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.     

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.     

Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.     

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.     

Twist2 regulates CD7 expression and galectin-1-induced apoptosis in mature T-cells

In the periphery, a galectin-1 receptor, CD7, plays crucial roles in galectin-1-mediated apoptosis of activated T-cells as well as progression of T-lymphoma. Previously, we demonstrated that NF-κB downregulated CD7 gene expression through the p38 MAPK pathway in developing immature thymocytes. However, its regulatory pathway is not well understood in functional mature T-cells. Here, we show that CD7 expression was downregulated by Twist2 in Jurkat cells, a human acute T-cell lymphoma cell line, and in EL4 cells, a mature murine T-cell lymphoma cell line. Furthermore, ectopic expression of Twist2 in Jurkat cells reduced galectin-1-induced apoptosis. While full-length Twist2 decreased CD7 promoter activity, a C-terminal deletion form of Twist2 reversed its inhibition, suggesting an important role of the C-terminus in CD7 regulation. In addition, CD7 expression was enhanced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate, which indicates that Twist2 might be one of candidate factors involved in histone deacetylation. Based on these results, we conclude that upregulation of Twist2 increases the resistance to galectin-1-mediated-apoptosis, which may have significant implications for the progression of some T-cells into tumors such as Sezary cells.     

Characterization of galectin-9-induced death of Jurkat T cells

Galectin-9, a mammalian lectin with affinity for beta-galactosides, is known as an apoptosis inducer of activated T lymphocytes. In the present study, we examined the properties of galectin-9-mediated cell death of Jurkat T cells. Galectin-9NC (wild-type), consisting of two CRDs (N-terminal and C-terminal carbohydrate recognition domains), and derivatives of it, galectins-9-NN and -9-CC, induced Jurkat T-cell apoptosis. However, a single CRD (galectin-9NT or -CT) had no effect, suggesting the stable dimeric structure of two CRDs is required for the activity. The apoptosis was inhibited by pretreatment with an N-glycan synthesis inhibitor, indicating that the expression of N-glycans in the cells is essential for galectin-9-induced apoptosis. We previously showed that the apoptosis of MOLT-4 cell is mediated by galectin-9 via a Ca(2+)-calpain-caspase-1-dependent pathway. In Jurkat cells, the cell death by galectin-9, was insufficiently suppressed by caspase inhibitors, Ca(2+)-chelator or calpain inhibitor. Furthermore, we observed the loss of mitochondrial membrane potential and significant AIF release in galectin-9-treated cells. These findings suggest that caspase-dependent and-independent death pathways exist in Jurkat cells, and the main pathway might vary with the T-cell type.     

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.     

T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R- T4 cell subsets

The immunoregulatory functions of human T4 cell subpopulations defined by mAb to the CD45R molecule (2H4) were examined. Both CD45R- and CD45R+ T4 cells that had been treated with mitomycin C (CD45R- and CD45R+ T4-mito) provided help for the generation of Ig-secreting cells (ISC) in cultures stimulated by PWM or by immobilized mAb to CD3 (64.1). IL-2 enhanced the generation of ISC in PWM-stimulated cultures and in anti-CD3-stimulated cultures containing CD45R+ T4-mito. The generation of ISC was maximal in cultures containing anti-CD3-activated CD45R- T4-mito and was not increased by IL-2. By contrast, CD45R+ T4 cells that had not been treated with mitomycin C suppressed B cell responses in cultures stimulated with PWM or anti-CD3, whereas CD45R- T4 cells suppressed the generation of ISC only in cultures stimulated with anti-CD3. IL-2 enhanced suppression by anti-CD3, but not PWM, activated CD45R- T4 cells. Suppression by CD45R+ T4 cells was maximal and not increased by IL-2. CD45R+ T4-mito were more effective suppressor-inducers in PWM-stimulated cultures, promoting the differentiation of suppressor-effector cells from CD8+ T cells. However, both CD45R+ and CD45R- T4-mito exerted comparable suppressor-inducer function in anti-CD3-stimulated cultures. Moreover, in anti-CD3-stimulated cultures, T8 cells could function as both suppressor-effector cells and suppressor-inducer cells. One of the functions of suppressor-inducer cells in this system appeared to involve the production of IL-2. Thus, the addition of IL-2 facilitated the induction of suppressor-effector T8 cells by CD45R- T4-mito in PWM-stimulated cultures. Although IL-2 production by the T cell subsets varied widely depending on the nature of the stimulus, these differences could not entirely explain their capacity to function as helper cells, suppressor-effector cells or suppressor-inducer cells. These results indicate that both CD45R+ and CD45R- T4 cells can help or suppress B cell responses, as well as induce suppressor-effector T8 cells. Moreover, suppressor-inducer function of T cells is not limited to the T4 cell population, but rather can also be accomplished by T8 cells. The results indicate that both T4 cell subsets and T8 cells exert multiple regulatory effects on human B cell function, with the nature of the activating stimulus playing a major role in determining the functional capacity of various T cell subsets.     

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.     

Coordinate regulation of T cell activation by CD2 and CD28

T cell activation requires co-engagement of the TCR with accessory and costimulatory molecules. However, the exact mechanism of costimulatory function is unknown. Mice lacking CD2 or CD28 show only mild deficits, demonstrating that neither protein is essential for T cell activation. In this paper we have generated mice lacking both CD2 and CD28. T cells from the double-deficient mice have a profound defect in activation by soluble anti-CD3 Ab and Ag, yet remain responsive to immobilized anti-CD3. This suggests that CD2 and CD28 may function together to facilitate interactions of the T cell and APC, allowing for efficient signal transduction through the TCR.     

Beta 1 integrin (CD29) expression on human postnatal T cell subsets defined by selective CD45 isoform expression

The integrin beta 1 (CD29) is a marker for total very late activation Ag integrins on cells, and exhibits considerable fluctuation in cell surface density at various stages of T cell development. We have analyzed beta 1 integrin expression on subsets of human thymus, and on T cells from healthy babies and children, in comparison to healthy adults aged 26 to 75. T cells from adult peripheral blood include a CD29-, a CD29lo, and a CD29hi set. Compared with adults, PBMC T cells from children have reduced numbers of both CD29lo and CD29hi subsets but equivalent numbers of CD29- T cells. The number of CD29hi T cells increases gradually with age, achieving adult levels only at about 26 yr of age; in aged adults (69 to 75 yr), nearly all T cells have a CD29hi phenotype. Most thymocytes and cord blood T cells, in contrast, have a single peak of CD29 staining that is intermediate to the two peaks seen in adults. Multi-negative progenitor and CD45RO- thymocytes (presumptive thymic generative line-age) are 98% CD29hi. Progenitor thymocytes and adult PBMC T cells express equivalent amounts of beta 1 and alpha 4, but progenitors are alpha 5hi, whereas PBMC T cells are alpha 5lo. T cells from children have reduced beta 1hi and alpha 5lo, but nearly comparable numbers of alpha 4hi. This suggests that the major very late activation Ag integrins during childhood may be alpha 5 beta 1 and alpha 4 complexed with an alternate beta chain. In children, the majority of CD29hi cells are also CD45RAhi, in contrast to the pattern in adults, in whom the majority of CD29hi T cells are CD45RA-. This suggests that in children, the main defense against infection may reside in the CD29hi45RAhi T cells, which have not yet made the transition to CD45RO and to bona fide memory status. The proliferative response to tetanus toxoid of 4- to 6-mo-old babies correlates with the number of CD29hi45RAhi T cells, suggesting that it derives at least in part from cells that do not express a "memory" phenotype. These observations show a pattern of alternating high and low density CD29 during T cell development, which is consistent with the idea that CD29 is a marker for functionally defined T cell sets. Analysis of the CD29 expression of CD29hi thymocytes developing in vitro supports this view. We suggest that the intensity of CD29 expression on a T cell varies, dependent upon the microenvironmental interactions required by a differentiating T cell.     

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.     

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.     

RIP-in CD95-induced cell death: The control of alternative death receptors pathways by cIAPs

Comment on: Geserick P, et al. J Cell Biol 2009; 187:1037-54.     

Restoration of T cell receptor-mediated signal transduction by transfection of CD45 cDNA into a CD45-deficient variant of the Jurkat T cell line.

The TCR is a multimeric structure comprised of distinct Ag recognition and signal transduction components. Although none of the molecules that make up the TCR possess intrinsic protein tyrosine kinase (PTK) activity, stimulation of T cells via the TCR results in the rapid appearance of newly tyrosine phosphorylated proteins in cell lysates. Evidence suggests ligation of the TCR induces activation of a PTK that may be a member of the src family. One early consequence of this TCR-mediated PTK activation is the phosphorylation of the gamma 1 isoform of phospholipase C. This phosphorylation event is associated with increased enzymatic activity resulting in the hydrolysis of phosphatidylinositol 4,5 bisphosphate into two second messengers, inositol 1,4,5 trisphosphate and diacylglycerol. Recently, our laboratory and others have isolated mutant T cells that lack surface expression of CD45, the major surface tyrosine phosphatase expressed on lymphoid cells. Stimulation of the TCR on these cells fails to result in the expected activation events. We demonstrate that reconstitution of surface expression of the 180-kDa isoform of CD45 by gene transfer into a CD45-deficient mutant of the Jurkat T cell leukemic line restores the ability of the TCR to couple fully to its signal transduction machinery. These results support the role of CD45 tyrosine phosphatase activity in regulating the TCR-activated PTK.     

CD45 isoform expression on human neonatal T cells: expression and turnover of CD45 isoforms on neonatal versus adult T cells after activation.

Neonatal T cells are phenotypically similar to "naive" T cells from adult donors in the CD45 isoform expression. Despite the phenotypic similarity, large differences were found between neonatal and adult T cells when T cells were activated. After activation with PHA, adult CD45RA+ T cells began to express CD45RO and no loss of CD45RA expression had yet occurred at Day 3 post-stimulation. Three days after activation, CD45RA+ neonatal T cells also coexpressed CD45RO; however, in contrast to adult T cells, a marked loss of CD45RA was observed. We analyzed the rapid loss of CD45RA found in neonatal T cells. The de novo synthesis of CD45 isoforms in neonatal T cells was essentially the same as that in the adult T cells. Turnover of the CD45RA was very rapid in both resting adult and neonatal T cells. After activation with PHA, the turnover of CD45RA on adult T cells was decreased significantly, while the turnover of CD45RA on neonatal T cells was not changed after activation. Therefore, the regulation of CD45 isoform expression not only involves switches in alternative splicing, but also involves different regulation of turnover of these isoforms from the cell membrane.     

Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death

Galectins are a family of mammalian beta-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.