Mechanisms of TLR signaling and cooperation in B lymphocytes

B lymphocytes play important roles in antibody production, cytokine production, and antigen presentation to T cells. Ligation of Toll-like receptors (TLRs) on B cells stimulates cellular activation and B cell effector functions. Synergistic activation of other receptors such as CD40 or the B cell receptor (BCR) with TLR ligation further enhances B cell activation and effector functions. The tumor necrosis factor receptor associated factor (TRAF) family of proteins act as cytoplasmic signaling adaptor molecules and moderate downstream signaling from both the tumor necrosis factor receptor (TNFR) superfamily of proteins, including CD40, and the IL-1R/TLR superfamily of proteins.

To date, only TRAFs 3 and 6 have been shown to be involved in TLR signaling, with TRAF6 providing positive regulation and TRAF3 providing negative regulation of TLR signaling in B cells. Deficiency in another TRAF family member, TRAF5, has been implicated in the development of atherosclerosis, a disease developed in part due to TLR dysregulation. Here, we addressed the hypothesis that TRAF5 is a negative regulator of TLR signaling.

We found that TRAF5 negatively regulated TLR-mediated cytokine and antibody production in B lymphocytes. The enhanced cytokine production seen in TLR-stimulated TRAF5 KO B cells was not attributable to altered cellular survival or proliferation, but instead more cytokine was produced on a per-cell basis, likely due to enhanced MAPK pathways after TLR ligation. Additionally, TRAF5 deficiency did not dramatically affect cytokine production in TLR-stimulated bone marrow-derived macrophages or dendritic cells, suggesting that TRAF5 plays a greater role in TLR signaling in lymphoid versus myeloid cells. TRAF5 associated with the TLR signaling proteins MyD88 and TAB2, and negatively regulated the association of TAB2 with its binding partner TRAF6.

Furthermore, we manipulated B cell activation via ligation of various TLRs, CD40, and/or the BCR in order to activate the cells to effectively present antigen. Activated B cells pulsed with antigen served as an effective cellular vaccine and offered protection against both an infectious pathogen (Listeria monocytogenes) and a model of murine melanoma. We identified two candidate activation criteria for B cell vaccines (Bvacs): stimulation through the BCR and TLR7, and stimulation through CD40 and TLR4. Additionally, we found that high IL-6 production by the activated Bvac was essential for inducing optimal CD8+ T cell memory. These B cell activation protocols offer significant advantages over those currently being tested for clinical use. Understanding B cell activation through TLRs is a critical step in developing new therapies against cancer and infectious disease.