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.     

Phorbol-ester stimulated lysis of weak and nonspecific target cells by cytotoxic T lymphocytes

We have investigated the effects of short-term incubation of cloned and in vivo-produced cytotoxic T lymphocytes (CTL) with phorbol esters on their lytic activity against weak and nonspecific targets. These experiments demonstrate that 4 beta-phorbol-12-myristate, 13-acetate (PMA), 4 beta-phorbol-12,13-dibutyrate, and 4 beta-phorbol-12,13-didecanoate, but not the 4 alpha-phorbol-12,13 didecanoate esters stimulate the lytic apparatus. The stimulation is specific for the CTL rather than the target and appears to be nearly instantaneous in action. This rapid stimulation of the CTL lytic process is consistent with previously reported effects of phorbol esters associated with T cell activation in other functional assays.     

Demonstration of a calcium influx in cytolytic T lymphocytes in response to target cell binding

By using the Ca2+-sensitive dye indo-1, an antigen-specific increase in intracellular Ca2+ in cloned cytolytic T lymphocytes (CTL) was measured under conditions that were permissive for T cell-mediated cytolysis. To synchronize lethal hit delivery in a suspension of effector and target cells, a modification of the cation pulse method in which Ca2+ is added to preformed conjugates of CTL and target cells was used. Conjugate formation was unaffected by the absence of extracellular Ca2+ under these conditions. Lytic activity of these cloned CTL was markedly reduced in the absence of extracellular Ca2+ and was restored upon Ca2+ repletion. When indo-1-loaded CTL were preincubated with target cells in the absence of extracellular Ca2+, a marked antigen-specific increase in indo-1 fluorescence, indicative of an increase in intracellular Ca2+, was observed after repletion of extracellular Ca2+. This increase in intracellular Ca2+ was shown to be due solely to changes in the CTL and not the target cell within the time course of the experiment, and results from the influx of extracellular Ca2+. Antibody to the T cell receptor for antigen also evokes a similar increase in intracellular Ca2+ in CTL under these conditions. This method provides a means for the direct examination of the response of CTL to cellular antigen as well as soluble antibody and is a versatile and valuable tool for the study of CTL function.     

Retrovirus-induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes

Retrovirus infection of murine fibroblasts was found to alter the expression of major histocompatibility complex (MHC) antigens. Fibroblasts infected with Moloney murine leukemia virus (M-MuLV) exhibited up to a 10-fold increase in cell surface expression of all three class I MHC antigens. Increases in MHC expression resulted in the increased susceptibility of M-MuLV-infected cells to lysis by allospecific cytotoxic T lymphocytes (CTL). M-MuLV appears to exert its effect at the genomic level, because mRNA specific for class I antigens, as well as beta 2-microglobulin, show a fourfold increase. Fibroblasts infected with the Moloney sarcoma virus (MSV):M-MuLV complex show no increase in MHC antigen expression or class I mRNA synthesis, suggesting that co-infection with MSV inhibits M-MuLV enhancement of MHC gene expression. Quantitative differences in class I antigen expression on virus-infected cells were also found to influence the susceptibility of infected cells to lysis by H-2-restricted, virus-specific CTL. Differential lysis of infected cells expressing varied levels of class I antigens by M-MuLV-specific bulk CTL populations and CTL clones suggests that individual clones may have different quantitative requirements for class I antigen expression. The MSV inhibition of MHC expression could be reversed by interferon-gamma. Treatment of MSV:M-MuLV-infected fibroblasts with interferon-gamma increased their susceptibility to lysis by both allogeneic and syngeneic CTL. The data suggest that interferon-gamma may function in the host's immune response to viral infections by enhancing MHC antigen expression, thereby increasing the susceptibility of virus-infected cells to lysis by H-2-restricted, virus-specific CTL.     

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.     

Specificity, phenotype, and precursor frequency of primary cytolytic T lymphocytes specific for class II major histocompatibility antigens

Most cytolytic T lymphocytes (CTL) recognize class I rather than class II MHC determinants, and relatively little is known about those CTL that do recognize class II MHC determinants. The present study was undertaken to document the specificity, phenotype, and precursor frequency of primary class II allospecific CTL. It was found that class II-allospecific CTL could be consistently generated in vitro from unprimed spleen or thymus populations in the presence of exogenously added helper factors. The class II MHC specificity of both the precursor and CTL effectors activated in primary cultures by Ia-disparate stimulator cells was documented both by blocking experiments with anti-Ia mAb and by the use of L cell transfectants. The mechanism by which primary allospecific CTL effectors lysed their targets appeared to involve direct cell-cell contact, because they failed to lyse bystander target cells. The frequency in unprimed spleen populations of precursor CTL specific for class II alloantigens was examined by limiting dilution analysis and was found to be as high as 1/15,000 splenocytes and approximately 10% of the frequency reported for primary class I allospecific CTL. Finally, the Lyt phenotype of primary class II allospecific CTL precursors and effectors was determined. It was found that anti-class II CTL derive from at least two distinct precursor subpopulations that are either L3T4+Lyt-2- or L3T4-Lyt-2+, and that the Lyt phenotype expressed by the CTL effectors are concordant with that of their precursors. No correlation was found between the I subregion gene products recognized by CTL effectors and the Lyt phenotype they expressed in that both I-A- and I-E-specific CTL were both L3T4+Lyt-2- and L3T4-Lyt-2+.     

Expression of antigen on mature lymphocytes is required to induce T cell tolerance by gene therapy

Expression of a retrovirally encoded allogeneic MHC class I gene in bone marrow-derived cells can be used to induce tolerance to the product of the retrovirally transduced gene. In this work we examined whether expression of a retrovirally transduced allogeneic MHC class I gene in bone marrow-derived cells from recombinase-activating gene-1 (RAG-1)-deficient mice was sufficient to induce tolerance when transplanted into conditioned hosts together with bone marrow from MHC-matched wild-type mice. Reconstitution of mice with either MHC-matched RAG-1-deficient or wild-type bone marrow transduced with the allogeneic MHC class I gene H-2K(b) led to long-term expression of K(b) on the surface of bone marrow-derived hematopoietic lineages. T cells from mice reconstituted with H-2K(b)-transduced wild-type bone marrow were tolerant to K(b). In contrast, expression of K(b) in the periphery of mice reconstituted with a mixture of retrovirally transduced RAG-1-deficient bone marrow and mock-transduced wild-type bone marrow fell below detectable levels by 4 wk after transplantation. T cells that developed in these mice appeared to be hyporesponsive to K(b), demonstrating that expression of K(b) on bone marrow-derived APCs was not sufficient to induce tolerance. Our data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes that populate the host thymus.     

Killing of mouse blastocyst stage embryos by cytotoxic T lymphocytes directed to major histocompatibility complex antigens

A new assay, mixed embryo leukocyte interaction assay, in which the ability of cytotoxic T lymphocytes (CTL) to kill preimplantation mouse embryos could be investigated, is described. CTL were generated both in vitro and in vivo to the H-2b and H-2d haplotypes. The specificity of the CTL was verified by using EL-4 (H-2b) and P815 (H-2d) target cells in a 51Cr-release assay. The cytolytic effect of the CTL on mouse blastocysts was measured by assessing blastocoel retention and inhibition of [3H]thymidine incorporation by the embryos. It was shown that CTL kill blastocyst stage embryos from C57BL/6J (H-2b) and B10.D2 (H-2d) mice with the zona pellucida removed, but not with the zona pellucida intact. These results demonstrate that the H-2 antigens present on mouse blastocysts can be recognized by CTL. It is suggested that one biologic role for the zona pellucida is the prevention of cell-mediated destruction of preimplantation embryos in utero.     

Recognition and lysis of target cells by cytotoxic T lymphocytes

A single cytotoxic T lymphocyte (CTL) is capable of performing the two most fundamental functions of an immune response, recognition and elimination of foreign antigens. It is now clear that in a CTL these two functions are linked via the antigen-specific, heterodimeric receptor. We review here some experimental approaches that justify this conclusion and provide the means for further examination of the mechanisms by which CTLs lyse their target cells. When antireceptor antibodies serving as antigen substitutes are attached to various cells, they trigger the lytic activity of particular CTLs, which results in lysis of the antibody-modified cell. In the process, a novel serine esterase, which is located within cytolytic granules of the CTL, is released. The presence of this enzyme and a complement-like protein, perforin, in granules of a CTL has led to the suggestion that CTLs and complement have similar cytolytic mechanisms. However, the resistance of some CTLs to lysis by other CTLs, but not to lysis by antibody-activated complement, suggests fundamental differences between cytolytic mechanisms of CTLs and complement.     

Quantitative ultrastructure of cytolytic lymphocytes mediating allograft rejection in the mouse. I. Cellular alterations in T lymphocytes during specific target cell lysis

A quantitative ultrastructural analysis of cytolytic T lymphocytes (CTL) is presented which allows both the distinction of these cells from normal T lymphocytes and permits the demonstration of ultrastructural alterations of putative CTL following interaction with target cells (TC). Alloreactive CTL were generated in C57BL/10 mice receiving intraperitoneal fibroblastic allografts and target-binding splenic lymphocytes (TBSL) were concentrated by specific immunoadsorption on fibroblast monolayers. TBSL were subjected to ultrastructural quantification either at the onset of TC interaction or following 30 or 60 min incubation at 37 degrees C. By means of simple stereological relationships it was shown that, in comparison with normal, non-cytolytic splenic T lymphocytes, TBSL were slightly larger cells, displaying around 60% more cytoplasm, a similarly-sized nucleus and approximately triple the volume of Golgi apparatus. During the first 30 min of interaction with TC, the target binding surface of the TBSL plasma membrane decreased in area. This change was accompanied by a polarization of the TBSL towards the target. Incubation of lymphocytes with TC for a further 30 min resulted in a general polarization of lymphocytic cellular constituents away from the TC. These results were only attainable by objective quantitative analysis and are discussed in relation to possible mechanisms of CTL-mediated lysis.     

Dissociation of alloantigen recognition from self major histocompatibility complex-restricted recognition of cytolytic T lymphocytes by monoclonal antireceptor antibodies

Two monoclonal antibodies (mAb) directed to the dual reactive cytolytic T lymphocyte clone OH8 (Db + H-Y and H-2d) were established. Analysis by cell surface staining and immunoprecipitation of radiolabeled surface molecules of OH8 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that both mAb recognized an identical heterodimeric, clonotypic structure on OH8 cells, i.e., T cell receptor. However, although the MR3-2 mAb inhibited the lysis of either Db + H-Y or H-2d targets by OH8, the MR3-6 mAb inhibited the lysis of H-2d target cells, but not that of Db + H-Y target cells. Modulation of T cell receptor by either MR3-2 or MR3-6 mAb rendered the OH8 cytolytic T lymphocyte incapable of killing both Db + H-Y and H-2d target cells. These findings suggest that different epitopes of OH8 T cell receptor were involved for the recognition of self + antigen and alloantigen.     

Non-H-2 histocompatibility antigens encoded by Moloney-murine leukemia virus in Mov mouse strains are detectable by skin grafting and cytolytic T lymphocytes

The integration and expression of Moloney-murine leukemia virus (M-MuLV) into the germ line of Mov mouse strains on the C57BL/6 background results in the expression of a cell-surface Ag with characteristics expected from non-H-2 histocompatibility Ag: the ability to stimulate graft rejection and generation of CTL. However, both the previously studied Mov-3 and Mov-14 strains differ from the coisogenic C57BL/6 strain by different length segments of chromosome derived from the ICR strain in addition to the integrated M-MuLV genome. To conclusively demonstrate that an Ag encoded by M-MuLV is solely responsible for rejection of Mov skin grafts by coisogenic recipients, we have studied additional Mov strains that differ from coisogenic 129 or BALB/c backgrounds only by integration of an M-MuLV genome. A total of 129 strain recipients reject skin grafts from two viremic Mov strains, Mov-17 and Mov-18. A total of 129 strain hosts primed with either 1) multiple sets of Mov-17 and Mov-18 skin grafts or 2) single injections of Mov-17 and Mov-18 spleen cells produce M-MuLV-specific CTL that could be boosted in primary mixed lymphocyte culture. Generated CTL were reactive with Con A-stimulated lymphoblasts from all tested viremic Mov strains on the B6 and 129 backgrounds as well as B6 lymphomas. Further, we have observed that 129 strain mice reject Mov-9 skin grafts if these skin grafts are transplanted to virgin 129 recipients which have not received prior skin grafts from non-viremic Mov donors. In addition, skin grafts were transplanted from two viremic Mov strains, Mov-15 and Mov-16, to coisogenic BALB/c recipients; rejection of both sets of grafts was observed. However, BALB/c responders did not generate specific CTL after priming in vivo, with either multiple sets of allogeneic grafts or spleen cell injections, and boosting in vitro. These observations confirm the ability of integrated and expressed M-MuLV genomes to encode what is operationally defined as a non-H-2 histocompatibility Ag.     

Kinetics of cytolytic T lymphocyte binding to target cells in suspension

Cytolytic T lymphocytes (CTL) were able to specifically bind and lyse allogeneic P815 tumor cells and LPS blast cells in suspension. An assay was developed to measure the rate of target cell binding in suspension independent of the rate of lysis. Target cell binding was found to plateau within 3 hr in suspension. The presence of free, functional CTL and targets at these plateaus was demonstrated, indicating that target cell binding was an equilibrium process. Scatchard plots were used to derive values for Kd (apparent affinity) and bmax (maximum binding). Target cell binding in suspension could not be blocked by purified plasma membranes. Target cell binding was compared for CTL generated by secondary in vitro stimulation with intact cells or with purified membranes. These 2 CTL populations yielded distinct values for Kd and bmax. Implications of this kinetic difference for CTL recognition of purified plasma membranes are discussed.     

Capacity of thymic cells to effect target cell lysis following treatment with concanavalin A

The capacity of lymph node, thymus and bone marrow cells to mediate a cytolytic effect on target monolayers in the presence of concanavalin A was investigated. It was found that freshly prepared rat lymph node cells, but not thymus or bone marrow cells, lyse mouse fibroblasts in cultures treated with concanavalin A. A population of thymic cells did, however, manifest cytolytic capacity in the presence of concanavalin A after having been first sensitized and selected on mouse fibroblasts. Thus, sensitized thymus cells possess a lytic potency which can be activated under proper conditions.     

Poly-(ADP-ribose) polymerase partially contributes to target cell death triggered by cytolytic T lymphocytes.

We hypothesized that CTL-induced target cell (TC) death is partially due to processes that follow the DNA damage in target cells and include the activation of poly-ADP-ribose transferase (PADPRT) by DNA strand breaks. According to this model, the activated PADPRT is expected to deplete NAD, ATP, and to contribute to the TC death. We used inhibitors of PADPRT and a PADPRT-deficient cell mutant, as well as other nucleated TC and SRBC to test the role of PADPRT in CTL-induced cytotoxicity. It is found that inhibitors of PADPRT (3-aminobenzamide, benzamide (aromatic amides)) and nicotinamide all inhibit the CTL-mediated lysis of both Ag-specific TC and of Ag-nonbearing TC. The effect of PADPRT inhibitors was not due to inhibition of the lethal hit delivery by CTL, because in parallel control experiments, the same inhibitors did not interfere with CTL-induced lysis of SRBC, cells that are devoid of nuclei and PADPRT. Moreover, the effect of inhibitors of PADPRT did not affect earlier stages of lethal hit delivery because 3-aminobenzamide and benzamide did not interfere with CTL-induced DNA fragmentation in TC at concentration which protected TC lysis. Importantly, a PADPRT-deficient cell line was also much more resistant to CTL-induced lysis as tested in retargeting (4 and 8 h) assays; this was expected if activation of PADPRT is indeed involved in TC death. Control experiments reveal that the relative resistance of the PADPRT-deficient cell mutant to CTL-induced lysis was not related to its impaired ability to form conjugates and to trigger CTL (as tested in granule exocytosis assay). In addition, PADPRT-deficient cells were as susceptible to CTL-induced DNA fragmentation as were the control cells; yet, they were resistant to CTL-induced 51Cr-release. Control cells and PADPRT-deficient mutant were equally susceptible to antibody+C'-mediated lysis. Our data support the view that the activation of PADPRT can contribute to the CTL-induced cytolysis of some TC, but is not involved in lysis of other TC, as evidenced by the ability of CTL to efficiently lyse SRBC. These data suggest that there could be multiple molecular pathways of TC death in CTL-mediated cytotoxicity and the relative contribution of PADPRT and/or other enzymes will reflect the individual make-up of a particular TC.     

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.     

Loss of effector function of human cytolytic T lymphocytes is accompanied by major alterations in N- and O-glycosylation

Most human tumors are not eliminated by the immune system, and therapeutic vaccination shows poor results, a fact that can be explained at least partially by an immunosuppressive tumor microenvironment that is abundant in galectin-3. On cytolytic T lymphocyte (CTL) clones, maintained in culture by regular stimulation, recently activated CTLs present low effector functions. However, these functions are restored after a short treatment with LacNAc. The latter, which is in agreement with the glycoprotein-galectin lattice concept involving reduced motility, poses the question why galectin-3 ligands improve effector functions. We employed ultrasensitive MALDI-TOF-MS on resting and recently activated CTL clones combined with various glycosidase digestions and GC-MS linkage analyses. Our results showed that compared with the resting CTLs, the N-glycans of the recently activated CTLs consisted of (i) larger LacNAc oligomers of which a significant portion was longer than four-units and (ii) more multi-antennary structures. Interestingly, our results showed that the poly-LacNAc appeared to be equally distributed on all available N-glycan branches and not selectively enriched on a specific branch. The above structural alterations in the recently activated CTLs are expected to increase the galectin-3-LacNAc lattices and multivalent interactions and, therefore, reduce the motility of surface glycoproteins, such as the T-cell receptor. These findings suggest that the loss of effector functions on CTLs may be linked to reduced motility of surface glycoproteins. In addition, our results showed that recently activated CTLs had a reduced abundance of NeuAcα2,6-linked N-glycans and an increased abundance of disialylated core 1 and monosialylated core 2 O-glycan structures.     

Inhibitory effect of herpes simplex virus infection to target cells on recognition of minor histocompatibility antigens by cytotoxic T lymphocytes

Target cell lysis by CTL specific for minor histocompatibility Ag (minor HA), which were generated in (C3H/He x BALB/c)F1 mice immunized with A/J mouse spleen cells, was dramatically reduced by infection of HSV to Neuro-2a (A/J mouse origin) cells as target. The reduction was apparent at 5 h after infection of HSV to target cells, when many viral proteins were produced in the cells. Conversely, MHC-restricted HSV-specific CTL-mediated cell lysis increased time dependently. Using an RNA virus, vesicular stomatitis virus, significant reduction of minor specific CTL-mediated target cell lysis was also found. During the time when this reduction of target cell lysis by HSV occurred, the surface expression of class I H-2Dd molecules was maintained, and anti-H-2a allo-MHC-specific CTL lysed HSV-infected Neuro-2a cells as strongly as uninfected Neuro-2a cells. When HSV-infected or uninfected Neuro-2a cells were treated with Brefeldin A that selectively blocks transportation of newly synthesized proteins out of endoplasmic reticulum, both HSV- and minor HA-specific CTL-mediated cell lyses were blocked. These observations demonstrated that minor HA are continuously synthesized and associated with class I molecules at pre-Golgi and transported via trans Golgi system with quick turnover, and that newly synthesized HSV Ag, which are also associated with class I molecules and transported via the same system, should take the place of intrinsic minor HA and be presented on the surface of the cells to be recognized by MHC-restricted CTL.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. II. Responsible factors for nonspecific destruction

Cell-free supernatants of thoracic duct lymphocyte cultures which were stimulated in vitro by horse serum on syngeneic fibroblast monolayers are demonstrated to be cytotoxic on syngeneic embryonic fibroblasts by means of a direct cell count using microtest plates. Experimental supernatants showed up to 100% suppression of fibroblast growth at $\text{1}\text{3}$ dilution and up to 96% suppression at $\text{1}\text{4}$ dilution as compared to the control supernatants. Evidence is presented indicating that lymphocytes cultured on mosaic monolayers, which were comprised of syngeneic and xenogeneic fibroblasts, were reacting both to xenogeneic cells and horse serum in the medium at the cellular level. A hapten-to-carrier type relationship is suggested between xenogeneic antigen and horse serum. Absence of horse serum in the test cultures using these lymphocytes resulted in the abrogation of nonspecific toxic activity of lymphocytes while the specific activity, though diminished, remained. This again indicates the difference in the mechanisms underlying the specific and nonspecific target cell destruction by T cells.