B cell stimulatory function was evaluated by measuring T cell proliferation and T cell cytokine secretion after 5 days of co-incubation. from peripheral blood were stimulated in vitro with CP-870,893, in the presence or absence of the toll like receptor 9 (TLR9) PF-06256142 ligand CpG oligodeoxynucleotide IL1F2 (ODN). B cell surface molecule expression and cytokine secretion were evaluated using flow cytometry. Activated B cells were used as stimulators in mixed lymphocyte reactions to evaluate their ability to induce allogeneic T cell responses. Results Incubation with CP-870,893 activated B cells, including both memory and na?ve B cells, as demonstrated by upregulation of CD86, CD70, CD40, and MHC class I and II. CP-870,893-activated B cells induced T cell proliferation and T cell secretion of effector cytokines including IFN-gamma and IL-2. These effects were increased by TLR9 co-stimulation via a CpG ODN identical in sequence to a well-studied clinical grade reagent. Conclusion The CD40 mAb CP-870,893 activates both memory and na? ve B cells and triggers their T cell stimulatory capacity. Simultaneous TLR9 ligation augments the effect of CP-870,893 alone. These results provide further rationale for combining CD40 and TLR9 activation using available clinical reagents in strategies of novel tumor immunotherapy. Background The activation status of host antigen presenting cells (APC) critically determines the quality and effectiveness of T cell immune responses. Resting APC may drive T cell tolerance and anergy, but fully activated APC – classically termed “licensed APC” – autonomously trigger effective and productive T cell responses [1]. This paradigm holds true for both dendritic cells (DC) and B cells. PF-06256142 Among the many microenvironmental factors now appreciated to contribute to APC licensing, ligation of the cell surface molecule CD40 on the surface of both DC and B cells is usually fundamental, particularly for tumor immunity [2-8]. CD40 is a member of the tumor necrosis factor receptor (TNF) superfamily and is broadly expressed by immune and other normal cells [9]. CD40 itself lacks intrinsic signal-transduction activity and mediates its effects via downstream adapter molecules that regulate gene expression. CD40-ligand (CD40L), also known as CD154, is the chief ligand for CD40 and is expressed primarily by activated T cells and platelets [10,11]. The conversation of CD40 and CD40L represents a major component of T cell help. Ligation of CD40 on DC, for example, induces increased surface expression of costimulatory and MHC molecules, production of proinflammatory cytokines, and enhanced T cell triggering [11,12]. CD40 ligation on resting B cells increases antigen-presenting function and proliferation [11,12]. In mice, agonist CD40 antibodies have been shown to mimic the signal of CD40L and substitute for the function of CD4+ helper T cells in experimental systems testing T cell-mediated immunity [2-4]. In tumor-bearing mice, agonist CD40 antibodies overcome T PF-06256142 cell tolerance, evoke effective cytotoxic T cell responses, and enhance efficacy of anti-tumor vaccines [5-7]. Toll-like receptor (TLR) signalling can cooperate with CD40 activation in this regard; for example, co-administration of CD40 and TLR9 ligands in mice elicits a more effective anti-melanoma response than either ligand alone [13]. Despite these landmark studies, the clinical translational of CD40 activation in cancer patients has been limited, owing primarily to the lack of an appropriate and PF-06256142 available drug. CP-870,893 is usually a fully human, selective agonist CD40 mAb and has shown early clinical promise in phase I trials, particularly in patients with advanced melanoma [14]. Little direct evidence is available regarding its mechanism of action and in particular, its biological effects on patient APC. The primary clinical side effect of CP-870,893 infusion has been moderate to moderate cytokine release syndrome, manifesting as transient fever, chills, and rigor within minutes to hours after the end of the CP-870, 893 infusion and associated with acute elevations in serum IL-6 and TNF-alpha [14]. The primary pharmacodynamic effect.