectin-1-induced clustering of CD45 on the T cell surface, an initial step in galectin-1 cell death. Thus, regulation of glycoprotein sialylation may control susceptibility to cell death at specific points during T cell development and peripheral activation.     

Restricted receptor segregation into membrane microdomains occurs on human T cells during apoptosis induced by galectin-1.

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.     

Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways

The galectin family of lectins regulates multiple biologic functions, such as development, inflammation, immunity, and cancer. One common function of several galectins is the ability to trigger T cell death. However, differences among the death pathways triggered by various galectins with regard to glycoprotein receptors, intracellular death pathways, and target cell specificity are not well understood. Specifically, galectin-9 and galectin-1 both kill thymocytes, peripheral T cells, and T cell lines; however, we have found that galectin-9 and galectin-1 require different glycan ligands and glycoprotein receptors to trigger T cell death. The two galectins also utilize different intracellular death pathways, as galectin-9, but not galectin-1, T cell death was blocked by intracellular Bcl-2, whereas galectin-1, but not galectin-9, T cell death was blocked by intracellular galectin-3. Target cell susceptibility also differed between the two galectins, as galectin-9 and galectin-1 killed different subsets of murine thymocytes. To define structural features responsible for distinct activities of the tandem repeat galectin-9 and dimeric galectin-1, we created a series of bivalent constructs with galectin-9 and galectin-1 carbohydrate recognition domains connected by different peptide linkers. We found that the N-terminal carbohydrate recognition domain and linker peptide contributed to the potency of these constructs. However, we found that the C-terminal carbohydrate recognition domain was the primary determinant of receptor recognition, death pathway signaling, and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency make distinct contributions to the specific effects of different galectins in initiating T cell death.     

Altered T cell surface glycosylation in HIV-1 infection results in increased susceptibility to galectin-1-induced cell death

The massive T cell death that occurs in HIV type 1 (HIV-1) infection contributes profoundly to the pathophysiology associated with AIDS. The mechanisms controlling cell death of both infected and uninfected T cells ("bystander" death) are not completely understood. We have shown that HIV-1 infection of T cells results in altered glycosylation of cell surface glycoproteins; specifically, it decreased sialylation and increased expression of core 2 O-glycans. Galectin-1 is an endogenous human lectin that recognizes these types of glycosylation changes and induces cell death of activated lymphocytes. Therefore we studied the possible contribution of galectin-1 in the pathophysiology of AIDS. O-glycan modifications were investigated on peripheral lymphocytes from AIDS patients. Oligosaccharides from CD43 and CD45 of CEM cells latently infected with HIV-1 were chemically analyzed. Consistent with our previous results, we show that HIV-1 infection results in accumulation of exposed lactosamine residues, oligosaccharides recognized by galectin-1 on cell surface glycoproteins. Both latently HIV-1-infected T cell lines and peripheral CD4 and CD8 T cells from AIDS patients exhibited exposed lactosamine residues and demonstrated marked susceptibility to galectin-1-induced cell death, in contrast to control cultures or cells from uninfected donors. The fraction of cells that died in response to galectin-1 exceeded the fraction of infected cells, indicating that death of uninfected cells occurred. Altered cell surface glycosylation of T cells during HIV-1 infection increases the susceptibility to galectin-1-induced cell death, and this death pathway can contribute to loss of both infected and uninfected T cells in AIDS.     

Galectin-1 induces nuclear translocation of endonuclease G in caspase- and cytochrome c-independent T cell death

Galectin-1, a mammalian lectin expressed in many tissues, induces death of diverse cell types, including lymphocytes and tumor cells. The galectin-1 T cell death pathway is novel and distinct from other death pathways, including those initiated by Fas and corticosteroids. We have found that galectin-1 binding to human T cell lines triggered rapid translocation of endonuclease G from mitochondria to nuclei. However, endonuclease G nuclear translocation occurred without cytochrome c release from mitochondria, without nuclear translocation of apoptosis-inducing factor, and prior to loss of mitochondrial membrane potential. Galectin-1 treatment did not result in caspase activation, nor was death blocked by caspase inhibitors. However, galectin-1 cell death was inhibited by intracellular expression of galectin-3, and galectin-3 expression inhibited the eventual loss of mitochondrial membrane potential. Galectin-1-induced cell death proceeds via a caspase-independent pathway that involves a unique pattern of mitochondrial events, and different galectin family members can coordinately regulate susceptibility to cell death.     

CD99 signals caspase-independent T cell death

Death signaling by Fas and TNF receptors plays a major role in the control of activated mature T cells. However, the nature of the death receptors, which may be used by the immune system to control T cells that have not acquired susceptibility to Fas ligand or TNF, is not established. In this study, we demonstrate that engagement of distinct epitopes on CD99 rapidly induces T cell death by a novel caspase-independent pathway. A new mAb to these CD99 epitopes, Ad20, induces programmed cell death of transformed T cells as determined by morphological changes, phosphatidylserine exposure on the cell surface, and uptake of propidium iodide. In general, ligation of CD99 induced kinetically faster and more profound death responses as compared with the impact of anti-Fas and TNF-related apoptosis-inducing ligand (TRAIL). Ad20-induced programmed cell death was observed with seven of eight T cell lines examined, and notably, only two of these were distinctly responsive to anti-Fas and TRAIL. CD99-mediated death signaling proceeded independently of functional CD3, CD4, CD45, and p56(lck), revealed distinctions from CD47-mediated T cell death responses, and was not influenced by interference with CD47 signaling. In contrast to the effect on transformed T cell lines, Ad20-induced death responses were not observed with normal peripheral T cells. Thus, our data suggest that CD99 is linked to a novel death pathway that may have biologic relevance in control of early T cells.     

T cell activation via Leu-23 (CD69)

The CD69 (Leu-23) activation Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer that is rapidly induced after lymphocyte activation. CD69 is not present on the surface of peripheral blood resting T cells, but is constitutively expressed by CD3bright thymocytes. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters directly induces CD69 expression on T cells. In the attempt to elucidate the function of CD69 we investigated the ability of the CD69 glycoprotein to transmit an activation signal. Cross-linking of CD69 by mAb induced a prolonged elevation of intracellular [Ca2+], mostly due to an influx of extracellular Ca2+. This signal alone was unable to effectively activate PKC. When PKC was simultaneously activated by PMA, stimulation of CD69 induced IL-2 and IFN-gamma gene expression, enhancement of CD25 expression, and ultimately IL-2-dependent T cell proliferation. Both CD4+ and CD8+ peripheral T cells responded to CD69-mediated activation. Stimulation of CD69 induced proliferation of thymocytes as well as peripheral T cells, but both required independent PKC activation by PMA. Cyclosporin A, which does not prevent PKC-induced CD69 expression, completely suppressed CD69-induced IL-2 and IFN-gamma gene expression. Although the signal delivered by the CD69 initiates T cell proliferation, it is unable to trigger cytotoxicity programs in CD69+-activated T cells or T cell clones.     

Expression of a specific glycosyltransferase enzyme regulates T cell death mediated by galectin-1

Galectin-1 induces apoptosis of immature thymocytes and activated T cells, suggesting that galectin-1 regulates cell death in the thymus during selection and in the periphery following an immune response. Although it is known that galectin-1 recognizes lactosamine (Gal-GlcNAc) as a minimal ligand, this disaccharide is ubiquitously expressed on a variety of cell surface glycoproteins. Thus, susceptibility to galectin-1 may be regulated by the presentation of lactosamine on specific oligosaccharide structures created by specific glycosyltransferase enzymes. The core 2 beta-1, 6-N-acetylglucosaminyltransferase (core 2 GnT) creates a branched structure on O-glycans that can be elongated to present multiple lactosamine sequences. In the thymus, the core 2 GnT is expressed in galectin-1-sensitive thymocyte subsets. In the periphery, an oligosaccharide epitope created by the core 2 GnT is expressed on galectin-1-sensitive activated T-cells. In this report, we demonstrate that expression of the core 2 GnT was necessary and sufficient for galectin-1-induced death of murine T cell lines. In addition, overexpression of the core 2 GnT in mice increased the susceptibility of double positive thymocytes to galectin-1. These data demonstrate that expression of a specific glycosyltransferase can control susceptibility to galectin-1, suggesting that developmentally regulated glycosyltransferase expression may be a mechanism to modulate cell death during T cell development and function.     

CD47 signals T cell death.

Activation-induced death of T cells regulates immune responses and is considered to involve apoptosis induced by ligation of Fas and TNF receptors. The role of other receptors in signaling T cell death is less clear. In this study we demonstrate that activation of specific epitopes on the Ig variable domain of CD47 rapidly induces apoptosis of T cells. A new mAb, Ad22, to this site induces apoptosis of Jurkat cells and CD3epsilon-stimulated PBMC, as determined by morphological changes, phosphatidylserine exposure on the cell surface, uptake of propidium iodide, and true counts by flow cytometry. In contrast, apoptosis was not observed following culture with anti-CD47 mAbs 2D3 or B6H12 directed to a distant or closely adjacent region, respectively. CD47-mediated cell death was independent of CD3, CD4, CD45, or p56lck involvement as demonstrated by studies with variant Jurkat cell lines deficient in these signaling pathways. However, coligation of CD3epsilon and CD47 enhanced phosphatidylserine externalization on Jurkat cells with functional CD3. Furthermore, normal T cells required preactivation to respond with CD47-induced apoptosis. CD47-mediated cell death appeared to proceed independent of Fas or TNF receptor signaling and did not involve characteristic DNA fragmentation or requirement for IL-1beta-converting enzyme-like proteases or CPP32. Taken together, our data demonstrate that under appropriate conditions, CD47 activation results in very rapid T cell death, apparently mediated by a novel apoptotic pathway. Thus, CD47 may be critically involved in controlling the fate of activated T cells.     

Galectin-3 but not galectin-1 induces mast cell death by oxidative stress and mitochondrial permeability transition

Galectin-1 and galectin-3 are the most ubiquitously expressed members of the galectin family and more importantly, these two molecules are shown to have opposite effects on pro-inflammatory responses and/or apoptosis depending on the cell type. Herein, we demonstrate for the first time that galectin-3 induces mast cell apoptosis. Mast cells expressed substantial levels of galectin-3 and galectin-1 and to a lesser extent the receptor for advanced glycation end products (RAGE) on their surfaces. Treatment of cells with galectin-3 at concentrations of > or =100 nM for 18-44 h resulted in cell death by apoptosis. Galectin-3-induced apoptosis was completely prevented by lactose, neutralizing antibody to RAGE, and the caspase-3 inhibitor z-DEVD-fmk. Galectin-3-induced apoptosis was also completely abolished by dithiothreitol and superoxide dismutase, but not inhibited by catalase. Moreover, galectin-3 but not galectin-1 induced the release of superoxide, which was blocked by lactose, anti-RAGE, and dithiothreitol. Finally, galectin-3-induced apoptosis was blocked by bongkrekic acid, an antagonist of the mitochondrial permeability transition pore (PTP), while atractyloside, an agonist of the PTP, greatly facilitated galectin-1-induced apoptosis. These data suggest that galectin-3 induces oxidative stress, PTP opening, and the caspase-dependent death pathway by binding to putative surface receptors including RAGE via the carbohydrate recognition domain.     

Tumor cell programmed death ligand 1-mediated T cell suppression is overcome by coexpression of CD80

Programmed death ligand 1 (PDL1, or B7-H1) is expressed constitutively or is induced by IFN-γ on the cell surface of most human cancer cells and acts as a "molecular shield" by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma), we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1-mediated immune suppression by tumor cells and restores T cell activation. Mechanistically, CD80 mediates its effects through its extracellular domain, which blocks the cell surface expression of PDL1 but does not prevent intracellular expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating antitumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression.     

Selective regulation of TCR signaling pathways by the CD45 protein tyrosine phosphatase during thymocyte development

In CD45-deficient animals, there is a severe defect in thymocyte-positive selection, resulting in an absence of mature T cells and the accumulation of thymocytes at the DP stage of development. However, the signaling defect(s) responsible for the block in development of mature single-positive T cells is not well characterized. Previous studies have found that early signal transduction events in CD45-deficient cell lines and thymocytes are markedly diminished following stimulation with anti-CD3. Nevertheless, there are also situations in which T cell activation and TCR signaling events can be induced in the absence of CD45. For example, CD45-independent TCR signaling can be recovered upon simultaneous Ab cross-linking of CD3 and CD4 compared with cross-linking of CD3 alone. These data suggest that CD45 may differentially regulate TCR signaling events depending on the nature of the signal and/or on the differentiation state of the cell. In the current study, we have assessed the role of CD45 in regulating primary thymocyte activation following physiologic stimulation with peptide. Unlike CD3-mediated stimulation, peptide stimulation of CD45-deficient thymocytes induces diminished, but readily detectable TCR-mediated signaling events, such as phosphorylation of TCR-associated zeta, ZAP70, linker for activation of T cells, and Akt, and increased intracellular calcium concentration. In contrast, phosphorylation of ERK, which is essential for positive selection, is more severely affected in the absence of CD45. These data suggest that CD45 has a selective role in regulating different aspects of T cell activation.     

Developmentally regulated changes in glucosidase II association with, and carbohydrate content of, the protein tyrosine phosphatase CD45

Glucosidase II (GII) stably interacts with the external domain of CD45 in a carbohydrate-dependent manner. We have found that the association occurs in immature cells, but is significantly reduced in mature T cells. Using mannose-binding protein (MBP), in both FACS analysis and pull-down assays, we find that MBP can specifically recognize cell surface CD45 from immature, but not mature T cells. Analysis of thymocytes reveals increased MBP binding and GII association with CD45 in double-positive thymocytes compared with either double-negative or single-positive thymocytes. As well, the same pool of CD45 recognized by MBP can also associate with GII. Initial analysis of the basis of the interaction between CD45 and MBP suggests MBP binds two different glycoforms of CD45 based on the differential competition with glucose. Finally, inhibition of GII activity in cells that do not normally express MBP ligands results in significant increases in cell surface MBP ligands, including CD45. Taken together, these data suggest that the glucose content of the cell surface CD45 changes as thymocytes undergo maturation to mature T cells, and may be regulated by GII interactions. Such changes in the cell surface carbohydrate on CD45 may affect the development of thymocytes, perhaps via binding of CD45 on thymocytes to lectins on stromal cells.     

Human galectin-2: novel inducer of T cell apoptosis with distinct profile of caspase activation

Galectin-2 is structurally closely related to galectin-1, but has a distinct expression profile primarily confined to the gastrointestinal tract. Prominent differences in the proximal promoter regions between galectins-2 and -1 concern Sp1-, hepatocyte NF-3, and T cell-specific factor-1 binding sites. Of note, these sequence elements are positioned equally in the respective regions for human and rat galectins-2. Labeled galectin-2 binds to T cells in a beta-galactoside-specific manner. In contrast to galectin-1, the glycoproteins CD3 and CD7 are not ligands, while the shared affinity to beta1 integrin (or a closely associated glycoprotein) accounts for a substantial extent of cell surface binding. The carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Fluorogenic substrate and inhibitor assays reveal involvement of caspases-3 and -9, in accordance with cleavage of the DNA fragmentation factor. Enhanced cytochrome c release, disruption of the mitochondrial membrane potential, and an increase of the Bax/Bcl-2 ratio by opposite regulation of expression of both proteins add to the evidence that the intrinsic apoptotic pathway is triggered. Cell cycle distribution and expression of regulatory proteins remained unaffected. Notably, galectins-1 and -7 reduce cyclin B1 expression, defining functional differences between the structurally closely related galectins. Cytokine secretion of activated T cells was significantly shifted to the Th2 profile. Our study thus classifies galectin-2 as proapoptotic effector for activated T cells, raising a therapeutic perspective. Of importance for understanding the complex galectin network, it teaches the lesson that selection of cell surface ligands, route of signaling, and effects on regulators of cell cycle progression are markedly different between structurally closely related galectins.     

HIV-1 gp120-mediated apoptosis of T cells is regulated by the membrane tyrosine phosphatase CD45

The molecular mechanism of the human immunodeficiency virus type 1 (HIV-1) gp120-induced apoptosis of bystander T cells is not well defined. Here, we demonstrate that CD45, a key component of the T cell receptor pathway, plays a crucial role in apoptosis induced by HIV-1 gp120. We observed that HIV-1 gp120-induced apoptosis was significantly reduced in a CD45-deficient cell line and that reconstitution of CD45 in these cells restored gp120-induced apoptosis. However, expression of a chimeric protein containing only the intracellular phosphatase domain was not able to restore the apoptotic function in the CD45-negative clone, indicating an important role for the extracellular domain of CD45 in this function. The role of CD45 in gp120-induced apoptosis was further confirmed in T cell lines and peripheral blood mononuclear cells using a selective CD45 inhibitor as well as CD45-specific small interfering RNA. We also observed that gp120 treatment induced CD45 association with the HIV coreceptor CXCR4. Further elucidation of downstream signaling events revealed that CD45 modulates HIV-1 gp120-induced apoptosis by regulating Fas ligand induction and activation of the phosphoinositide 3-kinase/Akt pathway. These results suggest a novel CD45-mediated mechanism for the HIV envelope-induced apoptosis of T cells.     

GPR30 contributes to estrogen-induced thymic atrophy

The mechanisms by which prolonged estrogen exposures, such as estrogen therapy and pregnancy, reduce thymus weight, cellularity, and CD4 and CD8 phenotype expression, have not been well defined. In this study, the roles played by the membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), and the intracellular estrogen receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta), in 17beta-estradiol (E2)-induced thymic atrophy were distinguished by construction and the side-by-side comparison of GPR30-deficient mice with ERalpha and ERbeta gene-deficient mice. Our study shows that whereas ERalpha mediated exclusively the early developmental blockage of thymocytes, GPR30 was indispensable for thymocyte apoptosis that preferentially occurs in T cell receptor beta chain(-/low) double-positive thymocytes. Additionally, G1, a specific GPR30 agonist, induces thymic atrophy and thymocyte apoptosis, but not developmental blockage. Finally, E2 treatment attenuates the activation of nuclear factor-kappa B in CD25(-)CD4(-)CD8(-) double-negative thymocytes through an ERalpha-dependent yet ERbeta- and GPR30-independent pathway. Differential inhibition of nuclear factor-kappaB by ERalpha and GPR30 might underlie their disparate physiological effects on thymocytes. Our study distinguishes, for the first time, the respective contributions of nuclear and membrane E2 receptors in negative regulation of thymic development.     

Presentation of galectin-1 by extracellular matrix triggers T cell death

Apoptotic elimination of T cells at sites of inflammation or infiltration into tumors limits an effective immune response. T cell apoptosis can be initiated by a variety of triggers, including galectin-1, a soluble, secreted lectin that binds to oligosaccharide ligands on cell surface glycoproteins, or to oligosaccharide ligands on extracellular matrix glycoproteins in tissue stroma. Although galectin-1 has no transmembrane domain and is secreted from cells that make it, it is not clear if galectin-1 functions as a soluble death trigger in vivo. We examined the ability of stromal cells secreting galectin-1 to kill T cells. Although the stromal cells synthesized abundant galectin-1, the majority of the galectin-1 remained bound to the cell surface, and stromal cell-associated galectin-1 killed bound T cells. In contrast, insufficient amounts of functional galectin-1 were released from the stromal cells into the media to kill T cells in the absence of contact with stromal cells. However, when stromal cells were grown on Matrigel, a mixture of extracellular matrix proteins, or on permeable membranes above Matrigel, secreted galectin-1 bound to Matrigel and killed T cells without stromal cell contact. Ten-fold less galectin-1 on Matrigel was sufficient to kill adherent T cells compared with soluble galectin-1. These results demonstrate that galectin-1 in extracellular matrix is able to directly kill susceptible T cells. Because increased galectin-1 deposition in tumor stroma occurs with tumor progression in various types of cancer, galectin-1 in stroma may act locally in the apoptotic elimination of infiltrating T cells during an immune response.