Immune interferon production by lymphoid cells: role in the inhibition of herpesviruses.

Bovine peripheral blood lymphocytes (PBL) obtained from infectious bovine rhinotracheitis (IBR) virus- and tuberculin-immunized animals produced large quantities of interferon within 24 h of in vitro stimulation by IBR and purified protein derivative antigens. Separation of PBL into populations enriched in T lymphocytes or B lymphocyte provided the antigen-specific step for immune interferon production. A 2- to 10-fold increase in interferon occurred when lymphocytes were combined with autologous macrophages. Although macrophages, even if treated with antilymphocyte serum to remove any contaminating lymphocytes, could produce some interferon, the augmented interferon produced by macrophage-lypmhocyte cultures was not dmpocytes. Direct physical contact between macrophages and lymphocytes was required for the production of enhanced levels of interferon. Antigen-antibody complexes of irradiated virus-infected cells in the presence of antibody were as efficient or better at stimulating interferon than was free antigen. Because IBR virus was inhibited by interferon levels stimulated in cultures by IBR antigen, it was suggested that the local production of interferon by immune cells might play a similar role in curtailing virus dissemination in vivo, thus leading to recovery from disease.     

Lymphocyte transformation and interferon production in human mononuclear cell microcultures for assay of cellular immunity to herpes simplex virus.

Interferon production and transformation in response to herpes simplex virus antigen were studied in microcultures of human mononuclear cells. Mononuclear cells consisting of monocytes and both T and B lymphocytes were purified by Ficoll-Hypaque gradients. Lymphocytes, predominantly T with 5% B, were obtained by passage of buffy-coat cells through nylon fiber columns. For some experiments, autochthonous macrophages and column-purified lymphocytes were stimulated with herpesvirus antigen. The effect of specific antibody and cell concentration on reactivity is described. Crude and purified antigens were compared as cell culture stimulants. Significant differences in transformation and interferon were observed between donors with a history of herpes labialis and donors with no detectable antibody, both in cultures prepared by Ficoll-Hypaque gradients and by column purification of lymphocytes. Cultures from seronegative donors prepared by Ficoll-Hypaque gradients produced interferon but did not transform when stimulated by herpes simplex antigen. "Immune" interferon production, that is, type II as opposed to type I, occurred only with autochthonous macrophage and column-purified lymphocyte cultures. Interferon produced by Ficoll-Hypaque-purified mononuclear cultures was type I, and its production was unrelated to immune status. Similarly, column-purified lymphocytes responded to herpes simplex virus antigen with type I interferon if obtained from a seropositive donor.     

Immune-specific production of gamma interferon in human lymphocyte cultures in response to mumps virus.

The production of interferon (IFN) was investigated in human peripheral lymphocyte cultures stimulated by mumps virus. IFN-alpha and IFN-gamma were produced in lymphocyte cultures from immune donors. However, no IFN-gamma was produced in lymphocyte cultures from nonimmune donors. IFN-alpha was produced by B lymphocytes, and IFN-gamma was produced by T lymphocytes. An increase in the production of IFN-gamma resulted from the mixture of autologous macrophages in T-lymphocyte cultures. The production of IFN-gamma by T lymphocytes was found to depend on prior, possibly cellular, immunity.     

Replication of rubella virus in human mononuclear blood cells.

Rubella virus was capable of replicating in both unstimulated and phytohemagglutinin-stimulated cultures of human mononuclear blood cells. Monocyte-derived macrophages were the main cell type responsible for viral replication. The susceptibility of macrophages increased during cultivation. Phytohemagglutinin-stimulated lymphocytes were able to support replication to a limited degree. No viral replication was detected in unstimulated lymphocytes. Both stimulation and viral replication in phytohemagglutinin-treated lymphocyte cultures were enhanced by the addition of murine macrophages. Human leukocyte interferon depressed the production of virus in these combined cultures. The finding that rubella virus is able to replicate in human lymphocytes as well as in macrophages may contribute to understanding the mechanisms of the suppressive effect of the virus on in vitro lymphocyte phytohemagglutinin responsiveness and in vivo immune functions.     

Production of gamma interferon in mice immune to Rickettsia tsutsugamushi.

C3H/He mice immunized by subcutaneous infection with Rickettsia tsutsugamushi Gilliam were examined for the production of immune interferon after intravenous administration of irradiated strain Gilliam antigen, in supernatants of immune lymphocytes stimulated with specific antigen, and after a secondary challenge with viable rickettsiae. Mice administered various doses of irradiated whole-organism antigen 28 days after immunization showed circulating levels of interferon which peaked 4 h after inoculation and were antigen dose dependent. The interferon produced was pH 2 sensitive and stable at 56 degrees C for 1 h and was neutralized by antiserum directed against immune, but not against alpha/beta, interferon. The production of another lymphokine, macrophage migration inhibition factor, paralleled that of interferon. The interferon produced by cultures of spleen cells obtained from immune animals was antigen specific and dose dependent. Peak levels were obtained 48 to 72 h after the addition of antigen. The interferon produced by spleen cell cultures after stimulation with Gilliam antigen was characterized as immune interferon by the same physical and antigenic criteria used for serum interferon. Interferon was produced in vitro by the Thy-1.2+ lymphocyte and required the presence of a spleen-adherent cell population. Immune mice produced high circulating levels of immune interferon after intraperitoneal challenge with viable rickettsiae, which suggested a possible role for interferon in the resistance of immune mice to rechallenge with R. tsutsugamushi.     

Chronic lymphocytic leukaemia. Studies on mitogen-stimulated lymphocyte interferon as a new technique for assessing T lymphocyte effector function

The present study employs a new technique for the study of human T-cell effector function in patients with chronic lymphocytic leukaemia: mitogen-stimulated interferon. Cultures of macrophages, T cell-enriched lymphocytes, or macrophages and lymphocytes combined were prepared from the blood of fourteen normal donors and five patients with chronic lymphocytic leukaemia. The effects of the mitogens, phytohaemagglutinin and pokeweed, on interferon production and lymphocyte transformation were studied and the following observations made: (a) T-cell effector and proliferative functions were depressed as evidenced by the absence of interferon and proliferative response to PHA and PWM at 3 days in vitro; (b) Three out of five patients showed no interferon or proliferative response at 6 days, thus indicating a B lymphocyte abnormality as well; (c) macrophages from both normal and leukaemic subjects augmented mitogen-stimulated lymphocyte interferon production and lymphocyte transformation. However, the addition of normal allogeneic macrophages to cultures of lymphocytes prepared from the patients did not restore the proliferative and interferon responses to normal levels.     

Breast milk lymphocyte response to K1 antigen of Escherichia coli.

Comparison milk and blood lymphocyte blastogenic responses to the K1 antigen of Escherichia coli and lipopolysaccharide (LPS) from E. coli O127,B8 were examined in 16 postpartum women by [3H]thymidine uptake. Rabbit hemolysincoated sheep erythrocyte monolayers were used to deplete macrophages from milk lymphocyte preparations and to enrich for T lymphocytes in order to make milk preparations more comparable to blood preparations. Response was defined as a stimulation index of greater than or equal to 2.0. There was no evidence of selective response to K1 antigen by milk lymphocytes, since both blood and milk lymphocytes responded in four women and neither blood nor milk lymphocytes responded in nine. Milk lymphocytes alone responded to K1 in one woman, whereas blood lymphocytes alone responded in two women. Additional nonpaired milk or blood cultures were available from three women. None of these responded to K1 antigen. Corresponding lymphocyte cultures were stimulated with LPS. A positive K1 response was always accompanied by an LPS response, and the LPS response correlated with the K1 response in 17 of 19 women. Stool cultures examined with an antiserum agar showed no correlation between the presence of K1 E. coli in the stool and milk or blood lymphocyte response to K1 antigen. In the system used here, no selectivity of response of breast milk lymphocytes to K1 antigen was noted.     

Hybridoma-derived human suppressor factors: differential effects on mouse lymphocytes

We have recently identified a family of suppressor factors produced by certain human T-T cell hybridomas that we developed (references 1 and 2) and by the Jurkat T cell line. These suppressor factors significantly inhibited proliferative responses to mitogens and allogeneic cells in mixed lymphocyte culture and antibody production by human peripheral blood mononuclear cells. We investigated and report here the effect of these suppressor factors on certain in vitro murine immune responses. Suppressor factors produced by certain of these hybrids, such as 153, 160, 170 and by the Jurkat T-cell line were able to inhibit: (1) proliferative responses to mitogens of mouse thymocytes and splenocytes; (2) proliferative responses of mouse splenocytes to allogeneic cells in mixed lymphocyte cultures; (3) primary in vitro antibody responses of mouse spleen lymphocytes to sheep erythrocytes; (4) primary in vitro antibody responses of mouse spleen lymphocytes to a T-cell independent antigen (TNP-Ficoll). Inhibition of murine immune responses in vitro by these suppressor factors was regular and reproducible and it was observed in a large number of experiments. In contrast, suppressor factors produced by the 169 and by the 77(38F3) hybrids did not suppress the murine immune responses. The basis for these differences are not known at the present. The ability of human suppressor factors to inhibit effectively mouse immune responses provides an additional opportunity for the characterization of the properties of these factors in vivo using mouse models of human disease.     

Immune interferon. I. Production by lymphokine-activated murine macrophages.

Unfractionated murine spleen cells produce immune interferon (type II) upon stimulation with antigen or mitogen. When spleen cells were passed over glass bead columns, interferon production decreased whereas the mitotic response to the stimulants drastically increased. When these cells were further purified over nylon wool columns, interferon production was totally abolished whereas thymidine incorporation in stimulated cultures was invariably high. Interferon production by nylon wool column-purified lymphocytes could be restored with macrophages grown from bone marrow cultures or spleen cells but not with macrophages from proteose peptone-induced peritoneal exudate cells. It was also found that pure macrophage cultures from spleens of BCG-immunized mice consistently produced interferon activity without any further stimulation. When culture supernatants of activated T lymphocytes, which did not contain any interferon activity, were transferred to macrophage cultures from different sources and incubated for 45 h, interferon activity could be detected in supernatants of macrophage cultures from bone marrow and spleen but not in those from proteose-induced peritoneal exudate cells. It is concluded that certain macrophage populations can be induced to produce interferon activity whereas others are refractory to this induction which appears to be linked to their differentiation state.     

Immunoregulatory Function of Human Cord Blood Lymphocytes on Immunoglobulin Production

ABSTRACT: The functional maturity of human umbilical cord blood B lymphocytes and the immunoregulatory activity of cord T lymphocytes were assessed by measuring the in vitro immunoglobulin production by B cells from either cord or adult blood. Supernatants from 48-hr pokeweed-mitogen (PWM) stimulated cord or adult lymphocyte cultures were added to cord or adult B cell cultures in the presence of PWM; a significant amount of immunoglobulin was produced in adult B cell cultures only. Adult B or T cells were then cocultured with cord T or B cells; a significant amount of immunoglobulin was again found only in adult B cell cultures. These results indicated that cord B cells were functionally immature and that cord helper T cell function was adequate but masked by excessive suppressor activity. Indeed, addition of cord T cells but not of allogeneic adult T cells to PWM stimulated adult lymphocyte cultures inhibited their immunoglobulin production; this confirmed cord T cells' increased suppressor activity. Cord T cells were not intrinsically suppressive since they failed to suppress immunoglobulin production by Epstein-Barr Virus (EBV) transformed B cells. They could be activated, however, by PWM or allogeneic cells (in mixed lymphocyte cultures) and their effect was mediated via soluble factor(s) as demonstrated by the suppressor effect of these culture supernatants on immunoglobulin production by unfractionated adult lymphocytes. In contrast, when these supernatants were added to T cell-depleted adult lymphyocyte cultures, enhancement rather than suppression was observed. These results indicated that the soluble factor(s) released by Cord T lymphocytes was not suppressing per se but induced suppression through activation of suppressor cells.     

Production of alpha- and gamma-interferons by human central lymph cells in different pathological states

The IF-producing capacity of human lymphocytes obtained by drainage of thoracic duct of surgical patients was studied. The lymph cells incubated with the appropriate inducer produced interferon of alpha and gamma types. The level of IF production, particularly of the gamma type, was negatively influenced by the storage of lymph at +4 degrees C and recovery of the cells in ficoll-hypaque gradient. The time course of IF production was found on the whole to be similar to that in the peripheral blood lymphocytes. The level of alpha-IF synthesis was higher for blood cells, and that of gamma-IF for lymph cells. The synthesizing capacity of the thoracic duct lymphocytes decreased in the acute stage of the disease and increased during convalescence which was especially typical of gamma-IF production.     

Enzymatic induction of interferon production by galactose oxidase treatment of human lymphoid cells.

Human lymphocyte cultures produced large amounts of interferon after treatment with the enzyme galactose oxidase. Interferon production was detectable as early as 3 h after enzymatic treatment and reached a level of about 10(4) reference units 20 to 24 h later. Galactose oxidase-induced interferon appeared to be immune interferon on the basis of acid lability, lack of neutralization by antibody to leukocyte interferon, and slow kinetics of activation of the cellular antiviral state. Interferon production was inhibited to the same extent (99%) by pretreatment of the cells with beta-galactosidase or with neuraminidase followed by beta-galactosidase, suggesting that the critical event for activation of interferon production is the oxidation of exposed galactose residues on lymphocyte membrane.     

Cells binding the antigen keyhole limpet haemocyanin in the peripheral blood and in the lymphocyte cultures of non-immune and immunized human subjects

Cells binding the antigen Keyhole limpet haemocyanin (KLH) were detected among human peripheral blood leucocytes and antigen-stimulated cultured lymphocytes. They were detected by their formation of rosettes with antigen-coated human O red blood cells. In the peripheral blood 0·13% of the lymphocytes of non-immune and 0·80% of the lymphocytes of immune subjects were antigen binding. In KLH-stimulated cultures of the lymphocytes of non-immune subjects there were 0·1% antigen-binding cells and in those of immune subjects there were 8·5% antigen-binding cells. Binding was specific. Thus, lymphocytes from phytohaemagglutinin- and streptolysin O-stimulated cultures did not bind KLH-coated red blood cells and did not form rosettes with KLH-stimulated lymphocytes. Incubation of KLH-stimulated lymphocytes with heterologous anti-immunoglobulin serum revealed that the KLH receptor was related to IgM. Addition of metabolic blocking agents (puromycin, cycloheximide, actinomycin-D and arabinosyl cytosine) to KLH-stimulated lymphocyte cultures revealed that the generation of antigen-binding cells required both DNA and protein synthesis. After primary immunization of normal human subjects, antigen-binding cells were detected in their appropriate lymphocyte cultures at 7 days and reached their maximum level at 21 days, in five of the six kinetically followed individuals and at 14 days in the other. This data represents the first report of the generation of antigen-binding cells in lymphocyte cultures demonstrated by the rosette method. These methods should be useful in the study of a variety of immunological problems in man.     

Cellular co-operation in the production of human leucocyte inhibitory factor by lymphocyte subpopulations stimulated with antigens and allogeneic cells.

The ability of antigens and allogeneic cells to induce lymphokine synthesis and cellular co-operation in lymphokine production was investigated. Human peripheral blood lymphocytes were separated into T- and B-cell populations by sheep red blood cell rosette formation and centrifugation on Ficoll--Isopaque. The cells were then stimulated with PPD, SK-SD, candida and with allogeneic cells. The presence of leucocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose migration method. The results indicated that T lymphocytes produced LIF after stimulation with antigens and allogeneic cells. In addition, B cells responded to PPD. Except for PPD the stimulants did not induce significant T-cell LIF production in the absence of monocytes. Only autologous monocytes enhanced LIF synthesis after antigenic stimulation, whereas in mixed lymphocyte cultures allogeneic monocytes were as effective as autologous ones. The monocyte helper effect was mediated by soluble factors in mixed lymphocyte cultures and by soluble factors and direct cell--cell contact in antigen-stimulated cultures. No co-operation between T and B lymphocytes was found. B cells did not enhance LIF production by T cells, nor could T cells induce a B-cell response to antigens or allogeneic cells.     

In vitro augmentation of natural killing activity by OK-432

OK-432, a streptococcal preparation, augmented the natural killing (NK) activity of peripheral blood lymphocytes of normal donors and cancer patients against both NK sensitive and resistant human target cells in vitro. The enhancement of NK activity was evident after 4 h pretreatment and maximum by 16-24 h. The manifestation of OK-432 induced augmentation required active cell metabolism, RNA and protein synthesis but no DNA synthesis of lymphocytes. The supernatants produced by OK-432 stimulated lymphocyte cultures had no enhancing substance nor interferon. Anti-interferon antibodies did not inhibit boosting activity of OK-432. Large granular lymphocytes were involved in both spontaneous and OK-432 induced cytotoxic activity. The proportion of lymphocytes conjugating to target cells was not changed by OK-432. These results suggest that OK-432 augments cytotoxic activity of large granular lymphocytes having ability to recognize target cells independent of interferon induction.     

Human peripheral null lymphocytes. II. Producers of type-1 interferon upon stimulation with tumor cells, Herpes simplex virus and Corynebacterium parvum

Human blood lymphocytes, exposed for 6 to 24 h in vitro to tumor cells (K 562, IGR3, L1210), Herpes simplex virus type 1 (HSV) or Corynebacterium parvum (CP), produced high levels of anti-viral activity which was identified as type-1 interferon (IF). In mixed lymphocyte tumor cell cultures (MLTC), the generated type-1 IF was definitely shown to originate from the lymphocytes and not from the tumor cells. Supplementation of leukocyte cultures with 10% fetal calf serum instead 10% human AB serum had little influence on tumor cell-induced IF production, but strongly reduced CP-induced IF production. Lymphocyte fractionation procedures involving iron/plastic treatment, nylon wool columns, Ig-anti-Ig columns and rosette (E, EA) separation led to the identification of null cells as highly efficient producers of type-1 IF. T cells obtained by different ways (E-rosette sedimentation, passage through 1 nylon and 2 Ig-anti-Ig columns, or thoracic duct lymphocytes) were poor IF producers in response to tumor cells, HSV and CP, but secreted anti-viral activity when stimulated with phytohemagglutinin. In MLTC, the level of generated type-1 IF roughly stimulated with phytohemagglutinin. In MLTC, the level of generated type-1 IF roughly paralleled nautral killer (NK) cell activity. Evidence is presented that type-1 IF can be produced by an Fc receptor-negative null cell subset, whereas NK activity requires Fc receptor-positive cells. It is suggested that production of type-1 IF represents one of the earliest functions in the differentiation process of mononuclear phagocytes and is likely to develop before the appearance of Fc receptors, diffuse esterase staining and latex phagocytosis.     

Alloresponses of cord blood cells in primary mixed lymphocyte cultures

The aim of this study was to compare the alloreactive responses against HLA antigens of cord blood cells with those of adult peripheral blood cells. In primary mixed lymphocyte cultures and bulk cell-mediated lympholysis experiments cord blood cells demonstrated significantly decreased proliferation and cytotoxicity. Experiments analyzing the specificity of anti-HLA cytotoxic T lymphocytes (CTL) revealed that cord blood (CB) CTL reacted only partially with third-party cells expressing the stimulating HLA antigens. Lower frequencies of IL-2 producing helper, cytotoxic T-cell precursors and IL-4 producing CB cells were found, whereas the frequencies of IFN-gamma producing cells, as determined by ELISpot experiments, were equivalent to the frequencies of adult IFN-gamma producing cells. Our results imply that, although CB cells have significantly decreased proliferative and cytotoxic alloresponses in bulk mixed lymphocyte cultures, their IFN-gamma production is comparable with that of adult mononuclear cells. Preserved production of IFN-gamma may be a risk factor for the development of graft-versus-host disease and should be taken into consideration when evaluating the possibility for stem cell transplantation with HLA-mismatched CB.     

Ability of human polymorhonuclear blood cells to produce interferon after induction with phage double-stranded RNA.

The ability of human peripheral blood leukocytes to produce interferon in response to phage double-stranded (ds) RNA was studied. Under the conditions used, interferon was produced not only by lymphocytes but also by polymorphs and monocytes. These "pure" cultures showed no marked differences in the degree of interferon production as compared with the mixed leukocyte culture. The involvement of polymorphs in the production of interferon induced by phage ds RNA is discussed.     

Immune depression in trypanosome-infected mice. I. Depressed T lymphocyte responses.

Using a wide range of experimental conditions, several kinds of T lymphocyte responses in spleen cell populations from trypanosome-infected mice were studied. Lymphocyte stimulation after culture with the mitogen concanavalin A or with histoincompatible cells differing at H-2 or minor lymphocyte-stimulating loci was reduced or abolished in spleen cells from infected mice when compared with responses of spleen cells from uninfected controls. In addition, cytotoxic lymphocytes were not generated in mixed lymphocyte cultures which contained spleen cells from infected animals. Allogeneic skin grafting experiments performed with normal and infected mice showed that a decreased T lymphocyte response also occurs in vivo. The depressed immune responses were not simply due to low numbers of T lymphocytes in spleens of infected animals, but reflected a generalized immune depression which was not antigen-specific.     

Impaired autologous mixed lymphocyte reactivity in Hodgkin's disease

Summary In patients with Hodkin's disease, the impaired immune reactivity, especially of the thymus dependent system, is well established. This decreased immune response of the lymphocytes from the peripheral blood contrasts to an increased lymphocytopoiesis in the lymphatic organs with a hyperplasia of these tissues. We studied the reactivity of peripheral T lymphocytes from 20 patients with Hodgkin's disease and 26 healthy control persons against autologous and allogeneic non T cells respectively in the mixed lymphocyte culture (MLC). Our experiments show an extremely depressed autologous mixed lymphocyte reactivity (MLR) of T lymphocytes from patients with Hodgkin's disease compared to those from normal donors. In the allogeneic MLC, the proliferation of the patients' T cells was stronger than in the autologous MLC, but significantly lower than the proliferation of normal T lymphocytes when stimulated by normal non T cells. Patients' non T cells stimulated T lymphocytes from healthy donors as well as non T lymphocytes from normals did. Finally, the autologous MLR of normal lymphocytes was significantly suppressed by 18 of 23 sera from Hodgkin's patients when these sera were substituted for normal AB serum in the cultures. These results demonstrate an impaired function of T lymphocytes from patients with Hodgkin's disease in the autologous MLC and the presence of one or more factors in their serum which inhibit the proliferation of normal lymphocytes in the autologous MLC. The role of suppressor cells and their factors will be discussed.Supported by the Deutsche Forschungsgemeinschaft