Number Of Plaque-forming Cells Research Articles

The Chicken's Femoral-Lymph Nodules: T and B Cells and the Immune Response

Femoral lymph nodules (FLN), which are barely perceptible in normal birds, after a footpad injection of sheep red blood cells (SRBC) may either significantly enlarge (responder) or remain reduced in size (nonresponder). There were approximately 38% T cells and 53% B cells in the FLN of responder chickens. Significantly more plaque-forming cells (PFC) developed in the FLN than in the spleen after a footpad injection of SRBC. Total antibody, mercaptoethanol- (ME) resistant, and ME-sensitive fractions were significantly higher in birds given i.v. than in those given footpad injections. There were no differences in PFC and agglutinin titers between FLN-responders and nonresponders. The number of PFC in FLN exceeded the number of splenic PFC previously reported. The high PFC response of the FLN may reflect the large percentage of B cells in this lymphomyeloid tissue or the presence of antigen-experienced B cells in the FLN. Although FLN may influence a systemic immune response its major role appears to be restricted to a local response.

Lipopolysaccharide and lipid A‐induced human blood lymphocyte activation as detected by a protein A plaque assay

Various purified cell wall lipopolysaccharides (LPS) from gram-negative bacteria and derivatives of these LPS were tested for their stimulatory capacity for human peripheral blood cells. Immunoglobulin (Ig) production was tested by an indirect plaque-forming cell assay using Staphylococcus aureus protein A-coupled erythrocytes and specific anti-Ig as developing serum. This method allows the detection of the majority of cells secreting Ig of a single class, and the number of plaque-forming cells detected are approximately 100-1000 times the amount obtained using normal sheep red cells as targets. LPS containing the O antigen-specific chain, as well as mutant products only containing lipid A and ketodeoxyoctonate trisaccharide, could induce cell division and antibody synthesis. The polypeptide antibiotic polymyxin B was found to inhibit LPS-induced activation. Furthermore, purified lipid A, complexed with bovine serum albumin, was also found to activate human peripheral blood B cells. These findings demonstrate that human peripheral blood lymphocytes can be activated by LPS and also indicate that lipid A is the active part of these molecules.

The Immune Response to the Alpha 1–6 Epitope of Dextran is Determined by a Gene Linked to the IgCH Locus

There is a marked strain variability in the immune response against the alpha 1-6 epitope of dextran B512. Responsiveness was independent of the H-2 genotype of the strains but completely determined by the IgCH allotypes of the strains. Thus, IgCHb and IgCHj allotypes characteristic of C57BL and CBA strains, respectively, determined high responsiveness, whereas strains possessing all other allotypes studied were low responders. Backcross analysis indicated that a gene(s) determined responsiveness and that this gene was linked to allotypes of the IgCH locus. F1 hybrids between high and low responder strains always gave a response, but the number of plaque-forming cells was approximately half of that obtained in the high responder strains.

An immunostimulatory substance in the ascites fluid of patients with cancer metastatic to the peritoneum

The ascitic fluids from patients with cancer metastatic to the peritoneum contain a factor(s) which stimulates the primary antibody response to sheep red blood cells (SRBC) in vitro. This enhancement is manifested by an increase in the number of plaque-forming cells per culture and a slight increase in plaque size. This factor has a molecular weight in the range 30,000–100,000 as determined by Sephadex gel filtration. The factor, which we have called “stimulatory factor” (SF), will completely replace the requirement for fetal calf serum in the Mishell-Dutton type of assay. Enhancement of the antibody response is most apparent at suboptimal culture conditions. SF does not increase the number of plaque-forming cells to the T-independent antigen Escherichia coli but there is a marked increase in the size of the plaques produced to the lipopolysaccharide using coated SRBC as targets. The stimulation induced by this factor is not due to endotoxin contamination since endotoxin is heat stable and the SF is heat inactivated at 80 °C for 10 min. In addition endotoxin does not act in a manner similar to SF. Thus, the SF appears to influence both T and B cells. With thymus-dependent antigen the factor results in increased numbers of antibody cells being generated; with thymus-independent antigen the factor results in increased quantity of antibody being produced.

Enhanced Immune Response Late in Primary Cytomegalovirus Infection of Mice

Murine cytomegalovirus (MCMV) infection has been previously shown to depress humoral and cell-mediated functions to non-MCMV antigens. In this report we show that in C3D2 mice undergoing nonlethal primary infection the depressed anti-sheep RBC plaque-forming cell (PFC) response is followed by an enhanced PFC response. Infected mice often generated twice the number of PFC per spleen than that of control mice. Total numbers of spleen cells as well as the recovery of virus from spleens of infected mice did not distinguish the depressed from the enhanced phase of the response. Investigation of the kinetics of the response revealed a defect in shutdown regulation. This enhanced PFC response during primary MCMV infection was not reflected in measurements of serum hemagglutinin. These findings suggest that MCMV induces an impairment of immunoregulation.

In vitro development of a primary antibody response with lymph node cells

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10 6 recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.

Cellular immune response in rainbow trout Salmo gairdneri Richardson to Yersinia ruckeri O‐antigen monitored by the passive haemolytic plaque assay test

Abstract. The specificity and kinetics of the immune response of rainbow trout (Salmo gairdneri) to single injections of an O‐antigen extracted from the bacterial pathogen Yersinia ruckeri, which causes enteric redmouth in fish, were investigated by the passive haemolytic plaque assay and serum antibody quantitation. Doses ranging from 5 ng to 500 mg in 10‐fold increments were injected intraperitoneally into groups of trout held at 17 × 1°5°C. The occurrence of plaque forming cells (PFC) and humoral antibody was followed for 35 days after injection. Trout gave an immune response to doses of 500 ng and above. Seven days after injection no humoral antibody was detected, but PFC were found in the spleen. The maximum PFC numbers occurred 11 days after injection. On day 21, few PFC were found, whereas serum antibody titres were highest. The antibody from immunized trout showed little or no cross‐reactions with sheep red blood cells passively labelled With antigens from other fish pathogens.

Different Effects of the Capsular Polysaccharide of Klebsiella pneumoniae on the Functions of Various Subpopulations of B Cells in the Immune Response of Mice to T‐Dependent Antigen

AbstractUsing the capsular polysaccharide of Klebsiella pneumoniae (CPS‐K) as a polyclonal B‐cell activator (PBA) and sheep red blood cells (SRBC) as a T‐dependent antigen, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T‐dependent antigen. Antibody‐forming cells (AFC) of IgM and IgG types were estimated as anti‐SRBC direct and indirect plaque‐forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette‐forming cells (RFC) of the B‐cell type. Stimulation of normal mice by CPS‐K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS‐K‐treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS‐K‐untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS‐K than that to generate direct PFC. When CPS‐K was injected into normal mice simultaneously with SRBC, CPS‐K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC‐primed mice, the number of RFC was markedly decreased following injection of CPS‐K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS‐K‐treated SRBC‐primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS‐K‐untreated SRBC‐primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS‐K than that to generate the secondary direct PFC response. When CPS‐K was injected into SRBC‐primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS‐K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T‐dependent antigen in different ways, and acts to regulate the levels of B‐cell responses to the antigen‐mediated positive signal.

Immune Response to Levan

Abstract Pretreatment of BALB/c mice with antisera to a cross-reactive idiotype (E109IdX) expressed on many anti-bacterial levan (BL) and anti-inulin (Inu) antibodies leads to a prolonged suppression in production of IdX-bearing molecules in response to BL immunization. There is a comparable suppression in numbers of plaque-forming cells secreting IdX-bearing anti-BL and anti-Inu molecules. Furthermore, spleen cells from anti-E109IdX pretreated mice are unable to transfer to irradiated recipients the ability to produce IdX-bearing anti-BL and anti-Inu antibodies. These results indicate that the suppressive effect is at the precursor level and not simply a clearance of antibodies bearing the IdX. Suppression of IdX production can be achieved by pretreating nu/nu BALB/c mice with anti-E109IdX antibodies. Furthermore, spleen cells from pretreated mice do not inhibit the capacity of spleen cells from normal mice transferred to irradiated recipients to produce E109IdX in response to BL. This indicates that the suppression of IdX production in the anti-BL system is T independent and probably represents direct inhibition of precursors by anti-IdX.

Restoration of T-cell function in aged mice with long-term administration of levamisole

The in vivo effect of levamisole on antibody production against sheep red blood cells (SRBC) in aged mice was examined. Aged C57BL/6 mice were treated with levamisole, 2.5 mg/kg per os, for 5 months. The long-term administration of levamisole restored the ability of immune response to SRBC. The effect of levamisole on B cells was examined by an in vitro coculture technique. There was no change in the number of plaque-forming cells (PFC), indicating that levamisole did not act on B cells. Suppressor function of T cells after long-term administration of levamisole was also examined. Concanavalin A (Con A)-treated spleen cells from aged mice previously treated with levamisole suppressed the number of PFC of cocultured spleen cells from young mice, but Con A-treated spleen cells from aged control mice (no levamisole treatment) failed to suppress the number of PFC. The in vitro effect of levamisole was assessed by the number of PFC. The number of PFC was not altered significantly when levamisole was added to the culture medium. These results indicate that long-term administration of levamisole to aged mice can restore helper as well as suppressor T-cell function, and suggest that levamisole itself is not active in vitro but exerts its effect through metabolites in vivo.

The effect of antigen priming on immunological response regulation in aging

The primary anti-sheep red blood cells response and the response of antigen primed CBA mice aged 3–29 months were compared. There was a 30-fold decline with age in the primary immunological response, tested by the direct plaque-forming cell number. In young and young adult mice, priming resulted in a fall of antibody production and of number of Ig-positve spleen lymphocytes. During the lifespan the response of primed mice increased, reaching and then exceeding the level of primary response in senescence as much as 20-fold. Negative correlation was established between antibody-forming and nuclear spleen cell numbers of old primed mice. The adoptive transfer of spleen cells of primed young adult mice suppressed the antibody production in immunized recipients, whereas that of old primed spleen cells produced no reaction, the response of the recipients of old primed spleen cells being thus higher. The addition of young bone marrow to old primed mice enhanced the response and abolished the negative correlation between plaque-forming cell and nuclear spleen cell numbers. The data obtained showed a decreased suppressor activity of the antigen primed spleen cells with aging. This phenomenon seems to reflect certain features of the immune response regulation in old age.

Suppressive effect of catecholamines on 19-S antibody formation in mouse spleen fragments in vitro.

Spleen fragments of Balb/C mice were immunized with sheep red blood cells in vitro and hemolytic plaque forming cells (PFC) were assayed on the 5th day of immunization. Epinephrine in culture medium diminished the number of PFC dose-dependently over the range from 10(-6) M to 2.7 X 10(-4) M. Incubation with 9 X 10(-5) M epinephrine diminished PFC response only when the epinephrine was present on the first day of the culture. No suppression was found for treatment during any other 24-hr periods. Three beta-adrenergic stimulating catecholamines, epinephrine, isoproterenol, and norepinephrine, suppressed PFC response in similar degree. It is concluded that beta-adrenergic stimulation suppressed 19-S antibody formation mainly by inhibiting induction of immune response.

Immunosuppresive effect of alpha-fetoprotein at different stages of pregnancy in mice

A possible immunoregulatory role of alpha-fetoprotein (AFP), both as contained in amniotic fluid and in isolated form, was investigated at different stages of pregnancy. Virgin, as well as pregnant mice on days 6, 9, 12 or 16 of gestation were immunized by a single intraperitoneal injection of sheep red blood cells (SRBC). Antibody responsiveness in these animals treated with mouse amniotic fluid (MAF) or isolated AFP, was assessed by measuring YM splenic plaque-forming cells (PFC) 4 days after primary immunization. There was a significant suppression of plaque-forming activity in MAF and AFP treated non-pregnant mice. Similar suppression was evident also in animals in which SRBC were injected on days 6 or 9 of pregnancy followed by treatment with MAF or AFP. Conversely, the same treatment did not result in suppression of antibody response to SRBC, injected on days 13 or 16 of gestation. One of the possible explanations of such lack of AFP effect could be increasing production of endogenous AFP which itself already restrained the mother's immune reactivity. The decreased number of PFC in the last week of gestation in non-treated mice may support this assumption.

Autoimmune Response to DNA, Red Blood Cells, and Thymocyte Antigens in Association with Polyclonal Antibody Synthesis During Experimental African Trypanosomiasis

Abstract In vivo induction of autoantibodies by polyclonal B cell activators has been suggested to be the direct consequence of the polyclonal B lymphocyte activation. Since the appearance of various autoantibodies represents a common feature of some parasitic infections, the possibility that such autoantibodies develop as a result of the stimulation of polyclonal antibody synthesis was tested in mice infected experimentally with Trypanosoma brucei brucei (T. brucei). Polyclonal antibody production in the spleen was measured by the Cunningham plaque-forming cell (PFC) assay with SRBC, TNP-SRBC, and FITC-SRBC as indicator cells. Compared with normal mice, T. brucei-infected animals displayed a marked increase in the number of PFC to TNP, FITC, or SRBC. The level of polyclonal antibody synthesis was correlated to the number of T. brucei injected and to the number of parasites in circulating blood. The observed B lymphocyte activation is not dependent upon the influence of T cells since infection of athymic nu/nu mice also led to polyclonal antibody synthesis. Stimulation of polyclonal antibody synthesis was also observed after T. brucei infection of C3H/HeJ mice, a strain of mice that is nonresponsive to bacterial lipopolysaccharides. Therefore, it is unlikely that the lipopolysaccharides component of endogenous Gram-negative bacteria play an important role in the observed proliferation and differentiation of B lymphocytes. Autoimmune responses to DNA, red blood cells, and thymocyte antigens were observed in mice infected with T. brucei and the kinetics of their formation was parallel to the development of the polyclonal B cell activation.

Plaque-Forming Cells and Humoral Antibody in Rainbow Trout (Salmo gairdneri) Induced by Immersion in a Yersinia ruckeri O-Antigen Preparation

Rainbow trout (Salmo gairdneri) were exposed to the O-antigen of Yersinia ruckeri by various immunization regimens. The passive hemolytic plaque assay was used to show specific splenic plaque-forming cells (PFC) and passive hemagglutination demonstrated humoral antibody titers in fish injected with or immersed in the antigen preparations. Preceding antigen immersion with a 2-min immersion in either a 5.2 or a 2.6% NaCl solution did not affect the numbers of PFC or levels of antibody. Fish held for 2 min in ethyl m-aminobenzoate methanesulfonate (MS-222®) before antigen immersion showed no significant difference from the control fish. Dosage studies showed that the minimal concentration of antigen for induction of PFC by immersion was 5.0 μg/mL. Key words: plaque-forming cells, immunization, rainbow trout immersion, Yersinia ruckeri

Polyclonal Activation of Human B Lymphocytes in Vitro by Pokeweed Mitogen: a Simple Technique for the Simultaneous Assessment of Cell Proliferation, Generation of Plasma Cells, Plaque‐forming Cells and Immunoglobulin Production

A simple technique has been worked out for the simultaneous assessment of cell proliferation, generation of plasma cells, plaque-forming cells and immunoglobulin production from single cultures of 1 X 10(6) human peripheral blood lymphocytes stimulated with pokeweed mitogen (PWM). Kinetic studies showed that number of cells, thymidine incorporation rate, number of plaque-forming cells against fluorescein-isothiocyanate-haptenated sheep erythrocytes and concentration of IgM and IgG in the supernatant peaked on the average between day 5 and 7 of culture. This technique is particularly suitable for the analysis of hypogammaglobulinaemias in infancy.

Induction of haemolytic plaque-forming cells with sheep lymphoid cells in vitro

The in vitro induction of specific primary and secondary immune responses in sheep lymph node cell suspensions is described and suitable culture conditions determined. The induction of primary immune responses required supplementation of the culture medium with antigen-absorbed homologous serum or lymph, whereas the requirements for the induction of a secondary response were less stringent. The addition of 2-mercaptoethanol to the medium was required. The amounts of heterologous erythrocytes used for immunization were critical and optimal responses were obtained when 50 μl of a 1% suspension were added to 1 ml cultures. Lymphocyte densities of about 5 × 10 6/ml were found optimal in primary immune responses in vitro. Less than 2 × 10 6 cells/ml rarely gave rise to plaque-forming cell (PFC) generation, whereas densities of 10 × 10 6 and above reduced the number of PFC obtained per number of cultured cells. Lymphocytes obtained from the efferent lymph draining lymph nodes previously immunized with heterologous erythrocytes were found to generate PFC in vitro when specific antigen was added to the cultures, but attempts to generate PFC in vitro with cells from efferent lymph draining non-immunized nodes failed.

Spontaneous rosette-forming lymphocytes in sheep lymph: A marker for antigen-binding cells

We have determined the frequency of rosette-forming cells (RFC) in lymphocytes from the efferent lymph of sheep. Very low numbers of RFC were present in the efferent lymph of non-immunized lymph nodes, but after immunization with heterologous erythrocytes the frequencies of specific RFC and of plaque-forming cells (PFC) increased. The number of RFC remained elevated following the decline and ultimate disappearance of PFC in the lymph. Although most PFC were found to be RFC, the converse was not true. The number of RFC in efferent lymph always exceeded the number of PFC. Most RFC were found in nylon wool-adherent purified B cell fractions, whereas the highest number of PFC were regularly found in the cell fraction which passed through the nylon wool.

Differential sensitivity of memory cell subpopulations to anti-immunoglobulin and complement.

Evidence is presented for the unique sensitivity of memory cells bearing surface immunoglobulin G1 (IgG1) to functional elimination with anti-immunoglobulin (anti-Ig) sera and complement (C). Treatment of cells for adoptive transfer with C and anti-gamma1, anti-kappa, or anti-Ig significantly reduces the number of plaque-forming cells (PFC) of only the IgG1 isotype found in adoptive recipients. An increase in PFC of other isotypes accompanies the decrease in IgG1 PFC; there is no net change in the total PFC response. The depletion of IgG1 PFC requires treatment of transferred cells with both specific antisera and C; antisera directed against other isotypes show no significant effects. The maintenance of the magnitude of PFC response, compensation, is discussed.

Formation of heterophile antibodies by human tonsilar lymphocytes: I. In vitro stimulation with heterologous antigens

Short term cultures of human tonsilar lymphocytes (HTL) in a medium with human AB serum were studied for production of plaque forming cells (PFC) against bovine (BRBC) and sheep red blood cells (SRBC) following in vitro stimulation by various antigens. Stimulation by BRBC or SRBC resulted in production of significant number of PFC against BRBC and SRBC, 100 to 500/10 6 HTL, 4 to 8 days after initiation of the cultures. Seven of 10 HTL specimens stimulated by BRBC produced PFC against both BRBC and SRBC and three gave negative results. Of 12 SRBC-stimulated HTL samples, five produced PFC against both SRBC and BRBC, four against SRBC only and the remaining three gave negative results. Evidence was presented that E rosetting cells (T) as well as plastic-adhering cells were required for the in vitro PFC responses. Stimulation of HTL by murine thymocytes or by murine lymphoma EL-4 cells resulted also in formation of PFC against both BRBC and SRBC. Antibodies secreted by the PFC were studied by inhibition tests with solubilized heterophile antigens. These studies demonstrated that Hanganutziu-Deicher antibodies were secreted by PFC produced by HTL stimulated by BRBC as well as by SRBC and that Forssman antibodies were secreted by PFC formed by SRBC-stimulated HTL. Demonstration of P-B antibodies was not unequivocal. Stimulation by Hanganutziu-Deicher antigen induced in seven of 19 HTL specimens formation of PFC against BRBC and SRBC and stimulation by Paul-Bunnell antigen resulted in formation of PFC against BRBC or against BRBC and SRBC in four of 25 specimens.