Functional glycan-free adhesion domain of human cell surface receptor CD58: design, production and NMR studies.
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A general strategy is presented here for producing glycan-free forms of glycoproteins without loss of function by employing apolar-to-polar mutations of surface residues in functionally irrelevant epitopes. The success of this structure-based approach was demonstrated through the expression in Escherichia coli of a soluble 11 kDa adhesion domain extracted from the heavily glycosylated 55 kDa human CD58 ectodomain. The solution structure was subsequently determined and binding to its counter-receptor CD2 studied by NMR. This mutant adhesion domain is functional as determined by several experimental methods, and the size of its binding site has been probed by chemical shift perturbations in NMR titration experiments. The new structural information supports a 'hand-shake' model of CD2-CD58 interaction involving the GFCC'C" faces of both CD2 and CD58 adhesion domains. The region responsible for binding specificity is most likely localized on the C, C' and C" strands and the C-C' and C'-C" loops on CD58. (+info)
Inhibition of Rho at different stages of thymocyte development gives different perspectives on Rho function.
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Development of thymocytes can be staged according to the levels of expression of the cell-surface markers CD4, CD8, CD44, CD25 and CD2. Thymocyte development is regulated by a complex signalling network [1], one component of which is the GTPase Rho. The bacterial enzyme C3 transferase from Clostridium botulinum selectively ADP-ribosylates Rho in its effector-binding domain and thereby abolishes its biological function [2,3]. To explore the function of Rho in thymocyte development, we previously used the proximal promoter of the gene encoding the Src-family kinase p56lck to make transgenic mice that selectively express C3 transferase in the thymus [4,6]. In these mice, which lack Rho function from the earliest thymocyte stages, thymocyte numbers are reduced by approximately 50- to 100-fold. Here, we describe transgenic mice that express C3 transferase under the control of the locus control region (LCR) of the CD2 gene; this regulatory element drives expression at a later stage of thymocyte development than the lck proximal promoter [7]. In these mice, thymocyte numbers were also reduced by 50- to 100-fold, but unlike the lck-C3 mice, in which the reduction predominantly results from defects in cell survival of CD25(+) thymocyte progenitors, the CD2-C3 transgenic mice had a pre-T-cell differentiation block at the CD25(+) stage after rearrangement of the T-cell receptor (TCR) beta chains. Analysis of CD2-C3 mice demonstrated that Rho acts as an intracellular switch for TCR beta selection, the critical thymic-differentiation checkpoint. These results show that Rho-mediated survival signals for CD25(+) pre-T cells are generated by the extracellular signals that act on earlier thymocyte precursors and also that temporal cell-type-specific elimination of Rho can reveal different functions of this GTPase in vivo. (+info)
Structure of a heterophilic adhesion complex between the human CD2 and CD58 (LFA-3) counterreceptors.
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Interaction between CD2 and its counterreceptor, CD58 (LFA-3), on opposing cells optimizes immune recognition, facilitating contacts between helper T lymphocytes and antigen-presenting cells as well as between cytolytic effectors and target cells. Here, we report the crystal structure of the heterophilic adhesion complex between the amino-terminal domains of human CD2 and CD58. A strikingly asymmetric, orthogonal, face-to-face interaction involving the major beta sheets of the respective immunoglobulin-like domains with poor shape complementarity is revealed. In the virtual absence of hydrophobic forces, interdigitating charged amino acid side chains form hydrogen bonds and salt links at the interface (approximately 1200 A2), imparting a high degree of specificity albeit with low affinity (K(D) of approximately microM). These features explain CD2-CD58 dynamic binding, offering insights into interactions of related immunoglobulin superfamily receptors. (+info)
Dependence of T cell antigen recognition on the dimensions of an accessory receptor-ligand complex.
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The T cell antigen receptor (TCR) and its ligand peptide-major histocompatibility complex (MHC) are small (approximately 7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23-50 nm). We have proposed that molecules at the T cell/antigen-presenting cell (APC) interface segregate according to size, with small "accessory" molecules (e.g., CD2, CD4, CD8, CD28, and CD154) contributing to the formation of a close-contact zone, within which the TCR engages peptide-MHC, and from which large molecules are excluded (Davis, S.J., and P.A. van der Merwe. 1996. Immunol. Today. 17:177-187). One prediction of this model is that increasing the size of these small accessory molecules will disrupt their function. Here, we test this prediction by varying the dimensions of the CD2 ligand, CD48, and examining how this affects T cell antigen recognition. Although the interaction of CD2 on T cells with wild-type or shortened forms of CD48 on APCs enhances T cell antigen recognition, the interaction of CD2 with elongated forms of CD48 is strongly inhibitory. Further experiments indicated that elongation of the CD2/CD48 complex inhibited TCR engagement of peptide-MHC, presumably by preventing the formation of sufficiently intimate contacts at the T cell/APC interface. These findings demonstrate the importance of small size in CD2/CD48 function, and support the hypothesis that T cell antigen recognition requires segregation of cell surface molecules according to size. (+info)
Tumor therapy with bispecific antibody: the targeting and triggering steps can be separated employing a CD2-based strategy.
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For tumor therapy with unprimed effector cells, we developed a novel combination of a CD2 x tumor Ag bispecific targeting Ab and an anti-CD2 triggering Ab. These Ab constructs were derived from two novel CD2 mAbs, termed M1 and M2 that, together, but not individually activate T cells. Unlike many other CD2 Abs, M1 and M2 do not interfere with TCR/CD3 triggering nor do they inhibit binding of CD2 to its ligand CD58, thus preserving the physiological functions of these important effector cell molecules. M2 was chemically conjugated with an Ab recognizing the epidermal growth factor-receptor (EGF-R). Incubation of unprimed peripheral blood mononuclear cells with the bispecific F(ab')2 construct (M2xEGF-R) in the presence of trigger Ab M1 led to efficient selective lysis of EGF-R-positive targets by CTL and NK cells. Importantly, the need for trigger Ab M1 for effector cell stimulation allowed to separate targeting from triggering steps in vitro and should thus enable to focus immune responses to sites of target Ag expression in vivo. (+info)
Ly49A inhibitory receptors redistribute on natural killer cells during target cell interaction.
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When T effector cells meet antigen-bearing target cells, there is a specific accumulation of T-cell receptors, co-receptors and structural proteins at the point of cell-cell contact. Ly49 inhibitory receptors bind to murine major histocompatibility complex (MHC) class I molecules and prevent natural killer-(NK) cell cytotoxicity. In this study we have tested whether inhibitory receptors accumulate at the point of cell-cell contact when NK cells encounter target cells bearing MHC class I ligands for those inhibitory receptors. We have used RNK-16 effector cells that express Ly49A receptors and have found that there was a specific accumulation of Ly49A receptors at the point of NK cell-target cell contact when the target cells expressed H-2Dd. We also observed that engagement of Ly49A on NK cells resulted in an altered redistribution of potential triggering receptors CD2 and NKR-P1. These data indicate that inhibitory receptors, like activating receptors, may specifically aggregate at the point of cell-cell contact which may be necessary for them to mediate their full inhibitory effect. (+info)
Human endothelial cells augment early CD40 ligand expression in activated CD4+ T cells through LFA-3-mediated stabilization of mRNA.
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Human endothelial cells (EC) augment CD40 ligand (CD40L) expression on PHA-activated CD4+ T cells at early times (e.g., 4-6 h). Fixed EC, devoid of mRNA, are comparable to living EC in their capacity to augment early CD40L expression on CD4+ T cells. Fixed EC increase T cell mRNA expression of both IL-2 and CD40L compared with PHA alone at 6 h. EC are unable to increase the rate of transcription of CD40L compared with PHA alone as measured with a promoter-reporter gene, although they do increase transcription of an IL-2 promoter-reporter gene. Fixed EC prolong the half-life of CD40L mRNA >2-fold. Inclusion of anti-human LFA-3 (CD58) mAb or pretreatment of EC with an LFA-3 antisense oligonucleotide blocks EC-induced increases in CD40L expression, whereas mAb to ICAM-1 or pretreatment with ICAM-1 antisense oligonucleotide does not. Moreover, mAb to LFA-3 reverses the capacity of EC to prolong the half-life of CD40L mRNA, whereas mAb to ICAM-1, even in combination with mAb to ICAM-2, does not. We conclude that EC use LFA-3 to increase early CD40L protein expression on newly activated CD4+ T cells by stabilizing CD40L mRNA. (+info)
Better-surviving liver grafts by the injection of anti-CD2 antibody: the important roles of host CD8+ and CD2+CD28+ T cells in chronic graft rejection and beta type platelet-derived growth factor receptor (PDGFR-beta) expression on apoptotic liver grafts.
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Syngeneic liver grafts were implanted in the livers of 22 LEW/Sea strain rats. To prolong the graft survival, anti-CD2 monoclonal antibody (MAb) or anti beta type platelet-derived growth factor receptor (PDGFR-beta) antibody (Ab) was injected, or splenectomy was performed in the rats which were then followed until 10 to 11 weeks posttransplantation. The 22 rats with chronic graft rejection showed increased CD8a-like antigen (probably Fas ligand) on the peripheral blood T cells. All the liver grafts had both necrosis and apoptosis. The liver graft apoptosis was indicated by histopathological abnormalities, and by DNA strand breaks and hemosiderin depositions in the cytoplasm. PDGFR-beta expression in the apoptotic liver graft was demonstrated immunohistochemically. Among the 17 rats injected with anti-CD2 MAb, CD2 signaling on host T cells was effectively suppressed by the injection of anti-CD2 MAb in 4 rats with better-surviving liver grafts. In these 4 rats, CD28 antigen on thymic lymphocytes was down-modulated and high numbers (136-233-positive cells per lobe) of the epithelial reticular cells with apoptotic lymphocytes were counted. Anti-PDGFR-beta Ab caused high pulmonary secretions of growth factors and reticular fibrosis in the lungs of 5 rats injected with the Ab. Anti-PDGFR-beta Ab injection reduced the host cell apoptosis in the lung and thymus, but did not prolong the survival of liver grafts. In the 9 rats with both splenectomy and anti-CD2 MAb injection, pulmonary apoptosis was induced with the 6-16% reductions of CD4+ lymphocytes. Prolonged graft survival was observed in only one of the 9 rats. Anti-CD2 MAb was effective for prolonging the liver graft survival with suppressed CD28 antigen, but anti-PDGFR-beta Ab and splenectomy were not. (+info)