Immunoglobulin class and subclass distribution of antibodies reactive with the immunodominant antigen of Actinobacillus actinomycetemcomitans serotype b

The aims of this study were to determine the immunodominant antigens of Actinobacillus actinomycetemcomitans serotype b (Aab) for the different immunoglobulin (Ig) classes and subclasses and to determine the relative levels of these different Igs in serum. Seropositive early-onset periodontitis patients were sampled, and the Ig classes IgG, IgA, and IgM and subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 were studied. Reactivity with Aab antigens was assessed by using the Western blot (immunoblot) in limiting dilution analysis and radioimmunoassay with sera from 13 early-onset periodontitis subjects. A smeared antigen in the upper portion of the immunoblots, typical of high-molecular-weight LPS, was immunodominant for IgG, IgA, IgM, IgG1, IgG2, IgG3, IgA1, and IgA2. This smeared antigen was present in every patient for all of these Igs at the endpoint. A few additional antigens were also present at the endpoint in some patients, but none were present in more than half of the subjects. The distribution of antibody titers by Ig classes reactive with the Aab immunodominant antigen was IgG > IgA > IgM. The distribution of antibody titers by IgG subclass was IgG2 > IgG1 approximately IgG3. Further quantitation by radioimmunoassay revealed that the mean concentration of IgG2 (65.7 micrograms/ml) was significantly greater than that of IgG1 (8.8 micrograms/ml). The IgA subclass distribution was IgA1 > IgA2, with IgA1 apparently being second only to IgG2. Therefore, the Aab antigen eliciting the highest antibody level in virtually all Ig classes and subclasses appeared to be lipopolysaccharide, and IgG2 was markedly elevated over all other serum Ig classes or subclasses reactive with Aab.     

Antibodies to Klebsiella pneumoniae lipopolysaccharide in patients with ankylosing spondylitis

The role of microbial lipopolysaccharides (LPS) in the aetiopathogenesis of ankylosing spondylitis (AS) is a matter of continuing debate. In this study, class-specific IgG, IgA and IgM antibodies against Klebsiella pneumoniae, Escherichia coli, Salmonella typhimurium and Salmonella enteritidis LPS were measured by enzyme-linked immunosorbent assay (ELISA) in 100 AS patients, 50 rheumatoid arthritis (RA) patients and 50 healthy control subjects. The AS patients had significantly elevated levels of IgG and IgA antibodies against K. pneumoniae LPS (P < 0.001) and IgA antibodies against E. coli LPS (P < 0.05) compared to healthy controls. There were no significant elevations of antibody levels against S. typhimurium and S. enteritidis in the three study groups. In addition, there was a correlation between IgG and IgA anti-K. pneumoniae LPS antibody levels and the acute-phase reactant C-reactive protein (P < 0.001).     

Polyclonal immunoglobulin response of thymic, hepatic and splenic lymphocytes from fetal, germ-free and conventionally reared pigs to different B-cell activators

Immunoglobulin (Ig) response to different polyclonal B-cell activators was measured by ELISA in cell culture media of thymocytes, splenocytes and liver cells isolated from pig fetuses, 8-d-old germ-free piglets and conventionally reared pigs. Both in fetal and in postnatal life polyclonally stimulated lymphocytes were found to produce predominantly the IgM isotype; the first IgM formation was detected in 50-d-old fetal liver (gestation in pigs lasts 114 d). Surprisingly, 73-d-old fetal thymic cells were shown to be induced to Ig synthesis and secretion. In contrast to splenocytes of the same age, which secreted exclusively IgM, fetal thymocytes produced IgM, IgG and IgA. Polyclonally stimulated splenic cells as compared with thymic cells started to produce IgA later in fetal ontogeny, whereas the IgG response was not detectable in splenic cell culture media during the whole embryonal development and appeared only after birth. The earliest and the highest Ig stimulation was found after cultivation of lymphocytes with Nocardia delipidated cell mitogen. Interestingly, the moderate stimulatory effect of 65-kDa heat shock protein (Hsp-65) in polyclonal IgM response of fetal splenocytes was observed. We showed that thymic B lymphocytes represent probably the first maturing B cell population detectable in fetal life, which is able to differentiate after polyclonal stimulation into IgM as well as IgA and IgG producing cells.     

Effects of type I/type II interferons and transforming growth factor-beta on B-cell differentiation and proliferation. Definition of costimulation and cytokine requirements for immunoglobulin synthesis and expression

In this report, we sought to determine the role of selected type I interferons [interferon-alpha (IFN-alpha) and interferon-tau (IFN-tau)], IFN-gamma and transforming growth factor-beta (TGF-beta) in the regulation of bovine antibody responses. B cells were stimulated via CD40 in the presence or absence of B-cell receptor (BCR) cross-linking. IFN-alpha enhanced IgM, IgG2 and IgA responses but did not enhance IgG1 responses. BCR signalling alone was more effective at inducing IgG2 responses with IFN-alpha than dual cross-linking with CD40. Recombinant ovine IFN-tau was less effective at inducing IgG2 responses when compared with IFN-alpha, though IgA responses were similar in magnitude following BCR cross-linking. At higher concentrations, IFN-tau enhanced IgA responses greater than twofold over the levels observed with IFN-alpha. Previous studies have shown that addition of IFN-gamma to BCR or pokeweed mitogen-activated bovine B cells stimulates IgG2 production. However, following CD40 stimulation alone, IFN-gamma was relatively ineffective at stimulating high-rate synthesis of any non-IgM isotype. Dual cross-linking via CD40 and the BCR resulted in decreased synthesis of IgM with a concomitant increase in IgA and similar levels of IgG2 production to those obtained via the BCR alone. We also assessed the effects of endogenous and exogenous TGF-beta on immunoglobulin synthesis by bovine B cells. Exogenous TGF-beta stimulates both IgG2 and IgA production following CD40 and BCR cross-linking in the presence of IL-2. Blocking endogenous TGF-beta did not inhibit the up-regulation of IgG2 or IgA by interferons.     

The effects of periodontal therapy on serum antibody levels to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis (part II)

Levels of IgG and IgM antibodies were estimated against Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were determined by enzyme linked immunosorbent assay (ELISA) in 17 patients with juvenile periodontitis, 15 with adult periodontitis and 24 healthy controls at the beginning of treatment and 3 to 8 months after periodontal therapy. After treatment, antibodies to A. actinomycetemcomitans and P.gingivalis had decreased in patients, but the levels were still significantly higher than in healthy controls. Whether or not an of antibody level against a specific bacteria changes after periodontal treatment is however, still debatable.     

CD40 cross-linking inhibits specific antibody production by human B cells

Ligation of CD40 on B cells is a co-stimulatory signal for proliferation, antibody secretion, heavy chain switching and rescue from apoptosis after somatic mutation in the germinal centre. The importance of these manifold responses to CD40 activation for humoral immunity is exemplified by the inability of boys with X-linked hyper IgM syndrome to make IgG, IgE or IgA due to a mutation in in the gene coding for CD40 ligand (CD40L). In the present study, we have investigated the effect of CD40 ligation on specific antibody production by human B cells to influenza virus. The antibody response was T cell dependent and specific for the strain of influenza virus used as antigen. Addition of either CD40 mAb or recombinant trimeric CD40L profoundly inhibited specific antibody production. Antibody production by unseparated tonsillar mononuclear cells and by T-depleted B cells stimulated with antigen in the presence of T cell replacing factor were equally inhibited with CD40 antibody showing that the effect was due to ligation of CD40 on B cells rather than blocking of T cell help. The specific antibody detected in these experiments was mostly IgG with little or no IgM and was obtained from surface IgM B cells consistent with activation of a secondary (memory) response. Co-stimulation of tonsillar B cells with CD40 antibody and anti-IgG induced proliferation of IgG+ B cells. These results suggest that CD40 ligation can inhibit specific antibody responses and stimulate proliferation in the same IgG+ (memory) B cell subpopulation. Addition of CD40 antibody during the first 24-48 h of the response was required for inhibition, suggesting that the effect was on early B cell activation and/or proliferation required for antibody production. There was no correlation, however, between the ability of CD40 mAb to stimulate proliferation and inhibit antibody production. We suggest that early activation of CD40 in the specific antibody response inhibits the formation of plasma cells and promotes instead the generation of memory cells.     

Comparison of cardiac findings at necropsy in octogenarians, nonagenarians, and centenarians

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2196-561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2196-561 as the antigen substrate. The analyzed sera were divided according to T. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG+ IgA- IgM-; n = 35), group B (IgG+ IgA+ IgM+; n = 21), group C (IgG+ IgA+ IgM-; n = 5), and group D (IgG+ IgA- IgM+; n = 16). Twenty-six T. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2196-561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2196-561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.     

Studies on translocation of immunoglobulins across intestinal epithelium. II. Immunoelectron-microscopic localization of immunoglobulins and secretory component in human intestinal mucosa

To define mechanisms involved in the transport of immunoglobulins into intestinal fluids, we localized IgM, IgA, IgG, and secretory component (SC) in human intestinal mucosa by the peroxidase-labeled antibody technique. At the light microscopic level, immunocytes containing IgA, IgM, or IgG were found in the lamina propria. IgA, IgM, and SC were prominent in the epithelium of gland crypts; IgG was limited to a few cells at tips of villi. At the electron-microscopic level, SC was localized to perinuclear spaces, endoplasmic reticulum, saccules associated with Golgi complexes, cytoplasmic vesicles, and lateral and basal plasma membranes of columnar epithelial cells. IgA and IgM, but not IgG, also were localized to plasma membranes and cytoplasmic vesicles of these cells. Neither the immunoglobulins nor SC was found within other types of epithelial cells (Paneth, goblet, endocrine). The findings provide evidence that (1) the site of SC synthesis in intestinal epithelium is secretory columnar cells, principally those in gland crypts; (2) the polymeric immunoglobulins IgM and IgA are translocated through such SC-containing cells by a process that involves formation of cytoplasmic vesicles; (3) IgM and IgA could combine with SC during transcellular transport (likely sites are lateral or basal plasma membranes or supranuclear cytoplasm); (4) the monomeric immunoglobulin IgG does not share the transepithelial cell route involved in IgM and IgA transport.     

Serum rheumatoid factor induced by intraperitoneal administration of periodontopathic bacterial lipopolysaccharide in mice

Serum rheumatoid factor (RF) level and peritoneal and splenic CD5+B (B-1) cells in mice were examined after intraperitoneal administration of purified lipopoly-saccharides (LPS) from oral periodontopathic bacteria; Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum and Capnocytophaga ochracea. F. nucleatum and C. ochracea LPS induced higher levels of serum IgM- and IgG-RF, while P. gingivalis LPS showed the least induction. In addition, wet weights of spleen and serum IgM and IgG concentration were markedly increased in F. nucleatum LPS injected group. On the other hand, the proportion of CD5+ B cells to lymphocytes in the peritoneal cavity and spleen did not increase. The reason for this was not clear but conventional B cells (CD5- B cells) might increase more rapidly with splenic enlargement than CD5+ B cells. These results suggested that RF induced by bacterial LPS may modulate immune responses against bacteria and plays an important role for defence and destruction of periodontal tissue.     

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. I. Correlation of graft survival with antidonor IgG antibody subclasses

We have recently demonstrated that cardiac allograft rejection in the PVG.R8-to-PVG.1U rat strain combination involves the recognition of a isolated class I (RT1.Aa) molecules as peptides in the context of the recipient MHC molecules. Three synthetic peptides (P1, P2, and P3) corresponding to the alpha-helices of the RT1.Aa molecule served as T-cell epitopes for graft rejection. In this study, we demonstrate that two of these peptides (P2 and P3) are sufficient to induce immune nonresponsiveness (median survival time >237 days) to cardiac allografts when presented to the recipient immune system in the thymus 7 days before transplantation. This effect was time dependent, as intrathymic inoculation 60 days before transplantation did not prolong graft survival (median survival time=12 days). Previous studies have demonstrated a critical role for alloantibody responses in mediating graft rejection in this rat strain combination. We, therefore, studied the role alloantibody responses may play in the observed immune nonresponsiveness. The titers of alloantibody in serum samples harvested from graft recipients at different times after transplantation were measured. We used recipient primary aortic endothelial cells genetically manipulated to express the donor RT1.Aa molecule as targets in an enzyme-linked immunosorbent assay. High titers of anti-RT1.Aa IgM antibody were detected in unmanipulated controls at the time of graft rejection. The IgM antibody switched to high IgG titers in intrathymically inoculated rats with accelerated or delayed rejection. Graft rejection in intrathymically manipulated recipients that had achieved a transient state of immunological nonresponsiveness correlated with higher titers of the IgG2b alloantibody. In marked contrast, the long-term graft survivors expressed undetectable or low levels of the IgG2b antibody and moderate to high levels of the IgG1 and IgG2a subclasses. These data suggest that the IgG2b alloantibody may contribute to the rejection reaction, whereas IgG1 and IgG2a may be involved in active enhancement of graft survival.     

IFN-gamma is a potent inducer of Ig secretion by sort-purified murine B cells activated through the mIg, but not the CD40, signaling pathway

IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were >99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.     

Mitogenic activation of human lymphocytes: a protein A plaque assay evaluation of polyclonal B-cell activators

Using the protein A plaque assay, the capacity of various polyclonal B cell activators to induce differentiation in human B lymphocytes was investigated. Dextran sulphate and native dextran were both virtually devoid of mitogenic properties. Lipopolysaccharide, however, was found to be a potent mitogen in human cells that, although giving rise to low DNA synthetic response, induced high numbers of immunoglobulin-synthesizing cells. Mean plaque-forming cell (PFC) numbers in healthy blood donors assayed on the optimal day (days 5-7) were 23,493 IgM/10(6) cells, 11,288 IgG/10(6) cells, and 2643 IgA/10(6) cells. Values obtained in spleen cells, peaking at days 4-6, were slightly higher. Purified protein derivative (PPD) was equally or even more effective than lipopolysaccharide (LPS) in generating PFC of different subclasses in peripheral blood with mean of 29,241 IgM/10(6), 21,269 IgG/10(6), and 3681 IgA/10(6). PPD furthermore induced a marked DNA synthetic response in human lymphocytes. These data suggest that LPS and PPD may both be used as functional markers in human cells when analysing patients with a suspected immunodeficiency state. It is suggested that cultures should be assayed using the protein A plaque assay, thereby being able not only to investigate the individual immunoglobulin classes but also to avoid the possible hazards involved in measuring antigen-specific responses in patients whose prior immunization to the antigen tested can never be totally excluded.     

Production and characterization of a set of mouse-human chimeric immunoglobulin G (IgG) subclass and IgA monoclonal antibodies with identical variable regions specific for Pseudomonas aeruginosa serogroup O6 lipopolysaccharide

The heavy- and light-chain variable regions from a murine monoclonal antibody that recognize Pseudomonas aeruginosa serogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies to P. aeruginosa and will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy.     

Induction of long-term potentiation at perforant path dentate synapses does not affect place learning or memory

B lymphocytes are crucial participants in pulmonary immune defense. However, excess local antibody production is associated with accelerated lung destruction in several types of lung disease. The purpose of the current study was to study the potential role of alveolar macrophages (AM) in the local regulation of immunoglobulin (Ig) production in the lung in response to a direct B cell mitogen, Staphylococcus aureus Cowan strain (SAC). AM, when added to peripheral blood mononuclear cells, caused a dose-dependent inhibition of IgG and IgM, while not affecting IgA production in response to SAC. The mechanism of the AM-induced inhibition included both membrane-bound and soluble signals. The inhibition was not abrogated by indocin and catalase, or reversed by blocking antibodies to transforming growth factor-beta or interferon-gamma. Mononuclear cells isolated from human lung parenchyma displayed a reduced response to SAC compared with blood cells. However, depletion of macrophages from the parenchymal cells was associated with a restoration of IgG production in response to SAC. The results demonstrate that AM inhibit B cell responses to direct B cell mitogen and suggest that the effect of AM is selective for IgM and IgG.     

Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyer's patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.     

B and T lymphocytes are enhanced in the gut of piglets fed heat-treated soyabean proteins

Gut immune responses have been suspected in food hypersensitivity reactions such as those to soyabean proteins in early-weaned piglets. The present study examines the lymphoid cell subset distribution in piglets fed heat-treated (HTSP) or ethanol-treated soyabean proteins (ETSP). Duodenal cryosections of 4-week-old HTSP piglets (n = 10) and ETSP piglets (n = 8) were analysed for IgA, IgM, IgG1 and IgG2 positive cells, CD2, CD4, CD8, WC1 T cell positive antigens using immunohistochemical peroxidase reactions. Densities of IgM+ and IgA+ cells were three times and, IgG1+ and IgG2+ six times higher in the lamina propria of HTSP piglets compared with ETSP (P < 0.05). Increased CD2+ T cells were accounted for by a rise in both CD4+ and CD8+ T cell subsets in the lamina propria (P < 0.01) as well as in the epithelium of the duodenal mucosa of piglets fed HTSP. The density of the WC1+ T cell subset in the epithelium was significantly higher in HTSP than in ETSP piglets (P < 0.01). Immune reactions in the duodenal mucosa, involving both B and T lymphocytes may be related to atrophy of the duodenal villi in HTSP piglets.     

The induction and expression of IgA anti-DNP antibodies in rat bile and secretions

Pregnant rats were immunized with dinitrophenylated type III-pneumococcal vaccine by the intravenous, gastric, or intramammary routes. Anti-DNP antibody responses in the IgA, IgG and IgM isotypes were measured in serum, secretions and bile. Gastric intubation was most effective at eliciting IgA antibody responses in bile and secretions, whereas the other routes were more effective at inducing IgG responses in serum and bile. IgM antibody responses were infrequent and were found in fluids most closely associated with the immunization route. Isoelectric focusing studies of IgA antibodies appearing in secretions and bile revealed that the gastric route consistently elicited antibody spectrotypes with shared components. Intravenous and intramammary immunization resulted in IgA spectrotypes possessing less homology, suggesting that these protocols lead to independent antibody responses triggered in spleen, draining lymph nodes, or secretory sites. After gastric stimulation, the appearance of IgA antibodies with identical spectral components in secretions and bile favours the concept that IgA precursor cells with identical clonotype potential migrate from the gastrointestinal area to secretory sites. Antibody expression in bile appears to result from the selective transfer of IgA populations gaining access to serum after synthesis at a secretory site.     

Autologous bone marrow support and bone disease in metastatic breast cancer

Production of IgM, IgG and IgA was induced from human blood B lymphocytes by culturing with a CD40 MoAb and IL-2 for 9 days. Replacement of IL-2 by IL-10 markedly enhanced production of all three isotypes. High levels of immunoglobulin production also occurred when activated irradiated autologous T cells replaced the CD40 MoAb, and when IL-10 replaced IL-2 in these cultures a spectacular increase in IgG production occurred. The effectiveness of the T cell stimulus depended on the mode of purification of the T cells and the nature of the stimulant used to activate them. Differences in the kinetics and level of expression of CD40L on the various T cell preparations were observed, but did not account for variations in immunoglobulin-inducing efficiency. Immunoglobulin production from sIgD+ and sIgD- B cells was investigated. IgG and IgA were found in sIgD+ cultures, indicating that some isotype switching had occurred, but the major part of the IgG and IgA secreted was from cells already committed to these isotypes. Anti-IgD or anti-IgM MoAbs enhanced the proliferation of B cells induced by anti-CD40 antibody, but immunoglobulin production was not enhanced. Factors affecting the balance of proliferation and differentiation are discussed.     

Helper activity of chicken V beta 1+ and V beta 2+ alpha beta T cells for in vitro IgA antibody synthesis

Chickens have only two T cell receptor variable beta gene families: V beta 1 and V beta 2 (1). In our previous work we found that IgA production was almost completely suppressed in chickens depleted of V beta 1+ alpha beta T cells by treatment with a TCR V beta 1-specific monoclonal antibody (2), while IgM and IgG production was not affected. Our present results indicate that, in vitro, both V beta 1+ and V beta 2+ chicken cecal tonsil T cells provide help for the differentiation of cecal tonsil IgA B cells, suggesting that the failure of V beta 1+ T cell-depleted chickens to produce IgA is not caused by the inability of V beta 2+ T cells to provide help for IgA production by B cells, but rather by the scarcity of these T cells in mucosal tissues (3), where most IgA responses are induced (4).