Natural Killer Cell Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity to Herpes Simplex Virus-Infected Cells in Human Pregnancy

ABSTRACT: Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a ⁵¹Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.     

Effect of herpes simplex virus infection on murine antibody-dependent cellular cytotoxicity and natural killer cytotoxicity.

Mice intraperitoneally inoculated with a sublethal dose of herpes simplex virus (HSV) produced immunoglobulin G antibody-dependent cellular cytotoxicity (ADCC) and radioimmunoassay (RIA) antibody as early as 3 days after infection. There was a rise in natural killer cytotoxicity (NKC) to infected and uninfected target cells 1 to 3 days postinfection mediated by nonadherent peritoneal cells (PC) in mice inoculated with HSV, but also with other substances commonly used in tissue culture media. HSV caused the highest and most consistent increase in NKC. PC-NKC, as ADCC, was inhibited by latex and silica, both macrophage inhibitors. PC-ADCC markedly declined 3 to 8 days after HSV inoculation. This was not due to a soluble or cellular suppressor factor, was not reversed by incubation or trypsin treatment of PC, was not associated with a change in PC Fc receptors, adherence, or acridine orange staining characteristics, and could not be induced by inactivated HSV. In vitro inoculation of PC with HSV similarly caused a reduction in the ability of PC to mediate ADCC to HSV-infected target cells. These data demonstrate the complex stimulatory and inhibitory interactions between virus and host defense mechanisms.     

Prostacyclin release and cytotoxicity of peritoneal cells are inversely related in pregnant and non-pregnant mice infected with herpes simplex virus

Cytotoxicity of peritoneal cells in a HSV-infected murine model is attenuated in late pregnancy. Prostacyclin (PGI2) is elevated at this time in reproductive tissues and has been implicated in the regulation of the immune response. The purpose of this study was to estimate PGI2 in the peritoneal wash or culture supernatants of peritoneal cells obtained from uninfected and HSV-infected pregnant and virgin mice using a radioimmunoassay for 6-keto-prostaglandin F1 alpha. The peritoneal wash of uninfected pregnant and virgin mice contained high levels of 6-keto-PGF1 alpha, 505 +/- 51 pg/100 microliters, (mean +/- S.E., n = 15), and 200 +/- 19 pg/100 microliters, (n = 30), ad did peritoneal effector and target cell cultures (1,159 +/- 118 pg/100 microliters, n = 6, and 1,057 +/- 207 pg/100 microliters, n = 7), respectively. HSV-infection induced in vitro cytotoxicity and suppressed the release of 6-keto-PGF 1 alpha (r = -0.897, P less than 0.05, n = 18). Its concentration was significantly higher (14-fold, P less than .05) in the peritoneal wash, but not in the cell culture, of pregnant (212 +/- 29 pg/100 microliters, n = 19) as compared to virgin mice (18.5 +/- 3.4 pg/100 microliters, n = 27). The levels of 6-keto-PGF1 alpha were inversely correlated (P less than .05) with the combined effects of HSV-infection and cytotoxicity.     

Murine cellular cytotoxicity to syngeneic and xenogeneic herpes simplex virus-infected cells.

Cellular cytotoxicity of C57BL/6 adult mice peritoneal cells to xenogeneic (Chang liver) and syngeneic (BL/6-WT3) herpes simplex virus (HSV)-infected cells was analyzed in a 6-h 51Cr release assay. There was no difference in antibody-dependent cellular cytotoxicity to either target. There was no natural killer cytotoxicity to targets with cells from uninfected mice except at very high effector cell ratios. HSV-infected (2 X 10(4) PFU intraperitoneally 1 day previously) mice mediated significantly higher antibody-dependent cellular cytotoxicity and required less antibody (10(-5) versus 10(-2) dilution), fewer cells, and less time to kill than cells from uninfected mice. HSV-infected mice mediated natural killer cytotoxicity but preferentially killed syngeneic HSV-infected cells. Stimulation of cytotoxicity was not virus specific since influenza-infected mice mediated similar levels of cytotoxicity to HSV-infected targets. There was no difference in morphology (95% macrophage) or in the percentage of FcR-positive cells, but infected mice had more peritoneal cells and generated higher levels of superoxide in response to opsonized zymosan or phorbolmyristate acetate. These data demonstrate nonspecific virus-stimulated metabolic and effector cell function which may enhance clearance of virus in an infected host.     

Human antibody-dependent cellular cytotoxicity and natural killer cytotoxicity to herpes simplex virus-infected autologous and allogeneic cells.

Using cultured skin shavings, human cellular cytotoxicity to uninfected and herpes simplex virus (HSV)-infected autologous and allogeneic fibroblasts and Chang liver cells was analysed in a 51Cr release assay. The effector cell requirements and characterization, time kinetics and antibody requirements were similar using each HSV-infected target cell in an antibody-dependent cellular cytotoxicity (ADCC) system. There was lower natural killer cytotoxicity (NKC) to uninfected autologous cells than unrelated cells in an 18 hr assay. NKC to infected autologous and unrelated fibroblasts was similar to that mediated against Chang liver cells. Thus NKC to uninfected fibroblasts correlated with the relationship of effector and target cells while NKC to infected cells correlated with the intrinsic lytic potential of the effector cells. The autologous system offers little advantage in the analysis of ADCC or NKC in normal individuals to virus-infected cells, but is probably crucial for the detection of HLA-restricted T-cell cytotoxicity. The demonstration of autologous anti-viral ADCC and NKC lends further credence to the in vivo importance of the mechanisms.     

In Vitro Cell-Mediated and Complement-Mediated Cytotoxicity to Murine Testicular Cells

ABSTRACT: Male BALB/c mice at 8 to 14 weeks of age were divided into three groups: group 1 was immunized with an emulsion of testicular cells (TCs) (10⁷/mouse), complete Freund's adjuvant (CFA), and extract of Bordetella pertussis (BP); group 2 was given CFA and BP injections; and group 3 was given sterile saline injections. Suspensions of TCs and spleen cells (SCs) from each mouse were prepared 4 weeks after the first immunization for a cytotoxic T lymphocyte (CTL) assay. For the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) assays, TCs and SCs were prepared from normal male and female mice, respectively. Targets were labeled with Na₂⁵¹ CrO₄. The interactions of targets (TCs) and effectors (SCs) were conducted at 32.5°C (for CTL, ADCC, and ACC assays) or 37°C (for ACC assay). In the CTL assay, SCs from group 1 and group 2 caused significantly more killing than those from group 3. Specific cytotoxicity in the ADCC assay was only detected in the serum (maximum specific lysis 47.6%) of one mouse. No other cytotoxicity was detectable in 61 serum samples from group 1 (n = 25), group 2 (n = 17), and group 3 (n = 19). In the ACC assay, no significant specific cytolysis was found at different incubation temperatures (32.5 and 37.0°C) in 44 serum samples from the three groups. These results suggest that CTLs are important in the pathogenesis of experimental allergic orchitis (EAO). Adjuvant alone, probably because of breakdown of the blood-testis barrier, causes significant T lymphocyte cytotoxicity to TCs. ADCC and ACC are not important mechanisms in the immunopathogenesis of murine EAO.     

Immune modulation of natural killer cell cytotoxicity against herpes infected target cells in pregnancy

Natural killer cell cytotoxicity (NKC) is a nonspecific, primary immunodefense system active against a variety of pathogens, including herpes simplex virus (HSV). Evidence suggests that during pregnancy, NKC is attenuated. The regulatory mechanisms for this immune attenuation have yet to be defined. We examined two cytokines (interleukin-2 [IL-2] and alpha interferon [IFN]) for their ability to alter NKC responsiveness during pregnancy, utilizing an HSV-infected target cell model. Peripheral mononuclear effector cells were isolated from 19 pregnant and 19 nonpregnant subjects by Ficoll-Paque separation. These cells were incubated with IFN, IL-2, or media alone, and analyzed for %NKC by an 18 h chromium release assay. The percentage of NKC was lower using the effector cells from the pregnant subjects as compared to nonpregnant controls. Incubation with either IFN or IL-2 resulted in a significant augmentation of NKC in both the pregnant and nonpregnant derived cells. There were no differences in IL-2 dose requirements or levels of cytotoxicity achieved (43.1 +/- 6.8% vs. 44.4 +/- 6.8%, respectively) between pregnant and nonpregnant derived cells. The IFN-mediated augmentation of NKC was somewhat blunted in pregnancy both in terms of absolute levels of cytotoxicity achieved (26.1 +/- 3.9% vs. 37.2 +/- 4.9%, respectively) and dose response curves generated. These results demonstrate that NKC against HSV infected cells is attenuated during pregnancy and can be immunoregulated with the use of either IFN and IL-2. The restoration of NKC responsiveness with IFN, however, remains incomplete during pregnancy, suggesting that this cytokine's mechanism of action differs from that of IL-2.     

Cytotoxicity reactions against target cells infected with rabies virus

Some aspects of the cytotoxicity reactions were studied in the rabies system. The antibodydependent complement cytotoxicity (ADC), the cellular cytotoxicity (CC), and the antibody-dependent cellular cytotoxicity (ADCC) are shown, being the cytotoxic effect as evidenced by the ⁵¹Cr released from the cells infected with the Pasteur strain of rabies virus. Some parameters such as time of cellular infection, the amount of infected cells, the concentration of complement, and the incubation time of the ADC reaction, which help to increase the performance of this reaction, are discussed. The detection and the level of the cellular response against the Pasteur strain of rabies virus in immunized mice is shown. Evidence is presented that in the ADCC test, specific human antibodies and non-immune human lymphoid cells are able to mediate in vitro lysis of cells infected with rabies virus. A comparison of the ADCC test with serum neutralization and immunoenzymatic tests is shown.     

Deficient Antibody-Dependent Cellular Cytotoxicity against Human Immunodeficiency Virus (HIV)-Expressing Target Cells in Perinatal HIV Infection

Peripheral blood mononuclear cells (PBMC) of human immunodeficiency virus (HIV)-infected children, age-matched HIV-seronegative controls, and HIV-infected asymptomatic and symptomatic adults were compared for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell-mediated cytotoxicity against target cells expressing HIV or herpes simplex virus (HSV) antigens. Target cells consisted of CD4 lymphocytes purified from PBMC of HIV-seronegative adults and incubated with the IIIB strain of HIV, HUT78 cells chronically infected with IIIB, and HSV-infected human fibroblasts. PBMC of asymptomatic HIV-infected adults were generally able to lyse CD4 cells expressing HIV antigens. Direct correlation was found between the magnitude of lysis and absolute CD4 cell counts in these individuals. In contrast to these results, PBMC from HIV-infected children were generally unable to lyse IIIB-expressing CD4 cells, regardless of the children’s clinical status, age, or absolute CD4 cell counts. Cells from HIV-seronegative adults and children did not directly lyse these target cells either but, in contrast to cells of HIV-seropositive children, were able to mediate cell lysis when serum from an HIV-seropositive adult was added. However, effector cells from these HIV-infected children were able to mediate both ADCC against HSV-infected fibroblasts and NK cell-mediated cytotoxicity against IIIB-infected HUT78 cells. Reduced ability of PBMC from vertically HIV-infected children to mediate ADCC against HIV antigen-expressing CD4 cells may contribute to rapid progression to AIDS.     

Failure to Detect Complement-Mediated Antibody-Dependent Cytotoxicity Against Human Orbital Tissue Cells in the Serum of Patients with Thyroid-Associated Ophthalmopathy

Antibodies which are cytotoxic to human eye muscle cells and orbital fibroblasts in antibody dependent cell-mediated cytotoxicity (ADCC) are routinely detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In the present study sera from patients with TAO, most of which were shown to be positive in ADCC against eye muscle cell targets, were tested for complement-mediated antibody-dependent cytotoxicity (CMAC) against human and pig eye muscle cells, human (abdominal) skeletal muscle cells and human orbital fibroblasts, in ⁵¹ Cr release assays. Several different assay protocols and complement sources were used and patient and age, sex matched normal sera compared. In preliminary studies tests were always negative when eye muscle cells, other skeletal muscle cells, or orbital fibroblasts were used as targets, regardless of the complement source, or concentration, or assay conditions used. When larger numbers of patients and normals were tested in a single assay mean (± SE) % specific lysis for patients with TAO was not significantly different from that for normals for either eye muscle cells or other skeletal muscle cells and taking the upper limit of normal as mean + 2 SD for the normals, tests were positive in no patient with either target. On the other hand ADCC tests were positive in 57 % of the same sera tested with the same eye muscle cell targets. When human thyroid cells were used as targets, tests were positive in 10 of the 14 patients tested and monoclonal antibodies, in enzyme-linked immunosorbent assay, reactive with eye muscle antigens gave positive lysis of eye muscle cells.

Although eye muscle reactive autoantibodies have been well described in TAO, particularly IgG₃ subclass antibodies against a 64 kDa membrane antigen, their significance in the pathogenesis of the eye muscle component of this disorder is unclear. In contrast to thyroid cells where microsomal, and perhaps other membrane reactive antibodies, are cytotoxic in both ADCC and CMAC, antibodies associated with TAO appear to be cytotoxic only in ADCC. Although the reason for this discrepancy is unknown one possible mechanism is failure of IgG₃ subclass antibodies, which are usually cytotoxic, to activate complement when the target antigen is in too low a density on the cell surface.     

Analysis of Cell-Mediated Cytotoxicity of CD18-Deficient Bovine Neutrophils

Killing activity (KA) and antibody-dependent cell cytotoxicity (ADCC) of neutrophils from normal cattle and cattle with bovine leucocyte adhesion deficiency (BLAD) were compared in order to evaluate the cytotoxicity of CD18-deficient bovine neutrophils. The killing activity of CD18-deficient neutrophils was significantly lowered (p<0.01) when compared to normal neutrophils, whereas ADCC activity was increased. Fc receptor for immunoglobulin G (FcyR-IgG) mediated ADCC of neutrophils appeared to be enhanced in BLAD-neutrophils.     

Experimental infection of inbred mice with Herpes Simplex Virus

Summary Peritoneal exudate cells (PEC) of DBA/2 mice, after 7 days ofin vitro preculture and consisting of virtually 100 per cent macrophages, were able to support the replication of Herpes Simplex Virus type 1 strain WAL (HSV). Using a standard medium based on Dulbecco's Modified Eagle Medium (D-MEM), no virus replication was observed in freshly isolated PEC. However a medium based on RPMI1640 consistently yielded higher virus titres in precultured PEC than the D-MEM medium, and also allowed virus replication in freshly isolated PEC. Macrophages derived from the spleens or the bone marrow, and precultured in the same way as PEC represented a highly pure population and were permissive for infection with HSV. Titres of about 10⁶ PFU HSV were observed in PEC 48 hours after infection with 10³ or 10⁶ PFU. However, whereas a complete destruction of the cell monolayer was observed 24 hours after infection with 10⁶ PFU, complete cytopathogenicity in PEC infected with 10³ PFU required at least twice this time.In the latter situation, plaque formation was observed 24 hours after infection. PEC of different strains of mice were compared. Of these, PEC of all mice that are susceptible to HSV infectionin vivo replicated HSV to the same degree as PEC of DBA/2 mice, whereas PEC of resistant C57 BL/6 and C3H/HeJ mice produced 1000 fold lower titres of viral progeny. Whereas the number of infectious centres were equal in PEC of DBA/2 and C57 BL/6 mice, the plaques observed after infection of confluent PEC with a low MOI were considerable smaller in cells from C57 BL/6 mice. Furthermore, significantly higher titres of interferon were measured in the supernatants of HSV-infected C57BL/6 macrophages than in those of DBA/2 macrophages, and the former were made fully susceptible by thein vitro addition of an anti-interferon serum.With 3 Figures     

Cytotoxicity of leukocytes from normal and Shigella-susceptible (opium-treated) guinea pigs against virulent Shigella sonnei.

Intraepithelial lymphocytes were collected from the ileum of adult Hartley strain guinea pigs and used as effector cells in a 60-min bactericidal assay with virulent Shigella sonnei as target cells. Natural killer cytotoxicity (NKC) and antibody-dependent cellular cytotoxicity (ADCC) were measured and correlated with the resistance of the animals to infection by S. sonnei. Normal guinea pig intraepithelial lymphocytes exhibited mean NKC and ADCC values of 22.8 +/- 5.0 and 34.1 +/- 13.6, respectively. These animals were resistant to oral challenge with virulent S. sonnei. Intraepithelial lymphocytes from guinea pigs which were fasted for 4 days demonstrated NKC and ADCC values similar to those of normal animals (31.0 +/- 8.1 and 41.7 +/- 6.7, respectively). These animals also were resistant to oral challenge. Intraepithelial lymphocytes from guinea pigs which were given 1 ml of deodorized tincture of opium 2 h before cell collection demonstrated deficient NKC (4.7 +/- 4.2) and ADCC (5.3 +/- 4.9) values but remained resistant to infection by S. sonnei. When guinea pigs were fasted for 4 days and given opium, deficient NKC (2.0 +/- 2.0) and ADCC (1.3 +/- 1.3) values were demonstrated; this group of animals was susceptible to infection by S. sonnei (P less than 0.04). These experiments demonstrated that opium treatment depresses one form of gut immunity. When combined with starvation, opium treatment may increase susceptibility to infection by shigellae by modulation of immunity in addition to the effects on gut motility and bacterial flora.     

The Effects of Cyclophosphamide on Antibody Dependent Cell-Mediated Cytotoxicity in Mice

Abstract

The effect of cyclophosphamide (CY) on antibody dependent cell-mediated cytotoxicity (ADCC) was evaluated. The results indicated that a single injection of CY markedly increased ADCC activity of murine splenic cells. This effect was dose dependent and was maximal on day 7 after the drug injection. The number of lymphoid cells in treated mice decreased to approximatedly 50%; however the percentages of EA rosettes were similar to those of control animals suggesting that the augmented cytotoxic activity was not due to a relative increase in Fc receptor bearing cells. CY treatment also enhanced delayed type hypersensitivity, but suppressed antibody production to sheep red blood cells. The possibility that CY eliminates a suppressor cell subpopulation that normally regulates ADCC levels is discussed.     

Effects of Soluble Tumor Necrosis Factor (TNF) on Antibody-Dependent Cellular Cytotoxicity of Therapeutic anti-TNF-α Antibody

Anti-TNF antibodies are major therapeutics for rheumatoid arthritis and have been approved for marketing in many countries. Antibody-dependent cellular cytotoxicity (ADCC) is considered to be a potential mechanism of action of anti-TNF antibodies, since some anti-TNF antibodies have been confirmed to induce cytotoxic effects on TNF-producing cells via ADCC and complement-dependent cytotoxicity (CDC) in in vitro experiments. In this study, we established a new stable effector cell line expressing human FcγRIIIa, CD16:KHYG-1, and compared the performance of this cell line with that of peripheral blood mononuclear cells (PBMCs) in ADCC assays against CHO-derived target cells expressing protease-sensitive pro-TNF. Although an inhibitory effect of soluble TNF released from pro-TNF expressing cells on ADCC activity was seen, clear dose-responsive ADCC activities were observed even in the presence or absence of TNF-α converting enzyme (TACE) inhibitor. However, significant differences in the ADCC activities in the presence or absence of TACE inhibitor were only noted when CD16:KHYG-1 cells were used as the effector cells. Our findings indicate that soluble TNF may influence ADCC activity of anti-TNF antibody. Moreover, the fact that the influence was able to be detected only in the case using stable effector cell also suggests that the stable effector cell established this time enable highly accurate ADCC measurement.     

Modulation of Antibody-Dependent Cellular Cytotoxicity Against Chicken Erythrocyte Targets by Adriamycin and Daunorubicin

Abstract

Peritoneal exudate cells isolated from Adriamycin (AM) or daunorubicin (DM)-treated mice demonstrated an increased ability to mediate antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated chicken red blood cell targets. Following initial suppression of this cell-mediated function 3 days after a single injection, increased effector cell efficiency occurred to an equal degree in both groups of drug treated mice by day 10 compared to controls. This increase in ADCC activity occurred in parallel with a decrease in the total number of peritoneal cells recovered. It is hypothesized that the drugs acted to modulate ADCC in two ways: 1) suppression by induction of suppressor cell activity, and 2) enhancement by elimination of suppressor cells which resulted in increased effector activity of the remaining cells.     

Azathioprine suppression of natural killer activity and antibody-dependent cellular cytotoxicity in renal transplant recipients

The relative effects of azathioprine (AZA) and prednisone (PRED) on natural killer (NK) activity and the antibody-dependent cellular cytotoxicity (ADCC) of K (killer) cells and the number of FcR and other lymphoid cells were examined in renal transplant recipients. In addition to both long-term (>6 months) and short-term (<6 months) transplant recipients receiving conventional AZA-PRED therapy, an important group of long-term recipients receiving PRED but not AZA was studied for the first time. Both NK activity and ADCC are profoundly reduced in the long-term AZA-PRED group but are normal in the long-term PRED-alone (no-AZA) group. The short-term AZA-PRED group exhibits NK and ADCC levels significantly lower than normal but not as low as those of the long-term AZA-PRED group. Patient groups with low NK and ADCC also have low circulating Fc receptor-bearing (FcR) cells. A single patient in the long-term AZA-PRED group was removed from AZA therapy, and approximately 3 months was required for the patient's suppressed NK and ADCC to return to normal. These findings indicate that AZA rather than PRED is the major drug important in suppressing ADCC and NK activity in renal transplant recipients. Several months are required for combination AZA-PRED therapy to reduce these cytotoxic activities. Similarly, several months are required for suppressed ADCC and NK activity to return to normal upon discontinuation of AZA.     

The Mechanism of the Inhibitory Effect of Progesterone on Lymphocyte Cytotoxicity: II. Relationship Between Cytotoxicity and the Cyclooxygenase Pathway of Arachidonic Acid Metabolism

ABSTRACT: In the previous paper we have demonstrated that progesterone-treated lymphocytes of healthy pregnant women produce a 34,000 MW protein that inhibits cytotoxic activity and prostaglandin F₂α synthesis. Since recently it has been shown that certain leukotrienes have a stimulatory effect on natural killer activity, in this study an attempt was made to determine whether there is a relationship between cytotoxicity and PGF₂α synthesis or if alterations in the values of these parameters are independent. Arachidonic acid increased cytotoxic activity of healthy pregnant women's peripheral blood mononuclear cells (PBMC) in a concentration-dependent manner. Exogenous arachidonic acid was able to counteract the blocking effect of the above-mentioned protein produced by progesterone-treated lymphocytes. To determine whether the products of the cyclooxygenase or the lipoxygenase pathway of arachidonic acid metabolism are responsible for increased cytotoxicity, both enzyme systems were blocked separately. Both indomethacin and the lipoxygenase inhibitor BW 755C reduced cytotoxicity in a dose-dependent fashion. However, the lipoxygenase inhibitor prevented prostaglandin synthesis to the same extent, or even more than indomethacin, in all concentrations used; so, its blocking effect cannot be considered as supportive evidence for the role of leukotrienes in cytotoxicity. On the other hand, lipopolysaccharide, with a selective stimulatory effect on prostaglandin synthesis, increased cytotoxicity. Lipopolysaccharide had no effect on progesterone-pretreated PBMC. The above data allow the assumption that besides leukotrienes, cyclooxygenase products may also increase cytotoxicity.     

Herpes simplex virus thymidine kinase expression in trigeminal ganglion infection: correlation of enzyme activity with ganglion virus titer and evidence of in vivo complementation

Experimental trigeminal ganglion and corneal infection in mice was studied with three thymidine kinase-positive (TK⁺) strains of herpes simplex virus type 1 (HSV-1) and eight TK^? HSV-1 mutants. Viruses were extensively tested in cell culture to determine whether any were temperature sensitive (ts) for virus replication or for TK activity. TK^? mutants were no more ts than were TK⁺ viruses. By arabinosylthymine testing and measurement of thymidine phosphorylation, it was apparent that some TK^? mutants were, in fact, intermediate for TK activity (TK^±). After corneal inoculation of individual viruses it was observed with one exception that TK^?, TK^±, and TK⁺ viruses replicated in ocular tissues (3 days postinoculation, 2.2 × 10³–1.1 × 10⁵ PFU/eye). However, TK^? mutants were rarely isolated from trigeminal ganglia (<2.5–<5.6 × 10^?1 PFU/mg), whereas TK⁺ and to a lesser degree TK^± viruses were frequently (1.2 × 10³–1.2 × 10⁴ PFU/mg and 3.2 × 10⁰–2.1 × 10³ PFU/mg). In vivo complementation studies were performed by corneal inoculation of TK^??TK^? and TK^??TK^? virus mixtures. TK⁺ HSV complemented TK^? virus since significant amounts of TK^? HSV were isolated from trigeminal ganglia (complementation indices were >60–>1600). In addition, after inoculation of certain TK^?–TK^? pairs, complementation in ganglia was observed (complementation indices were >2.4–>120). These studies support the hypothesis that HSV-1 TK expression is necessary for sensory ganglion (neuron) infection in three ways: HSV-1 TK mutants that replicated in ocular tissues and were not ts mutants did not replicate in vivo in trigeminal ganglia; (ii) there was a correlation between level of viral TK activity and trigeminal ganglion virus titer; and (iii) when complemented by TK⁺ or TK^? HSV, TK^? virus replicated in trigeminal ganglia.     

Two Distinct Mechanisms of Cytotoxity Mediated by Herpes Simplex Virus-Specific CD4+Human Cytotoxic T Cell Clones

The present study was undertaken to elucidate the mechanisms responsible for the cytotoxicity of herpes simplex virus (HSV)-specific CDA4⁺human cytotoxic T lymphocyte (CTL) clones, focusing on perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-β). Two HSV-specific CDA4⁺CTL clones, which expressed both perforin and membrane-bound LT, exerted HSV-specific cytotoxicity and cytotoxicity against LT-sensitive L929 cells. These CDA4⁺CTL clones lysed HSV-infected cells directly in an HLA class II-restricted manner and did not exhibit "bystander killing." The culture supernatants of these clones stimulated with HSV antigen showed no cytotoxicity against HSV-infected cells or L929 cells, suggesting that adhesion to target cells is essentail to their antigen-specific and antigen-nonspecific cytotoxicities. The cytotoxicities of these clones against HSV-infected autologous cells were inhibited by an anti-CD3 monoclonal antibody but not by an anti-LT antibody. Conversely, their cytotoxicities against L929 cells appeared to be partially inhibited by the anti-LT antibody but not by the anti-CD3 monoclonal antibody. Furthermore, target cell DNA fragmentation induced by these CDA4⁺CTL clones was apparently observed in L929 cells but only faintly detected in HSV-infected autologous cells. L929 cell DNA fragmentation was also inhibited by adding the anti-LT antibody to CDA4⁺CTL cultures. these data suggest that some CDA4⁺CTL possess at least two cytolytic mediators, i.e., perforin and membrane-bound LT simultaneously, and can exert both antigen-specific and antigen-nonspecific cytotoxicity via two distinct mechanisms, necrosis and apoptosis.