Spontaneous T cell antigen receptor variants of a human T leukemia cell line

We have sought to address the question of clonal variation of TCR within a human T leukemia cell line, HPB-ALL. To do so, a panel of anti-idiotypic antibodies was produced and the cell line examined for variants. We isolated both spontaneous idiotype and receptor-negative variants without applying mutagens or any selective pressure other than sorting the cells. These sorted and cloned populations are all clonally related to each other as shown by their beta-TCR locus gene rearrangements. The idiotype variants have alpha-chains which are differentially glycosylated, but they have the same size core protein after treatment with peptide N-glycosidase F to remove their carbohydrate side chains. This probably accounts for their idiotypic difference, since the antibody that distinguishes them appears dependent upon glycosylation for its binding, as shown by immunoprecipitation in the presence versus the absence of tunicamycin, which inhibits glycosylation from occurring. The idiotype variants differed from one another in variable region sequences by only a single amino acid substitution in the beta-chain, which is likely not important for the idiotypic difference. The receptor-negative variant produces both alpha- and beta-mRNA and cytoplasmic protein for TCR, but fails to transport this protein to the cell surface. We conclude that idiotype and receptor-negative variants of a T cell clone can occur in the absence of appreciable somatic mutation.     

Antigen-specific T cell activation results in an increase in cytoplasmic free calcium

Free intracellular calcium acts as a messenger in response to extracellular stimuli, including those that result in cellular proliferation. For example, mitogenic lectins have been shown to increase intracellular calcium concentration ([Ca+2]i) during proliferation of T lymphocytes. To determine if similar changes in [Ca+2]i occur when T cells are activated by nominal antigen, [Ca+2]i was measured in murine T cells from a bovine insulin-specific, major histocompatibility-restricted T hybridoma by using the calcium-sensitive fluor quin-2. Quin-2-loaded T hybridoma cells were activated by incubation with antigen-pulsed antigen-presenting cells (APC) and [Ca+2]i determined by measurement of quin-2 fluorescence. T cell [Ca+2]i rose sharply within 20 min after incubation with APC. Incubation of T cells with unpulsed APC resulted in [Ca+2]i not significantly different from resting levels. Further evidence that this activation was antigen specific was demonstrated at the level of both the APC and the T cell. Incubation of quin-2-loaded T cells with APC pulsed with the inappropriate antigen, porcine insulin, did not result in an increase in [Ca+2]i. Additionally, pretreatment of T cells with a monoclonal antibody against the T cell antigen receptor abrogated the [Ca+2]i increase. Finally, the antigen-induced rise in [Ca+2]i could be blocked by pretreatment of APC with appropriate but not inappropriate Ia monoclonal antibodies. These results suggest that a rapid rise in [Ca+2]i is an early event in the antigen-specific activation of the T cell and may be related to later steps, such as the secretion of lymphocyte monokines.     

Aluminum fluoride induces phosphatidylinositol turnover, elevation of cytoplasmic free calcium, and phosphorylation of the T cell antigen receptor in murine T cells

Antigen activation of murine T lymphocytes leads to phosphorylation of three subunits of the murine T cell antigen receptor (L.E. Samelson, M.D. Patel, A.M. Weissman, J.B. Harford, and R.D. Klausner. 1986. Cell 46:1083). Two kinases are activated in this process: protein kinase C which leads to phosphorylation of the gamma and, to a lesser extent, the epsilon subunits on serine residues and a tyrosine kinase which phosphorylates the p21 subunit (M.D. Patel, L.E. Samelson, and R.D. Klausner. 1987. J. Biol Chem. 262:5831). We sought to determine whether treatment of these cells with NaF could simulate any of these antigen-induced events. Indeed NaF treatment resulted in breakdown of polyphosphoinositides and production of phosphoinositols. This treatment also resulted in a rise in cytosolic free Ca2+. EGTA failed to block this rise suggesting that NaF liberated intracellular stores of Ca2+. Finally NaF treatment resulted in phosphorylation of the gamma and epsilon chains of the T cell receptor indistinguishable from the effects of phorbol esters. The NaF effect was potentiated by addition of A1Cl3 consistent with the view that the active moiety is A1F4-. The A1F4--induced phosphorylations were abolished in cells in which protein kinase C was depleted by prior treatment with phorbol myristate acetate. All of these observations are compatible with the interpretation that the A1F4- phosphorylation is mediated by protein kinase C. Antigen and anti-receptor antibody-induced receptor serine phosphorylation and phophatidylinositol turnover are blocked by raising intracellular levels of cyclic adenosine monophosphate. In contrast, A1F4--induced effects were insensitive to cyclic adenosine monophosphate.     

A novel pathway of human T cell activation via a 103 kD T cell activation antigen

A novel triggering signal for human proliferating and cytotoxic T lymphocytes defined by a 103 kD T cell-specific activation antigen (Tp103) is described. Tp103 is expressed on all proliferating normal T cells but is not present, or present only in low amounts, on resting peripheral blood T lymphocytes. Cross-linking of T cell and Fc receptor-positive accessory or target cells by an antibody against Tp103 leads to activation of the T cell. The proliferative response is due to an autocrine IL 2-dependent mechanism and can be inhibited by antibodies against the IL 2 receptor or by Cyclosporin A. Resting Tp103-positive T cells also respond to anti-Tp103. Although Tp103 is not linked to the antigen receptor/T3 complex, triggering via Tp103 can be inhibited by modulation of the T3 molecule. Thus, Tp103 defines a new antigen-independent pathway of T cell activation that can be regulated via other T cell surface structures.     

Regulation of cytoplasmic free calcium by plant cell membranes

The free calcium concentration in the cytoplasm of higher plant cells is believed to be about 10⁻⁷M. The role of various membrane-borne calcium transporters present in plant tissues, together with chloroplasts and mitochondria, in maintaining this calcium concentration is reviewed, together with the role of various organelles in providing transient calcium fluxes upon stimulation.     

Immunosuppressive effect on T cell activation by interleukin- 16-cDNA-transfected human squamous cell line

It is well known that it is difficult to induce an immunotolerance with allogeneic skin transplantation. We attempted to find the immunosuppressive protocol for prolonging skin allograft rejection by using interleukin-16 because IL-16 is considered one of the natural ligands to CD4 molecules. First we examined whether synergistic immunosuppressive effects of recombinant IL-16 plus anti-CD4 mAbs are induced in mixed lymphocyte reaction (MLR). Next we used IL-16-cDNA-transfected OSC-20 (human oral squamous cell carcinoma cell line) as an in vitro model of the epidermal keratinocyte equivalent and examined whether this transfectant could inhibit the activation of allogeneic T cells. Our data indicated that IL-16 clearly inhibited human MLR and that IL-16 increased synergistically the immunosuppressive effect of anti-CD4 mAb. We also used IL-16 transfectant and this produced more than 50 ng/ml of IL-16 in the supernatant by which human MLR was significantly inhibited. Furthermore, this transfectant also inhibited the activation of allogeneic lymphocytes stimulated directly with transfectant cells. These results indicated that the IL-16-producing allogeneic skin graft might have a local immunosuppressive action that would prolong graft survival.     

The T cell antigen receptor zeta chain is tyrosine phosphorylated upon activation

The T cell antigen receptor is composed of at least seven chains derived from six different gene products. Upon stimulation, several chains can be phosphorylated. Two of these, CD3-gamma and CD3-epsilon are phosphorylated on serine residues. In addition, a 21-kDa nonglycosylated receptor component is phosphorylated, upon activation, on tyrosine residues. We have referred to this phosphoprotein as p21 because we have previously not been able to assign the tyrosine phosphorylation to any of the described receptor subunits (Samelson, L. E., Patel, M. D., Weissman, A. M., Harford, J. B., and Klausner, R. D. (1986) Cell 46, 1083-1090). In this paper, we demonstrate that it is the 16-kDa zeta chain which is the tyrosine phosphorylated subunit, and thus the p21 nomenclature can be replaced. This phosphorylation results in a shift of the apparent Mr of zeta to 21 kDa. Proof that p21 is tyrosine phosphorylated zeta was afforded by a number of approaches. Specific anti-zeta antibodies directly precipitated phospho-p21. Metabolically labeled protein corresponding to p21 could only be observed after activation. When this 21-kDa band was isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment with alkaline phosphatase, its migration was identical with that of zeta. Furthermore, peptide mapping of metabolically labeled p21 (after gel isolation and dephosphorylation) showed it to be indistinguishable from p21. Thus, one of the early events of T cell activation is the tyrosine phosphorylation of the zeta chain of the T cell antigen receptor.     

Caspase-3 activation is an early event and initiates apoptotic damage in a human leukemia cell line

Many apoptotic pathways culminate in the activation of caspase cascades usually triggered by the apical caspases-8 or -9. We describe a paradigm where apoptosis is initiated by the effector caspase-3. Diethylmaleate (DEM)-induced apoptotic damage in Jurkat cells was blocked by the anti-apoptotic protein Bcl-2, whereas, a peptide inhibitor of caspase-3 but not caspase-9 blocked DEM-induced mitochondrial damage. Isogenic Jurkat cell lines deficient for caspase-8 or the adaptor FADD (Fas associated death domain) were not protected from DEM-induced apoptosis. Caspase-3 activation preceded that of caspase-9 and initial processing of caspase-3 was regulated independent of caspase-9 and Bcl-2. However, inhibitors of caspase-9 or caspase-6 regulated caspase-3 later in the pathway. We explored the mechanism by which caspase-3 processing is regulated in this system. DEM triggered a loss of Erk-1/2 phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression. The phorbol ester PMA activated a MEK-dependent pathway to block caspase-3 processing and cell death. Constitutively active MEK-1 (CA-MEK) upregulated XIAP expression and exogenous XIAP inhibited DEM-induced apoptotic damage. Thus, we describe a pathway where caspase-3 functions to initiate apoptotic damage and caspase-9 and caspase-6 amplify the apoptotic cascade. Further, we show that MEK may regulate caspase-3 activation via the regulation of XIAP expression in these cells.     

The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain.

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.     

The interference of T cell activation by calcium channel blocking agents

Calcium has been identified as having an important role as a transmembrane messenger in the activation signal for lymphocytes. To additionally examine this model, we evaluated the effect of calcium channel blocking drugs (verapamil, nifedipine, and diltiazem) on lymphocyte activation. In these studies we found that the drugs inhibit, in a dose-dependent fashion, the proliferation of T cells and the appearance of certain activation antigens after mitogen stimulation. This appears to result from the marked decrease in mitogen-induced 45calcium (45Ca+2) influx secondary to the addition of these agents. In addition, T cell proliferation resulting from IL 2 binding to its receptor is also suppressed by the calcium channel blocking drugs. These data suggest that the passive calcium channel plays a pivotal role in both the initial activation of T cells after ligand-receptor interaction and the ongoing signal for proliferation provided by IL 2 binding to its receptor.     

Accessory cell requirement for activation antigen expression and cell cycle progression by human T lymphocytes

Stringent accessory cell (AC) depletion by a three-step procedure--plastic adherence, nylon wool adherence, followed by simultaneous treatment with two anti-AC monoclonal antibodies + complement--has allowed the demonstration of several AC-dependent stages in the T cell activation pathway. Simultaneous analysis of DNA content and cell surface immunofluorescence (correlation of activation antigen expression with cell cycle position) or DNA and RNA content (cell cycle position) of cultured cells was accomplished by dual parameter flow cytometry. AC-depleted, PHA-stimulated human peripheral blood T lymphocytes (PBTL) failed to exhibit "early" indicators of activation, including increased RNA content, expression of three activation-associated cell surface proteins (IL 2 receptor, transferrin receptor, and 4F2 protein), and the production of IL 2. The AC-depleted PBTL that failed to express these "early" markers of activation also failed to progress into the "late" phase of activation, DNA synthesis. All indicators of PHA responsiveness were fully replenished upon addition of AC but were only reconstituted to intermediate levels by addition of excess quantities of either highly purified IL 1 or crude AC-conditioned medium with lymphocyte-activating factor activity. These data suggest that the AC membrane plays a key and as yet undefined role in the stimulation of T cells by PHA.     

Growth inhibition of human T cells by antibodies recognizing the T cell antigen receptor complex

Monoclonal antibodies that bind to the T cell MHC-antigen recognition complex (anti-T3 or anti-Ti) are known to either mimic ligand binding and activate T cells or block ligand binding, leading to an inhibition of T cell activation. In the present experiments, we demonstrate a direct inhibitory effect on the growth of human T cells by anti-T3 or anti-Ti antibodies. The proliferation of human peripheral blood T cells preactivated by exposure to PHA was inhibited in a specific manner by anti-T3. Colony formation in soft agar by REX cells, a leukemic cell line of early T cell phenotype, was completely inhibited by anti-T3 or anti-Ti antibodies, whereas isotype-matched antibodies to a variety of other T cell markers had no effect. Growth of REX cells in suspension culture was not affected by anti-T3 or anti-Ti. A cell line, T3.N1, was established from an agar colony of anti-T3-resistant REX cells. T3.N1 was phenotypically identical to REX except for failure to express any detectable T3 or Ti surface antigen. T3.N1 colony formation in soft agar was not inhibited by anti-T3 or anti-Ti. There was no rise in [Ca2+]i of T3.N1 cells after anti-T3 or anti-Ti exposure. These results indicate that in addition to the well-known positive regulatory effects of ligand binding to the T3/Ti complex, T3/Ti binding can also result in a down-regulatory signal for human T cell growth.     

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.     

Identification and characterization of a B cell activation factor (BCAF) produced by a human T cell line

A human T cell line, Peer, that expresses the T cell helper phenotype produces discrete activation and growth factors for tonsillar B cells. The B cell activation factor produced by Peer is biochemically and physiologically distinct from other lymphokines known to enhance B cell proliferation, namely, interleukin 1, interleukin 2, interferon, and previously characterized B cell growth factors (BCGF). The BCGF produced by Peer is functionally similar to previously described BCGF but has a m.w. of approximately 30,000 daltons. The identification and characterization of a T cell-derived activation factor that can induce apparently resting (Go phase) B cells to enter S phase in the absence of an exogenous first signal has important implications in the additional dissection of the complex steps in the human B cell cycle.     

Unique T cell Ia antigen expressed on a hybrid cell line producing antigen-specific augmenting T cell factor

Hybrid cell lines were established by fusion between keyhole limpet hemocyanin(KLH) binding T cells of A/J mice and an AKR T cell tumor line, BW5147. Hybrids were selected for the presence of Ia antigen and KLH-specific augmenting activity of their extracts in the secondary antibody response. The detailed phenotypic and functional analysis of 1 of these clones, FL10, is reported here. The hybrid was positive for both Thy1.1 and Thy1.2 antigens and possessed the Lyt-1+,2-,3- phenotype. Both VH and Ia determinants were detected on their cell surface. The IA locus was mapped in the I-A subregion, but the Ia specificities were serologically distinct from those of B cell Ia antigen. This was demonstrated by the fact that anti-Ia antiserum preabsorbed with B cells could react with the hybrid cells, whereas none of the monoclonal anti-Ia specific for private and public determinations of Iak could. The extract from the cell line specifically augmented the in vitro secondary antibody response against dinitrophenylated KLH, and this activity was removed by absorption with antigen and conventional anti-Ia antisera. The results indicate that the cell line, FL10, carries Ia antigen unique to the T cell, which is associated with the antigen-specific augmenting molecule.