IgG anti-tetanus toxoid antibody production induced by Epstein-Barr virus from B cells of human marrow transplant recipients

This investigation shows that Epstein-Barr virus (EBV)-activated human B cells from marrow transplant recipients can produce in vitro IgG anti-tetanus toxoid antibody (anti-TT) without booster immunizations with tetanus toxoid (TT). Purified B cells (E-rosette negative) from 8 normal subjects, 6 healthy long-term marrow graft recipients, and 15 long-term marrow graft recipients with chronic graft-vs-host disease (GVHD), were stimulated for 12 days with EBV to induce anti-TT production in culture supernatants. The amount of anti-TT in culture supernatants was quantitated using a enzyme-linked immunosorbent assay. B cells from all 8 normal controls produced in vitro IgG anti-TT after EBV stimulation. Five of 6 healthy recipients had B cells that produced anti-TT after EBV stimulation. Four of 15 recipients with chronic GVHD had B cells capable of producing anti-TT after EBV stimulation. The number of cultures making anti-TT responses was less in those with chronic GVHD than in those without chronic GVHD or normal individuals (P less than 0.001). B cells from patients with chronic GVHD had fewer responses exceeding the overall median of 0.7 ng/ml when compared with the other two groups (P less than 0.03). These data show that B cells of donor origin can produce in vitro IgG anti-TT antibody to tetanus toxoid antigen in a T-independent fashion.     

Characterization of peripheral blood acetylcholine receptor-binding B cells in experimental myasthenia gravis

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.     

Idiotypic determinants on human T cells and modulation of human T cell responses by anti-idiotypic antibodies

The role of idiotypic anti-idiotypic interactions in the regulation of the human T cell response to tetanus toxoid (TT) antigen was examined in three subjects. Rabbit anti-idiotypic (anti-Id) antisera were raised against IgG (Fab')2 anti-TT obtained 7 to 10 days after booster immunization with TT. F(ab')2 fragments of rabbit-anti-Id IgG were used in conjunction with fluorescein-conjugated goat anti-rabbit Ig in an indirect immunofluorescence assay to determine the frequency of Id-positive cells in T cell-enriched preparations. This frequency was 24, 29, and 38 per 10,000, respectively, in the three subjects studied. Significant contribution of contaminating B cell to fluorescence-staining was ruled out by capping experiments using goat anti-human Ig (GAHIG) and by double staining experiments using rhodamine-conjugated GAHIG. Absorption of anti-Id antisera with Epstein Barr virus (EBV)-transformed B cell lines from the IgG (Fab')2 anti-TT donor, but not with EBV-B cell lines from unrelated donors, removed their reactivity with the T cells. Rabbit anti-Id IgG caused minimal proliferation (two-threefold) of T cells and had no effect on T cell proliferation in response to TT antigen when added to the cultures. Preincubation of T cells for 48 hr with rabbit anti-Id IgG (Fab')2, but not with preimmune rabbit IgG (Fab')2, resulted in the generation of antigen-specific suppressor cells that inhibited T cell proliferation in response to TT, but not in response to diphtheria toxoid (DT). These cells also inhibited the synthesis of IgG anti-TT in response to in vitro stimulation with TT antigen, but not the synthesis of IgG anti-DT in response to DT antigen. Adsorption of T cells over plates coated with rabbit anti-Id IgG (Fab')2 enhanced the proliferative response of the T cells to TT, but not to DT antigen, and enhanced the helper activity of the T cells for the in vitro synthesis of IgG anti-TT but not of IgG anti-DT antibodies. These results suggest that idiotypic-anti-idiotypic interactions play a role in the human T cell response to antigen.     

Specific activation of lymphocytes against acetylcholine receptor in the thymus in myasthenia gravis

In 13 patients with myasthenia gravis, spontaneous in vitro production of antibody to acetylcholine receptor (AChR) by thymic cells was observed in seven patients, by bone marrow cells in nine, by peripheral blood cells (PBL) in six, and by lymph node cells in nine. The rate of anti-AChR production in culture closely correlated with the serum anti-AChR level. Specific activity of the immunoglobulin (Ig) G spontaneously produced (anti-AChR/total IgG) was about 10-fold higher in the thymus than in bone marrow, peripheral blood, or lymph node cultures. Pokeweed mitogen (PWM) enhanced anti-AChR production only by PBL. With neither thymus nor lymph node cells did PWM stimulate anti-AChR production, although it greatly enhanced total IgG production. In bone marrow, it depressed both, and it appeared that the anti-AChR was derived from long-lived plasma cells that may be responsible for delaying the fall of serum anti-AChR levels after thymectomy. The results suggest that AChR-specific cells are selectively activated in the thymus, and this may help to explain the benefits of thymectomy in myasthenia gravis.     

The induction and suppression of in vitro IgG anti-tetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunizations

This report presents a new approach that by-passes booster immunizations with tetanus toxoid (TT) before in vitro studies of antibody (Ab) production. The methodology for optimal TT-induced synthesis of specific IgG anti-tetanus toxoid Ab (IgG anti-TT) by peripheral blood mononuclear cells (PBMC) from randomly selected TT immune individuals without recent booster immunizations is described. PBMC from most normal immune subjects could be repeatedly induced to produce in vitro IgG anti-TT; PBMC from subjects with high TT titers are not required for this new approach. This approach uses high cell concentrations in multiple replicate microcultures and TT washout to obtain optimal IgG anti-TT synthesis. Washed cultures produced more Ab than nonwashed cultures (p less than or equal to 0.005). The readdition of TT (2.5 to 250 ng/ml) to the culture media after washout of TT on day 4 suppressed specific Ab formation, whereas diphtheria toxoid added at comparable doses did not inhibit specific Ab formation. Suppression of antibody synthesis mediated by T cells could be induced by TT per se, and was not due to binding of synthesized Ab to TT in the latter 8 days of culture. In addition, suppression could not be induced in the first 4 days of culture by IgG anti-TT, IgG, or IgM. This approach permits the analysis of antigen-specific regulatory circuits in the steady and activated immune states, and the evaluation of in vivo and in vitro effects of biologic response modifiers on specific Ab production.     

Ecto-5'-nucleotidase expression during human B cell development. An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia

Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in human B cells at different stages of development. Ecto-5'-NT activity of B cell preparations from fetal spleen and cord blood was 5.08 and 5.59 +/- 2.8 nmol/hr/10(6) cells, respectively; that of B cell preparations from adult peripheral blood, spleen, or lymph node was fivefold to sixfold higher (27.9 +/- 12, 29.2 and 33.8 nmol/hr/10(6) cells, respectively). The increased enzyme activity in B cell preparations from adult peripheral blood as compared with cord blood paralleled increased percentages of 5'-NT+ cells (69 +/- 12% vs 32 +/- 17%) and an average of twice as much enzyme activity per positive cell. Small, resting B cells that cannot synthesize Ig in vitro in response to pokeweed mitogen (PWM) were isolated from adult peripheral blood by mouse erythrocyte rosetting. Total ecto-5'-NT activity and the percentage of 5'-NT+ cells were equivalent in total B cells and the mouse erythrocyte rosette-positive subpopulation. Thus, ecto-5'-NT activity is acquired before B cells gain the ability to differentiate into Ig-secreting plasma cells in response to PWM. Ecto-5'-NT activity was also measured in B cell preparations from eight patients with common variable immunodeficiency. Six had reduced ecto-5'-NT activity (2.83 to 15.4 nmol/hr/10(6) cells), and two had normal activity (34.7 and 58.2 nmol/hr/10(6) cells). B cells from all six patients with low ecto-5'-NT activity failed to synthesize Ig when cultured with PWM and normal irradiated T cells. Of the two patients with normal B cell ecto-5'-NT activity, one also had B cells unresponsive to PWM, but B cells from the other patient appeared to more normal, in that they synthesized IgM and IgG when cultured with PWM plus irradiated allogeneic T cells. Thus, measurement of B cell ecto-5'-NT activity allows the subclassification of patients who have a common inability to synthesize immunoglobulin in vitro response to PWM. B cells with low ecto-5'-NT activity are presumably blocked at an earlier stage in development than B cells with normal ecto-5'-NT activity. Evaluation of ecto-5'-NT activity along with the expression of other B cell surface antigens should aid in the definition of discrete stages of B cell development.     

Analysis of the antigen- and mitogen-induced differentiation of B lymphocytes from asymptomatic human immunodeficiency virus-seropositive male homosexuals. Discrepancy between T cell-dependent and T cell-independent activation

Five asymptomatic human immunodeficiency virus (HIV)-seropositive male homosexuals were immunized with the recall antigens tetanus toxoid (TT) and the three types of poliovirus present in diphtheria, tetanus, and polio vaccine. Four weeks after immunization, the in vivo response to booster immunization, the in vitro pokeweed mitogen (PWM)-induced IgG secretion, and the in vitro T cell-dependent and T cell-independent antigen-induced antibody response were assayed. Increase in serum antibody titer to TT and poliovirus was low and normal, respectively. In all five subjects studied, a high rate of spontaneous IgG production, including antibodies directed toward HIV was observed. Addition of PWM to the cultures induced suppression of the spontaneous IgG secretion. Only one donor showed a slightly increased IgG production after stimulation with PWM. Peripheral blood mononuclear cells of four of the five HIV-seropositive individuals did not produce TT, or poliovirus-specific antibodies when stimulated with the respective T cell-dependent antigens. However, stimulation of these peripheral blood mononuclear cells with TT coupled to agarose beads, which was shown to be T cell-independent, resulted in the generation of IgG anti-TT antibody-forming cells.     

Pathophysiologic analysis of peripheral blood lymphocytes from patients with primary immunodeficiency. I. Ig synthesis by peripheral blood lymphocytes stimulated with either pokeweed mitogen or Epstein-Barr virus in vitro

In the present study 5 patients with common variable hypogammaglobulinemia (CVH) and 4 patients with selective IgA deficiency (IgA-D) were analyzed for the cellular defects responsible for impaired Ig synthesis with use of peripheral blood lymphocytes stimulated with either PWM or EBV in vitro. By the use of co-culture with PWM, all the patients examined had intrinsic B cell defects restricted to the synthesis of Ig class corresponding to the low or absent Ig class(es) in the sera. Two types of excessive suppressor T activity were found, which were abrogated by irradiation. One was isotype-nonspecific and the other was IgA-specific. Moreover, failure of IgA-specific helper T activity was demonstrated. The use of EBV as an agent that polyclonally activates B cells independently of T cells and monocytes should allow a clearer delineation of the level of the B cell defects. When co-cultured with EBV, B cells from 3 patients with CVH produced normal to subnormal quantities of IgM although they could produce no IgM upon co-culturing with normal T cells and PWM. B cells from 2 patients with CVH could produce IgM normally by stimulation with either PWM or EBV; however, there was no restoration to produce IgG or IgA in these patients. In addition, B cells from 2 patients with IgA-D produced not only IgG and IgM but also IgA almost normally at 4 days after in vitro stimulation with EBV.     

Transgenic expression of IL-10 in T cells facilitates development of experimental myasthenia gravis

Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8(+)-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8(+)-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-gamma, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.     

The limitation of IL-10-exposed dendritic cells in the treatment of experimental autoimmune myasthenia gravis and myasthenia gravis

Dendritic cells (DC) are highly specialized antigen presenting cells that play critical roles as instigators and regulators of immune responses including B cell function, antibody synthesis and isotype switch. In this study, we compared immunotherapeutic effect of IL-10-treated DC (IL-10-DC) via both intraperitoneal (i.p.) and subcutaneous (s.c.) delivery in rats with incipient experimental autoimmune myasthenia gravis (EAMG). Spleen DC were isolated from onset of EAMG on day 39 post-immunization, exposed in vitro to IL-10, and then injected into incipient EAMG at dose of 1 x 10(6) cells/rat on day 5 after immunization. Intraperitoneal administration of IL-10-DC suppressed clinical scores, anti-acetylcholine receptors (AChR) antibody secreting cells, antigen-specific IL-10/IFN-gamma production and T cell proliferation compared to control EAMG rats. Importantly, IL-10-DC, if given by s.c. route, failed to ameliorate clinical sign of EAMG. Simultaneously, T cell proliferation, anti-AChR antibody secreting cells and IL-10/IFN-gamma production had no alteration, as compared to control EAMG rats. Both in vitro and in vivo experiments showed that treatment of IL-10 inhibited the migration of DC toward MIP-3beta and lymph node, indicating that in vitro manipulation of DC with IL-10 alters the migration of DC that influences the therapeutic effect in the treatment of autoimmune diseases. In MG patients, neither the improvement of clinical symptom nor the alteration of immunological parameter was observed through s.c. delivery of IL-10-DC, suggesting the limitation of IL-10-DC in the treatment of MG patients.     

Human polysaccharide-specific B cells are responsive to pokeweed mitogen and IL-6

The responsiveness of polysaccharide-specific B cells to PWM was examined in vitro. Spleen cells from six patients immunized with Haemophilus influenzae type b-diphtheria toxoid, pneumococcal and meningococcal vaccines were T cell-depleted and separated by Percoll density gradient centrifugation. In each B cell fraction, spontaneous antibody production was demonstrated to capsular polysaccharides as well as diphtheria toxoid. The peak of spontaneous antibody production was demonstrated to be five to seven days after immunization. When T cells and PWM were added, the total Ig secretion increased in all B cell fractions. PWM also enhanced IgG antibody directed to each of three polysaccharide Ag measured. This enhancement was most noticeable for nonresting B cells. The PWM effect was not confined to IgG, as IgM and IgA to Neisseria meningitidis type C were measured and also enhanced. The kinetics of the PWM response demonstrated the most IgG antibody to polysaccharide Ag from spleens immunized five to seven days before splenectomy. When the patients were immunized either 2 days or 4 mo before splenectomy, no spontaneous IgG antibody to polysaccharides was detected although PWM induced small amounts of antibody. Finally, anti-IL-6 antibody blocked PWM-induced total and polysaccharide-specific antibody production. We conclude that human polysaccharide-specific B cells are responsive to PWM and IL-6. We suggest that polysaccharide B cells are not truly "T cell-independent" and may respond to T cell lymphokines and thus are similar to protein-specific B cells.     

IgG subclasses of anti-tetanus toxoid antibodies in adult and newborn normal subjects and in patients with systemic lupus erythematosus, Sjogren's syndrome, and drug-induced autoimmunity

The IgG subclasses of anti-tetanus toxoid (anti-TT) antibodies were quantitated in normal sera and sera from patients with rheumatic disease. Detection relied on a set of four mouse monoclonal antibodies, each of which showed specificity for the respective isotype, independent of gamma-chain allotype or light chain class of the human antibody. Approximately 90% of the total anti-TT activity in normal adults and patients with Sjogren's syndrome was IgG1. In addition, IgG4 antibodies were detected in one-half the samples, but IgG2 and IgG3 antibodies were observed in only two out of 36 sera. However, antibodies elicited in children immunized with TT were exclusively IgG1 and IgG3, with IgG4 antibodies detectable only at birth (presumably due to transplacental passage of antibody) in three of 12 children. In contrast to normal adults, patients with systemic lupus erythematosus (SLE) and drug-induced autoimmunity (DIA) had a more promiscuous isotype profile. IgG2 and/or IgG3 anti-TT antibodies were detected in 13 of 22 SLE patients and IgG3 antibodies in six of 11 patients with DIA. IgG4 anti-TT antibodies were predominant in seven of these 33 patients. These findings suggest that IgG isotypes may depend on the frequency of the stimulus, but global alterations in immunologic status as reflected in systemic autoimmune disease may override the homeostatic mechanisms that control isotype restriction.     

Development of an antigen-specific CD8 suppressor effector clone in man

A long-term cultured IL-2-dependent keyhole limpet hemocyanin (KLH)-specific CD8 (T8) suppressor clone (5B9) was generated from a healthy donor hyperimmunized with KLH. The 5B9 clonal population suppressed in vitro anti-KLH antibody response but did not suppress anti-TT antibody response or PWM-driven IgG synthesis. Moreover, 5B9 cells could not suppress anti-TT antibody response even in the presence of free KLH. 5B9 cloned cells suppressed the anti-KLH antibody response of B cells cultured with CD4+4B4+ cells without requiring the presence of CD8+ cells. This KLH-specific CD8 suppressor clone is an effector type rather than an inducer type of suppressor T cell. The cloned cells expressed alpha- and beta-TCR proteins (defined by WT-31 antibody) on their cell surface. More importantly, the CD3:TCR complex was functionally important in the suppression induced by this clone, because after CD3 antigen modulation from its cell surface, the suppressor effector function was abolished.     

Interferon acts directly on human B lymphocytes to modulate immunoglobulin synthesis

At different times of exposure, interferon (IFN) enhanced and suppressed pokeweed mitogen- (PWM) induced IgG synthesis by human peripheral blood lymphocytes (PBL). Pretreatment of PBL and IFN frequently increased antibody production by more than 100% when compared with that by untreated PBL. Results of experiments in which PBL were separated into T and B subpopulations indicated that IFN preparations acted directly on B cells. Thus, mixtures of IFN-treated B cells and untreated T cells from 5 of 7 persons tested produced 81% to 500% more IgG than untreated, matched control cells. However, IFN-treated monocytes mixed with untreated B and T cells or IFN-treated T cells mixed with untreated B cells failed to enhance IgG production significantly in similar assays. In contrast to the pretreatment protocol, when IFN was present in the incubation mixture throughout the PWM assay, IgG production decreased. Sephadex chromatography of the IFN and tests of the resulting fractions indicated that the IgG production-enhancing activity was located in the fraction carrying the antiviral activity.     

Differential synthesis of IgM and IgA anti-tetanus toxoid antibody in vitro following in vivo booster immunization of humans.

The in vitro syntheses of IgM and IgG anti-tetanus toxoid antibody by human peripheral blood leukocytes were compared prior to and at various intervals following in vivo booster immunization with soluble tetanus toxoid. Prior to booster immunization, the in vitro synthesis of IgG anti-tetanus toxoid antibody by combinations of B cells and irradiated T lymphocytes was negligible following pokeweed mitogen stimulation. Within 2 weeks after booster immunization, the quantity of IgG anti-tetanus toxoid antibody synthesized in vitro increased 5- to 20-fold. There was no comparable increase in total IgG synthesis. In contrast to the synthesis of IgG antibody, in vitro synthesis of IgM anti-tetanus toxoid antibody occurred prior to booster immunization and did not increase significantly following booster immunization. This dichotomy in anti-tetanus antibody production was further demonstrated in an individual with common variable hypogammaglobulinemia whose lymphocytes synthesized normal quantities of total IgG, IgM, and IgM anti-tetanus toxoid antibody in vitro, but failed to synthesize IgG anti-tetanus antibody following in vivo booster immunization.     

The relationship between immunization and circulating antigen-specific plaque-forming cells

The relationship between the appearance of cells producing antibody to tetanus toxoid (TT) in the circulation and the serum titers of anti-TT IgG following booster immunization has been studied. It was found that cells producing anti-TT antibody can be detected in the circulation in a hemolytic plaque assay using sheep red blood cells (SRBC) coated with TT by the chromic chloride method. In symmetric inhibition studies using cells from TT or keyhole limpet hemocyanin (KLH)-immune donors, the homologous antigen inhibited 100% of the PFC with no cross-inhibition. Thus, the plaque-forming cells (PFC) detected in this assay are specific for the immunizing antigen. No evidence of polyclonal B-cell activation in response to TT was found, as shown by a failure to detect any PFC against unmodified or KLH or human serum albumin-treated SRBC. In addition, the increase in total Ig-secreting cells observed in a staphylococcal protein A reverse hemolytic plaque assay was always accounted for by the number of anti-TT antibody-producing cells observed. The peak number of anti-TT antibody-producing cells varied between donors, but the kinetics of their appearance was highly reproducible--none before Day 5, peak numbers between Days 6 and 8, and a sharp decline with only rare anti-TT Ig-secreting cells in the circulation by Day 15 postimmunization. Anti-TT antibody-producing cells appeared in the circulation prior to any detectable increase in serum anti-TT antibody titers, and following the disappearance of PFC from the circulation, there was no further increase in serum IgG anti-TT levels. These observations demonstrate a marked specificity of B-cell activation on boosting with a recall antigen, and a parallelism between the appearance of activated B cells in the circulation and of IgG anti-TT synthesis by the subject as a whole.     

Direct Proof of the In Vivo Pathogenic Role of the AChR Autoantibodies from Myasthenia Gravis Patients

Several studies have suggested that the autoantibodies (autoAbs) against muscle acetylcholine receptor (AChR) of myasthenia gravis (MG) patients are the main pathogenic factor in MG; however, this belief has not yet been confirmed with direct observations. Although animals immunized with AChR or injected with anti-AChR monoclonal Abs, or with crude human MG Ig fractions exhibit MG symptoms, the pathogenic role of isolated anti-AChR autoAbs, and, more importantly, the absence of pathogenic factor(s) in the autoAb-depleted MG sera has not yet been shown by in vivo studies. Using recombinant extracellular domains of the human AChR α and β subunits, we have isolated autoAbs from the sera of four MG patients. The ability of these isolated anti-subunit Abs and of the Ab-depleted sera to passively transfer experimental autoimmune MG in Lewis rats was investigated. We found that the isolated anti-subunit Abs were at least as efficient as the corresponding whole sera or whole Ig in causing experimental MG. Abs to both α- and β-subunit were pathogenic although the anti-α-subunit were much more efficient than the anti-β-subunit ones. Interestingly, the autoAb-depleted sera were free of pathogenic activity. The later suggests that the myasthenogenic potency of the studied anti-AChR MG sera is totally due to their anti-AChR autoAbs, and therefore selective elimination of the anti-AChR autoAbs from MG patients may be an efficient therapy for MG.     

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.     

DNA and immunoglobulin synthesis by rabbit peripheral blood lymphocytes in vitro: complete and incomplete stimulation

Activation of resting (G0) rabbit peripheral blood lymphocytes (PBLs) into DNA synthesis and IgG synthesis was studied using sheep anti-rabbit IgG (SARIgG), protein A, pokeweed mitogen (PWM), and lipopolysaccharide (LPS). DNA synthesis was assayed by [125I]iododeoxyuridine incorporation. IgG synthesis was measured by determination of Ig in culture supernatants by an ELISA assay. Rabbit PBLs cultured with SARIgG or protein A for 48 hr and then without these reagents for 72 hr showed both DNA synthesis and Ig synthesis, whereas PWM and LPS had very little, if any, effect. PBLs stimulated with SARIgG for 6 hr and then without SARIgG for subsequent 114 hr did not become activated into DNA synthesis or IgG synthesis. However, PBLs prestimulated with SARIgG for 6 hr and then with PWM for 114 hr showed prominent DNA and IgG synthesis. LPS also maintained activation of PBLs after prestimulation of these cells with SARIgG, but the effect was much smaller than that of PWM. No evidence was found for production of factors by SARIgG-stimulated PBLs that could, by themselves, either stimulate resting cells or maintain activation of SARIgG-prestimulated cells. These results suggest that anti-IgG and protein A are complete activating mitogens for resting rabbit B cells to proliferate and differentiate into IgG-producing cells, whereas PWM and LPS are not able to activate G0 cells directly, but have a sustaining effect after activation of resting B cells with anti-IgG, either directly or via production of factors by accessory cells.     

Role of interleukin 2, interleukin 4 and interleukin 5 in the T helper cell-driven B cell polyclonal differentiation in the elderly.

There is evidence for an impaired T cell-mediated B cell response during senescence. In thirty aged donors, pokeweed mitogen (PWM)-driven immunoglobulin (Ig) synthesis by B cells co-cultured with autologous enriched CD4+ lymphocytes and low amounts of monocytes, was evaluated. Under such experimental conditions, elderly cultures displayed a reduced IgG and/or IgM production when compared with the younger counterpart. Moreover, interleukin (IL)-2 and/or IL-5 addition to cultures led to an enhancement of Ig release. In contrast, IL-4 supplementation failed to positively modulate B cell differentiation. At the same time, aged cells cultured in the presence of IL-2 + IL-5 exhibited an increased Ig synthesis, while the addition of IL-2 + IL-4 or IL-4 + IL-5 mixtures did not induce any significant effect in comparison with homologous untreated samples. The results suggest a critical role for IL-2, IL-4 and IL-5 in the modulation of T helper cell-driven B cell polyclonal responsiveness in the elderly.