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.     

A mutation in a non-MHC murine cell surface antigen detectable by cytotoxic T lymphocytes

During the course of screening new T-H-2 region congenic strains of mice constructed from the C57BL/6 and B6-H-2k strains, a new cell surface polymorphism, designated dtc-1, was identified by cell-mediated lympholysis techniques. The dtc-1 antigen can be found on both Con A- and LPS-stimulated lymphoblasts, peritoneal macrophages, and SV40-transformed mouse embryo fibroblasts. Lysis of dtc-1+ targets by CTL is H-2Dk restricted. All inbred strains tested are dtc-1+, with the exception of the B6-H-2k strain, which is dtc-1-, and several congenic strains directly derived from B6-H-2k. Because B6/Boy and AKR/Boy, the parents of the B6-H-2k strain, are dtc-1+, the dtc-1- phenotype may be the result of mutation in the locus specifying the cell surface molecule that carries this antigen. Segregation analysis of the dtc-1+/dtc-1- polymorphism demonstrated that this locus is not linked to T or H-2. The dtc-1 antigen thus identifies yet another cell surface polymorphism and adds another immunologically defined genetic marker to the murine genome. Furthermore, the dtc-1 system indicates the need for reevaluation and restandardization of congenic strains of mice derived from the B6-H-2k congenic strain.     

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.     

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.     

Cellular localization of perforin 1 in murine cloned cytotoxic T lymphocytes

The localization of perforin 1 (P1) in cytotoxic cells was studied by immuno-electron microscopy by using a monospecific rabbit antiserum against highly purified mouse P1 and protein A gold as a second ligand. P1 was found in specific granules of cloned cytotoxic T lymphocytes (CTL). Within the granules, P1 antigen was localized in the fine granular matrix, whereas the vesicular compartment remained free of gold particles. The amount of P1 antigen detectable by immuno-electron microscopy varied between different CTL clones. CTL with NK-like activity had the highest level of P1 antigen. A cytotoxicity loss CTL mutant had no detectable P1 antigen, suggesting an important role of P1 during cell-mediated cytolysis. P1 antigen was undetectable also in bone marrow macrophages, indicating a different cytolytic mechanism of these cells.     

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.     

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.     

Mechanisms for generating cell membrane antigens that are recognized by cytotoxic T lymphocytes

Studies with many viruses have revealed that viral specific protein synthesis is an obligatory step in generating antigens on target cells for antiviral cytotoxic T lymphocytes. This has been most clearly demonstrated with DI particles, virions that are structurally complete but lack infectious RNA. Adsorption of such particles onto target cell membranes does not render these cells susceptible to lytic attack by antiviral effector cells, unless some viral protein synthesis transpires. However, some viruses, such as Sendai virus, circumvent the requirement for viral protein synthesis via fusion of the viral envelope with the target cell membrane, a process mediated by a specialized fusion protein. Once inserted into the lipid bilayer, it is likely that viral components and self H-2 noncovalently associate so that the complex can be recognized by antiviral cytotoxic T cells. This idea is supported by the demonstration that viral proteins and H-2 containing membrane proteins, incorporated into reconstituted membrane vesicles or liposomes are recognized by cytotoxic T cells. These data further show that native rather than altered viral and H-2 molecules are the moieties recognized. Associations between antigen and H-2 have been detected by a variety of techniques and in some cases are not random but selective; that is, viral antigens perferentially associate with some H-2 alleles and not others. In summary, these findings indicate that although viral antigens are present in the mature virions, these components are not recognized by antiviral killer cells until integrated into the plasma membrane. This may be achieved either through direct fusion of the viral envelope with the target cell or following viral protein synthesis and insertion of viral antigens into the plasma membrane.     

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.     

The mechanism of anti-Lyt-2 inhibition of antibody-directed lysis by cytotoxic T lymphocytes

Bifunctional antibodies specific for a determinant within the T cell receptor (TcR) complex of cytotoxic T lymphocytes (CTL) and a determinant expressed on the surface of the target cell will effectively mediate cytolysis. In such a lytic system anti-Lyt-2 antibody can block cytolysis. We have observed that the amount of inhibition varies considerably from clone to clone and surprisingly correlates well with inhibition of conjugate formation as mediated by bifunctional antibody. This implies that inhibition of antibody-mediated killing occurs as the result of reduction of the avidity of the effector cell for its target, the same mechanism responsible for inhibition of receptor-mediated lysis by anti-Lyt-2. In light of the similarity between the mechanism of inhibition by anti-Lyt-2 of receptor-mediated and antibody-mediated cytolysis, we compared the ability of anti-Lyt-2 to inhibit cytolysis in these two different assay systems by using a number of different CTL clones. Whereas the majority of secondary CTL clones (presumed to have high affinity TcR) are inhibited equally in both assay systems, most primary CTL (presumed to have low affinity TcR) are more susceptible to inhibition by anti-Lyt-2 in their receptor-specific than their antibody-directed cytolysis. These results, taken together with an apparent correlation between the amount of Lyt-2 expressed on the cell surface and susceptibility to inhibition, suggest anti-Lyt-2 may block CTL function by sterically inhibiting mobility of the TcR complex.     

Fas-ligand-mediated lysis of erbB-2-expressing tumour cells by redirected cytotoxic T lymphocytes

A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor γ chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-γ clones, and lysis of erbB-2⁺ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-γ, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL. Received: 23 July 1998 / Accepted: 5 October 1998     

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.     

Target cell lysis and IL-2 secretion by gamma/delta T lymphocytes after activation with bacteria

Peripheral blood T lymphocytes from healthy donors were stimulated with Mycobacterium tuberculosis in vitro and afterward analyzed phenotypically. Marked expansion of the gamma/delta T cell population (3- to 21-fold) was observed in 15/21 donors 7 to 10 days after stimulation. In addition to M. tuberculosis, Mycobacterium leprae (six of eight) as well as the gram-positive bacteria, Staphylococcus aureus (two of six), group A streptococci (seven of nine), and Listeria monocytogenes (four of eight) augmented gamma/delta TCR expression in peripheral blood T cells of many donors. gamma/delta T lymphocytes expressed IL-2R and secreted IL-2 upon restimulation with M. tuberculosis. Stimulation with M. tuberculosis evoked specific cytolytic activities in gamma/delta T lymphocytes because: gamma/delta T cells lysed M. tuberculosis pulsed but not unpulsed targets; high concentrations of TCR delta 1 mAb facilitated killing of unpulsed target cells; and low doses of anti-TCR delta 1 mAb blocked killing of pulsed targets. Furthermore, gamma/delta T cells from four donors, after activation with M. tuberculosis or with group A streptococci, respectively, only lysed targets pulsed with the homologous agents, whereas in other donors some cross-reactivity was observed. We conclude that, upon contact with mycobacteria and perhaps other microorganisms, gamma/delta T cells are activated which contribute to immunity against infection via IL-2 secretion and specific target cell lysis.     

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.     

Stable expression of cytolytic activity by influenza virus-specific cytotoxic T lymphocytes

Influenza-specific immune cytotoxic T lymphocyte (CTL) populations maintain a constant level of in vitro cytolytic activity. This is demonstrable with both heterogeneous populations of anti-viral CTL from immune donors and long-term CTL clones derived from primed CTL precursors. Cytolytic machinery is stably expressed by these CTL populations under a variety of in vitro cultivation conditions. This finding is in contrast to results with alloreactive CTL generated by stimulation of primed CTL precursors that lose cytolytic activity on a per cell basis with time after stimulation. The results indicate that virus-specific, cloned CTL that stably express cytolytic activity are representative of the heterogeneous populations from which they are derived and further suggest a qualitative difference in the regulation and expression of cytolytic machinery between heterogeneous populations of influenza-specific CTL and alloreactive CTL.     

Modulation of the recognition and lysis of EL4 tumor target cells by cytotoxic T lymphocytes

When the EL4 targets were harvested from the peritoneal cavity (in vivo), they had less than half as much cell-surface sialic acid as EL4 cells harvested from tissue culture (in vitro), apparently due to the presence of a neuraminidase activity in the peritoneal cavity. Both the recognition and the lysis of either EL4 in vivo or EL4 in vitro target cells by allogeneically primed cytotoxic T lymphocytes were enhanced upon removal of cell-surface sialic acid by neuraminidase treatment. However, even after neuraminidase treatment, there still remained a difference in the lytic profile when using EL4 targets that were harvested in vivo versus in vitro. Both conjugate formation between the target and the T cells and anti-H-2D^b adsorption by the target cells were unaffected by the culture conditions of the target line. However, antibody-induced capping and exocytosis of vesicles differed between the differently cultured target cells, suggesting that there was a membrane organizational difference between them that was detected by the cytotoxic T cells. These data are consistent with the idea that cell surface sialic acid as well as the membrane organization can influence T-cell recognition and lysis of target cells.     

Direct cross-priming by th lymphocytes generates memory cytotoxic T cell responses

Under optimal Ag stimulation, CTL become functional effector and memory T cells. Professional APCs (pAPC) are considered essential for the activation of CTL, due to their unique capacity to provide costimulation and present exogenous Ags through MHC class I molecules. In this study, we report a novel means by which Th lymphocytes acquire and present MHC class I determinants to naive CTL. Although previous studies have looked at T cell Ag presentation to activated T cells, this study presents the first example of Ag presentation by Th cells to naive CTL. We report that activated Th cells can function as effective pAPC for CTL. Our results show that: 1) In addition to acquisition of cell surface molecules, including MHC class I/peptide complexes, from pAPC, Th cells can acquire and present MHC class I-binding peptides through TCR-MHC class II interactions with pAPC; 2) the acquired Ag can be functionally presented to CTL; and 3) Ag presentation by Th cells induces naive CTL to proliferate and preferentially differentiate into cells that phenotypically and functionally resemble central memory T cells. These findings suggest a novel role of Th cells as pAPC for the development of memory immune responses.     

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.