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The Ig heavy chain locus contains a number of binding sites for the transcriptional activator, c-Rel. In this study, we evaluated the capacity of B cells from mice made genetically deficient in the C-terminal, transactivation domain of the c-Rel protein (delta c-Rel) to undergo Ig class switching. Flow-cytometric and digestion circularization PCR analyses revealed that delta c-Rel B cells failed to switch to IgG3 in response to LPS alone, or to IgG1 or IgE in response to LPS + IL-4. This failure to switch to IgG3 or IgG1 was associated with a corresponding loss of germline CH gamma 3 or CH gamma 1 RNA. However, the defective switching to IgE in delta c-Rel B cells was associated with normal levels of germline CH epsilon RNA relative to control B cells. The ability of delta c-Rel B cells to switch to IgG1, in response to LPS + IL-4, could be restored through the action(s) of additional stimuli, and this was associated with induction of normal levels of germline CH gamma 1 RNA relative to controls. In contrast, LPS-activated B cells from delta c-Rel mice underwent normal switching to IgA in the presence of TGF-beta, relative to control B cells. This was associated with equivalent steady state levels of germline CH alpha RNA between the two B cell populations. These data are the first to demonstrate a key and selective role for c-Rel in the regulation of Ig class switching. Furthermore, distinct differences are revealed in the Ig isotype induction profiles of B cells lacking c-Rel activity vs those deficient in p50/nuclear factor-kappa B.

The humoral immune response of neonates to T cell-independent type 2 (TI-2) Ags is markedly defective. We previously demonstrated that multivalent membrane Ig cross-linking, using dextran-conjugated anti-Ig Abs (anti-Ig-dextran), is an in vitro model for membrane Ig-dependent TI-2 induction of Ig secretion. In this work, we demonstrate that highly purified neonatal B cells are intrinsically defective in IgM secretion in response to anti-Ig-dextran and cytokines in vitro, as well as other modes of B cell activation, relative to adult B cells. However, costimulation of anti-Ig-dextran-activated neonatal B cells with either CD40-ligand, a recombinant bacterial lipoprotein, or LPS restores the IgM secretory response of neonatal B cells to adult levels. Analysis of Ig isotype secretion indicates that neonatal B cells have an enhanced capacity to secrete IgE and IgA relative to other Ig isotypes. These data suggest that neonatal B cells are competent to secrete Ig in response to TI-2 Ags if adequate costimuli are provided, and thus may have particular relevance for the design of vaccine strategies in the immunodeficient host. The data also suggest that neonatal B cells are programmed to secrete relatively enhanced amounts of IgE and IgA, which may be relevant for antimicrobial resistance at mucosal surfaces.

A number of distinct functional abnormalities have been observed in B cells derived from p50/ NF-kappa B or c-rel knockout mice. RelB, another member of the NF-kappa B/Rel family of transcription factors, is expressed during the latter stages of B cell maturation and can bind to regulatory sites within the Ig heavy chain locus. Therefore, we tested the ability of B cells from relB knockout mice (relB-/-) to proliferate, undergo maturation to IgM secretion, and switch to the expression of downstream Ig isotypes in response to distinct activators including LPS, anti-CD40 mAb or CD40 ligand, and/or dextran anti-IgD antibodies in combination with various cytokines, including IL-4, IL-5, IFN-gamma, and TGF-beta. B cells lacking RelB showed up to 4-fold reductions in DNA synthesis in response to LPS, CD40, and membrane Ig-dependent activation relative to controls. However, relB-/- B cells were comparable to control B cells in their ability to undergo maturation to IgM secretion and switch to the expression of IgG3, IgG1, IgG2b, IgG2a, IgE, and/or IgA under all activation conditions tested. Thus, RelB, like c-Rel and p50/NF-kappa B, plays a role in B cell proliferation. However, in contrast to c-Rel and p50/ NF-kappa B, it is not critically involved in maturation to Ig secretion or expression of Ig isotypes.

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.

Sort-purified resting murine B cells proliferate in response to dextran-conjugated anti-IgD Abs (alpha delta-dex) but fail to secrete significant amounts of Ig even after the addition of IL-1 + IL-2. We show that either IL-3 or granulocyte-macrophage CSF (GM-CSF) stimulates 10- to 50-fold enhancements in IgM secretion by sort-purified B cells treated with alpha delta-dex + IL-1 + IL-2, and that the combined actions of IL-3 and GM-CSF are typically greater than additive. Both IL-3 and GM-CSF act primarily as B cell differentiation factors, although IL-3 induces a modest enhancement in cellular outgrowth. The enhancing effects of IL-3 and GM-CSF require multivalent Ag receptor cross-linkage, mediated by alpha delta-dex, as neither cytokine induces IgM secretion in the presence of unconjugated anti-IgD Abs. Although both alpha delta-dex and IL-1 + IL-2 are required for optimal IL-3- and GM-CSF-mediated IgM secretion, both IL-3 and GM-CSF stimulate a modest IgM secretory response by cells activated with alpha delta-dex alone. In this regard, supernatant from either an activated CD4+ Th1 or Th2 clone potently induces IgM secretion by alpha delta-dex + IL-1 + IL-2-activated B cells and this is due, in large part, to the presence in these supernatants of either IL-3 and/or GM-CSF. Neither IL-3 nor GM-CSF stimulates significant IgM secretion by B cells activated through the CD40 signaling pathway alone, although the combination of CD40 and membrane Ig signaling leads to a strong enhancement of the IL-3 + GM-CSF-mediated IgM synthesis above that obtained with membrane Ig signaling alone. The demonstration that IL-3 and GM-CSF act directly as differentiation factors for B cells activated through their Ag receptor establishes a novel cytokine pathway for induction of humoral immunity.

We previously demonstrated that dextran-conjugated anti-IgD antibodies (alpha delta-dex) induce proliferation of small, B cell-enriched murine spleen cells (Be cells), and in the presence of IL-2, stimulate Ig secretion in vitro. We have shown that alpha delta-dex-stimulated B cells provide an in vitro model for studying B cell activation by T cell-independent type 2 (TI-2) Ag, as exemplified by the bacterial polysaccharides. We now show that highly purified resting B cells, obtained by electronic cell sorting (Bsp cells), fail to secrete Ig in the presence of alpha delta-dex + IL-2. The alpha delta-dex + IL-2-induced Ig secretory response of Bsp cells is restored upon addition of splenic non-B, non-T cells or a pure population of in vitro-generated NK cells. Similarly, pretreatment of Be cells with anti-AsGm-1 plus complement inhibits Ig secretion in response to alpha delta-dex + IL-2. An IL-2-induced NK cell supernatant (NKSN) is equally potent at stimulating Ig secretion by alpha delta-dex-activated Bsp cells, indicating that cell contact between Bsp and activated NK cells is not required for this effect. IL-2 stimulates not only NK cells, but B cells as well, since addition of anti-IL-2 + anti-IL-2R antibodies to Bsp cell cultures, in the presence of alpha delta-dex + NKSN, inhibits Ig secretion. These data describe a novel animal model for NK cell-induced B cell maturation to Ig secretion and suggest a pathway for Ig production in response to T1-2 Ag.