SummaryT cell-independent type 2 (TI-2), in contrast to T-dependent, antigens stimulate the production of murine IgG3. To investigate a possible role for cytokines in mediating the induction of this IgG subclass, we established an in vitro polyclonal model system for studying TI-2 antigen-mediated B cell activation by using dextran-conjugated anti-IgD antibody (o~6-dex). We demonstrate that interferon "r (IFN-7) stimulates, and interleukin 4 inhibits, the expression of IgG3 by c~&dex-activated cells. The production of IFN-'r by non-T cells in response to bacterial products, possibly capsular polysaccharides, may provide an explanation underlying the ability of TI antigens, which are unable to directly stimulate T cell-derived cytokines to induce Ig isotype switching.ttle is known regarding the parameters that regulate the humoral immune response to T cell-independent type 2 (TI-2) I antigens. Such antigens, exemplified by the haptenated polysaccharides, are typically abundant in bacterial cell walls. Immunization of mice with this class of antigen, in contrast to T cell-dependent antigens, stimulates a significant increase in serum levels of antigen-specific IgG3 (1). Since Ig class switching is widely viewed as a process that is regulated by the release of T cell-derived cytokines, the ability of TI-2 antigens to stimulate IgG3 production is especially intriguing. In this regard, it is of interest that no cytokine has been described that regulates its synthesis in a selective and positive manner.The study of B cell responses to TI-2 antigens has been hampered by the low frequency of B cells specific for a given immunizing antigen. We recently described a model system in which resting murine mlgD + B cells are polyclonally activated in vitro in a manner similar to the specific antibody response mediated by the prototypical TI-2 antigen, TNPFicoll (2, 3). Thus, like TNP-Ficoll, multiple anti-IgD mAb molecules covalently linked to a high molecular weight dextran backbone induce resting B cells to proliferate in the absence of significant Ig production. The addition of a differentiation factor, such as IL-5, stimulates the secretion of large amounts of polyclonal Ig by dextran-conjugated anti-IgD an-1 Abbreviations used in this paper: o~-dex, dextran-conjugated anti-IgD antibody; TI-2, T cell-independent type 2.

Bacterial lipopolysaccharide (LPS) has been reported to induce immunoglobulin (Ig)G2b class switching, yet we observed strain differences in IgG2b secretion in response to this mitogen. Specifically, BALB/c B cells, unlike those from DBA/2, synthesized relatively low amounts of IgG2b relative to IgG3, IgG1, or IgM. This report demonstrates that transforming growth factor (TGF) beta 1, previously shown to induce IgA class switching, selectively stimulates IgG2b secretion by BALB/c resting B cells activated with LPS. This activity was specifically reversed with a neutralizing anti-TGF-beta 1 antibody. The ability of TGF-beta 1 to act directly on highly purified membrane (m)IgM+ mIgG2b- cells to stimulate IgG2b production, stimulate an increase in IgG2b-secreting cells, and selectively increase the steady-state levels of germline gamma 2b RNA, suggests that it promotes IgG2b class switching. In this regard, addition of anti-TGF-beta antibody to cultures of DBA/2-derived resting B cells activated by LPS, alone, led to selective reduction in IgG2b secretion, indicating that endogenous TGF-beta 1 accounts for the high IgG2b secretory response observed in that strain. Finally, TGF-beta 1 failed to stimulate IgG2b secretion by B cells activated with dextran-conjugated anti-IgD antibody. We propose that TGF-beta 1 is a switch factor for the murine IgG2b subclass for appropriately activated B cells. In combination with other data, this would show that all six non-IgM, non-IgD isotypes in the mouse can be selectively induced by specific cytokines.

The parameters necessary for induction of high-rate IgA class switching are unknown. Thus, although TGF-beta is switch factor for the IgA class, the percentage of membrane (m)IgA+ cells generated in vitro in response to TGF-beta and various individual modes of B cell activation is limited to 1 to 2% of the total B cell population, a percentage far below that observed within Peyer's patches. In this report we determined a set of parameters that act synergistically to generate up to 15 to 20% mIgA+ cells in vitro. A dual mode of B cell activation is required whereby signaling through CD40 or in response to LPS stimulation must occur in concert with multivalent Ag receptor crosslinking. A complex cytokine requirement is also revealed in that both IL-4 and IL-5 must be present with TGF-beta for high-rate IgA class switching to occur. By contrast, IFN-gamma, a known antagonist of IL-4, strongly suppresses the induction of mIgA+ cells in response to these stimuli. This novel cellular system should serve as a powerful tool for studying the molecular mechanisms that underly the IgA class switch and may provide insight into the physiologic parameters that induce it.