The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d–specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-γ production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

Blockade of the CD40 pathway with anti-CD40 mAb is immunosuppressive in a large animal, preclinical renal transplant model. The potential effect of this therapy on viral immune responses will be important to consider for the design of safe clinical trials.

Interactions between gp39 (CD40L, TRAP, T-BAM) on activated T cells and CD40 on antigen-presenting cells play an important role in regulating antibody production by B cells, cytokine production by monocytes, and other immune responses which require T cell "help". Using structure-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD40 and gp39 surface residues which are important for receptor-ligand binding. Binding studies with CD40 or gp39 proteins containing single and double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y145 are important for CD40 binding. Analysis of the location of amino acid substitutions in the naturally occurring gp39 mutants expressed by the X-linked hyper-IgM (X-HIM) patients studied to date indicated the E129/G substitution found in the S128/R-E129/G double mutant affects a solvent-accessible residue which might participate in CD40/gp39 binding. Binding studies with E129/G and E129/A gp39 point mutants showed that this residue does not contribute directly to CD40/gp39 binding but that its substitution with a glycine disrupts the gp39 structure. Comparison of the gp39 and CD40 residues involved in receptor-ligand contacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues from contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction.

Activated leukocyte cell adhesion molecule (ALCAM)was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain. Ig supergene family (IgSF)1 members have been shown to interact with a wide variety of other molecules, including integrins, cytokines, and other IgSF members. Many of these interactions are mediated through protein-protein contacts, although a subset of these proteins, known as sialoadhesins, recognize sialic acid (1). Recently, we have reported on a novel interaction between an IgSF member, activated leukocyte cell adhesion molecule (ALCAM), and a member of the scavenger receptor cysteine-rich (SRCR) family of proteins, CD6 (2). Soluble recombinant proteins consisting of the extracellular domains of either ALCAM or CD6 fused to human IgG1 constant domains were shown to specifically bind to COS cell transfectants expressing CD6 or ALCAM, respectively.ALCAM is a type I membrane protein whose extracellular domain is composed of five Ig-like domains: two amino-terminal V set Ig domains followed by three domains of the C2 set, a hydrophobic transmembrane domain, and a short cytoplasmic anchor sequence (2). ALCAM is also known as SC-1/DM-GRASP/ BEN in the chicken (3-5) and as KG-CAM in the rat (6). The chicken counterpart of ALCAM is a neural adhesion molecule capable of supporting neurite outgrowth (4, 5). Data from the chicken indicate that ALCAM is capable of homophilic interactions (4, 5), and the possibility of such interactions has also been suggested on the basis of molecular modeling (7). We have previously reported that COS cells that expressed CD6 were able to bind to ALCAM positive thymic epithelial cells, which suggested that CD6 and ALCAM binding can mediate adhesive interactions between thymocytes and thymic epithelial cells (2).CD6, also a type I membrane protein (8), is expressed by thymocytes, T cells, a subset of...

Binding studies with a CD6 immunoglobulin fusion protein (CD6 Rg) resulted in the identification and cloning of a CD6 ligand. This ligand was found to be a member of the immunoglobulin supergene family and was named ALCAM (activated leukocyte cell adhesion molecule). Cell adhesion assays showed that CD6-ALCAM interactions mediate thymocyte-thymic epithelium cell binding. ALCAM is also expressed by activated leukocytes and neurons and may be involved in interactions between T cells and activated leukocytes and between cells of the immune and nervous systems, respectively. Herein we describe the preparation of domain-specific murine CD6 Rg fusion proteins and show that the membrane-proximal SRCR (scavenger receptor cysteine-rich) domain of CD6 contains the ALCAM binding site. We also show that mAbs which bind to this domain preferentially block CD6-ALCAM binding. These results demonstrate that the membrane-proximal SRCR domain of CD6 is necessary for CD6 binding to ALCAM and provide the first direct evidence for the interaction of an SRCR domain with a ligand.

The interaction between gp39 (CD40L, TRAP, T-BAM) on activated T cells and mast cells and CD40 on antigen-presenting cells modulates immune responses. Gp39 and CD40 are homologous to tumor necrosis factor (TNF) and its receptor (TNFR), respectively. The TNF-beta/TNFR interaction has been analyzed on the basis of mutagenesis experiments and crystal structures. Using the interaction of TNF-beta/TNFR as a guide, we previously reported a site-directed mutagenesis study in which we identified residues in gp39 (K143, Y145) and CD40 (Y82, D84, N86) involved in gp39/CD40 interactions. Here we describe the use of the TNF-beta/TNFR complex crystal structure as a template to prepare molecular models of gp39, CD40, and their approximate interaction. The application of these models has allowed us to extend our mutagenesis analysis of gp39/CD40 interactions. These experiments have led to the identification of additional gp39 (Y146, R203, Q220) and CD40 (E74, E117) residues that contribute to the gp39/CD40 interaction. We also further explored the importance of gp39 residue Y145 and CD40 residue Y82 for the gp39/CD40 interaction by conservatively replacing these residues with Phe. The results of these studies have enabled us to approximately outline the binding sites in gp39 and CD40. It appears that the gp39/CD40 interaction is centered on at least two clusters of residues and involves residues of two adjacent gp39 monomers. The molecular regions involved in the gp39/CD40 interaction essentially correspond to those in the homologous TNF-beta/TNFR system.

Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen CD6. Herein, we report on the isolation and characterization of cDNA clones encoding mouse ALCAM (mALCAM). Comparison of the predicted amino acid sequence of mALCAM and human ALCAM (hALCAM) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of mALCAM showed that the CD6 binding site is located in the N-terminal Ig-like domain and that mALCAM is capable of binding both human and mouse CD6. Mutagenesis studies on hALCAM suggested that residues critical for CD6 binding map to the predicted A'GFCC'C" beta-sheet of ALCAM's N-terminal binding domain. Residue differences in the N-terminal domains of mALCAM and hALCAM were analyzed with the aid of a molecular model of ALCAM. All residues critical for CD6 binding are conserved in both mALCAM and hALCAM, whereas residue differences map to the predicted BED face which is opposite the CD6 binding site on hALCAM. These findings provide a molecular rationale for the observed cross-species CD6/ALCAM interaction and the apparent inability to generate monoclonal antibodies (mAb) against the CD6 binding site. RNA blot analysis showed that mRNA transcripts encoding mALCAM are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for mALCAM was produced and by two-color immunofluorescence studies was shown to bind to both activated CD4+ and CD8+ T cells.

Human thymus-dependent lymphocytes (T cells) are defined by their ability to form spontaneous rosettes (E-rosettes) with sheep erythrocytes (SRBC) (1-3). Recently, several murine monoclonal antibodies that recognize distinct differentiation antigens on human T cells have been described (4-11). We report here a monoclonal antibody, designated 9.6, that identifies a 50,000-dalton surface protein that appears to be present on all E-rosette-forming cells. Blocking and lysostripping experiments indicate that this monoclonal antibody reacts either with the E receptor itself or with a closely associated structure. Materials and MethodsCells. Mononuclear cells from peripheral blood (PBL) or bone marrow were obtained from normal volunteers and isolated by centrifugation over Ficoll-Hypaque (LSM; Litton Bionetics Inc., Kensington, Md.). T cells were enumerated by rosetting with 2-aminoethylisothiouronium bromide hydrobromide (AET)-treated SRBC (SRBC^v..r) in medium (RPMI-1640) with 12% fetal calf serum (12). For the isolation of purified T cells, PBL were passed over nylon wool (11) and then incubated with SRBCA~--v. Rosette-forming T cells (E +) were separated from non-T cells (E-) by centrifugation over Ficoll-Hypaque. T cells were recovered from the rosettes by lysis of SRBC in Tris-buffered 0.83% ammonium chloride. Normal human thymocytes were prepared from thymus specimens obtained in the course of corrective open heart surgery from children < 14 yr old. Long-term-cultured T cells, stimulated by alloantigen, were maintained in medium supplemented with T cell growth factor (TCGF) (13).A selected panel of cultured lymphoid cell lines was used for serological analysis. Included were leukemic T cell lines (CEM, HSB2, 8402, Jurkat, Molt-4F, and KE37), the leukemic pre-B cell line NALM-6, Epstein-Barr virus-transformed B-lymphoid cell lines SB, 8392, HA, Swei), and the Burkitt's lymphoma ceil line Daudi.Leukemic blasts from peripheral blood of patients with acute lymphocytic leukemia (ALL), were separated over Ficoll-Hypaque, and cryopreserved. Cells were obtained either at presentation or relapse when the leukocyte count was >20,000/mm s and >90% of the cells were blasts. Subdivision of ALL into T and null cell types was based on the clinical criterion of a thymic mass, and on determination of whether the leukemic blasts formed E-rosettes or expressed ialike antigen.