Cytotoxic activity against rubella-infected cells in the supernatants of human lymphocyte cultures stimulated by rubella virus.

Supernatant fluids of lymphocyte cultures from rubella-seropositive donors, stimulated with inactivated rubella virus, showed cytotoxic activity against rubella-infected target cells (NYU 32 line of human embryonic fibroblasts) but not against uninfected fibroblasts. The time of appearance of cytotoxic activity in rubella-stimulated lymphocyte cultures correlated with increased rate of DNA synthesis as measured by thymidine uptake. No such cytotoxic activity became detectable in the supernatants of lymphocyte cultures from rubella-seronegative donors cultured in the presence of rubella virus, or in unstimulated lymphocyte cultures from seropositive or seronegative donors. The cytotoxic activity was lost at 60degreesC in 30 min. In contrast to this rubella virus-induced cytotoxic activity, cytotoxin produced in mitogen-stimulated lymphocyte cultures from rubella seropositive and seronegative donors was equally cytocidal against rubella-infected and uninfected human fibroblasts. Although the nature of cytotoxic activity remains to be characterized, it is suggested that it is associated with a lymphokine released immune-specifically from rubella virus-stimulated lymphocytes.     

Cell-mediated immunity to Herpes simplex in humans: lymphocyte cytotoxicity measured by 51-Cr release from infected cells.

We assessed cell-mediated immunity to herpes simplex virus type 1 antigen in patients suffering from recurrent cold sores and in a series of healthy controls. Paradoxically, all those subject to recurrent herpetic infections had, without exception, evidence of cell-mediated immunity to herpes antigens. This was demonstrated by lymphocyte transformation and specific 51Cr release from infected human amnion cells after incubation with peripheal blood mononuclear cells. Where performed, skin tests with herpes antigen were also positive. In addition, serum from these patients specifically sensitized herpesvirus-infected cells to killing by nonimmune, control mononuclear cells. These tests were negative in the control patients except in a few cases, and it is suggested that these latter may be the asymptomatic herpesvirus carriers previously recognized or that they may have experienced a genital infection.     

Cytokine production in varicella zoster virus-stimulated limiting dilution lymphocyte cultures.

Human blood lymphocytes were stimulated with varicella zoster virus (VZV) antigen in limiting dilution cultures and the amounts of interferon-gamma (IFN-gamma) and IL-4 measured in the supernatants. The results indicate that up to 85% of proliferating cells of young adults produce IFN-gamma and up to 10% make IL-4. At limiting dilution, few if any wells were positive for both IFN-gamma and IL-4. The amount of IFN-gamma per well increased in the presence of antibody to IL-4, but anti-IFN-gamma not increase IL-4 production. The frequency of wells containing IFN-gamma was lower in subjects < 19 or > 55 years of age, and the amounts of IFN-gamma in positive wells was significantly lower in cultures of the older subjects' lymphocytes. The frequency of IL-4-making cells did not fall significantly with age. The data suggest that the age-related decline in the frequency of blood T cells which responds to VZV affects mainly the cells with a Th1-like cytokine phenotype.     

Cell-mediated immunity assayed by 51Cr release test in mice infected with mouse adenovirus.

Immune spleen cells (ISC) from mice immunized with a sublethal dose of mouse adenovirus (M-Ad) were shown by the 51Cr release test to be cytotoxic to target mouse embryonic cells or lymphoid cells infected with M-Ad. The number of ISC required for release of statistically significant amounts of 51Cr from target cells varied from one sample to another, ranging from 5 to greater than or equal to 30 ISC per target cell. When 24-h-infected mouse embryonic cells were used as targets, the release of 51Cr became evident in 6 h after the addition of ISC to the cells, gradually increased with time, and then leveled off. Cytolytic activity of M-Ad ISC is specific for M-Ad, since ISC do not lyse mouse embryonic cells infected with human adenovirus type 12 and vice versa. Kinetic study of the development of cell-mediated immunity to M-Ad assayed by 51Cr release showed that cytolytic activity of ISC in infected mice became detectable 4 days postinfection, reached its peak level about 7 to 10 days postinfection, and fell to undetectable levels about 10 days thereafter. This is consistent with the data obtained by inhibition of intracellular viral antigen synthesis or by the macrophage migration inhibition test in our prevous reports.     

Cell-mediated immunity in periodontal disease; cytotoxicity, migration inhibition and lymphocyte transformation studies

Cultures of peripheral blood lymphocytes from patients with gingivitis or mild periodontitis were stimulated with ultrasonicates of Veillonella alcalescens to yield an increase of ¹⁴C-thymidine incorporation and of cytotoxicity against ⁵¹Cr-labelled chicken red cells. The cell-free supernatant from the antigen-stimulated cultures inhibited migration of guinea-pig peritoneal macrophages. In patients with severe periodontitis lymphocytes showed only weak transformation but strong cytotoxic and migration-inhibitory activity. These data suggest that cell-mediated immunity against an oral Gram-negative micro-organism plays some part in the pathogenesis of periodontal disease. The advanced stage of disease, however, revealed a dissociation between lymphocyte transformation and cytotoxicity or macrophage inhibition.     

Cell-mediated anti-embryo cytotoxicity in human pregnancy.

The microcytotoxicity assay has been used to study human maternal cell-mediated immunity against foetal lung cells derived from foetal autopsies and amnion cells from full-term deliveries. Cytotoxicity against semi-allogeneic foetal lung cells but not against adult skin fibroblasts was detected in fourteen cases. No cytotoxicity was detected in six mothers tested during the first 15 weeks of gestation whereas 58% of mothers in the 15-17 weeks of gestation (twelve cases) and 88% with more advanced pregnancies (eight cases) were specifically cytotoxic. Cross-reactions against allogeneic foetal lung cells were mainly produced by effector cells from patients with 16-17-week-old pregnancies. No cytotoxicity was detected against semi-allogeneic amnion cells by effector cells from mothers after full-term deliveries. Cytotoxicity was blocked by autologous post-abortum sera in two out of seven cases. The effect of autologous post-abortum sera on non-cytotoxic effector cells was nonspecifically arming in four out of ten cases.     

Cell-mediated immunity in human cytomegalovirus infection.

The direct leukocyte migration inhibition test, in response to cytomegalovirus stimulation, was used to study cell-mediated immunity in a group of children with cytomegalovirus infection. The test was impaired in children with chronic disease associated with cytomegaloviruria. In those cases with no viruria at the moment of the test, leukocyte migration inhibition was normal. Our data suggest that the acquired chronic cytomegalovirus infection may be sustained by a state of specific cellular desensitization, as already demonstrated for congenital infection.     

Detection of long-term cellular immunity to Coxiella burneti as assayed by lymphocyte transformation.

Delayed hypersensitivity to the antigens of Coxiella burneti, Nine Mile strain, was demonstrated in human subjects with various past histories of exposure to the organism by using lymphocyte transformation assays. Individuals with histories indicating exposure to C. burneti up to 8 years before the study demonstrated marked lymphocyte transformation in vitro to whole-cell antigens consisting of formalin-killed C. burneti phase I and phase II. These individuals also demonstrated a marked lymphocyte response to the trichloracetic acid-soluable phase I antigen. One individual who acquired Q fever during the study and one individual who received an experimental Q fever vaccine 4 years earlier were also evaluated by the lymphocyte transformation assay. It was also found that phase I trichloroacetic acid-soluble material was capable of acting as an antigen in the assay, whereas the phase II trichloroacetic acid-soluble material did not contain any antigenic material capable of causing lymphocyte transformation. The complete phase I trichloroacetic acid-soluble antigen, which was found to consist of protein and carbohydrate, was chemically fractionated into monospecific fractions. The fraction treated to eliminate carbohydrate was the only fraction found to elicit an in vitro response.     

Cell-mediated immunity to Chlamydia pneumoniae measured as lymphocyte blast transformation in vitro.

The purpose of the present study was to analyze Chlamydia pneumoniae-induced, antigen-specific, cell-mediated immunity. Peripheral blood mononuclear cells of four persons infected with C. pneumoniae Kajaani 6 and 17 healthy volunteers were stimulated with antigen composed of whole elementary bodies of C. pneumoniae Kajaani 6 (CP-Ag). Definitive antigen-specific lymphoproliferation (LP) responses were developed after recent infection. The LP responses of healthy people to CP-Ag varied considerably. There was no clear correlation between LP responses to CP-Ag and those to an antigen prepared from Chlamydia trachomatis serotype L2 (r > or = 0.50, P < 0.1). A larger study is required to demonstrate whether the LP responses to CP-Ag can be used for the diagnosis of C. pneumoniae infection.     

Low level rubella immunity detected by ELISA and specific lymphocyte transformation

Summary The level of humoral and cell mediated immunity in persons with low or undetectable hemagglutination-inhibition (HAI) rubella titers was investigated by ELISA, IgG presence in sucrose gradient separated serum fractions and lymphocyte transformation. The study population consisted of persons with stated history of natural rubella infection and rubella vaccinees. Persons with natural rubella infection and HAI titers of 1:8 or ±1:8 (i.e., incomplete HAI at serum dilution of 1:8) were all ELISA positive and the stimulation index (SI) of specific lymphocyte transformation was higher than 2.5. Among the 20 persons with HAI titers of <1:8, 8 were found to be ELISA positive and their SI was also >2 and IgG was detected in their serum. Rubella vaccinees with HAI titers of 1:8 or ±1:8 were likewise ELISA positive. Their SI was lower: none higher than 3, but none lower than 1.5. Among 23 HAI negative vaccinees, 14 were found ELISA positive. This serum fraction contained IgG and the SI was >1.5. It appears that ELISA test is able to detect antibodies where the HAI test fails. The positive outcome of ELISA test in this case was confirmed by the presence of IgG in serum fractions and by the lymphocyte response to rubella specific stimulation.With 1 Figure     

Cell-mediated cytotoxicity in recovery from poxvirus infections

The availability of mutant and gene targeted knockout mice with defects in components of cellular cytotoxicity mediated by either the Fas or the exocytosis pathway permitted an analysis of their role in recovery from poxvirus infections. Ectromelia (EV), a natural mouse pathogen causing mousepox, the closely related orthopoxviruses cow pox (CPV) and vaccinia virus (VV), each encode serpins that inhibit Fas mediated apoptosis and lysis of target cells. Nevertheless, distinct differences were seen when the three viruses were inoculated into perforin-deficient mice: highly resistant C57Bl/6 mice became susceptible to low doses of EV; resistance to CPV increased whereas there was no effect on VV infections. Absence of the cytolytic granule associated granzymes (gzm) A and B rendered C57Bl/6 mice increasingly more susceptible to EV infections. Lack of both gzms rendered them as susceptible as perforin deficient mice, despite the presence of functionally active perforin. Elevated EV titres in liver and spleen of gzmA x B deficient mice, early after infection and before cytotoxic T cells were detectable, strongly suggests that these two gzms exert an antiviral effect by a mechanism distinct from effector molecules of NK and cytotoxic T cells.     

Histamine-releasing activity of lymphocyte supernatants of guinea pig spleen cell cultures

We demonstrated the production of a histamine releasing factor (HRF) by 24-h cultures of guinea pig spleen cells which were stimulated or not with specific antigen (ovalbumin, OA) or mitogen (phytohemagglutinins or concanavalin A). HRF induced the release of histamine from homologous mesenteric mast cells in a dose-dependent fashion. The HRF-induced histamine release was not high compared to the release induced by calcium ionophore A23187, but higher than that induced by compound 48/80, polymyxin B and con canavalin A. The mast cells from sensitized guinea pigs released histamine when challenged with OA. We found that HRF-induced histamine release was additive to that induced by antigen, when both agents were added simultaneously to sensitized mast cells. The phenomenon was most significant when a suboptimal dose of antigen was used. Moreover, we did not observe any differences in the magnitude of HRF-induced histamine release between the mast cells from nonsensitized and sensitized guinea pigs. The time course of histamine release induced by HRF was significantly slower than that with specific antigen (10 min and 45 sec, respectively). Our results may suggest that HRF acts on mast cells through a different not immunological mechanism.     

Cell-mediated immunity in anorexia nervosa.

Twelve patients with anorexia nervosa were studied for cell-mediated immunity in terms of delayed hypersensitivity reactions to recall antigens, lymphocyte transformation responses to T-cell mitogens, and numbers of circulating leucocytes and T-cell subpopulations. Compared to controls, all patients had reduced cutaneous reactions and four were anergic. There was a mild leucopenia in patients and both T4+ and T3+ numbers were slightly reduced. Mean peak transformation responses for patients were slightly lower than controls for phytohaemagglutinin, but not for concanavalin A; however, patients required greater doses of mitogens to elicit peak transformation responses. Plasmas from patients did not contain inhibitors of transformation responses. We conclude that there are functional cellular abnormalities associated with the under-nutrition of anorexia nervosa.     

Suppression of polyclonal B cell proliferation mediated by supernatants from human myeloma bone marrow cell cultures.

For functional characterization and semi-quantitative estimation of soluble regulator factors influencing polyclonal B cell proliferation and differentiation, we established two assays. One of the assays measures enhancement or inhibition of proliferation from purified human spleen B lymphocytes, and the other one the effect of soluble factors on CESS-cell differentiation. We found no difference concerning regulator factors for B cell differentiation between bone marrow cell culture supernatants from multiple myeloma (MM) patients and from controls, whereas significantly higher suppressor activity on polyclonal B cell proliferation could be detected in the former group of supernatants. The extent of such determined suppressor activity in vitro correlated with the amount of polyclonal serum IgM of the corresponding patients. These results indicate that one or several soluble suppressor factors may be involved in immunoregulatory mechanisms responsible for the humoral immunodeficiency observed in MM patients.     

Activation of human lymphocytes by supernatants from human thymic epithelium.

Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS.     

Cell-mediated immunity to varicella-zoster virus demonstrated by viral inactivation with human leukocytes.

Cell-mediated immunity to varicella-zoster (V-Z) virus in persons immune to varicella has been demonstrated, using a tissue culture technique. Cell-mediated immunity was reflected by the ability of peripheral leukocytes (lymphocytes and monocytes) from human donors to inactivate V-Z virus. Leukocytes were stimulated by the addition of noninfectious V-Z antigen to cultures newly infected with V-Z virus. Several days leter, the V-Z virus in these cultures was titered. When leukocytes from donors immune to caricella were used, a significant decrease in V-Z titer, compared with controls, was noted. When leukocytes from donors susceptible to varicella were tested, no decrease in V-Z virus titer was found. A mixed population of lymphocytes and monocytes from immune donors was required to demonstrate inactivation of V-Z virus. The development of specific cell-mediated immunity to V-Z virus may play a role in termination of varicella and in prevention of second attacks of this disease.     

Cell-mediated immunity in human herpesvirus infection: analysis by monoclonal antibodies

Peripheral blood lymphocytes (PBL) were obtained from heterosexual patients during the first or recurrent episodes of genital herpes simplex type 2 (HSV-2) infections. These cells were incubated with a panel of lymphocyte-specific monoclonal antibodies and examined by indirect immunofluorescence and flow cytometry. Lymphocyte proliferative responses to inactivated HSV-2 (UV-HSV-2) were also determined on repeat occasions using PBL obtained from patients with first episodes of infection. Flow cytometry analysis indicated that PBL obtained within 1 week after the onset of symptoms of first episode genital herpes exhibited significantly lower OKT 4 helper/OKT 8 suppressor T-cell ratios (1.26 +/- 0.12) than cells from either normal controls (2.44 +/- 0.3, P less than 0.01) or patients with recurrent genital HSV-2 infection (2.58 +/- 0.40, P less than 0.01). This lowered ratio was due to a decrease in OKT 4-positive helper T cells present early during initial infection (30.2 +/- 2.9, P less than 0.01 compared to recurrent disease patients or controls) and was not caused by a concomitant increase in OKT 8-positive T cells. No variation was observed during the course of infection in the proportion of total T cells as determined by EAET rosette formation or reactivity with the T-cell specific monoclonal antibody 9.6. PBL obtained from patients within 1 week after symptom onset exhibited a poor proliferative response to UV-HSV-2. Maximal proliferative responses occurred 6-9 weeks after disease onset and required the presence of OKT 4-positive T lymphocytes. The results of these experiments indicate that at different stages of disease, HSV infection is accompanied by fluctuations in the ratio of helper to suppressor T cells and alterations in antigen-specific lymphocyte proliferation. These findings suggest that variations in lymphocyte proliferative responses during the course of first episode genital herpes infection may reflect disease related variations in either the number or activity of circulating OKT 4-positive T lymphocytes.     

Effect of supernatants from PHA-stimulated and non-stimulated lymphocyte cultures on thromboxane B2 release by polymorphonuclear leukocytesin vitro

The present study involves the determination of the effect of supernatants obtained from mitogen-induced lymphocytes of human tonsils and containing lymphotoxins on human peripheral blood polymorphonuclear leukocytes (PMNs). PMNs treated with supernatants from phytohemagglutinin-stimulated lymphocytes were found to increase the release of thromboxane B₂, compared to normal control levels. This increase was blocked by the preincubation of PMNs with indomethacin. The production or increase in production of thromboxanes may be induced by lymphotoxins since these substances have been reported to activate phospholipase A₂ in target cell membranes, which may result in release of the thromboxane precursor, arachidonic acid.