We present evidence that DOCK8 functions as an adaptor that links TLR9 via MyD88 to a Pyk2-Src-Syk-STAT3 signaling cascade, and we demonstrate that this pathway is essential for TLR9-driven B cell proliferation and immunoglobulin production.
DOCK8-deficient patients had impaired ability to sustain a protective antibody response, similar to DOCK8 mutant mice, and lacked circulating CD27+ memory B cells. DOCK8-deficient B cells failed to proliferate and secrete IgM and IgG in response to CpG. This defect is cell-autonomous, specific to CpG and not accounted for by the lack of memory B cells. Because of constraints in obtaining sufficient amounts of blood from children for B cell purification, our experiments used PBMCs, which were stimulated with the B cell selective TLR9 ligand CpG ODN2006, and/or EBV-B cells. Whenever possible, purified B cells from at least two patients were also used.
CpG stimulation normally upregulated the expression of AICDA
, CD23 and CD86 in DOCK8-deficient B cells and resulted in normal activation of NF-κB and p38, and normal IRF7-dependent secretion of IFN-α in DOCK8-deficient PBMCs, indicating that these events occur independently of DOCK8. Unexpectedly, given the reported GEF activity of DOCK proteins for small GTPases21
, CpG activated Rac1 independently of DOCK8. CpG activation of Rac1 in pDCs uses a non-TLR9 sensor and DOCK2 (ref. 34
). A similar pathway could be operative in B cells.
A central finding in this study is that CpG causes STAT3 phosphorylation in B cells in a DOCK8-dependent manner. Impaired STAT3 phosphorylation in DOCK8-deficient B cells was specific for CpG stimulation, and was not secondary to the lack of memory B cells. The critical role of STAT3 in CpG-driven B cell proliferation and differentiation was demonstrated by the observation that these responses were impaired in patients with dominant-negative mutations in STAT3
. The failure of IL-6, which caused STAT3 phosphorylation in DOCK8-deficient B cells, to correct their defective response to CpG indicates that other DOCK8-dependent signals are also required for CpG-driven B cell proliferation and IgG secretion. These may include signals delivered by Pyk2, Src and Syk which trigger the activation of phospholipase C-γ, phosphatidylinositol-3-OH kinase and B cell linker protein (BLNK)44–46
Syk was shown to play a critical role in CpG-driven STAT3-dependent B cell proliferation and differentiation. CpG stimulation of B cells caused Syk phosphorylation, which was severely impaired in DOCK8-deficient B cells, placing Syk downstream of DOCK8. The Syk selective inhibitor SYKINH-61 blocked CpG-driven STAT3 phosphorylation, placing Syk upstream of STAT3, and inhibited CpG-driven B cell proliferation and IgG secretion. CpG stimulation of B cells caused DOCK8-dependent phosphorylation of Pyk2 and Src. DOCK8 was demonstrated to link TLR9-MyD88 to a Pyk2-Src-Syk-STAT3 cascade in B cells, which was shown to be essential for TLR9-driven B cell activation. The observation that CpG-driven phosphorylation of Pyk2, Src, Syk and STAT3, and B cell proliferation and differentiation were dependent on TLR9 and MyD88, indicate that CpG engagement of TLR9-MyD88 results in DOCK8-dependent B cell activation. CpG-A DNA has been reported to induce tyrosine phosphorylation of proteins, including Syk, in monocytes and macrophages independently of TLR9 and MyD88 (ref. 47
). Differences in the ODNs and target cells used may account for the difference in the requirement for TLR9 and MyD88.
DOCK8 was found to exist in a complex with MyD88 and Pyk2. MyD88 was not essential for DOCK8-Pyk2 association, and DOCK8 was not essential for MyD88-Pyk2 association, although it was essential for Pyk2 phosphorylation following TLR9 ligation. Following CpG stimulation, DOCK8 became more strongly associated with MyD88 and Pyk2, underwent tyrosine phosphorylation and associated with Src and/or Lyn. We propose the following model of DOCK8 dependent TLR9 signaling. Ligation of TLR9 by CpG causes recruitment and stabilization of a preexisting MyD88-Pyk2-DOCK8 complex, which results in autophosphorylation and activation of Pyk2. Pyk2 then phosphorylates DOCK8 causing it to recruit Src kinases, including Lyn, via their SH2 domain, releasing them from auto-inhibition. Src then activates Syk, which drives STAT3 activation. The proposed pathway is likely a simplification. Src and Syk can phosphorylate Pyk2 (ref. 40
), and Src and Pyk2 can synergize to cause STAT3 phosphorylation48, 49
. Future experiments will determine whether the DOCK8-dependent TLR9-MyD88 signaling pathway we have identified is used by other receptors that signal via MyD88 in B cells and other cells. Preliminary data indicates that this pathway is activated by TLR4 ligation in PBMCs (data not shown).
DOCK8 deficiency results in impaired immunological synapse in B cells22
and may impair the ability of T cells and DCs to drive antibody production by B cells. Mice with selective DOCK8 deficiency in B cells, and mice in which the interaction between MyD88 and DOCK8 is disrupted will help define the contribution of DOCK8-dependent MyD88 signaling in B cells to the impaired serologic memory in DOCK8 deficiency. Given that TLR9 ligands are vaccine adjuvants and the role of TLR9 in autoantibody responses to self DNA50
, the TLR9 signaling pathway we have described may be important for developing better vaccines and understanding and treating autoantibody-mediated diseases.