Natural and immune cytolysis of canine distemper virus-infected target cells.

Natural and immune cytolysis of canine distemper virus (CDV)-infected target cells in vitro is described. Lymphocytes expressing natural cytotoxicity were found in specific-pathogen-free beagle dogs and in beagle-coonhound crosses before vaccination with CDV and indefinitely after vaccination, when the ephemeral immune lymphocyte-mediated cytotoxicity (ILMC) had declined. In contrast to the natural lymphocyte-mediated cytotoxicity, the ILMC was genetically restricted, could not be blocked by CDV-specific antibody, and was effective against measles virus-infected as well as CDV-infectd target cells. Lymphocyte populations were depleted of Fc receptor and surface immunoglobulin-bearing cells by rosetting techniques and tested in comparison. An antibody-dependent cell-mediated cytotoxicity was demostrated against CDV-infected target cells that were preincubated with CDV antibody when Fc receptor-bearing lymphocytes were not removed. The ILMC was measurable for approximately 10 days beginning at 6 days post-vaccination. In contrast, CDV antibody measured by virus neutralization and humoral cytotoxicity was detectable by 6 days postvaccination and persisted at peak levels for at least 5 months.     

Increased intracellular cAMP renders HL-60 cells resistant to cytotoxicity of taxol.

The treatment of advanced cancers with paclitaxel (taxol) is hindered by the development of drug resistance. Resistance to taxol is known to be associated with multidrug resistance (MDR) and a mutation affecting either the alpha- or beta-subunit of tubulin. In this study, we demonstrated that an intracellular cAMP level may also play an important role in resistance to taxol in HL-60, acute promyelocytic leukemia cells. Exposure of HL-60 cells to various doses of taxol for 18 hr resulted in cell death. However, pretreatment of the cells with cAMP analogs such as N6:O2-dibutyl cAMP (Db-cAMP), 8-(4-chlorophenylthio) cAMP (CPT-cAMP) and 8-bromo-cAMP (8-Br-cAMP) or an intracellular cAMP elevating agent such as forskolin apparently rendered HL-60 cells more resistant to taxol, but not with dimethyl sulfoxide (DMSO) or retinoic acid (RA), well known differentiating agents. To investigate whether protein kinase A (PKA) activated by an increase in intracellular cAMP level could be involved in increased taxol resistance of the cells, we examined the effects of PKA inhibitors, including H-89 and KT5720, on taxol resistance induced by Db-cAMP. The PKA inhibitors significantly abolished Db-cAMP-induced taxol resistance. These results suggest that cAMP analogs may render tumor cells more resistant to taxol via PKA activation.     

Mycobacterium leprae renders Schwann cells and mononuclear phagocytes susceptible or resistant to killer cells.

Acquired resistance to Mycobacterium leprae, the etiologic agent of leprosy, crucially depends on cellular immune mechanisms. In addition to interleukin-mediated helper functions, killer mechanisms seem to be involved. This study addresses the question of how M. leprae render mononuclear phagocytes and Schwann cells, its natural targets, susceptible or resistant to killer cells. Killer activities were stimulated in peripheral blood mononuclear cells from healthy individuals by incubation with mycobacteria plus interleukin-2. These cells lysed Schwann cells and mononuclear phagocytes which had been pulsed with dead M. leprae, while unpulsed targets remained virtually unaffected. Importantly, targets infected with viable M. leprae were not lysed; furthermore, infection with viable M. leprae as well as gamma interferon stimulation or heat shock caused resistance in otherwise susceptible targets which had been pulsed with dead M. leprae. Thus, M. leprae markedly influenced the effect of killer cells on Schwann cells and mononuclear phagocytes.     

Constitutive synthesis of polyoma antisense RNA renders cells immune to virus infection.

Mouse fibroblasts were stably transfected with expression plasmids in which sequences of the early region of polyomavirus were inserted both in sense and antisense orientation. The cell lines that synthesize in the antisense orientation, a 1195-bp viral genome fragment covering the Ori, Cap, ATG, and all of the early mRNA splicing sites acquire resistance to viral infection. Smaller fragments covering Ori, Cap, and ATG sites or the splicing sites, as well as fragments cloned in sense orientation, failed to confer cell immunity to polyoma infection. The resistance proved to be directly dependent upon the specific antisense RNA and to be inversely proportional to the multiplicity of infecting polyoma.     

Altered cell-fusion capacity in lines of KB cells resistant to Sendai virus-induced cytolysis.

Lines of KB cells resistant to Sendai virus-induced cytolysis (sil cells) have been isolated and characterized. Acquisition of resistance to the cytolysis was found to be associated with changes in the cell fusion capacity. The ability of sil cells to adsorb Sendai virus was essentially the same as that of KB cells, although they were found to have a decreased amount of sialic acid compared to KB cells. The elution of adsorbed virus from sil cells was increased over that from KB cells. The penetration of the virus was markedly reduced at high multiplicities of infection, while it proceeded normally at low multiplicities of infection. The consequences of these results are discussed.     

Cortisone-resistant thymocytes as target cells in lymphocyte-mediated cytolysis

⁵¹Cr-labeled cortisone resistant thymocytes (CRT) can be used as target cells for measuring lymphocyte-mediated cytotoxicity (LMC) in vitro. Since target cell lysis is highly specific and since CRT are available from any mouse strain, they are particularly suited for specificity experiments in cytotoxic reactions.     

Macrophage activation for cytolysis of virally infected target cells

Bone-marrow-culture-derived macrophages killed virally infected cells but not uninfected cells. This activity could be enhanced by preexposure of the macrophages to lipopolysaccharide (LPS), and/or purified interferons. The ability to kill virally infected targets was not restricted to a single cell type or virus. Comparing the ability of activators to induce activity against virally infected targets or tumor (P815) targets, it was found that much lower levels of LPS or alpha/beta-interferon were able to induce cytolytic activity for virally infected cells than were needed for tumor targets. Further, while the antitumor activity did not change significantly with an increase in the time of exposure to activating stimuli from 4 to 24 h, the activity against virally infected cells decreased dramatically with the longer exposure to stimuli.     

Cyclosporin A regulates human NK cell apoptosis induced by soluble HLA-I or by target cells

Human natural killer (NK) cells are effectors of innate immunity, capable of killing transformed or virus-infected cells and producing pro-inflammatory cytokines. Soluble molecules of HLA-I (sHLA-I), which are significantly increased in the serum of patients affected by auto-immune or infectious or neoplastic diseases, induce NK cell apoptosis interacting with its ligands, such as CD8 or the activating isoforms of members of inhibitory superfamily receptors (IRS). This cell death is accompanied by the release of large amounts of interferon-gamma. NK cells can kill autologous target cells, including antigen presenting cells or infected or tumor cells, by engaging the natural cytotoxicity receptors (NCR) NKp30, or NKp44 and NKp46. Again, the binding between NCR on NK cells and their putative ligands on targets leads to NK cell apoptosis. FasL produced and secreted by NK cells is responsible for the NK cell apoptosis induced by either HLA-I receptors or NCR. Interestingly, cyclosporin A (CsA) blocks NK cell death consequent to interaction with target cells or with sHLA-I, without affecting the activation of cytolysis. This would indicate that CsA can maintain NK cell-dependent innate immunity by prolonging NK cell survival in an hostile environment in the presence of sHLA-I or target cells.     

Cdk4 disruption renders primary mouse cells resistant to oncogenic transformation,leading to Arf/p53-independent senescence

A large number of human cancers display alterations in the Ink4a/cyclin D/Cdk4 genetic pathway, suggesting that activation of Cdk4 plays an important role in oncogenesis. Here we report that Cdk4-null mouse embryonic fibroblasts are resistant to transformation in response to Ras activation with dominant-negative (DN) p53 expression or in the Ink4a/Arf-null background, judged by foci formation, anchorage-independent growth, and tumorigenesis in athymic mice. Cdk4-null fibroblasts proliferate at normal rates during early passages. Whereas Cdk4(+/+)Ink4a/Arf(-/-) cells are immortal in culture, Cdk4(-/-)Ink4a/Arf(-/-) cells undergo senescence during continuous culture, as do wild-type cells. Activated Ras also induces premature senescence in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Thus, Cdk4 deficiency causes senescence in a unique Arf/p53-independent manner, which accounts for the loss of transformation potential. Cdk4-null cells express high levels of p21(Cip1/Waf1) with increased protein stability. Suppression of p21(Cip1/Waf1) by small interfering RNA (siRNA), as well as expression of HPV-E7 oncoprotein, restores immortalization and Ras-mediated transformation in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Therefore, Cdk4 is essential for immortalization, and suppression of Cdk4 could be a prospective strategy to recruit cells with inactive Arf/p53 pathway to senescence.     

Codon optimized expression of HPV 16 E6 renders target cells susceptible to E6-specific CTL recognition

The early proteins E6 and E7 of the cancer-related human papillomavirus type 16 (HPV 16) are constitutively expressed in cancer cells thus are targets for immune therapeutic approaches. Whereas previous studies have mainly focussed on the immunogenicity of E7 protein little is known about E6. In order to evaluate E6-specific DNA immunization strategies in a preclinical mouse model C57BL/6 mice were injected with plasmid pTHampE6 and analyzed for E6-specific CTL induction. CTL specific for the H2-K(b)-restricted E6-derived epitope E6 48-57, were readily detectable among splenocytes of immunized animals, however, these CTL showed a differential recognition pattern on various E6-expressing target cells. Using a newly generated E6-specific monoclonal antibody we found that most cell lines expressing E6 encoded by the natural gene showed undetectable protein amounts and were ignored by E6-specific CTL. However, transfection of a codon optimized version of the E6 gene (E6opt) strongly enhanced protein expression levels within these cells turning them into susceptible target cells. Surprisingly, we found that E6-positive TC-1 cells, although recognized by E6-specific CTL, were totally devoid of any detectable E6 protein. Inhibition of proteasomal function by lactacystin treatment diminished E6-specific CTL recognition of TC-1 cells and RMA/E6opt transfectants accompanied by intracellular accumulation of E6 protein as observed in RMA/E6opt transfectants, but not in TC-1 cells. These data suggest that in TC-1 cells rapid degradation processes might prevent stable expression of E6 protein yet generate precursor peptides in amounts sufficient for MHC class I restricted antigen presentation. Thus, the results presented in this paper show that: (i) use of optimized codons in transfection experiments can improve susceptibility of target cells to E6-specific CTL recognition and (ii) lack of detectable protein within a cell does not necessarily indicate the absence of epitope presentation. Both findings are of potential relevance for the design of tumor vaccines.     

Assay of immune cytolysis of lymphocytes and tumour cells by automatic determination of cell volume distribution.

Immune cytolysis (lysis) of cells due to the action of antibody in the presence of complement is usually substantiated by the uptake of vital dye by the cells, or by the escape of radiolabel from the cells. Immune cytolysis has now been assayed by determination of cell volume distribution with a Coulter multi-channel particle size analyser used in conjunction with a Coulter counter. For Ehrlich ascites and sarcoma-180 cells, volume degradation corresponding to vital staining was obtained only if trypsin (final concentration 625 microgram/ml) was added immediately after the usual 1 h incubation period for cells, antibody and complement. For L1210 leukaemia cells, trypsin was added at 0 degrees just 1 min before Coulter evaluation, to avoid potentiation of antibody-mediated cell lysis by trypsin. Immune cytolysis of mouse thymic, splenic and lymph node lymphocytes required addition of pronase (final concentration 625 microgram/ml) at 0 degrees for further disruption of antibody-damaged cells, prior to determination of cell volume distribution in the Coulter equipment. Scanning electron micrographs of L1210 cells undergoing immune cytolysis illustrated the changes in cell volume recorded by the Coulter apparatus. This new method for determination of immune cytolysis provides detailed information about the volume distribution of target cells, which permits detection of subtle changes and gives insight into the process of cytolysis. It is not intended to displace other procedures in routine use, except that complete automation of the present method is possible in future.     

Influence of cell type and virus upon virus-specific immune cytolysis.

Immune cytolysis was measured by release of absorbed radioactive chromium from infected cells that were incubated with antiviral antibody and complement. The presence of virus-specific antigens detected in this manner on the surface of several types of cells infected with Japanese encephalitis (JE) virus did not correlate in each instance with the maturation of infectious virus. JE-infected LLC-MK-2 and Vero cells could not be lysed until long after the first appearance of released virus, and the lysis was minimal in that only a small amount of chromium was released. However, JE-infected BHK-21 and chicken embryo cells could be lysed as soon as new virus was detected in the culture medium, and the lysis reached maximum levels before the time that maximum levels of infectious virus were found in the culture fluids. This phenomenon was restricted to JE virus since BHK-21 cells infected with dengue-2 virus (another group B arbovirus) could not be lysed until well after the appearance of new virus in the culture medium.     

Virion antigens introduced exogeneously into the cell membrane render syngeneic target cells susceptible for T cell-mediated cytolysis.

Non-infectious sendai virus renders H-2 matched target cells susceptible to the lytic effect of sendai virus immune cytotoxic T lymphocytes. This observation suggests that exogeneous insertion of virion antigen in the membrane of the target cell is sufficient for T cell cytotoxicity. The finding is incompatible with the concept that H-2K or H-2D gene products of the target cells must be altered in their primary structure (pretranslational effect of the virus infection) for T cell-mediated cytolysis to occur.     

Cyclosporin A can switch the immune response induced by antigen from a humoral to a cell-mediated mode.

Cyclosporin A (CsA) is known as a nontoxic inhibitor of the immune response that is primarily employed in humans to prevent the rejection of allografts. We report here on a novel activity for this drug as an immunomodulator. CsA can either inhibit or facilitate the induction of delayed-type hypersensitivity (DTH) depending on the class of immune response that would be induced in the absence of the drug. The drug inhibits the in vivo and in vitro induction of DTH when antigen is given under conditions that normally result in the induction of this response. The same concentration or dose of CsA inhibits the in vitro and in vivo induction of an antibody response and the antigen induces DTH instead. An explanation for these different effects of CsA on the induction of DTH is proposed. This novel activity of CsA in modulating the immune response from an antibody to a cell-mediated mode may have clinical applications.     

Expression of yeast but not human apurinic/apyrimidinic endonuclease renders Chinese hamster cells more resistant to DNA damaging agents

Abasic sites represent ubiquitous DNA lesions that arise spontaneously or are induced by DNA-damaging agents. They block DNA replication and are considered to be cytotoxic and mutagenic. The key enzymes involved in the repair of abasic sites are apurinic/apyrimidinic (AP) endonucleases which process these lesions in an error-free mechanism. To analyze the role of AP endonuclease in the protection of mammalian cells against DNA damaging agents, we have transfected both the human (APE) and the yeast (APN1) AP endonuclease in Chinese hamster cells and compared the effects of expression of these genes in stable transfectants as to survival of cells and formation of chromosomal aberrations. Although APE was markedly expressed on RNA and protein level, nuclear extracts of human APE transfectants did not show a higher AP endonuclease activity than the parental line and became not more resistant to the cell killing and clastogenic effect of methyl methanesulfonate (MMS) and hydrogen peroxide (H₂O₂). In contrast, cells transfected with the yeast APN1 gene expressed higher AP endonuclease activity and became clearly more resistant to the cytotoxic and chromosome breakage inducing activity of the agents. The results indicate that the excision repair capacity and correspondingly the mutagen resistance can be elevated by introducing, in mammalian cells, a yeast DNA repair gene and verify that AP sites are both cytotoxic and clastogenic lesions.     

Possible role of Fc receptors on cells infected and transformed by herpesvirus: escape from immune cytolysis.

Receptors for the Fc portion of nonimmune immunoglobulin G were demonstrated on B103 rat brain neuroma cells infected with herpes simplex virus type 1 (HSV-1) KOS by a radioimmunoassay using 125I-labeled heat-aggregated Fc fragments. Immune F(ab')2 fragments specific for HSV antigens competed efficiently for Fc binding sites, suggesting that the binding of Fc fragments to infected cells is specific for viral cell-surface antigens. It has been suggested that the binding of immune complexes to Fc receptors on the surfaces of tumor cells in vivo plays a role in protecting these cells from immune destruction. In vitro evidence is presented for the ability of aggregated immunoglobulin G molecules bound to cell-surface Fc receptors to protect both HSV-infected and HSV-transformed cells against complement-dependent and cell-mediated immune lysis.     

Cell membrane-mediated cytolysis by membranes from noncytolytic cells.

Cytolysis was obtained with plasma membrane fractions prepared not only from cytolytic T cells, but also from nonimmune lymphoid cells and, moreover, from cell lines of nonlymphoid origin displaying no lytic ability. Furthermore, no relationship exists between the genetic specificty of T cell-mediated cytolysis and the activity of membrane preparations. If anything, it appears that certain cell types may be unusually sensitive to membrane-mediated cytolysis. Our observations argue against a mechanistic relationship between T cell-mediated and membrane-mediated cytolysis.     

Artificial binding of macrophages to syngeneic cells elicits cytostasis but not cytolysis.

Artificial binding of mouse peritoneal macrophages to syngeneic embryonic fibroblasts by means of rabbit anti-mouse anti-macrophage serum or Con-canavalin A inhibited the proliferation of syngeneic target cells without destroying them. It is suggested that an intimate membranal contact between macrophages and target cells triggered cytostasis, whereas cytotoxicity requires an additional step of foreign recognition.