SummaryThe dose of foreign antigen can influence whether a cell-mediated or humoral class ofirm'nune response is elicited, and this may be largely accounted for by the development of CD4 + T helper cells (Th) producing distinct sets of cytokines. The ability of antigen dose to direct the development of a Thl or Th2 phenotype from naive CD4 + T cells, however, has not been demonstrated. In this report, we show that the antigen dose used in primary cultures could directly affect Th phenotype development from naive DOl1.10 TCR-et[3-transgenic CD4 + T cells when dendritic cells or activated B cells were used as the antigen-presenting cells. Consistent with our previous findings, midrange peptide doses (0.3-0.6 p,M) directed the development of Th0/Thl-like cells, which produced moderate amounts of interferon ~/ (IFN-y). As the peptide dose was increased, development of Thl-like cells producing increased amounts of IFN-'y was initially observed. At very high (>10 p,M) and very low (<0.05 b~M) doses of antigenic peptide, however, a dramatic switch to development of Th2-1ike cells that produced increasing amounts of interleukin 4 (IL-4) and diminishing levels of IFN-y was observed. This was true even when highly purified naive, high buoyant density CD4 + LECAM-1 hi T cells were used, ruling out a possible contribution from contaminating "memory" phenotype CD4 + T cells. Neutralizing anti-IL-4 antibodies completely inhibited the development of this Th2-like phenotype at both high and low antigen doses, demonstrating a requirement for endogenous IL-4. Our findings suggest that the antigen dose may affect the levels of endogenous cytokines such as IL-4 in primary cultures, resulting in the development of distinct Th cell phenotypes.

SummaryThe specifidty of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of slg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, nontolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efl~dent activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the slg signaling pathway which prevented activation of receptor-assodated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this slg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering slg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of slg signaling as a mechanism for controlling self-reactive B cells.

Despite extensive research, the role of CD40 signaling in B cell terminal differentiation remains controversial. Here we show that CD40 engagement arrests B cell differentiation prior to plasma cell formation. This arrest is manifested at a molecular level as a reduction in mRNA levels of secretory immunoglobulin gene products such as mu(s) and J chain as well as the loss of the transcriptional regulator BLIMP-1. Furthermore, the inhibition of B cell differentiation by CD40 engagement could not be overcome by either mitogens or cytokines, but could be reversed by antibodies that interfere with the CD40/gp39 interaction. These data suggest that secretory immunoglobulin is not produced by B cells that are actively engaged by gp39-expressing T cells.

Two rat IgG2a antibodies which define distinct epitopes on murine CD40 have been generated. These antibodies specifically bind recombinant murine CD40 expressed on L cells, and the soluble extracellular domain of murine CD40 coated onto microtiter plates. Both antibodies bind B220+ but not B220 murine spleen cells, and immunoprecipitate a 45-kDa protein from the surface of purified murine splenic B cells. These antibodies exhibit separate functional properties, consistent with the notion that they define two distinct CD40 epitopes. One of the monoclonal antibodies (designated 1C10) directly induces a specific proliferative response from mature murine B cells, up-regulates several B cell surface antigens, and rescues immature B lymphoma cells from anti-IgM-induced growth arrest. The other monoclonal antibody (designated 4F11) exhibits none of these properties, but is capable of synergizing with suboptimal amounts of either anti-IgM antibodies or the 1C10 agonistic anti-CD40 antibody to produce an optimal proliferative response of purified small dense B cells. Furthermore, 4F11 antibody synergizes with suboptimal amounts of 1C10 antibody to rescue B lymphoma cells from anti-IgM-induced growth arrest. The 1C10 and 4F11 antibodies were unable to cross-block each other's binding to recombinant CD40 expressed in L cells, providing strong support for the notion that the antibodies recognize distinct epitopes on CD40. The potential implications of two functionally distinct CD40 epitopes are discussed.