Nancy J. Dooley

Activation of Suppressor T Cells during Epstein-Barr-Virus-Induced Infectious Mononucleosis

Infectious mononucleosis is caused by the Epstein-Barr virus (EBV), an unusual human pathogen because it preferentially infects B lymphocytes and consequently activates them to produce immunoglobulins. When cultures of lymphocytes from patients with infectious mononucleosis were stimulated with polyclonal activators, unseparated cells failed to produce immunoglobulins, whereas purified B cells responded normally. Cocultures demonstrated profound suppressor T-cell activity in blood from patients with infectious mononucleosis. Early in this disease, circulating immunoglobulin-secreting cells were elevated, but during the second week their number was strikingly depressed. These data indicate that during infectious mononucleosis, EBV causes polyclonal activation of B cells, reflected by hypergammaglobulinemia and increased circulating immunoglobulin-secreting cells. Next, suppressor T cells become activated and inhibit further B-cell activation. Thus, activation of suppressor T cells in infectious mononucleosis provides a unique additional mechanism of host defense because these T cells inhibit the activation and proliferation of an important target of the causative virus.

B Cell Differentiation and Immunoregulatory T Cell Function in Human Cord Blood Lymphocytes

The functional maturity of T and B lymphocyte populations from human newborns was evaluated using a reverse hemolytic plaque assay to detect immunoglobulin-secreting cells generated in in vitro cultures stimulated with pokeweed mitogen (PWM), a T cell-dependent polyclonal activator, and the Epstein-Barr virus (EBV), a T cell-independent B cell activator. Cord blood lymphocytes failed to produce immunoglobulin in response to PWM, but did respond with immunoglobulin synthesis to stimulation with EBV. Co-culture experiments demonstrated that cord blood T cells would inhibit immunoglobulin production by adult cells stimulated with PWM, but not with EBV. Cord blood T cells did suppress immunoglobulin production by cord blood B cells when stimulated with a mixture of EBV and PWM, indicating that cord blood, in contrast to adult blood, contains a population of suppressor T cell precursors that are easily activated by PWM. Irradiation of the cord blood T cells with 2,000 rad eliminated the suppressor activity and revealed normal helper function for immunoglobulin (Ig) G, A, and M when these T cells were co-cultured with adult B cells. Cord blood B cells co-cultured with adult T cells or irradiated cord blood T cells did produce immunoglobulin in response to PWM, but the response was significantly lower than that of adult B cells, and only IgM was produced in these cultures. These studies demonstrate that both the T and B cells of the human newborn have significant functional differences compared with the functions of T and B lymphocyte populations in adults.

Immunoglobulin production induced in vitro by glucocorticoid hormones: T cell-dependent stimulation of immunoglobulin production without B cell proliferation in cultures of human peripheral blood lymphocytes.

The direct effects of steroid hormones on the production of immunoglobulins and DNA synthesis by human T and B lymphocytes was evaluated in cultures of peripheral blood mononuclear cells. As detected by a reverse hemolytic plaque assay, the addition of 0.1 mM to 10 nM hydrocortisone to lymphocytes in culture in the absence of other stimulants or mitogens, resulted in the dramatic induction of immunoglobulin production with responses comparable to those seen in similar cultures stimulated with pokeweed mitogen. Steroid-stimulated immunoglobulin production was first seen after 48 h and peaked at 8-10 d of culture. The production of IgG, IgA, and IgM was induced following incubation with steroid. Glucocorticoids, but not estrogens or androgens, were capable of mediating this effect, and only compounds with affinity for the glucocorticoid receptor were active. The induction of immunoglobulin production was dependent on both T cells and monocytes; cultures depleted of either cell type did not produce immunoglobulin when stimulated with glucocorticoid hormones. Proliferation of B cells or T cells could not be detected by [3H]thymidine incorporation or total cell recovery from steroid-stimulated cultures, even though such cultures demonstrated marked increases in immunoglobulin production. The mechanism responsible for this functional maturation of B cells to become high rate immunoglobulin producing cells is as yet undefined, although it appears to involve more than merely steroid mediated inactivation of suppressor T cells.

Infectious Agammaglobulinemia: Suppressor T Cells with Specificity for Individual Immunoglobulin Classes

Immunoregulatory cells with potent suppressive effects on immunoglobulin or specific antibody production have been described in many experimental systems. In the chicken, bone marrow cells from adult birds rendered agammaglobulinemic by bursectomy and irradiation at hatching will induce agammaglobulinemia in recipients when transplanted into normal adult chickens of the same strain (1). This “infectious agammaglobulinemia” is induced by donor suppressor T cells which appear in the donor birds in detectable quantities by about 10 weeks of age. Suppressor cells can be demonstrated in spleen, thymus and peripheral blood in addition to bone marrow. The immunodeficiency which develops in the recipient chickens is characterized by their failure to produce specific antibody after challenge with any of several thymic dependent or thymic independent antigens, the rapid disappearance of existing serum IgG, IgA and IgM, the persistence of an intact bursa of Fabricius, and the persistence of a normal number of B lymphocytes in the spleen of these birds at least until after serum immunoglobulin has become undetectable (2, 3, 4).

Differential effects of ouabain on human cell-mediated cytotoxicity: I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets☆

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed 51Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10−4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.

Differential effects of ouabain on human cell-mediated cytotoxicity: II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets

Abstract We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10 −5 M . Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10 −9 M ) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.

IMMUNOGLOBULIN CLASS SPECIFIC SUPPRESSOR T CELLS

Publisher Summary This chapter describes a few studies demonstrating suppressor cells specific for each of the major immunoglobulin classes as revealed by limiting dilution transfer experiments. Inbred line 6, subline chickens were surgically bursectomized and given 550R whole body irradiation on the day after hatching. Agaranaglobulinemia develops in approximately 90% of the birds by 6 weeks of age. Suspensions of spleen, thymus, bone marrow, and peripheral blood mononuclear cells were obtained from these aγ birds at 4 months of age and were given in various dilutions intravenously to normal 2- or 20-week old chickens of the same strain. Serum immunoglobulin levels were measured in the recipient birds using radial diffusion in agar, and the presence of B lymphocytes in the spleens of these birds was measured by immunofluorescence with FITC conjugated rabbit antichicken immunoglobulin antisera. It was found that 20-week old recipient chickens had normal levels of IgG, IgA, and IgM prior to the injection of aγ lymphoid cells. Within 24 h of cell transfer, all classes of immunoglobulin began to disappear from the serum of these birds so that total agammglobulinemia was present by 45 days after transplant.