Specific interaction of lymphocyte function-associated antigen 3 with CD2 can inhibit T cell responses.

Accessory cell surface molecules, such as T cell antigen CD2 and its ligand lymphocyte function-associated antigen 3 (LFA-3; CD58), are critical costimulatory pathways for optimal T cell activation in response to antigens. Interaction of CD2 with cell surface LFA-3 not only increases T cell/accessory cell adhesion, but also induces signal transduction events involved in the regulation of T cell responses. In this report, we show that specific interactions of LFA-3 with CD2 can result in T cell unresponsiveness to antigenic or mitogenic stimuli in vitro. By deletion of certain regions of the extracellular domain of LFA-3, we localized the CD2 binding site to the first domain of LFA-3. We then demonstrated that a soluble, purified first domain-LFA-3/IgG1 fusion protein (LFA3TIP) interacts with CD2 and binds to the same CD2 epitope as purified multimeric or cell surface-expressed LFA-3. LFA3TIP inhibits tetanus toxoid, hepatitis B surface antigen, anti-CD3 mAb, Con A, and phytohemagglutinin P-induced T cell proliferation, as well as xenogeneic and allogeneic mixed lymphocyte reactions (MLR). Unlike anti-LFA-3 or anti-CD2 monoclonal antibodies (mAbs) which inhibit T cell responses by blocking LFA-3/CD2 binding, LFA3TIP is capable of rendering T cells unresponsive to antigenic stimuli in situations where T cell activation is independent of CD2/LFA-3 interactions. Furthermore, LFA3TIP, but not blocking anti-CD2 mAbs, is capable of inducing T cell unresponsiveness to secondary stimulation in allogeneic MLR. This inhibition of T cell responses by LFA3TIP occurs through a different mechanism from that of mAbs to LFA-3 or CD2.

BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

CD4 T cell subsets in the mucosa are CD28+Ki-67-HLA-DR-CD69+ but show differential infection based on alpha4beta7 receptor expression during acute SIV infection.

BACKGROUND: CD4 T cell depletion in the mucosa has been well documented during acute HIV and SIV infections. The demonstration the HIV/SIVcan use the alpha4beta7 receptor for viral entry suggests that these viruses selectively target CD4 T cells in the mucosa that express high levels of alpha4beta7 receptor. METHODS: Mucosal samples obtained from SIV infected rhesus macaques during the early phase of infection were used for immunophenotypic analysis. CD4 T cell subsets were sorted based on the expression of beta7 and CD95 to quantify the level of SIV infection in different subsets of CD4 T cells. Changes in IL-17, IL-21, IL-23 and TGFbeta mRNA expression was determined using Taqman PCR. RESULTS: CD4 T cells in the mucosa were found to harbor two major population of cells; -25% of CD4 T cells expressed the alpha4(+)beta7(hi) phenotype, whereas the rest of the 75% expressed an alpha4(+)beta7(int) phenotype. Both the subsets were predominantly CD28(+)Ki-67(-)HLA-DR(-) but CD69(+), and expressed detectable levels of CCR5 on their surface. Interestingly, however, alpha4(+)beta7(hi)CD4 T cells were found to harbor more SIV than the alpha4(+)beta7(int) subsets at day 10 pi. Early infection was associated with a dramatic increase in the expression of IL-17, and IL-17 promoting cytokines IL-21, IL-23, and TGFbeta that stayed high even after the loss of mucosal CD4 T cells. CONCLUSIONS: Our results suggest that the differential expression of the alpha4beta7 receptor contributes to the differences in the extent of infection in CD4 T cell subsets in the mucosa. Early infection is associated dysregulation of the IL-17 network in mucosal tissues involves other non-Th-17 cells that likely contributes to the pro-inflammatory environment in the mucosa during acute stages of SIV infection.

Anti-retroviral therapy fails to restore the severe Th-17: Tc-17 imbalance observed in peripheral blood during simian immunodeficiency virus infection.

BACKGROUND: Human immuno deficiency virus and simian immunodeficiency virus infections are characterized by a severe loss of Th-17 cells (IL-17(+)CD4(+) T cells) that has been associated with disease progression and systemic dissemination of bacterial infections. Anti-retroviral therapy (ART) has led to repopulation of CD4(+) T cells in peripheral tissues with little sustainable repopulation in mucosal tissues. Given the central importance of Th-17 cells in mucosal homeostasis, it is not known if the failure of ART to permanently repopulate mucosal tissues is associated with a failure to restore Th-17 cells that are lost during infection. METHODS: Dynamics of alpha4(+)beta7(hi) CD4(+) T cells in peripheral blood of SIV infected rhesus macaques were evaluated and compared to animals that were treated with ART. The frequency of Th-17 and Tc-17 cells was determined following infection and after therapy. Relative expression of IL-21, IL-23, and TGFbeta was determined using Taqman PCR. RESULTS: Treatment of SIV infected rhesus macaques with anti-retroviral therapy was associated with a substantial repopulation of mucosal homing alpha4(+)beta7(hi)CD4(+) T cells in peripheral blood. This repopulation, however, was not accompanied by a restoration of Th-17 responses. Interestingly, SIV infection was associated with an increase in Tc-17 responses (IL-17(+)CD8(+) T cells) suggesting to a skewing in the ratio of Th-17: Tc-17 cells from a predominantly Th-17 phenotype to a predominantly Tc-17 phenotype. Surprisingly, Tc-17 responses remained high during the course of therapy suggesting that ART failed to correct the imbalance in Th-17 : Tc-17 responses induced following SIV infection. CONCLUSIONS: ART was associated with substantial repopulation of alpha4(+)beta7(hi) CD4(+) T cells in peripheral blood with little or no rebound of Th-17 cells. On the other hand, repopulation of alpha4(+)beta7(hi) CD4(+) T cells was accompanied by persistence of high levels of Tc-17 cells in peripheral blood. The dysregulation of Th-17 and Tc-17 responses likely plays a role in disease progression.

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.

Vascular cell adhesion molecule (VCAM)-Ig fusion protein defines distinct affinity states of the very late antigen-4 (VLA-4) receptor.

The Very Late Antigen-4 receptor (VLA-4) (alpha 4 beta 1) is constitutively expressed on leukocytes and plays a role in cell trafficking, activation and development through its interaction with two alternative ligands, Vascular Cell Adhesion Molecule (VCAM-1) and fibronectin (FN). VLA-4-dependent cell adhesion is augmented by various stimuli, such as divalent cations, certain beta 1-specific monoclonal antibodies (mAbs) and cell activation. However, the steps of the adhesive process which they affect are currently undefined. In order to investigate whether or not these stimuli affect the primary step, VLA-4/ligand binding, we employed a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for VLA-4. Using this soluble ligand, we have directly demonstrated that the VLA-4 receptor can exist in at least three different affinity states on the cell surface. Two distinct high affinity states are induced on normal peripheral blood T cells, one by the anti-beta 1 mAb TS2/16, and one of 15-20 fold higher affinity by the divalent cation Mn2+. Interestingly, activation through the T cell receptor (TcR), through CD31 or by the Macrophage Inflammatory Protein-1 beta chemokine (MIP-1 beta) do not detectably increase VLA-4 affinity although they do augment VLA-4 dependent cell adhesion in vitro. Thus, VCAM-Ig binding defines high affinity VLA-4 receptors, revealing unique effects of the TS2/16 mAb and Mn2+ cations in vitro, and distinguishes VLA-4/VCAM interactions from subsequent steps in cell adhesion.

Expression and functional characterization of a soluble form of endothelial-leukocyte adhesion molecule 1.

Endothelial leukocyte adhesion molecule 1 (ELAM1) is a leukocyte adhesion molecule induced on human venular endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for ELAM1 has been constructed, stably expressed in Chinese hamster ovary cells, and the secreted recombinant soluble form of ELAM1 (rsELAM1) purified to homogeneity by immunoaffinity chromatography. rsELAM1, when immobilized on plastic, is fully functional as an adhesion protein, and selectively binds only cells known to bind cell-surface ELAM1 expressed on human endothelial cells, including the myelomonocytic cell line HL60 and the colon carcinoma cell line HT29. Immobilized rsELAM1 also binds human PMN, monocytes, NK cells, and T cells. T cell subset analyses indicate preferential binding of CD4+ T memory cells. However, rsELAM1 is only a weak inhibitor of ELAM1-mediated adhesion. rsELAM1 should prove valuable for the further study of the role of ELAM1 expressed on the vascular wall during the inflammatory response.

Identification of a palmitic acid-modified form of human Sonic hedgehog.

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the alpha-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D.

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM /=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.