Signaling by Toll-like Receptors 8 and 9 Requires Bruton's Tyrosine Kinase

Toll-like receptors (TLRs) are a primary surveillance system for the detection of pathogens and are crucial to the activation of host defense. TLR7 and TLR8 sense single-stranded RNA from viruses or host ribonucleoproteins and synthetic imidazoquinolines such as R848, whereas TLR9 senses unmethylated CpG motifs in viral and bacterial DNA and in host DNA. Here we report the endogenous interaction between Brutons's tyrosine kinase (Btk) and human TLR8 and TLR9 in the monocytic cell line THP1. We also show that R848, single-stranded RNA, and CpGB-DNA activate Btk in THP1 cells as shown by phosphorylation of the tyrosine 223 residue of Btk and also by increased autokinase activity. We demonstrate that Btk is required for NFκB activation, participating in the pathway to increased phosphorylation of p65 on serine 536 activated by TLR8 and TLR9. Finally we demonstrate that peripheral blood mononuclear cells from patients with X-linked agammaglobulinaemia (XLA) that have dysfunctional Btk are impaired in the induction of interleukin-6 by CpGB-DNA. This study therefore establishes Btk as a key signaling molecule that interacts with and acts downstream of TLR8 and TLR9. Lack of functioning Btk in XLA patients downstream of TLR8 and TLR9 might explain the susceptibility of XLA patients to viral infections. Toll-like receptors (TLRs) are a primary surveillance system for the detection of pathogens and are crucial to the activation of host defense. TLR7 and TLR8 sense single-stranded RNA from viruses or host ribonucleoproteins and synthetic imidazoquinolines such as R848, whereas TLR9 senses unmethylated CpG motifs in viral and bacterial DNA and in host DNA. Here we report the endogenous interaction between Brutons's tyrosine kinase (Btk) and human TLR8 and TLR9 in the monocytic cell line THP1. We also show that R848, single-stranded RNA, and CpGB-DNA activate Btk in THP1 cells as shown by phosphorylation of the tyrosine 223 residue of Btk and also by increased autokinase activity. We demonstrate that Btk is required for NFκB activation, participating in the pathway to increased phosphorylation of p65 on serine 536 activated by TLR8 and TLR9. Finally we demonstrate that peripheral blood mononuclear cells from patients with X-linked agammaglobulinaemia (XLA) that have dysfunctional Btk are impaired in the induction of interleukin-6 by CpGB-DNA. This study therefore establishes Btk as a key signaling molecule that interacts with and acts downstream of TLR8 and TLR9. Lack of functioning Btk in XLA patients downstream of TLR8 and TLR9 might explain the susceptibility of XLA patients to viral infections. Toll-like receptors (TLRs) 2The abbreviations used are:TLRToll-like receptorTIRToll/IL-1RIL-1interleukin-1IL-1IL-1 receptorBMDMbone marrow-derived macrophageBtkBruton's tyrosine kinaseIκBinhibitor of κBNFκBnuclear factor κBLPSlipopolysaccharidePBMCperipheral blood mononuclear cellXLAX-linked agammaglobulinaemiassRNAsingle-stranded RNAXidX-linked immune deficiencyISREinterferon-stimulated response elementCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acidELISAenzyme-linked immunosorbent assay. 2The abbreviations used are:TLRToll-like receptorTIRToll/IL-1RIL-1interleukin-1IL-1IL-1 receptorBMDMbone marrow-derived macrophageBtkBruton's tyrosine kinaseIκBinhibitor of κBNFκBnuclear factor κBLPSlipopolysaccharidePBMCperipheral blood mononuclear cellXLAX-linked agammaglobulinaemiassRNAsingle-stranded RNAXidX-linked immune deficiencyISREinterferon-stimulated response elementCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acidELISAenzyme-linked immunosorbent assay. are a family of receptors in innate immunity that play an important role in the initiation of host defense. They activate signaling pathways that culminate in the induction of immune and inflammatory genes, including those encoding co-stimulatory molecules, antibodies, cytokines, chemokines, and adhesion molecules. There are 10 TLRs in humans. The best characterized is TLR4, which recognizes the Gram-negative product lipopolysaccharide (LPS). The other antibacterial TLRs are TLR2, which in combination with TLR1 recognizes triacylated lipopeptides, or in combination with TLR6, recognizes diacylated lipopeptides, and TLR5, which recognizes bacterial flagellin. The antiviral TLRs are TLR3, which recognizes double-stranded RNA, TLR7 and TLR8, which recognize single-stranded RNA (ssRNA), and TLR9, which recognizes unmethylated CpG motifs that exist in both viral and bacterial DNA (1O'Neill L.A. Bowie A.G. Nat. Rev. Immunol. 2007; 7: 353-364Crossref PubMed Scopus (1942) Google Scholar). All TLRs contain extracellular leucine-rich repeat domains and a cytoplasmic Toll/IL-1R (TIR) domain, which is crucial for signaling downstream of the TLR (2O'Neill L.A. Dinarello C.A. Immunol. Today. 2000; 21: 206-209Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Once activated, TLRs trigger a cascade of cellular signals, culminating in the eventual activation of the Rel family transcription factor NFκB (3Doyle S.L. O'Neill L.A. Biochem. Pharmacol. 2006; 72: 1102-1113Crossref PubMed Scopus (504) Google Scholar). NFκB binds to discrete nucleotide sequences in the upstream regions of genes that produce pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6, thereby regulating their expression (4Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4563) Google Scholar). Regulation of NFκB following stimulation with LPS occurs via activation of two pathways. The best characterized of these regulates the release of NFκB from its inhibitory protein IκBα, subsequently allowing NFκB to translocate to the nucleus and bind its target genes. However, this nuclear translocation of NFκB is not sufficient to activate NFκB-dependent gene transcription alone. The second pathway, which involves post-translational modifications, regulates the transactivating ability of the p65 subunit of NFκB once bound to its consensus sequence, i.e. the ability to recruit the transcriptional apparatus and stimulate target gene expression (5Schmitz M.L. Bacher S. Kracht M. Trends Biochem. Sci. 2001; 26: 186-190Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Toll-like receptor Toll/IL-1R interleukin-1 IL-1 receptor bone marrow-derived macrophage Bruton's tyrosine kinase inhibitor of κB nuclear factor κB lipopolysaccharide peripheral blood mononuclear cell X-linked agammaglobulinaemia single-stranded RNA X-linked immune deficiency interferon-stimulated response element 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid enzyme-linked immunosorbent assay. Toll-like receptor Toll/IL-1R interleukin-1 IL-1 receptor bone marrow-derived macrophage Bruton's tyrosine kinase inhibitor of κB nuclear factor κB lipopolysaccharide peripheral blood mononuclear cell X-linked agammaglobulinaemia single-stranded RNA X-linked immune deficiency interferon-stimulated response element 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid enzyme-linked immunosorbent assay. Bruton's tyrosine kinase (Btk), a cytoplasmic non-receptor tyrosine kinase, is a member of the Tec family of protein kinases. It was first identified in the male immune disorder X-linked agammaglobulinaemia (XLA). The XLA syndrome is manifested by severe defects in early B cell development, resulting in a nearly complete absence of peripheral B cells and immunoglobulins of all classes (6Bradley L.A. Sweatman A.K. Lovering R.C. Jones A.M. Morgan G. Levinsky R.J. Kinnon C. Hum. Mol. Genet. 1994; 3: 79-83Crossref PubMed Scopus (90) Google Scholar). Various mutations in the human btk gene were identified as being responsible for the XLA phenotype (7Hashimoto S. Tsukada S. Matsushita M. Miyawaki T. Niida Y. Yachie A. Kobayashi S. Iwata T. Hayakawa H. Matsuoka H. Tsuge I. Yamadori T. Kunikata T. Arai S. Yoshizaki K. Taniguchi N. Kishimoto T. Blood. 1996; 88: 561-573Crossref PubMed Google Scholar). These include mutations in both the kinase domain and the pleckstrin homology domain, which affect kinase activity and translocation to the membrane, respectively. The pleckstrin homology domain is important in the process of Btk activation since it localizes Btk to the membrane through its interaction with phosphatidylinositol 3,4,5-triphosphate. In mice, a naturally occurring mutation in the pleckstrin homology domain has been mapped to arginine 28 (R28C), which causes X-linked immune deficiency (Xid) in these mice. Mutated Btk in Xid mice is prevented from associating with the membrane, thus inactivating it (8Park H. Wahl M.I. Afar D.E. Turck C.W. Rawlings D.J. Tam C. Scharenberg A.M. Kinet J.P. Witte O.N. Immunity. 1996; 4: 515-525Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). We have previously described a key role for Btk in NFκB transactivation in response to LPS (9Doyle S.L. Jefferies C.A. O'Neill L.A. J. Biol. Chem. 2005; 280: 23496-23501Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The ability of Btk to associate with the TIR domain of TLR4 was also demonstrated (10Jefferies C.A. Doyle S. Brunner C. Dunne A. Brint E. Wietek C. Walch E. Wirth T. O'Neill L.A. J. Biol. Chem. 2003; 278: 26258-26264Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). In addition, however, we found that Btk can interact with the TIR domains of TLR8 and TLR9. This suggested a possible role for Btk in TLR8 and TLR9 signaling, although this has never been examined directly. Btk-deficient mice have exhibited high susceptibility to acute mouse adenovirus type-1 (MAV-1) in contrast to controls, and XLA patients are highly susceptible to enteroviral infections, indicating a possible role for Btk in preventing virus-induced disease (11Moore M.L. McKissic E.L. Brown C.C. Wilkinson J.E. Spindler K.R. J. Virol. 2004; 78: 5584-5590Crossref PubMed Scopus (43) Google Scholar, 12Winkelstein J.A. Marino M.C. Lederman H.M. Jones S.M. Sullivan K. Burks A.W. Conley M.E. Cunningham-Rundles C. Ochs H.D. Medicine (Baltimore). 2006; 85: 193-202Crossref PubMed Scopus (416) Google Scholar). This could be due to a role for Btk in TLR8 or TLR9 signaling. Both TLR8 and TLR9 have been shown to be expressed in B cells, and their activation has been shown to induce antibody production and proliferation (13Chuang T.H. Ulevitch R.J. Eur. Cytokine Netw. 2000; 11: 372-378PubMed Google Scholar, 14Schlaepfer E. Audige A. Joller H. Speck R.F. J. Immunol. 2006; 176: 2888-2895Crossref PubMed Scopus (99) Google Scholar, 15Dasari P. Nicholson I.C. Hodge G. Dandie G.W. Zola H. Cell. Immunol. 2005; 236: 140-145Crossref PubMed Scopus (98) Google Scholar). Given these aspects and the apparent interaction between Btk and TLR8 and TLR9, we have examined a role for Btk in TLR8 and TLR9 signaling. We provide the first evidence for an interaction between Btk and these TLRs endogenously and for potent activation of Btk by TLR8 and TLR9, and we demonstrate that Btk is required for transactivation by p65. We also demonstrate impaired induction of IL-6 by TLR9 in PBMCs prepared from XLA patients. Our data therefore identify for the first time a key role for Btk in TLR8 and TLR9 signaling. Cell Culture—The HEK293 cell line and THP1 cell line were obtained from European Cell Culture Collection and were maintained as described previously (16Jefferies C.A. O'Neill L.A. Immunol. Lett. 2004; 92: 15-22Crossref PubMed Scopus (60) Google Scholar). Female and Male CBA/CaHN-BtkJ mice were gifts from Stephanie Vogel. We used female CBA/CaHN-BtkJ that do not exhibit the disease and male CBA/CaHN-BtkJ that are affected by the mutation. BMDMs were prepared from CBA/CaHN-BtkJ mice and maintained as described previously (9Doyle S.L. Jefferies C.A. O'Neill L.A. J. Biol. Chem. 2005; 280: 23496-23501Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Two male patients diagnosed with XLA, according to the World Health Organization (WHO) classification, and two healthy age- and sex-matched controls, were included in this study after informed consent was obtained. This study was approved by the St. James's Hospital/Adelaide and Meath Hospital research ethics committee. Both patients were receiving regular and were of the time of blood blood were to the of PBMCs were as described previously T.H. J.P. J. T. J. Immunol. 2006; 176: PubMed Scopus Google Scholar). and following were the and Btk and of the encoding p65 subunit of NFκB to the domain and the gene The following were and and and and and The following were the and the IL-6 All other were obtained from with the of the and and cells were as described previously for both the and the the interferon-stimulated response element HEK293 cells were to and of of and the of were according to the were as described previously (9Doyle S.L. Jefferies C.A. O'Neill L.A. J. Biol. Chem. 2005; 280: 23496-23501Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). cells were and in to a of BMDMs were in cells were in and 10 and of cell were by the assay. of protein were The were to with primary as were with the were by according to the was and was to the protein and expressed as in the cells were a of to of cells was used and cells were with or for the time and in of high 10 and or of 10 and for on were by for with with the which been to protein or with of the was to that the protein of was this was to the and for the the immune were with All was and were in of The were for by and by Btk cells were prepared for autokinase as described previously (10Jefferies C.A. Doyle S. Brunner C. Dunne A. Brint E. Wietek C. Walch E. Wirth T. O'Neill L.A. J. Biol. Chem. 2003; 278: 26258-26264Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). IL-6 PBMCs were and with or of and time as were and the of IL-6 was by according to the was on a of Btk with TLR8 and a we found that a to for possible interaction for Btk a encoding human TLR8 (10Jefferies C.A. Doyle S. Brunner C. Dunne A. Brint E. Wietek C. Walch E. Wirth T. O'Neill L.A. J. Biol. Chem. 2003; 278: 26258-26264Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). It was found that Btk could also interact with the TIR domains of TLR4, TLR6, and TLR9. However, the interaction was found with the TIR domain of TLR8 (10Jefferies C.A. Doyle S. Brunner C. Dunne A. Brint E. Wietek C. Walch E. Wirth T. O'Neill L.A. J. Biol. Chem. 2003; 278: 26258-26264Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). this we first We THP1 monocytic cells as a system since this cell line expressed both TLR8 and TLR9 and was to the TLR8 and the TLR9 CpGB-DNA as shown by TLR8 or TLR9 was from prepared from THP1 cells with or time and TLR from time was with a interaction between Btk and TLR8 was both and after However, we found that the interaction to be and after stimulation in with of an interaction between Btk and TLR9 was in cells and cells with to the between TLR8 and the interaction to CpG stimulation with an apparent increased interaction after and of of stimulation In both were for Btk to and second The of these was in THP1 by the ability of the mouse with Btk to that of the TLR were the in the and These were also for Btk to and Btk and TLRs interact in and THP1 and THP1 cells were with or for the time were in and TLR8 TLR9 or mouse and was by were with Btk antibody to an interaction and cell were also for Btk and second and are of of TLR8 and TLR9 Btk examined the ability of and to induce tyrosine phosphorylation on which is an of Btk were from prepared from THP1 cells with time protein from time was for Btk was after of stimulation with were also for Btk to early for Btk is Btk which on T. Immunol. PubMed Scopus Google therefore the ability of Btk to in which is an of its autokinase was examined an in kinase assay. of THP1 cells with a in Btk This was from were also for Btk to The were on THP1 cells with R848, with from prepared from THP1 cells with time and for Btk Btk after of stimulation were also for Btk to of THP1 cells with a in the activity of which was of stimulation were also for Btk to The on Btk has been identified as (8Park H. Wahl M.I. Afar D.E. Turck C.W. Rawlings D.J. Tam C. Scharenberg A.M. Kinet J.P. Witte O.N. Immunity. 1996; 4: 515-525Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). We therefore examined the of CpG and on this response a antibody on phosphorylation of Btk was after of stimulation with with phosphorylation and first also increased phosphorylation on the being after of stimulation with R848, with phosphorylation and a in phosphorylation from second We also the TLR7 for its ability to Btk phosphorylation on response was after of stimulation with phosphorylation and a in phosphorylation from In all were also for Btk to and In as shown in and were were Btk on the from TLR8 and TLR9 to ability of both and to activate Btk that Btk might be an important signaling downstream of TLR8 and TLR9. We have previously shown that Btk is activated downstream of TLR4 and a role in the phosphorylation of the NFκB p65 subunit on which is for the transactivation of NFκB in response to LPS (9Doyle S.L. Jefferies C.A. O'Neill L.A. J. Biol. Chem. 2005; 280: 23496-23501Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). We therefore Btk a role in TLR8 and TLR9 signaling to we examined the of two of Btk which is to to the membrane, and Btk a of on the induction of an NFκB-dependent gene by TLR8 and TLR9. The of TLR8 and TLR9, and the extracellular domain to the cytoplasmic domains of the are can be in of in HEK293 cells to the induction of the NFκB gene This activation was by the two of Btk and Btk However, whereas of in HEK293 cells also to the induction of the gene this activation was by of Btk or Btk of in HEK293 cells to the induction of the NFκB gene This activation was also by the two of Btk However, as in the of TLR8, whereas of in HEK293 cells also to the induction of the gene this activation was by of Btk or Btk The of on the an important in the role of Btk in TLR8 and TLR9 signaling. We examined the of Btk and Btk on the system HEK293 cells to for a role for Btk downstream of TLR8 and TLR9 in the p65 transactivation HEK293 cells with a encoding activation of the system This ability of to activate was cells were with a encoding Btk or Btk was HEK293 cells, the system was This ability of to activate was cells were with a encoding Btk or Btk we previously shown that Btk is in the phosphorylation of p65 on in response to we Btk a role in response to or The phosphorylation of serine 536 in response to was in BMDMs from female and male Xid mice. Xid mice a of In this the female BMDMs as the due to Xid being an X-linked disease with being increased p65 phosphorylation This response was impaired in BMDMs from male Xid mice Xid BMDMs were with R848, was also increased p65 which from by This response was also impaired in BMDMs from male Xid mice to IL-6 in to TLR8 and TLR9 we examined the induction of IL-6, a pro-inflammatory to be the transcriptional of in PBMCs from XLA patients. with to induce IL-6 production by after both and with PBMCs We also examined PBMCs with a Btk inhibitor IL-6 production by was impaired in cells with with cells not with the inhibitor We were to in XLA PBMCs since response was in which is to be due to expression of data therefore an important role for Btk in signaling by TLR8 and TLR9. In this we demonstrate that endogenous TLR8 and TLR9 interact with Btk by and that of TLR8 or TLR9 in the activation of Btk in a TLR8, and TLR9 to a of of TLR8, and TLR9 in response to their on and of and to with P. H. H. T. H. G. K. S. H. S. Eur. J. Immunol. 2003; PubMed Scopus Google Scholar). TLR8, and TLR9 a the TLR family that recognizes in the of an unmethylated bacterial and viral is by TLR9, and the for TLR7 and TLR8 are and H. H. C. S. G. H. S. 2004; PubMed Scopus Google Scholar, H. T. T. S. H. M. K. H. K. S. 2000; PubMed Scopus Google Scholar, T. H. S. C. 2004; PubMed Scopus Google Scholar). and with and early E. A. T. J. 2004; PubMed Google have identified the that TLR8, and TLR9 are expressed in as the which localizes to E. A. T. J. 2004; PubMed Google and P. H. H. T. H. G. K. S. H. S. Eur. J. Immunol. 2003; PubMed Scopus Google Scholar, E. A. A. E. T. Nat. Immunol. 2004; PubMed Scopus Google have also shown that signaling is in these as by the of to in response to and for TLR7 and or We have demonstrated by that Btk interacts with TLR8 and TLR9 endogenously and that increased the Given that we have also shown that Btk can interact with (10Jefferies C.A. Doyle S. Brunner C. Dunne A. Brint E. Wietek C. Walch E. Wirth T. O'Neill L.A. J. Biol. Chem. 2003; 278: 26258-26264Abstract Full Text Full Text PDF PubMed Scopus (237) Google it is possible that Btk to the is via We examined a role for Btk in TLR8 and TLR9 We previously shown that Btk is activated LPS stimulation of TLR4 (10Jefferies C.A. Doyle S. Brunner C. Dunne A. Brint E. Wietek C. Walch E. Wirth T. O'Neill L.A. J. Biol. Chem. 2003; 278: 26258-26264Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Our data demonstrate that Btk is also tyrosine and subsequently activated in response to both and in the human monocytic THP1 cell Our data identify Btk as an important tyrosine kinase activated by TLR8 and TLR9. Given that TLR8, and TLR9 activate both Btk and NFκB and previously shown a role for Btk in NFκB activation (9Doyle S.L. Jefferies C.A. O'Neill L.A. J. Biol. Chem. 2005; 280: 23496-23501Abstract Full Text Full Text PDF PubMed Scopus (129) Google we Btk a role in and NFκB of an gene by TLR8 and TLR9 was by of the of Btk on activation by TLR8 or TLR9, indicating that Btk acts upstream of NFκB It is that phosphorylation of the p65 subunit the nuclear transcriptional of NFκB and is required for transcriptional This phosphorylation of p65 is to be required for transactivation of gene expression serine in the transactivation domains of p65 NFκB-dependent gene transcription in M.L. Bacher S. Kracht M. Trends Biochem. Sci. 2001; 26: 186-190Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). we previously Btk downstream of TLR4 in the transactivation of the p65 subunit of we examined Btk was in and p65 TLR8 and TLR9 activated a p65 transactivation and of Btk this we show for the first time that and phosphorylation of p65 on and that BMDMs in Btk were in this the of have not been phosphorylation of the p65 subunit of NFκB has as a by which transcriptional activity of NFκB is in an However, it is suggested that to of the transcriptional the role of phosphorylation in p65 to or of the transcriptional has been H. E. S. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, H. A. M.L. E. K. Kracht M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). that Btk to play a role downstream of TLR8 and TLR9 in the transactivating of it that a of Btk might on the induction of NFκB-dependent genes in response to or We found a in IL-6 induction in response to in PBMCs from XLA patients to The Btk inhibitor also induction of IL-6 by CpG in We were therefore to a key role for Btk in TLR9 signaling in human We found that PBMCs not to R848, and we were to TLR8 in a However, impaired IL-6 and tumor necrosis factor-α production has been in cells from patients with XLA K. J. J. A. Blood. 2007; PubMed Scopus Google Scholar). it that this is due to TLR8 signaling in cells with These are important in to XLA patients although regular to of antibodies, XLA patients with and severe and enteroviral infections, by In enteroviral are the of in XLA patients J.A. Marino M.C. Lederman H.M. Jones S.M. Sullivan K. Burks A.W. Conley M.E. Cunningham-Rundles C. Ochs H.D. Medicine (Baltimore). 2006; 85: 193-202Crossref PubMed Scopus (416) Google Scholar, S.L. Rev. PubMed Scopus Google Scholar). patients with XLA are to be susceptible to viruses such as and 2006; PubMed Scopus Google Scholar). viruses have been shown to contain unmethylated CpG motifs M. M. Sci. S. A. PubMed Scopus Google and according to study on XLA of all viral H.M. J.A. Medicine (Baltimore). PubMed Scopus Google Scholar, J. Virol. 2000; PubMed Scopus Google Scholar). in TLR9 signaling due to the in Btk explain this Our data are also in to inflammatory such as the disease B cell receptor signaling with or stimulation of or TLR9 with host in immune have been to the and of S. K. A. J. C. G. Sci. S. A. 2006; PubMed Scopus Google Scholar). Given the role of Btk in signaling by TLR8 and TLR9 Btk might be in the of We the XLA patients in this study and for blood We also Stephanie for the gifts of the Xid mice.