To examine the role of the TNF-R superfamily signaling protein TRAF2 in mature B cell development and NF-kappaB activation, conditionally TRAF2-deficient mice were produced. B cells lacking TRAF2 expression in these mice possessed a selective survival advantage, accumulated in the lymph nodes and splenic marginal zone, were larger in size, and expressed increased levels of CD21/35. These TRAF2-deficient B cells could not proliferate or activate the canonical NF-kappaB pathway in response to CD40 ligation. By contrast, noncanonical NF-kappaB activation was constitutively hyperactive, with TRAF2-deficient B cells exhibiting close to maximal processing of NF-kappaB2 from p100 to p52 and high levels of constitutive p52 and RelB DNA binding activity. These findings establish TRAF2 as a multifunctional regulator of NF-kappaB activation that mediates activation of the canonical pathway but acts as a negative regulator of the noncanonical pathway. This dual functionality explains the contrasting roles of TRAF2 in B cell maturation and activation.

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Tumor necrosis factor (TNF) superfamily receptors typically induce both NF-B and JNK activation by recruiting the TRAF2 signal transduction protein to their cytoplasmic domain. The type 2 TNF receptor (TNFR2), however, is a poor activator of these signaling pathways despite its high TRAF2 binding capability. This apparent paradox is resolved here by the demonstration that TNFR2 carries a novel carboxyl-terminal TRAF2-binding site (T2bs-C) that prevents the delivery of activation signals from its conventional TRAF2-binding site (T2bs-N). T2bs-C does not conform to canonical TRAF2 binding motifs and appears to bind TRAF2 indirectly via an as yet unidentified intermediary. Specific inactivation of T2bs-N by site-directed mutagenesis eliminated most of the TRAF2 recruited to the TNFR2 cytoplasmic domain but had no effect on ligand-dependent activation of the NF-B or JNK pathways. By contrast, inactivation of T2bs-C had little effect on the amount of TRAF2 recruited but greatly enhanced ligand-dependent NF-B and JNK activation. In wild-type TNFR2 therefore, T2bs-C acts in a dominant fashion to attenuate signaling by the intrinsically more active T2bs-N but not by preventing TRAF2 recruitment. This unique uncoupling of TRAF2 recruitment and signaling at T2bs-N may be important in the modulation by TNFR2 of signaling through coexpressed TNFR1.

TNF, lymphotoxin (LT) and their receptors are expressed constitutively in the thymus. It remains unclear whether these cytokines play a role in normal thymic structure or function. We have investigated thymocyte differentiation, selection and thymic organogenesis in gene targeted mice lacking LTα, TNF, or both (TNF/LTα-/-). The thymus was normal in TNF/LTα-/- mice with regard to cell yields and stromal architecture. Detailed analysis of αβ and γδ T cell-lineage thymocyte subsets revealed no abnormalities, implying that neither TNF nor LT play an essential role in T cell differentiation or positive selection. The number and distribution of thymic CD11c+ dendritic cells was also normal in the absence of both TNF and LTα. A three-fold increase in B cell numbers was observed consistently in the TNF/LTα-/- thymus. This phenotype was due entirely to the LTα deficiency and associated with changes in the hemopoietic compartment, rather than the thymic stromal compartment of LTα-/- mice. Finally, specific Vβ8+ T cell deletion within the thymus following intrathymic injection of staphylococcal enterotoxin B (SEB) was TNF/LT independent. Thus, despite the presence of these cytokines and their receptors in the normal thymus, there appears no essential role for either TNF or LT in development of organ structure or for those processes associated with T cell repertoire selection.