OX40 is a member of the TNF/NGF-receptor family expressed on activated T cells, whose ligand is found on activated T and B cells. In the present study, we show that cross-linking of OX40L on CD40L-stimulated B cells, alpha IgD dextran-stimulated B cells, or both results in a significantly enhanced proliferative response with no change in the cell survival rate. Furthermore, OX40 stimulation increases immunoglobulin heavy chain mRNA levels and immunoglobulin secretion, which could not be blocked by anti-cytokine antibodies. In additional molecular studies, we show that OX40L cross-linking results in the down-regulation of the transcription factor BSAP. This, in turn, leads to a change in the in vivo binding pattern of the immunoglobulin heavy chain gene 3' alpha enhancer, suggesting its activation. This effect may thus be one mechanism for OX40-induced increase in immunoglobulin secretion. In conclusion, our data suggest that the OX40-OX40L interaction is a novel pathway in T cell-dependent B cell proliferation and differentiation.

The tumor necrosis factor family molecule Ox40-ligand (Ox40L) has been identified as a potential costimulatory molecule and also has been implicated in T cell homing and B cell activation. To ascertain the essential functions of Ox40L, we generated and characterized Ox40L-deficient mice. Mice lacking Ox40L exhibit an impaired contact hypersensitivity response, a dendritic cell-dependent T cell-mediated response, due to defects in T cell priming and cytokine production. In contrast, Ox40L-deficient mice do not have defects in T cell homing or humoral immune responses. In vitro, Ox40L-deficient dendritic cells are defective in costimulating T cell cytokine production. Thus, Ox40L has a critical costimulatory function in vitro and in vivo for dendritic cell:T cell interactions.

CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. Studies with human B cells show that the binding of CD154 (gp39, CD40L) to CD40 recruits TNF receptor– associated factor 2 (TRAF2) and TRAF3 to the receptor complex, induces the downregulation of the nonreceptor-associated TRAFs in the cell and induces an increased expression of Fas on the cell surface. Combined signaling through the interluekin 4 receptor and CD40 induces an increased expression of Fas with a commensurate increase in the level of TRAF2, but not TRAF3, that is recruited to the receptor complex. In contrast, engagement of the membrane immunoglobulin and CD40 limits Fas upregulation and reduces the recruitment of TRAF2, relative to TRAF3, to the CD40 receptor complex. These studies show that the TRAF composition of the CD40 receptor complex can be altered by signals that influence B cell differentiation.

Dendritic cells (DC) for the immunotherapy of cancer and infectious disease require the appropriate maturation and activation signals to effectively present antigen to drive a proinflammatory response. Here we present a comparison of 4 different maturation protocols for antigen-encoded mRNA electroporated DC. Two protocols rely on cytokine-induced maturation given either preelectroporation or postelectroporation. In addition to the cytokine treatment, 2 further maturation protocols use coelectroporation of CD40L mRNA, with antigen-encoding RNA, to deliver CD40 signals. There were no significant differences in expression of costimulatory molecules such as CD80, CD83, and CD86 or the levels of expression of major histocompatibility complexes. However, results indicate that delivery of an inflammatory signal that includes interferon-gamma before the CD40 signal results in high levels of expression of interleukin-12 that was not seen in the absence of CD40L mRNA. All 4 preparations could induce expansion of primary MART-1-specific CD8+ T cells from healthy donors in vitro, but only the 2 processes receiving CD40L could induce interferon-gamma expression by those responder cells. Only DC electroporated with CD40L RNA after delivery of the inflammatory signal (PME-CD40L DC), could drive the long-term expansion of MART-1-reactive cells that displayed a CD28+/CD45RA- effector/memory phenotype with strong cytolytic activity.