CD5 antibodies increase intracellular ionized calcium concentration in T cells

The binding of a variety of monoclonal antibodies to the CD5 (T, gp67) pan T cell differentiation antigen has been shown to potentiate T cell proliferation. In this paper we show that CD5 monoclonal antibodies cause increased intracellular free calcium concentration ([Ca2+]i) in T cells. An increase in [Ca2+]i occurred within 1 min in indo-1-loaded PBMC after the addition of CD5 monoclonal antibodies and cross-linking with a second step anti-mouse kappa light chain antibody. Cross-linking of CD5 was effective when done directly on the cell surface or by the administration of preformed soluble complexes that contained CD5 antibodies. Calcium mobilization induced by suboptimal concentrations of CD3 antibodies was specifically augmented and sustained by CD5 antibodies, although the enhancement was modest in magnitude. When cell surface phenotype was correlated with calcium mobilization, it was found that the CD5 response was restricted to CD5+/CD3+ cells, and that approximately 90% of CD5+ cells had responded. CD5-induced calcium mobilization was found to differ from CD3 stimulation in that EGTA entirely ablated the CD5 response, whereas the CD3 response was resistant to EGTA, indicating that the CD5-induced increased [Ca2+]i is derived primarily or entirely from extracellular calcium. CD5-stimulated calcium mobilization also differed from CD3 in that the CD5 response was inhibited by pretreatment with phorbol myristate acetate, whereas the CD3 response was not, suggesting that depletion of protein kinase C causes an uncoupling of signal transduction between CD5 and calcium channels. Finally, experiments were done with T cells after antigenic modulation of the CD3 or CD5 molecules. Unexpectedly, both the CD5 and the CD3 responses were ablated on CD3-modulated cells, whereas only the CD5 response was ablated on CD5-modulated cells. In addition, several Cd5+/CD3- T cell leukemia lines also failed to respond to CD5 stimulation, providing further evidence which indicates that the CD5 response depends on the cell surface expression of CD3 or a CD3-associated structure. These findings suggest that one mechanism for CD5-induced augmentation of mitogen-stimulated T cell proliferation involves increased [Ca2+]i which is distinct from but interdependent with that induced by stimulation of the CD3 molecule.     

Tissue distribution and biochemical and functional properties of Tp55 (CD27), a novel T cell differentiation antigen

Two monoclonal antibodies (CLB-CD 27/1 and CLB-CD 27/2) were raised against a novel determinant on human T lymphocytes. One of these antibodies, CLB-CD 27/1 (clone 9F4), was grouped by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens together with three other monoclonal antibodies (VIT 14, OKT 18A, and S152) in the new cluster CD27. In this paper we show that antibodies belonging to this cluster recognize an antigen present on a large subset of peripheral T lymphocytes and most medullary thymocytes. At least two different nonoverlapping epitopes were identified with directly labeled monoclonal antibodies. Immunoprecipitation studies indicate that the target antigen of CD27 antibodies is a polypeptide of 55 kDa, which appears in the form of a disulfide-linked homodimer on the T lymphocyte membrane (Tp55). Stimulation of T cells via the T3/T cell antigen-receptor complex, with either phytohemagglutinin or CD3 monoclonal antibodies, resulted in a fivefold increase in the membrane expression of Tp55, whereas activation by phorbol myristate acetate caused a marked down-regulation. Moreover, an additional molecule of 32 kDa was precipitated from the membrane of activated but not of resting T cells. Addition of CD27 antibodies to cultures stimulated with either phytohemagglutinin or CD3 monoclonal antibody led to enhanced proliferation, whereas no effect was observed in phorbol myristate acetate or interleukin 2-stimulated cultures. The possible role of the Tp55 antigen in T cell activation is discussed.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and potential for idiotypic mimicry

Human immunodeficiency virus (HIV), the retrovirus that causes the acquired immunodeficiency syndrome, is cytopathic for CD4+ T cells and binds to these cells via a complex of the 110,000 m.w. viral-envelope glycoprotein, gp110, and the CD4 molecule. We treated virus with several physical, chemical, and enzymic agents to determine their effect on the capacity of HIV to bind to the CD4+ T cell line, CEM. Reduction and alkylation (but not alkylation alone) and trypsin digestion (but not glycolytic enzyme digestions) of HIV destroyed its capacity to bind. If the tertiary protein structure conferred by disulfide bonding is not disrupted, the tertiary and secondary conformations dependent on noncovalent forces appear to be thermodynamically favored, because treatment with denaturants such as sodium dodecyl sulfate, 8 M urea, alcohol, or heat (56 degrees C or 65 degrees C for 30 min) followed by removal of the denaturants did not affect binding. Irreversible denaturation and loss of binding occurred after heating at 100 degrees C for 10 min. HIV binding to CD4+ T cells was inhibited either by murine monoclonal antibodies to the CD4 molecule or by human polyclonal or murine monoclonal antibodies to the gp110 molecule. On the basis of results of binding inhibition obtained with a panel of alpha-CD4 monoclonal antibodies, the receptor site for virus on the CD4 molecule was mapped to the amino-terminal portion of the molecule. Four candidate alpha-CD4 monoclonal antibodies that were potent inhibitors of virus binding (OKT4A, OKT4D, OKT4F, and Leu-3a) were examined for the possibility that their binding sites (idiotopes) might share structural and conformational similarity with the CD4-binding site on gp110. Polyclonal human or rabbit anti-HIV sera (that reacted with gp110 and inhibited virus binding) did not react with or inhibit the binding of these four alpha-CD4 monoclonal antibodies. Conversely, rabbit anti-idiotypic sera raised against each of the four candidate CD4 monoclonal antibodies did not react with virus or inhibit virus binding to CD4+ T cells. Further search or different approaches may yet yield an idiotype that is a structural and conformational "internal image" of the CD4-binding site of virus.     

Antagonist properties of monoclonal antibodies targeting human CD28

Antagonist antibodies targeting CD28 have been proposed as an alternative to the use of CD80/86 antagonists to modulate T cell responses in autoimmunity and transplantation. Advantages would be the blockade of CD28-mediated co-stimulatory signals without impeding the co-inhibitory signals dependent on CD80 interactions with CTLA-4 and PD-L1 that are important for the control of immune responses and for the function of regulatory T cells. Anti-CD28 antibodies are candidate antagonists only if they prevent access to the CD80/86 ligands without simultaneously stimulating CD28 itself, a process that is believed to depend on receptor multimerization. In this study, we evaluated the impact of different formats of a potentially antagonist anti-human CD28 antibody on T cell activation. In particular, we examined the role of valency and of the presence of an Fc domain, two components that might affect receptor multimerization either directly or in the presence of accessory cells expressing Fc receptors. Among monovalent (Fab’, scFv), divalent (Fab’2), monovalent-Fc (Fv-Fc) and divalent-Fc (IgG) formats, only the monovalent formats showed consistent absence of induced CD28 multimerization and absence of associated activation of phosphoinositol-3-kinase, and clear antagonist properties in T cell stimulation assays. In contrast, divalent antibodies showed agonist properties that resulted in cell proliferation and cytokine release in an Fc-independent manner. Conjugation of monovalent antibodies with polyethylene glycol, α-1-antitrypsin or an Fc domain significantly extended their in vivo half-life without modifying their antagonist properties. In conclusion, these data indicate that monovalency is mandatory for maintaining the antagonistic activity of anti-CD28 monoclonal antibodies.     

Efficient expansion of tumor-infiltrating lymphocytes from solid tumors by stimulation with combined CD3 and CD28 monoclonal antibodies

Combined CD3 and CD28 monoclonal antibodies (mAb) may initiate efficient activation and expansion of tumor-infiltrating lymphocytes (TIL). In this study we compared phenotypical and functional characteristics of TIL from a group of 17 solid human tumors, stimulated either by high-dose recombinant interleukin 2 (rIL-2, 1000 IU/ml) or by a combination of anti-CD3 and anti-CD28 monoclonal antibodies in the presence of low-dose rIL-2 (10 IU/ml). Compared to activation with high-dose rIL-2, stimulation of TIL with CD3/CD28 mAb induced significantly stronger proliferation and yielded higher levels of cell recovery on day 14. Following the CD3/CD28 protocol, expansion of an almost pure population of CD3⁺ cells was obtained. Whereas CD4⁺ cells dominated in the first week of culturing, within 4 weeks the CD8⁺ population increased to over 90%. The specific capacity to kill autologous tumor cells was not increased as compared to the high-dose rIL-2 protocol, but all cultures showed high cytotoxic T cell activity as measured in a CD3-mAb-mediated redirected kill assay. These studies show that combined CD3 and CD28 mAb are superior to rIL-2 with respect to the initiation of expansion of CD8⁺ cytolytic TIL from solid tumors. Stimulation with specific tumor antigens at a later stage of culturing may further augment the expansion of tumor-specific cytolytic T cells.     

The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies

Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.     

The anti-idiotypic antibody 1F7 selectively inhibits cytotoxic T cells activated in HIV-1 infection

Circulating CD8+ T lymphocyte numbers rise substantially following infection with HIV-1. This expanded CD8+ T cell population includes HIV-specific CTL and CTL that kill activated uninfected CD4+ lymphocytes. Experimental, epidemiological and clinical evidence supports the possibility that expansion of CD8+ CTL contributes to CD4+ T cell depletion and disease progression in human HIV infection. Therefore, modulation of CD8+ T cell numbers or of certain CD8+ CTL activated in HIV-infected individuals may be beneficial. It was found that 1F7, a mAb against an idiotype common to anti-HIV and anti-simian immunodeficiency virus (SIV) antibodies, selectively inhibited both anti-HIV CTL and CTL against uninfected CD4+ T cells. Alloantigen-specific CTL and NK cells from either HIV-infected individuals or controls were unaffected by 1F7. Prolonged incubation of CD8+ T cells from HIV-infected individuals with 1F7 induces apoptosis, which was shown to be reflected functionally in reduced total CTL activity and in especially reduced CTL activity against uninfected CD4+ lymphocytes. The selective reactivity of 1F7 with certain CD8+ CTL could be applied towards the modulation of CD8+ T cell responses involved in AIDS pathogenesis.     

Immunochemical analysis of CD107a (LAMP-1)

CD107a, also known as the lysosome associated membrane protein-1 (LAMP-1), is expressed largely in the endosome-lysosome membranes of cells, but is also found on the plasma membrane (1-2% of total LAMP-1). LAMP-1 has been implicated in a variety of cellular functions, including cancer metastasis. It has been proposed as a therapeutic agent for some cancers, and is a marker for lysosomal storage disorders and different cell types such as cytotoxic T cells. In light of this diversity of applications, it is important to have well characterized immune-reagents for the detection and quantification of LAMP-1. We have compared a new monoclonal antibody 80280 against LAMP-1 to an existing monoclonal antibody BB6 and a rabbit polyclonal antibody. While all antibodies gave similar results by immunofluorescence, the monoclonal antibody 80280 showed no epitope reactivity to LAMP-1 peptides, suggesting the possibility of a carbohydrate epitope. Western blotting revealed a weaker activity of the monoclonal antibody 80280 relative to either the BB6 monoclonal or the polyclonal antibodies. The monoclonal antibody 80280 is distinct from BB6, providing an additional reagent for CD107a analysis.     

A single injection of recombinant measles virus vaccines expressing human immunodeficiency virus (HIV) type 1 clade B envelope glycoproteins induces neutralizing antibodies and cellular immune responses to HIV

The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.     

Major histocompatibility complex-unrestricted cytolytic activity of human T cells. Analysis of precursor frequency and effector phenotype

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. These conditions were shown to expand a mean of 96% of cells cultured. All of the 198 clones generated by this method were T cells (CD2+, CD3+, CD4+ or CD2+, CD3+, CD8+) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. In addition, the activity was not inhibited by monoclonal antibodies directed against class I or class II nonpolymorphic MHC determinants. Killing, however, was inhibited by soluble monoclonal antibodies against the CD3 complex. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur or K562 was not mediated by a soluble factor secreted by the clones. Some of the clones retained their cytotoxic activity when grown in rIL-2 alone for 4 to 6 wk, whereas others exhibited markedly diminished cytotoxicity after maintenance in this manner. Clones that exhibited diminished or no cytotoxic activity after prolonged maintenance in rIL-2 could be induced to kill by stimulation with immobilized but not soluble monoclonal antibodies to CD3 in the absence of lectin. All of the clones examined expressed NKH1 and CD11b but none were CD16 positive. The degree of cytotoxicity of resting or activated clones could not be correlated with expression of these markers. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.     

Hydrolysis of phosphatidylinositol 4,5-bisphosphate and increase in cytosolic free Ca2+-concentration induced in a human T cell leukemia line, JURKAT, by monoclonal antibodies against the T3 complex

The monoclonal antibodies against the T3 complex on human T lymphocytes, anti-Leu-4, OKT3, and T3, induced an accumulation of inositol phosphates in a human T cell leukemia line, JURKAT, in the presence of LiCl. The monoclonal antibodies also induced an increase in the cytosolic free Ca2+-concentration ([Ca2+]i) in JURKAT. The accumulation of inositol phosphates and the increase in [Ca2+]i were specifically induced by the monoclonal antibodies against the T3 complex. Other monoclonal antibodies against differentiation antigens on human T lymphocytes were not active in inducing these responses in JURKAT. Stimulation of JURKAT by anti-Leu-4 induced a rapid and immediate decrease in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and an increase in the 32P-labeling of phosphatidic acid, which occurred after a short lag period. An analysis of inositol phosphates formed in the anti-Leu-4-stimulated JURKAT indicated the formation of inositol trisphosphate. These results strongly suggested that the T3 complex or T3/antigen receptor (Ti) complex functions as a receptor which transduces antigen signal, presented by either antigen-presenting cells or target cells, into the hydrolysis of PtdIns(4,5)P2. Fetal bovine serum at a dose of 1-20 microliters/ml induced a marked and transient [Ca2+]i increase in JURKAT immediately after addition. However, the level of formation of inositol phosphates was very small in cells stimulated by fetal bovine serum. Fetal bovine serum induced an immediate increase in the 32P-labeling of phosphatidic acid in JURKAT. These and other results suggested that serum increased [Ca2+]i in JURKAT by a mechanism different from that for the anti-Leu-4-induced [Ca2+]i response.     

Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli

Engineered single chain antibodies have become a powerful source of immunotherapy against a wide range of diseases. Here, we present the generation of human CD28 single-chain antibody gene (CD28-ScFv), which contained variable fragments of heavy chain and light chain (VH and VL) of the anti-CD28 antibody, and a linking peptide (Gly4Ser)3 inserted in the middle of VH and VL. The fused gene CD28-ScFv was successfully expressed in BL21 (DE3) cells and confirmed by western blotting assay. The molecular weight of CD28-ScFv was 43 kDa and the major fraction was expressed as an insoluble body. By dissolving the insoluble bodies, renaturing in vitro and purifying with a Ni-NTA affinity column, highly purified expression products of CD28-ScFv were obtained. This product could recognize and bind to CD28+ positive T cells. The proliferation capacity of peripheral blood T cells was increased by purified CD28-ScFv. In this study, we improved orthodox renaturing techniques by combining the dilution renaturation with phase gradient dialysis. With this new method, highly purified CD28-ScFv products were developed and biological activity of the products was similar to that of the mouse monoclonal anti-human CD28 antibody.     

Superantigen-mediated human monocyte-T lymphocyte interactions are associated with an MHC class II-, TCR/CD3-, and CD4-dependent mobilization of calcium in monocytes.

The present study was designed to examine the potential involvement of calcium ions as second messengers in the mediation of the staphylococcal enterotoxin A (SEA)/MHC class II-induced activation of human monocytes. Treatment of monocytes with a monomeric form of SEA failed to induce detectable changes in the level of intracellular calcium in either monocytes or THP-1 cells. However, cross-linking of SEA with biotin-avidin induced a rapid and transient increase in calcium levels in monocytes and in INF-gamma-treated THP-1 cells. This artificial cross-linking system was reproduced by natural physiologic ligands expressed on the surface of T lymphocytes. Delayed, transient, and concentration (cell as well as toxin)-dependent increases in the cytoplasmic level of free calcium in SEA-treated monocytes were observed upon the addition of autologous resting T cells or purified CD4+ cells, but not of CD8+ cells, B cells, or neutrophils. Antibodies against MHC class II Ag, TCR/CD3, and CD4 molecules inhibited the SEA-dependent interaction between monocytes and T cells as indicated by significant decreases in the rise of calcium levels observed in monocytes. Anti-CD8 and anti-class I antibodies did not affect the interaction between the monocytes and the T cells and failed to alter the calcium response. Taken together, these results suggest that the SEA-induced, T cell-dependent calcium mobilization in monocytes requires physical interactions between SEA-MHC class II, TCR/CD3, and CD4 molecules. The ability to mediate a T cell-dependent calcium increase in monocytes was shared by several enterotoxins including staphylococcal enterotoxin B and toxic shock syndrome toxin-1. The characteristics of the SEA-mediated calcium mobilization in monocytes strongly support the hypothesis that this response is an integral part of the signal transducing machinery linked to MHC class II molecules.     

Evidence for the involvement of three distinct signals in the induction of IL-2 gene expression in human T lymphocytes

The regulation of IL-2 gene expression during T cell activation and proliferation has been investigated in primary cultures of purified human peripheral blood T cells. Prior results indicated that stimulation of T cells by anti-CD28 mAb plus PMA could induce IL-2 expression and T cell proliferation that was entirely resistant to cyclosporine. The present studies examined whether CD28 augments IL-2 expression by a unique pathway or merely acts at a point common to CD3-induced proliferation but distal to the effects of cyclosporine. The induction of maximal IL-2 gene expression required three signals provided by phorbol ester, calcium ionophore, and anti-CD28 mAb. Stimulation of cells by optimal amounts of calcium ionophore and PMA induced IL-2 mRNA that was completely suppressed by cyclosporine. The addition of anti-CD28 to T cells stimulated with PMA plus calcium ionophore induced a 5- to 100-fold increase in IL-2 gene expression and secretion that was resistant to cyclosporine. The CD28 signal was able to increase steady state IL-2 mRNA levels even in cells treated with maximally tolerated amounts of calcium ionophore and PMA. The three-signal requirement did not reflect differential regulation of lymphokine gene expression between the CD4 and CD8 T cell subsets or differences in the kinetics of IL-2 mRNA expression. The signal provided by CD28 is distinct from that of CD3 because although anti-CD28 plus PMA-induced proliferation is resistant to cyclosporine, anti-CD3 or anti-CD3 plus PMA-induced IL-2 expression is sensitive. Thus, these studies show that three biochemically distinct signals are required for maximal IL-2 gene expression. Furthermore, these studies suggest that lymphokine production in T cells is not controlled by an "on/off" switch, but rather, that CD28 regulates a distinct intracellular pathway which modulates the level of IL-2 production on a per cell basis. The observation that CD28 stimulation results in IL-2 concentrations that exceed 1000 U/m1 in tissue culture supernatants suggests that a role in vivo for CD28 might be to amplify immune responses initiated by the CD3/T cell receptor complex. Finally, the observation that CD28 interacts with the signals provided by PMA and calcium ionophore shows that the function of CD28 is not merely to act as a scaffold to stabilize or enhance signalling through the CD3/TCR complex.     

Activation of human T cells is associated with tyrosine phosphorylation of several cellular proteins

Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.     

Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes

The aggregation of a specific class of lymphocyte surface molecules results in patching, capping, and surface modulation of the aggregated ligand. Both CD4, an associative recognition structure found on helper T lymphocytes, and CD3, a component of the T cell receptor complex, are members of this functional subgroup. When 125I-labeled monoclonal antibodies reactive with either CD4 (19Thy 5D7) or CD3 (RW24B6) were bound to T lymphocytes, the subsequent addition of goat anti-mouse Ig resulted in their rapid, temperature-dependent internalization. Whereas the binding of 125I-19Thy 5D7 (anti-CD4) was inhibited by greater than 90% in the presence of unlabeled 19Thy 5D7, no inhibition occurred in the presence of unlabeled antibody reactive with CD3 (RW28C8). We took advantage of the fact that these antibodies were of different isotypes (19Thy 5D7:IgG2a; RW28C8:IgGl) to determine whether the internalization of CD3 induced the comodulation of CD4. T lymphocytes preincubated with 125I-19Thy5D7 (anti-CD4) and unlabeled RA28C8 (anti-CD3) were treated with goat anti-mouse IgGl under conditions shown to quantitatively internalize CD3. After 1 h at 37 degrees C, T lymphocytes had internalized 10.5 +/- 2.6% (n = 3) of their antibody-bound cell surface CD4. After similar incubations with media alone or with goat anti-mouse IgGl in the absence of prebound RW28C8 (anti-CD3), no internalization of CD4 could be detected. Control antibodies reactive with CD45R (2H4, IgGl) also failed to induce the internalization of CD4. Similar results were obtained by using a helper T cell clone (T4C1) that internalized 9.6 +/- 2.8% (n = 3) of its antibody-bound cell surface CD4 in response to CD3 modulation. In a reciprocal experiment, 125I-anti-CD3 (RW24B6, IgG2b) was preincubated with T4Cl cells together with unlabeled anti-CD4 (12T4D11, IgG1) prior to the addition of goat anti-mouse IgGl. The quantitative modulation of CD4 induced the co-internalization of 4.6 +/- 0.6% (n = 3) of cell surface CD3. These results suggest that approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes are specifically associated with CD4. Furthermore, our results suggest that an average of two CD4 molecules associate with each CD3:T cell receptor complex.     

Effects of cytokines and prolactin on the replication of Toxoplasma gondii in murine microglia

In the central nervous system, cytokine-activated microglia play a crucial role in host defence against Toxoplasma gondii infections. In this study, the effect of recombinant tumor necrosis factor (rTNF)-alpha and prolactin (PRL) on T. gondii infection in microglia was examined. Pretreatment of microglia with rTNF-alpha and PRL induced toxoplasmastatic activity, the intracellular killing of T. gondii and the release of interleukin (IL)-1 beta IL-3 and IL-6: 50% of the intracellular killing was abrogated by anti-ICAM-1 monoclonal antibodies, whereas more than 54 or 87% of toxoplasmastatic activity was reversed by anti-IL-3 or IL-6 monoclonal antibodies. In addition, the treatment of microglia with either rIL-3 or rIL-6, in the absence or presence of rTNF-alpha significantly limited T. gondii replication. Inasmuch as either NMA or S-M-ITU affected cytokine-activated toxoplasmastatic activity during the infection phase, the NO-dependent pathway itself appears not to be directly involved in the parasitostatic activity. These findings suggest that TNF-alpha and PRL up-regulate the expression of ICAM-1 and the production of endogenous IL-6 and IL-3 by microglia, which could induce anti-parasitic functions against T. gondii infection in the brain.     

Functional studies with anti-CD3 heavy chain isotype switch-variant monoclonal antibodies. Accessory cell-independent induction of interleukin 2 responsiveness in T cells by epsilon-anti-CD3

A series of heavy chain isotype switch-variant anti-CD3 monoclonal antibodies (mAb) was used to study the proliferation requirements of purified T cells. None of the variant antibodies was able by itself to induce proliferation. In the presence of exogenous interleukin 2 (IL-2) strong mitogenesis was observed upon stimulation with epsilon-anti-CD3, whereas gamma 1, gamma 2b, gamma 2a, and alpha-anti-CD3 failed to induce T cell proliferation. All variant antibodies induced vigorous proliferation in combination with phorbol myristate acetate. Purified T cells, cultured in the presence of epsilon-anti-CD3, in the absence of IL-2, did not express detectable amounts of TAC-antigen (CD25). The binding of the variant antibodies to the CD3 antigen was evaluated in cross-blocking experiments. It was demonstrated that the epsilon-anti-CD3 antibody, in comparison with the other variant mAb, has a relatively low avidity for the CD3 antigen. In modulation experiments, the IgE variant antibody was unable to induce a substantial loss of CD3 antigen. T cell triggering was investigated at the level of Ca2+ mobilization by means of the dye Indo-1. In contrast to the gamma 1, gamma 2b, gamma 2a, and alpha mAb, which induced a rapid and high rise in the free intracellular calcium level, epsilon-anti-CD3 caused a slow and low rise. These studies indicate that the epsilon-anti-CD3 antibody has a low avidity for the CD3 antigen, compared with the other variant mAb, possibly as a result of monovalent binding. Apparently, the avidity and/or valency of CD3 antigen binding not only has a major influence on CD3 modulation and anti-CD3-induced Ca2+ mobilization, but also sets T cell requirements for IL-2 responsiveness.