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.     

Inhibition of H-Y cell-mediated cytolysis by monoclonal H-Y-specific antibody

Assays of H-Y-specific, cell-mediated cytolysis (CMC) in vitro were carried out with B6 female effector cells and B6 male target cells. Monoclonal H-Y antibody was added to the lytic assay to test whether the antigenic determinant(s) involved in H-Y-specific CMC was distinct from the serologically detected H-Y antigen. Significant blocking was observed, suggesting that the H-Y antigen detectable serologically is similar to H-Y antigen recognized by cytotoxic T cells.Abbreviations used in this paper B6 C57BL/6 - BALB BALB/c - CMC cell-mediated cytolysis - E effector cells - T target cells     

Regulation of the alternative pathway of T cell activation by anti-T3 monoclonal antibody

T cell activation may be triggered either through the T3-Ti antigen receptor complex or via an alternative macrophage-independent pathway involving the 50KD T11 sheep erythrocyte-binding glycoprotein. Monoclonal antibodies anti-T11(2) and anti-T11(3), directed at distinct epitopes of the T11 molecule, trigger mature T cells to proliferate and express their functional programs, and induce expression of IL 2 receptors on both T3+ and T3- thymocytes. We now show that a non-mitogenic anti-T3 antibody blocks activation via the T11 pathway of not only peripheral blood T cells, but also T3+ thymocytes. Anti-T3 does not affect surface expression of T11 or the rapid augmentation of T11(3) expression after incubation of cells with anti-T11(2). However, anti-T3 inhibits generation of IL 2 receptors and production of IL 2 by T lineage cells cultured with anti-T11(2) plus anti-T11(3). In contrast, modulation of the T11 molecule by a non-mitogenic anti-T11 antibody does not inhibit activation of T cells by a mitogenic anti-T3 antibody. The ability of anti-T3 to block expression of IL 2 receptors on both thymocytes and mature T cells activated by the T11 pathway suggests that a regulatory interaction may be important during T cell ontogeny to provide a mechanism for inhibiting expansion of autoreactive clones.     

Competitive inhibition in vivo and skewing of the T cell repertoire of antigen-specific CTL priming by an anti-peptide-MHC monoclonal antibody.

We have recently described a mAb, KP15, directed against the MHC-I/peptide molecular complex consisting of H-2D(d) and a decamer peptide corresponding to residues 311-320 of the HIV IIIB envelope glycoprotein gp160. When administered at the time of primary immunization with a vaccinia virus vector encoding gp160, the mAb blocks the subsequent appearance of CD8(+) CTL with specificity for the immunodominant Ag, P18-I10, presented by H-2D(d). This inhibition is specific for this particular peptide Ag; another H-2D(d)-restricted gp160 encoded epitope from a different HIV strain is not affected, and an H-2L(d)-restricted epitope encoded by the viral vector is also not affected. Using functional assays and specific immunofluorescent staining with multivalent, labeled H-2D(d)/P18-I10 complexes (tetramers), we have enumerated the effects of blocking of priming on the subsequent appearance, avidity, and TCR Vbeta usage of Ag-specific CTL. Ab blocking skews the proportion of high avidity cells emerging from immunization. Surprisingly, Vbeta7-bearing Ag-specific TCR are predominantly inhibited, while TCR of several other families studied are not affected. The ability of a specific MHC/peptide mAb to inhibit and divert the CD8(+) T cell response holds implications for vaccine design and approaches to modulate the immune response in autoimmunity.     

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.     

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.     

Streptococcus iniae inhibition of apoptosis of nonspecific cytotoxic cells: a mechanism of activation of innate immunity in teleosts

Nonspecific cytotoxic cells (NCC) may provide innate anti-bacterial resistance against Streptococcus iniae infections in tilapia. The mechanism of immunity would be elaboration and release of various cytokines, augmentation of inflammation and amplification of increased antigen processing. To investigate bacterial regulation of NCC function, 2 different processes of cellular pathology were examined: apoptosis and necrosis. Different isolates of S. iniae from diseased teleosts, a dolphin and a human were tested. All isolates were examined for their ability to produce apoptosis and/or necrosis on freshly purified tilapia NCC and on a tilapia continuous cell line (i.e. TMB-8 cells). Two different isolates (9033 and 173) inhibited the outer membrane expression of phosphatidylserine (PS) by NCC, an early sign of apoptosis. This occurred at 4 h post-treatment and lasted throughout the 24 h treatment period. All other isolates either did not differ from control levels or produced a small increase in PS expression by NCC. The early reduction in PS expression occurred concomitantly with increased necrosis associated with nonspecific DNA fragmentation. Two-color flow cytometry (Annexin-V vs propidium iodide staining) demonstrated the specificity of Annexin-V binding. Experiments were also done to determine the effects of S. iniae on TMB-8 cells. Treated TMB-8 cells did not produce appreciable Annexin-V binding. Compared to the ATCC strain, 9033 produced high levels of necrosis-associated DNA fragmentation of TMB-8 cells at 4 and 8 h post-treatment. These data indicated that different isolates of S. iniae may regulate NCC anti-bacterial resistance by causing reduced levels of programmed cell death (PCD), increased necrosis and associated enhancement of inflammatory responses. Understanding the relevance of these bacterial effects on NCC may be an important consideration in the evaluation of isolates used in vaccine/ bacterin production.     

Inhibition of T cell-mediated cytolysis by monoclonal antibodies directed against Lyt-2: heterogeneity of inhibition at the clonal level

The inhibitory effect of monoclonal anti-Lyt-2 antibodies on T cell-mediated cytolysis has been investigated at the clonal level. In agreement with previous reports from several laboratories, populations of cytolytic T lymphocytes (CTL) generated in vitro in mixed leukocyte cultures (MLC) were reversibly inhibited by monoclonal anti-Lyt-2 antibodies in a dose-dependent fashion. However, when alloimmune peritoneal exudate lymphocytes (PEL) were used as a source of CTL, little or no inhibitory effect of anti-Lyt-2 antibodies on cytolysis was observed. A series of CTL clones derived from MLC or PEL populations was also tested for inhibition of cytolysis by anti-Lyt-2 antibodies. In agreement with results obtained at the population level, most MLC-derived clones (81%) were strongly inhibited by the reagent, whereas few PEL clones (15%) were inhibited. Several of these clones were expanded and maintained in culture without loss of their "inhibition phenotype." Flow cytofluorometric analysis using the same monoclonal anti-Lyt-2 antibodies further revealed that both inhibited and uninhibited clones expressed comparable amounts of Lyt-2 antigen. These results provide direct evidence that inhibition of CTL by anti-Lyt-2 antibodies is heterogeneous at the clonal level. The possibility that this heterogeneity may be related to avidity of antigen receptors is discussed.     

Characteristics of a T lymphocyte-enhancing Ab-specific monoclonal antibody

We recently described the production and characterization of an A^b-specific monoclonal antibody (mAb), designated K14.83-11, which specifically enhanced the alloreactivity of normal T lymphocytes, as well as highly selected A^b-specific T-cell lines and clones against the appropriate A^b molecule. In the present report, we have studied both the binding specificity and immunoenhancing activity of K14.83-11 in a number of genetic combinations to better characterize the possible modes through which this mAb achieves its T cell-enhancing activity. Results reported here (1) confirm and extend previous findings revealing the possible importance of the immunoglobulin G₃ isotype of K14.83-11 in its immunoenhancing activity, and (2) show a distinction in the determinant binding and subsequent ability to elicit an enhancing effect. Surprisingly, K14.83-11 proved ineffective in enhancing autologous or A^b-restricted reactions. This system, then, may provide the means of comparing the functional importance of A^b expression in self-restricted and allogeneic reactions.     

Characterization of monoclonal nonspecific suppressor factor (MNSF) with the use of a monoclonal antibody

The secretion of immunoglobulin (Ig) from cultured mononuclear cells by lipopolysaccharide (LPS) stimulation is inhibited by monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma. In an attempt to develop a murine monoclonal antibody (MAb) with specific reactivity against MNSF, a cell fusion technique that incorporated immune murine splenocytes and HAT-sensitive murine myeloma cells was used. Cross-reactivity experiments confirmed that the MAb (MO6) does not bind to unrelated proteins such as bovine serum albumin, mouse IgG, and murine interferon-gamma (IFN-gamma). There are no effects when anti-IFN-gamma antibodies are used with MNSF. As far as biological activity is concerned, MO6 inhibits in vitro the activity of MNSF in terms of the Ig secretion from cultured lymphocytes. By using MO6, affinity chromatography and immunoblotting were performed. The MNSF on the SDS-PAGE showed a band with m.w. of approximately 70,000, indicating the formation of an aggregate in saline; but after treatment with 0.4 M pyridine-acetic acid buffer, separate bands of 24,000 and 16,000 daltons were evident. Therefore MO6 recognizes 70,000 and both 24,000 and 16,000 daltons. Thus we confirmed by using this MAb and affinity chromatography, the existence of human counterpart, human nonspecific suppressor factor (hNSF), in supernatant from concanavalin A-stimulated T cells. When hNSF was fractionated by high pressure liquid chromatography (HPLC), the activity was found in a region corresponding to 70,000 daltons. However, when fractionated in pyridine-acetic acid buffer, hNSF activity was distributed in a slightly wider range of 15,000 to 30,000 daltons. Physicochemical analysis showed that the purified hNSF is resistant to either heating at 56 degrees C or to 2-mercaptoethanol treatment; however, it is labile to acidification at pH 2.0 and is also sensitive to protease treatment, the characteristics of which were similar to those of murine MNSF. Thus MO6 was confirmed to be a pertinent tool for isolation of hNSF, as well as for murine MNSF.     

Irreversible inhibition of a monoclonal antibody by a nitrophenyl ester

TEPC-15 is a phosphorylcholine-binding mouse myeloma protein which reacts with an ester-containing phosphorylcholine, thep-nitrophenyl ester of 6-(phosphorylcholine)hexanoic acid (PEPCH). The rate of nitrophenolate release mediated by the antibody ispH-dependent and increases with increasingpH. The antibody becomes inactive during the reaction with the ester. The inactive antibody is not reactivated even after treatment with hydroxylamine. Antibody activity is associated with the Fab' fragment. These observations together with thepH profile of the reaction suggest that the ester acylates a lysine side chain near the antibody-binding site.A preliminary discussion of this work was presented at the 78th Annual Meeting of the American Society of Biological Chemists, Philadelphia, Pennsylvania, 1987.     

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.     

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.     

Platelet-collagen adhesion: inhibition by a monoclonal antibody that binds glycoprotein IIb

To identify platelet surface structures involved in adhesion to collagen, the effect of 16 murine antiplatelet membrane hybridoma antibodies were tested in a defined, in vitro assay. Four of these antibodies inhibited platelet-collagen adhesion and reacted with a polypeptide with Mr approximately 125,000, as determined by immunoblots after gel electrophoresis under reducing conditions. Through detailed studies with one of these antibodies, the monoclonal antibody PMI-1, the relevant antigen was identified as platelet glycoprotein IIb alpha, based upon (a) co-migration with this glycoprotein in two-dimensional gel electrophoresis and (b) co-purification by immunoaffinity chromatography with a protein with apparent Mr identical to that of glycoprotein III, under conditions in which glycoproteins IIb and III form a complex. Univalent antibody fragments prepared from monoclonal antibody PMI-1 inhibited greater than 80% of platelet-collagen adhesion, and inhibition was completely blocked by the immunopurified antigen. These results indicate that glycoprotein IIb participates in some aspect of platelet-collagen adhesion. In contrast, the purified antigen only partially neutralized a polyclonal antiserum that blocked platelet-collagen adhesion, to a maximum of approximately 25%, at saturating antigen concentrations. Thus, by these immunological criteria, glycoprotein IIb is not the only molecule involved in this process.     

Studies on the cytolytic attack mechanism of the cytotoxic T lymphocyte (CTL): preparation of antisera against cellfree cytosolic extracts of a CTL clone capable of blocking the lethal hit stage of CTL cytolysis and analysis of the cytolytic structure

Rat antiserum (as well as purified IgG and F(ab')2 fragments) raised against cellfree cytosolic extracts (CFE) of an alloimmune cytotoxic T lymphocyte (CTL) clone (B6.1.SF.1) is a potent inhibitor of CTL-mediated cytotoxicity. Inhibition by this antiserum (termed alpha CTLL) occurred during the postbinding lethal hit stages of cytolysis, because it did not inhibit target cell binding, nor did it prematurely dissociate CTL-target cell conjugates; inhibition was observed regardless of the H-2 haplotype of the target cell or CTL employed; inhibition was reversible when pretreated, and washed CTL were used as effectors; and in Ca++ pulse experiments alpha CTLL inhibited cytolysis beyond the Ca++-dependent (lethal hit) stage of cytolysis. This antiserum did not inhibit lysis of P815 cells by activated murine macrophages or by human cytotoxic cells, and extensive absorption of the antiserum on viable thymocytes, normal spleen cells, or CTL did not reduce its blocking activity. CFE prepared from several sources of CTL, including in vivo elicited peritoneal exudate lymphocytes (PEL), secondary MLC-generated CTL, alloimmune splenic T cells, and CTL clones, contained material(s) that inhibited the ability of alpha CTLL to block CTL-mediated cytolysis. The inhibitory activity was not detected in CFE from a variety of noncytotoxic cell sources, including thymocytes, normal C57BL/6 spleen cells, EL4 or P815 tumor cells, macrophages, and helper T cell clones. It was also absent in CFE prepared from human CTL cells. Furthermore, although alpha CTLL neutralizing activity was not detectable in CFE prepared from memory CTL, it rapidly appeared in CTL parallel to the development of cytolytic activity during secondary MLC cultures. The inhibitory material in CTL-CFE appeared to be specific for alpha CTLL antibody, as it did not affect the CTL blocking activity of anti-Lyt-2 or anti-target cell antisera. Finally, CTL-CFE did not contain proteases that degraded the alpha CTLL antibody. By the use of a soluble-phase immunoabsorbent assay, the biochemical properties of materials present CFE derived from CTL and reactive with alpha CTLL antibody were examined. CTL cytosolic material(s) reactive with alpha CTLL IgG was unstable to brief heating (50 degrees C) or acidic pH, but not to high ionic strength buffers. The material was inactivated by treatment with pronase but not by DNase, collagenase, or trypsin. Gel filtration chromatography of CTL-CFE revealed multiple peaks of alpha CTLL neutralizing activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preferential elimination of NK and CTL functions by anti-D44 monoclonal antibody

Monoclonal antibodies reactive with T cells at various stages of maturation were used in negative selection experiments to study their effects on NK function in the presence of complement. Anti-D47 and anti-A50, respectively, directed against corticothymocytes and mature peripheral E+ cells were without effect. Anti-D66 reactive with an epitope of the T cell E receptor inhibited up to 60% of NK activity. Anti-D44, which primarily recognizes corticothymocytes and 60 to 80% of the E(+)-PBL was found to abrogate NK activity together with alloreactive CTL reactivity but to leave intact most of the MLR and PHA proliferative responses. Therefore D44 appears as a discrete antigen allowing preferential elimination of NK cells and CTL from PBL.     

抗CD3/CD28单克隆抗体联合PHA对T淋巴细胞活化和增殖的影响

目的探讨抗人CD3/CD28单克隆抗体联合植物血凝素(PHA)对人外周血T淋巴细胞活化增殖的影响。方法通过Ficoll密度梯度离心法分离健康成人外周血单核细胞后,用CD3免疫磁珠分选T淋巴细胞,抗CD3/CD28单抗、PHA及重组人白介素-2(recombinant human IL-2, rhIL-2)刺激T淋巴细胞。用CCK-8试剂盒(CCK-8 cell counting kit)检测细胞增殖情况, ELISA测定细胞上清液干扰素γ(interferon-γ, IFN-γ)的含量。结果 CCK-8结果显示在增殖3 d时,CD3/CD28+PHA组A_(450 nm)值与PHA组、IL-2组与空白组比较,差异有统计学意义(P<0.05);比较各组在6、9、12 d的增殖情况,CD3/CD28+PHA组的A_(450 nm)值与其他各组差异均有统计学意义(P<0.05)。刺激3 d各组分泌的IFN-γ质量浓度是最高的。刺激9 d时,CD3/CD28+PHA组、CD3/CD28组、PHA组上清液中IFN-γ浓度分别为(235.27±27.80)、(189.23±24.79)和(124.66±3.39)pg/mL,CD3/CD28+PHA组上清液中IFN-γ的质量浓度高于其余2组(P<0.05)。结论抗CD3/CD28单抗联合PHA比单用抗CD3/CD28单抗、PHA或rhIL-2能更好地激活T细胞及持久地促使T细胞增殖。     

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.