TNP-specific IgM Research Articles

Affinity-Driven Site-Specific High Mannose Modification Determines the Structural Polymerization and Function of Tetrameric IgM in a Primitive Vertebrate.

Teleost tetramer IgM is the predominant Ig in the immune system and plays essential roles in host defense against microbial infection. Due to variable disulfide polymerization of the monomeric subunits, tetrameric IgM possesses considerable structural diversity. Previous work indicated that the teleost IgM H chain was fully occupied with complex-type N-glycans. However, after challenge with trinitrophenyl (TNP) Ag, the complex N-glycans in the Asn-509 site of Oreochromis niloticus IgM H chain transformed into high mannose. This study, therefore, was conducted to examine the functional roles of the affinity-related high-mannose modification in tilapia IgM. The TNP-specific IgM Ab affinity maturation was revealed in tilapia over the response. A positive correlation between TNP-specific IgM affinity and its disulfide polymerization level of isomeric structure was demonstrated. Mass spectrometric analysis indicated that the relationship between IgM affinity and disulfide polymerization was associated with the Asn-509 site-specific high-mannose modification. Furthermore, the increase of high mannose content promoted the combination of IgM and mannose receptor (MR) on the surface of phagocytes. Moreover, the increased interaction of IgM and MR amplified the phagocytic ability of phagocytes to Streptococcus agalactiae. To our knowledge, this study demonstrates that site-specific high-mannose modification associates with IgM Ab affinity and its structural disulfide polymerization and amplifies the phagocytosis of phagocytes by the combination of IgM and MR. The present study provides evidence for understanding the association of IgM structure and function during the evolution of the immune system.

Relative content detection of oligomannose modification of IgM heavy chain induced by TNP-antigen in an early vertebrate through nanoLC-MS/MS

N-glycan modification is reported to be important in regulating the structure and function of immunoglobulins in mammals. While, the study on teleost immunoglobulin glycosylation is still limitted. In this study, we constructed a TNP-antigen driven model, and detected the site-specific N-glycans of PBS-immunized and TNP-specific Oreochromis niloticus serum IgM through 18O-labeling and nanoLC-MS/MS. These methods are widely used for peptide enrichment and protein modification identification, but rarely used in detecting the level of N-glycosylation in teleost Igs that driven by specific antigen. The results revealed that there are four N-glycosylation sites in O.niloticus IgM heavy chain, namely, the Asn-315 site in the CH2 domain, the Asn-338 site in the CH3 domain, and the Asn-509 and Asn-551 sites in the CH4 domain, All of the four residues were efficiently N-glycosylated. After immunized with TNP-antigen, the signal strength of oligomannose in the TNP-specific IgM in primary mass spectrometry was significantly higher than that in the PBS-immunized IgM. Notably, the TNP-specific IgM had an Asn-509 site fully occupied with oligomannose, while only a small amount of oligomannose was found in the PBS-immunized IgM of this site. N-glycans in other sites were mainly complex-type with a low content of fucosylation and sialylated. The oligomannose in TNP-specific IgM was further verified to be essential for the binding of IgM and MBL. These results demonstrated that the TNP-antigen induced the site-specific oligomannose modification of O.niloticus IgM heavy chain, and played an important role in the interaction of IgM and MBL, which provided insights into the evolutionary understanding of the IgM oligomannose modification and function.

Functional Adaptive Immune Responses in Hematopoietic Chimeric Mice After Umbilical Cord Blood Cell Transplantation.

Abstract 2995 Introduction:An increasing number of clinical trials have demonstrated the usefulness of cord blood as a source of hematopoietic stem cells for reconstitution of the hematopoietic system. Nevertheless, due to a lack of convenient animal models, information about the immunological competence of umbilical cord blood cell (UCBC)-derived lymphocytes has been relatively limited. Recently, we have established a murine model of UCBC transplantation, which reconstitutes the hematopoietic system of immunodeficient mice, and studied the correct immunological functions of UCBC-derived lymphocytes generated in an allogeneic environment. Materials and Methods:UCBC prepared from C57BL/6 (H-2b) mice were transferred into lethally irradiated BALB/c (H-2d) mice lacking T- and B-lymphocytes (RAG2 knockout). In order to evaluate the adaptive immune response in the recipient mice, rejection of skin grafts from third-party C3H/He (H-2k) mice and antibody responses to immunization with a T-dependent antigen, 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), were assessed. Additionally, the responsiveness of generated T cells was verified by mixed lymphocyte reaction (MLR) assay, cytotoxic T-lymphocyte (CTL) assay and phytohemagglutinin (PHA) blast formation assay. Results:In this UCBC transplantation model, the overall survival of the recipient mice was dose-dependent, and all surviving recipient mice were hematopoietic chimeras possessing all lineages. These allogeneic chimeras specifically rejected skin grafts from third-party C3H/He mice (rejection time; 9–16 days) while showing tolerance to skin grafts from both donor-type and recipient type, although with a significant delay in comparison to normal BALB/c mice (8–12 days) and C57BL/6 mice (9–11 days). Although accurate antibody responses of the allogeneic chimeras against TNP-KLH immunization were not anticipated, substantial amounts of TNP-specific IgG were detected in their sera. Interestingly, the level of TNP-specific IgM was significantly higher than that of normal BALB/c mice after TNP-KLH immunization, indicating retarded immunoglobulin class-switching in the allogeneic chimeras. The mechanism responsible for the antibody production by allogeneic chimeras still remains unclear. CTL and MLR assay revealed cell-mediated third-party-specific activity of killer and helper T cells, consistent with the tendency for the skin graft rejection and antibody production capability of the allogeneic chimeras to be inferior to those of normal mice. T cells of the allogeneic chimeras also proliferated non-specifically in response to PHA stimulation. The T-cell proliferation indices of the allogeneic chimeras when those of normal mice were taken as 100 were 21 in the MLR assay and 48 in the PHA blast formation assay, suggesting relative inferiority of the recognition and response system of T cells generated in an allogeneic environment. In any event, it may be important to note that T-dependent antigen-specific antibody production and alloantigen-specific rejection were substantial in the allogeneic chimeras. Conclusions:Overall, the present findings suggest that UCBC-derived lymphocytes generated in HLA-mismatched recipients are immunologically functional and competent. Regarding the relative inferiority of immune responses in the allogeneic chimeras, we predict the causes originate from narrowing of the T- and B-cell receptor repertoire by the allogeneic environment, and this possibility is now being examined. Disclosures:No relevant conflicts of interest to declare.

Reconstitution of B-1a Cells in Murine Allogeneic Transplantation of Umbilical Cord Blood Cells: A New Advantage of the Umbilical Cord Blood-Derived Hematopoietic System.

Abstract 2427Poster Board II-404 Background:Umbilical cord blood cells (UCBCs) have been used for the treatment of various hematological diseases. In clinical situations, perfect major histocompatibility complex (MHC) matching cannot be expected in UCBC transplantation because UCBC transplantation essentially involves non-related donor-recipient combinations. Although even MHC-mismatched UCBCs can be used because of the low content of mature T cells, the extent of recovery of immune function in lymphocytes derived from MHC-mismatched UCBC transplantation has remained unclear. To resolve this issue, we examined the functional maturation of lymphocytes derived from a fully MHC-mismatched UCBC transplantation in a murine model system. For comparison, bone marrow cell (BMC) transplantation was also performed. In this study, we first found that B-1a cells, which produce natural IgM antibodies against pathogens such as Streptococcus pneumoniae and the influenza virus, can be generated from UCBCs but not from BMCs. Methods:RAG2(-/-)BALB/c [H-2d] mice exposed to 8-Gray irradiation were transplanted with 3 different doses (1×105, 5×105, or 1×106 cells) of UCBCs or BMCs, which had been depleted of mature T cells, obtained from green fluorescent protein-transgenic C57BL/6 (B6) [H-2b] mice. More than 16 weeks after transplantation, we observed successful reconstitution of immune cells in the recipients, as shown by flow cytometric analysis. In addition, the function of T and B cells developed from UCBCs and BMCs was evaluated by examining the rejection of skin grafts from third-party donors and the production of antibodies against intraperitoneally injected 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), respectively. Furthermore, the ability of UCBCs and BMCs to repopulate natural IgM antibody-producing B-1a cells was examined using flow cytometry and enzyme-linked immunosorbent assay. Results:At 16 weeks after transplantation, a similar survival rate (40-100%), which was dose-dependent, was observed between recipient mice with UCBC and BMC transplants. Phenotypically mature fluorescent cells of donor origin, including T cells, B cells, monocytes, and granulocytes, were detected in the recipients' peripheral blood, even after MHC-mismatched UCBC and BMC transplantation. Functional analysis showed that mice with allogeneic UCBC and BMC transplants accepted skin grafts from both BALB/c and C57BL/6 mice. However, these chimeric mice completely rejected skin grafts from third-party C3H/HeJ [H-2k] mice, indicating that both CD8+ killer and CD4+ helper T cells were functionally mature in the recipients. In addition, TNP-KLH-immunized mice with UCBC and BMC transplants produced both TNP-specific IgM and IgG antibodies. These findings indicate the ability of recipient mice to develop antibody responses with Ig class switching to T-dependent antigens, thereby confirming that both B cells and CD4+ helper T cells derived from allogeneic UCBC were immunologically competent. In contrast, surprisingly, flow cytometric analysis revealed the presence of peritoneal B-1a cells only in UCBC recipients but not in BMC recipients; the presence of these cells appeared to correlate with the bacterial polysaccharide-reactive natural IgM level in UCBC recipients. Conclusion:Although UCBCs and BMCs essentially have similar ability for reconstituting the hematopoietic system, this study showed the promising potential of UCBCs in reconstituting B-1a cells even in an allogeneic environment, thereby replenishing polysaccharide-reactive IgM as a front-line defensive factor against invading pathogens. On the basis of our findings, we strongly recommend the use of UCBCs in hematopoietic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

Spleen Vagal Denervation Inhibits the Production of Antibodies to Circulating Antigens

BackgroundRecently the vagal output of the central nervous system has been shown to suppress the innate immune defense to pathogens. Here we investigated by anatomical and physiological techniques the communication of the brain with the spleen and provided evidence that the brain has the capacity to stimulate the production of antigen specific antibodies by its parasympathetic autonomic output.Methodology/Principal FindingsThis conclusion was reached by successively demonstrating that: 1. The spleen receives not only sympathetic input but also parasympathetic input. 2. Intravenous trinitrophenyl-ovalbumin (TNP-OVA) does not activate the brain and does not induce an immune response. 3. Intravenous TNP-OVA with an inducer of inflammation; lipopolysaccharide (LPS), activates the brain and induces TNP-specific IgM. 4. LPS activated neurons are in the same areas of the brain as those that provide parasympathetic autonomic information to the spleen, suggesting a feed back circuit between brain and immune system. Consequently we investigated the interaction of the brain with the spleen and observed that specific parasympathetic denervation but not sympathetic denervation of the spleen eliminates the LPS-induced antibody response to TNP-OVA.Conclusions/SignificanceThese findings not only show that the brain can stimulate antibody production by its autonomic output, it also suggests that the power of LPS as adjuvant to stimulate antibody production may also depend on its capacity to activate the brain. The role of the autonomic nervous system in the stimulation of the adaptive immune response may explain why mood and sleep have an influence on antibody production.

Genistein suppresses antigen-specific immune responses through competition with 17β-estradiol for estrogen receptors in ovalbumin-immunized BALB/c mice

Objective The aim of this study was to determine the effects of phytoestrogen genistein on antigen (Ag)-specific immune responses and elucidate the mechanisms underlying those effects. Methods Ovalbumin (OVA)-immunized BALB/c mice were administered genistein for 35 d, and OVA-specific immune responses were examined by measuring OVA-specific proliferative responses, production of cytokines, and antibody responses. To assess the effect of genistein on antibody responses to thymus-independent Ag, mice were immunized with 2,4,6-trinitrophenyl (TNP)-Ficoll instead of OVA. Effect of genistein on the functions of CD11c + dendritic cells was also examined. Finally, to determine the contribution of estrogen receptor to genistein-mediated immune regulation, mice that had been administered genistein were treated with the estrogen receptor antagonist ICI 182,780 and OVA-specific proliferative responses were examined. Results OVA-specific proliferative responses and interferon-γ production levels were decreased in mice administered 20 mg/kg genistein compared with those in control mice without reduction in responses to anti-CD3 monoclonal (m)antibody. The level of OVA-specific immunoglobulin (Ig)G1 was also decreased in mice administered genistein. Levels of OVA-specific IgG2a and IgG2b production and interleukin-4 production in response to OVA were not significantly different but tended to decrease in genistein-treated mice. Genistein administration did not influence the TNP-specific IgM and IgG levels. Furthermore, genistein did not affect the Ag-presenting activity of CD11c + dendritic cells. Treatment with ICI 182,780 decreased OVA-specific proliferative responses, but genistein did not suppress these responses synergistically in mice treated with ICI 182,780. Conclusions The results of this study suggest that genistein suppresses Ag-specific immune responses. The mechanism underlying the suppression is responsible for the competition of genistein with endogenous 17β-estradiol for estrogen receptors.

Anti-inflammatory and Immune Regulatory Properties of 5-Androsten-3β, 17β-Diol (HE2100), and Synthetic Analogue HE3204: Implications for Treatment of Autoimmune Diseases

5-Androsten-3beta, 17beta-diol (HE2100), and a synthetic analogue HE3204 are regarded as immune-regulating hormones, because both induce changes in the reporter antigen-popliteal lymph node assay (RA-PLNA). Mice were injected in the footpad with either HE2100 or HE3204 (0.01-3 mg), and a nonsensitizing dose of trinitrophenyl ovalbumin (TNP-OVA) was used as bystander reporter antigen. Seven days later, nodes were removed and numbers of cells (CD3, CD4, CD8, CD19; flow cytometry), TNP-specific IgM, IgG1, and IgG2a antibody-forming cells (AFCs; ELISPOT assay), and cytokines (interleukin-4 [IL-4], interferon-gamma [IFN-gamma]; ELISA) were measured. HE2100 and HE3204 increased cell numbers in a dose-dependent fashion. T (helper and suppressor) cells and B cells were increased (>5-fold). HE3204 was apparently twice as potent as HE2100. Both increased the B/T ratio (fivefold), increased TNP-specific IgM and IgG1 ( approximately 50-fold), and induced IgG2a AFCs. Both increased IL-4 and IFN-gamma secretion (up to threefold). Both displayed anti-inflammatory activity in the murine model of carrageenan-induced pleurisy, as evidenced by reduced neutrophil numbers and exudate volumes. Our observations suggest that both HE2100 and HE3204 are immune-regulating steroid hormones that exhibit anti-inflammatory properties. HE2100 (1 mg/mouse per day) provided significant benefit when given at disease onset in the SJL/J female mouse model of experimental autoimmune encephalomyelitis. These compounds and their analogues are candidates for further testing in autoimmune diseases.

γ-Aninobutyric acid (GABA) suppresses antigen-specific immune responses in ovalbumin γ(OVA)-immunized BALB/c mice

γ-Aminobutruic acid (GABA) is known to be a ubiquitous inhibitory neurotransmitter in the central nervous system. Furthermore, recent studies have shown that GABA modulates cardiovascular and respiratory functions. In this study, we examined the effect of GABA on antigen (Ag)-specific immune responses and elucidated the mechanisms underlying those effects in ovalbumin (OVA)-immunized BALB/c mice. OVA-specific proliferative responses were decreased in mice that had been administered 20 mg/kg GABA compared to those in control mice without reduction in responses to anti-CD3 monoclonal (m)Ab. The levels of OVA-specific IgG, IgG1 and IgG2a were also decreased in mice that had been administered GABA. However, GABA administration did not influence the TNP-specific IgM and IgG levels. Ag-presenting activity of CD11c+ dendritic cells from GABA-treated mice was less than that of cells from control mice. These results suggest that the suppressive effect of GABA on Ag-specific immune responses contributes to the reduction of antigen-presenting function on CD11c+ dendritic cells.

Selective enhancement of B cell antigen receptor-mediated antigen presentation by treatment with transforming growth factor-beta.

TGF-beta1 was examined for the ability to regulate Ag-presentation by B cells, using A20-HL B lymphoma cells bearing TNP-specific IgM receptors. Treatment of A20-HL cells with TGF-beta1 at 1 ng/ml, a concentration that inhibited proliferation, enhanced presentation of Ag internalized via surface IgM (sIgM), but not via fluid-phase pinocytosis. TGF-beta1-treatment slightly enhanced surface expression of sIgM, but not of MHC class II molecules. The treatment accelerated recovery of sIgM expression after its removal by ligation with TNP-OVA, and induced prolonged intracellular residence of TNP-OVA internalized via sIgM, which co-localized with intracellular MHC class II molecules. TGF-beta1-treatment increased accumulation of newly synthesized intracellular MHC class II molecules that were localized in compartments positive for lysosome-associated membrane protein 1, although cellular protein synthesis was decreased by the treatment. The accumulated intracellular MHC class II molecules were triggered to the cell surface by ligation of sIgM. Finally, TGF-beta1-treatment induced Igalpha-phosphorylation in response to lower concentrations of TNP-OVA. On the basis of these findings, we conclude that TGF-beta1-treatment of A20-HL cells selectively enhances the ability to present Ag internalized via sIgM, not via fluid-phase pinocytosis, through accelerating sIgM recovery, increasing accumulation of intracellular MHC class II molecules and enhancing the ability of sIgM ligation to induce Igalpha-phosphorylation.

Differential Sensitivity to Antigenic Competition in Antigen-Specific and -Nonspecific Antigen Presentation by B Cells

We have previously shown that a specific Ag presentation by B cells is different from a nonspecific one in the sensitivity to protein synthesis inhibition. In the present study we have compared the sensitivity of these Ag presentations to antigenic competition. A20HL cells expressing TNP-specific IgM were pulsed with anti-mouse IgM goat IgG (aMGG) or trinitrophenylated goat IgG (TNP-NGG) as an Ag internalized through Ag receptor or NGG as an Ag internalized by fluid-phase pinocytosis. The pulsed cells induced IL-2 production by NGG-specific cloned T cells. The presence of dog IgG during pulsing A20-HL cells severely inhibited the presentation of NGG but not of aMGG or TNP-NGG. The presence did not decrease the internalization of 125I-NGG into A20-HL cells, suggesting that the inhibition was localized into the complex formation of antigenic peptides with MHC class II molecules. Thus, a specific Ag presentation by A20-HL cells is different from a nonspecific one in its sensitivity to antigenic competition.

High-frequency loss of specific immunoglobulin production in hybridoma cell lines bearing a chromosomal immunoglobulin kappa gene modified by homologous recombination.

We have examined the stability of trinitrophenyl (TNP) -specific IgM production in hybridoma cell lines in which homologous recombination was used to change the variable region of an endogenous chromosomal immunoglobulin kappa gene to one specific for TNP. Mutant hybridomas that have lost TNP-specific IgM production are detected with a frequency of approximately 1%. Characterization of the mutant cells reveals a variety of gross rearrangements in the recombinant kappa TNP gene as well as in the endogenous kappa and muTNP genes.

Effect of diisopropylfluorophosphate on the antibody response

The impact of the in-vivo administration of diisopropylfluorophosphate (DFP) on the antibody response in mice was examined. The antigen-specific plaque-forming cell (PFC) response of mice injected with DFP (4 mg/kg) at the time of immunization with a macrophage-dependent T-cell antigen, trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) or sheep red blood cells (SRBC), was significantly depressed. In contrast, the injection of a similar dose of DFP had no effect on the PFC response of mice immunized with a macrophage-independent B-cell antigen, lipopolysaccharide (LPS). This differential effect suggests a possible detrimental effect of DFP on cells involved in antigen processing and/or presentation which are required for the antibody response to SRBC and TNP-KLH. These results, however, do not exclude T cells as a possible target and the cellular targets of DFP action remain to be established. The DFP-mediated depression of the PFC response to SRBC was still evident when DFP was given as early as 1 day before, but not 1 day after, the injection of antigen, suggesting that DFP may well affect an early event of the antibody response. For the secondary TNP-specific IgM PFC response, a significant depression was observed only when DFP was administered at the time of antigen challenge. The injection of DFP at the time of antigen priming, however, was effective in depressing the IgG, but not the IgM, PFC response. Therefore, DFP may also interfere with the generation of antigen-specific memory cells for the secondary IgG response.

Enhanced transcription in a pre-B-like cell line transformed with cloned antibody genes after antigen-specific stimulation.

The transducing vector, pSV2-neo, carrying the rearranged immunoglobulin (Ig) heavy (mu) and light (kappa) chain genes specific for the hapten 2,4,6-trinitrophenyl (TNP) was introduced into a pre-B cell line. The transformants expressed the TNP-specific IgM receptor on the surface. Furthermore, the addition of TNP-bovine serum albumin (hapten-carrier conjugate) to the culture media activated the expression of the transferred Ig genes and several endogenous genes such as v-abl and beta-tubulin. However, expression of the beta2-microglobulin gene was not affected. The results presented in this paper show that transfection of cloned Ig genes into B cells is a useful system for establishing monoclonal B cell lines expressing functional Ig receptor molecules with defined hapten specificity.

Expression of I-A and I-E,C Region-Coded Ia Antigens on Functional B Cell Subpopulations

Abstract Ia antigens from specific subregions have been examined on functional B cell populations. Expression of both I-A and I-E,C region antigens was demonstrated on cells required for both lipopolysaccharide mitogenesis and polyclonal activation. Similar I-A and I-E,C subregion expression was found on cells required for response to the T-independent antigen, polyvinylpyrrolidone. TNP-specific IgM and hen egg lysozyme-specific IgG plaque-forming cells also express I-A and I-E,C region antigens. No evidence was found for an Ia- population responsive in the systems tested. Further, no evidence of preferential expression of I-A or I-E,C region antigens was observed in any system examined. Therefore, it appears that B cells express both I-A and I-E,C region-coded Ia antigens.