CD154 variants associated with hyper-IgM syndrome can form oligomers and trigger CD40-mediated signals.

X-linked hyper-IgM syndrome is a rare immunodeficiency disorder resulting from mutations in the gene encoding the CD40 ligand (CD154) molecule. These mutations are very heterogeneous, ranging from a single point mutation to a large deletion in the open reading frame. To investigate the molecular mechanisms that are responsible for the functional defect of these mutants, we examined the biochemical properties of 14 hyper-IgM-related CD154 mutant proteins produced by transient expression in COS7 cells. We show that deletion mutants lacking a significant portion of the tumor necrosis factor homologous domain cannot be stably produced. In contrast, point mutants can be detected as oligomers. Surprisingly, gene products of two point mutants, Thr-211 --> Asp and Met-36 --> Arg, can bind to the receptor, CD40. For Thr-211 --> Asp variant, it is comparable to the wild-type protein in its surface expression level, biochemical structure, and functional activities. Thus, it appears that this mutation is a polymorphism of CD154 gene. For Met-36 --> Arg variant, although it is interactive with CD40, it has a much lower surface expression level than wild-type protein. We propose that Met-36 --> Arg mutant represents a prototype of a defective CD154 family whose low cell surface expression of intrinsically active protein is simply insufficient to trigger productive signals through CD40.

A humanized form of a CD4-specific monoclonal antibody exhibits decreased antigenicity and prolonged plasma half-life in rhesus monkeys while retaining its unique biological and antiviral properties.

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.

Heteromultimeric complexes of CD40 ligand are present on the cell surface of human T lymphocytes.

CD40 ligand (CD40L), a 33-kDa type II membrane glycoprotein expressed primarily on activated CD4+ T lymphocytes, is responsible for the helper function of T cells on resting B cells in a non-antigen-dependent, non-major histocompatability complex-restricted fashion. Interaction of CD40L with its receptor CD40 induces proliferation of and isotype switching in B lymphocytes. Recently we solved the x-ray structure of recombinant soluble CD40L and showed that, similar to other members of the tumor necrosis factor family, CD40L indeed exists as a trimer. We now report that, under normal physiological conditions, CD40L molecules exist as heteromultimeric complexes. These CD40L complexes, made of the full length and smaller fragments of CD40L, are present on the cell surface of T lymphocytes and are capable of interacting with CD40 molecule. A prominent fragment with a mass of 31 kDa accounts for as much as half of the CD40L on the surface of Jurkat cells. N-terminal sequence data revealed that this fragment lacks the cytoplasmic tail. A minor 18-kDa fragment of CD40L was also characterized which lacks the cytoplasmic tail, transmembrane region, and stalk region of the extracellular domain. The presence of CD40L heteromultimeric variants implies an additional regulation of the functional activity of this ligand complex.

Vascular cell adhesion molecule-Ig fusion protein selectively targets activated alpha 4-integrin receptors in vivo. Inhibition of autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice.

The alpha 4 beta 1-integrin (CD49d, CD29) constitutively expressed on leukocytes regulates cell migration to inflammatory sites, cell activation, and development through its interactions with two alternate ligands, vascular cell adhesion molecule-1 (VCAM-1; CD106) expressed on cytokine-activated endothelium, dendritic and stromal cells, and the extracellular matrix protein fibronectin. Another alpha 4-integrin receptor, alpha 4 beta 7, expressed on leukocytes also binds VCAM-1 and fibronectin (FN), and controls homing to mucosal tissues through its interactions with mucosal vascular addressin MAdCAM-1. In vitro studies have shown that alpha 4-dependent cell adhesion is regulated by the activation state of the cell and by divalent cations. However, the existence and role of cells with different alpha 4 activation states in vivo have not been defined. Herein we show that a soluble ligand with the two N-terminal domains of human VCAM-1 fused to a human IgG1 constant region, VCAM-Ig, binds selectively to activated alpha 4-receptors on murine cells, such as those induced by Mn2+ in vitro. To determine whether the cells identified by VCAM-Ig were required under physiologic conditions, we assessed its anti-inflammatory effect. We show that VCAM-Ig is not bound to the majority of murine alpha 4+ cells after in vivo administration, yet it significantly delays the onset of adoptively transferred autoimmune diabetes. Thus, soluble VCAM-Ig can modify alpha 4-dependent disease progression, apparently by its selective action on cells with activated alpha 4-integrin receptors, thereby providing evidence for distinct alpha 4 activation states in vivo.

Characterization of surface lymphotoxin forms. Use of specific monoclonal antibodies and soluble receptors.

Lymphotoxin (LT) is a cytokine related to TNF, found in human systems in both secreted and membrane bound forms. The well characterized secreted form is a trimer of a single protein, LT-alpha, whereas the surface form is composed of a complex between two related molecules, LT-alpha and LT-beta. Because there is a distinct receptor for the complex, the membrane form is believed to signal via events different from those elicited by TNF and secreted LT-alpha. By using a battery of anti-LT-alpha and LT-beta mAbs, it is clear that two LT surface forms exist on the surface of PMA-activated II-23 cells, a human T cell hybridoma. Assuming that these surface forms are trimers, a minor form appears early after induction having an apparent stoichiometry of LT-alpha 2/beta 1 and is recognized by one group of anti-LT-alpha mAbs and the p55-TNF receptor. The second and predominant form has an apparent LT-alpha 1/beta 2 composition and is recognized by a second group of pantrophic anti-LT-alpha mAbs and the LT-beta receptor. Neither of the heteromeric forms nor a putative LT-beta homotrimeric form were found to be secreted. The properties of surface LT on the II-23 cell system were similar to those of the surface LT forms on Chinese hamster ovary cells transfected with both LT-alpha and LT-beta genes and a number of lymphoid tumor lines. These experiments point toward the LT-alpha 1/beta 2 complex as the predominant membrane form of LT on the lymphocyte surface, and this complex is the primary ligand for the LT-beta receptor.

A lymphotoxin-beta-specific receptor.

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LT-alpha) are members of a family of secreted and cell surface cytokines that participate in the regulation of immune and inflammatory responses. The cell surface form of LT-alpha is assembled during biosynthesis as a heteromeric complex with lymphotoxin-beta (LT-beta), a type II transmembrane protein that is another member of the TNF ligand family. Secreted LT-alpha is a homotrimer that binds to distinct TNF receptors of 60 and 80 kilodaltons; however, these receptors do not recognize the major cell surface LT-alpha-LT-beta complex. A receptor specific for human LT-beta was identified, which suggests that cell surface LT may have functions that are distinct from those of secreted LT-alpha.