Immunoglobulin (Ig) mu, kappa transgenic mice express transgenic idiotype on endogenously rearranged IgM and IgA molecules by secretion of chimeric molecules.

The sera of C57BL/6 mice transgenic for a mu a allotype heavy (H) chain and kappa light chain gene contained endogenous nontransgene immunoglobulin (IgM) (mu b allotype) and IgA molecules which carried the idiotype expressed by the transgenically encoded IgM (mu a) molecule. Serological analysis demonstrated that the presence of the transgenic idiotype on endogenous IgM and IgA was caused by the secretion of chimeric molecules that carried both chains encoded by the mu a transgene and products of endogenously rearranged Ig mu b or alpha genes. These and other results suggest that allelic exclusion of Ig gene rearrangement in mu, kappa transgenic mice is not absolute, that B cells can secrete Igs composed of more than a single (H) chain type, and that endogenous isotype switching does not result in a complete silencing of transgene expression.

T cell independent antigens.

T cell independent antigens type 2 (TI-2), which are represented predominantly by polysaccharide antigens, stimulate humoral antibody responses in the absence of T-cell help. We and others have recently reported that natural killer cells and/or natural killer cell derived lymphokines may provide a form of 'help' that is necessary for the induction and maintenance of TI-2 responses. Two natural killer cell derived lymphokines, interferon-gamma and granulocyte-macrophage colony-stimulating factor, show synergistic stimulatory activity in inducing Ig secretion in B cells stimulated by a multivalent ligand that mimics TI-2 antigens. The recent finding that natural killer cells have receptors for various classes of polysaccharides supports a role for these cells in regulating responses to TI-2 antigens.

Analysis of anti-insulin antibodies using time-resolved fluoroimmunoassay.

This paper describes the analysis of both monoclonal and polyclonal murine insulin antibodies with time-resolved fluoroimmunoassay (TRFIA). This assay is based on the binding of antiserum by coated isotype-specific capture antibodies and the detection of insulin binding using europium-labeled human insulin (HI-Eu). This reagent was shown to have a detection limit of 10(-16) mol. Among various applications this technique permitted an isotype-specific determination of the affinity distribution of polyclonal antibodies from individual mice.

Substantially increased sensitivity of the spot-ELISA for the detection of anti-insulin antibody-secreting cells using a capture antibody and enzyme-conjugated insulin.

This paper describes an antibody capture spot-ELISA for the detection of anti-insulin antibody-secreting cells. The assay is based on the binding of secreted antibodies by immobilised isotype-specific capture antibodies and subsequent detection of insulin-specific antibodies with a conjugate of human insulin and alkaline phosphatase (HI-AP). Compared with the conventional approach, using antigen for coating and employing an enzyme-linked detecting antibody, this technique improved the detection of murine cells secreting anti-insulin antibodies of different IgG subclasses.

The effect of divalent and univalent binding on antibody titration curves in solid-phase ELISA.

This paper describes the influence of antigen coating concentration, epitope density per antigen molecule and anti-immunoglobulin reagents on antibody titration curves in solid-phase ELISA. Based on results obtained with fluorescein as the hapten and monoclonal anti-fluorescein antibody, which were confirmed in another antigen-antibody system, it is concluded that: (a) Antibody titration curves are independent of antigen-coating concentration in a limited range of concentrations only. (b) The complex between one antibody and two epitopes ('divalent binding') is more stable than the complex between one antibody and one epitope ('univalent binding). The ratio between divalent and univalent binding depends on the epitope density per antigen molecule and on the antigen-coating concentration. (c) The prozone phenomenon can be explained by an increased instability of plate bound antibodies due to a shift from divalent to univalent binding. (d) In solid-phase ELISA a correct evaluation of the antiserum specificity can be performed only if it is ascertained that all target antigens are coated under saturating conditions.

Terasaki-ELISA for murine IgE. III. Determination of concentration and functional affinity by sequential equilibrium binding analysis.

A simple Terasaki tray-based ELISA technique with a fluorescent detecting system has been used to determine the affinity of murine IgE antibodies. The system was shown to be sensitive enough to measure affinities in the range of 10(-6)-10(-10) M as well as detect IgE antibodies down to a limit of 0.1 ng/ml. The results, expressed as arbitrary fluorescence units (AFU), were compared with those obtained using equilibrium dialysis for several DNP-specific IgE monoclonal antibodies of known affinities yielding KD values. The relationship between KAFU and KD established a conversion factor which could then be used to compute KD from KAFU, provided the detection system remained identical. Based on the equations proposed, an alternative method for the quantitation of murine IgE is described which is independent of the affinity of IgE for the coated antigen.

Mice deprived of exogenous antigenic stimulation develop a normal repertoire of functional T cells.

The development of T cells and the selection of the TCR repertoire in the absence of exogenous antigenic stimulation were investigated. For this purpose germfree BALB/c mice fed an ultrafiltered solution of chemically defined low m.w. nutrients (GF-CD) were used. Previous studies on B cell development and differentiation in GF-CD mice have demonstrated a high reduction in the number of cells secreting Ig of the non-IgM isotypes but an Ig-VH gene usage and a B cell specificity repertoire that is substantially different from that observed in conventional adult mice and more closely resembles that of neonatal conventional mice. In contrast, the present comparison of the various lymphocyte populations in the thymus, lymph nodes, and spleen from GF-CD and conventional mice using flow cytometry analysis revealed no significant differences. Analysis of the TCR-V beta expression on both mature thymocytes and lymph node T cells showed a high degree of similarity between GF-CD and conventional mice. These findings indicate a marked difference in the influence of exogenous antigenic stimulation on the development of B and T cells. Additionally, development in an environment free of exogenous antigenic stimulation allows for full functional maturation of T cells to occur, because MLC showed that GF-CD splenic T cells could mount allogeneic responses in a way similar to T cells generated in a conventional environment. Most importantly, full Th cell function is generated, because activation of GF-CD spleen cells by cross-linking with mAb against CD3 resulted in the induction of cells secreting IFN-gamma and Ig of the non-IgM isotypes, which cannot be detected in GF-CD sera. These findings demonstrate that functional T and B cells develop in mice that have not been exposed to exogenous Ag, and that the TCR repertoire, in contrast to the B cell compartment, is predominantly shaped by endogenously expressed Ag.

Heterogeneity in the ability of cytotoxic murine NK cell clones to enhance Ig secretion in vitro.

We recently described a panel of cytotoxic murine NK cell clones that also enhanced Ig secretion by B cells activated in an in vitro model of T cell-independent type 2 (TI-2) responses. We employed dextran-conjugated anti-IgD (alphadelta-dex) as a model antigen. Here we study the mechanism of Ig induction by these clones. Addition of the various NK clones to sort-purified B cells stimulated with alphadelta-dex and IL-2 resulted in a markedly heterogeneous increase in Ig secretion, which varied from 3-fold, as mediated by clone PKO 56, to 15-fold, as induced by clone PKO 101. The other NK cells showed intermediate levels of Ig induction. Furthermore, while addition of as few as 0.04% of PKO 101 cells stimulated significant increases and 1% induced near maximum Ig production, a 3% addition of PKO 56 cells was required for significant enhancement of Ig secretion. Supernatant material collected from the NK clones mediated Ig production at levels that mirrored the induction by the corresponding cells. Cytokine analysis showed that while all members of the NK panel produced IFN-gamma only two secreted granulocyte macrophage colony stimulating factor and that the levels of Ig induction mediated by the NK clones correlated only with their levels of IFN-gamma secretion. Culture of B and NK cells in the presence of anti-IFN-gamma demonstrated that IFN-gamma was the critical cytokine in NK-induced Ig production. These findings establish heterogeneity in the ability of NK cells to increase Ig secretion in vitro and show that NK-produced IFN-gamma is an important factor in determining this heterogeneity.

T(h)1 versus T(h)2 cytokine profile determines the modulation of in vitro T cell-independent type 2 responses by IL-4.

We have previously demonstrated that stimulation of B cells by multivalent membrane Ig cross-linking, using dextran-conjugated anti-IgD mAb (alpha delta-dex), in the presence of cytokines, is an in vitro model for T cell-independent type 2 (TI-2) Ig secretory responses. Earlier studies have shown that IL-4 enhances IgM secretion upon stimulation with alpha delta-dex plus IL-5 and induces IgG1 isotype-switching, without altering the proliferative response to alpha delta-dex. Here we show that IL-4 can have both stimulatory and inhibitory effects on alpha delta-dex-induced Ig secretion. Both the kinetics and time of exposure to IL-4, and the nature of the cytokine additions, T(h)1 versus T(h)2, determine whether stimulation or inhibition is observed. Preincubation of sort-purified B cells with IL-4 caused a 6- to 8-fold increase in Ig secretory responses to subsequent stimulation with alpha delta-dex plus IL-1, IL-2 or a combination of both. However, the continued presence of IL-4 during B cell stimulation suppressed responses to all cytokine combinations tested, except for those which included IL-5. Of 11 cytokines tested, only IL-4 showed this dual effect of enhancement and suppression. The stimulatory effect of IL-4 required a minimum of 4 h of preincubation and could be inhibited by the addition of IFN-gamma. Thus stimulation of non-MHC class II-dependent T or non-T cells by multivalent antigens to secrete IL-4 may regulate the response to these antigens, such that early and brief exposure of B cells to IL-4 will enhance a subsequent TI-2 response in the presence of T(h)1-dependent cytokines, while continuous exposure will result in inhibition of the response.

B-cell activation by T-cell-independent type 2 antigens as an integral part of the humoral immune response to pathogenic microorganisms.

Antigens that are expressed on the surface of pathogens in an organized, highly repetitive form can activate specific B cells by cross-linking of antigen receptors in a multivalent fashion. B cells respond to these multivalent antigens in the absence of MHC class II-restricted T-cell help by a mechanism that depends on the expression of a functional Bruton's tyrosine kinase (Btk). Accordingly, this class of immunogens has been designated T-cell-independent type 2 (TI-2) antigens. The unique properties of the B-cell response to TI-2 antigens are critically dependent on the formation of a small number of antigen receptor clusters, each of which contains approximately 10 to 20 antigen-bound membrane Ig (mIg) molecules. These clusters induce local membrane association of multiple activated Btk molecules, which results in long-term mobilization of intracellular ionized calcium. Such persistent calcium fluxes efficiently recruit transcription factors and thereby induce T-cell-independent B-cell activation and proliferation. While this first signal of multivalent mIg cross-linking can induce B-cell proliferation, we propose that a second signal is required for a TI-2 Ig secretory response. We have found that engagement of members of the Toll-like receptor (TLR) family could provide second signals that selectively induce Ig secretion in B cells that were activated by multivalent, but not by bivalent, antigen receptor engagement. This finding demonstrates a general mechanism by which TLRs recognize molecular motifs on the surface of pathogens and provide the TI-2-activated B cell with a second signal. In addition, TLR-dependent recognition of these non-self motifs by cells of the innate immune system can induce these cells to provide alternative and/or additional second signals in the TI-2 response. The complement system provides another link between the B cell and the innate immune system, and facilitates the mIg signal transduction by recruitment of CD21 in the immune response. Thus, the TI-2 response provides the host with a combination of "the best of both worlds": the recruitment of the fine specificity of the adaptive immune response and the utilization of both the speed of the innate immune system and the wealth of cytokines produced by its member cells upon stimulation by pathogenic organisms or their products. By combining these two pathways, the TI-2 response enables the host to rapidly produce antigen-specific Ig effector molecules that can be secreted at a sufficient rate to keep up with the rapid multiplication of invading infectious microorganisms, and will also prevent the intracellular spreading of a significant part of this population.

Enhanced and sustained activation of human B cells by anti-immunoglobulin conjugated to the EBV glycoprotein gp350.

We coupled a monoclonal anti-human IgD to the gp350 gylcoprotein of Epstein-Barr virus, which has been shown to bind to the complement receptor 2 (CR2), and compared its B cell stimulatory ability to that of anti-Ig and to a multivalent anti-Ig-dextran conjugate. The anti-Ig-gp350 conjugate stimulated higher levels of human B cell proliferation in vitro than did anti-Ig or anti-Ig conjugated to control viral protein, comparable to the proliferation stimulated by the multivalent anti-Ig-dextran. This enhanced proliferation was dependent on binding of the conjugate to CR2, inasmuch as an anti-CD2 antibody blocked the enhanced proliferative response. This enhanced proliferative response was associated with prolonged elevations of intracellular ionized calcium, which was comparable to the response stimulated by anti-Ig-dextran. These findings suggest the use of gp350 as a carrier molecule for weakly immunogenic peptides or antigens which, when bound to gp350, would enhance B cell clonal expansion and activation of antigen-specific B cells.

In vivo polysaccharide-specific IgG isotype responses to intact Streptococcus pneumoniae are T cell dependent and require CD40- and B7-ligand interactions.

In vivo Ig responses to soluble, haptenated polysaccharide (PS) Ags are T cell independent and do not require CD40 ligand (CD40L). However, little is known regarding the regulation of in vivo PS-specific Ig responses to intact bacteria. We immunized mice with a nonencapsulated, type 2 Streptococcus pneumoniae (R36A) and compared the parameters that regulated in vivo Ig isotype responses to the bacterial cell wall C-PS determinant, phosphorylcholine (PC), relative to Ig responses to the cell wall protein, pneumococcal surface protein A. Consistent with previous reports using soluble PS and protein Ags, the anti-PC and anti-pneumococcal surface protein A responses differed in that the anti-PC response was induced more rapidly, had a distinctive Ig isotype profile, and failed to demonstrate boosting upon secondary challenge with R36A. However, in contrast to previous studies, the IgG anti-PC response was TCR-alphabeta+ T cell dependent, required CD40L, and was blocked by administration of CTLA4 Ig. The nature of the T cell help for the anti-PC response had distinct features in that it was only partially blocked by CTLA4 Ig and was dependent upon both CD4+ and CD8+ T cells. Surprisingly, whereas the IgM anti-PC response was largely T cell independent, a strong requirement for CD40L was still observed, suggesting the possibility of an in vivo T cell-independent source for CD40L-dependent help. These data suggest that the regulatory parameters that govern in vivo Ig responses to purified, soluble PS Ags may not adequately account for PS-specific Ig responses to intact bacteria.

Modulation of B-lymphocyte and NK cell activities by glycoinositolphospholipid purified from Trypanosoma cruzi.

Glycoinositolphospholipids (GIPLs) are some of the major glycolipids of the Trypanosoma cruzi surface that were previously shown to activate B cells. In the present study, we investigated whether (i) T. cruzi GIPLs could induce immunoglobulin secretion from B cells in the absence of T cells and NK cells and whether (ii) NK cells are also stimulated by the GIPLs. B cells purified from mice deficient in both T and NK cells (CD3epsilon transgenic mice) secreted immunoglobulin in response to the GIPL. This response was increased by coculture with a murine NK cell line. The T. cruzi GIPL also increased the NK cell (interleukin-2 induced) proliferative response. Our data indicate that the T. cruzi GIPL has a direct stimulatory effect on NK cells and induces immunoglobulin secretion in the absence of T lymphocytes and NK cells. These findings suggest that this T. cruzi-derived molecule may be one of the stimulators that lead to NK cell activation during T. cruzi infection.

Phenotypic and functional characterization of a panel of cytotoxic murine NK cell clones that are heterogeneous in their enhancement of Ig secretion in vitro.

NK cells not only function as cytotoxic effector cells, but also have immunoregulatory roles including the enhancement of Ig secretion. To have a stable and uniform population of NK cells to study their role in Ig secretion, we generated murine NK clones. Thus, culture of splenocytes from mice that were homozygous for a mutation in the p53 tumor suppressor gene (p53-KO) with IL-2 and poly(IC) resulted in a long-term NK line, from which four stable clones were derived. This approach also yielded a long-term NK line from splenocytes of normal C57BL/6 mice. Identification of the clones as members of the NK lineage was based on large granular morphology, expression of NK-TR and absence of TCR gene rearrangement. Flow cytometry revealed that all clones expressed IL-2R alpha and beta, chains and B220, but no CD3, NK1.1, DX5 or Ly-49. RT-PCR analysis showed heterogeneity in NK1.1 gene expression, and demonstrated expression of perforin and several granzymes in all clones. Three out of four clones lysed YAC-1, but not P815 target cells, corresponding to a pattern of NK specificity. All NK clones enhanced Ig secretion in an in vitro model for T cell-independent type 2 antigens, albeit to varying degrees. We found no correlation between the degree of helper activity of the NK clones and the level of their cytotoxic activity on YAC-1 targets. Thus, we established murine NK clones, and show that they mediate both cytotoxicity and enhancement of Ig secretion.

Multivalent cross-linking of membrane Ig sensitizes murine B cells to a broader spectrum of CpG-containing oligodeoxynucleotide motifs, including their methylated counterparts, for stimulation of proliferation and Ig secretion.

We have previously reported that B cells that are activated by multivalent but not bivalent membrane Ig cross-linking ligands synergize with various B cell activators culminating in enhanced B cell proliferation. In this study we asked whether B cells that are activated by a multivalent mIg cross-linking agonist could respond to oligodeoxynucleotides (ODN) containing non-stimulatory motifs. Earlier reports have shown that ODN containing a CpG motif in which the cytosine is unmethylated and is flanked by two 5' purines and two 3' pyrimidines induce high levels of B cell activation, while ODN whose CpG are methylated or flanked by sequences other than the optimal two 5' purines and two 3' pyrimidines were non-stimulatory. In this manuscript we show that when B cells are stimulated in vitro with dextran-conjugated anti-IgD antibodies (anti-IgD-dex), as the multivalent mIg ligand, their proliferation is enhanced and they can be induced to secrete Ig in response to ODN containing various non-optimal motifs, both methylated and non-methylated. Furthermore we could induce synergistic levels of proliferation with concentrations of anti-IgD-dex that were in the picomolar concentration range and with concentrations of ODN that were 10- to 100-fold less than previously reported to be necessary for mitogenic activity. These data provided a model to explain how low concentrations of a multi-epitope-expressing microorganism in the context of mammalian (methylated) or microorganism (non-methylated) DNA can lead to dysregulated B cell proliferation and Ig secretion.