Number Of Plaque-forming Cells Research Articles

Chlamydia trachomatis stimulates human peripheral blood B lymphocytes to proliferate and secrete polyclonal immunoglobulins in vitro.

Infectious Chlamydia trachomatis (LGV strain), obligate intracellular bacteria, stimulated human peripheral blood lymphocytes to proliferate and secrete immunoglobulins in vitro. In contrast, mock-infected preparations were unable to induce similar responses in peripheral blood lymphocytes. Although levels of immunoglobulin secreted into the media of LGV-stimulated cultures were greater than 10 micrograms/ml, we estimated that less than 1% of these molecules were directed against the bacteria itself, suggesting polyclonal antibody production. Since stimulation with Formalin-killed bacteria resulted in comparable numbers of plaque-forming cells (PFC) as infectious particles, we concluded that the polyclonal immunoglobulin response was not dependent on the in vitro chlamydial infectious process. The polyclonal PFC response induced by LGV was highly sensitive to monocyte inhibition. Although LGV induced proliferation of predominantly B cells, the numbers of generated PFC was increased by the addition of autologous T cells. Neither lymphocyte proliferation nor PFC responses of normal human volunteers correlated significantly with the presence or titer of antichlamydial antibodies in their sera.

The influence of a progressively growing MCA-induced fibrosarcoma on the humoral response to SRBS.

The effect of a progressively growing methylcholanthrene-induced tumour on the host’s immune response to sheep red blood cells was assessed. Total splenic lymphocyte counts were found to be significantly higher in humour bearing animals than controls at all stages of tumour growth. Tumour-bearing animals had greater numbers of plaque-forming cells per spleen than corresponding control animals. Serum IgM antibody titres, measured using a chromium release assay, were found to be depressed in tumour-bearing animals.

Kinetic Analysis of Subset Growth and B‐Cell Activation in Pokeweed Mitogen‐Stimulated Cultures of Peripheral Blood Mononuclear Cells Depleted of or Enriched for OKT8+ Cells

Peripheral blood mononuclear cells (PBMN) that were depleted of OKT8+ cells and stimulated with pokeweed mitogen (PWM) produced higher cell yields and higher numbers of plaque-forming cells than unfractionated PBMN. Conversely, OKT8-enriched PBMN, prepared by mixing unfractionated and OKT8+ cells in a ratio of 3:1, gave reduced cell growth and B-cell activation. In OKT8-depleted cultures, B cells, OKT4+ cells, OKT8+ cells, and OKM1+ cells increased in number between days 4 and 7 of culture by factors of 9.8, 5.9, 20.1, and 5.6 respectively, whereas growth rates for these subsets were 2.4, 1.0, 2.0, and 1.3 in unfractionated cultures and 0.9, 1.0, 1.2, and 0.6 in cultures enriched for OKT8+ cells. On day 7 of culture, 73 +/- 10% of B cells secreted immunoglobulin in unfractionated cultures, whereas only 21 +/- 10% of B cells were activated in OKT8-enriched cultures. Surprisingly, PWM stimulation of OKT8-depleted PBMN produced only 40 +/- 12% activated B cells.

T cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol.

Unprimed murine lymphocytes maintained in culture medium containing fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) developed very high levels of anti-trinitrophenyl (TNP) plaque forming cells (PFC). Both FCS and 2-ME contributed to the response. The development of anti-TNP PFC during culture was accompanied by a 10-fold expansion in the number of immunoglobulin-secreting cells, indicating polyclonal stimulation. However, the number of anti-TNP PFC was disproportionately high and not accompanied by a similar increase in plaques specific for sheep red blood cells. The TNP-specific plaques were not artifacts of the plaque assay since they were 98% inhibited by specific antigen. The in vitro induction of anti-TNP PFC by FCS and 2-ME was common to a number of mouse strains, although some genetic variation occurred. Nylon-wool-separated B cells, nude mouse spleen cells, and bone marrow cells all produced high levels of anti-TNP after culture with medium containing FCS and 2-ME. The addition of T cells to B-cell cultures increased the numbers of anti-TNP PFC by 1.5- to 2.5-fold. The presence of a TNP-cross-reacting antigen in FCS probably contributed to the unexpectedly high anti-TNP response. The response to the antigen in FCS was potentiated by the enhancing activity of 2-ME.

Spleen cell-mediated suppression of IgG production to a non-parasite antigen during chronic Trypanosoma cruzi infection in mice.

Splenic plaque-forming cell (PFC) responses to TNP-BGG (thymus-dependent) and TNP-Ficoll (thymus-independent) were measured during acute and chronic T. cruzi infections produced in C57BL/10 mice. The number of anti-TNP PFC to both antigens was suppressed as has been shown. Approximately 40% of untreated mice survived acute disease to enter chronic T. cruzi infection characterized by a decrease in parasitemia, a reduction in spleen size, a return to normal of the IgM responses to TNP-BGG and TNP-Ficoll, persistant polyclonal activation, and continued suppression of the IgG responses to TNP-BGG. Mice that were drug-treated during the acute disease had high survival rates and similar immune response patterns, ie., suppressed IgG PFC responses to TNP-BGG and normal IgM PFC responses to TNP-BGG and TNP-Ficoll. The selective suppression of the IgG response was transferred to nonirradiated syngeneic recipients by Thy-1.2-positive cells present in the spleens of chronically infected mice. These observations may be interpreted to suggest the persistence of nonspecific suppressor T cells during chronic T. cruzi infections.

Regulation of naturally occurring autoantibody secretion by a radiosensitive lymphocyte: Initial characterization and ontogeny

Low level irradiation (400–500 R) of normal mice or of murine spleen cells resulted in the detection of an enhanced number of plaque-forming cells against bromelain-treated autologous red cells (Br MRBC) 1 day later. The mechanism responsible for the increased numbers of plaques is apparently the elimination of a suppressor T cell since the addition of thymocytes or of Lyt 1 +2 + splenic cells to cultures of irradiated cells reversed the radiation-induced increase. Studies on the ontogeny of the phenomenon indicate that anti-Br MRBC plaques can be formed by spleen cells taken from mice shortly after birth although adult levels are not reached until after 3 weeks of age. Radiation-induced increases in the number of plaques were not seen until 3 weeks of age, thus, suggesting a temporal developmental sequence of the ability to produce autoantibodies and to regulate such production.

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.

Synthetic Analogues of the N-Terminal Lipid Part of Bacterial Lipoprotein are B-Lymphocyte Mitogens in vitro and in vivo

Two analogues of the N-terminal part of bacterial lipoprotein, S-[2,3-bis(palmitoyloxy)-(2 RS)propyl]-N-palmitoyl-( R)-cysteine methyl ester («tripalmitoyl cysteine») and S[-2,3-bis(palmitoyloxy)-(2 RS)-propyl]-N-palmitoyl-( R)-cysteinyl-( S)-seryl-( S)-seryl-( S)-asparaginyl-( S)-alanine («tripalmitoyl pentapeptide») were synthesized and tested for mitogenic activity. The compounds were potent mitogens towards mouse spleen cell cultures, as measured by 3H-thymidine incorporation and by hemolytic plaque assays. This activity was not dependent on the presence of serum. Tripalmitoyl pentapeptide had little, if any, effect on thymocytes. When injected intravenously into Balb/c mice, the synthetic compounds induced splenomegaly and polyclonal B-cell activation, the latter was evident from an increase in the number of plaque-forming cells against trinitrophenylated sheep red blood cells. Thus, a synthetic fragment of a bacterial surface component was shown to exhibit marked biological activity in vitro as well as in vivo.

Activation of T cells in tumor-bearing mice

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly1 +2 − cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating nonspecific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.

Polyclonal activation of immunoglobulin secretion without prior DNA synthesis in human B lymphocytes induced by Klebsiella pneumoniae

The capacity of various cell preparations of Klebsiella pneumoniae K43 (Klebs) to induce [ 3H]thymidine uptake and immunoglobulin (Ig) secretion by human mononuclear blood cells (MNC) and their lymphocyte subpopulations was investigated. All Klebs preparations were virtually devoid of mitogenic properties, in contrast to control preparations of pokeweed mitogen (PWM) and group A streptococcal cell membranes (A-ScM). Klebs induced differentiation of B cells into Ig-secreting cells. B-cell populations that were sufficiently depleted of T cells to be unresponsive to A-ScM (“highly purified B cells”) showed a marked response to Klebs. Similarly, the number of plaque-forming cells (PFC) in Klebs-driven cultures did not change after restitution of T cells, whereas the presence of restituted T cells augmented the B-cell response to PWM and A-ScM. Radical removal of adherent MNC (“monocytes”), however, completely abrogated the PFC response and [ 3H]thymidine uptake of both MNC activated by Klebs and MNC activated by PWM or A-ScM.

B-cell differentiation in multiple sclerosis and the effect of intravenous ACTH.

B-cell differentiation was studied in patients with MS and in age- and sex-matched controls, using a pokeweed mitogen (PWM)-stimulated in vitro culture system. Peripheral blood lymphocytes were obtained and separated into T-cell and non-T-cell fractions. Autologous and allogeneic combinations of T cells and B cells were cultured in the presence of pokeweed mitogen for 7 days. Numbers of plaque-forming cells (PFC) were measured at the end of the culture period. T cells from MS patients before and after a 10-day course of adrenocorticotropic hormone (ACTH) were able to cooperate fully in the generation of PWM-generated PFC. B cells from MS patients showed a decreased ability to differentiate into PFC in the presence of either autologous or allogeneic T cells. No significant change in differentiation was observed after a 10-day course of intravenous ACTH. We were thus unable to demonstrate any alteration in T-cell function in MS but were able to demonstrate a decreased ability of MS B cells to differentiate into immunoglobulin-secreting cells. ACTH had no significant effect on these abnormalities.

Alteration of the immunoglobulin G subclass responses in mice to lipopolysaccharide: effects of nonbacterial proteins and bacterial membrane phospholipids or outer membrane proteins of Proteus mirabilis

The immunoglobulin M (IgM) and the IgG1, IgG2ab, and IgG3 subclasses of plaque-forming cells (PFC) specific for lipopolysaccharide (LPS) were measured after immunization of mice with LPS alone and compared with the responses to LPS in combination with nonbacterial proteins and with bacterial membrane phospholipid vesicles or two major outer membrane proteins from Proteus mirabilis. The relative numbers of IgG PFC belonging to the IgG1, IgG2, or IgG3 subclasses induced by immunization with LPS alone depended upon the type of LPS administered. Phospholipids and the proteins effected characteristic alterations in not only the strength but also the subclass of the IgG responses to LPS. The results suggest that the hydrophobic-hydrophilic nature or state of aggregation of the preparations plays a role in the induction of IgG1 and IgG2 subclasses of PFC specific for LPS. Complex formation with LPS and adjuvant was apparently necessary to obtain these effects.

Polyclonal activation of B-lymphocytes in vivo by Salmonella typhimurium lipoprotein.

Lipoprotein prepared from the outer membrane of Salmonella typhimurium is a polyclonal activator of murine B-lymphocytes. It was shown to be mitogenic for splenic cultures, stimulating increased incorporation of [3H]thymidine into DNA. When injected intravenously into mice, the lipoprotein induced splenomegaly and polyclonal B-cell activation. The latter was evident from an increase in the number of plaque-forming cells against trinitrophenylated sheep erythrocytes. Similar results were obtained with Escherichia coli lipoprotein.

Spontaneous reverse haemolytic plaque formation. II. The role of T cells and monocytes in regulating immunoglobulin secretion by human peripheral blood B cells.

A sensitive reverse haemolytic plaque assay was used on freshly isolated blood cells from normal human subjects to show that T lymphocytes and monocytes were both necessary for immunoglobulin production by unstimulated B cells cultured only for the time necessary to form plaques. When lymphocyte preparations were fully depleted of T cells by E-rosette formation overnight on ice followed by Ficoll-Hypaque centrifugation, the number of plaque-forming cells was reduced by up to 94%; this reduction was reversed by the replacement of T cells, although excess T cells suppressed plaque formation. Moreover, when T-cell function was blocked by 10-1000 ng of two monoclonal anti-T-cell antibodies, OKT3 or UCHT1, this significantly reduced or abolished spontaneous IgG plaques, and higher concentrations of either OKT3 or UCHT1 reduced the numbers of IgA and IgM plaques formed by B cells. The role of monocytes in spontaneous plaque formation was investigated. The removal of plastic-adherent cells from mononuclear cell preparations did not consistently result in a reduction in the numbers of plaques, but complement-mediated lysis of monocytes with either of two monoclonal antibodies with specificity for monocytes, OKM1 and FMC17, reduced by 50% the number of IgG, IgA and IgM plaques. This effect was reversed by addition of as few as 1% plastic-adherent cells. Decreased plaque formation by B cells, resulting from either blocking of T-cell function with monoclonal antibody or complement-mediated lysis of monocytes, or both, was fully reversed by soluble factors present in cell-free conditioned medium from lectin-activated T cells. Thus spontaneous plaque formation by human peripheral blood B cells requires T cells and a small number of monocytes, and the major function of these cells is to help B cells by the production of soluble factors.

Use of nuclear antigen-coated red cells in hemolytic plaque assays

By studying antinuclear antibody production at the cellular level, we can better understand the problems of immunoregulation in individuals with systemic lupus erythematosus. To date, the use of hemolytic plaque assays to detect B cells secreting antinuclear antibodies has been hampered by an inability to achieve reliable coating of red cells by nuclear antigens. Because the chronic chloride technique has proved ineffective for coupling nucleic acids and/or nuclear antigens to sheep red blood cells (SRBC) in our laboratory, we have developed a method of coupling SS DNA, DS DNA, poly(I)·poly(C), Sm, and ENA to red cells pretreated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ECDI) and poly( L-lysine) (PLL). Coated red cells were agglutinated by specific antisera and not by normal sera and were then used in hemolytic plaque assays to detec antinuclear plaque-forming cells (PFC) in spleens from various strains of mice with lupus-like syndromes. PFC specific for SS DNA, DS DNA, poly(I)·poly(C), Sm, and ENA were found in MRL/lpr and NZB×W mice, and the number of anti-SS DNA and anti-DS DNA PFC correlated with the age of the animals. Indirect (IgG) PFC specific for nuclear antigens increased dramatically in female NZB×W mice between 11 and 13 months, a time when more than 50% of the animals usually die. Preliminary studies have shown that PFC specific for nuclear antigens can be detected in peripheral blood from patients with lupus erythematosus. Pretreatment of sheep red cells with ECDI and PLL thus allowed the coupling of selected nuclear antigens to these cells and provided the first demonstration of IgM and IgG PFC specific for a variety of nuclear antigens.

Histamine-induced suppressor factor (HSF): inhibition of helper T cell generation and function.

The effect of histamine-induced suppressor factor (HSF) on the humoral immune response was examined with the model of polyclonal B cell activation induced during a primary mixed lymphocyte culture (MLC). The number of plaque-forming cells (PFC) generated during MLC was measured by a protein A plaque assay. HSF was produced by incubating lymphocytes from normal subjects with 10(-4) M histamine. The addition of HSF on day 0 to MLC-induced plaques reduced the mean number of IgG, IgM, and IgA PFC by 60 to 80%. HSF supernatants were active at a titer of 1/1000 and suppressed IgG, IgM, and IgA isotypes equally. To study the effect of HSF on the T helper cell component of this reaction, purified T lymphocytes were activated in undirectional MLC and subsequently combined with unprimed B cells to induce a polyclonal PFC response. HSF present during the generation phase of activated T cells or only at the time of co-culture inhibited the total PFC response by 83 +/- 13% and 76 +/- 23%, respectively. The expression of "Ia" and autologous DR antigens normally detected on 50 to 62% of activated T cells generated during MLC were reproducibly reduced to 20% in the presence of HSF. Similarly, the polyclonal B cell response inducible by MLC-derived helper factors (1500 IgG PFC/10(6)) was markedly inhibited. Thus, HSF inhibits MLC-induced polyclonal B cell activation by interfering with the generation and effector function of T helper cells as well as the B cell response to preformed helper factors.

Regulation of the development of plaque-forming cells to bromelain-treated syngeneic mouse erythrocytes in bone marrow cell cultures.

The development of plaque-forming cells (PFC) to bromelain-treated syngeneic mouse red blood cells (Br-MRBC) was studied in bone marrow cell (BMC) cultures. It was found that the number of marrow PFC to Br-MRBC does not show the typical spontaneous increase observed in spleen cell (SPC), or peritoneal cell (PC) cultures. The number of anti-Br-MRBC PFC was markedly increased by lipopolysaccharide (LPS), even in conditions in which cell proliferation was blocked by mitomycin C, suggesting the presence of high numbers of Br-MRBC-specific precursor cells, potentially capable of differentiating into autoantibody-producing cells, in the marrow. Moreover, the low levels of anti-Br-MRBC PFC were further reduced in the presence of concanavalin A (Con A). The addition of Con A-activated BMC to BMC, SPC, or PC cultures actively suppressed the development of anti-Br-MRBC PFC. Con A-activated BM suppressor cells were found to be Thy 1.2-negative, Ig-negative, nonadherent cells. A possible role for the BM suppressor cell in tolerance to self antigens is discussed.

Effects in mice of rat bromelain-treated RBC and lipopolysaccharide on autoantibody production against bromelain-treated isologous RBC.

Autoantibody production against mouse bromelain-treated (brom) red blood cells (RBC) was significantly increased in mice injected with rat brom RBC. These autoantibodies were not adsorbed by rat brom RBC in serological assays and did not lyse rat brom RBC in plaque-forming cell (PFC) assays using mixtures of rat brom RBC and mouse brom RBC as targets. These data suggest that the increased response induced by rat brom RBC is not due to the presence of common, or similar, antigens on the two types of RBC. The spleens of mice injected with lipopolysaccharide (LPS), or with both LPS and rat brom RBC, had a markedly increased number of PFC lysing mouse brom RBC. About 20% of the PFC induced by LPS and rat brom RBC also lysed rat brom RBC. The autoimmune response was not increased in mice injected twice with rat brom RBC and the secondary response induced by two injections of LPS was lower than that induced by one injection of LPS. However, injection of LPS after an initial challenge with rat brom RBC induced an autoimmune response similar in size to that induced by LPS alone. The decreased secondary response against mouse brom RBC following a second injection of rat brom RBC was associated with decreased production of antibodies of various specificities as detected in a reverse PFC assay. These results do not support the hypothesis that the poor secondary responses against mouse brom RBC following a second injection of rat brom RBC are due to the exhaustive differentiation of autoimmune B cells as part of a fail-safe mechanism to prevent autoimmunity.

Synthesis of IgM, IgG and IgA in rheumatoid arthritis.

We studied the production of immunoglobulins by lymphocytes separated from the blood of 15 rheumatoid arthritis (RA) patients, of 12 patients suffering from other connective tissue diseases (CTD), and of 18 healthy controls. The production of IgM, IgG and IgA in pokeweed-mitogen-stimulated cultures was measured by counting the number of plaque-forming cells (PFC) and by determining the concentration of secreted immunoglobulins by means of an enzyme immunoassay. Synthesis of immunoglobulins, particularly IgM and IgG, was lower than in other CTD patients or controls. The IgM response of RA patients was 20% and 29% (PFC and Ig concentrations) that of the controls. The respective figures for IgG were 33% and 53% and for IgA 61% and 72%.

Evaluation of B-cell, T-helper-cell, and T-suppressor-cell function in patients with Graves' disease before and after treatment with anti-thyroid drugs

Spontaneous and pokeweed mitogen (PWM)-induced immunoglobulin-secreting B cells were evaluated in 20 newly diagnosed Graves' disease patients, and 11 of the patients were retested when euthyroid after treatment for 6–10 months with anti-thyroid drugs. Numbers of spontaneous plaque-forming cells (PFC) in patients tested before or after treatment were not significantly different from age- and sex-matched controls, but PWM-induced IgG-, IgA-, and IgM-PFC were significantly reduced in the pretreatment group to approximately one-half the mean control value. After treatment, numbers of PFC were midway between pretreatment and control values. Reduced B-cell activation was not due to circulating in vivo activated suppressor cells nor reduced T helper function. Concanavalin A-induced T suppressor function was normal in all but 2 20 pretreatment and 1 11 post-treatment patients.