Plaque-forming Cell Assay Research Articles

Anti-immunoglobulin antibodies VI. Age-dependent isotype and auto anti-immunoglobulin variation during secondary immune response in 129 mice

The secondary and tertiary immune responses to TNP-KLH and the autoanti-immunoglobulin response were studied in 1-, 3- and 6-month-old 129/J and 129/Sv mice. A profound inability of aging 129/Sv mice (lifespan: 9 months) to produce γ2a anti-TNP antibodies relative to their normal counterparts was observed. However, this markedly low response could not be related to clearance of antibody by RFs since all 129 mice studied produced significant amounts of RFs after immunization with this T-dependent antigen. Furthermore, the results of the plaque forming cell assays indicated that young (1 month old) 129/Sv mice have an accelerated immune response while older mice produce significantly low direct and indirect responses compared to the control groups. The data suggest a fundamental dysfunction of B and/or T cells in 129/Sv mice and that spontaneous production of RFs in older mice may affect the ability of B and T cells to cooperate in a secondary response.

Impairment of T immunoregulatory activities in the induction of antibody specific response in aged humans

T helper (Th) and T suppressor (Ts) functions on the induction of specific antibody response have been studied in 80 aged individuals by means of a plaque-forming cell assay. Of the subjects 45.2% exhibited a reduction of Ts activity on Ig production by adding Concanavalin A (Con A) to cultures on day 0, while 35.7% of aged donors showed a decrease of Th functions by supplementation of Con A on day 2. A small number of individuals displayed a combined deficit (Th + Ts). Furthermore, these defects seem to be related to soluble suppressive factors which might adhere to cell surface. In fact, preincubation of peripheral blood mononuclear cells (PBMC) before their addition to cultures and resuspension in fresh medium normalized the immunoregulatory defects. On the other hand, overnight supernatants from old PBMC transferred to young PBMC cultures induced the same deficit observed in the aged cell suspensions. Finally, Zinc chloride supplementation to cultures was able to correct the deficient Th activity only. These data suggest an additional defect of immunoregulation in the elderly.

Sézary syndrome: phenotypic and functional characterization of the neoplastic cells.

Phenotypic properties of the neoplastic cells in skin, blood and lymph node specimens from 5 patients with the Sézary syndrome were examined by immuno-enzymatic and -fluorescence labelling of cells and tissue sections with a monoclonal antibody panel. In 3 cases, the in vitro functional properties of the neoplastic cells (isolated from blood specimens) were also analysed using a reverse plaque-forming cell assay. 3 different immunological categories were identified as follows: T-helper/inducer neoplasms (3 patients); T-suppressor/cytotoxic neoplasms (1 patient); and neoplastic T-cells demonstrating characteristics consistent with a concept of their derivation from inducible suppressor T-cells (1 patient). These data provide conclusive evidence that Sézary syndrome is heterogeneous with respect to the immunological characteristics of the neoplastic cells.

Suppression of primary antibody response by a single exposure to cadmium in mice

Suppression of primary antibody response to sheep red blood cells (SRBC) by a single intraperitoneal (i.p.) injection of cadmium into mice was investigated by the methods of in vitro plaque-forming cell (PFC) assay. BALB/c mice were given 1.8 mg cadmium kg body weight, and 1, 2 or 7 days later, spleen cells from exposed and control mice were cultured with SRBC. PFC responses of all exposed groups were significantly suppressed compared to those of control groups. Addition of control adherent cells to spleen cells from exposed mice failed to recover control level. In the cell-reconstitution experiments, the activity of B-cell function from the exposed group was suppressed more by cadmium than that of T-cell function. These results suggest that the suppression of primary PFC response by cadmium exposure may be caused by the inactivation of B-cells.

Effect of 2,5-hexanediol on immunocompetence of mice

A preliminary study on immunotoxicologic evaluation of 2,5-hexanediol (one of the principal metabolites of n-hexane), involving multiple immunological parameters, was carried out in mice. Mice were exposed to 2,5-hexanediol at a 1 5 LD 50 dose level for 7 days. Pathotoxicological changes such as marked reduction in absolute and relative lymphoid organ weights, histological abnormalities in thymus, spleen, and adrenal, and reduction in cellularity of lymphoid organs were found. Immune function tests such as delayed hypersensitivity reactions, plaque-forming cell assays, serum antibody titer against SRBC, and resistance to endotoxin shock were also markedly impaired. Results obtained in this study showed that 2,5-hexanediol causes impairment to immunocompetence in mice.

Effect of thyroidectomy and thyroxine on 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced immunotoxicity

Radiothyroidectomy protected against 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD_-induced immunotoxicity in rats as assessed by the spleen anti-SRBC plaque-forming cell assay. Thyroxine (T 4) replacement therapy partially reversed the effects of thyroidectomy on T 4 and triiodothyronine (T 3) serum levels, body weight and immune function as well as restored TCDD-induced immunotoxicity. Thus, hypothyroidism induced by TCDD exposure can be viewed as a protective response of the organism to reduce the insult caused by TCDD.

Glucocorticoids Enhance the in Vitro Ig Synthesis of Pokeweed Mitogen‐Stimulated Human B Cells by Inhibiting the Suppressive Effect of T8+ T Cells

The in vitro effects of three potent glucocorticoids (GC) (dexamethasone, prednisolone, cortisone) on human lymphocyte functions were investigated. In pharmacological concentrations GC strongly suppressed lymphocyte transformation induced by T-cell mitogen (concanavalin A and phytohaemagglutinin). After pokeweed mitogen (PWM) stimulation GC enhanced the number of immunoglobulin (Ig)-producing cells without affecting the proliferative response. Mineralocorticoid aldosterone showed no effect. Addition of 3 X 10(-8)-3 X 10(-6) mol/l of different GC to PWM cultures significantly increased the number of Ig-secreting cells, measured by the plaque-forming cell assay. Experiments conducted with fractionated defined lymphocyte subpopulations showed that the T8+, a radiosensitive T suppressor cell, is more sensitive than the T4+ T helper cell to GC effects. It is concluded that GC in pharmacological concentrations display a dual effect on human lymphocyte functions in vitro: an inhibition of lectin-induced T lymphocyte proliferation and a rather selective inhibition of T suppressor cell function which leads to an enhanced B-cell maturation and Ig synthesis in PWM-stimulated cultures. No measurable direct effect on the B lymphocytes was noticed.

Plaque-forming cell capability in the senescent

The number of plaque-forming cells (PFC) developed in pokeweed mitogen (PWM)-activated unfractionated or T/B separated, 4:1 reconstituted cultures of peripheral blood lymphocytes (PBL) with well-characterized subpopulations obtained from healthy, aged subjects was compared to that of young blood donors. The absolute number of PBL in the aged was reduced by 36%, and the percentage of sheep erythrocyte-rosette-forming cells (E-RFC) by 27%, compared to the percentage obtained in young donors. The IgM-, IgG- and IgA-immunoglobulin (Ig) secretion was monitored with a protein A PFC assay. The number of PFC in PBL cultures of the aged was 58% of the number found in cultures of the young controls. The number of PFC generated in cultures of autologous irradiated T and untreated B cells showed a 104% increase in the aged whereas a 63% increase was obtained using cells from young individuals. Co-cultures of young B cells with untreated or irradiated young or aged T cells showed a significant rise in the PFC response in cultures with irradiated aged T cells, while an equal number of PFC was generated in cultures of young B cells with young or aged untreated T cells. Our results demonstrate a decreased number of PBL, especially T cells, an impaired B cell function and a pronounced enhancement of the PFC response in cultures of irradiated aged T cells and young or aged B cells, whereas the T helper function of untreated cells was found to be normal. The influence of monocytes on the PFC response did not differ in the two groups.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity

The selective toxicity of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) for the thymus, consisting primarily of immature T-cells, led us to search for an analogous selective toxicity for the immature B-lymphocytes in the bone marrow. In the dose-response study C57B1/6 male mice were injected with either vehicle alone (corn oil), 30, 60, or 120 micrograms/kg of TCDD i.p. The mice were killed by cervical dislocation 7 days later. In the time-response study, mice were injected with either saline or 120 micrograms/kg i.p. TCDD, 3, 7, 14, or 21 days before killing. In both studies, the following were analyzed: change in body weight, thymus weight, spleen and bone marrow cellularity, and spleen and marrow B-lymphocyte function, measured using the in vitro B-lymphocyte colony forming unit in culture assay, with the mitogen lipopolysaccharide (LPS) from Salmonella typhosa, and the in vitro plaque forming cell assay, with the thymus independent antigen, TNP-LPS. In the dose-response study there was a reduction in thymic weight, spleen B-cell functional response (per spleen), and bone marrow B-cell functional response to 14%, 35-54%, and 20-32% of control, respectively, at a dosage of 120 micrograms/kg. In the time-response study, thymic weight and bone marrow B-cell functional response (per femur) were reduced to 6% and 18% of control, respectively, at day 21. The results indicate that TCDD was selectively more toxic to the immature B-cells in the bone marrow than the more mature B-cells in the spleen. This immunotoxicity was dose-dependent.

Enumeration of (auto)antibody producing cells in human using the “spot-ELISA”

An enzyme-linked immunosorbent assay (spot-ELISA) for individual immunoglobulin secreting cells, which became recently available, was applied to the enumeration of human B lymphocytes secreting specific antibodies of thyroglobulin. Polyclonally activated B cells from patients with auto-immune thyroid disease are incubated in thyroglobulin coated plates. After removal of the cells specific antibodies are visualized by means of an immunoenzyme technique employing agarose to localize converted substrate. Individual specific antibody secreting cells are counted as blue spots using an inverted microscope. Numbers and isotype of spots correlate well with the amount and isotype of secreted antibody as detected with a conventional ELISA. This easy to perform, complement-independent technique offers a useful alternative to conventional plaque forming cell assays.

Effects of tick infestation on the plaque-forming cell response to a thymic dependent antigen

Strain-2 guinea-pigs were given two five-day infestations with Dermacentor andersoni larvae. Each exposure consisted of 100 larvae, and the first and second infestations were separated by a seven-day tick-free period. Tick-exposed animals were given an intravenous injection with sheep red blood cells (SRBC) at selected times during and after infestation: (a) the last, fifth day, of a first exposure, (b) the second day of a second infestation, (c) the fifth day of a second infestation and (d) four days after termination of the second infestation. Ability of these animals to respond immunologically to the SRBC injection was assessed by the direct haemolytic plaque-forming cell assay, a very sensitive test used to determine the number of spleen cells producing IgM to SRBC target cells. Strain-2 animals given SRBC at the end of an initial infestation, or during a second tick exposure, produced significantly fewer direct haemolytic plaque-forming cells than did uninfested controls given a similar SRBC immunization regimen. Spleen cells of animals administered SRBC on the fourth day after termination of a second infestation displayed a haemolytic plaque-forming cell response which did not differ significantly from that of uninfested controls.

Hidden autoantibodies against common serum proteins in murine systemic lupus erythematosus. Detection by in vitro plaque-forming cell assay

Hidden autoantibodies against common serum proteins in murine systemic lupus erythematosus. Detection by in vitro plaque-forming cell assay

Helper T cell requirements for T15 idiotype expression on phosphorylcholine-specific antibodies.

The requirement for idiotype-specific T cells was investigated in the T15 idiotype-dominant T cell-dependent response of unprimed BALB/c and (BALB/c X C57BL/6)F1 B cells to phosphorylcholine (PC). It was first demonstrated that cloned keyhole limpet hemocyanin (KLH)-specific, major histocompatibility complex (MHC)-restricted T helper (Th) cells as well as heterogeneous KLH-primed Th populations were capable of generating PC-specific antibody responses in T-depleted unprimed B cell populations cultured in the presence of PC-KLH. The PC-binding antibody responses generated under these conditions were indistinguishable when assayed for carrier-hapten linkage requirements, immunoglobulin isotype (predominantly IgM) or PC affinity. Further, it was observed that the PC-binding antibodies which were generated in the presence of these two T cell populations expressed equivalently high levels of T15 idiotype. Assaying antibody and idiotype by either enzyme-linked immunosorbent assay or plaque-forming cell assay yielded similar results. Since monoclonal MHC-restricted, KLH-specific Th cells presumably lack any additional T cell populations, these results argue against an absolute requirement for anti-idiotypic Th cells in the generation of T15-dominant antibody responses.

The cellular immune response of rainbow trout ( [formula omitted][formula omitted] Richardson) to sheep red blood cells

The cellular immune response of rainbow trout, Salmo gairdneri, to sheep erythrocytes was investigated. Both the primary and secondary responses were measured using the migration inhibition factor (MIF), antigen-binding (ABC), and plaque-forming cell (PFC) assays. These immune function assays provide measures of both T and B cell activity. The kinetics of these three responses at 16°C were determined by sampling fish over an 18 day period for the primary response and a ten day period for the secondary response. The peak MIF response occurred two days after injection, while the primary peak PFC reponse was observed 14 days post-injection. Two ABC peaks were observed in the primary response, one at four days and one at ten days after injection. In the secondary response the peak ABC response was observed four days and the peak PFC response six days post-inoculation. The possible interrelationships of the various cell populations are discussed.

Peritoneal B cells differentiate without proliferation into autoantibody secretors under the influence of factors released by other cells.

After 3-5 days of in vitro culture, peritoneal cells from untreated C3H mice produce autoantibodies specific for autoantigens in the membranes of mouse erythrocytes. The autoantigens are exposed in vitro by treating mouse erythrocytes with the proteolytic enzyme bromelain. Limiting-dilution cell culture techniques were used to determine the frequency of the autoreactive B cells. Cells were cultured in Terasaki trays at 10-200 cells/well. Autoantibody production was assayed with an in situ plaque-forming cell (PFC) assay. On average, one autoantibody precursor cell was detected for every 150-200 peritoneal cells cultured. This precursor frequency was increased to 1 in 50 by the addition of lipopolysaccharide (LPS) and dextran sulphate (DS) to the culture medium. The addition of a culture supernatant from an EL4 lymphoma subline also induced a high proportion of the peritoneal cells to secrete autoantibodies. However, it was not possible to determine the frequency of PFC accurately because at limiting numbers of peritoneal cells the effects of EL4 affected more than one limiting variable. Significant cell division was observed in cultures to which LPS/DS had been added, in contrast to untreated cultures to which EL4 supernatant was added. The results show that high numbers of autoreactive B cells committed to self antigens are present in the peritoneal cavity and that these cells under the influence of appropriate cytokines can differentiate in vitro, even without proliferation, into autoantibody secretors. The cell type or types releasing the cytokines remain to be identified.

Antigen-induced plaque-forming cell responses in cultures of peripheral blood mononuclear cells of human neonates and infants

Human cord blood mononuclear cells (CBMC) were stimulated in vitro with a number of T cell-dependent antigens. Antigen-induced B cell activation was measured applying a plaque-forming cell assay for the detection of antigen-specific IgM-secreting B cells. With the exception of diphtheria toxoid, the antigens ovalbumin, sheep red blood cells, Helix pomatia hemocyanin, burro red blood cells, and tetanus toxoid elicited an IgM-plaque-forming cell response in cultures of CBMC to levels obtained for peripheral blood mononuclear cells (PBMC) from adult controls. However, for each antigen used, the antigen dose optimal for the induction of a response was consistently found to be a hundred to a thousand times lower than the concentration of the corresponding antigen optimal for adult PBMC. Longitudinal studies on PBMC obtained from infants between 2 and 30 months of age revealed that a shift of the antigen dose toward concentrations needed to induce plaque-forming cells in cultures of adult PBMC occurs at approximately age 8 months. Our data indicate that various antigens can be used for the in vitro analysis of antigen-specific B cell activation and regulatory T cell functions in studies concerning the ontogeny of the humoral immune response in humans.

Immune characteristics of the beige-nude mouse. A model for studying immune surveillance.

Double immunodeficient mice, homozygous for both the beige and the nude genes, were developed on C57BL/6N-beige and N:NIH(S)-nude background through five steps of mating. The animals were healthy and had a life span comparable to that of regular nude mice. Beige and regular nude mice showed a significant difference in natural killer (NK) activity, with mean +/- SE values of 6 +/- 1.0% and 25 +/- 2.8%, respectively. The response to the T-cell mitogens phytohaemagglutinin and concanavalin A was similar in beige and NIH mice of the same genotypes for the nude genes. Both groups of nu/nu mice also showed some response in these assays, as well as in the plaque-forming cell assay, indicating that nude mice are not completely devoid of functional T or T-like cells. The nude genes were found to increase the response to the B-cell mitogen lipopolysaccharide in both the beige and NIH animals, whereas the response was reduced in mice of the beige genotypes. The viable, low NK beige-nude mice here reported on may be a valuable tool for studying factors involved in host defence against tumours. Interestingly, no spontaneous tumours have so far been observed in such mice.

Plaque-forming cells in man. III. Generation of plaque-forming cells in allogeneic in vitro cultures of HLA-D/DR-incompatible B and T lymphocytes.

We have investigated the ability of allogeneic, irradiated T lymphocytes to induce proliferation and immunoglobulin (Ig) secretion in untreated peripheral blood B lymphocytes. Non-mitogen-activated co-cultures of isolated T and B lymphocytes from selected, full-house HLA-A,B and D/DR antigen-phenotyped donors were reconstituted in a ratio of 4:1. Proliferation was assessed on day 5-6 of culture by the 3H-thymidine incorporation technique, and the Ig secretion was monitored on day 6 with a protein A plaque-forming cell (PFC) assay. B lymphocytes were able to differentiate into PFC, and the number of plaques was significantly higher in cultures of cells with two HLA-D/DR antigen incompatibilities than in those sharing one antigen. In cultures of peripheral blood lymphocytes with no HLA-D/DR antigen difference, only a few PFC developed. HLA-A and B antigens had no influence on the response. Further, monocytes were not an absolute requirement for allogeneic activation of B cells. Sonicated T cells and culture supernatants from allogeneic T- and B-cells cultures were not able to induce PFC formation in B lymphocytes. Our results indicate that the PFC response obtained in non-mitogen-activated cultures of allogeneic T and B lymphocytes is dependent on HLA-D/DR disparity or on genes encoded in the HLA-D/DR region.

NEONATAL B CELL DIFFERENTIATION (RIFF)

Perinatal factors influencing diff of cord blood B lymphocytes into immunoglobulin secreting plasma cells was studied in 126 neonates with gestational ages(GA) from 20-44 wks. A plaque forming cell assay measured background B cell diff and diff in response to pokeweed mitogen(PWM)plus hydrocortisone(HC). 8 infants had a GA less than 27.9 wks, 24 had GA's 28-32.9 wks, 30 had GA's 33-37.9 wks, 51 had GA's 38-41.9 wks and 13 had GA's above 42wks. The mean±SD GA was 36±5wks and B.Wt. 2400±980gm. The mean±SD background plaque formation was 98±253 and in response to PWM+HC was 7505±11874/106 cord blood mononuclear cells. B cell diff was observed in some neonates in all GA groups. The magnitude of in vitro neonatal B cell diff undergoes a continuous and significant(p<.002)reduction as GA increases. The influence of intrauterine nutritional deprivation(IUGR)on B lymphocyte diff was studied, and compared to GA matched controls with a normal intrauterine nutritional status. IUGR was not associated with a decrease in B cell responsiveness. Cesarean section and low one minute Apgar scores were associated with a significantly(p<.05)increased B lymphocyte diff. Although prolonged rupture of maternal amniotic membranes(PROM)was not singularly related to a significant increase in B cell diff, the combination of PROM and low Apgar scores(stress)was associated with a more significant increase(p<.003)than either factor alone. Thus, short term neonatal stress such as the combination of PROM and low Apgar scores has as much influence on neonatal B cell function as GA.

IMPAIRED B LYMPHOCYTE FUNCTION DURING OPEN-HEART SURGERY: Effects of Anaesthesia and Surgery

B lymphocyte function in vitro was measured in patients undergoing open-heart surgery. Conventional balanced anaesthesia, or high-dose fentanyl anaesthesia was used. Pokeweed mitogen (PWM) induced lymphocyte transformation was depressed at the end of the operation, but the response to formalinized Staphylococcus aureus Cowan I (StaCw) was not. The numbers of immunoglobulin producing and secreting cells measured by an indirect protein A plaque-forming cell assay decreased after PWM-stimulation, but remained unchanged after StaCw stimulation at the end of the operation. IgG, IgM and IgA secretion by PWM- and StaCw-stimulated lymphocytes, into the culture medium, was depressed in the period after operation. Depressed immune functions occurred after open-heart surgery, but not in association with anaesthesia alone before surgery. The decreases were not mediated by hydrocortisone-sensitive suppressor cells. Minor differences between the two anaesthetic techniques were found in lymphocyte proliferative responses.