Identification of monoclonal antibodies specific for the T cell receptor complex by Fc receptor-mediated CTL lysis

Monoclonal antibodies (mAb) directed at the T cell receptor complex (TcR) on cloned T cells have generally been identified by their ability to inhibit the clone's antigen-specific function. Because such inhibition is highly dependent on antibody concentration and affinity, detection of anti-clonotypic antibodies to murine alloreactive T cells has been very difficult. In this report, an alternative method is described on the basis of the ability of antibodies specific for the TcR complex to activate T cells in an antigen-independent manner. The assay is based upon the observation that soluble antibodies to human T3 promote lysis of irrelevant, Fc receptor-positive targets by a human CTL line. By using this approach, an anti-TcR mAb has been identified among a panel of murine mAb generated against an alloreactive CTL clone. Induction of lysis by soluble anti-TcR mAb has been shown to require both the expression of Fc receptors on the target cell and conjugate formation between the effector and the target cell. This assay provides a screening procedure that is much more sensitive than inhibition of function, and it preferentially detects antibodies specific for cell surface molecules involved in T cell activation.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. II. Electrolyte permeability increase in the target cell membrane concomitant with programming for lysis.

In a previous study of the mechanism of specific target cell lysis by alloimmune cytolytic T lymphocytes (CTL), we established that the target cell becomes irreversibly programmed to lyse within a few minutes after contact with the CTL. We here show that at each point in time, the level of specific release of the potassium analog, 86Rb equals the percentage of target cells which have been programmed to lyse. It is also shown that specific release of 86Rb is more rapid than that of a small metabolite of similar weight, 14C-nicotinamide, which in turn is specifically released more rapidly than 51Cr. Thus, an electrolyte-permeable lesion is produced in the target cell membrane within minutes of contact with the CTL. Since measurements of 86Rb release, unlike measurements of programming for lysis, do not involve exposure of the cells to EDTA and vigorous shearing forces, the present observations corroborate and extend, by an independent and gentler method, our previous conclusion that the CTL effects crucial and irreversible changes in the target cell within minutes after contact. The present results are consistent with the possibility that the first, and perhaps the only damage administered directly by the CTL is a membrane lesion permeable to electrolytes and possibly to small molecules.     

Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen

Three mouse hybridomas secreting antibodies against the undecapeptide Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr, corresponding to the carboxy terminus of simian virus 40 large T antigen, were isolated and cloned. A sensitive enzyme-linked immunosorbent assay was used to characterize the properties of the monoclonal antibodies. All three hybridomas, designated KT1, KT3, and KT4, produced antibodies that immunoprecipitated large T. The antibodies differed in their affinities for the peptide and for the native protein. Antibodies from KT3 precipitated large T better than those from KT1 or KT4. KT3 antibodies also had the highest affinity for the free peptide (5.2 X 10(6) M-1) as determined by radioimmunoassay; KT1 and KT4 antibodies had ca. 5- and 1,000-fold lower affinities, respectively. Inhibition studies with shorter peptides, overlapping the undecapeptide, revealed the approximate regions recognized by the different monoclonal antibodies. KT3 antibodies bound to a region within the carboxy-terminal six amino acids of large T. Antibodies from KT1 and KT4 reacted with sequences located further towards the amino terminus of the undecapeptide. Surprising results were obtained with KT4 antibodies. Their binding to the undecapeptide was completely inhibited by the undecapeptide itself or the carboxy-terminal hexapeptide. The carboxy-terminal pentamer, on the other hand, slightly enhanced binding, and the carboxy-terminal tetramer, Glu-Pro-Glu-Thr, was strongly stimulatory. A model for this effect is proposed. Using the enzyme-linked immunosorbent assay, we confirmed previous studies (W. Deppert and G. Walter, Virology 122:56-70, 1982) which found that antiserum against sodium dodecyl sulfate-denatured large T reacts strongly with the carboxy terminus of large T. By inhibition studies, we identified the approximate region within the undecapeptide recognized by anti-sodium dodecyl sulfate-denatured large T and compared this region with the region identified by antipeptide serum.     

Antigen-dependent release of IFN-gamma by cytotoxic T cells up-regulates Fas on target cells and facilitates exocytosis-independent specific target cell lysis

Effector cytolytic T (Tc) lymphocytes, deficient in the exocytosis-mediated pathway of target cell lysis, induce Fas on target cells and, in turn, delayed cell death and apoptosis via the Fas ligand-Fas interaction. The induction of Fas can be blocked by anti- IFN-gamma Abs. This Fas up-regulation on initially Fas-negative target cells is not mediated by TCR-MHC/peptide signaling per se, but by secreted IFN-gamma from Tc cells after Ag engagement. The Fas up-regulation by Tc cells can be mimicked by treatment of target cells with rIFN-gamma. Tc cells from IFN-gamma knockout mice do not induce Fas expression on target cells. Tc cell-mediated Fas expression on third party, bystander, target cells does not enhance their susceptibility to lysis by these nominal effector cells. The results are discussed as to the possible relevance of the phenomenon in efficiency and regulation of the Tc cell response to infections by viruses.     

Induction of antibodies to a tumor-associated antigen by immunization with a whole melanoma cell vaccine

Summary Urinary-tumor-associated antigen (U-TAA) is a glycoprotein present in the urine of melanoma patients. Previous studies have addressed the role of U-TAA in immunoprognosis. The present investigation was undertaken to determine whether the administration of whole melanoma cell vaccine (MCV) could induce the formation of anti-(U-TAA) antibodies in melanoma patients. The subjects of this study were stage II and III melanoma patients receiving MCV alone or in conjunction with cyclophosphamide. Anti-(U-TAA) IgM and IgG antibody levels were determined by enzyme immunoassay in sequential serum samples from 15 stage II and III melanoma patients receiving MCV. U-TAA purified from the urine of a melanoma patient was used as a target in this assay. The mean anti-(U-TAA) IgM titer prior to vaccination was similar to that of a non-vaccinated melanoma control group (1:1138±214, n=15 vs 1:1334±254, n=7; P=0.375) but prevaccination IgG levels were generally higher than in the control group (1:3984±602 vs 1:2595±423; 0.1>P>0.05). While only 6 of the 15 patients demonstrated a rise in levels of IgG antibodies (mean 1:2964±1047 pre-MCV to 1:9958±2677 post MCV, P<0.01), 11 of the 15 patients demonstrated a greater than twofold rise in their anti-(U-TAA) IgM titers following vaccination (1:1051±259 pre-MCV to 1:2518±576 post-MCV; P<0.005). In addition, patients with visceral metastases consistently elicited anti-(U-TAA) responses equivalent to those with more limited disease. Concomitant administration of cyclophosphamide did not affect the response rates or peak antibody levels. The possibility that these antibody responses were actually against histo-compatibility locus antigens (HLA) (contaminating our U-TAA preparation) was ruled out because the target antigen (U-TAA) was devoid of HLA, and because the induction of anti-(U-TAA) antibodies did not correlate with the induction of anti-HLA antibodies. These results demonstrate augmentation of anti-(U-TAA) IgM and IgG antibodies by immunization with the MCV.     

Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T antigen.

We isolated 16 new monoclonal antibodies that recognize large T antigen of simian virus 40 and mapped the epitopes to three distinct regions of the large T antigen. Also, 3 of the 16 recognized the large T antigen of the human papovavirus BKV.     

Soluble trinitrophenylated proteins and trinitrophenylated cells as specific inhibitors of target cell lysis by cytotoxic T lymphocytes.

Bovine serum albumin (BSA) substituted in 12 to 15 amino groups with 2,4,6-trinitrophenyl (Tnp-BSA) or carbobenzoxy (Cbz-BSA) or acetyl (Ac-BSA) groups was tested as inhibitor of the reaction in which anti-Tnp cytotoxic T lymphocytes (CTLs) lysed syngeneic ⁵¹Cr-labeled Tnp-modified spleen cells [concanavalin A (Con A) blasts]. Inhibition was observed with some consistency only with Tnp-BSA at extremely high concentration (50 mg/ml). To explore the significance of this observation, inhibition of anti-Tnp CTLs was also tested with Tnp-modified cells on which products of the major histocompatibility loci H-2K and H-2D were lacking or different from those on the stimulator cells used to elicit the CTLs. Only those Tnp cells with the same H-2 products as the stimulators were inhibitory, even though all the Tnp cells tested had essentially the same surface density of Tnp (ca 1 × 10⁸ groups/cell). It is concluded that effective specific inhibitors of anti-Tnp CTLs have both Tnp groups and the correct H-2 products on the same particle and that the specific inhibitory activity of soluble Tnp-BSA was probably due to its adsorption onto cells in the suspensions used to assay cytotoxicity.     

Relationship between surface H-2 concentration, size of different target cells, and lysis by cytotoxic T cells

We have examined the parameters of target cell cross-sectional area and surface H-2 concentration in relation to their susceptibility to cytotoxic T-cell-mediated lysis, using a series of commonly used murine target cells of the H-2k haplotype. We used a sensitive immunoferritin labeling technique and electron microscopy to estimate relative cell diameter and H-2 concentration combined with standard 51Cr-release assays for cytotoxicity. We found that susceptibility to cytotoxic T-cell lysis was not related consistently to either factor alone, but was related to a combination of the two, such that above a certain value for the product of the two factors, no further increase in cytotoxic T-cell efficiency was seen. The information presented here should be of value to workers seeking to select a target cell type that will maximize the sensitivity of 51Cr-release-based assays for the detection of cytotoxic T cells.     

Contribution of antigen processing to the recognition of a synthetic peptide antigen by specific T cell hybridomas

Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the alpha-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the alpha-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.     

Receptor-mediated B cell antigen processing. Increased antigenicity of a globular protein covalently coupled to antibodies specific for B cell surface structures

Helper T cell recognition of globular protein antigens requires the intracellular processing of the native molecule by an antigen-presenting cell and subsequent presentation of a peptide fragment, containing the antigenic determinant, on the cell surface where it is recognized by the specific T cell in conjunction with Ia. B lymphocytes can function as antigen-presenting cells and, when antigen is bound by their surface Ig, are greatly enhanced in this capacity. In this report it is demonstrated that pigeon cytochrome c covalently coupled to antibodies directed toward either B cell surface immunoglobulin, class I or class II are effectively processed and presented by B cells to cytochrome c-specific T cells, requiring up to 1000-fold less cytochrome c as compared with cytochrome c alone or cytochrome c coupled to nonspecific immunoglobulin. The potent activity of the cytochrome c-antibody conjugates appears to be due to the ability of B cells to concentrate the antigen when the process becomes receptor mediated rather than to a signal provided to the B cell by the conjugate binding, because cytochrome c was not more effectively presented in the presence of unconjugated antibodies as compared with cytochrome c alone. Furthermore, the binding of the native antigen to B cell surfaces is not alone sufficient for T cell activation, in that the cytochrome c-antibody conjugates require processing and are major histocompatibility complex restricted. The results presented here indicate that surface immunoglobulin is not unique in its ability to facilitate antigen processing and/or presentation and that Ig, class I and class II are capable of transporting the cytochrome c to a cytoplasmic vesicle where proteolysis occurs yielding the required peptide, minimally of 10 amino acids. Cytochrome c coupled to monovalent fragments of anti-Ig-antibodies was nearly as effectively presented as cytochrome c coupled to bivalent antibodies, indicating that phenomena mediated by bivalent binding, such as patching and capping of the surface Ig, were not required for effective antigen presentation. The cytochrome c-antibody conjugates, which allow antigen processing to be initiated by receptor-mediated endocytosis, may provide the necessary tools to unravel the intracellular processes by which protein antigens are processed and presented by B lymphocytes.     

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.     

Characterization by monoclonal antibodies of a target cell antigen complex recognized by nonspecific cytotoxic cells

Fish nonspecific cytotoxic cells (NCC)3 recognize and lyse a large variety of human and mouse transformed cells. In an effort to determine the Ag recognized by NCC on these targets, mAb were raised against NC-37 target cells. Four anti-NC-37 mAb were chosen for further characterization based on their effects on NCC lysis of target cells. Purified mAb 18C2 and 1E7 (IgM isotype) inhibited NCC killing of the following targets: U937, MOLT-4, K562, HL-60, DAUDI, NC-37, P815, and YAC-1. The dose-dependent inhibitory activity occurred at the target cell level and ranged from 50 to 70% at a concentration of 50 micrograms/well when compared to noninhibitory mAb 7C6 and 1D4 (IgG isotype). Similarly, mAb 18C2 protected the fish parasite Tetrahymena pyriformis from lysis by NCC when compared to mAb 7C6. Adsorption experiments demonstrated that the inhibitory effect on NC-37 lysis by NCC could be removed in a titratable fashion by incubation of mAb 1E7 with any one of the other target cell lines, but it could not be removed by incubation with effector cells. The inhibitory activity of mAb 1E7 and 18C2 was shown to be caused by the inhibition of conjugate formation between effector and NC-37 target cells. The relative membrane concentration of the antigenic determinants recognized by these mAb on the target cells was studied by flow cytometry using FITC-labeled mAb. These experiments showed that all four mAb bound to the surface of the cells tested. Biochemical analysis with Western blots and immunoprecipitation showed that mAb 18C2 and 1E7 recognize two Ag in NC-37 lysates: a larger protein of around 80 kDa and a smaller one of 42 kDa.     

The kinetics of target cell lysis by cytotoxic T lymphocytes: a description by Poisson statistics.

The kinetics of T cell killing are analyzed with the assumption that encounters between killer and target cells occur at random. The application of Poisson statistics leads to a number of theoretical predictions which were then tested experimentally. Good agreement between data and theory was found.     

Anti-T cell receptor antibodies fail to inhibit specific lysis by CTL clones but activate lytic activity for irrelevant targets

In this report, we describe the functional effects of anti-T cell receptor antibodies on a panel of MHC-restricted, influenza virus-specific CTL clones. Approximately 25 to 30% of these clones are recognized by KJ16-133, an anti-T cell receptor monoclonal antibody presumably specific for products of the V beta 8 gene family, and an antibody with similar specificity, F23.1. In contrast to most previous reports, both KJ16-133 and F23.1, over a wide range of antibody concentrations, fail to inhibit the antigen-specific effector function of these CTL. Instead, the antibodies activate the CTL to kill without regard for the MHC haplotype of the target cells or the presence of the appropriate viral antigen. This anti-T cell receptor antibody-induced cytolysis by our clones does not appear to be mediated by Fc receptors on target cells. Nuclear destruction of target cells as a result of antibody-induced lysis suggests that it occurs via a mechanism similar to antigen-specific lysis by CTL. In addition, both soluble bivalent F23.1 and F23.1 coupled-Sepharose beads are able to induce the secretion of interferon-gamma from these CTL clones.     

Features of target cell lysis by class I and class II MHC-restricted cytolytic T lymphocytes

The lytic activity of influenza virus-specific murine cytolytic T lymphocyte (CTL) clones that are restricted by either H-2K/D (class I) or H-2I (class II) major histocompatibility (MHC) locus products was compared on an influenza virus-infected target cell expressing both K/D and I locus products. With the use of two in vitro measurements of cytotoxicity, conventional 51Cr release, and detergent-releasable radiolabeled DNA (as a measure of nuclear disintegration in the early post-lethal hit period), we found no difference between class I and class II MHC-restricted CTL in the kinetics of target cell destruction. In addition, class II MHC-restricted antiviral CTL failed to show any lysis of radiolabeled bystander cells. Killing of labeled specific targets by these class II MHC-restricted CTL was also efficiently inhibited by unlabeled specific competitor cells in a cold target inhibition assay. In sum, these data suggest that class I and class II MHC-restricted CTL mediate target cell destruction by an essentially similar direct mechanism.     

A novel antigenic cell surface protein associated with T200 is involved in the post-activation stage of human NK cell-mediated lysis

A monoclonal antibody, 9.1C3, was used to investigate the mechanism of natural killer (NK) cell-mediated lysis. In addition to blocking NK cell function, the antibody blocked antibody-dependent cellular cytotoxicity against the K562 target cell at the effector cell level. The stage at which 9.1C3 antibody inhibited cytolysis was established with a Ca++ pulse technique, whereby it was shown that the antibody inhibited killing at a discrete step after the Ca++-dependent programming for lysis. The 9.1C3 antigen appeared to be associated with the T200 glycoprotein complex. Thus the 66 and 77 Kd proteins detected by 9.1C3 were also precipitated with a monoclonal antibody to T200, and in sequential immunoprecipitations, 9.1C3 antibody removed these bands from immunoprecipitates with antibody to T200. Also, in co-modulation studies, it was found that antibody to T200 co-capped the 9.1C3 antigen but that capping with 9.1C3 antibody did not induce co-modulation of the T200 antigen. Expression of the 9.1C3 and T200 antigens on different cell types, however, was not identical, and the 9.1C3 antibody did not immunoprecipitate high m.w. proteins in the region of 200 Kd. Functionally, in NK cell killing studies, the antibody to T200 used alone did not block but was synergistic with the 9.1C3 antibody. The differential effect of the enzymes pronase and trypsin on the cell surface expression of the 9.1C3 and T200 antigens reflected the ability of these enzymes to inhibit NK cell killing. These data suggest that the 9.1C3 antigen participates in a late event in the cytolytic pathway.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VIII. Kinetics of lysis of target cells bound by more than one cytotoxic T lymphocyte

We measured the effects of having multiple cytotoxic T lymphocytes (CTL) bound to one target cell by using the single-cell cytotoxicity in agarose assay. We found that even though there is variability in the time at which individual target cells are lysed, we can identify a general trend: the mean rate of lysis increases with the number of CTL bound per target cell, reaching a maximum when the CTL-target cell ratio is three. Combining a quantitative model for the rate of lethal hitting in multicellular conjugates with a multi-event model for the rate of target cell disintegration, we developed a new multistage kinetic model for predicting the rate of target cell lysis in multiple lymphocyte-target cell conjugates. The variability in the time at which target cells are hit and the variability in the time until they disintegrate are incorporated into the model. By analyzing our measured data in the context of the multistage kinetic model, we were able to estimate via nonlinear least squares regression the target cell disintegration rate, but not the lethal hitting rate. Lethal hitting appeared to be too fast, when compared with disintegration, to significantly affect the time of target cell lysis. By using previously determined values of the lethal hitting rate for single lymphocyte-target cell conjugates and by postulating that lymphocytes act independently of each other in delivering lethal hits, we were able to estimate the rate at which target cells are hit in multiple-lymphocyte single target cell conjugates. By using this estimate of the lethal hitting rate and the regression estimate of the disintegration rate, the multistage kinetic model gave a quantitative fit to our data. From this analysis, we found that the rate at which a target cell disintegrates after being lethally hit increases with the number of CTL per conjugate. This result is quite surprising, because once the first hit has been received, a target cell can disintegrate in a killer cell-independent manner. Under the conditions of our experiment, it appears as if target cell disintegration is not killer cell-independent. Furthermore, our analysis of the time course of target cell disintegration suggests that the process is not governed by simple first order kinetics, but rather by a more complex multistep mechanism.