Polyclonal Antibody Synthesis Research Articles

Mathematical Model to Predict Polyclonal T-Cell-Dependent Antibody Synthesis Responses

Mathematical models are becoming indispensable tools to explore the complexities of biological systems at cellular levels. We present a model to explore the baseline immune cell interactions for in vitro polyclonal antibody synthesis via B-cells regulated by helper and regulatory T-cells. The model incorporates interactions of antigen-presenting cells, T-cells, regulatory T-cells, and B-cells with each other and predicts time-dependent trajectories of these cells and antibody synthesis stimulated by pokeweed mitogen. We used an ordinary differential equation-based approach to simulate the dynamic changes in the cells and cytokines numbers due to the cellular and humoral response to pokeweed mitogen stimulation. The parameters of the ordinary differential equations model are determined to yield a normal immune response as observed in the pokeweed mitogen-stimulated in vitro antibody synthesis via normal T, B, and antigen-presenting cells. The dose effects of antigen load and basal values of regulatory T-cells on the profiles of various immune response variables are also evaluated.

Erythema nodosum leprosum is associated with up-regulation of polyclonal IgG1 antibody synthesis.

Erythema nodosum leprosum (ENL) is a serious complication of lepromatous (L) disease in leprosy. We have previously shown that of the four IgG subclasses, IgG1 and IgG3 Mycobacterium leprae-specific antibodies are significantly lower in leprosy patients during ENL reaction compared with untreated L patients. To see if this decrease results from a down-regulation of antibody synthesis during ENL, the frequency of antibody-secreting B cells (ABSC) in the blood compartment was determined by ELISPOT and related to serum immunoglobulin concentrations (microgram/ABSC). Control groups consisted of 16 patients with stable L disease and 32 healthy endemic controls (EC). Paired samples were analysed during acute ENLS (n = 13) and after the reaction had subsided to identify changes associated with ENL. Polyclonal (PC) IgG1 was elevated in L patients compared with EC (325 micrograms versus 180 micrograms). Interestingly, patients during acute ENL showed concentrations higher than L patients (419 micrograms), which decreased after the reaction had subsided (260 micrograms), indicating the transient nature of the antibody response. IgG2 antibodies showed the reverse trend and were lower during ENL and increased after the reaction had subsided. The mean concentrations for PC IgG3 and IgG4 antibodies were similar during ENL and after the reaction had subsided. Thus, decrease in M. leprae-specific IgG1 and IgG3 antibodies is not related to down-regulation of B cell responses. Identification of factors which regulate PC IgG1 antibody synthesis may provide additional insights into determinants of ENL reactions.

Antibody production in autoimmune BXSB mice. I. CD40L-expressing B cells need fewer signals for polyclonal antibody synthesis.

Male, but not female, BXSB mice develop severe lupus associated with multiple immune system defects. It was recently shown that one immunological abnormality found in male BXSB mice encompasses B cell expression of CD40 ligand (CD40L) by an expanded population of large B cells. The present study was undertaken to determine how the CD40L-expressing large B cells in male BXSB mice compared with size-matched B cells from female mice in terms of their ability to secrete antibody. It was shown that the large B cells from female mice, similar to the small B cells from either male or female mice, required CD40 signalling, immunoglobulin cross-linking and cytokines for optimal antibody synthesis. In contrast, large B cells from male BXSB mice produced high levels of antibody when stimulated with only two of the three signals, and made significantly more total IgM and IgG, and anti-ssDNA antibody than size-matched B cells from female mice when stimulated with IL-4/IL-5 alone, IL-4/IL-5 plus low levels of anti-IgD-dextran, or IL-4/IL-5 plus anti-CD40 MoAb. The ability of the large B cells from male mice to produce antibody under suboptimal stimulatory conditions correlated with their expression of CD40L, and was inhibited by CD40-immunoglobulin. Taken together, these findings suggested that large CD40L-expressing B cells from male BXSB mice may be able to bypass a need for CD40 signalling from T cells, thus contributing to autoimmune disease by promoting antibody production in the absence of cognate T cell help.

Circulating antibodies directed against tryptophan-like epitopes in sera of patients with human African trypanosomiasis.

Human African trypanosomiasis is often associated with an intense proliferation of B lymphocytes, leading to polyclonal antibody synthesis. Using a modified enzyme-linked immunosorbent assay method, we have found highly significant levels of circulating anti-conjugated tryptophan-like epitope antibodies in sera of patients with sleeping sickness. These antibodies were immunoglobulins (Ig) of the M isotype. There was no correlation between immunologic binding and the Ig levels found in sera of patients with human African trypanosomiasis. Higher antibody levels in stage II of the disease than in stage I may be related to damage to the central nervous system. The specificity of this immunologic binding was evaluated by 1) comparison with that obtained with other related conjugates and 2) serum titration. Anti-conjugated tryptophan-like epitope antibodies were not found in other neurologic diseases tested. Their involvement in this pathology remains unknown.

Immunologic response of adenoidal lymphocytes to respiratory syncytial virus.

Adenoidal lymphocytes obtained from 43 subjects with serum antibodies to respiratory syncytial virus (RSV) were established in culture in vitro and analyzed for immunoglobulin (Ig) and RSV-specific antibody synthesis. Spontaneous synthesis of Ig was consistently observed in culture supernatants. The ratios of IgA to IgG and IgM to IgG in adenoidal lymphocyte culture supernatant were higher than in serum. In cell cultures stimulated with RSV or pokeweed mitogen, RSV antibody activity was detected in 25 of 43 (58.1%) for IgG, 5 of 43 (11.6%) for IgA, and 4 of 43 (9.3%) for IgM. Also, RSV-specific IgG was detected in some supernatants from unstimulated cultures. In seven cases the cultures of autologous tonsillar lymphocytes were also investigated. Autologous organs exhibited similar polyclonal Ig production and RSV-specific antibody synthesis. These observations demonstrate that both adenoids and palatine tonsils are continuously engaged in synthesis of local antibodies to viral pathogens available to the nasopharynx and respiratory mucosa.

Suppression of C3H/HeJ cell activation by lipopolysaccharide endotoxin.

Earlier studies in our laboratory showed that the lipopolysaccharide (LPS) of Salmonella typhi, which fails to activate B lymphocytes of C3H/HeJ mice, can suppress proliferation and polyclonal antibody synthesis by these cells when they are stimulated by polyclonal activators. In order to determine what stage of the cell cycle was blocked, resting B cells from C3H/HeJ spleens were activated by using different mitogens in the presence of inhibitory concentrations of LPS and analyzed by flow cytometry, using acridine orange to stain DNA and RNA. LPS was found to inhibit the progression of cells into the G1 stage of the cell cycle. Furthermore, [3H]uridine uptake studies showed that RNA synthesis is inhibited during the early phase of activation. These results indicate that inhibition by LPS of the signalling process occurs during a critical period of the cell cycle when the cells become susceptible to the inhibitory effects of LPS. To examine whether LPS acts only on B cells or whether it can suppress other immunocompetent cells from C3H/HeJ mice, studies were carried out on activated thymocytes and macrophages. LPS was found to inhibit thymocyte proliferation stimulated by concanavalin A or the combination of phorbol myristate acetate and ionomycin. Prostaglandin E2 synthesis by macrophages was also blocked by LPS. Thus, LPS is a potent inhibitor of the functioning of the major immunocompetent cells of C3H/HeJ mice.

Thymus‐Independent B‐Cell Activation: Passively Incorporated Membrane Antibodies Serve to Focus the Antigen but Cannot Transmit Activation Signals

We have activated B cells with membrane-incorporated monoclonal antibodies against the hapten trinitrophenyl (TNP) using various concentrations of the polyclonal activator lipopolysaccharide (LPS) haptenated with TNP. We found that anti-TNP-decorated B cells were polyclonally activated by 1000-fold lower concentrations of TNP-LPS than untreated B cells or B cells decorated with antibodies of other specificities. In order to test whether the passively incorporated anti-TNP monoclonals could mediate activation signals or only served to focus the polyclonal activator TNP-LPS to the B cells, we compared the response of anti-TNP-decorated B cells from normal C3H/He mice and from the LPS-unresponsive strain C3H/HeJ. We found that B cells from C3H/HeJ mice could not be activated to polyclonal antibody synthesis, in contrast to B cells from the LPS-responsive strain C3H/He. Thus, contrary to what was previously suggested, the incorporated anti-TNP antibodies could not mediate activation signals, but only served to passively bind and concentrate TNP-LPS to the membrane of the B cells, thereby increasing the concentration of the polyclonal B-cell activator LPS to the B cells.

Inhibition of activated nonresponder C3H/HeJ lymphocytes by lipopolysaccharide endotoxin.

The B lymphocytes and macrophages of lipopolysaccharide (LPS) nonresponder C3H/HeJ mice were found to respond to certain R types of LPS endotoxin in a fashion resembling that ordinarily seen with the cells from normal responder mice. DNA synthesis, polyclonal antibody synthesis, and interleukin-1 activity were stimulated by Bordetella pertussis LPS and Salmonella minnesota R595 LPS, although to a lesser extent than with responder cells. Mitogenesis stimulated by both LPSs was inhibited by polymyxin B; this finding provided evidence that any trace endotoxin-associated proteins were not responsible for the activity. Of particular interest was the finding that wild-type smooth LPS actually inhibited activation of the C3H/HeJ B cells not only by the LPS but also by mitogenic proteins, including purified protein derivative of tuberculin. The nonspecific nature of this inhibition and the fact that maximal inhibition occurred some 9 to 12 h into the culture period suggested that the proliferation of the B cells was affected by smooth-type LPS in a manner heretofore unrecognized. These findings permit a new approach to the study of how LPS endotoxin affects cells of the immune system.

In vitro production of antibody in cultures of human peripheral blood lymphocytes

This chapter describes in vitro techniques for the induction of polyclonal antibody synthesis. One of these techniques involves the use of human helper/inducer T cell clones that is also able to induce type-E immunoglobulin (IgE) antibody synthesis. The in vitro induction of antibody synthesis in murine systems has been a powerful technique available for many years; it has allowed investigators to dissect the requirements for the activation of B lymphocytes. The induction of antibody synthesis in human B cells has been more difficult, mostly because of the difficulty in immunizing human subjects. In induction of immunoglobulin synthesis, peripheral blood mononuclear cells (PBMC) from normal subjects can be stimulated in vitro with pokeweed mitogen (PWM) to produce antibody of the IgG, IgM, and IgA isotopes. The technique is relatively simple but IgE isotype responses cannot be induced. The FCS should be screened for the ability to support in vitro induction of antibody synthesis.

Polyclonal response of human lymphocytes to Chlamydia trachomatis.

The mitogenicity of Chlamydia trachomatis was investigated. Umbilical cord blood cells from healthy newborns were used. These cells were stimulated in cultures with partially purified C. trachomatis elementary bodies. Proliferation of cultured lymphocytes and secreted immunoglobulins and leukocyte migration inhibitory factor were measured in the culture supernatants. Our results showed that C. trachomatis is able to elicit polyclonal antibody, lymphokine, and DNA synthesis.

Suppression of polyclonal immunoglobulin production by a soluble factor produced by a human thymus hybridoma

A human thymus cell hybridoma was established using thymus cells obtained from a patient with common variable hypogammaglobulinemia and associated thymoma. This hybridoma secreted a suppressor factor for polyclonal antibody synthesis. Supernatants of this hybrid showed 40–80% suppression of both IgM and IgG synthesis by pokeweed mitogen-stimulated human peripheral blood lymphocytes. Hybridoma supernatants were suppressive for immunoglobulin production only if added within the initial 48 h of the seven-day culture period. Suppression of antibody production by the hybridoma supernatant was prevented by preabsorption with T lymphocytes. Further, the suppressor factor was shown to inhibit antibody production in reconstructed cultures containing T4+ cells and B cells, yet the suppression could be abrogated by increasing the number of T4+ cells. The hybrid supernatant had no affect on the proliferation of human mononuclear cells in response to pokeweed mitogen, lipopolysaccharide, concanavalin A or alloantigen but inhibited phytohemagglutinin-induced proliferation. The target cell population for the inhibition of phytohemagglutinin responsiveness was shown to be a T4+ lymphocyte (helper inducer T cell). These results suggest that thymus hybridoma cells can produce immunoregulatory products that act through the modulation of T4+ lymphocyte function. To our knowledge this is the first human thymus cell hybridoma to be reported. Studies on such cell lines may provide important information on immunoregulatory thymic factors.

Salivary gland lymphocytes in primary Sjogren's syndrome lack lymphocyte subsets defined by Leu-7 and Leu-11 antigens.

Primary Sjogren's Syndrome (SS) is an autoimmune disease characterized by dry eyes and dry mouth due to lymphocytic infiltration of lacrimal and salivary glands. Biopsies of their salivary glands provided an opportunity to characterize the phenotypic and functional properties of inflammatory site lymphocytes. We found that the salivary gland lymphocytes (SGL) of SS patients differed from the peripheral blood lymphocytes of the same patients because: a) SGL lacked lymphocytes reactive with anti-Leu-7 and anti-Leu-11 monoclonal antibodies; b) SGL lacked natural killer (NK) activity; and c) SGL lacked the ability to suppress polyclonal B cell responses in the presence of complement fragment C3a, a function that requires the presence of Leu-7+ cells. These studies also showed that the SGL of SS patients differed from tonsillar lymph node (LN) lymphocytes of immunologically normal individuals because tonsillar LN contained Leu-7+ T cells, and tonsillar LN could suppress polyclonal B cell responses in the presence of the complement fragment C3a. The absence of this regulatory subset in the salivary glands of SS patients may contribute to pathogenesis, because these cells may be important in the suppression of polyclonal antibody synthesis and in the elimination of neoplastic or viral infected cells.

The B cell repertoire revealed by major histocompatibility complex-specific helper T cells. I. Frequencies of a genetically defined V region marker among mitogen- and T helper cell-reactive B lymphocytes in normal and immunized mice.

The aim of the present work was to analyze the frequencies of a genetically defined variable (V) region marker in the B cell subset sensitive to T cell help. To this end we used an alloreactive T cell line that has the property of inducing B cells of the appropriate haplotype to exponential growth and polyclonal antibody synthesis. The frequency obtained with this helper line was also directly compared to that obtained with lipopolysaccharide (LPS). We found that in normal BALB/c mice the frequency of M460-positive clonotypes was respectively, 1/100 and 1/1,000 among the T helper- and LPS-sensitive B cell subsets. In mice immunized with antiidiotype coupled to a thymus-dependent antigen, the differences in the numbers of idiotype-positive precursors were even more accentuated, i.e. 1/20 in the B cell subset triggered by T helper cells and 1/800 in those cells responsive to LPS. The frequencies of the M460 determinant in mice immunized with anti-idiotypes coupled to thymus-independent antigens were not significantly different, in either B cell subset, from those obtained with spleen cells of normal nonimmunized animals. Taken as a whole, our results imply that the V gene repertoire revealed by LPS includes precursor distribution, as this distribution occurs during the early stage of B cell development (potential repertoire), while the repertoire revealed by T helper cells includes the V region distribution of those clones that are selected in the periphery of the functional immune system.

Modification of lipopolysaccharide non-responder cells in low cell density culture.

Lipopolysaccharide (LPS) fails to induce polyclonal antibody responses in LPS non-responder C3H/HeJ mice in vivo and also in in vitro cultures of 10(7) cells/ml of C3H/HeJ spleen cells. However, this study shows that LPS elicits polyclonal antibody synthesis in low cell density cultures of C3H/HeJ cells by activation of a translational process without an increase in total RNA and messenger RNA synthesis. Polyclonal antibody synthesis by LPS non-responder cells in low cell density culture was significantly increased by LPS in either serum-free medium or medium containing serum. LPS-induced antibody production by LPS non-responder cells in low cell density was not due to the relative elevation of LPS concentrations.

Carrageenans, Highly Sulfated Polysaccharides and Macrophage-Toxic Agents: Newly Found Human T Lymphocyte Mitogens

1. CGNs were toxic to human monocytes, cytotoxicity varying from 40 % to 60 %, as judged by trypan blue dye exclusion test. 2. CGNs were proved to be newly found mitogens for human T cells, whereas CGNs are polyclonal B-cell activators in mice and induced DNA synthesis that peaked at days 6 or 7. In addition, L CGN induced an early peak of 3H-thymidine uptake at day 1, which found out to be cytoplasmic uptake of 3H-thymidine. 3. Only λ and ι CGN induced slightly higher DNA synthesis in purified B cells, but both failed to induce polyclonal antibody synthesis in B cells. 4. In spite of the fact the CGNs are macrophage-toxic agents, CGNs could induce II-1 production by the surviving monocytes. Thus CGN-induced human T-cell activation requires a relatively smaller number of monocytes. 5. The mitogenic responses of T cells induced by ϰλand ι CGN were significantly inhibited by cyclosporin A (CyA) treatment (250 ng/ml) and Con A and PHA responses were also inhibited by CyA addition. It was concluded that CGNs were newly found human T-cell mitogens, which is in sharp contrast with polyclonal B-cell activators in mice, and the mechanism of T-cell activation induced by CGNs was the same as that by Con A or PHA, though there may be another possibility of inhibition mechanism by CyA.

Role of Fc receptors in lymphocyte activation: deficiency in T- and B-lymphocytes from aged animals

Fc fragments derived from human immunoglobulin possess the ability to induce B-cell proliferation. polyclonal antibody responses, and augment cell-mediated and T-dependent humoral responses. However, aged animals display much lower responses to Fc fragment stimulation. Proliferation and polyclonal antibody synthesis are reduced two-five-fold in aged animals compared to the young-adult responses. Furthermore, Fc fragments are unable to potentiate plaque-forming cell (PFC) development in aged animals. Aged B-lymphocytes are deficient in responding to Fc fragments, as their admixture with young-adult T-cells does not restore polyclonal antibody formation. T-cells from aged animals are also ineffective in promoting the polyclonal response when mixed with young-adult B-cells. The T-cell lesion has been further defined as a deficiency in production of an Fc fragment induced T-cell-replacing factor [(Fc)TRF]. The inability of Fc fragments to potentiate anti-SRBC responses in aged animals is also due to a T-cell defect which can be corrected by supplementation with interleukin-2 (IL-2).

Activation and immunoregulation of antigen-specific human b lymphocyte responses: multifaceted role of the monocyte.

The multifaceted role of the monocyte in the induction and modulation of antigen-specific antibody responses by human B cells was delineated. Monocytes were absolutely required for the induction of specific antibody responses to both TT and KLH in an antigen-induced in vitro assay. Monocytes were also required for the PWM induction of specific antibody in immunized subjects. Pulsing monocytes with specific antigen or with PWM consistently stimulated proliferation of T cells in absence of added antigen and could also stimulate specific antibody synthesis although less consistently. Stimulation of specific antibody responses with antigen required fewer numbers of monocytes than did stimulation of specific antibody responses with PWM. Polyclonal antibody synthesis induced by PWM was also dependent on monocytes. However, polyclonal antibody synthesis induced by supraoptimal concentrations of antigen was usually optimal in the absence of monocytes and was actually suppressed when increased numbers of monocytes were added to monocyte-depleted cultures. Monocyte supernatants could not replace the absolute requirements for monocytes in the induction of specific antibody synthesis. However, monocyte supernatants could profoundly modulate the antigen-specific as well as the polyclonal Ig response of lymphocytes to either antigen or PWM stimulation in a manner closely resembling monocytes themselves. Thus, we demonstrated that monocytes and their products play a critical role in the activation and immunoregulation of antigen-specific antibody responses of human B cells.

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.

Polyclonal antibody synthesis by bone marrow cells of New Zealand Black mice. II. Cellular basis of PAS activity of NZB bone marrow cells.

In the preceding report, enhanced polyclonal antibody synthesis (PAS) activity of bone marrow cells (BMC) of NZB mice was shown. Cells involved in its activity among BMC population were explored in this report. Antibody-forming cells (AFC) already existing in the bone marrow were not responsible for this activity since depletion of AFC by passage through a Sephadex G-10 column did not affect the numbers of anti-TNP AFC and immunoglobulin-secreting cells (IgSC) in the spleen of BMC-reconstituted mice when assayed 7 days after the transfer. Thy-1-positive cells among BMC population and host thymus were not relevant to PAS. Suppressor cells seemed to play little role as observed in transfer of mixed BMC population from NZB and BALB/c mice. PAS activity was prominently observed in surface immunoglobulin-positive cells. Significance of enhanced PAS activity was discussed with reference to autoantibody production.

Serum-mediated suppression of nonspecific B cell activation. II. Relative resistance of B cells from the NZB mouse strain to regulation by a natural inhibitor in normal mouse serum.

Recent work has shown that normal mouse serum (NMS) and plasma contain inhibitory substance(s), termed NMS-In, that suppress proliferative and polyclonal antibody responses elicited in vitro with various B cell mitogens. In this study, the NZB mouse, which has a high degree of spontaneous polyclonal B cell activity, was examined for possible reduced amounts of NMS-In. Also, the capacity of NZB B cells to be inhibited by NMS-In was determined. It was found that sera from NZB mice had normal amounts of NMS-In, as assessed by inhibition of mitogenesis of spleen cells in culture induced by bacterial endotoxin (ET). However, B cells of the NZB mouse were found to be abnormal, requiring approximately 4 to 7 times more NMS-In to inhibit polyclonal antibody synthesis and mitogenesis elicited by ET than B cells of more immunologically normal mice. Removal of T cells by treatment with anti-thy 1.2 and complement did not change the sensitivity of the NZB B cells to NMS-In. Insensitivity to NMS-In was expressed in B cells from relatively young NZB mice, e.g., 9 to 15 wk of age. The relative resistance of B cells of the NZB mouse to regulation by a natural inhibitor in NMS could partially serve to explain their high level of spontaneous antibody synthesis. Furthermore, the fact that B cells of the NZB mouse were ultimately susceptible to inhibition by relatively large amounts of NMS-In affords the opportunity in the future to suppress polyclonal antibody activity in the NZB mouse with purified NMS-In in an attempt to ameliorate autoimmune disease.