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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.

The mAb 1B11 has been characterized as recognizing the activation-associated glycoform of murine CD43, a heavily O-glycosylated protein implicated in leukocyte homing. When hemopoietic cells from CD43-/- mice were stained with 1B11, CD43-independent binding of 1B11 was observed on peripheral CD8 T cells and at low levels on thymocytes, while no binding was detected on CD4 T cells, B cells, or bone marrow cells. Levels of 1B11 staining were comparable in lymph node CD8+ T cells from both CD43-/- mice and CD43+/+ mice. We sought to identify the CD43-independent target of 1B11 expressed on CD8 T cells. Previous work had demonstrated that neuraminidase treatment of lymph node cells (LNC) enhanced 1B11 binding on CD43+/+ LNC; this enhancement was also observed in CD43-/- LNC. We show that neuraminidase-enhanced 1B11 binding in CD43-/- LNC and EL4 thymoma cells is CD43 independent and that 1B11 detects a novel target of apparent mass of approximately 200 kDa identified as a hyposialylated form of CD45RB preferentially expressed on peripheral CD8, but not CD4, T cells. Our data also show that the recognition of CD43 and CD45RB by 1B11 is differentially affected by O-linked glycosylation and sialic acid. Whereas 1B11 recognition of CD43 on activated T cells required both core 2 O-glycan branching and sialic acid, 1B11 recognition of CD45 only occurred in the absence of both core 2 glycosylation and sialic acid.

CD43, a large highly glycosylated molecule, is arguably the most abundant molecule on the surface of T cells. Nevertheless, the function of CD43 remains unclear. Utilizing fluorescence microscopy, we find that CD43 is excluded from the T cell-APC contact site. This exclusion is Ag dependent since optimal CD43 exclusion requires Ag-pulsed APC, and since signaling through CD3, in the absence of any other receptor ligand interactions, can induce the modulation of CD43. These data suggest that CD43 may function as a barrier to nonspecific T cell-APC interactions that is removed as a result of T cell activation. Exclusion from the interaction site is a unique feature of CD43 and not universally found for all large highly glycosylated molecules since CD45 is not excluded. Thus, CD43 may represent a novel regulatory molecule on the T cell surface that can direct T cell interactions by changing its location on the cell surface.

CD43 is a major membrane sialoglycoprotein expressed by cells of hematopoietic origin. One property of CD43 is its ability to interfere with heterotypic and homotypic cellular adhesion. To determine whether CD43 expression can affect cell functions requiring intercellular adhesion, we compared a CD43-positive human T cell line (CEM) and its CD43-negative counterpart derived by gene targeting for susceptibility to cell-mediated lysis. CD43-negative CEM cells were more susceptible than CD43-positive cells to lysis by allospecific T cell lines derived from several donors. Induction of CD43 expression on transfected HeLa cells also imparted resistance to lectin-mediated lysis by a CD8+ T cell clone. The effect of CD43 expression on reducing susceptibility to lysis was more pronounced in short-term cytotoxicity assays and tended to disappear as the time of contact between the effector cell and its target increased. The enhanced susceptibility of CD43-negative cells to lysis was not associated with increased expression of adhesion molecules known to mediate antigen-independent cellular adhesion. Sialic acid residues on CD43 contributed to the CD43 protective effect. These results suggest that either diminished CD43 expression or incomplete sialylation may render hematopoietic cells more susceptible to T lymphocyte-mediated cytolysis.

CD43 is a major leukocyte surface glycoprotein thought to have important functions for T lymphocyte adhesion and activation. We investigated the function of CD43 by using gene targeting to eliminate CD43 expression in the human T lymphocyte line CEM and then testing their adhesive phenotype. Loss of CD43 expression by the CEM cells enhanced their homotypic adhesion and binding to two distinct ligands, fibronectin and HIV-1 gp120. The enhanced homotypic adhesion was blocked specifically by an anti-beta 1 integrin mAb, and the enhanced binding to fibronectin and gp120 was blocked specifically by anti-beta 1 integrin and anti-CD4 mAb, respectively. Partial reconstitution of CD43 expression in the CD43-negative cells resulted in a corresponding reversion to a less adhesive phenotype. These data suggest that CD43 interferes with T lymphocyte adhesion and that CD43 can regulate lymphocyte adhesion by providing a threshold that must be overcome for cell-cell and cell-ligand interactions to occur.

We previously found that murine leukemia cells of T cell, B cell, and erythroid ontogeny express a cell membrane antigen that cross-reacts with an idiotype of an anti-retroviral antibody. In the present study, the expression of this antigen (termed AVID, for anti-viral idiotype) by murine erythroleukemia (MEL) cells was examined during chemically induced differentiation. AVID expression by MEL cells was found to be lost when they were treated with either dimethyl sulfoxide or hexamethylene bisacetamide, two chemicals that induce MEL cells to terminally differentiate. The kinetics of disappearance of AVID during inducer treatment reflected the kinetics with which the inducers caused MEL cell commitment to terminal differentiation. Loss of AVID expression by inducer-treated cells was inhibited by dexamethasone, which inhibits commitment and MEL cell differentiation. The subset of inducer-treated cells that expressed the least amount of AVID contained the greatest number of cells committed to differentiate. These results indicate that AVID identifies a novel differentiation antigen of MEL cells.