Plaque-forming Cell Assay Research Articles

Immunosuppressive activity of sera from gastric cancer patients.

Sera from 60 gastric cancer patients and 20 patients with benign gastric diseases and 8 healthy controls were tested for inhibitory effects on the humoral response to sheep erythrocytes (SRBC) by the plaque forming cell assay (PFC R.I.) using mouse spleen cells and on the phytohemagglutinin (PHA)-induced blastogenesis of normal mouse spleen cells (PHA S.R.). Gastric cancer patient sera showed a significantly lower PFC R.I. than did sera from benign gastric disease patients and from the healthy controls. However, there was no appreciable interstage difference in the degree of depression. The PHA-induced blastogenesis of normal spleen cells was also decreased in the presence of sera from cancer patients, as compared to that in the presence of sera from benign disease patients and from the healthy controls. The depression progressed with advancing stage of cancer. The PHA S.R. showed significant negative correlations with serum levels of IAP, IS, alpha 1-acid glycoprotein and alpha 1-antitrypsin, but there were no such correlations between PFC R.I. and these glycoproteins in serum. There was also no correlation between the values of the PHA S.R. and the PFC R.I. These results suggest that these two assays may depict immunosuppressive activities operating through entirely different mechanisms.

Mitogen-induced hyperproliferation response of peripheral blood mononuclear cells from patients with severe generalized periodontitis: Lack of correlation with proportions of T cells and T-cell subsets

Severe generalized periodontitis (SGP) is a localized inflammatory disease which differs clinically from common periodontitis in that it leads to remarkable extensive alveolar bone loss in relatively young adults. There is evidence that B-cell responses to bacterial substances may play a major role in the pathogenesis of this disease. In the present report, we show that a B-cell mitogen from Actinomyces viscosus (AVIS) bacteria provokes a hyperproliferation response of peripheral blood mononuclear cells (PBMNC) from these patients. In addition, AVIS-stimulated PBMNC from SGP patients proliferate for longer periods in culture than do PBMNC from control subjects. There were, however, no differences between patients and controls in the numbers of immunoglobulin-secreting cells in these cultures as determined by an indirect plaque-forming cell assay. The possibility that differences in numerical proportions of regulatory T-cell subsets may play a role in the mitogen-induced hyperproliferation phenomenon is examined. PBMNC were stained with fluorescein isothiocyanate-conjugated monoclonal antibodies OKT3, OKT4, and OKT8 in order to identify, respectively, total T cells, helper/inducer, and suppressor/cytotoxic subsets. Flow cytometric analysis of such specifically stained cell preparations from 14 control subjects and 14 SGP patients did not reveal any significant differences between the proportions of total T cells or T-cell subsets of the two groups. Furthermore, there were no statistically significant correlations between the magnitude of proliferation responses and the proportions of total T cells or either of the T-cell subsets.

Influence of methimazole on murine thyroiditis. Evidence for immunosuppression in vivo.

Thionamide drugs are immunosuppressives in vitro. To examine this action in vivo, A/J mice were immunized with human thyroglobulin (hTg) (0.5 mg intraperitoneal injections for 5 d) beginning on days 6, 24, and 43 with or without methimazole (M) (0.05%) and l-thyroxine (T4) (0.1 micrograms/ml to prevent thyroid hypertrophy) in their water supply. Groups (n = 8) were killed on days 37, 42, and 59. Spontaneous splenic IgG-secreting cells determined by Staphylococcus protein A-linked sheep erythrocytes (SRBC) via indirect plaque-forming cell (PFC) assay indicated polyclonal stimulation induced by the hTg exposure (controls = 2,285 +/- 599, hTg-only = 5,570 +/- 470 PFC per 10(6) spleen cells), but this was significantly reduced in the M plus T4-treated group (3,640 +/- 415 PFC, P = 0.05). hTg antibody was measured by specific PFC assay using hTg-linked SRBC. Anti-hTg PFC were absent in controls and were 147 +/- 41, 25 +/- 8, and 173 +/- 58 PFC per 10(6) spleen cells in the hTg-only groups on days 37, 42, and 59, respectively. Anti-hTg PFC results in the M plus T4-treated animals were significantly reduced to 0, 15 +/- 5, and 63 +/- 30 anti-hTg PFC. Histological examination revealed a marked thyroiditis in hTg-only animals and a significantly reduced degree of mononuclear cell infiltration and follicular destruction in the M plus T4-treated groups (graded 1.9 compared with 3.6 in hTg-only P = less than 0.01). Examination of IgG deposition using fluorescent anti-mouse IgG revealed a similar granular pattern and degree of staining in both immunized groups. Control animals that received concurrent T4 administration alone showed similar hTg-induced murine thyroiditis to non-T4-treated animals and could not explain the apparent immunosuppression observed. In conclusion, these data demonstrated that M reduced both the splenic immune response and the degree of thyroiditis after heterologous Tg immunization, while a quantitative difference in the circulating and intrathyroidally deposited Tg antibody was not detected.

Autoregulation of autoantibody synthesis in mercuric chloride nephritis in the Brown Norway rat. II. Presence of antigen-augmentable plaque-forming cells in the spleen is associated with humoral factors behaving as auto-anti-idiotypic antibodies.

Plaque-forming cell (PFC) assays were used to investigate in vitro the immunoregulatory mechanism operating in the self-limiting anti-glomerular basement membrane (GBM) autoantibody response of Brown Norway (BN) rats given HgCl2. The peak splenic PFC response occurred at day 9; thereafter the response fell sharply and was rarely detected after day 12. In specificity studies, incorporation of soluble GBM in the PFC assays of animals at day 9 had two distinct effects. In some animals the PFC response was inhibited in a dose-dependent fashion; however, in others an augmented number of PFC was observed. Furthermore, addition of GBM to the PFC mixture from certain animals studied at day 12 (or after) revealed large numbers of GBM-specific PFC when originally no GBM-specific PFC had been observed in the standard PFC assay. Sera from such animals, with and without antigen-augmentable PFC, were incorporated in the PFC mixture containing cells taken from day 9 animals. Sera from animals with revealed plaques could inhibit the GBM-specific PFC response of day 9 animals, whereas sera from animals without revealed plaques could not. Thus sera, from BN rats whose own antibody levels were falling, could inhibit the GBM-specific plaque-forming capability of cells from animals at an earlier stage of the autoimmune response and showed the potential importance of humoral factors, putatively antiidiotypic antibodies, in effecting autoregulation of autoantibody formation in this model.

Selective inhibition of various mitogen responses in human lymphocytes.

Adenosine and deoxyadenosine are known inhibitors of the in vitro proliferative response of peripheral blood lymphocytes(PBL) to various mitogens(1–7). Erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride (EHNA), an inhibitor of the enzyme adenosine deaminase(ADA), potentiates the inhibitory effects of both adenosine and deoxyadenosine. The incorporation of3H-leucirie into protein appeared to be a more sensitive index of adenosine toxicity than the incorporation of3H-thymidine(1). A differential sensitivity of cultured human T and B lymphoblasts towards the toxic effects of adenosine and deoxyadenosine has been observed: T cells are more sensitive than B cells(8,9). On the basis of3H-thymidine incorporation, mouse splenic cells stimulated by lipopolysaccharide(LPS) were less sensitive to the addition of adenosine than the concanavalin A(ConA) stimulated cells (10). Using a plaque forming cell assay, a biphasic effect of adenosine was seen on the in vitro antibody production in mice; concentrations of adenosine around 1mM stimulated antibody production in vitro, whereas concentrations above 1.5 mM had inhibitory effects (10).

Evaluation of the acyclovir-induced modulation of the plaque-forming cell response of human peripheral blood lymphocytes.

The increasing use of the antiviral agent acyclovir has focused the attention to the possible side-effects on cellular and humoral immunological processes. The drug has preclinically been tested extensively but most investigations have been carried-out in animal models. The influence of acyclovir on the proliferation and the plaque-forming capability of pokeweed mitogen (PWM) activated cultures of unfractionated and isolated subpopulations from human peripheral blood lymphocytes (PBL), was investigated. These parameters were monitored with the 3H-thymidine incorporation technique and a protein A plaque-forming cell assay. Acyclovir had little inhibitory effect at physiological concentration whereas a detrimental decrease was noted at higher concentrations.

The role of B cell surface Ia antigen recognition by T cells in B cell triggering. Analysis of the interaction of cloned helper T cells with normal B cells in differing states of activation and with B cells expressing the xid defect.

Two discrete mechanisms of T-B cell collaboration appear to exist. In cognate recognition, B cell triggering results from a direct recognition of antigen and MHC determinants at the B cell surface. Alternatively, B cells can be triggered by transstimulation, in which the Th cell is activated by an antigen-presenting cell to produce soluble factors which in turn trigger the B cell. This report addresses the question of whether antigen recognition at the B cell surface in association with Ia determinants delivers a signal to the B cell, which is qualitatively different from the signals delivered by the soluble mediators released by the activated Th cell. Previous reports from a number of laboratories suggest that cognate recognition is obligatory for the triggering of small resting B cells and B cells of the Lyb-5- phenotype, whereas enlarged B cell blasts and the Lyb-5+ subset can be triggered solely by soluble mediators. Contrary to these findings, the experiments described here indicate that B cells isolated in different states of activation from normal spleens on the basis of their buoyant density in Percoll density gradients, or unfractionated B cells from mice differing genetically due to the xid defect [Lyb-5- B cells from (CBA/N X BALB/c)F1 male mice], do not discriminate between the two modes of Th cell function. In both stimulation modes, the high density B cells, and the B cells from xid mice made very poor immunoglobulin secretory responses measured in terms of reverse plaque formation on protein A-coupled erythrocytes. When the responses of different density fractions of B cells were compared under conditions where stimulation occurred either directly or indirectly via transstimulation, the following hierarchy of responsiveness in both the proliferative and plaque-forming cell (PFC) responses was observed in the density fractions 60% greater than 65% greater than 70% greater than 75%. The hierarchy was the same in both modes of interaction and the deficiency of the high density, small B cells was far more marked in the PFC assay than in the proliferative assay. We conclude that the initial proliferative response of the resting B cell can be triggered comparably in vitro under conditions of direct or transstimulation. Thus, recognition of B cell surface Ia by Th cells is not obligatory for B cell activation and does not transfer an essential transmembrane signal to the B cell.

Multiple mechanisms of B cell immunoregulation in man after administration of in vivo corticosteroids.

Despite the extensive clinical use of corticosteroids (CS) in the treatment of immune-mediated diseases, relatively little is known concerning the effects of CS on B cell function as measured by in vitro assays. The effects of single-dose vs several days of in vivo CS therapy on the spontaneous and mitogen-induced Ig production by human peripheral blood B cells are reported here. Spontaneous Ig production by individual B cells was enhanced by in vivo CS as measured by an in vitro plaque-forming cell (PFC) assay. The same increased response was also observed with a brief in vitro exposure of the B cells to CS, which suggests that a mere brief exposure to an active CS analogue is all that is required to produce the enhanced response. The immunoregulatory effects of in vivo CS on mitogen-induced Ig production are more complex. Pokeweed mitogen-induced PFC responses were suppressed 4 to 5 hr after a single in vivo pharmacologic dose of CS, with complete recovery by 24 hr. In contrast, after a 5-day course of CS, the suppressed PFC response did not recover until 60 hr after the last dose. Moreover, several mechanisms of suppression were operative. Ten hours after completing the 5-day course of CS, there was a relative enrichment in the peripheral blood compartment of lymphocytes bearing the OKT8 suppressor/cytotoxic T cell phenotype that coincided with a depressed PFC response. At 36 hr after the last dose, the T lymphocyte profile returned to normal while B cell function remained suppressed. The complex, multifaceted modulation of the immune response, resulting from redistribution of cell subsets as well as altered cell functions, vary with time-dose parameters of in vivo CS administration. These observations should provide additional insights into the heterogeneity of CS-induced therapeutic effects.

Synovial fluid and blood monocytes/macrophages in rheumatoid arthritis. Influence on polyclonal activation of autologous B lymphocytes.

The regulatory role of synovial fluid monocytes/macrophages from patients with rheumatoid arthritis in terms of B lymphocyte activation was evaluated by a reverse haemolytic plaque-forming cell (PFC) assay. Macrophage-depleted blood mononuclear cells (BMC) failed to respond to pokeweed mitogen (PWM). With autologous synovial fluid macrophages added, the PFC responses of macrophage-depleted BMC increased, and optimal concentration for full restoration of the PFC responses ranged from 8 to 35%. Synovial fluid mononuclear cells (SMC) as well as macrophage-depleted SMC were not able to respond to PWM. Addition of irradiated autologous blood macrophages to SMC did not increase the SMC PFC responses. It is concluded that the regulatory properties of synovial fluid macrophages do not explain the low PFC response of SMC to PWM.

Systemic effects of methylcholanthrene in mice. II. Immune responses to sheep red cells.

Assays of splenic plaque-forming cells (PFC) for sheep red cells (SRC), done in parallel in control and methylcholanthrene (MCA)-injected mice of strains C3H/eB and C57BL/6, disclosed immunodepression, the degree of which depended on strain and sex of mice, and time elapsed between injection of MCA and performance of test. The most severe and long-lasting effects were observed in male C3H/eB mice, and the least impairment was seen in female C57BL/6 mice. After surgical splenectomy for assay of PFC, MCA-injected mice were observed for appearance of induced tumors up to 52 weeks after administration of carcinogen. In most groups of C57BL/6, but only rarely in C3H/eB mice, a correlation was found in individual mice between immunodepression and appearance of tumor. Since, however, the incidence of induced tumors was higher in C57BL/6 than in C3H/eB mice, immunodepression cannot be considered a universally obligatory step in carcinogenesis, but its involvement may be limited to particular phenotypes.

Evaluation of circulating immunoglobulin‐secreting cells in infants and children detected by protein A plaque forming cell assay

Evaluation of circulating immunoglobulin‐secreting cells in infants and children detected by protein A plaque forming cell assay

A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells

A solid-phase enzyme-linked immunosorbent assay (ELISPOT) is described for enumeration of cells secreting specific antibody. Spleen cells from immunized mice are incubated in antigen-coated polystyrene plates. After removal of the cells, bound antibodies are demonstrated by means of an immunoenzyme procedure in which enzyme-substrate reactions are performed in agarose. Dark-brown circular zones (spots), localized in areas of the dish where antibody production has occurred, are enumerated with the naked eye. Spectrophotometric estimation of enzyme-bound activity may be performed by substituting the gel for a liquid buffer, allowing accurate estimation of the total amount of secreted antibody. Versatile, sensitive and very easy to perform, this new assay provides a useful alternative to conventional plaque-forming cell assays.

Plaque-forming cells in man. I. Basic technical results obtained with the protein A technique.

This paper describes the results obtained using an indirect protein A plaque-forming cell (PFC) assay applied to human peripheral blood lymphocytes (PBL) activated with pokeweed mitogen (PWM). A maximal response was obtained after 6 days of culture with regard to the three major classes of immunoglobulin (Ig) investigated. No difference was found between females and males. T and B lymphocytes mixed in ratios varying from 1:8 to 8:1 and unfractionated cells were investigated. A maximal PFC response of isolated lymphocytes was found in cultures of 1:4 T/B reconstituted suspensions of untreated cells, whereas the maximal response of untreated B and 2000-rad-irradiated T lymphocytes was found in cultures with a 4:1 T/B ratio. The number of plaques developed in cultures of unfractionated cells exceeded the response of 4:1 T/B reconstituted untreated lymphocytes but was far below the number of PFC developed in cultures of untreated B and 2000-rad-irradiated T lymphocytes. Normal donors developed approximately 70 X 10(3) PFC/10(6) cells. The viability was 85% after the incubation period. No difference was found in the PFC response of cultures of separated, reconstituted T/B lymphocytes set up in autologous and allogeneic combinations.

Defective B cell function associated with inherited interstitial deletion of the short arm of the X chromosome.

The B cell function of a patient with low serum immunoglobulin (Ig) levels and with an interstitial deletion in the short arm of one of her X chromosomes (del(Xp] and the B cell function of her relatives were analyzed by indirect protein A plaque-forming cell (PFC) assay and by measuring immunoglobulin secretion in vitro by ELISA. B cells were activated by pokeweed mitogen (PWM) with or without hydrocortisone (HC) to inhibit HC-sensitive suppressor cells. The patient, her mother, and one sister, all of them having del(Xp), generated very few PFC induced by PWM, even in the presence of HC, whereas normal PFC responses were found in the patient's cytogenetically normal sisters. The B cells of the subjects with del(Xp) secreted very low amounts, if any, of IgA, IgG, and IgM. Co-culture experiments with B cells from del(Xp) subjects and normal OKT 4+ cells revealed no or very low Ig production. The function of the del(Xp) subjects' OKT 4+ cells was slightly reduced, whereas the activity of their OKT 8+ cells was normal. The cell subset analysis in the peripheral blood revealed decreased OKT 4+:8+ ratios in all del(Xp) subjects. The results indicate an intrinsic B cell defect with a possible concomitant immunoregulatory defect associated with del(Xp). Moreover, the results support the hypothesis that antibody production is at least partially controlled by genes located in the short arm of the X chromosome.

Storage of protein A-conjugated sheep erythrocytes for use in plaque-forming cell (PFC) assays

Sheep red blood cells conjugated with protein A from Staphylococcus aureus were frozen in liquid nitrogen. After thawing these cells were used as indicator cells in a plaque-forming cell assay visualizing human B lymphocytes secreting IgG, IgM or IgA. The thawed erythrocytes performed as well in the assay as erythrocytes freshly conjugated with protein A. Recovery of conjugated erythrocytes after freezing and thawing averaged 66%. By the method described indicator cells for a number of PFC assays may be prepared in bulk, frozen in suitable portions and thawed when needed. This prevents possible batch-to-batch variations and saves time.

IgG rheumatoid factor-secreting lymphocytes in rheumatoid arthritis: evaluation of a haemolytic plaque-forming cell technique.

A haemolytic plaque-forming cell (PFC) assay detecting human B lymphocytes secreting IgG rheumatoid factor (RF) was established using sheep erythrocytes (SRBC) sensitized with rabbit IgG, developing rabbit anti-human IgG, and complement. IgG-RF PFC were only demonstrated with IgG-depleted guinea-pig serum as the source of complement. Cells spontaneously secreting IgG-RF were found among synovial fluid mononuclear cells (mean, 134/10(6)) and synovial tissue mononuclear cells (mean, 1775/10(6)) from patients with rheumatoid arthritis, whereas few were recorded among blood lymphocytes (mean, 3/10(6)). The experiments revealed that the RF-IgG PFC were protein-synthesizing B lymphocytes. The antibody specificity of the secreted IgF-RF was verified by the inhibitory effect of exogenous human and rabbit IgG on PFC formation.

A plaque assay for assessment of immune responses to Streptococcus mutans cell wall carbohydrate

A plaque-forming cell (PFC) assay has been developed for measurement of single cell responses to the serotype carbohydrate antigen of the Streptococcus mutans cell wall. Serotype g carbohydrate was purified from a mutanolysin (M1) enzyme digest (and designated M1 g) of S. mutans 6715 cell walls by ion exchange and gel filtration chromatography. M1 g carbohydrate was esterified by stearoylization (sM1 g), and subsequently coated to sheep erythrocytes (sM1 g-SRBC). This coating antigen was then used for enumeration of IgM anti-M1 g PFC responses from spleens of either mice or rats immunized with S. mutans 6715 antigen. Good splenic IgM anti-M1 g PFC responses were seen in either mice or rats given S. mutans whole cells or cell walls, while cell wall lysates or M1 g were lowly immunogenic. Of interest was the finding that sM1 g induced good IgM anti-M1 g PFC responses in mice and in murine spleen cell cultures, in vitro. This study describes a method for assessment of individual antibody-producing cells to a major S. mutans cell wall determinant which should facilitate studies directed to determine mechanisms involved in the induction of immune responses to this important bacterium.

Effect of thyroxine on immune response in C57Bl/6J mice.

The effect of T4 treatment (40 micrograms, 5 times/week) on immune function in C57Bl/6J mice was studied. The primary antibody synthesis as measured by the plaque-forming cell (PFC) assay demonstrated significant suppression in mice treated with T4 for 30 days. Mitogen assays using PHA and Con A demonstrated significant suppression of cellular immune responses as measured by [3H]thymidine incorporation. The effect was reversed when the treatment was discontinued and a normal response reappeared after 14 days. Allogeneic skin grafts (donor A/J mice) showed prolonged survival (median survival time, 13.5 days) in T4-treated mice as compared to control untreated mice (median survival time, 11 days). Further experiments using Chromium 51 assay for delayed type hypersensitivity also showed suppressed response. These results suggest that treatment with T4, in the above doses and duration, cause immunosuppression in C57Bl/6J mice.

A case of K cell deficiency with diabetes mellitus and Graves' disease

The following immunological functions were studied in a case of insulin dependent diabetes mellitus with Graves' disease: (1) Lymphocyte subpopulations, (2) mitogen response, (3) immunoglobulin producing cells by the plaque forming cell assay, (4) cell-mediated cytotoxicity, and (5) natural killer activity were normal. The patient lacked antibody-dependent cell-mediated cytotoxicity. No conclusion could be drawn as to whether effector cells mediating natural (NK cell) and antibody-dependent cell-mediated cytotoxicity (K cell) are identical or different. However, the existence of such a case strongly suggests that the K cell is distinct from the NK cell. To resolve the question of whether or not a loss of antibody-dependent cell-mediated cytotoxicity activity plays an important role in the development of diabetes mellitus and Graves' disease, further studies on a large number of cases are necessary.

Intratumor chemoimmunotherapy with mitomycin C and BCG in C3H/He mice transplanted with MH134.

Experiments were performed to explore the influence of local chemoimmunotherapy by intratumoral administration of mitomycin C (MMC) and/or BCG on the survival rate, lymph-node metastasis, growth pattern of rechallenge tumor, and host immune function, using a host-tumor system consisting of C3H/He mice and syngeneic tumor MH134. Combined intratumoral regimens of MMC plus BCG yielded a significantly higher survival rate than those achieved with BCG or MMC alone. Furthermore, the incidence of metastatic involvement of regional lymph nodes was also remarkably reduced in the combined regimen group. The group given the combined MMC-BCG regimen also showed a marked suppression of the growth of rechallenge tumor after surgical removal of the primary tumor and showed a greater induction of tumor-specific immunity than that seen in the group given intratumoral BCG injection alone. Both the delayed-type hypersensitivity, as measured by the footpad swelling assay, and the antibody response in spleen lymphocytes, as estimated by the plaque-forming cell assay, were found to be substantially depressed following a systemic injection of MMC, whereas the intratumoral administration of MMC had little or no effect on these parameters.