TNF receptor (TNFR) superfamily members, CD40, and BAFFR play critical roles in B cell survival and differentiation. Genetic deficiency in a novel adaptor molecule, Act1, for CD40 and BAFF results in a dramatic increase in peripheral B cells, which culminates in lymphadenopathy and splenomegaly, hypergammaglobulinemia, and autoantibodies. While the B cell-specific Act1 knockout mice displayed a similar phenotype with less severity, the pathology of the Act1-deficient mice was mostly blocked in CD40-Act1 and BAFF-Act1 double knockout mice. CD40- and BAFF-mediated survival is significantly increased in Act1-deficent B cells, with stronger IkappaB phosphorylation, processing of NF-kappaB2 (p100/p52), and activation of JNK, ERK, and p38 pathways, indicating that Act1 negatively regulates CD40- and BAFF-mediated signaling events. These findings demonstrate that Act1 plays an important role in the homeostasis of B cells by attenuating CD40 and BAFFR signaling.
T cells and cytokines were used to activate highly enriched populations of 2,4,6-trinitrophenyl (TNP)-binding B cells (TNP-ABC). TNP-ABC did not proliferate or differentiate when they were cultured with thymus-dependent (TD) antigen, even in the presence of supernatants known to contain B-cell growth and differentiation factors. However, purified TNP-ABC did proliferate and differentiate when they were cultured with TD antigen in the presence of carrier-primed T cells and antigen (TNP-keyhole limpet hemocyanin)-i.e., linked recognition. TNP The induction of maximal B-cell plaque-forming cell (PFC) responses to certain soluble antigens requires the participation of H-2-restricted antigen-specific T helper cells [hence the antigens are called "thymus-dependent" (TD)] and the appropriate hapten-carrier conjugates (linked recognition) (1-5). The role of cytokines in inducing or sustaining the proliferation and differentiation of B cells responding to TD antigens has not been extensively studied. In general, supernatants containing T cellderived lymphokines that act on B cells have been described in polyclonal systems (6-18) or in systems using erythrocytebound antigens (7,8).In this study we have utilized a population of purified 2,4,6-trinitrophenyl (TNP)-antigen-binding B cells (TNP-ABC) in a TD antigen-driven system to analyze the effect(s) of T cells and cytokines. The major advantage of this system, compared to the use of non-antigen-enriched B-cell populations, is that B-cell growth as well as differentiation can be assessed. In addition, because the B cells are >80% TNP specific, responding TNPspecific B-cell blasts can be visualized and isolated. As described previously (19), when T cells primed to keyhole limpet hemocyanin (KLH) are cultured with antigen (TNP-KLH) and TNP-binding B cells from unprimed mice, B-cell proliferation and differentiation occur.The data presented in this communication indicate that cytokines (with or without antigen) do not induce the differentiation or proliferation of "resting" TNP-ABC. However, when the TNP-ABC are first activated by means of linked recognition, the resultant blasts both proliferate and differentiate in response to cytokines; when activation occurs by means of nonlinked recognition, the blasts proliferate but do not differentiate in response to the same cytokines. Preparation of Cells. The methods used for the isolation of TNP-specific B cells have been described (20). T cells were obtained from the popliteal lymph nodes of mice immunized 5-8 days previously in the hind footpads with 50-75 Ag of KLH (Calbiochem-Behring). Between 90% and 95% of the B cells were removed by adherence to Petri dishes coated with affinity-purified rabbit anti-mouse Ig (RAMIg) (21). Immediately prior to use, the T cells were irradiated (1,500 rads; 1 rad = 0.01 gray).Culture Conditions. Culture conditions have been described (19).Generation of Activated TNP-ABC (TNP-ABC*). TNP-ABC were cultured with primed T cells (3-4 X 105 cells per well in 0.2 ml) and antigen [TNP-KLH or f...
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