Hierarchy of cytotoxic T cell clones. I. Reversal of resistance to lysis and killing between anti-hapten cytotoxic T cells

Cells from clones of anti-hapten cytotoxic T lymphocytes (CTL) can act as both effector cells and, when treated with the specific hapten, as target cells. Individual clones can kill haptenated cells only from other clones that are less efficient killers. Clones specific for both fluorescein and trinitrophenol could be ordered in a single hierarchy in which resistance to lysis correlated with lytic efficiency. When the killing efficiency was reduced with phorbol myristate acetate (PMA) or the colchicine analogue, Colcemid, the degree of resistance to lysis was also reduced. The use of PMA-treated fluoresceinated targets greatly enhanced intraclonal killing and similarly lead to a repositioning of clones within the hierarchy of normal cells. By the haptenation of appropriate clones, efficient CTL could kill cells from other clones in a direction apparently opposite to recognition. The results demonstrate that effects other than antigen recognition of the target cell may result in variations in the nature of T cell immune responses.     

Requirements for triggering of lysis by cytolytic T lymphocyte clones

Cloned murine cytolytic T lymphocytes (CTL) having defined specificity were triggered by the phorbol ester together with a calcium ionophore (either A23187 or Ionomycin) to lyse syngeneic or third party target cells efficiently. Neither phorbol 12-myristate 13-acetate (PMA) nor calcium ionophore alone induced efficient lysis. The characteristics of the lytic process induced by these signals are similar to those of antigen-specific or lectin-facilitated lysis by CTL. Lysis is calcium and temperature dependent and shows kinetics which are not grossly different from lysis mediated via the antigen receptor. Two helper T lymphocyte clones were not induced to lyse efficiently EL-4 target cells by concanavalin A or PMA + ionophore. Triggering of lysis induced with PMA plus ionophore by the CTL clone L3 differed from antigen-mediated lysis in specificity and in the susceptibility to inhibition by cytochalasin B. Properties of the target cell determine which cell surface associative recognition structures are important in the efficient lysis of these cells. Anti-LFA-1 monoclonal antibodies inhibited efficiently both antigen-mediated and PMA + ionophore-induced lysis of P-815 or EL-4 target cells which are of hematopoietic origin. However, anti-LFA-1 antibodies do not inhibit antigen-mediated, lectin-facilitated, or PMA + Ionomycin-induced CTL cytolysis of target cells derived from the L cell fibroblast line. We conclude that two intracellular signals, which can be provided by the combination of PMA + ionophore, are required for efficient lysis by antigen-specific murine CTL clones. When the T cell receptor for antigen is bypassed using PMA + ionophore to trigger lysis, we show that Lyt-2 and LFA-1 molecules may be required for efficient lysis. These associative recognition structures appear to play an important role in postactivation steps leading to efficient delivery of the lethal hit to the target cell.     

The requirements for triggering of lysis by cytolytic T lymphocyte clones. II. Cyclosporin A inhibits TCR-mediated exocytosis by only selectively inhibits TCR-mediated lytic activity by cloned CTL

TCR-mediated granule exocytosis, as measured by the release of serine esterase activity, has been implicated in the lytic process of Ag-specific CTL. Exocytosis appears to be the mechanism of release of other lysis-relevant molecules including cytotoxic lymphokines and proteins that have the capacity to induce membrane lesions as measured by the hemolysis of non-nucleated SRBC. In the studies presented here, we assessed the contribution of exocytosis and lymphokine production in CTL lysis of nucleated and non-nucleated target cells by using a panel of murine CTL clones. Ag-mediated activation of cytolysis, lymphokine production, and exocytosis could be mimicked by mAb against the TCR/CD3 complex, or by stimulation with the combination of PMA + calcium ionophore, which appear to bypass the TCR (neither PMA nor calcium ionophore alone induced these functions efficiently in our CD8+ CTL clones). Although lysis, IFN-gamma production and exocytosis of N-alpha-benzyloxycarbonyl-L-lysin esterase (BLTE) activity were induced by either stimulus, we were able to identify distinct activation requirements for each of these functions. We found that lymphokine production, exocytosis, and cytolysis could be selectively inhibited. Cycloheximide inhibited IFN-gamma production, but did not inhibit exocytosis of BLTE activity or cytolysis. In addition we showed that cyclosporine A (CsA) profoundly inhibited IFN-gamma production as well as exocytosis induced by stimulation through the Ag receptor or by PMA + calcium ionophore. In contrast, CsA had little or no effect on lysis of nucleated target cells that bear the relevant Ag. These findings indicate that our CTL clones can lyse target cells by a mechanism independent of exocytosis or (de novo) lymphokine production. To directly assess the capacity of our CTL clones to lyse target cells without inducing nuclear damage we developed a system of coating non-nucleated SRBC with anti-CD3 mAb for use as stimuli and as targets for lysis. We found that our cloned CTL were indeed activated to produce IFN-gamma by SRBC that were coated with anti-CD3 mAb, and, furthermore, they were able to lyse the SRBC in a short term cytolytic assay. Thus our CD8+ CTL are capable of lysing certain target cells by a mechanism independent of DNA degradation, presumably by inducing a membrane lesion. In addition, CsA did inhibit lysis of the non-nucleated SRBC targets as well as exocytosis of BLTE activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of syngeneic cytotoxic T lymphocytes against a B cell tumor. II. Characterization of anti-idiotypic CTL lines and clones.

In an earlier communication we showed that idiotypic immunoglobulin (Id+ Ig) of a B cell hybrid, 2C3, can induce cytotoxic T lymphocytes (CTL) in the spleens of mice that are hyperimmunized with the irradiated tumor cells. To understand the extent of heterogeneity in the splenic CTL population, stable anti-idiotypic CTL lines and clones were established from 2C3-primed splenocytes. One representative CTL line A102 which exhibited the phenotype of CD3+, CD4-, and CD8+, has been maintained in long-term culture for more than 18 months. Cytotoxic specificity of A102 was determined by cold target inhibition assay using a panel of syngeneic and allogeneic B cell tumors. The CTL line A102 was highly cytotoxic to 2C3, only weakly to other syngeneic tumors, but not at all to allogeneic B cell tumor CH12. Furthermore, CTL-mediated cytolysis was significantly abrogated by blocking 2C3 cells with anti-idiotypic monoclonal and polyclonal antibodies. These results clearly show that 2C3 Id represents the immunodominant epitope(s) recognized by the CTL line A102. To isolate a highly Id-specific effector population, A102 was repeatedly subcloned by limiting dilution. One such clone 102.F5 exhibited considerable specificity toward Id+ 2C3 while another clone 102.E10 showed no such specificity in a competitive cytotoxicity assay. This was further confirmed by the inhibition studies with anti-Id mAb. Thus, hyperimmunization with irradiated 2C3 cells evokes a spectrum of anti-2C3 cytotoxic effector cells, of which a major population is reactive to the idiotypic determinants associated with 2C3 Ig.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. III. Analysis of viral glycoproteins recognized by CTL clones by using recombinant herpes simplex viruses

Human cytotoxic T cell (CTL) clones specific for herpes simplex virus (HSV) type 1- and type 2-infected cells were generated and were analyzed with regard to the viral glycoproteins they recognize on autologous HSV-infected cells. By use of target cells infected with wild-type HSV strains, a gC deletion mutant of HSV-1, and HSV-1 X HSV-2 intertypic recombinants, some HSV-1-specific CTL clones were found to be directed against L region-encoded gA/B-1, and others against S region-encoded glycoproteins (gD-1 or gE-1). Some HSV-2-specific clones were found to be directed against L region-encoded gC-2, whereas others were directed against S region-encoded glycoproteins (gD-2, gE-2, or gG). These findings provide direct evidence that several HSV glycoproteins that are expressed on the surface of HSV-infected cells serve as recognition structures for human HSV-specific CTL.     

Induction of I region-restricted hapten-specific cytotoxic T lymphocytes.

Murine cytotoxic T lymphocytes (CTL) reactive to TNP-conjugated syngeneic target cells do lyse to a moderate but significant extent TNP-conjugated, I region compatible but H-2K or H-2D region incompatible target cells. Antibody inhibition experiments and "cold inhibition" experiments indicate that some CTL clones recognize TNP-conjugated targets in association with syngeneic I region determinants independently of H-2K or H-2D gene products. The data suggest that besides TNP-conjugated H-2K or H-2D gene products, in principle, also TNP-conjugated I region determinants do stimulate TNP-specific CTL precursor cells and act as target antigens of TNP-specific CTL.     

Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: demonstration of soluble mediators released from H-2-restricted CTL clones

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells. YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.     

Lack of target cell participation in cytotoxic T lymphocyte-mediated lysis.

Data on the subject of cell-mediated cytotoxicity suggest that no single mechanism is likely to provide a satisfactory explanation of this process. Lytic pathways have been proposed that involve both the effector cell and the target cell as active participants. In this report we describe a system in which the target cell is rendered unable to participate in its own demise. Using sheep E derivatized with CD3 antibodies, we show that metabolic inhibition of SRBC by depleting intracellular ATP with iodoacetamide, or even conversion of SRBC to "ghosts" by hypotonic lysis and resealing, has no effect on cytolysis. In the presence of EGTA or cholera toxin, both of which inhibit CTL degranulation, there is a strong suppression of both serine esterase release and cytolysis. These data show clearly that in some situations CTL are able to lyse target cells without any active participation by the target cells themselves.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). II. Genuine class II specificity of Lyt-2+ CTL clones

Class II-specific allogeneic cytolytic T lymphocytes (CTL) consist of two types of cells, i.e., Lyt-2+L3T4- and Lyt-2-L3T4 T cells. The Lyt-2+L3T4- class II-specific CTL population constitutes a conspicuous exception to the general correlation observed between the class of major histocompatibility complex antigen recognized and the type of accessory molecules expressed by T cells. In order to examine the specificity of such an exceptional T cell population, CTL clones were established by limiting dilution of a bulk CTL line developed in an I region incompatible combination of mouse strains, B10.QBR anti-B10.MBR. These CTL lines showed single genetic specificity indicating their clonal nature with respect to CTL activities. Lyt-2+L3T4- (2+4-), Lyt-2-L3T4+ (2-4+) and Lyt-2-L3T4- (2-4-) clones were obtained. Among many CTL clones showing a spectrum of genetic specificities, 2+4- and 2-4+ clones with apparent I-Ak-specificity, were studied further and four lines of evidence confirmed their class II specificity: 1) genes encoding the target antigen for these CTL clones were mapped within the I-A subregion by simple genetics; 2) an I-Ak-specific monoclonal antibody readily blocked specific cytolysis by these clones; 3) the clones failed to react with cells expressing mutated I-Ak antigens; and 4) a B cell tumor transfected with alpha- and beta-chain genes of I-Ak was specifically lysed by these CTL clones. These data therefore establish the existence of Lyt-2+ CTL with genuine class II specificity. All 2-4+ CTL were sensitive to the blocking effect of an antibody to L3T4, whereas none of the 2+4- class II-specific CTL were sensitive to blocking by an anti-Lyt-2 antibody, indicating that class II-specific CTL with "wrong phenotype" is not dependent on the function of the accessory molecule. Besides true class II-specific CTL clones, 2+4- clones with a spectrum of genetic specificities were obtained, including clones recognizing a combination of an I-Ak product and the Kb molecule. Two 2-4- clones were also specific for the combination of Kb + I-Ak. These clones most likely recognize an allogeneic class II antigen in the context of a class I antigen and therefore would more appropriately be included in the class I-restricted T cell population.     

Target cell lysis by cytotoxic T lymphocytes redirected by antibody-coated polystyrene beads

Cytotoxic T lymphocytes were found to mediate rapid lysis of target cells not normally recognized in the presence of small polystyrene beads coated with a combination of anti-T3 and antitarget cell antibodies. Lysis was not seen with beads bearing one of these antibodies alone, nor with a mixture of two types of beads each coated with a single antibody. The effector cells mediating this lysis include long term allospecific human CTL, and both human and mouse CTL clones recognizing mouse class I MHC Kb Ag. TNP-modified mouse tumor cells, a human lymphoblastoid line, and human red cells were found to be good targets for this cytotoxicity. Polystyrene beads with diameters of 3 to 15 mu caused target lysis, with a dose-response curve which typically went through a maximum and declined at high bead numbers. Maximal bead-redirected lysis by CTL was less efficient than that mediated by soluble antibody heteroconjugates of the same two antibodies. Bead-redirected target lysis was calcium dependent. These results are interpreted as a form of bystander lysis induced by the beads, since the target cell membrane is not directly crosslinked to the region of CTL activation. These observations thus favor a mechanism of lysis involving the polarized secretion of a locally acting lytic agent by CTL.     

Regulation of the hapten-specific T cell response. I. Preferential induction of hyporesponsiveness to the D-end of the major histocompatibility complex in the hapten-specific cytotoxic T cell response

In this paper we have examined the phenomenon of hapten-specific tolerance in the cytolytic T lymphocyte (CTL), using the trinitrophenyl (TNP) and azobenzenearsonate haptens. We found that the H-2 K and H-2 D-end restricted CTL in H-2a mice are differentiable in the ease with which they are tolerized to the TNP hapten. With TNP modified syngeneic spleen cells (TNP-SC), or low amounts of trinitrobenzylsulfonic acid as tolerogen, preferential hyporesponsiveness of D-end restricted CTL can be observed. Larger doses of hapten, e.g. a higher amount of trinitrobenzylsulfonic acid, will tolerize both K- and D-end restricted TNP-specific CTL in H-2a mice. The phenomenon of preferential D-end restricted CTL hyporesponsiveness is not observed in H-2d, H-2k, or H-2b mice, nor is it observed in H-2a mice with respect to the azobenzenearsonate hapten. We have also shown that the clones of CTL responsible for lysis of TNP-modified allogeneic targets (cross-reactive lysis) in H-2a mice probably overlap with the D-end restricted TNP-specific CTL since D-end restricted hyporesponsiveness induced by intravenous injection of TNP spleen cells also results in the elimination of cross-reactive lysis of TNP-modified allogeneic targets. The possible mechanisms of preferential D-end hyporesponsiveness to the TNP hapten in the H-2a mice as well as its significance and relationship to previous work in this area are discussed in this paper.     

Cloned cytotoxic T lymphocytes as target cells. II. Polarity of lysis revisited

The original polarity of lysis experiments suggested that CTL are themselves sensitive to whatever mechanism it is that CTL use to lyse their targets. This concept has placed certain limitations on possible mechanisms of lysis by CTL. Recently, we found in studies with cloned CTL as targets that cloned CTL are in fact highly resistant to lysis by other CTL, as well as to their cytotoxic granule proteins. We show here that although cloned CTL are extremely resistant to lysis by primary and cloned CTL, they are readily inactivated functionally by all primary CTL and by at least one CTL clone. Moreover, cloned CTL are also functionally inactivated by cytotoxic granule proteins. The activation of CTL, which we call inhibitin, is Ca2+ insensitive and distinct from hemolytic activity, and is, thus, unlikely to be perforin. These experiments suggest a possible alternative interpretation of the original polarity of lysis experiments.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

Measles virus-specific human T cell clones. Characterization of specificity and function of CD4+ helper/cytotoxic and CD8+ cytotoxic T cell clones

PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.     

Epidermal cells as accessory cells in the generation of allo-reactive and hapten-specific cytotoxic T lymphocyte (CTL) responses

The capacity of epidermal cells (EC) to stimulate T cell activation is a Langerhans cell (LC)-dependent phenomenon. In all in vitro assays probed, LC subserve antigen-presenting cell functions in that they display surface-bound foreign or altered-self structures and thereby activate T cell responses. In contrast, attempts to demonstrate accessory cell (ACC) function of LC-containing EC have yielded negative results, i.e., EC lacking foreign cell surface antigens were not able to restore cytotoxic T lymphocyte (CTL) responses in Ia+ adherent cell-depleted cultures. Reasoning that the ACC function of EC might be critically linked to cluster formation between LC and other cell types involved, we tested the ACC function of EC under experimental conditions that allow a close physical contact between the cell types involved (round-bottomed microtiter plates and brief centrifugation of culture plates). By using these modifications, the failure of highly purified B6 T cells to develop alloreactive CTL activity when stimulated with either highly purified, mitomycin C-treated C3H or B6CF1 T cells was restored by the addition of B6 EC. The CTL thus generated produced significant lysis of Con-A-stimulated C3H or BALB/c, but not B6, spleen cell targets. In a similar fashion, TNP- or FITC-specific CTL were generated when (in a syngeneic system) mitomycin C-treated TNP- or FITC-modified stimulator T cells and responder T cells were co-cultured in the presence, but not in the absence, of unmodified EC. The capacity of EC to restore CTL activity in a culture system depleted of Ia-bearing cells was not dependent upon their H-2 type, but was critically linked to the presence of Ia-bearing LC. We therefore conclude that LC-containing EC can subserve the ACC function in the generation of H-2-restricted CTL, provided that culture conditions are chosen that allow a close physical contact between the cell types involved.     

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.     

Target cell lysis by cytotoxic T lymphocytes that lack detectable hemolytic perforin activity

When mouse target cells are subjected to cytolytic attack by mouse CTL cell lines that have been cultured for many months in high levels of IL-2, and have abundant perforin-rich secretory granules, they exhibit two prominent changes: 1) rapid and massive increase (greater than 10-fold) in intracellular Ca2+ concentration and 2) fragmentation of DNA into nucleosome-sized fragments. We show here that when the same target cells are subjected to cytolytic attack by perforin-deficient CTL, either human CTL or primary mouse CTL from peritoneal exudates, the same changes are observed, suggesting that perforin-rich and perforin-deficient CTL kill their target cells by similar (if not identical) mechanisms. It is possible that perforin-deficient CTL produce enough perforin to destroy target cells but not enough to be detected by currently available methods.     

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.     

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.