Number Of Plaque-forming Cells Research Articles

10 Antigen-specific helper and suppressor T cell factors in man

Outbred rabbits were immunized intravenously with 109 sheep erythrocytes (SRBC) and/or horse erythrocytes (HRBC) and sacrificed 1 to 60 days later. At the height of the immune response (Day 7 postimmunization), antibody-forming cells identified by their ability to form hemolytic plaques in the plaque-forming cell (PFC) assay were detected only in the spleen. The splenic PFCs were totally inhibited if they were first incubated with the autologous thymus cells (immune thymus suppressor cells or ITSCs) for 4 hr prior to assaying for PFCs. Incubation of the spleen mononuclear cells with cells of any of the other lymphoid organs of the immunized rabbit for up to 6 hr did not inhibit the PFCs to any significant degree. There is no MHC restriction in the suppressor activity of the ITSCs since they could totally suppress the PFCs of autologous and allogeneic 7-day immune spleen cells. Neither thymus cells nor cells of any of the other lymphoid organs of the unimmunized rabbit could inhibit allogeneic 7-day immune splenic PFCs. The ITSCs from rabbits immunized with SRBC could be isolated by rosetting with the immunizing antigen, SRBC, but not with the non-cross-reacting antigens HRBC, human erythrocytes (HuRBC), and rabbit erythrocytes (RRBC), and vice versa, thus demonstrating the antigenic specificity of the suppressor cells. The thymus cells, but not the cells of any of the other lymphoid organs of the SRBC-immunized rabbit, secreted a factor referred to as immune thymus suppressor factor (ITSF) during incubation for 4 hr at 37°C which could, by itself, totally suppress the splenic PFC. ITSF could be absorbed out of solution by incubation with the immunizing antigen, SRBC, but not with the non-cross-reacting antigens HRBC, HuRBC, and RRBC. Similarly, the ITSF secreted by the thymus cells of the HRBC-immunized rabbit could be absorbed out of solution by incubation with the immunizing antigen, HRBC, but not with the SRBC, HuRBC, and RRBC. ITSF is therefore antigen specific as is its parent ITSC. The cells of the lymphoid organs of unimmunized rabbits did not secrete a suppressor factor during incubation in vitro. ITSCs capable of totally inhibiting the PFCs were detected in the immunized rabbits by Day 5 post-primary-immunization; the ITSCs were capable of secreting ITSF which could totally inhibit the PFCs by Day 7 post-primary-immunization. Although the ITSCs were detected in a suppressive state up to Day 40 post-primary-immunization, they lost their capacity to secrete ITSF by Day 21 post-primary-immunization. ITSCs could no longer be detected in the thymus or any other lymphoid organ by Day 60 post-primary-immunization. The ITSCs are T cells with receptors for the Fc of IgG since they rosette with EA(G) and not with EAC and are lysed following short-term incubation with horse anti-rabbit T-cell antiserum and complement but not with horse anti-rabbit B-cell antiserum and complement. Since the splenic MNCs assayed 7 days post-iv-primary-immunization are actively syntehsizing and secreting antibodies as confirmed by the large number of PFCs, it may be concluded that the antigen-specific ITSCs and ITSF are capable of inhibiting the PFCs by inhibiting the secretion of antibodies from the antibody-synthesizing cells.

Microinjection of macromolecules into normal murine lymphocytes by means of cell fusion. II. Enhancement and suppression of mitogenic responses by microinjection of monoclonal anti-cyclic AMP into B lymphocytes.

Reproducible methods are now available for introducing protein molecules such as antibodies into normal murine lymphocytes by fusion with protein molecule-containing erythrocyte ghosts. Monoclonal antibodies against cyclic AMP were raised by hybridoma technique and packed into erythrocyte ghosts. Then, monoclonal anti-cyclic AMP containing ghosts were fused with splenic B lymphocytes by polyethylene glycol-mediated fusion at various intervals after LPS stimulation. This method made it possible for us to quantitatively microinject antibodies into B lymphocytes. Microinjection of anti-cyclic AMP antibody molecules into lymphocytes at a very early stage of LPS stimulation resulted in a marked enhancement of DNA synthetic responses as well as increased numbers of plaque-forming cells. Intracellular cyclic AMP levels were found to be markedly decreased after microinjection of monoclonal anti-cyclic AMP, suggesting that lowering the intracellular cyclic-AMP level in the B lymphocytes at an early stage of stimulation might have induced the enhanced proliferative as well as differentiative responses to LPS. Similar enhancing effects on cell proliferation were obtained when antibodies were injected 18 hr after stimulation. Microinjection of anti-cyclic AMP at 12 hr after culture, however, inhibited the DNA synthetic responses, and induction of plaque-forming cells was suppressed when anti-cyclic AMP was injected 6 hr after LPS stimulation. The present data suggest the biphasic regulatory roles of cyclic AMP at the early stage of B lymphocyte activation. This approach may be useful in identifying regulatory molecules in B lymphocyte induced by mitogenic or antigenic stimulation.

The differential effects of PGE on the immune response in normal and immunosuppressed mice

The effect of 16,16-dimethyl-PGE 2-methyl ester (di-M-PGE 2) on humoral and cellular immunoresponsiveness has been compared in normal mice and in mice immunosuppressed by splenectomy and thymectomy plus antithymocyte serum (ATS). Splenectomy resulted in immunosuppression manifested by augmentation of B-16 melanoma growth; this stimulatory effect was reversed by di-M-PGE 2. In animals immunosuppressed by thymectomy plus ATS, di-M-PGE 2 augmented the humoral and cellular immune responses; this was manifested by slowing of the growth of B-16 melanoma and by stimulating the number of plaque-forming cells, hemagglutinin titers, and delayed-hypersensitivity reactions to sheep erythrocytes. In contrast, in normal (nonthymectomized) mice, di-M-PGE 2 was mildly immunosuppressive. Finally, adriamycin-immunosuppressed normal mice and this suppression were reversed by the addition of di-M-PGE 2 to the treatment regimen.

In vitro generation of cells producing antierythrocyte autoantibodies in normal mice

The production of antierythrocyte autoantibodies was studied by a plaque assay. Plaque-forming cells (PFC) and hemagglutinating antibodies were produced upon in vitro culture of murine spleen cells in the presence of mouse red blood cells (MRBC). Kinetic experiments revealed that the plaque response reached a maximum after 5 to 6 days of culture. The generation of PFC required the presence of MRBC at the beginning of the culture. PFC numbers were highly increased when 1% horse serum was added to the culture medium. Horse serum was found to sensitize the plaque assay by interacting directly with MRBC. After filtration of horse serum on Sephadex G-200 columns, only the fraction eluting in the void volume increased PFC numbers and interacted with MRBC.

Induction of immunoglobulin-producing human peripheral blood lymphocytes in serum-free medium

Induction of immunoglobulin-secreting cells from human peripheral blood lymphocytes in a serum-free culture medium was studied. Albumin, transferrin, insulin and fibronectin can replace serum entirely for support of pokeweed mitogen (PWM)-stimulated B lymphocytes, measured by a reverse hemolytic plaque assay using protein A-coated red cells. In this serum-free system, growth and maturation to IgM and IgG secretion occur at the same or higher efficiency as in conventional serum-containing medium, with maximum numbers of plaque-forming cells on day 6 at optimal dose of PWM, 0.5 ∼ 5 μg/ml. This system can be used to avoid the interference from undefined serum components.

High incidence of spontaneous Ig-producing lymphocytes in peripheral blood and synovial fluid of patients with active seropositive rheumatoid arthritis.

Numbers of in vitro spontaneous IgG, IgM and IgA plaque-forming cells (PFC) as assessed by a modification of the protein A haemolytic plaque assay were determined in the blood and synovial fluid of patients with seropositive rheumatoid arthritis (RA) and compared with those of control groups. The total numbers of PFC were significantly higher in the peripheral blood of patients with active seropositive RA than in that of normal controls. In addition, most B lymphocytes in the synovial fluid of patients with active seropositive RA were active immunoglobulin (Ig) producers, whereas synovial fluid lymphocytes from patients with inactive seropositive RA and seronegative arthritis were not. In general, IgA PFC were relatively high in blood, whereas IgG PFC dominated in the synovial fluid. IgM PFC appear to be relatively low in blood and synovial fluid. However, a relative increase of IgG PFC was noted in the peripheral blood of patients with active RA. To test for polyclonality of the increased Ig synthesis, we tested the sera of patients and controls for the presence of polyclonal antibodies against sheep erythrocytes (SRBC) and SRBC modified by fluorescein isothiocyanate (FITC) and trinitrophenyl (TNP). No differences were observed with SRBC and TNP-SRBC agglutinin titres between patients and controls, but patients with RA had higher titres of FITC-SRBC agglutinins than normal sera. This finding supports the concept of a polyclonal nature of antibody production in RA patients.

Thyroglobulin production by rat thyroid cells in culture: a study at the level of single cells.

The production of thyroglobulin (TG) by cultured rat thyroid cells was demonstrated at the level of a single cell using a modified reverse plaque-forming assay. Thyroid cells were cultured in the presence of sheep erythrocytes (SE) coated with hybrid antibody specific for both SE and TG. Upon the addition of anti-TG serum and complement, hemolytic plaques appeared around TG-producing cells. The kinetics of plaque formation and the distribution of plaque dimensions were investigated. The number of plaqueforming cells reached a peak after 48–72 h of culture, and at this time, 40–60% of the cells were producing TG; the number of plaque-forming cells declined thereafter, reaching a plateau after 5–7 days of culture. This behavior could be related to changes in TG production during the cell cycle. Most plaques showed a single thyroid cell in the center, thus suggesting that, at least in vitro, follicular organization is not an absolute requirement for TG secretion.

PGE restores the immune response in chemotherapy-treated, tumor-bearing mice

Two hundred seventy-one B-16 melanoma-bearing mice were randomized and treated for 4 days with either control diluent, 10 μg of 16, 16-dimethyl-PGE 2-methyl-ester (di-M-PGE 2), chemotherapy, or chemotherapy plus di-M-PGE 2. The chemotherapeutic regimens included adriamycin (7.5 mg/kg), 5-fluorouracil (250 mg/kg), nitrogen mustard (5 mg/kg), and vincristine (0.5 mg/kg). The number of plaque-forming cells and hemagglutinin titers in response to sheep erythrocytes were used as measures of humoral immunity while cellular immunity was assessed by evaluation of delayed hypersensitivity. As we previously reported, the presence of subcutaneous B-16 tumors induced substantial immunosuppression and this suppression was reversed by treatment with di-M-PGE 2. Treatment with all four chemotherapeutic agents induced profound immunosuppression. Similarly, the addition of di-M-PGE 2 to the chemotherapy protocols resulted in significant augmentation of cellular and humoral immunity.

Specific elimination of the T lineage cells: Effect of in vitro treatment with anti-Thy 1 serum without complement on the adoptive cell transfer system

Thymocytes from C57BL/6(B6) mice treated with anti-Thy 1 antiserum without complement in vitro were transferred to lethally irradiated AKR mice. Five days following transfer, the proportion of Thy 1.2(+) cells recovered from the recipient spleen was significantly lower (7%) than that from the control mice which had received untreated cells (64%). The B6 spleen cells were treated in the same manner and transferred with SRBC (T-dependent antigen) or DNP-Ficoll (T-independent antigen) to irradiated syngeneic recipients. The recipients developed a response to SRBC which was significantly lower than that observed in control mice, but showed the same number of plaque-forming cell (PFC) against TNP-SRBC as the control group of mice which had received untreated B6 spleen cells. These results clearly show that in vitro pretreatment of lymphocytes with anti-Thy 1 serum without complement specifically resulted in elimination or inactivation of the T lineage cells in the host environment. The mechanisms of the elimination are discussed in this study.

Immunoteratology of chlordane: Cell-mediated and humoral immune responses in adult mice exposed in utero

The functional status of the immune system of adult mice prenatally exposed to the chlorinated hydrocarbon pesticide chlordane was evaluated by assessing cell-mediated and humoral immune responses. Gravid BALB c mice fed 0, 0.16, or 8.0 mg/kg chlordane daily throughout gestation produced viable, overtly normal offspring. At 101 days of age cell-mediated immune (CMI) responses were measured by the contact hypersensitivity response to oxazolone. Offspring were sensitized by application of oxazolone to the denuded flank and 5 days later challenged by percutaneous application of oxazolone to the ear. Degree of induration was determined by daily micrometer measurement of ear thickness for 3 days after challenge. Offspring in the higher dose group had a significantly ( p < 0.01) depressed CMI response when compared to offspring of the control or lower dose groups. The primary humoral response to sheep red blood cell (sRBC) innoculation was determined at 101 days of age using the hemolytic plaque assay. Subjects were immunized with washed sRBC. IgM antibody released from the spleen cells was detected by addition of complement which caused lysis of the sRBC's. No significant differences existed between the number of plaque-forming cells (PFC) produced by spleen cells from either chlordane-treated group or the vehicle-control group. Results of this study revealed two significant findings: (1) severe depression of the functional CMI response in apparently normal treated offspring, but (2) no effect on the T-cell-dependent humoral immune response. The effect of chlordane on CMI and PFC responses might be explained by a reduction in the activity or number of T effector cells.

Lack of involvement of auto-anti-idiotypic antibody in the regulation of oscillations and tolerance in the antibody response to levan

Bacterial levan (BL) induces a cyclic (oscillatory) antibody response in both euthymic and athymic BALB/c mice. Significant numbers of plaque-forming cells (PFC) making auto-anti-idiotypic antibody directed against a major cross-reactive idiotype expressed on antibodies specific for BL were detected in euthymic—but not athymic—mice. This suggests that auto-anti-idiotypic antibody, the formation of which requires the participation of mature T cells, does not play a decisive role in generating the cyclic patterns observed. However, such antibody might still influence the proportion of PFC making antibody of complementary idiotype. No relationship between the suppressive property of auto-anti-idiotypic antibody and either the induction or maintenance of immunological tolerance to BL was evident.

Increase in the number of splenic plaque forming cells with length of gestation in untreated pregnant mice

The number of plaque forming cells (PFC) in spleens from untreated virgin and syngeneically pregnant CBA mice has been evaluated by use of two different hemolytic plaque assays. The total number of splenic IgM- and IgG-PFC in virgin and 16 day pregnant mice was determined by protein A-sheep red blood cell (SRBC) plaque assay. The number of both IgM- and IgG-PFC was significantly increased in spleens from pregnant mice, but the total number of IgM-PFC was several times higher than that of the IgG-PFC. In the same experiment the number of splenic anti-trinitrophenyl (TNP) PFC was determined simultaneously. The values obtained for anti-TNP PFC in the different animals were found to correlate with the values for IgM-PFC in the protein A-SRBC plaque assay. Finally, the number of splenic anti-TNP PFC was determined at different occasions during pregnancy (day 4, 8, 12, and 16). It was found that the number of anti-TNP PFC was elevated as early as the 4th day of pregnancy, and that the number of PFC per spleen increased with the length of gestation.

Plaque-forming cells in human cord blood: a soluble factor suppressing differentiation but not proliferation of B cells.

Cord blood-derived T cells have been shown to suppress differentiation of B cells into immunoglobulin-secreting cells and proliferation of mononuclear cells (MNC). We report about a soluble factor present in the supernatant of pokeweed mitogen (PWM)-stimulated cord blood MNC which was able to suppress significantly the number of plaque-forming cells (PFC) in adult MNC populations, but had no effect upon proliferation of adult MNC in response to PWM and allogeneic MNC. The number of PFC was not suppressed by supernatant from unstimulated cord blood MNC or by PWM-stimulated adult MNC.

Polyclonal antibody synthesis by bone marrow cells of New Zealand black mice. I. Assessment of activity in an adoptively transferred histocompatible host.

The activity of polyclonal antibody synthesis (PAS) of bone marrow cells (BMC) was assessed by enumerating anti-trinitrophenyl (TNP) plaque-forming cells (PFC) or immunoglobulin-secreting cells (IgSC) generated in the spleen of BMC-reconstituted syngeneic or H-2-compatible hosts. BMC of NZB mice generated large numbers of anti-TNP PFC or IgSC as compared with those of C57BL/6 mice or H-2-compatible, nonautoimmune strains of mice (BALB/c, DBA/2 and B10 . D2 mice). Causes for the enhanced PAS activity were explored. Susceptibility to known B cell mitogens was examined and it was shown that spleen cells of the recipients of NZB BMC were refractory to the mitogens while PAS was apparent and acquired responsiveness after 15 days as those from nonautoimmune strains of mice. Furthermore, environmental factor(s) did not seem to be responsible for the enhanced PAS, since BMC from nonautoimmune BALB/c mice showed little PAS activity when transferred into heavily irradiated NZB mice and those of NZB mice showed enhanced PAS activity when transferred into B10 . D2 or BALB/c mice. It seems most likely that enhanced PAS activity of BMC of NZB mice is a genetically inherent phenomenon. PAS activity of BMC and spleen cells of NZB mice increased significantly with increasing age of mice.

Influence of sera from tumor bearing mice on immune reaction.

Immunopotentiation abilities of sera from mice bearing fibrosarcoma or lymphoma for various periods of time were assayed using the plaque forming cell (PFC) technique. The influence of such sera on cell proliferation, evaluated by the spleen index, was determined in lethally irradiated mice reconstituted with syngeneic bone marrow cells. Sera from tumorous animals stimulated the number of PFC significantly, fibrosarcoma sera being more effective in the advanced stage and lymphoma in the initial phase. Sera from mice with both tumors in the early phase increased spleen indices significantly, and sera from mice with advanced tumors were less effective. The possible role of lymphokines in these events is discussed.

Suppression of IgM-producing cells by administration of anti-IgM serum in vivo before sheep red blood cells injection.

The primary immune response in young adult mice to sheep red blood cells was studied by following the effects of anti-IgM or anti-Fc sera on the number of plaqueforming cells for IgM (dPFC) and IgG (iPFC) in the spleen. The number of dPFC in the case of administration of anti-IgM serum at 1 h before antigen injection was significantly decreased to 3.7±0.9/106 spleen cells in comparison with the value of control serum (69.0±15.7/106 spleen cells). However, the numbers of iPFC in the cases of both anti-IgM or control sera were similar. From these results, we suppose that progenitors of IgM-producing cells are different from progenitors of IgG-producing cells in adult mice.

Evidence for a spontaneous activity and a weak helper function in cord blood lymphocytes.

Human lymphocytes from cord blood (CBL) and adult peripheral blood were studied for suppressive and stimulatory effects. Using a double chamber assay we found that via soluble substance(s) cells from approximately 90% of all cord bloods and about 25% of the tested adults could diminish the proliferative response and the IgM and IgG plaque-forming cell numbers of phytohemagglutinin (PHA) stimulated adult cell cultures. CBL were spontaneously suppressive, whereas cells from 'suppressive adults' required activation with PHA to exert suppression. Co-cultures of T cells and B cells were stimulated with pokeweed mitogen. Cord B lymphocytes were able to secrete not only IgM but also IgG. The Ig response of CBL was weaker than that of adult cells, which argues for an immaturity of cord helper T lymphocytes, and possibly also of cord B lymphocytes.

Primary antibody response of rabbit blood lymphocytes in vitro

This paper describes a method for in vitro induction of a primary response of rabbit peripheral blood lymphocytes to sheep red blood cells. The response is measured by visualizing and enumerating the plaque-forming cells (PFC). Removal of an adhering suppressor cell and use of a low cell concentration in culture are among the crucial requirements. Maximum response was usually reached after 10–15 days of culture. The number of PFC then decreased or stayed at roughly plateau level at least up to the fourth week of culture, when most of the experiments were terminated. In several instances the response had a cyclical character with repeating peaks of PFC. Only plaques of the direct type were found.

Activated T cells in human peripheral blood: quantitation with a reverse hemolytic plaque assay.

Rabbit antiserum to culture supernatant of concanavalin A-stimulated peripheral blood mononuclear cells was used in a reverse hemolytic plaque assay to detect activated T lymphocytes in human peripheral blood. The number of plaque-forming cells in fresh, unstimulated lymphocyte preparations was approximately 400/10(6) cells, and increased 7- to 14-fold after stimulation with a variety of mitogens and antigens. The kinetics of the increase paralleled 3H-thymidine incorporation, with a maximum on day 3 of culture. Plaques were eliminated by treatment of cells with anti-Leu-I + complement or by depletion of E-rosettes. The activated T cells were not restricted to a given inducer, suppressor, or Ia+ T cell subset, however. Both mitogen-stimulated culture supernatants and mitogen-activated lymphocytes, but not resting lymphocytes, were effective in absorbing the capacity of the rabbit antiserum to develop plaques. This suggested that the predominant specificity of this antiserum was directed toward surface membrane determinant(s) of T cells actively shed into the medium. This process was shown to require ongoing protein synthesis, and, in mitogen-stimulated lymphocyte preparations, DNA synthesis as well. This approach has enabled detection of a surprisingly large number of endogenously activated T cells in normal human blood, and should be useful for the analysis of immunoregulatory events at the single-cell level.

Isolation of helper T cells from the peripheral blood of normal donors, using serum from a patient with agammaglobulinemia

Serum from a patient with agammaglobulinemia was found to contain antibody reactive with a subset of normal T lymphocytes. This subset, designated AGG +, was shown to express the properties associated with helper T cells. The addition of AGG + cells to B cells caused an increase in the number of plaque-forming cells, whereas the AGG − population was unable to enhance Ig production. In addition, the AGG + population was unable either to recognize or to kill allogeneic cells, with recognition and killing events being carried out by AGG − T cells. The relationship of this subset to other subsets defined by reactivity with auto- or heteroantibody was examined.