The repertoire of anti-TNP antibodies in mice neonatally suppressed with anti-IgM

Previous studies have shown that mice immunized with TNP-Ficoll when young, adult, or aged expressed different repertoires of anti-TNP antibodies. The aim of the present study was to find out whether this age-related nonrandom progression was driven by antigen, and whether it was regulated by the immune network through surface-Ig receptors on B lymphocytes. The approach utilized was to block receptor expression on B lymphocytes of mice by the chronic administration of anti-IgM from birth for approximately 1 year, and then compare their subsequent antibody response to that of age-matched control animals. The results obtained have shown that the age-dependent shift in the anti-TNP repertoire expressed could take place in animals whose B lymphocytes were blind to antigen and anti-id for the greater part of their lives and thus suggest that the regulatory events responsible for this shift may be (surface Ig) receptor independent.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

Suppression of polyclonal antibody production in Trypanosoma cruzi-infected mice by treatment with anti-L3T4 antibodies

Acute Trypanosoma cruzi infection of mice results in a very marked polyclonal activation of B and T lymphocytes, accompanied by high numbers of Ig-secreting PFC and lectin-dependent effector CTL. Treatment of mice with monoclonal anti-L3T4 antibodies from the time of infection (days 0, 4, and 8) completely suppresses the polyclonal PFC response and CTL generation. Treatment of nude mice with antibody does not alter the lipopolysaccharide-induced polyclonal PFC response, and it only modulates the isotypic profile of the PFC response to T. cruzi infection, without reducing its magnitude. Furthermore, antibody-treated, T. cruzi-infected euthymic mice do not develop the typical B cell blastogenic response, but show high numbers of activated Lyt-2+ lymphoblasts in the spleen. These results indicate that effector cell generation in T. cruzi-infected mice is predominantly helper T cell-dependent.     

B cell dependent T cell function blocked by perinatal exposure of mice to anti-IgM antibodies

Mice born to mothers deprived of B lymphocytes by their chronic treatment with anti-IgM antibodies (Su/N) do not possess naturally occurring anti-ids (present in sera of normal mice at 2 weeks of age) up to 10 weeks, despite the presence of normal levels of B cells and serum Ig (in these animals). Su/N mice of the same age also lack a T cell subset which together with anti-ids are thought to participate in an antisuppressor regulatory pathway. It is suggested that early development of these T cells may be linked and be dependent on the presence of these anti-ids synthesized early in ontogeny, providing one explanation for a selective T cell deficiency of B cell-deprived mice.     

Suppression of polyclonal B cell activation in scrapie-infected C3H/HeJ mice.

A consistent modification in B lymphocyte activation has been observed 1 month after infection of C3H/HeJ mice with scrapie. The mitogenic response to lipopolysaccharide of splenocyte cultures from experimental mice was reduced 30 to 60% as compared to controls. This reduction in mitogen responsiveness was transient but coincided with the onset of detectable splenomegaly and with the reported recovery of maximum yield of infectious scrapie agent in the spleen. The DNA synthetic response to lipopolysaccharide stimulation of splenocytes from scrapie-infected C3H/HeJ mice was depressed relative to controls only between 20 and 40 days after intracerebral inoculation. At all other times, experimental and control responses were identical. Scrapie-associated decreases in mitogenesis were found whether the spleen cell cultures contained splenocytes from individual mice, splenocytes pooled from several mice, or gradient-purified mononuclear cells. The responses of C3H/HeJ splenocyte cultures to phytohemagglutinin or concanavalin A stimulation were unaffected by scrapie infection.     

Characterization of small lymphocytes in the bone marrow of mice after prolonged treatment with anti-IgM antibodies.

In mice treated with anti-IgM antibodies from birth, small lymphocytes in the bone marrow and spleen have been characterized by their incidence, surface markers, and turnover. Essentially complete elimination of IgM-bearing small lymphocytes followed injections of anti-IgM but this treatment did not deplete the IgM-negative small lymphocytes in the marrow. These IgM-negative cells also lacked other markers of B lymphocytes, such as receptors for Fc and complement; they failed to bind anti-mouse T-lymphocyte serum and they formed a rapidly renewing population within the marrow. This population may represent cells whose normal differentiation has been aborted by the anti-IgM antibodies or, alternatively, they may be “null” cells, distinct from B lymphocytes.     

Triple immunofluorolabeling with two rabbit polyclonal antibodies and a mouse monoclonal antibody allowing three-dimensional analysis of cotton wool plaques in Alzheimer disease.

We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Abeta42 or anti-Abeta40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Abeta40 or anti-Abeta42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 micro m were reconstructed to allow three-dimensional (3D) observation of these three epitopes. Abeta40 was localized to core-like structures, mainly in layers I-III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers I-VI, were labeled with anti-Abeta42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Abeta deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.     

Characterization of T cell proliferative responses induced by anti-Qa-2 monoclonal antibodies.

The MHC class I Qa-2 Ag are attached to the cell surface by a glycanphosphatidylinositol (GPI) anchor. Crosslinking of Qa-2 and several other cell surface Ag attached by the GPI linkage has been shown to lead to cell activation. We have developed 10 new anti-Qa-2 mAb and characterized their capacity to induce proliferation of spleen cells. In the absence of anti-Ig-mediated crosslinking, none of the mAbs alone could induce activation. However, mAb 23.1 which reacts with the alpha 3 domain of Qa-2, when combined with most of the other mAbs (alpha 1, alpha 2 domain reactive), activated cells in the absence of anti-Ig crosslinking. The mAb pair 23.1 plus 24.16 was the most proficient and induced proliferation in the absence of any exogenous second signals. Responses were greatly enhanced and equivalent to those seen with anti-CD3 by the addition of phorbol myristate acetate (PMA). Ionomycin, rIL-2, or rIL-4 also potentiated anti-Qa-2 responses but less efficiently than PMA. Significant strain variation in the magnitude Qa-2-mediated proliferative responses was observed correlating with the levels of Qa-2 expressed on the cell surface. Crosslinking of Qa-2 molecules by the mAb combinations was required because monovalent Fab fragments failed to activate cells. F(ab')2 fragments of mAb 23.1 plus 24.16 induced vigorous proliferation indicating that accessory cell presentation of the mAb via Fc receptors was not required. Immobilized (plate bound) anti-Qa-2 mAb induced proliferation suggesting that the Qa-2 pathway may be distinct from that of other GPI molecules such as Thy-1 and Ly-6. Populations enriched for T cells (approximately 95%) responded as well as whole spleen cells, whereas B lymphocytes failed to proliferate to anti-Qa-2. Both CD4+ and CD8+ cells were activated following crosslinking of Qa-2. Finally, T cell activation mediated by Qa-2 induced elevation of [Ca2+]i, IL-2R expression, and the release of IL-2. These data demonstrate that crosslinking of Qa-2 on T lymphocytes represents a potent pathway for inducing cell activation.     

Antibody-mediated regulation of T cell responses. I. Characterization of a monoclonal antibody which specifically regulates contact hypersensitivity to DNFB in BALB/c mice

Contact hypersensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB) in BALB/c mice is regulated by autoanti-idiotypic antibody. This report describes the preparation and characterization of a monoclonal antibody, 2-16.1, which has characteristics previously described for the serum anti-idiotypic antibodies. Monoclonal 2-16.1 was prepared by fusing lymph node (LN) cells from optimally sensitized BALB/c mice to the P3X myeloma. The monoclonal product of the cloned hybridoma is an IgM (K) immunoglobulin which does not bind to DNP-protein but which does bind to other immunoglobulins with anti-DNP specificity, primarily of the IgM class. Functionally, 2-16.1 inhibits the efferent limb of the CS reaction as measured by passive transfer of immunity. This inhibition is antigen-specific and appears to require the presence of a subset of Ia+ T cells in the DNFB-immune LN cell population. Suppression of transfer of immunity is strain-specific. Finally, suppression occurs only in the absence of complement, indicating that a lytic mechanism is not involved and that 2-16.1 does not recognize determinants expressed on the effector T cells of the CS reaction. Collectively, these results indicate that 2-16.1 is a monoclonal anti-idiotypic antibody, and that the hybridoma CSDNP 2-16.1 represents a clone of B cells which is stimulated during the primary CS response to DNFB and whose antibody product is involved in the endogenous, active regulation of this T cell-mediated response.     

Characterization of IL-6 receptor expression by monoclonal and polyclonal antibodies

mAb and polyclonal antibodies against human IL-6R were prepared by using a murine transfectant cell line expressing the human IL-6R and a synthetic oligopeptide made on the basis of the deduced amino acid sequence as immunogens. Immunoprecipitation of radiolabeled IL-6R with these antibodies showed that the Mr of a mature IL-6R was 80 kDa and its value was reduced to 50K after treatment with O- and N-glycanase and neuraminidase, indicating that IL-6R is a glycoprotein. Two mAb recognizing different epitopes were prepared. One, PM1 inhibited the binding of 125I-IL-6 to the receptor and blocked the IL-6-dependent growth of a T lymphoma line, KT3. PM1 could not bind to IL-6R when it was saturated with IL-6, indicating that this antibody recognizes the IL-6 binding or the adjacent site on IL-6R. The other, MT18 was not inhibited by IL-6 for its recognition of IL-6R, therefore, this could be used for cytofluorometric staining of normal cells. Nonstimulated B cells expressed undetectable amount of IL-6R regardless of the expression of surface IgD. However, after the stimulation with PWM, IL-6R was observed on IgD- B cells with a relatively large size, but subtly on IgD- small B cells and not on IgD+ B cells, fitting the function of IL-6 which acts on activated B cells to induce Ig production. In contrast, IL-6R was detected on non-stimulated CD4+/CD8- and CD4-/CD8+ T cells. The level of IL-6R on both T cell subpopulations was not significantly changed after stimulation with phytohemagglutinin.     

Characterization of rat T cell subset antigen by monoclonal antibody

6B2-B8 T cell hybridoma cells were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated RTH-7, reacted with 89.5% of rat thymocytes, 30.2% of rat spleen cells, and 42.3% of rat lymph node cells. The RTH-7 reacted with a subset of rat T cells but not with B cells. Double staining analysis demonstrated that RTH-7 stained a rat T cell subset distinct from R1-10B5-positive cells that were known to be equivalent to mouse Lyt-2. It was revealed that RTH-7 and W3/25 recognize different antigenic epitopes on the same molecule. The RTH-7 as well as W3/25 substantially inhibited the production of interleukin 2 by cells in mixed lymphocyte reaction and the lymphocyte proliferation induced by mixed lymphocyte reaction. The RTH-7 inhibited the lymphocyte proliferation induced by Con A whereas W3/25 failed to do so. The RTH-7 defined antigen has a molecular weight of 53,000 under reducing condition and 47,000 under nonreducing condition. The RTH-7 defined antigen showed a wide range of heterogeneity in pI (6.2-8.8). The associated molecule of approximate molecular weight of 27,000 was occasionally detected with the RTH-7 defined antigen in 6B2-B8 T cell hybridoma cells as well as peripheral T cells but not in thymocytes. Thus, RTH-7 detects a cell surface antigen of a functional T cell subset of rat origin.     

Antigenic determinants on rat metallothionein: fine epitope mapping for a murine monoclonal antibody and rabbit polyclonal antisera.

In order to determine the epitope of metallothionein (MT) to a murine monoclonal antibody (MT 189-14-7) which had been produced by immunization with rat MT 2 (Kikuchi et al. (1988) Mol. Immunol. 25, 1033-1036), various lengths of synthetic oligopeptides were tested for their inhibitory activities in competitive radioimmunoassay (RIA). The amino-terminal acetylated pentapeptide, AcMDPNC, exhibited an inhibitory activity comparable to that of native MTs, whereas the acetylated tetrapeptide, AcMDPN, and the deacetylated heptapeptide, MDPNCSC, were much less inhibitory. The results suggest that the major part of the epitope structure of MT to the MT 189-14-7 monoclonal antibody is located within the amino-terminal acetylated pentapeptide, AcMDPNC. The specificities of polyclonal rabbit anti-MT antisera raised against the same immunogen were also determined by using various animal MTs and synthetic peptides as inhibitors in the RIA. Among three antisera tested, two reacted with several amino-terminal oligopeptides similarly to the MT 189-14-7 antibody. The major epitope structures to these polyclonal antibodies were shown to be located within the acetylated tetrapeptide, AcMDPN. Another antiserum contained at least two different populations of antibodies: one consisted of antibodies reactive with the amino-terminal synthetic peptides, while the other was not reactive with them. These results suggest that, in the rabbit also, the amino-terminal region common to various animal MTs can be an epitope to antibodies raised against rat MT, as shown in the mouse. Moreover, the results indicate that the synthetic amino-terminal peptides are useful for determination of the specificity of polyclonal rabbit anti-MT antibodies, which have been widely used for the quantification of MTs.     

Regulation of lymphocyte production in the bone marrow. I. Turnover of small lymphocytes in mice depleted of B lymphocytes by treatment with anti-IgM antibodies

To examine the concept that the genesis of lymphocytes in the bone marrow may be regulated by homeostatic feedback signals from peripheral B lymphocytes or their products, lymphocyte production was measured in mice selectively depleted of B lymphocytes by repeated administration of anti-IgM antibodies from birth. The turnover of small lymphocytes was quantitated radioautographically after DNA labeling by continuous infusion of 3H-thymidine. In the femoral marrow of anti-IgM-treated mice, the number of small lymphocytes was reduced and their turnover time was shorter than in control mice, presumably reflecting the premature elimination from the marrow of maturing cells about to express surface IgM. The absolute number of small lymphocytes being produced per femur in unit time, however, was identical in anti-IgM-treated and control mice. Lymphocyte production in the thymus was also unaffected by anti-IgM suppression whereas in the spleen the turnover of small lymphocytes was reduced due to the lack of young immigrant B lymphocytes from the bone marrow. The results demonstrate that the normal large-scale production of lymphocytes in mouse bone marrow is independent of the magnitude of the peripheral pool of B lymphocytes or the level of circulating immunoglobulins, suggesting the process is not subject to feedback control. Some implications for the genesis and diversity of primary B lymphocytes are discussed.     

Laminin biosynthesis in the extracellular matrix-producing cell line PFHR9 studied with monoclonal and polyclonal antibodies.

The biosynthesis of the basement-membrane glycoprotein laminin in the mouse teratocarcinoma cell line PFHR9 was studied by immunoelectron microscopy and pulse-chase experiments using monoclonal and polyclonal antibodies. By immunoelectron microscopy, most of the protein was found to be aggregated on the outer cell surface. Cytoplasmic stainings were rare and were located next to the intracellular side of the plasma membrane. Sequential immunoprecipitations of cell extracts with a monoclonal antibody (4C12) sensitive to the laminin native conformation and with a polyclonal antibody enables laminin, the B1 subunit and a 410 kDa molecule to be distinguished. Most of the laminin is of the A(B1B2) type, and the 410 kDa molecule appears to be a B1B2 heterodimer. The assembly of laminin from subunits is completed in less than 1 h, and B chains are incorporated via the formation of the B heterodimers. The B2 and A chains are not found as free forms, so their levels appear to be the rate-limiting factors for the assembly of the dimers and laminin respectively. The formation of an uncross-linked A(B1B2) complex as a short-lived intermediate in the biosynthetic process is possible. Together with immunoelectron microscopy, the present study suggests that the protein is rapidly exported after assembly to accumulate on the outer side of the cell membrane. The biosynthesis of laminin in the PFHR9 cell line appears to be similar to that in other matrix-producing cell lines.     

Anti-CD45 inhibition of human B cell proliferation depends on the nature of activation signals and the state of B cell activation. A study with anti-IgM and anti-CDw40 antibodies

The proliferation of human peripheral and tonsillar B cells stimulated with the anti-CDw40 mAb 626.1 and/or anti-IgM antibody (Ab) in the presence of anti-CD45 mAb A.1.1 was investigated. The anti-CD45 mAb suppressed the anti-CDw40-stimulated proliferation of peripheral blood B cells but had no effect on the proliferation of unfractionated tonsillar B cells stimulated similarly. When tonsillar B cells were separated according to their sizes, the anti-CDw40-induced proliferation of small tonsillar B cells was inhibited by the anti-CD45 mAb, whereas large tonsillar B cells were resistant. In contrast, anti-IgM-induced proliferation of human B cells was always significantly inhibited by the anti-CD45 mAb regardless of cell size and tissue origin. The anti-CD45 mAb also inhibited the anti-IgM-induced initial rise in intracellular [Ca2+] and the G0-G1 cell cycle transition of small tonsillar B cells. However, co-stimulation with anti-IgM/anti-CDw40 Ab resulted in the resistance to the anti-CD45 inhibitory effect on proliferation of peripheral blood B cells and the majority of tonsillar B cells. In contrast, B cell proliferation co-stimulated with anti-IgM Ab/and B cell growth factors were always suppressed by the anti-CD45 mAb. These results demonstrate that certain activational signal mechanisms utilized by anti-CDw40/anti-IgM Ab and anti-IgM Ab/B cell growth factors are different in that B cells stimulated with these agents differ in their sensitivity to the anti-CD45 mAb. Moreover, both the activational state of human B cells and the nature of activation signals given determine their response to the inhibitory signals delivered by the anti-CD45 mAb.     

Tissue distribution of cytochrome c oxidase isoforms in mammals. Characterization with monoclonal and polyclonal antibodies

Monoclonal and polyclonal antibodies specific to the two isoforms of subunit VIa of bovine cytochrome c oxidase were generated and used to study the tissue distribution of this subunit pair in beef, human and rat. The so-called H-(heart) form was found exclusively in heart and skeletal muscle, whereas the so-called L-(liver) form was the only isoform present in brain, kidney, liver and smooth muscle. Little or no L-form was detected in skeletal muscle. In bovine heart no subunit VIa-L was detected, while in human heart the subunit VIa-H and VIa-L isoforms were present in roughly equal proportions. These results imply that, in humans, the deficiency of a subunit VIa isoform may have a different effect on the physiology of heart then on the physiology of skeletal muscle.     

Activation of rat B lymphocytes. I. Characterization of anti-immunoglobulin responses and isotype density of rat B cells

Spleen cells of two rat strains, Lewis and Brown Norway (BN), have been activated by lectins and by antibodies specific for immunoglobulin isotypes embedded in their cell membranes. Optimal concentrations of antibodies specific for mu, gamma, or delta-chains of rat augments in vitro incorporation of 3H-TdR 5 to 18-fold in Lewis B lymphocytes and 1.5 to 4-fold in BN B lymphocytes. In addition, F(ab')2 fragments of anti-Ig reagents induced Lewis splenic B cells but not BN B cells to incorporate 3H-TdR. Responses to LPS and dextran sulfate, B lymphocyte mitogens, measured by radioactive uptake, were five to 10 times greater in Lewis B cell populations than in BN B cell populations. Density of surface Ig isotypes and capping kinetics were similar in the two rat strains, although the percentage of T cells, T cell subsets, B cells, and Ia+ B cells differed in the spleens of these strains of rats. Both T lymphocytes and macrophages were needed in culture to effect an optimal response. IL-2 restored the response in B cell cultures depleted of T cells and macrophages, and enhanced 3H-TdR uptake in whole spleen cells of Lewis but not BN rats. The strain-dependent responsiveness of B cells to specific anti-Ig reagents or B cell mitogens appears to be associated with inherent (genetic) defects in T cells and B cells or defects in T cell to B cell cooperation in BN rats.     

T helper cell-dependent, microbial superantigen-induced murine B cell activation: polyclonal and antigen-specific antibody responses.

Microbial superantigens (SA), bound to human B cell surface MHC class II molecules, have been shown to promote direct, "cognate" interaction with SA-reactive autologous Th cells, resulting in polyclonal Ig production. To investigate the potential for microbial SA to support Th cell-dependent, Ag-specific antibody responses, we have extended our studies to the murine system. BALB/c Th cell lines (TCL), specific for either the Mycoplasma arthritis-derived SA or the Staphylococcus aureus-derived toxic shock syndrome toxin-1) were generated. These TCL cells are SA-specific, functionally noncross-reactive, and utilize distinct TCR V beta gene families. Coculture of SA-reactive TCL cells and syngeneic B cells bearing the relevant SA results in B cell proliferation and polyclonal IgM and IgG production. In contrast, Ag-specific (SRBC-specific) antibody-forming cells are only generated in cultures that also contain SRBC. Thus, microbial SA-mediated Th-B cell interactions induce both polyclonal B cell activation and provide selective help for the proliferation and/or differentiation of B cells that have encountered specific Ag. In additional studies, we determined that the in vivo administration of toxic shock syndrome toxin-1 to young, athymic (nude) BALB/c mice results in SA binding to splenic B cells, rendering these B cells effective stimulators of and targets for SA-reactive helper TCL cells. Taken together, these results demonstrate that microbial SA mediate productive Th-B cell interactions analogous to those that occur during allospecific Th-B cell interactions in vitro and GVHD in vivo. These findings are consistent with the hypothesis that microbial SA represent environmental factors that may trigger autoimmune disease in the genetically susceptible host.