Identification of a novel non-carbohydrate molecule that binds to the ribosomal A-site RNA

We report the identification of a novel compound that binds to the Escherichia coli 16S ribosomal A-site. Binding by the compound was observed using nuclear magnetic resonance and mass spectrometry techniques. We show that the compound binds in the same position in the A-site RNA as occupied by the aminoglycoside class of antibiotics.     

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.     

1F7, a novel cell surface molecule, involved in helper function of CD4 cells

We have developed a monoclonal antibody, anti-1F7, that inhibits soluble Ag-driven T cell proliferation as well as PWM-driven IgG synthesis. Anti-1F7 antibody reacts with approximately 57% of unfractionated T cells, 62% of CD4+ cells, and 54% of CD8+ cells. Although the 1F7 Ag is widely distributed among lymphoid cells, this Ag on CD4+ cells is preferentially expressed on the CDw29(4B4+) helper population. Moreover, anti-1F7 antibody further subdivides the CD4+CDw29+ cell subset into CDw29+1F7+ and CDw29+1F7- populations. The CD4+CDw29+1F7+ population of cells maximally proliferates to recall Ag such as tetanus toxoid, whereas helper function for PWM-driven IgG synthesis by B cells belongs to both the CD4+CDw29+1F7+ and CD4+CDw29+1F7- population of cells. The most prominent structure defined by this antibody is a 110-kDa molecule that is different from the 135-kDa, 160-kDa, and 185-kDa glycoproteins identified by anti-CDw29 antibody and the 180-kDa glycoprotein identified by UCHL-1 antibody. It is, however, related to the molecule recognized by anti-Ta1, an activation Ag on T cells. Furthermore, although the Ta1 molecule is recognized by anti-1F7 mAb, the 1F7 family of structures also includes molecules distinct from Ta1.     

Capping of the surface OKT3 binding molecule prevents the T-cell proliferative response to antigens: evidence that this molecule conveys the activation signal

The human T-lymphocyte receptor for antigen appears to have been localized to a cluster of associated surface glycoprotein molecules, among which is the OKT3 binding molecule. We have tested the hypothesis that selective removal of the OKT3 binding molecule eliminates the cellular immune response to antigens by clearing the surface of the molecule that conveys the activation signal. Enriched T cells were obtained from donors immune to purified protein derivative (PPD), streptokinase-streptodornase (SK-SD), or keyhole limpet hemocyanin (KLH). T-3 molecules were cleared from the cell surface by capping with OKT3 and F(ab')2 goat anti-mouse IgG. Regeneration of surface molecules was prevented by culturing the T-3 capped cells with OKT3 625 ng/ml. The capacity of capped T cells to proliferate in culture with antibody in response to antigens, alloantigens, and the mitogens, PHA and ionophore A23187, was compared to uncapped cells pretreated with media and to capped cells permitted to regenerate the OKT3 binding molecule. T-3 capped cells cultured in the presence of antibody failed to proliferate to antigens or alloantigens. However, T-3 capped cells cocultured with antibody also did not significantly proliferate to PHA, but did respond to A23187. In contrast, both media-treated T cells and cells which had regenerated the OKT3 binding molecule proliferated to mitogens, antigens, or alloantigens. The requirement for the OKT3 binding molecule was determined by utilizing T-1, T-4, and T-8 capped cells. T-1, T-4, or T-8 capped cells cultured in the presence of OKT1, OKT4, or OKT8 proliferated in response to antigens, alloantigens, and mitogens. These results demonstrate that in the absence of the OKT3 binding molecule, antigens, alloantigens, and PHA failed to induce a significant cellular proliferative response. In the absence of this molecule, PHA cannot bind to its carbohydrate moiety, and therefore cannot activate T cells to proliferate. These data support the concept that the molecule binding OKT3 conveys the transmembrane signal resulting in cellular activation.     

Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes

We have cloned a previously undescribed adhesion molecule, VCAM-1, which is induced by cytokines on human endothelial cells and binds lymphocytes. Using a novel method requiring neither monoclonal antibodies nor purified protein, VCAM-1-expressing clones were selected by adhesion to human lymphoid cell lines. VCAM-1 mRNA is present in endothelial cells at 2 hr after treatment with IL-1 or TNF-alpha and is maintained for at least 72 hr; leukocyte binding activity parallels mRNA induction. Cells transfected with VCAM-1 bind the human leukemia lines Jurkat, Ramos, Raji, HL60, and THP1, but not peripheral blood neutrophils. VCAM-1, which belongs to the immunoglobulin gene super-family, may be central to recruitment of mononuclear leukocytes into inflammatory sites in vivo.     

Measles virus exploits dendritic cells to suppress CD4+ T-cell proliferation via expression of surface viral glycoproteins independently of T-cell trans-infection

Dendritic cells (DC) have been proposed to play a pivotal role in transient immune suppression induced by measles virus (MV) infection. In the present study, we show that DC-induced suppression of T-cell proliferation was not mediated by IL-10 or IFNalpha/beta, which are released following infection of DC, but required cell contacts between MV-infected DC and T cells. Human sera containing neutralizing anti-MV antibodies, as well as anti-MV hemagglutinin (HA) or fusion protein (F) mAbs, were found (i) to reverse suppression and (ii) to restore DC allostimulatory capacity. Interestingly, DC-induced T-cell suppression was associated with both phenotypic and functional DC maturation, as demonstrated by IL-12 production and chemotaxis to MIP-3beta. These data suggest that MV infection turns on the maturation program of DC allowing migration to draining lymph nodes, where potent T-cell immune suppression might be achieved via cell surface expression of HA and F glycoproteins, independently of T cell trans-infection.     

DEAF-1, a novel protein that binds an essential region in a Deformed response element.

A 120 bp homeotic response element that is regulated specifically by Deformed in Drosophila embryos contains a single binding site for Deformed protein. However, a 24 bp sub-element containing this site does not constitute a Deformed response element. Specific activation requires a second region in the 120 bp element, which presumably contains one or more binding sites for Deformed cofactors. We have isolated a novel protein from Drosophila nuclear extracts which binds specifically to a site in this second region. This protein, which we call DEAF-1 (Deformed epidermal autoregulatory factor-1), contains three conserved domains. One of these includes a cysteine repeat motif that is similar to a motif found in Drosophila Nervy and the human MTG8 proto-oncoprotein, and another matches a region of Drosophila Trithorax. Mutations in the response element designed to improve binding to DEAF-1 in vitro resulted in increased embryonic expression. Conversely, small mutations designed to diminish binding to DEAF-1 resulted in reduced expression of the element. Thus, DEAF-1 is likely to contribute to the functional activity, and perhaps to the homeotic specificity, of this response element. Consistent with this hypothesis, we have discovered DEAF-1 binding sites in other Deformed response elements.     

Identification of tissue transglutaminase as a novel molecule involved in human CD8+ T cell transendothelial migration

During inflammation, T lymphocytes migrate out of the blood across the vascular endothelium in a multistep process. The receptors mediating T cell adhesion to endothelium are well characterized; however, the molecules involved in T cell transendothelial migration (TEM) subsequent to lymphocyte adhesion to the endothelium are less clear. To identify receptors mediating TEM, mAbs were produced against human blood T cells adhering to IFN-gamma-activated HUVEC in mice and tested for inhibition of lymphocyte TEM across cytokine-activated HUVEC. Most of the mAbs were against beta(1) and beta(2) integrins, but one mAb, 6B9, significantly inhibited T cell TEM across IFN-gamma, TNF-alpha, and IFN-gamma plus TNF-alpha-stimulated HUVEC, and did not react with an integrin. 6B9 mAb did not inhibit T cell adhesion to HUVEC, suggesting that 6B9 blocked a novel pathway in T cell TEM. The 6B9 Ag was 80 kDa on SDS-PAGE, and was expressed by both blood leukocytes and HUVEC. Immunoaffinity purification and mass spectrometry identified this Ag as tissue transglutaminase (tTG), a molecule not known to mediate T cell TEM. Treatment of HUVEC with 6B9 was more effective than treatment of T cells. 6B9 blockade selectively inhibited CD4(-), but not CD4(+), T cell TEM, suggesting a role for tTG in recruitment of CD8(+) T lymphocytes. Thus, 6B9 is a new blocking mAb to human tTG, which demonstrates that tTG may have a novel role in mediating CD8(+) T cell migration across cytokine-activated endothelium and infiltration of tissues during inflammation.     

Regression of Epstein-Barr virus-induced B-cell transformation in vitro involves virus-specific CD8+ T cells as the principal effectors and a novel CD4+ T-cell reactivity

T-cell memory to Epstein-Barr virus (EBV) was first demonstrated through regression of EBV-induced B-cell transformation to lymphoblastoid cell lines (LCLs) in virus-infected peripheral blood mononuclear cell (PBMC) cultures. Here, using donors with virus-specific T-cell memory to well-defined CD4 and CD8 epitopes, we reexamine recent reports that the effector cells mediating regression are EBV latent antigen-specific CD4+ and not, as previously assumed, CD8+ T cells. In regressing cultures, we find that the reversal of CD23+ B-cell proliferation was always coincident with an expansion of latent epitope-specific CD8+, but not CD4+, T cells; furthermore CD8+ T-cell clones derived from regressing cultures were epitope specific and reproduced regression when cocultivated with EBV-infected autologous B cells. In cultures of CD4-depleted PBMCs, there was less efficient expansion of these epitope-specific CD8+ T cells and correspondingly weaker regression. The data are consistent with an effector role for epitope-specific CD8+ T cells in regression and an auxiliary role for CD4+ T cells in expanding the CD8 response. However, we also occasionally observed late regression in CD8-depleted PBMC cultures, though again without any detectable expansion of preexisting epitope-specific CD4+ T-cell memory. CD4+ T-cell clones derived from such cultures were LCL specific in gamma interferon release assays but did not recognize any known EBV latent cycle protein or derived peptide. A subset of these clones was also cytolytic and could block LCL outgrowth. These novel effectors, whose antigen specificity remains to be determined, may also play a role in limiting virus-induced B-cell proliferation in vitro and in vivo.     

Identification and characterization of rat AILIM/ICOS, a novel T-cell costimulatory molecule, related to the CD28/CTLA4 family

Activation-inducible lymphocyte immuno-mediatory molecule (AILIM) is an inducible cell surface glycoprotein expressed on thymocytes and activated lymphocytes. Specific monoclonal antibody to rat AILIM induced the cell aggregation of a rat thymoma cell line and ConA-activated splenocytes. In the present study, we identified the primary structure of two species of rat AILIM by expression cloning. We also cloned mouse and human AILIM homologues and the predicted amino acid sequences were identical to those of the inducible costimulator ICOS/CRP-1, which belongs to the CD28/CTLA4 family. Although the human and mouse AILIM/ICOS molecule is localized on T-cells, the major population of AILIM/ICOS-positive cells in rat splenocyte was CD45RA-positive B-cells. The expression level of AILIM/ICOS on T-cells was relatively low; however, its expression was drastically induced by the treatment with PMA plus Ca-ionophore or the engagement of CD3 and these costimulatory molecules. Almost all T-cells exhibited potency as to its expression. Functional analysis of AILIM/ICOS demonstrated that AILIM-mediated costimulation was relatively weak compared to that of human.     

CD4+ CD25+ regulatory T cell repertoire formation in response to varying expression of a neo-self-antigen

We have examined the development of self-peptide-specific CD4+ CD25+ regulatory T cells in lineages of transgenic mice that express the influenza virus PR8 hemagglutinin (HA) under the control of several different promoters (HA transgenic mice). By mating these lineages with TS1-transgenic mice expressing a TCR that recognizes the major I-E(d)-restricted determinant from HA (site 1 (S1)), we show that S1-specific T cells undergo selection to become CD4+ CD25+ regulatory T cells in each of the lineages, although in varying numbers. In some lineages, S1-specific CD4+ CD25+ regulatory T cells are highly abundant; indeed, TS1xHA-transgenic mice can contain as many S1-specific CD4+ T cells as are present in TS1 mice, which do not express the neo-self HA. In another lineage, however, S1-specific thymocytes are subjected to more extensive deletion and far fewer S1-specific CD4+ CD25+ regulatory T cells accumulate in the periphery. We show that radioresistant stromal cells can direct both deletion and CD4+ CD25+ regulatory T cell selection of S1-specific thymocytes. Interestingly, even though their numbers can vary, the S1-specific CD4+ CD25+ regulatory T cells in all cases coexist with clonally related CD4+ CD25- T cells that lack regulatory function. These findings show that the formation of the CD4+ CD25+ regulatory T cell repertoire is sensitive to variations in the expression of self-peptides.     

Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes

CD20 is a plasma membrane phosphoprotein expressed exclusively by B lymphocytes. mAb binding to CD20 alters cell cycle progression and differentiation, indicating that CD20 plays an essential role in B lymphocyte function. Whole-cell patch clamp and fluorescence microscopy measurements of plasma membrane ionic conductance and cytosolic-free Ca2+ activity, respectively, were used to directly examine CD20 function. Transfection of human T and mouse pre-B lymphoblastoid cell lines with CD20 cDNA and subsequent stable expression of CD20 specifically increased transmembrane Ca2+ conductance. Transfection of CD20 cDNA and subsequent expression of CD20 in nonlymphoid cells (human K562 erythroleukemia cells and mouse NIH-3T3 fibroblasts) also induced the expression of an identical transmembrane Ca2+ conductance. The binding of a CD20-specific mAb to CD20+ lymphoblastoid cells also enhanced the transmembrane Ca2+ conductance. The mAb-enhanced Ca2+ currents had the same conductance characteristics as the CD20- associated Ca2+ currents in CD20 cDNA-transfected cells. C20 is structurally similar to several ion channels; each CD20 monomer possesses four membrane spanning domains, and both the amino and carboxy termini reside within the cytoplasm. Biochemical cross-linking of cell-surface molecules with subsequent immunoprecipitation analysis of CD20 suggests that CD20 may be present as a multimeric oligomer within the membrane, as occurs with several known membrane channels. Taken together, these findings indicate that CD20 directly regulates transmembrane Ca2+ conductance in B lymphocytes, and suggest that multimeric complexes of CD20 may form Ca2+ conductive ion channels in the plasma membrane of B lymphoid cells.     

PDZ7 of glutamate receptor interacting protein binds to its target via a novel hydrophobic surface area

Glutamate receptor interacting protein 1 (GRIP1) is a scaffold protein composed of seven PDZ (Postsynaptic synaptic density-95/Discs large/Zona occludens-1) domains. The protein plays important roles in the synaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The interaction between GRIP1 PDZ7 and a Ras guanine nucleotide exchange factor, GRASP-1, regulates synaptic distribution of AMPA receptors. Here, we describe the three-dimensional structure of GRIP1 PDZ7 determined by NMR spectroscopy. GRIP1 PDZ7 contains a closed carboxyl group-binding pocket and a narrow alphaB/betaB-groove that is not likely to bind to classical PDZ ligands. Unexpectedly, GRIP1 PDZ7 contains a large solvent-exposed hydrophobic surface at a site distinct from the conventional ligand-binding alphaB/betaB-groove. NMR titration experiments show that GRIP1 PDZ7 binds to GRASP-1 via this hydrophobic surface. Our data uncover a novel PDZ domain-mediated protein interaction mode that may be responsible for multimerization of other PDZ domain-containing scaffold proteins.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.     

Induction of the immune response to cell surface antigens in vitro

Summary Two tissue culture incubation systems are described in which immune responses to cell surface antigens have been demonstrated In the one-way “mixed lymphocyte interaction” system, a specific stimulation of thymidine uptake was induced by a particulate membrane antigen fraction, the microsomal lipoproteins (MLP)when low levels (0.01 to 0.001 μg per ml) were incubated with spleen or lymph node cells from nonsensitized mice. No stimulation was seen when allogeneic MLP was used at high levels, 10 μg per ml, nor at any level with syngeneic MLP. Specific effectors were demonstrated after 72-hr incubation with stimulatory levels of allogeneic MLP in three separate in vitro assays, a plaque-forming cell reduction assay, a tumor target assay, and an antigen-binding cell assay. In the latter assay, [¹²⁵I]MLP was used as the source of antigen. This system has limited potential inasmuch as mouse spleen cells do not survive in it beyond the 4th day of culture. The second tissue culture system, the Marbrook system, has much greater possibilities because at least 25% of the inoculum is recovered 7days later. In this culture system a cell-free sheep erythrocyte membrane preparation can induce, plaque-forming cells in the absence of macrophages. Using a sensitive radioimmunoassay, frees specific antibody was detected in culture supernatant fluids. With the same culture system, allogeneic lymphocytotoxic cells (killer) have been induced with spleen cells from unprimed mice in strains differing at the major histocompatibility locus (H-2). Allogeneic MLP induced very significant “killer” cell activity with spleen cells from primed mice. In a syngeneic tumor systems, significant amounts of killer cell activity were induced with unprimed spleen cell inocula, and much larger amounts induced with spleen cells from immunized mice. Presented in the formal symposium on Carcinogenesis in Vitro, at the 25th Annual Meeting of the Tissue Culture Association, Miami Beach, Florida, June 3–6, 1974. This work was supported by Public Health Service Rescarch grants CA 07973 and CA 10815 from the National Cancer Institute.     

A novel monoclonal antibody, 1B3.1, binds to a new epitope of the VLA-1 molecule

A monoclonal antibody, 1B3.1, was raised against a cloned IL-2-dependent T cell line that expresses the T gamma delta T cell receptor. MoAb 1B3.1 reacted with long-term cultured T cell lines of both T gamma delta and T alpha beta lineage, and with in vivo-stimulated T cells, derived from synovial fluid, but not with resting or short-term activated T cells, B cells, or macrophages. Immunoprecipitation of the 1B3.1 target antigens showed that 1B3.1 recognizes a 200/110 kDa molecule that is identical to the VLA-1 heterodimer precipitated by MoAb TS2/7. 1B3.1, however, binds to an epitope of VLA-1 that is distinct from the TS2/7 binding site. This new MoAb could be useful in further studies of the functions of VLA-1, and of the cells that express this molecule.