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A substantial fraction of mature naïve B cells recognize endogenous antigens, and this autoreactivity must be controlled to prevent autoantibody secretion. Selective downregulation of the IgM BCR on autoreactive B cells has long been appreciated, and recent findings illustrate how this might impose tolerance. The BCR isotype maintained on autoreactive B cells, IgD, is less sensitive to endogenous antigens than IgM. This reduced sensitivity may be conferred by structural properties of IgD and/or differential association with activating and inhibitory co-receptors. Once activated, autoreactive B cells are normally excluded from rapid plasma cell responses, but they can enter the germinal center and lose their autoreactivity through a mutation-selection process termed clonal redemption., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The B cell antigen receptors (BCRs) play an important role in the clonal selection of B cells and their differentiation into antibody-secreting plasma cells. Mature B cells have both immunoglobulin M (IgM) and IgD types of BCRs, which have identical antigen-binding sites and are both associated with the signaling subunits Igα and Igβ, but differ in their membrane-bound heavy chain isoforms. By two-color direct stochastic optical reconstruction microscopy (dSTORM), we showed that IgM-BCRs and IgD-BCRs reside in the plasma membrane in different protein islands with average sizes of 150 and 240 nm, respectively. Upon B cell activation, the BCR protein islands became smaller and more dispersed such that the IgM-BCRs and IgD-BCRs were found in close proximity to each other. Moreover, specific stimulation of one class of BCR had minimal effects on the organization of the other. These conclusions were supported by the findings from two-marker transmission electron microscopy and proximity ligation assays. Together, these data provide evidence for a preformed multimeric organization of BCRs on the plasma membrane that is remodeled after B cell activation., (Copyright © 2015, American Association for the Advancement of Science.)

In humans, up to 75% of newly generated B cells and about 30% of mature B cells show some degree of autoreactivity. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model B-cell antigen receptor (BCR) transgenic systems have highlighted the critical role of functional unresponsiveness or ‘anergy’. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B-cell tolerance. However, it remains unclear whether the mature diverse B-cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which BCR signalling rapidly and robustly induces green fluorescent protein expression under the control of the Nur77 regulatory region, antigen-dependent and antigen-independent BCR signalling events in vivo during B-cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure, in turn, tunes the responsiveness of BCR signalling in B cells at least partly by downmodulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or anergy exists in the mature B-cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease.

Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca(2+) mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+) IgD(+) lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

Classical B lymphocyte activation is dependent on BCR cross-linking in combination with physical interaction with Th cells. Other B cell molecules that contribute to the activation are complement, cytokine, and TLRs recognizing specific pathogen-associated molecular patterns. Moraxella (Branhamella) catarrhalis is a common Gram-negative respiratory pathogen that induces proliferation in human IgD-expressing B cells independently of T cell help. The activation is initiated by the B cell superantigen Moraxella IgD-binding protein (MID) through a nonimmune cross-linking of IgD. However, IgD cross-linking alone is not sufficient to induce proliferation. In this study, we characterized the significance of TLRs in superantigen-dependent B cell activation using whole bacteria or rMID in the presence or absence of TLR ligands. IgD cross-linking by MID sensitized B cells obtained from children with tonsillar hyperplasia for mainly TLR9, whereas TLRs 1, 2, 6, and 7 were less important. The Moraxella-induced activation was inhibited when a dominant-negative TLR9 ligand was added. Interestingly, BCR-mediated endocytosis of whole Moraxella and degradation of live bacteria in naive B cells were observed with fluorescence, confocal, and transmission electron microscopy. This unique observation proved the strong intracellular TLR9 response as well as highlighted the Ag-presenting function of B cells. In conclusion, our findings suggest an important role of TLRs in the adaptive immune response and reveal novel insights into the T cell-independent B cell activation induced by bacteria.

Over the last several years, there has been a great deal of progress in characterizing the role of dendritic cells (DCs) in the activation and modulation of B cells. DC-secreted chemokines can induce B cell trafficking to the lymph nodes. DC-produced survival factors such as B cell-activating factor of the TNF family and a proliferation-inducing ligand have been shown to be essential for B cell maturation, but have also been implicated in class-switch recombination and B cell lymphoma survival. Recently added to this list of DC-derived factors effecting B cells is IgA-inducing protein (IGIP). In this study, we characterize production of IGIP by human DCs, and examine its capacity to induce IgA class switching and differentiation of naive B cells in vitro. Monocyte-derived DCs were cultured in vitro with TLR agonists (TLR3, 4, 5, and 9) and other factors, including CD40 ligand, GM-CSF, and IL-4 as well as the neuropeptide vasoactive intestinal peptide. Under in vitro stimulation with vasoactive intestinal peptide and CD40L, IGIP mRNA expression could be up-regulated as much as 35-fold above nonstimulated samples within 12-48 h. Naive B cells cultured with exogenous recombinant human IGIP produced IgA in greater quantities than nonstimulated controls. Finally, we demonstrate that IGIP stimulation drives the production of mu-alpha switch circles from IgM(+)IgD(+) naive human B cells, indicating its role as an IgA switch factor.

Contact sensitivity (CS) is related to delayed-type hypersensitivity and is a well-characterized prototype of T cell-mediated inflammation. However, the inflammatory response associated with CS is additionally dependent on Ag-specific IgM produced by a subpopulation of B cells in response to sensitization. Upon re-exposure to hapten, this IgM mediates rapid vascular activation and subsequent recruitment of proinflammatory T cells to the local site. Interference with this pathway prevents the full development of the classic delayed inflammatory response and is therefore termed the "CS initiation" pathway. In this study, we show that CS initiation is defective in mice deficient in activation-induced deaminase, an enzyme central to the process of somatic hypermutation. Using adoptive transfer experiments, we demonstrate that the defect is specific to a B-1-like population of B cells and that transfer of WT cells reconstitutes CS initiation mechanisms in deficient recipients. We went on to identify a novel subpopulation of Ag-binding B cells in the spleens of sensitized mice that possess initiation activity (CD19(+)CD5(+)Thy-1(int)IgM(high)IgD(high)) that we name "initiator B cells." Analysis of BCR H chain genes isolated from these cells revealed evidence of activation-induced deaminase-mediated somatic hypermutation. The sensitivity of CS initiation to very low amounts of sensitizing hapten suggests that the responsible B cells have increased IgM receptor gene mutations enabling selection to generate Abs with sufficient affinity to mediate the response.

ZAP-70 is a protein tyrosine kinase initially found in T and NK cells. Recently, this important signaling element was detected in leukemic B cells from a subgroup of patients with B cell chronic lymphocytic leukemia (B-CLL). In this study, ZAP-70 was detected in normal B cells from human tonsils, but not from peripheral blood. The cDNA sequence of B cell ZAP-70 was the same as that in T cells. Germinal center B cells and plasma cells had a substantial proportion of ZAP-70+ cells, while memory and follicular mantle B cells, which contain low numbers of activated B cells, expressed relatively little ZAP-70. A cell fraction of IgD+, CD38+ B cells, which are comprised of many in vivo activated B cells, exhibited the highest levels of ZAP-70. Density gradient fractionation of tonsil B cells confirmed that ZAP-70 was not expressed by resting B cells, but was expressed by buoyant, in vivo activated B cells. In these B cells, the expression of ZAP-70 correlated with that of CD38 and not with that of CD5, a hallmark of B-CLL cells. B-CLL cells are activated cells and their ZAP-70 expression reflects a normal property of activated B cells populations rather than a neoplastic aberration.

The induction of a humoral response depends upon efficient cross-linking by antigen of surface immunoglobulin on primary B lymphocytes. We demonstrate here the presence of a glycosylphosphatidylinositol-linked isoform of membrane IgD (mIgD) receptors on murine resting B cells. This subset was constitutively localized to cell membrane raft microdomains. Its stimulation resulted in the activation of cAMP-dependent signaling pathways, which integrated with signals derived from the transmembrane mIgD receptors. This, in turn, provided a mechanism by which the activation status of the target cells could be variably regulated. Thus, by partitioning receptor activity, preimmune B cells can moderate the extent to which they are activated, depending upon the strength of the antigenic stimulus.

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.

Effects of TGF-beta and IFN-gamma on locomotion of high density B cells from human tonsils were studied using polarization and filter assays. Culture with TGF-beta (10 pg to 1 ng/ml) induced a gradual increase in locomotor morphologies in 40 to 50% of B cells during overnight culture. This was not immediate (<30 min) suggesting that TGF-beta is not a chemotactic factor. The time course suggests that B cells acquired a locomotor phenotype during the first few hours of culture. B cells cultured in TGF-beta increased in size, but to a lesser extent than those cultured in IL-4 used as a control. More cells were polarized in TGF-beta + IL-4 than in either alone. Following culture in TGF-beta, B cells showed vigorous responses to anti-IgD used as a chemoattractant in short-term assays. In contrast, addition of IFN-gamma (20-100 U/ml) to B cells in culture in IL-4, anti-CD40, or TGF-beta inhibited activation of locomotor capacity by the latter agents, and IFN-gamma-cultured B cells showed an even lower response to anti-IgD in a short-term polarization or filter assay than those cultured in medium alone. IFN-gamma also inhibited uridine incorporation by cultured B cells, and cells cultured in IFN-gamma showed no size increase. We suggest that IFN-gamma prevents locomotor activation by inhibiting progress of B cells into the G1 phase of growth.

CHO cells permanently transfected with mouse Fc gamma RI alpha chain were prepared and used as a model to polyclonally activate murine B cells. The transfected CHO cells were treated with mitomycin C and placed into culture with varying quantities of anti-IgD. Using this model, murine splenic B cells (from BALB/c or C57Bl/6) were activated by mouse IgG2a-anti-IgD (10.4.22 or AF3.33) in a manner that is analogous to the activation of B cells seen with highly polyvalent anti-IgD (H delta(a)/1) prepared by chemical cross-linking to dextran. Efficient B cell activation was seen with nanogram quantities of anti-IgD. In the presence of IL-4 and IL-5, IgG1 production levels were equivalent to or better than seen when stimulation was with H delta(a)/1-dextran; however, IgE induction was not seen in either situation. The Ig production capacity was compared to that seen when B cells were activated with CD40L, using either CD40L-transfected CHO or a soluble CD40L construct. In the presence of IL-4 and IL-5, once a critical threshold of B cells was present, IgE and to a lesser extent IgG1 production was inversely proportional to B cell number when CD40L was the activating agent. In contrast, with Fc gamma RI-anti-IgD, IgM and IgG1 production was directly proportional to B cell number, while IgE production was never seen. Finally, when B cells were co-activated with immobilized anti-IgD and CD40L simultaneously, the IgE production from B cells induced by CD40L was strongly inhibited, while IgG1 and IgM production were not affected. Since B cell co-activation via sIg and CD40L would be a common scenario in secondary follicles, this inhibition of IgE production may be one of the reasons why serum IgE levels are much below IgG in normal immune situations.

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.

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.

Motheaten viable (mev) mice are deficient in the cytosolic protein tyrosine phosphatase, PTP1C, and exhibit severe B cell immunodeficiency and autoantibody production. The role of PTP1C in B cell selection and function was analyzed by breeding immunoglobulin transgenes specific for a defined antigen, hen egg lysozyme, into mev mice. Antigen triggered a greater and more rapid elevation of intracellular calcium in PTP1C-deficient B cells, indicating that this phosphatase negatively regulates immunoglobulin signaling. Elimination of self-reactive B cells carrying this signal-enhancing mutation was triggered during their development by binding a lower valency form of self-antigen than is normally required. These findings establish that activation of distinct repertoire-censoring mechanisms depends on quantitative differences in antigen receptor signaling, whose thresholds are determined by negative regulation through PTP1C.

Signaling through the T cell receptor must be accompanied by costimulatory signals for the differentiation of naive T cells to cytokine-producing effector T helper cells. The costimulatory signal through CD28 is required for T cell activation resulting in increased interleukin (IL)-2 production in vitro, but its role in the production of IL-4 and in the in vivo response is still unclear. We have examined the effects of blocking CTLA-4 (the CD28 homologue) ligand interactions on the in vivo development of IL-4-producing T helper effector cells during a primary mucosal immune response to the nematode parasite Heligmosomoides polygyrus and during a primary systemic immune response to immunogenic anti-IgD antibodies. Our results demonstrate that CD28 and/or CTLA-4 signaling is required for T cell priming leading to IL-4 cytokine production, B cell activation, and IgE secretion during both immune responses, suggesting that other signaling molecules do not substitute for these molecules in either of these two different immune responses. Furthermore, the CD28 ligands, B7-1 and B7-2, can substitute for each other in providing the required T cell costimulatory ligand interactions during the primary immune response to H. polygyrus. In contrast, memory T cells during the challenge immune response do not require CD28/CTLA-4 ligand interactions for IL-4 production and T helper effector function.

In vivo experiments were performed to determine if the cross-linking of mlg on antigen-presenting B cells could induce them to present Ag to naive T cells in a stimulatory rather than a tolerogenic fashion. Mice were injected with a foreign mAb to a B cell Ag (Fc epsilon RII or CR1), and/or a self-anti-IgD mAb. Injection of either mAb alone failed to induce an Ab response; however, simultaneous injection of the foreign anti-B cell mAb plus the self-anti-IgD mAb stimulated a large response. Inasmuch as injection of the anti-IgD mAb should not have facilitated transfer of the foreign mAb to dendritic cells, this observation suggests that cross-linking of B cell mlg can induce B cells to acquire the ability to present Ag to naive T cells in an activating manner. Furthermore, when injected with anti-IgD mAb, both foreign anti-B cell mAbs were more potent in inducing Ab responses in this system than were isotype-matched control mAbs, consistent with the hypothesis that T cells were activated by Ag-presenting B cells. Results of dose-response and cell transfer studies, however, suggested that stringent cross-linking of B cell mlg on large numbers of B cells is required for B cell Ag presentation to induce T cell activation rather than tolerance. Therefore, these observations suggest that professional APCs usually are required to activate naive T cells, and that B cell Ag presentation can only activate naive T cells under unusual circumstances.

In the present study, we examined the participation of CD40 ligand (L)-CD40 interaction in T cell-dependent B cell responses. To this end, purified B lymphocytes were cultured over irradiated CD4+ cloned T cells activated with immobilized anti-CD3 antibody. The anti-CD40 mAb 89 strongly blocked, in a specific fashion, both proliferation and Ig secretion of tonsil B cells. Interestingly, proliferation of surface (s)IgD+ B cell was significantly less inhibited by anti-CD40 than that of sIgD- cells. Preactivated T cells induced B cells to grow and secrete immunoglobulins preferentially in response to IL-2. This contrasts with the CD40 system where B cells are essentially responsive to IL-4 and IL-10 but not to IL-2 alone. Collectively, these data indicate that CD40L-CD40 interaction plays an important role in IL-2 mediated T cell-dependent B cell responses. However, the activation of a subset of sIgD+ cells may be independent of this interaction.

Cross-linking of surface IgM (sIgM) or sIgD by anti-IgM Ab or anti-IgD Ab, respectively, induced DNA synthesis in peripheral blood B cells (PBL-B). Cell division, determined by the increase in the number of M phase cells, was also induced when PBL-B were stimulated with anti-IgD Ab plus IL-4 or Staphylococcus aureus Cowan I (SAC), but far less by stimulation with anti-IgM Ab plus IL-4. Anti-IgM Ab did not suppress the DNA synthesis induced by SAC or anti-IgD Ab plus IL-4, but it did suppress the cell division induced by them. Thus, sIgM cross-linking generates both positive and negative signaling to B-cell proliferation. Cyclosporin A (CSA) and FK506 suppressed DNA synthesis and cell division at relatively high concentrations. On the other hand, CSA and FK506 at lower concentrations blocked the anti-IgM Ab-generated inhibition of cell division without affecting DNA synthesis. Low concentrations of CSA did not affect the cell division induced by anti-IgD Ab plus IL-4 but did increase the cell division induced by SAC or anti-IgM Ab plus IL-4, suggesting that stimulation with SAC, as well as with anti-IgM Ab plus IL-4, generates both positive and negative signals to cell division, whereas sIgD lacks the ability to transduce negative signaling.

Low concentrations of anti-Ig dextran conjugates that stimulate very high levels of B cell proliferation and Ig secretion stimulate no detectable increases in tyrosine phosphorylation. To study this point further, we compared tyrosine phosphorylation patterns induced by mitogenic and nonmitogenic anti-Ig antibodies. Whereas the mitogenic, strongly cross-linking, antibody H delta a/1 induced greater levels of tyrosine phosphorylation than did the nonmitogenic antibody FF1-4D5, the pattern of substrate phosphorylation was equivalent. At lower concentrations of H delta a/1, which were still mitogenic, the degree of phosphorylation that was induced was similar to that induced by high concentrations of FF1-4D5. Both antibodies stimulated comparable increases in the kinase activity of the three src-related kinases present in normal B cells and linked to the IgR, i.e., Blk, Fyn, and Lyn. These results suggest that the extent of tyrosine kinase activation is proportional to mIg cross-linking, that induction of B cell DNA synthesis may require little tyrosine kinase activation, and that activation of tyrosine kinase per se does not necessarily lead to B cell DNA synthesis.

A series of immunoglobulin (Ig)-transgenic mice were generated to study the functional capabilities of the IgM and IgD classes of B lymphocyte antigen receptor in regulating both cellular development and responses to specific antigen. B cells from Ig-transgenic mice expressing either hen-egg lysozyme (HEL)-specific IgM or IgD alone were compared with B cells from mice that coexpressed IgM and IgD of the same anti-HEL specificity. In all three types of Ig-transgenic mice, conventional B cells specific for HEL exhibited exclusion of endogenous Ig expression and matured to populate the usual microenvironments in peripheral lymphoid tissues. These peripheral B cells could be stimulated by HEL through either IgM or IgD antigen receptors to generate T cell dependent antibody production in vivo or to enhance T cell independent proliferative responses to lipopolysaccharide in vitro. Conversely, when HEL was encountered in vivo as a self-antigen, B cells expressing HEL-specific IgM or IgD alone were both rendered tolerant. In each case this occurred by clonal anergy in response to soluble autologous HEL, and clonal deletion when HEL was recognized as a membrane-bound self-antigen. Taken together, these findings indicate that IgM and IgD antigen receptors expressed alone on conventional B cells can support normal differentiation, antigen-dependent activation, and induction of self-tolerance, the only overt difference lying in a greater degree of receptor downregulation for IgM relative to IgD after induction of clonal anergy by soluble HEL.

The purpose of the experiments described in these studies was to determine the effects of 7,12-dimethylbenz[a]anthracene (DMBA) on B-cell activation produced by anti-IgD antibodies and interleukin-4 (IL-4). B and T cells are known to share many of the same biochemical pathways for cell activation by mitogen and antigen receptors. Previous studies in this laboratory have shown that DMBA inhibits mitogen-induced Ca2+ mobilization in murine and human T cells and produces an increase in intracellular Ca2+ in resting cells. The results of the present studies demonstrate that DMBA increases Ca2+ in resting B cells and inhibits B cell activation produced by anti-IgD antibodies, as measured by mobilization of free intracellular Ca2+ and [3H]thymidine incorporation. The proliferative response of B cells to insolubilized anti-IgD was suppressed only when cells were preexposed to DMBA. In contrast, IL-4 pathways of B-cell activation were insensitive to inhibition by DMBA, even when cells were preexposed. The induction of Class II MHC antigen (Ia) antigens on B cells by IL-4 was also found to be insensitive to DMBA treatment. These results suggest that DMBA suppresses only Ca(2+)-dependent pathways of B cell activation and indicate that altered Ca2+ homeostasis may be responsible for immunosuppression induced by this agent.

B lymphocytes, like macrophages and dendritic cells, can present antigen to CD4+ T cells. Antigen presentation by B cells is essential for the generation of an in vivo T cell dependent antibody response, and repeated antigen presentation by B cells to T cells is necessary to induce B cell clonal expansion. Presentation of antigen by resting B cells to unprimed T cells tolerizes T cells, while anti-IgD antibody activates B cells and allows B cell antigen presentation that productively activates T cells. However, activation is not all that is required for B cells to productively present antigen to T cells.

Insolubilized anti-IgD antibody markedly increased DNA synthesis in and cell division of normal peripheral blood B cells (PBL-B) when used in combination with IL-4. Anti-IgM antibodies also induced DNA synthesis of PBL-B, but their ability to induce cell division was less than that of anti-IgD antibodies even when used in combination with IL-4. Moreover, anti-IgM antibodies inhibited cell division of PBL-B stimulated with insolubilized anti-IgD antibody plus IL-4 without affecting DNA synthesis. Anti-IgM antibodies also inhibited Staphylococcus aureus Cowan I-induced cell division of PBL-B without affecting DNA synthesis. These results indicate that cross-linkage of surface IgM (sIgM) in mature B cells generates negative signals to inhibit cell division of mature B cells. Because anti-IgD antibodies did not inhibit cell division at all, the role of sIgD in the regulation of cell division of mature B cells may be quite different from that of sIgM. IFN-alpha/beta promoted cell division of PBL-B stimulated with insolubilized anti-IgD antibody plus IL-4. They also counteracted the inhibitory effect of anti-IgM antibody on cell division of PBL-B.

Stimulation of resting B lymphocytes with antibodies to surface immunoglobulin (sIgD or sIgM) induces protein tyrosine phosphorylation, implicating one or more B-cell protein-tyrosine kinases (PTKs) in sIg signal transduction. We have evaluated whether members of the src family of PTKs are involved in this process. Our results show that addition of antibodies to IgD or to IgM can stimulate the PTK activity of the blk, fyn, and lyn gene products. Additionally, all three PTKs were found to coimmunoprecipitate with sIg in digitonin lysates from resting B cells. In all stimulatory conditions, whether initiated through sIgD or sIgM, the blk gene product p56blk displayed the strongest activation index. The kinetics of activation of these kinases, particularly that of p56blk, paralleled the early appearance of newly tyrosine-phosphorylated B-cell proteins, suggesting that this group of kinases may account for some portion of the tyrosine kinase activity in sIg-activated B cells. These observations demonstrate a functional and possible physical association between the members of the src family of PTKs and the B-cell antigen receptors.

The injection of mice with a foreign, polyclonal antibody to IgD sequentially induces: 1) activation of B cells by cross-linking of their cell membrane (m) IgD; 2) B cell processing and presentation of the bound anti-IgD antibody to T cells; 3) activation of these T cells; and 4) T-dependent stimulation of B cell differentiation into IgG1 secreting cells. To determine whether the cross-linking of B cell membrane IgD and/or the resulting B cell activation that follows contribute to the generation of the polyclonal IgG1 response, we examined the abilities of three sets of anti-delta mAb or mAb fragments to stimulate polyclonal IgG1 production. Within each set mAb were matched for species and Ig isotypic determinants, but differed in avidity for IgD or in ability to cross-link IgD. In addition, experiments were performed to determine whether the anti-delta mAb had to be foreign to the immunized mouse to stimulate an IgG1 response. Results of these experiments indicate that: 1) recognition of the injected anti-delta antibody as foreign is required for the induction of a polyclonal IgG1 response; 2) the cross-linking of B cell membrane Ig, which directly activates B cells, can contribute considerably to the generation of in vivo IgG1 production; and 3) that even relatively weak cross-linking of membrane Ig by ligands that bind it with low avidity can make this contribution.

Injection of BALB/c mice with an affinity-purified goat antibody to mouse IgD (GaM delta) stimulates T cell-independent B cell activation as well as later T cell activation. Activated T cells then induce polyclonal differentiation of B cells into IgG1-secreting cells, which results in an approximately 100-fold increase in serum IgG1 level. It is not known whether the same B cells that are initially activated by GaM delta are the progenitors of the IgG1-secreting cells. To investigate this issue a system was developed in which CB20 mice, which are congenic to BALB/c mice but express Ig of the beta allotype rather than the BALB/c alpha allotype, were injected with GaM delta and simultaneously or subsequently also received BALB/c B cells. The IgG1 response generated by the donor BALB/c B cells was quantitated by an assay specific for IgG1 of the alpha allotype. Our experiments with this system indicate that: 1) BALB/c B cells transferred 2 days after CB20 mice were injected with GaM delta generate a much larger IgG1 response than do BALB/c B cells transferred simultaneously with GaM delta antibody; 2) B cells that express membrane IgD generate the great majority of this response; 3) differences in the magnitudes of the responses of BALB/c B cells transferred at different times after CB20 mice were injected with GaM delta antibody cannot be explained by differences in homing of the donor B cells to the host spleen or by short survival of donor BALB/c B cells after their transfer; and 4) the response made by donor BALB/c B cells transferred 2 days after CB20 mice were injected with GaM delta is proportionate to donor cell representation in the host spleen 1 day after their transfer, whereas the response made by donor cells transferred simultaneously with GaM delta is disproportionately small. These observations suggest that most of the IgG1 antibody made by GaM delta-injected mice is generated by newly produced, mIgD+ B cells that appear approximately 2 days after GaM delta injection, rather than by those B cells that are present in the spleen at the time of GaM delta injection, and support the view that signals that induce B cell secretion of Ig require an interaction with at least partially activated Th cells.

ECH408-1 is a murine B cell lymphoma expressing idiotypically and allotypically distinguishable transfected and endogenous IgD. Previously, we demonstrated that this cell line was not growth inhibited by antibodies directed at membrane IgD, but could be inhibited by antibodies which crosslink membrane IgM. Herein, we demonstrate that both anti-mu and anti-delta will cause calcium mobilization in this transfected cell line; this is followed by a period during which antibodies against the alternative isotype are unable to induce significant increases in intracellular calcium concentrations. This phenomenon, called "desensitization," is short-lived, lasting 20 min. We further demonstrate that acute desensitization of these cells by anti-delta has no effect on immediate growth inhibition which is elicited by anti-mu. These data confirm our earlier proposal that the rapid, initial calcium response seen in these lymphomas is not required for the negative signal for growth. Moreover, we also demonstrate that pretreatment of these lymphoma cells with phorbol myristate acetate (PMA) also renders these lymphoma cells temporarily incapable of manifesting a significant calcium signal. Nonetheless, PMA-pretreated B lymphoma cells are not altered in their subsequent sensitivity to anti-mu growth inhibition, nor are they affected in their resistance to inhibition by anti-delta. Our data confirm the proposal that neither the calcium signal nor protein kinase-C activation is involved in the modulation of B lymphoma growth.

Previous studies with BALB/c mice have established that the injection of a goat antibody to IgD (GaM delta) induces splenic B lymphocytes to undergo T-independent, polyclonal increases in cell surface (s) Ia expression, size, and DNA synthesis 1 day after injection, as well as T-dependent maintenance of this activation and differentiation into sIgG1+ cells and IgG1-secreting cells 6 to 7 days after injection. Because B lymphocytes from mice homozygous or hemizygous for the CBA/N X-linked immune defect fail to proliferate in vitro when cultured with soluble anti-Ig antibodies, yet can make in vivo primary antibody responses to T-dependent antigens, we studied the effects of injecting (CBA/N X DBA/2)F1 male mice (immune defective) and (CBA/N X DBA/2)F1 female mice (phenotypically normal) with 800 to 1600 micrograms of GaM delta. B cells from immune defective mice demonstrated definite but distinctly subnormal increases in B cell size, DNA synthesis, and sIa expression 1 to 5 days after GaM delta injection, but by day 7 showed increases in these parameters as well as in the percentages of splenic sIgG1+ cells and IgM-secreting cells that were similar to those exhibited by the immunologically normal mice. However, IgG1 secretion by the immune defective mice was at this time only one-fourth to one-half as great as that observed with normal mice. Our data raises the possibility that the differentiation of B cells into IgG1-secreting cells may be more dependent upon the GaM delta-induced activation steps that are defective in CBA/N mice than is the differentiation of B cells into IgM-secreting cells or into sIgG1 cells.

We have attempted to develop an in vitro system where polyclonal B lymphocyte responses could be induced in 'antigen-like' conditions, that is, where surface immunoglobulin-dependent binding mediates interaction with a mitogen. Monoclonal anti-mu and anti-delta antibodies were covalently bound to lipopolysaccharide (LPS) and these complexes were shown to display mitogenic activity. Polyclonal plaque-forming cell (PFC) responses, however, were diminished in cultures stimulated by anti-mu-LPS (but not by anti-delta-LPS) indicating that 'anti-mu inhibition' of terminal B-cell differentiation also applies to 'specific' antibody responses. Moreover, the analysis of the functional activity of monoclonal antibodies to major histocompatibility complex (MHC) class II molecules revealed a surprising synergy between low, non-stimulatory concentrations of anti-mu-LPS (but not anti-delta-LPS) with anti-I-A antibodies. These responses are T-cell dependent and synergy with anti-mu-LPS conjugates can also be obtained with 'naturally' activated CD4+ cells isolated from normal donors. A model of molecular and cellular interactions was derived, which accounts for the present findings and is applicable in antigen-dependent lymphocyte collaboration.

The effect of the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate) on normal human peripheral blood and tonsil B lymphocytes was investigated. A strong DNA-synthesis response with the maximum at day 4 was detected. This response was, however, inhibited by increasing concentrations of serum in the medium. The membrane Ig expression was changed with a rapid decrease in IgD expression and a slower decrease in IgM and IgG expression. TPA-induced Ig secretion was detected in 12 out of 22 tested donors and the response was found to be independent of T cells and macrophages. The expression of four monoclonal antibody-detected B cell activation and differentiation markers, B1, B2, LB1 and BB1, was followed. The results indicate activation and differentiation of the B cells.

The role of delta-positive cells in the immune response was studied by comparing the effects of treatment with allotype-specific IgD hybridoma antibody on homozygous BALB/c or SJL/J and heterozygous (BALB x SJL)F1 mice. Homozygous mice, injected from birth with the relevant anti-delta antibody, made primary or secondary immune responses to intravenously injected trinitrophenyl (TNP)-Brucella abortus, TNP-Ficoll, and TNP-keyhole limpet hemocyanin, which did not differ significantly from those of control mice, despite the fact that IgD+ cells were depleted and Ig+ cells were markedly reduced in the spleens of treated mice. Responses in nodes draining a local injection of TNP-Brucella abortus were, however, significantly suppressed. Heterozygous mice, injected from birth with either anti-Ig-5a or anti-Ig-5b, showed a marked reduction in the number cells producing IgG antibody of linked allotype specificity in the secondary response to intravenously injected sheep erythrocytes. A corresponding decrease in the amount of serum IgG2a of that allotype specificity was also noted. However, in agreement with the results obtained in homozygotes, heterozygotes injected simultaneously with anti-IgD directed against each of the allotypes made normal, if not enhanced, plaque-forming cell responses of both allotype specificities. Similarly, serum IgG2a levels were normal in all but one mouse treated in this fashion. These results indicate that IgD+ cells are not essential for an immune response in vivo. Although the delta-positive cell is used preferentially under normal conditions, it appears that an alternative mechanism exists by which, in the absence of these cells, the animal is able to make a normal immune response.

The majority of B lymphocytes in adult mice express both IgM and IgD on their surface (sIgM and sIgD). A small percentage of sIgM+ splenic B cells lack (or express very low levels of) sIgD. These cells have been termed "mu-predominant" (mu p) B cells. In neonatal mice (5 to 12 days of age), mu p B cells account for more than 50% of the sIg+ cells. There is conflicting evidence concerning the immunocompetency of mup cells in vitro. To study this question further, splenocytes from neonatal BALB/c mice were depleted of sIgD+ B cells by a panning procedure. A portion of the nonadherent (mu p) cell population was analyzed for residual sIgD+ cells by using indirect immunofluorescence in conjunction with the fluorescence-activated cell sorter (FACS). Such cells were then tested for their responsiveness to the thymus-independent (TI) antigen, trinitrophenyl Brucella abortus (TNP-BA), by using a limiting dilution culture system. Results indicate that the depletion of sIgD+ B cells and the decrease in the precursor frequency of splenocytes responding to TNP-BA are very similar, suggesting that virtually all of the responding B cells bear sIgD.

To investigate the properties which enable type 2 Ag, as exemplified by dextran and Ficoll, to stimulate high levels of antibody responses in the relative absence of T cells, we conjugated anti-IgD and anti-IgM mAb to both dextran and Ficoll and examined their B cell-activating properties. Such conjugated anti-Ig antibodies stimulated both early and later stages of B cell activation at picogram concentrations, which are at least 1000-fold lower than that required for B cell stimulation by unconjugated anti-Ig antibodies, and the level of proliferation they stimulated was on average 10-fold greater. Furthermore, concentrations of anti-Ig dextran (100 pg/ml) which modulated little sIgD from the B cell surface were strong inducers of enhanced B cell expression of MHC class II molecules. Conjugation of Fab fragments of anti-IgD or nonmitogenic anti-IgM mAb to dextran rendered them as mitogenic as dextran conjugated to strongly stimulatory anti-IgD or anti-IgM antibodies. The ability of dextran and Ficoll to serve as effective carrier molecules for anti-IgD was not related solely to their large m.w., because anti-IgD coupled to polymerized BSA (m.w. 1.5 X 10(6), was only 10- to 50-fold more potent than unconjugated anti-IgD antibodies at stimulating B cell DNA synthesis. These results suggest, therefore, that the unique ability of picogram concentrations of haptenated type 2 Ag to stimulate Ig secretion in the absence of T cells may be a function of their ability to promote effective cross-linking without resulting in the modulation of sIg. This would enable such Ag to mediate repetitive B cell signaling, a situation that cannot be achieved by unconjugated anti-Ig antibodies which result in modulation of sIg at their mitogenic concentrations. These compounds therefore may be employed to study B cell activation stimulated by sIg cross-linking at concentrations that may more closely reflect those which are achieved under physiologic conditions by type 2 Ag.

The effects of injecting monkeys with goat antiserum to IgD, the IgG fraction of that antiserum or normal goat serum (NGS) were compared. The subcutaneous injection of 4 ml/kg of the whole antiserum resulted in decreased percentages of lymphocytes with surface IgD or IgM lasting from day 1 through day 7 post-injection followed by substantial recovery on day 10. Lymphocytes from these animals were stimulated as indicated by the increased incorporation of 3H-TdR by cells placed in culture on days 7-21 post-injection. The increased blastogenesis occurred in rosette-depleted (B cell) populations and did not occur in rosette-enriched (T cell) preparations. Hypergammaglobulinaemia and increased concentration of serum IgG were first detected on day 10 postinjection, maximal on day 14 and were in decline by day 18. Injection of 4 ml/kg NGS did not alter the percentages of lymphocytes with surface immunoglobulins, result in hypergammaglobulinaemia, or stimulate the degree of blastogenesis observed after anti-IgD. Injection of the IgG fraction of the antiserum resulted in decreased lymphocytes with surface immunoglobulins but did not stimulate hypergammaglobulinaemia or increase blastogenesis. Injection of one monkey with the IgG fraction of anti-IgD combined with NGS resulted in increased serum IgG and increased blastogenesis. Both antibody to IgD and multiple antigenic challenge appear to be required for these responses.

Previous studies from this laboratory have shown that the injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) induces T-independent polyclonal increases in: 1) B cell DNA synthesis, and 2) expression of surface Ia antigen and receptors for T helper factors, 1 to 2 days after injection. In addition, T-independent polyclonal increases in B cell number and IgG1 secretion are observed 6 to 7 days after injection. The administration of normal goat IgG (GIgG) along with GaM delta has been shown to augment GaM delta-induced polyclonal IgG1 secretion. To obtain information about the characteristics of the T help that is required for polyclonal antibody production in this model system, we investigated: 1) the length of the period during which GaM delta must be present to induce day 7 polyclonal antibody production, and 2) the kinetics of the induction of splenic T cell DNA synthesis. We found that GaM delta can be neutralized 3 days after injection by the administration of IgD without decreasing day 7 polyclonal IgG1 secretion, as long as mice are given GIgG at the time that GaM delta is neutralized. In contrast, polyclonal IgG1 secretion is greatly inhibited if GaM delta is neutralized 1 to 2 days after injection or if GaM delta is neutralized 3 days after injection, but GIgG is not administered at this time. Because GIgG can stimulate activated GIgG-specific T cells to secrete helper factors, but, unlike GaM delta cannot focus GIgG-specific T help polyclonally onto B cells, these findings suggest that nonspecific T help, rather than antigen-specific T help, is required in this system after day 3 for the induction of polyclonal IgG1 secretion. Determination of the kinetics of the induction of T cell DNA synthesis in this system by in vivo [3H] thymidine incorporation studies, as well as dual laser fluorescence-activated cell sorter analysis of T and B cell DNA content, indicate that T cells are induced to synthesize DNA 2 days after GaM delta injection and reach plateau rates of DNA synthesis 3 days after injection. Taken together, the GaM delta neutralization experiments and DNA synthesis studies suggest that one reason that GaM delta is required for 3 days in this system is to allow maximal activation of GIgG-specific T cells, which when stimulated later by GIgG secrete nonantigen-specific helper factors that induce GaM delta-activated B cells to secrete IgG1.

Of 30 bacterial species tested 18 stimulated DNA synthesis in human blood lymphocytes. The maximum response was after 3-4 days of culture suggesting a mitogenic effect. This was confirmed by the induction of polyclonal antibody production shown by a plaque assay. Most bacterial species increased the DNA synthesis in B-enriched lymphocytes and unseparated lymphocytes but had negligible activity on T-enriched lymphocytes. Among bacteria with a mitogenic effect and ability to induce polyclonal antibody production are Staphylococcus aureus, Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Streptococcus group A and Streptococcus pneumoniae. In an attempt to define structure (s) on the B-lymphocyte surface responsible for the lymphocyte stimulation the binding of IgD, IgM, and HLA-A, -B and HLA-D antigens to different bacterial species was investigated. A high IgD binding to N. catarrhalis and H. influenzae and a moderate binding of IgD to streptococci was found. Binding studies employing radiolabelled IgD Fab- and Fc-fragments indicated that the binding probably involves the CHl-region of the IgD molecule. Three purified radiolabelled myeloma IgM M-components were all shown to be efficiently bound to many bacteria indicating that a part of the IgM molecule other than the antigen-combining site can be involved in attachment to bacteria. Highly purified detergent-solubilized HLA-A, -B and HLA-D antigens, when separately incorporated into liposomes, were bound efficiently to two strains of N. catarrhalis and to one strain of H. influenzae weakly to one strain of E. coli, but not at all to another strain E. coli. Preliminary experiments indicate that these bacteria-immunoglobulin and bacteria-HLA-antigen interactions lead to lymphocyte stimulation.

The injection of anti-IgD antibody into mice has been shown to increase the expression of Ia antigens on splenic B cells. These antigens are the most potent lymphocyte-activating determinants (LAD) that trigger proliferation in an H-2-defined mixed lymphocyte reaction (MLR) and may play a role in the recognition of minor lymphocyte-stimulating (MIs) determinants. Therefore, we wished to investigate a possible correlation between apparent quantitative alterations in B cell Ia expression after anti-IgD activation with changes in the functional capacity to present allogeneic major histocompatibility complex (MHC) and MIs antigens to responsive T cells. We observed that the capacity of splenocytes removed 24 hr after the in vivo injection of anti-IgD to stimulate T cell proliferation across an H-2 barrier was most frequently enhanced two- to fourfold when a suboptimal concentration of stimulator cells was used or an early time point in the MLR was examined. In contrast, the capacity of splenocytes to stimulate across an MIsa, d difference after exposure to heterologous or hybridoma anti-IgD antibody often was increased 10-fold or more. Optimal MLR stimulatory capacity was induced by injection of 100 to 200 micrograms of heterologous anti-IgD. Augmented MIs stimulatory capacity of spleen cells peaked 24 hr after such treatment and continued to decline from this value at day 3 and at day 7 after injection. In contrast, the H-2 stimulatory capacity increased 1 day after injection of anti-IgD and remained at that elevated level 3 and 7 days after injection. The spleen cells from B cell-defective (CBA/N x DBA/2)F1 male mice were unaffected in their capacity to stimulate across an MIsa barrier after in vivo anti-IgD treatment; however, spleen cells from phenotypically normal (DBA/2 x CBA/N)F1 male mice after exposure to anti-IgD did evidence a considerably enhanced ability to stimulate in an MIsa-defined MLR. Because anti-IgD antibodies presumably have their major initial effect on surface IgD-bearing B cells, these studies suggest that anti-immunoglobulin-activated B cells may have a role (direct or indirect via interaction with accessory cells) in the presentation of allogeneic MHC and MIs antigens to responsive T cells.

It has recently been demonstrated that the injection of adult mice with an affinity-purified goat antibody to mouse IgD (GaM delta) stimulates activation of the humoral immune system that resembles, on a polyclonal level, specific B cell activation by a T cell-dependent antigen. One to 2 days after adult BALB/c mice are injected with 200 micrograms of GaM delta, their splenic B lymphocytes undergo a series of T-independent activation steps that include increases in surface (s) Ia expression, cell size and DNA synthesis. Seven days after GaM delta injection, these cells undergo T-dependent activation steps, that include further proliferation as well as differentiation into IgG1-secreting cells. We have now studied the ontogeny of the T-independent (day 2) and T-dependent (day 7) activation steps by injecting 100-200 micrograms of GaM delta into 3-day- to 10-week-old BALB/c mice. GaM delta failed to induce increases in B cell sIa expression or size 2 days after injection of mice 2 weeks old or younger and failed to stimulate increased DNA synthesis 2 days after injection of 4-week-old mice. In contrast, increases in spleen cell sIa expression, size and DNA synthesis were seen 7 days after injection of 6- to 8-day-old mice. Furthermore, increases in the numbers of spleen cells with large amounts of intracytoplasmic IgG1 were seen at the same time, although these increases were much less than were seen in GaM delta-treated adult mice. Thus, the ability of GaM delta to induce T help and to act in concert with such help to stimulate B cell proliferation and differentiation precedes in ontogeny the ability of GaM delta to directly induce B cell proliferation and early differentiative events. In addition, the early activating events that we have studied are not required for T-dependent B cell proliferation and antibody production to occur, although they appear to contribute to the magnitude of clonal expansion and antibody production.

Monoclonal anti-mu and anti-delta antibodies and mixed lymphocyte reaction-derived T cell factors (MLR-TF) were examined alone and in combination for their effects on proliferation and differentiation of human B cells. MLR-TF induced proliferation and subsequent plasma cell differentiation of blood B cells without additional stimulation. The monoclonal anti-mu and anti-delta antibodies alone did not induce proliferation, but each was capable of augmenting B cell proliferation induced by MLR-TF. In contrast, the anti-mu antibody inhibited the MLR-TF induction of IgM plasma cell differentiation but did not affect the differentiation of IgG and IgA plasma cells. The anti-delta antibody had no effect on MLR-TF-induced plasma cell differentiation. Studies using density gradient separation of B cell subpopulations suggest that MLR-TF induce low-density B cells to proliferate and differentiate into plasma cells but that by themselves MLR-TF have little effect on B cells of relatively high density. The latter subpopulation of small resting B cells responded with proliferation to MLR-TF when combined with either the anti-mu or the anti-delta antibody, but these stimuli were insufficient for induction of terminal plasma cell differentiation.

Activation of murine B lymphocytes in a splenocyte stimulator population with affinity-purified goat anti-mouse IgD (G alpha M delta) antibody was previously shown by this laboratory to enhance the presentation of strongly stimulatory major histocompatibility complex (MHC) and minor lymphocyte-stimulating (Mlsa,d) determinants in a primary mixed lymphocyte reaction. In the present study, the G alpha M delta treatment of murine splenocytes was employed to enhance the detection of the weakly stimulatory non-MHC Mlsc determinant in order to study the role the MHC might play as a restricting element for the recognition of these minor antigens in a primary mixed lymphocyte reaction. Indeed, enhanced T cell proliferation to Mlsc determinants presented on G alpha M delta-treated splenocytes was observed when the responder and activated H-2-compatible stimulator cell shared certain MHC haplotypes. High responsiveness was associated with the H-2a,k,j,p haplotypes, intermediate responsiveness was associated with the H-2f,g haplotypes and low responsiveness was associated with the H-2b,s haplotypes. (Low X high responder)F1 T cells preferentially responded to the Mlsc determinants presented on G alpha M delta-treated stimulator cells of the F1 or parental high responder H-2 haplotype. When mitomycin C instead of irradiation was used to inactivate normal (non-IgD-treated) splenocytes, a similar preferential response of T cells to Mlsc determinants presented on stimulator cells of a high responder H-2 haplotype was also observed. The inability of G alpha M delta-treated splenocytes of the low responder haplotype to elicit substantial levels of T cell proliferation across an Mlsc difference could not be attributed to the failure of these stimulator cells to become activated by the anti-Ig antibody. In addition, co-culture experiments could not identify the poor T cell response to Mlsc determinants presented on certain MHC haplotypes as being caused by the induction of nonspecific suppressor cells. Presentation of Mlsc determinants caused by transgene product complementation was detectable in F1 mice derived by crossing one parent that had the Mlsc non-MHC genes and a poorly permissive H-2 haplotype with a parent that expressed a permissive H-2 haplotype but lacked the Mlsc non-MHC genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Human peripheral blood mononuclear cells were depleted of surface IgM+ or IgD+ cells and assayed for mitogen-induced differentiation to immunoglobulin-secreting cells (ISC) of IgM, IgG and IgA classes. Stimulatory agents included T cell-dependent pokeweed mitogen, B cell mitogen Staphylococcus aureus bacteria strain Cowan I, and a combination of the two which gives uniform, high levels of ISC from all normal donors. Depletion of either IgM- or IgD-bearing B lymphocytes resulted in loss of cells bearing the other Ig class and blocked most of the mitogenic reactivity to anti-IgM and anti-IgD. Proliferative responses to Cowan I in these depleted populations were about 20% that of unfractionated mononuclear cells. Depletion of T cells increased the mitogenic response to Cowan I and to the two antibody preparations, showing that they are T-independent mitogens. Depletion of IgD+ cells caused partial loss of mitogen-induced IgM ISC (22%-60% of unseparated controls) but no loss of IgG or IgA ISC. Depletion of IgM-bearing cells caused complete loss of IgM ISC, but no loss of IgG or IgA ISC. We previously demonstrated that anti-IgM ISC, but no loss of IgG or IgA ISC. We previously demonstrated that anti-IgM antibody blocked mitogen induction of Ig secretion of these three classes in spleen cells, but only IgM secretion in blood mononuclear cells. Together, the results suggest that the majority of cells in normal blood responding to mitogens to mature to IgG or IgA production belong to IgM-, IgD- B cell subjects, in contrast to precursors of secreting cells for these isotypes in the spleen. Thus, these blood precursors appear to be more mature than the corresponding spleen cells.

This study compares the effects of soluble intact (IgG) and F(ab')2 rabbit anti-mu and anti-delta antibodies on mouse B cells. The results show that while the F(ab')2 antibodies to both isotypes induce polyclonal B cell proliferation, the IgG antibodies are not mitogenic, but rather inhibit DNA synthesis induced by the homologous F(ab')2 fragments. Furthermore, intact anti-mu antibodies inhibit mitogenesis induced by F(ab')2 anti-delta, and vice versa. However, the intact antibodies to both isotypes promote early changes characteristic of B-cell activation, namely, increased Ia antigen expression, and priming for a facilitated proliferative response to F(ab')2 anti-Ig. In addition, F(ab')2 anti-mu and anti-delta both induce the breakdown of phosphatidylinositol phospholipids in B cells, an early consequence of Ig receptor cross-linking which may be involved in the induction of cell growth. These results therefore indicate that stimulating mature B cells via IgM or IgD receptors produces indistinguishable early effects, and that cross-linking Fc and surface Ig receptors by intact anti-Ig generates a dominant inhibitory signal, regardless of which isotype is involved.

Human tonsillar B cells were separated into three distinct subpopulations, Ba-/IgD+, Ba+/IgD+, and Ba+/IgD-, by using a B cell-specific monoclonal antibody (anti-Ba) that recognizes only activated B cells, and anti-IgD antibody. Stimulation of Ba-/IgD+ cells with anti-mu plus PHA-conditioned culture supernatant (PHA-sup) or TPA induced Ba+/IgD+ cells, which reverted to Ba-/IgD+ phenotype in the absence of continuous stimulation. Further stimulation of Ba+/IgD+ cells with several B cell activators, such as TPA plus anti-mu or PWM plus T cells, resulted in the loss of IgD expression. Three-color FACS analysis showed that the expression of transferrin receptor (TFR) was at its maximum in Ba+/IgD- cells, and the intensity of this expression was proportional to that of Ba expression in Ba+/IgD+ cells. PHA-sup induced maximum proliferation in Ba+/IgD- cells, and the degree of response was a function of the intensity of Ba expression in Ba+/IgD+ cells. PHA-sup or purified BCDF (BSF-2) induced Ig secretion preferentially in Ba+/IgD- cells. Taken together, these results show that resting B cells (Ba-/IgD+) are activated into Ba+/IgD+ cells, and then into Ba+/IgD- cells, under mitogenic stimulation, and BCDF induces the final maturation of Ba+/IgD- cells into Ig-secreting cells. Ba+/IgD- cells, which maximally expressed TFR as well as Ba and displayed maximum proliferative response to PHA-sup, did not express any Tac antigen. On the other hand, in vitro activated B cells expressed Ba and TFR as well as Tac antigen.

Branhamella catarrhalis initiated DNA synthesis in human blood or spleen cells enriched for B lymphocytes but did not activate T-lymphocyte-enriched fractions. Monoclonal antibodies were used to determine which B-cell surface molecules were of importance for the activation signal. The addition of monoclonal antibodies reactive with IgD, HLA class I antigens, and B2-microglobulin to B lymphocyte cultures selectively inhibited the B-lymphocyte response to B. catarrhalis. Antibody binding to IgD and class I antigens did not inhibit B-cell proliferation following stimulation with anti-IgM beads, Staphylococcus aureus, or Epstein-Barr virus. This suggests that surface IgD is of major importance for B-lymphocyte stimulation by B. catarrhalis. Since B. catarrhalis binds HLA-ABC containing liposomes it is suggested that a similar binding of B. catarrhalis to HLA-ABC on the surface of B lymphocytes serves as an accessory factor that stabilizes the binding of B. catarrhalis to surface IgD. Activation of human B lymphocytes by B. catarrhalis resulted in changes of cell surface molecules that were quantitatively and qualitatively similar to those that resulted from the activation by S. aureus. Therefore although these two bacteria appear to activate B cells in a similar manner, they induce B-cell proliferation through interactions with different cell surface structures.