The glycosidic antigen recognised by a novel monoclonal antibody, 75.12, is developmentally regulated on mouse embryonal carcinoma cells

Monoclonal antibody 75.12 raised against the human ovarian teratocarcinoma cell line PA1 detects a 'Y' or iso-leb glycosidic structure. Using the 75.12 antibody we have established that the Y antigen is expressed on some but not all mouse embryonal carcinoma (EC) lines. The Y or 75.12 antigen-positive EC cell lines F9 and PCC4 cease to express the antigen after differentiation induced with retinoic acid and this decreased expression parallels the morphological differentiation of the EC cells. These results support not only the idea that carbohydrate structures present on embryonic cells undergo marked alteration during differentiation, but also that established mouse EC cells may differ in their differentiation states.     

Requirements for T cell activation by OKT3 monoclonal antibody: role of modulation of T3 molecules and interleukin 1

The requirements for activation of human peripheral blood T cells by the mitogenic monoclonal antibody OKT3 were examined. OKT3 binds to a T cell molecule, T3, associated with the T cell antigen receptor and involved in T cell activation. Activation of T cells by OKT3 requires signals provided by accessory cells and is IL 2 dependent. In the presence of accessory cells, OKT3 induces loss of T3 molecules from the cell surface, production of IL 2, expression of IL 2 receptors, and proliferation. Modulation of T3 molecules by OKT3 can be induced in the absence of accessory cells with anti-mouse IgG. These T cells, however, are not induced to express IL 2 receptors or secrete IL 2. The addition of IL 1 induces expression of IL 2 receptors, but does not induce IL 2 secretion or proliferation. Thus, peripheral blood T cells appear to have different requirements for activation compared with antigen-specific T cell clones that can be induced to produce IL 2 when stimulated with OKT3 and IL 1. Expression of IL 2 receptors does not require modulation of T3 molecules, because the binding of OKT3 to T cells in the presence of IL 1 alone is sufficient to induce IL 2 receptor expression. The results suggest that IL 2 secretion depends on cross-linking and modulation of T3 molecules, and additional, as yet undefined, accessory cell signals. The expression of IL 2 receptors and proliferation of T cells can be induced in the absence of these signals when exogenous IL 2 is provided.     

A new determinant,Qa-m208, detected on T lymphocytes and transfected L cells by a Kb-specific monoclonal antibody

A K^b-specific monoclonal antibody, 6.3.4, defining a new class I specificity, m208, reacts with some K and D region products and with a Qa determinant present on T lymphocytes but not detectable on thymocytes and B lymphocytes. The strain distribution of reactions indicates that this determinant is controlled by the Qa-Tla region, and shows no concordance with the strain distribution pattern of any of the known Qa antigens. The antibody reacts also with L cells expressing a cloned H-2 ^b class I gene mapping in the Qa-Tla region.     

Differential immunomodulation by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell activation: dependence upon the individual anti-CD3 monoclonal antibody used for activation

Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent upon which anti-CD3 mAb was used as the stimulus, being more pronounced when the anti-CD3 mAb 454 was used as the stimulus than when either anti-CD3 mAb 147 or 446 was the stimulus. The effects of neuraminidase-treated sheep erythrocytes (which bind to CD2) were also more pronounced on mAb 454-induced proliferation than on mAb 147- or 446-induced proliferation. Furthermore, the effects of preincubation with anti-CD2 mAb depended upon the responder status of the donor to IgG1 anti-CD3 mAb. Preincubation of high-responder cells with anti-CD2 mAb had little effect on subsequent IgG1 anti-CD3-induced proliferation. In contrast, preincubation of low-responder cells with anti-CD2 mAb usually augmented the otherwise small proliferative response to IgG1 anti-CD3 mAb. Taken together, these observations suggest that interaction of surface CD2 with ligand alters the response of T cells to anti-CD3 mAb, but these effects depend upon the individual anti-CD3 mAb used for stimulation. These studies raise the possibility that perturbation of different parts of the CD3-T cell antigen receptor complex may lead to different sequelae, and, as a result, the T cell may respond to a given immunomodulator in different ways.     

Differential modulation of the CD-2 and CD-3 T cell activation pathways by a monoclonal antibody to Leu-13.

MAb anti-Leu-13 reacts with a 16-kDa-interferon-responsive lymphocyte-endothelial cell surface antigen and has been demonstrated to induce lymphocyte aggregation by an undefined adhesion pathway. While anti-Leu-13 inhibits proliferation triggered by CD3 antibodies it was found to consistently augment proliferation induced by a pair of CD2 antibodies at suboptimally mitogenic concentrations. The latter mechanism of T cell activation may represent an antigen-nonspecific activation pathway requiring extensive cell-cell interaction. Proliferation induced via the CD2 pathway was very sensitive to the presence of monocytes whose inhibitory effect was reversed by indomethacin. While the potent inhibitory effect of PGE2 on proliferation induced via the CD2 pathway was weakly antagonized by anti-Leu-13, the combined effects of anti-Leu-13 and PGE2 on the CD3 pathway were additive and very inhibitory. The possibility that the Leu-13 signal reflects a mechanism by which a monocyte/macrophage-sensitive T cell activation pathway might be selectively amplified in vivo is discussed.     

Rat peritoneal mast cells release regulated upon activation normal T cell expressed and secreted (RANTES) after TNF-alpha activation

Chemokines are a family consisting of at least ten distinct novel 8-10 kD cytokines. The cysteine-cysteine (C-C) chemokines are chemoattractant and activators for monocytes, T cells and mast cells. RANTES is the prototype of the C-C chemokine subfamily, purified from different sources with chemoattractant and activator properties. In this study we found that supernatants derived from TNF-alpha (scalar concentrations)-activated rat peritoneal mast cell cultures (5 x 10(5)/mL), incubated overnight, produced high levels of RANTES. This data describes an additional mode of generation of RANTES. Moreover, RANTES mRNA was not significantly produced in untreated cells, while it was dramatically increased by calcium ionophore A23187, LPS and TNF-alpha compared with the controls. These results underscore the importance of the presence of mast cells for the production of RANTES in the inflammatory process and contribute to an understanding of the mechanism by which RANTES profoundly affects inflammatory responses in vivo.     

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.     

Thy-1: a lymphoid cell subset marker capable of delivering an activation signal to mouse T lymphocytes

Monoclonal anti-Thy-1 antibodies are capable of activating mouse T cells in the absence of an antigen-specific signal. Therefore, Thy-1 appears to be connected to an alternative signal transduction pathway, operative in thymocytes as well as in neuronal cells, since this molecule is also present on brain. Biochemical data have shown that this molecule is differentially glycosylated with respect to its cellular distribution. Structure and sequence comparisons revealed a strong homology with the immunoglobulin primordial domain. In addition, the Thy-1 glycoprotein has the particularity of being anchored to the membrane via a glycophospholipid tail. Gene transfer experiments in different cell types have been performed to analyze the mechanism of the Thy-1 pathway of activation.     

A novel methodology for analysis of cell distribution in chimeric mouse organs using a strain specific antibody

Chimeric animals are very useful for analysis of cell lineage, homeostasis in tissue architecture, and cell-cell interactions during both organogenesis and carcinogenesis. However, there is not a generally effective means for marking cells of chimeric mice. We have therefore developed a polyclonal antibody that is useful for this purpose. This antibody specifically recognizes those cells derived from C3H strain mice. The specificity of this antibody was checked by both immunoblotting and immunoadsorption methods. The antigens were immunohistochemically detected in cytoplasm of both epithelial and mesenchymal cells of C3H/HeN strain mouse in many different organs, but not the corresponding cell types from BALB/c or C57BL/10 or several other mouse strains. The validity of these antibodies as markers for C3H cells was further checked by tissue recombination experiments and in mixed cultures of mouse and rat cells. In each case the antibody recognized only the C3H mouse cells. Next, chimeric mice were prepared between strains C3H/HeN and BALB/c, and C3H/HeN and C57BL/10 mice. Chimeras 2-mo old were examined for antigen distribution using the indirect immunofluorescence method. Many tissues in chimeric mice were composed of cells that were both stained and unstained by the anti-C3H specific antigen. The chimeric patterns were classified into four types, A-D. In well-defined structural units such as intestinal crypts, small intestinal villi, kidney convoluted tubules, exocrine gland acini, ovarian follicles, thyroid gland follicles, stomach glands, adrenal cortex, lingual papillae, etc., (A) each unit was composed entirely of either positive or negative cells, or else (B) in some organs each unit was composed of both types of cells. In the uniform tissues without such distinguishable units, such as stratified squamous epithelium, mesenchymal tissue, corpora lutea, pituitary gland, Islets of Langerhans, adrenal medulla etc., (C) the tissue was composed of definite small cell groups made entirely of either positive or negative cells, or else (D) the tissue was composed of both types of cells which were intermingled with one another. These findings strongly suggest that the chimeric patterns demonstrated here reflect the cell proliferative unit in each tissue. This cell marker system has proven useful for analysis of cell lineage and cell renewal systems in many organs of chimeric mice.     

A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2

A hybridoma-secreting monoclonal antibody was produced from the spleen cells of a mouse immunized with human thymocytes. This hybridoma antibody, termed OKT5, was reactive by indirect immunofluorescence with 80% of human thymocytes but only 20% of peripheral blood T cells. Moreover, OKT5 was unreactive with normal B cells, null cells, and macrophages at any dilution tested. A similar pattern of reactivity was seen with an equine antiserum to human thymocytes termed anti-TH2. Fluorescence-activated cell sorting demonstrated that the OKT5 antibody reactivity on peripheral T cells was restricted to the majority of the previously defined TH2+ subpopulation. In functional studies, the OKT5+ subset, like the TH2+ subset, proliferated well to the mitogen Con A and to alloantigens, and contained cytotoxic effector cells after sensitization in MLC, and suppressor effector cells after activation with Con A. In addition, like the TH2+ T cell, the OKT+ T cell was virtually unresponsive to soluble antigen. Thus, the OKT5 monoclonal antibody is reactive with the cytotoxic/suppressor T cell subset. OKT5 should provide an important probe to assess the status of suppressor cells in human disease.     

Inhibition of adhesion of F9 embryonal carcinoma cells to substratum by a novel monoclonal antibody, TEC-05, reactive with a developmentally regulated carbohydrate epitope

Embryonal carcinoma cells carry on their surfaces carbohydrate antigens that are also expressed in early embryonic cells. We report here the expression and properties of a new developmentally regulated carbohydrate epitope, which is defined by a monoclonal antibody TEC-05. This antibody was generated by immunization of a rat with mouse embryonal carcinoma cells P19S1801A1. By immunofluorescence, the TEC-5 epitope was first detected on 8-cell-stage mouse embryos and was present on all subsequent stages of preimplantation development. Absorption analysis revealed that TEC-5 epitope was expressed only on a limited number of adult mouse tissues. In the direct radioantibody binding assay, TEC-05 reacted strongly with OTF9-63 cells and with some of the mouse embryonal carcinoma cell lines tested. Its reaction with differentiated cell lines was weak or undetectable. In the course of differentiation of OTF9-63 cells induced by retinoic acid, the epitope disappeared with the onset of morphological differentiation. The binding of the antibody to OTF9-63 cells was inhibited to 50% by 10-50 microM N-acetyllactosamine and lactose. Immunolabelling of extracts from OTF9-63 cells separated by sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis revealed that TEC-5 epitope was carried by high-molecular-weight glycoconjugates (molecular weight greater than 100,000). Molecules, isolated from [3H]-fucose-labelled OTF9-63 cells by indirect immunoprecipitation with TEC-05 antibody, were degraded by extensive pronase digestion or mild alkaline treatment to large carbohydrate chains that were excluded from a Sephadex G-50 column. Direct evidence that TEC-05 antibody bound to embryoglycan was obtained using a modified Farr's assay. The antibody was found to inhibit adhesion of F9 and OTF9-63 cells to substratum. The inhibitory effect, which could be abrogated by lactose, seemed to be specific, because another IgM monoclonal antibody which also binds to embryoglycan had no effect. Combined data indicated that TEC-05 antibody recognizes a carbohydrate epitope which is involved in cell-substratum adhesion of F9 cells and which provides a new marker for structure-function studies of stage-specific embryonic antigens.     

A novel pathway of human T lymphocyte activation. Identification by a monoclonal antibody generated against a rheumatoid synovial T cell line

Substantial evidence indicates that compartmentalized infiltrates of T lymphocytes are central to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, but the mechanisms by which such cells become activated remain unknown. To define surface components of activation pathways important in the function of these cells, we have generated mAb against a rheumatoid synovial T cell line. One such antibody, termed anti-UM4D4, reacts with an Ag, termed UM4D4, which is strongly expressed on most rheumatoid synovial T cell lines and clones, and on a subset of peripheral blood T cells, resting or activated. Anti-UM4D4 is mitogenic in soluble form for PBMC and certain T cell clones, and is comitogenic with the phorbol ester PMA for purified resting T lymphocytes. These functional effects are similar to those previously observed with antibodies to epitopes of CD2 and CD3, surface Ag involved in two well defined pathways of human T cell activation. Binding of anti-UM4D4 to T cells is not, however, blocked by antibodies directed at various epitopes of CD2 and CD3. Moreover, UM4D4 does not comodulate with CD3, and is expressed on a T cell line that lacks CD2, CD3, and CD28. The data, therefore, indicate that anti-UM4D4 identifies a T cell activation pathway, distinct from those previously described, that could play a role in the pathogenesis of T cell-mediated autoimmune diseases.     

Heparan sulfate proteoglycans from mouse mammary epithelial cells: localization on the cell surface with a monoclonal antibody

Mouse mammary epithelial cells, of the normal murine mammary gland (NMuMG) cell line, bear a heparan sulfate-rich proteoglycan (HSPG) on their surfaces. A hybridoma (281-2) secreting a monoclonal antibody that recognizes this HSPG was produced by fusion of SP-2/0 myeloma cells with spleen cells from rats immunized with NMuMG cells. The 281-2 monoclonal antibody is directed against the core protein of the cell surface HSPG, as demonstrated by (a) recognition of the isolated proteoglycan but not its glycosaminoglycan chains, (b) co-localization of 281-2-specific antigen and radioactive cell surface HSPG on gradient polyacrylamide gel electrophoresis and on isopycnic centrifugation, and (c) abolition of immunofluorescent staining of the NMuMG cell surface by the intact, but not the protease-digested ectodomain of the cell surface HSPG. The antibody is specific for cell surface HSPG and does not recognize the HSPG that accumulates extracellularly beneath the basal cell surface. Therefore, the 281-2 antibody may be used to isolate the cell surface HSPG and to explore its distribution in tissues.     

Thx cells: a new subset in T helper cells, are they born to die

A new subset of T helper namely T helper x (Thx) was differentiated ex vivo from spleens by primary activation with α-CD3 and α-CD28 and later differentiated in the presence of α-IL-4, α-IFN and α-IL-12. Thx cells exhibit characteristic cell surface markers and intracellular cytokines patterns. These cells are extremely sensitive to reactivation-induced apoptosis. We further compared death pathways in all the T Helper cells that we had differentiated and found that Thl cells exhibited a Fas-FasL/ caspase dependent apoptotic pathway, Th2 cells died by a pathway independent of Fas/FasL and caspase 8. α-FasL antibody, ZVAD and TR6 were able to rescue Th1 cells from apoptosis confirming that Thl cells die by     

Lyb-8.2: A new B cell antigen defined and characterized with a monoclonal antibody

A DBA/1 B10.D2-specific monoclonal antibody (CY34) is described which defines a new murine B lymphocyte differentiation antigen designated Lyb-8.2. The ontogeny, strain distribution, and cell-surface density of the antigen were studied by radioimmunoassay and by fluorescence-activated cell sorter (FACS) analysis. Lyb-8.2 appears to be expressed on pre-B cells and on all mature B lymphocytes. Lyb-8.2 molecules immunoprecipitated from surface labeled B10.D2 spleen cells migrated in polyacrylamide gels with an apparent mol. wt. of 95000–105000 daltons and were bound by lentil lectin. The expression of Lyb-8.2 is controlled by a locus on chromosome 7 that is closely linked to Gpi-1 and RP-2. Added Lyb-8.2-specific antibody did not measurably impair B lymphocyte function in several in vitro systems studied.     

Identification,isolation, and characterization of a human T cell population by a monoclonal antibody

The hybridization of spleen cells from mice immunized with mononuclear leukocytes with the HAT-sensitive nonsecreting myeloma, NS1, resulted in the production of hybrid cell lines secreting monoclonal antibodies to lymphocyte surface antigens. One of these, anti-Ta, was shown by fluorescence-activated cell sorter analysis to be specific for a subpopulation of peripheral human T cells. Anti-Ta did not react with peripheral human B cells. Immunoprecipitation followed by two-dimensional gel analysis demonstrated that the T cell subpopulation-specific antigen recognized by this monoclonal antibody is part of, or firmly associated with, a protein of the plasma membrane.     

A monoclonal antibody that induces T cell aggregation reacts with vascular endothelial cells and placental trophoblasts

We have found that a mouse monoclonal antibody (alpha Leu-13) to a 16 kilodalton human lymphocyte surface antigen reacts with vascular endothelial cells as determined by immunoperoxidase staining of frozen tissue sections. In earlier studies, alpha Leu-13 was found to induce purified T cells to aggregate when added to cultures in nanogram concentrations. In the studies reported here, alpha Leu-13 stained vascular endothelial cells of arteries, capillaries, and veins in all organs examined from adults. It also reacted weakly with epithelial cells of proximal tubules of the kidney and with nonkeratinized basal epithelial cells of the cervix and esophagus. When a panel of tissues from a 14-wk-old fetus was examined, alpha Leu-13 was not found to react with endothelial cells of any specimen. However, it did stain medullary thymocytes and placental trophoblasts of this fetus. The implications of these findings to the possible function of the Leu-13 antigen in immune ontogeny are discussed.     

A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide

The alteration in the surface of endothelial cells (EC) in response to cytokines is likely to be of great importance to the regulation of cell migration and thereby to the evolution of inflammatory processes. We have generated three mAb against cytokine inducible Ag on EC. Whereas mAb 1.2B6 and 6.5B5 were found to react with ELAM-1 and ICAM-1, respectively, mAb 1.4C3 reacted with a novel molecule that showed a different pattern of expression from ELAM-1 or ICAM-1 after stimulation of EC by TNF, IL-1, or LPS. Like ELAM-1, the 1.4C3 Ag was minimally expressed on resting EC, whereas ICAM-1 was moderately expressed. After stimulation with IL-1, TNF, or LPS, ELAM-1 expression was maximal after 4 to 6 h, 1.4C3 Ag after 6 to 10 h, and ICAM-1 after 10 to 24 h. The duration of 1.4C3 expression was intermediate between ELAM-1 and ICAM-1, and was more prolonged in response to TNF than IL-1 or LPS. Whereas the expression of the three Ag showed a similar dose response to varying concentrations of IL-1 or LPS, EC required a 10-fold higher concentration of TNF for half maximal expression of ELAM-1 than for half maximal expression of 1.4C3 Ag or ICAM-1 (5 ng/ml compared to 0.5 ng/ml). Of the three Ag, only ICAM-1 was enhanced by IFN-gamma. SDS-PAGE under reducing conditions showed the 1.4C3 Ag to migrate as a single band with a relative molecular mass of approximately 95 kDa. mAb 1.4C3 adds to our understanding of the kinetics of the EC response to different cytokines and will be useful in studying the regulation of EC activation. Furthermore, the 1.4C3 molecule may have an important role in leukocyte-EC interactions.     

Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. Analysis of surface expression and human T cell activation

In this paper, we examined in detail the ability of anti-1F7 to modulate 1F7 (CD26) surface expression as well as analyzed the functional relationship between the surface expression of CD3, CD2, and CD26 and human T cell activation. We showed that anti-1F7-induced modulation is an energy-dependent process that occurs via capping and internalization of the Ag-antibody complex. Although the recovery rate for Ag reexpression of 1F7 following optimal modulation is relatively delayed, reexpression of 1F7 is greatly accelerated following phorbol ester treatment. Most importantly, we demonstrated that modulation of the CD26 Ag leads to an enhancement in the proliferative activity of modulated human T cells treated with anti-CD3 or anti-CD2, which is preceded by an enhancement in Ca2+ mobilization. CD26 modulation also led to an increase in anti-CD3- or anti-CD2-mediated T cell clone proliferation. Finally, whereas modulation of the CD26 Ag has an effect on CD3- or CD2-induced T cell activation, modulation of the CD3/TCR complex inhibits the proliferative response of T cells incubated with anti-CD3 plus anti-1F7 or anti-CD2 plus anti-1F7. However, modulation of the CD2 structure does not affect anti-CD3- plus anti-1F7-induced human T cell activation. The above results thus provide additional evidence that the CD26 Ag plays an integral role in the regulation of human T cell activation.