Enhancement of redirected target cell lysis by cytotoxic T lymphocytes in the presence of cytochalasin B

The cytochalasins are known secretogogues. Their function as such is examined in light of the granule exocytosis model for lymphocyte-mediated cytotoxicity. Cytochalasin B is found to enhance target cell lysis by cytotoxic T lymphocytes when antibody-coated polystyrene beads are used to bridge the cells. The pattern of lysis is found to be biphasic in its dependence on cytochalasin B. Secretion of the enzyme BLT-esterase from the effector cells parallels the cytochalasin concentration-dependent pattern of lysis. Cytochalasin D is also able to enhance lysis but at concentrations less than cytochalasin B. Cytochalasin B does not inhibit binding of breads to the effector cell. This is shown by the ability of fluorescent beads coated with antibody to bind with an appropriate specificity to cells. These studies indicate that cytochalasin B is not strictly inhibitory for the induction of target cell lysis but can enhance lymphocyte-mediated lysis at low drug concentrations. These results are compatible with the interpretation that target cell lysis is mediated through a secretion process from cytotoxic T lymphocytes.     

Definition of human cytomegalovirus-specific target antigens recognized by cytotoxic T cells generated in vitro by using an autologous lymphocyte system

We developed an in vitro system for the generation of human cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) that avoids the necessity of constituting a panel of HLA-typed fibroblasts. Autologous donor leucocytes were coated with CMV antigens and were used as both stimulator and target cells. With the use of this system, CMV-specific effector cells were efficiently generated from seropositive but not seronegative donors. These CMV-specific effectors were HLA-restricted and had characteristics of T cells. Maximum lymphoproliferation preceded the appearance of maximum CTL activity by 3 to 4 days, and a close correlation was seen between both activities. Mouse anti-CMV monoclonal antibodies were used in blocking experiments in an attempt to define target antigens recognized by CMV-specific cytotoxic lymphocytes. Monoclonal antibodies directed against an early CMV membrane antigen, against neutralization epitopes, or against nuclear inclusion body protein all specifically inhibited CMV-sensitized effector cell activity but did not affect influenza virus-specific lysis. Monoclonal antibodies directed against a normal cell determinant or against poliovirus did not affect CMV-specific CTL activity. CMV-immune cytotoxic T cells could be consistently and specifically inhibited in their lytic activity by pretreating antigen-coated target cells with monoclonal antibodies directed against CMV-related proteins.     

Inhibition of cytotoxic T cell lysis by anti-CD8 monoclonal antibodies: studies with CD3-targeted cytolysis of nominal antigen-negative targets

The function of the CD8 molecule in lympholysis mediated by cytotoxic T cells was investigated by examining possible contributions of ligands on the target cell to the inhibition of lysis observed with CD8-specific mAb. In order to evaluate a variety of target cells, including those not expressing the nominal Ag (NA) for which the CTL was specific, lysis was effected by cross-linking the CTL and the target cells with anti-CD3 mAb. Such CD3 redirected cytotoxicity was demonstrated to be inhibited by anti-CD8 mAb when low anti-CD3 mAb concentrations were used. The possibility that inhibition by anti-CD8 mAb resulted for competition for the FcR between the anti-CD3 mAb and anti-CD8 mAb was eliminated by targeting TNP-modified cells with an antibody heteroconjugate prepared from Fab fragments of anti-CD3 and anti-DNP antibodies. Inhibition of the lysis of target cells not expressing NA including those deficient in class I expression, demonstrated that neither NA nor class I expression was required for anti-CD8 mAb inhibition. Whether the anti-CD8 mAb inhibition required CD8 Ag interaction with any ligand on the target cell was further investigated by measuring exocytosis of enzyme granule from CTL activated with CD3-coated poly-styrene beads. CD8-specific mAb inhibited such CTL activation in this target cell-free system. A CD8(+), MHC class II-specific CTL clone, was used to show differential inhibition by anti-CD8 mAb, depending on the target cell, therefore providing evidence that anti-CD8 mAb binding does not generate an absolute off signal. These data are consistent with the hypothesis that anti-CD8 mAb affect the lytic process independent of the recognition of a ligand on the target cell by CD8.     

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.     

Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones

Human cytolytic T lymphocytes (CTL) clones and HLA-A2- and HLA-B7-transfected human, monkey, and mouse cell lines were used to investigate the basis for species-restricted antigen recognition. Most allospecific CTL clones obtained after stimulation with the human JY cell line (source of HLA-A2 and HLA-B7 genomic clones) recognized HLA antigens expressed in human and monkey cell lines but did not recognize HLA expressed in murine cells. By initially stimulating the responder cells with HLA-transfected mouse cells, two CTL clones were obtained that recognized HLA expressed in murine cells. Functional inhibition of these CTL clones with anti-class I monoclonal antibodies (MAb) indicated that clones reactive with HLA+ murine cells were of higher avidity than clones that did not recognize HLA+ murine target cells. MAb inhibition of accessory molecule interactions demonstrated that the LFA-1 and T8 surface molecules were involved in CTL-target cell interactions in all three species. In contrast, the LFA-2/CD2 molecule, previously shown to participate in a distinct activation pathway, was involved in the cytolysis of transfected human and monkey target cells, but not in the lysis of HLA+ murine cells. Thus transfection of HLA genes into different recipient species cell lines provides us with the ability to additionally delineate the functional requirements for allospecific CTL recognition and lysis.     

Role of accessory molecules in signal transduction of cytolytic T lymphocyte by anti-T cell receptor and anti-Ly-6.2C monoclonal antibodies

Accessory molecules present on the cell surface of cytolytic T lymphocytes (CTL) play an important role in their activation. Antigen-specific recognition by CTL is inhibited by antibodies against Lyt-2, L3T4, or LFA-1 molecules. Presently it is not known whether these molecules function by binding a ligand such as class I or class II on the target cell or by delivering a signal that down-regulates T cell activation. In the present study we utilized anti-T cell antibodies including anti-T3 and anti-T cell receptor (alpha/beta) as well as an anti-Ly-6.2C monoclonal antibody to activate CTL clones to kill irrelevant targets or secrete BLT esterase. The redirected lysis assay system utilizes the fact that heteroconjugates between anti-T3, and anti-T cell receptor, or anti-Ly-6.2C and anti-trinitrophenyl can trigger CTL lysis of trinitrophenyl-coupled targets that did not express antigen. In this system anti-Lyt-2 antibodies as well as anti-LFA-1 antibodies inhibited triggering via T cell receptor-related molecules but not via the anti-Ly-6.2C heteroconjugate. In addition, the anti-Lyt-2 was shown to inhibit conjugate formation in the heteroaggregate assay system suggesting that the anti-Lyt-2 antibodies acted early in inhibiting CTL activity. Similar results were observed in a system in which the CTL clones were triggered to secrete a BLT-esterase-like activity in the absence of target cells. Anti-T3 coated on plastic was shown to activate BLT-esterase secretion. This secretion was inhibited by anti-Lyt-2 and anti-LFA-1. Thus, it would appear that both the Lyt-2 molecule and the LFA-1 molecule act as signal-transducing elements involved in CTL activation. In particular, the Lyt-2 molecule appears to preferentially function in receptor-mediated T cell activation.     

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.     

Human neuroblastoma cell lines are susceptible to lysis by natural killer cells but not by cytotoxic T lymphocytes

The susceptibility of human neuroblastoma cells to direct cellular cytotoxicity has not been previously established. This is of particular interest because of their aggressive growth and low HLA expression. Neuroblastoma lines CHP 100 and CHP 126 were found to be excellent targets in 4-hr CML assays. Natural killer (NK) cells from fresh PBL and from an NK clone, 3.3, have high lytic activity against both cell lines. We also studied mixed lymphocyte culture-generated cytotoxic lines containing allo-specific cytotoxic T lymphocytes (CTL) directed against HLA antigens present on the neuroblastoma target cell lines. These lines did show excellent lytic activity, but cold target competition studies indicated that all of the lysis resulted from NK activity. This was verified by using inhibition studies with the use of monoclonal antibodies. OKT 3 and anti-HLA antibodies that block CTL function caused no reduction in kill. In contrast, anti-lymphocyte function antigen-1 (anti-LFA-1), which blocks both NK and CTL function, significantly inhibited lysis. These results serve as a functional confirmation of earlier findings of a very weak expression of HLA-A,B,C and beta 2-microglobulin on neuroblastoma cells.     

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.     

Temperature requirements and kinetics of T cell signaling. Velocity of triggering correlates with functional effects of anti-CD3 mAb

Antibody reactive with the CD3 complex on the surface of T lymphocytes can either: inhibit CTL lysis of target cells expressing Ag; or redirect CTL to lyse target cells expressing FcR in the absence of Ag expression. To investigate these phenomena we examined the effect of anti-CD3 mAb on two indicators of CTL activation, the release of esterase and target cell lysis. Esterase release by long term allo-reactive human CTL in response to target cells (JY or HLA transfected K562 cells) was found to be Ag specific and correlate with target cell lysis. Addition of anti-CD3 to either JY targets or K562 cells expressing FcR resulted in a high level of esterase release. Triggering of esterase release was found with both soluble intact and Fab fragment of anti-CD3 in the absence of cells expressing measurable FcR. This apparent FcR-independent triggering of esterase release occurred at 37 degrees C but not at 24 degrees C. In contrast esterase activity was released from CTL at both 24 and 37 degrees C in response to intact target cells, JY or K562 cells plus intact anti-CD3 mAb. Addition of anti-CD3 mAb, at a level capable of blocking target cell lysis by greater than 50%, resulted in an initial velocity of esterase release almost twice that found in response to JY target cells. With a low level of anti-CD3 mAb, able to block JY lysis by approximately 10%, the initial rate of esterase release was much slower than that found in response to target cells. In contrast when FcR+ cells, K562, were added along with a low level of anti-CD3 the initial velocity of esterase release was about twofold more than the velocity of esterase release triggered by soluble anti-CD3 alone. These results indicate that soluble antibody can trigger long term active CTL and the velocity of this triggering correlates with anti-CD3-mediated inhibition as well as redirected lysis.     

Gene mapping and somatic cell hybrid analysis of the role of human lymphocyte function-associated antigen-3 (LFA-3) in CTL-target cell interactions

LFA-3 is expressed on a wide variety of human cell lines, including those which have been used as recipients for gene transfer of human class I gene products, whereas a murine counterpart is either absent or significantly different such that the anti-LFA-3 monoclonal antibody (MAb) does not bind. By using a somatic cell genetic approach, we demonstrate that LFA-3 is not a major histocompatibility complex-encoded molecule, and that its gene locus maps to human chromosome 1. When LFA-3 and HLA-A2 are coexpressed on the mouse cell surface, anti-LFA-3 MAb interfered with specific recognition and lysis of these target cells by human CTL capable of lysing HLA-A2-expressing mouse transfectants. A significant contribution of the LFA-3 molecule to CTL reactivity was not observed, however, because the presence of LFA-3 did not restore recognition by CTL clones previously found incapable of lysing HLA-A2-expressing mouse transfectants, nor was it required by those human CTL that could lyse mouse cell transfectants. Thus, we have used genetic techniques to demonstrate that LFA-3 may serve a role in CTL-target cell interactions at the target cell level, but is not a molecule absolutely required for human allospecific CTL recognition of HLA antigens expressed on mouse cells. We suggest that LFA-3 may not participate directly in CTL function under normal circumstances, but delivers a more general inhibitory signal only when provoked by bound MAb.     

Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody

Heteroaggregates containing anti-T3 cross-linked to anti-target cell antibodies have been shown to cause human T cells to lyse target cells that express antigens recognized by the anti-target cell antibody. In this study, we test targeted human T cells for the ability to lyse human tumor cells as a first step toward the application of this phenomenon to tumor immunotherapy. Several monoclonal anti-human tumor antibodies were assayed for binding to a number of human tumor lines and for the ability to promote specific tumor cell lysis when cross-linked with anti-T3. We found that anti-T3 cross-linked to anti-tumor monoclonal antibodies caused cloned human T cells and fresh peripheral blood T cells to lyse the tumor cells with the same specificity as predicted by the binding studies. Peripheral blood T cells were then tested in the presence of various heteroaggregates for the ability to lyse single cell suspensions prepared from fresh tumor or fresh normal tissue. These studies showed that heteroaggregates containing anti-T3 cross-linked to anti-tumor antibody cause fresh human T cells to specifically lyse fresh tumor cells, but not (with one exception) fresh normal cells.     

Induction of nonspecific cytotoxicity by monoclonal anti-T3 antibodies

The effects of monoclonal anti-T3 antibodies on the effector phase of cytotoxic T lymphocytes (CTL) were studied with respect to antigen-specific and antigen-nonspecific lysis of different target cells. Anti-T3 antibodies inhibited the antigen-specific lysis by CTL generated in mixed lymphocyte cultures (MLC), but they concomitantly augmented the nonspecific killing of third-party cells such as the cell lines Daudi, Raji, and K562. This nonspecific cytotoxicity was induced by various anti-T3 antibodies, whereas antibodies reactive with other antigens expressed on the cytotoxic effector cells lacked any such activity. Anti-T3 antibodies induced nonspecific cytotoxicity only when activated T cells, obtained by primary MLC, by repeated restimulation, or after cloning, were used. The antibodies had no effect on unstimulated peripheral T lymphocytes or thymocytes. The inhibition of the antigen-specific lysis and the induction of nonspecific lysis by anti-T3 was dose dependent, and both effects occurred at the same concentration range of anti-T3. F(ab')2 fragments of anti-T3 inhibited the specific lysis but were not able to induce cytotoxic activity, indicating that this induction is an Fc-dependent process. When different target cells were tested, only Fc receptor-positive cells were susceptible for this nonspecific cytotoxicity. Thus, anti-T3 antibodies have a dual effect on effector CTL: they inhibit antigen-specific lysis and concomitantly induce nonspecific lysis in an Fc-dependent way.     

Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes.

The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist.     

Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells

The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens

In contrast to general findings that mouse and human cytotoxic T lymphocytes (CTL) are restricted in cytotoxic activity by major histocompatibility complex (MHC) class I antigens, we previously found that some herpes simplex virus (HSV) type I-infected cells that shared no HLA class I antigens with the HSV-1-stimulated lymphocytes were lysed. In this study, we addressed the question of the role of HLA antigens in human T cell-mediated lysis of HSV-1-infected cells by generating clones of HSV-1-directed CTL from two HSV-1-seropositive individuals. CTL clones that lysed autologous HSV-1-infected lymphoblastoid cell lines (LCL), but not natural killer-sensitive K562 cells or uninfected or influenza virus-infected LCL, were tested for cytotoxicity against a panel of allogeneic HSV-1-infected LCL. Clone KL-35 from individual KL lysed only HSV-1-infected LCL sharing the HLA class II MB1 antigen with KL. With all four CTL clones isolated from individual PM, only HSV-1-infected LCL sharing DR1 with PM were lysed. Monoclonal antibody s3/4 (directed against MB1 ), but not TS1/16 or B33 .1 (directed against a DR framework determinant), blocked lysis of autologous HSV-1-infected cells by KL-35. In contrast, B33 .1, but not s3/4, blocked lysis of autologous HSV-1-infected cells by the PM CTL clones but not by KL-35. Together, these results indicate that our five human CTL clones which are directed against HSV-1-infected cells, and which are all OKT3+, OKT4+, OKT8-, are restricted in lytic activity by HLA class II MB and DR antigens. These results suggest that the HLA D region-encoded class II antigens may be important in the recognition and destruction of virus-infected cells by human CTL.     

Human allospecific cytolytic T lymphocyte lysis of a murine cell transfected with HLA-A2

A variety of molecules are involved in the interaction of human allospecific cytolytic T lymphocytes (CTL) with target cells. Monoclonal antibodies specific for these molecules inhibit CTL-target conjugate formation and/or lysis. To further study recognition and lysis of targets by human CTL, we used a murine mastocytoma cell line transfected with the histocompatibility leukocyte antigen (HLA)-A2 gene (P815-A2+) as a target for human HLA-A2-specific CTL. We find that only a subset of human HLA-A2-specific CTL can lyse murine P815-A2+ cells, suggesting that the murine cells may lack one or more accessory molecules needed for CTL recognition and lysis.     

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. I. Cytotoxins produced by antigen-specific and natural killer-like CTL are dissimilar to classical lymphotoxins

Several cloned lines of IL 2-dependent human T cells derived from alloantigen, mitogen, or IL 2-stimulated peripheral blood lymphocytes were examined for their surface marker expression, cytolytic activity in a 51Cr-release assay, and capacity to release cytotoxic lymphokines. Thirty cell lines exhibiting either antigen-specific natural killer cell activity or lectin-dependent killer cell function, which expressed either the CD4 or CD8 surface differentiation markers, were capable of producing cytotoxin(s) in response to the lectins phytohemagglutinin and concanavalin A. Cytotoxin activity was detected on the murine L929 target cell in a 16-hr cytotoxicity assay. In contrast, several nonlytic T cell tumor lines failed to produce a soluble cytotoxin. Antibodies capable of neutralizing human alpha-lymphotoxin were completely ineffective in inhibiting the cytotoxin(s) produced by any of the cytotoxic T lymphocytes (CTL) cell lines. Comparative gel filtration and HPLC hydrophobic chromatography of alpha-lymphotoxin and CTL toxin produced by the CTL-830.B2 clone revealed significant differences in their elution profiles. The CTL-produced toxin and alpha-lymphotoxin exhibited similar kinetics of lysis of the L929 target cells, with 50% target cell lysis occurring at 10 hr. These data indicate human CTL produce a cytotoxin(s) antigenically distinct from alpha-lymphotoxin and imply that human cytolytic effector T cells are not the cellular source for the production of human alpha-lymphotoxin. The relationship of alpha-lymphotoxin and CTL toxin production was investigated in unseparated peripheral blood mononuclear cells stimulated with lectins or IL 2 for 1 and 5 days. Anti-alpha-lymphotoxin antibodies were capable of neutralizing only 30 to 50% of the cytotoxic activity in 24-hr supernatants. Cytotoxic activity in supernatants harvested after 120 hr stimulation with PHA or Con A was neutralized 70 to 100%, whereas the toxin(s) released from IL 2-stimulated lymphocytes was only neutralized 30%. These data suggest the observed heterogeneity of cytotoxic lymphokines produced by unseparated mononuclear cells depends in part on the subpopulations of effector cells responding to a given stimulus and the capacity of different subpopulations to produce distinct cytotoxins.     

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.     

Identification of a viral antigen recognized by H-2-restricted cytolytic T lymphocytes on a murine leukemia virus-induced tumor

Monoclonal antibodies were produced against protein p30, a structural protein of murine leukemia viruses (MuLV) coded by the gag gene of MuLV. Three monoclonal antibodies of different isotypes (i.e., IgG-1, IgG-2a, and IgG-2b) were chosen for extensive analysis. These three antibodies bound to mouse tumor cells induced by Friend, Moloney, Rauscher, and Gross MuLV, but not to noninfected normal mouse spleen cells. The ability of these monoclonal antibodies to inhibit cytolytic T lymphocyte (CTL) activity by masking the antigens recognized by CTL on the target cell surface was studied in various CTL systems. It was found that the only CTL that were consistently inhibited in their lytic activity came from BALB.B (H-2b) mice immunized against syngeneic Gross MuLV-induced B.GV cells. These results thus showed that a subpopulation of BALB.B anti-Gross MuLV CTL recognized a Gross MuLV gag gene product expressed on the surface of B.GV cells.