Intracellular adhesion molecule-1 modulates beta-chemokines and directly costimulates T cells in vivo.
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The potential roles of adhesion molecules in the expansion of T cell-mediated immune responses in the periphery were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the adhesion molecules intracellular adhesion molecule-1 (ICAM-1), lymphocyte function associated-3 (LFA-3), and vascular cell adhesion molecule-1 (VCAM-1) along with DNA immunogens, and we analyzed the resulting antigen-specific immune responses. We observed that antigen-specific T-cell responses can be enhanced by the coexpression of DNA immunogen and adhesion molecules ICAM-1 and LFA-3. Coexpression of ICAM-1 or LFA-3 molecules along with DNA immunogens resulted in a significant enhancement of T-helper cell proliferative responses. In addition, coimmunization with pCICAM-1 (and more moderately with pCLFA-3) resulted in a dramatic enhancement of CD8-restricted cytotoxic T-lymphocyte responses. Although VCAM-1 and ICAM-1 are similar in size, VCAM-1 coimmunization did not have any measurable effect on cell-mediated responses. These results suggest that ICAM-1 and LFA-3 provide direct T-cell costimulation. These observations are further supported by the finding that coinjection with ICAM-1 dramatically enhanced the level of interferon-gamma (IFN-gamma) and beta-chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated on activation normal T-cell expression and secreted (RANTES) produced by stimulated T cells. Through comparative studies, we observed that ICAM-1/LFA-1 T-cell costimulatory pathways are independent of CD86/CD28 pathways and that they may synergistically expand T-cell responses in vivo. (+info)
Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-A resolution.
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The binding of the cell surface molecule CD58 (formerly lymphocyte function-associated antigen 3) to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T cells. This was the first heterophilic cell adhesion interaction to be discovered and is now an important paradigm for analyzing the structural basis of cell-cell recognition. The crystal structure of a CD2-binding chimeric form of CD58, solved to 1.8-A resolution, reveals that the ligand binding domain of CD58 has the expected Ig superfamily V-set topology and shares several of the hitherto unique structural features of CD2, consistent with previous speculation that the genes encoding these molecules arose via duplication of a common precursor. Nevertheless, evidence for considerable divergence of CD2 and CD58 is also implicit in the structures. Mutations that disrupt CD2 binding map to the highly acidic surface of the AGFCC'C" beta-sheet of CD58, which, unexpectedly, lacks marked shape complementarity to the equivalent, rather more basic CD58-binding face of human CD2. The specificity of the very weak interactions of proteins mediating cell-cell recognition may often derive largely from electrostatic complementarity, with shape matching at the protein-protein interface being less exact than for interactions that combine specificity with high affinity, such as those involving antibodies. (+info)
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)
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)
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)
A triad of costimulatory molecules synergize to amplify T-cell activation.
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The activation of a T cell has been shown to require two signals via molecules present on professional antigen-presenting cells: signal 1, via a peptide/MHC complex; and signal 2, via a costimulatory molecule. Here, the role of three costimulatory molecules in the activation of T cells was examined. Poxvirus (vaccinia and avipox) vectors were used because of their ability to efficiently express multiple genes. Murine cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induced the activation of T cells to a far greater extent than cells infected with any one or two costimulatory molecules. Despite this T-cell "hyperstimulation" using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Results using the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell:T-cell ratios. Experiments using a four-gene construct also showed that TRICOM recombinants can enhance antigen-specific T-cell responses in vivo. These studies thus demonstrate for the first time the ability of vectors to introduce three costimulatory molecules into cells, thereby activating both CD4+ and CD8+ T-cell populations to levels greater than those achieved with the use of only one or two costimulatory molecules. This new threshold of T-cell activation has broad implications in vaccine design and development. (+info)
A space-time structure determination of human CD2 reveals the CD58-binding mode.
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We describe a procedure for a space-time description of protein structures. The method is capable of determining populations of conformational substates, and amplitudes and directions of internal protein motions. This is achieved by fitting static and dynamic NMR data. The approach is based on the jumping-among-minima concept. First, a wide conformational space compatible with structural NMR data is sampled to find a large set of substates. Subsequently, intrasubstate motions are sampled by using molecular dynamics calculations with force field energy terms. Next, the populations of substates are fitted to NMR relaxation data. By diagonalizing a second moment matrix, directions and amplitudes of motions are identified. The method was applied to the adhesion domain of human CD2. We found that very few substates can account for most of the experimental data. Furthermore, only two types of collective motions have high amplitudes. They represent transitions between a concave (closed) and flat (open) binding face and resemble the change upon counter-receptor (CD58) binding. (+info)
Characterization of activated lymphocyte-tumor cell adhesion.
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This study demonstrates the variable expression of ICAM-1 and leukocyte function antigen-3 (LFA-3) on four tumor cell lines (COLO526, K562, Daudi, and HT-29). In addition, phorbol ester (PMA) activation of lymphocytes modulated LFA-1 from a uniform to a clustered surface distribution; whereas after treatment with high levels of Mg2+ ions, the unique epitope for high-affinity LFA-1 was identified using clone Mab24. Using a flow cytometric adhesion assay it was demonstrated that PMA-activated lymphocytes formed conjugates with COLO526 and Daudi, and that these conjugates were inhibited by anti-CD2 with varying inhibition by LFA-1 clones MHM24 and 25.3.1. When lymphocytes were induced to express the high-affinity form of LFA-1, conjugates were identified with COLO526, K562, and Daudi and these conjugates were sensitive to the presence of both CD2 and LFA-1 antibodies. Further studies using confocal microscopy confirmed significant adhesion between peripheral blood lymphocytes pretreated with either PMA or high levels of Mg2+ and the adherent cell line COLO526. In conclusion, this unique study has demonstrated for the first time the important role of the active form of LFA-1 on the lymphocyte cell surface for conjugate formation with an ICAM-1-expressing tumor cell; also, two pathways of cell signaling were identified for conjugate formation to occur. (+info)