BANK1 Controls CpG-Induced IL-6 Secretion via a p38 and MNK1/2/eIF4E Translation Initiation Pathway Free

Human and mouse Bank1 encode the B cell scaffold protein with ankyrin repeats 1. During BCR activation BANK1 becomes extensively tyrosine phosphorylated and is capable of binding the Src family kinases Lyn and Blk (1, 2). Although apparently involved in BCR signaling, the function of BANK1 during signaling induced by CpG, an agonist of the major TLR, TLR9 expressed in B cells, is not known.

It has been proposed that BANK1 acts as an adaptor or scaffold protein in the same family as the B cell adaptor for PI3K (BCAP) and the Drosophila homolog Dof (2). Consistent with this hypothesis, our recent studies have shown that exon 2 of human BANK1 encodes a highly hydrophobic domain, which renders the protein susceptible to aggregation (3); scaffold and adaptor proteins are known to form complex structures to facilitate intracellular signaling at the proper time and differentiation stage. Furthermore, exon 2 also encodes a predicted N-terminal Toll/IL-1R (TIR) domain that is shared by BCAP (4) and used in the interaction of BCAP with the adaptors MyD88 and TIRAP. TLR9 is the major endosomal TLR in B cells that recognizes viral nucleic acids, and TLR9 signaling is thought to have an important role in autoimmunity (5). TLR9 signaling is stimulated by hypomethylated DNA oligonucleotides or CpG (6), leading to a proinflammatory response (7).

CpG-induced signaling activates MAPK pathways, including p38, JNK, and ERK. Stimulation of p38 and ERK signaling by growth factors, stress, or viral infections can induce transcriptional activation, but it can also induce two pathways of posttranscriptional regulation of protein synthesis: control of mRNA stabilization (8, 9) by the MAPK-activated protein kinase 2 MK2, and the transient formation of the heterotrimeric eIF4E/eIF4F/eIF4G translation initiation complex through phosphorylation of eIF4E (10, 11). In mice, the only kinases known to phosphorylate eIF4E are MNK1 and MNK2. MNK2 is constitutively active, whereas MNK1 is regulated by the MAPKs (12).

A second axis of control of eIF4E activation is through the AKT/mTORC1 pathway. This pathway regulates the phosphorylation of 4E-BP1, the eIF4E binding protein. Under nonphosphorylated conditions, 4E-BP1 retains eIF4E (13, 14). Once 4E-BP1 becomes phosphorylated by mTORC1, it releases eIF4E, which is in turn phosphorylated by MNK1/2 (15).

Because of the role of CpG-induced signaling in autoimmunity and the putative role of BANK1 as a TIR-containing adaptor, we hypothesized that BANK1 may participate in CpG-induced signaling. Our results establish a function for BANK1 in CpG-induced responses that could have important implications for the role of BANK1 in infections and autoimmunity, where BANK1 has been established as a susceptibility gene (16).

Bank1^−/− mice were provided by Dr. T. Kurosaki (Riken Research Centre for Allergy and Immunology, Kyoto, Japan) and were backcrossed nine generations onto the C57BL/6J background. C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen-free barrier conditions. This study was approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee.

Phospho-specific Abs to p38 (Thr¹⁸⁰/Tyr¹⁸², clone12F8, no. 4631), JNK (Thr¹⁸³/Tyr¹⁸⁵, clone 81E11, no. 4668), ERK (Thr²⁰²/Tyr²⁰⁴, clone D13.14.4E, no. 4370), IκBα (Ser³², clone 14D4, no. 2859), eIF4E (Ser²⁰⁹, no. 9741), MNK1/2 (Thr^197/202, no. 2111), 4E-BP1 (Thr^37/46, clone 236B4, no. 2855), mTOR (Ser²⁴⁴⁸, clone D9C2, no. 5536), and AKT (Ser⁴⁷³, clone 193H12, no. 4058), as well as phosphorylation state–independent Abs to p38 (no. 9212), JNK, ERK, IκBα, eIF4E (clone C46H6, no. 2067), MNK1 (clone C4C1, no. 2195), 4E-BP1 (clone 53H11, no. 9644), mTOR (clone 7C10, no. 2983), and AKT (no. 9272), were purchased from Cell Signaling Technology (Danvers, MA). Phosphorylation state–independent Ab for MNK2 (S-20) was purchased from Sigma-Aldrich (St. Louis, MO).

Splenic B cells were purified by negative selection using magnetic bead separation. Briefly, spleen cells from Bank1^+/+ and Bank1^−/− littermates were labeled with a mixture of biotin-conjugated Abs for 15 min. Cells were incubated an additional 15 min with anti-biotin microbeads (B cell isolation kit, mouse; Miltenyi Biotec, Auburn, CA) at 4°C. The labeled non–B cells were depleted by magnetic retention in a MACS column while unlabeled B cells were recovered. The purity of the resulting cell population was typically >95% B220⁺CD3⁻ as assessed by flow cytometry analysis.

Cell culture was performed with RPMI 1640 medium supplemented with 10% FBS, l-glutamine (2 mM), 2-ME (50 μM), and antibiotics for all experiments, except for p38 analyses and the Akt axis, where cells were serum-starved 2 h prior to stimulation. Purified splenic B cells (10⁶ cells/ml) were treated with 10 μg/ml F(ab′)₂ fragment of anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA), 2 μM CpG (oligodeoxynucleotide type B, 1668; InvivoGen, San Diego, CA), or a combination of anti-mouse IgM and CpG, 20 μg/ml TLR4 agonist LPS, or 1 μg/ml TLR7 agonist R848 (InvivoGen), or were left unstimulated. Supernatants were collected at 0, 12, 24, and 48 h, and at 72 and 96 h for anti-CD40 stimulation. Low-endotoxin, azide-free anti-mouse CD40 (BioLegend, San Diego, CA) was included (10 μg/ml final concentration) with or without CpG.

Stimulated splenic B cells were lysed in buffer containing 1% Triton X-100, 50 mM Tris (pH 7.4), 50 mM NaCl, 1 mM EDTA, 2 mM Na₃VO₄, and a protease inhibitor mixture (Roche Applied Science, Indianapolis, IN). LDS sample buffer and reducing agent (Life Technologies, Carlsbad, CA) were added. Samples were then boiled, and protein was separated with NuPAGE 4–12% Bis-Tris Gels (Life Technologies). Proteins were blotted onto polyvinylidene difluoride membranes, blocked with 5% skimmed milk, and the immunoblots were processed with specific Abs diluted with 5% BSA or 5% skimmed milk solution and detected with Novex ECL substrate (Invitrogen, Carlsbad CA).

Cytokine secretion by in vitro–stimulated B cells was measured using the ELISA kit for IL-6 and IL-10 purchased from BD Biosciences (San Jose, CA). For this, 96-well plates were coated with Ab against mouse IL-6 or mouse IL-10 overnight at 4°C. Plates were then blocked for 1 h at room temperature with 10% FBS in PBS. Supernatants were then added to the plate and left for 2 h at room temperature. Following washing, biotinylated anti–IL-6 or anti–IL-10 was added and plates were incubated for 1 h at room temperature. Streptavidin-alkaline phosphatase was then added following incubation at room temperature and washing with tetramethylbenzidine liquid substrate. Absorbance was read at 450 nm, from which absorbance was subtracted at 570 nm within 30 min of stopping reaction. Cytokine concentration was determined by extrapolation from the standard curve. Standard curve was generated using recombinant mouse IL-6 or recombinant mouse IL-10 (BD Biosciences).

Splenic B cells (0.5 × 10⁶ cells/well) were stimulated for 6 d and supernatants were analyzed for Ig isotype Abs as per the instructions for the BD Pharmingen mouse Ig isotype ELISA kit (BD Biosciences). We quantified IgG2a and IgG2c isotype Abs using a mouse IgG2a Ready-SET-Go! ELISA kit (eBioscience, San Diego, CA) and mouse IgG2c ELISA quantification set (Bethyl Laboratories, Montgomery, TX), respectively, and data are presented as nanograms per milliliter. For other Abs, only ODs are shown.

Purified splenic B cells were resuspended in prewarmed PBS/0.1% BSA, labeled with 2 μM CFSE (Life Technologies), and incubated at 37°C in a water bath for 10 min. Five volumes of culture medium were added for quenching. After incubating 5 min on ice, the cell pellet was collected by centrifugation, washed three times with fresh complete RPMI 1640 medium, and resuspended to the appropriate density. Cells were then stimulated with a 10 μg/ml F(ab′)₂ fragment of anti-IgM, 2 μM CpG, 10 μg/ml anti-CD40, 20 μg/ml LPS, and 1 μg/ml R848, respectively, or the shown combinations and cultured for 48, 72, and 96 h. After harvest, cells were stained with propidium iodide (PI; final concentration of 1 μg/ml; eBioscience) and acquired by LSR II or FACSCalibur (BD Siosciences) (36,000 events in total). For flow cytometry, debris and doublet cells were discriminated and viable cells (PI⁻) were gated and the percentage of cell viability was determined. The histograms shown with FL1 channel represent overlaid data from unstimulated CFSE-labeled B cells, stimulated Bank1^+/+ and Bank1^−/− B cells, and the y-axis is presented as percentage viability of maximum. The analysis was conducted using FlowJo software (Tree Star, Ashland, OR).

The MNK1 specific inhibitor, CGP57380 (4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine) was obtained from Calbiochem-Millipore (Billerica, MA), and MNK inhibitor cercosporamide and MK2 inhibitor PF-3644022 ((10R)-9,10,11,12-tetrahydro-10-methyl-3-(6-methyl-3-pyridinyl)-8H-[1,4]diazepino[5′,6′:4,5]thieno[3,2-f]quinolin-8-one hydrate) were purchased from Sigma-Aldrich. The purified splenic B cells (5 × 10⁵ cells/well) were seeded in complete RPMI 1640 medium in a 48-well tissue culture plate. Then, the cells were pretreated with or without inhibitor for 30 min or 1 h, respectively, before CpG stimulation for 24 h, at which time cells were harvested and subjected to determine percentage of viability by using PI staining (final concentration 1 μg/ml) and flow cytometry analysis. Supernatants were collected and stored at −80°C until use and IL-6 was measured using a capture ELISA. The control well contained CpG and cell culture grade DMSO (Pierce, Rockford, IL) only (17).

Total RNA was isolated with TRIzol (Invitrogen) from triplicate cultures of 5 × 10⁵ splenic B cells in the presence or absence of CpG treatment for the indicated times. After quantification and quality control of the RNA with a Nanodrop spectrophotometer (Thermo Scientific, West Palm Beach, FL), 400 ng RNA was subjected to first-strand cDNA synthesis for quantitative RT-PCR (Origene, Rockville, MD). cDNA/RNA (5 ng total) was used per reaction in TaqMan Fast Universal PCR Master Mix (Life Technologies). The quantitative RT-PCR reactions were performed on a 7900 HT Fast real-time PCR system. The primers and probes for mouse Tlr9 (assay ID: Mm00446193_m1), Prdm1 (Mm00476128_m1), and Il6 (Mm00446190_m1) and internal control gene 18S rRNA (4319413E) used were TaqMan Gene Expression Assays (Life Technologies).

For the Il6 mRNA stability assay, 5 × 10⁵ splenic B cells were cultured in triplicate and treated with 2 μM CpG for 20 h, followed by addition of 1 μg/ml actinomycin D (Sigma-Aldrich) for 0, 50, 100, and 200 min. RNA was isolated and Il6 transcription was determined with TaqMan quantitative RT-PCR using the primers as described above.

To discern the signaling cascades affected by BANK1, we tested whether BANK1 deficiency altered B cell proliferation after stimulation with CpG. Using purified splenic B cells from Bank1^−/− and littermate control (Bank1^+/+) mice, we did not observe any differences at any of the time points tested in proliferation with CpG or LPS, a TLR4 agonist (Fig. 1A). We also did not observe differences in cell proliferation when using anti-IgM, anti-IgM plus CpG, anti-CD40, CpG plus anti-CD40, R848, R848 plus anti-CD40, LPS, LPS plus anti-CD40, or anti-IgM plus LPS (Supplemental Fig. 1). We then tested the effects of BANK1 deficiency on IgM-induced activation of NF-κB by analyzing phosphorylation and degradation of IκBα, as well as activation of the MAPK pathways by studying phosphorylation of ERK and p38. BANK1 deficiency did not affect phosphorylation of MAPKs, or IκBα following stimulation with anti-IgM alone (Fig. 1B).

After CpG stimulation, importantly, p38 phosphorylation was reduced (Fig. 1C, 1D) but phosphorylation of ERK, JNK, and IκBα was unchanged in Bank1^−/− cells compared with cells from wild-type (WT) mice (Fig. 1C) or from Bank1^+/+ littermates (data not shown). For all pathways, stimulation with anti-IgM plus CpG (data not shown) yielded results identical to CpG alone (Fig. 1C). BANK1 deficiency had no effect on p38 signaling induced by LPS or R848 (Fig. 1E). Therefore, BANK1 specifically modulates CpG-induced p38 signaling in vitro.

It is known that the MAPK p38 signaling pathway regulates the production of several cytokines, including IL-6, TNF-α, IFN-γ, and IL-10 (18–20). Having shown that BANK1 deficiency reduced p38 phosphorylation after CpG stimulation, we asked whether reduction in p38 phosphorylation leads to modulation of IL-6 or IL-10 secretion by Bank1^−/− B cells. As expected (21–23), anti-IgM alone did not induce detectable IL-6 or IL-10 in splenic B cells from WT, Bank1^+/+, or Bank1^−/− littermates (data not shown), whereas CpG induced detectable levels of IL-10 and IL-6 production. Bank1^−/− B cells showed consistently reduced IL-6 but normal IL-10 secretion in response to CpG alone or the combination of CpG and BCR ligation (Fig. 2A).

In general, it has been observed that the combination of anti-CD40 and CpG leads to the most optimal secretion of IL-6 by B cells (24, 25), and BANK1 deficiency has been shown to lead to activation of Akt following stimulation through CD40. When we investigated production of IL-6 by combining anti-CD40 and CpG, we clearly observed that the amount of IL-6 secreted was importantly increased when adding anti-CD40 to CpG as compared with CpG alone in Bank1^+/+ B cells. Deficiency of BANK1 reduced the levels of secreted IL-6 (Fig. 2A). We tested whether stimulation with CpG would lead to a reduction in cell viability by BANK1-deficient cells and hence to reduced IL-6 secretion. This was not the case (Supplemental Fig. 1A). We did observe slightly fluctuated cell viability when combining CpG with anti-CD40 in BANK1-deficient cells, but proliferation was normal. Hence, we conclude that reduced secretion of IL-6 is not due to changes in cell viability in BANK1-deficient B cells.

CpG is a known agonist of Tlr9. To test whether the reduced expression of IL-6 and reduced phosphorylation of p38 could be due to reduced expression of Tlr9, we tested mRNA expression. Tlr9 gene expression was no different between Bank1^+/+ and Bank1^−/− mice (Fig. 2B).

Next, we examined whether BANK1 deficiency had an effect on the production of IL-6 induced by TLR7 and TLR4. However, LPS and the TLR7 agonist R848 induced subtle amounts of IL-6 (21, 22) as compared with CpG stimulation, and BANK1 deficiency had no effect (Fig. 2C). Taken together, these results show that BANK1 deficiency reduces secretion of IL-6 in response to CpG stimulation, overcomes the effect of anti-CD40, and is not due to changes in cell viability or to reduced Tlr9 expression.

We suspected that BANK1 deficiency could decrease IL-6 production by reducing Il6 translation initiation and/or mRNA stability, because p38 directly controls these via the kinases MNK1/2 and MK2, respectively (26, 27). MK2 inhibitors are known to reduce IL-6 secretion by human PBMCs (28). Consistent with this, we observed reduced IL-6 production in CpG-stimulated WT mouse B cells treated with the MK2 inhibitor PF-3644022 (Fig. 3A). Additionally, there was a significant and reproducible, although less marked, reduction of IL-6 secretion after treatment with the MNK1/2/eIF4E inhibitors CGP57380 and cercosporamide using doses that minimally affect cell viability (Fig. 3B–D). These results show that MNK1/2 and MK2 inhibitors can reduce IL-6 secretion in mouse B cells and confirm that both MNK1/2 and MK2 are important for CpG-stimulated IL-6 secretion from mouse splenic B cells.

Because CpG-induced IL-6 production was reduced in Bank1^−/− cells, we tested whether Il6 gene expression was altered by the deficiency of BANK1. As shown in Fig. 4A, Il6 gene expression showed no differences between BANK1-sufficient or BANK1-deficient B cells following CpG stimulation.

Because the p38/MK2 signaling pathway is responsible in maintaining mRNA stability, we tested the effect of BANK1 deficiency on MK2-mediated IL-6 secretion by testing the stability of Il6 mRNA. CpG-stimulated Bank1^+/+ and Bank1^−/− splenic B cells showed comparable Il6 mRNA stability (Fig. 4B). Next, we tested the effects of BANK1 on the activation of the p38/MNK1/2-mediated signaling pathway. MNK1/2 regulates the translation initiation factor eIF4E through phosphorylation (26, 29, 30). We therefore analyzed phosphorylation of MNK1/2 and eIF4E. CpG-induced MNK1/2 and eIF4E phosphorylation were consistently reduced in Bank1^−/− cells (Fig. 4C, 4D).

These results show that BANK1 influences IL-6 secretion by its effects on the p38-regulated MNK1/2/eIF4E/eIF4G pathway of translation initiation whereas BANK1 does not affect IL-6 secretion via the MK2 pathway.

Activation of eIF4E is also controlled via the AKT/mTORC1/4E-BP1 signaling cascade. Activation of this pathway leads to phosphorylation of 4E-BP1, which in turn releases eIF4E for it to be phosphorylated by MNK1/2 and initiate translation. Additionally, stimulation through CD40 has been reported to lead to increased AKT phosphorylation in Bank1^−/−-purified B cells (31). The cascade was induced with CpG alone (Fig. 5A), and the phosphorylation of AKT, mTOR, and 4E-BP1 was strongly induced when anti-CD40 was included alone or in combination with CpG (Fig. 5B, 5C). We did observe an increase, albeit weak, in phospho-AKT following anti-CD40 and anti-CD40 plus CpG treatment on Bank1^−/− B cells (Fig. 5B, 5C) (31). However, we did not observe any differences in the downstream mTORC1 to 4E-BP1 when we used anti-CD40 alone or combined with CpG between Bank1^−/− and Bank1^+/+ B cells or on phospho-AKT when using CpG alone (Fig. 5). Overall, our results show that BANK1 acts only in the MNK1/2 and eIF4E arm of p38 signaling to induce IL-6 secretion following CpG stimulation, and that absence of BANK1 does not affect activation of AKT, mTORC1, or 4E-BP1.

Finally, we tested whether BANK1 had an effect on the in vitro secretion of Abs following CpG stimulation. Our results show a nonsignificant tendency toward increased, rather than decreased, secretion of IgG2a and IgG2c Abs by Bank1^−/− B cells. Furthermore, we did not observe any difference in the expression of the Prdm1 gene that codifies for the transcription factor Blimp1 (Supplemental Fig. 2).

Our results suggest that secretion of IgG2a/2c Abs is not significantly affected by reduced secretion of IL-6 by Bank1^−/− B cells.

To our knowledge, we show for the first time that the B cell adaptor with ankyrin repeats BANK1 influences signaling, leading to the formation of the translation initiation eIF4E complex following stimulation with CpG, and that BANK1 deficiency results in reduction of the translation of the proinflammatory cytokine IL-6. We have shown that mRNA stability of Il6 is not affected, nor is the second axis of regulation of translation initiation via AKT, which is also induced by CpG and strongly amplified with anti-CD40 treatment. Clearly, the combination of CpG and anti-CD40 treatment leads to stronger signaling of the AKT axis followed by strong activation of mTOR and 4E-BP1. However, absence of BANK1 has no effect on the signaling pathway through this axis, except weak activation of AKT. Whereas the secretion of IL-6 is optimal (22, 23, 32) with the combination of CpG and anti-CD40, BANK1 deficiency leads to its reduction, and anti-CD40 cannot overcome this effect. Our results clearly show that overall BANK1 controls the production of IL-6 via the p38/MNK1/2/eIF4E pathway.

At the same time, activation of the CD40-induced pathway leads to strong phosphorylation of the AKT axis, which would eventually promote translation initiation of IL-6 through phosphorylation of 4E-BP1 and the release of eIF4E for it to become phosphorylated by MNK1/2. However, we clearly observe that this is not the case.

The involvement of BANK1 in modulating the IL-6 response after CpG stimulation has important implications in the pathogenesis of autoimmunity and viral infection.

Sera from autoimmunity-prone animals and cerebrospinal fluid from systemic lupus erythematosus (SLE) patients have elevated levels of IL-6 (33, 34), and peripheral blood cells from SLE patients spontaneously secrete increased levels of IL-6 (35). Autoimmunity is ameliorated by IL-6 ablation (36). Importantly, note that IL-6, coordinated with IL-21, has been reported to control the differentiation toward plasma cells (37). More recently, B cell–derived IL-6 was shown to function in an autocrine manner and trigger receptor revision through re-expression of RAG in the post–germinal center response (38).

IL-6 has also important roles in infectious diseases. It has been reported that B cells release IL-6 to promote a follicular helper T cell response to viral infection. B cell–derived IL-6 was necessary and sufficient to induce IL-21 from CD4⁺ T cells in vitro and to support follicular helper T cell development in vivo upon acute influenza virus infection (39). IL-6 is also involved in the induction but not maintenance of plasma cells (40). BANK1-deficient mice have normal T cell–dependent humoral responses following immunization with 4-hydroxy-3-nitrophenylacetyl hapten conjugated to chicken γ globulin (31). One explanation for the unchanged primary humoral response reported by Aiba et al. (31) in Bank1^−/− mice is likely to be the lack of TLR agonist challenge and IL-6 production by B cells in their in vivo system. Therefore, it will be worthwhile to address whether infection with DNA or RNA viruses, instead of 4-hydroxy-3-nitrophenylacetyl hapten conjugated to chicken γ globulin immunization, can initiate TLR9 or TLR7 activation that would probably result in reduced IL-6 production by Bank1^−/− B cells and suppressed germinal center responses. Regarding humoral responses, at this point we are unable to explain the tendency toward increased IgG2a/2c production induced by CpG, and we do not know whether this bears any relationship with the increased serum IgG2a observed by Aiba et al. (31).

BANK1 has been genetically associated with SLE and other autoimmune diseases, and in the present study we observe that BANK1 deficiency decreased IL-6 secretion by altering the translation initiation pathway upon CpG stimulation. Our results therefore suggest that BANK1 could be involved in controlling disease development through the control of IL-6 secretion. Although there are several mechanisms through which IL-6 production is regulated, it is clear that the production of IL-6 is controlled through a multitude of pathways and through different genes.

BANK1 was found to contain a conformational modular TIR domain at the N terminus, similar to its relative, the molecule BCAP. BCAP is required for TLR-mediated activation of PI3K and AKT in macrophages (4). In contrast, CpG-induced AKT activation is normal in Bank1^−/− B cells (Fig. 5A). The data suggest that BCAP, rather than BANK1, may play a critical role in TLR-mediated activation of AKT, and furthermore, BCAP and CD19 have complementary roles in BCR-mediated PI3K activation (41). Whereas BANK1 appears to mediate AKT activation upon CD40 ligation in B cells (Ref. 31 and Fig. 5B), our results support a specific role for BANK1 in transducing CpG-induced signals via p38/MNK1/2 and the translation initiation factor eIF4E in B cells. Thus, BANK1 and BCAP rather than playing redundant roles appear to have very different ones.

BANK1, with a putative TIR domain, would be prone to bind molecules containing TIR domains and explain the very specific role of BANK1 in p38 signaling and translation initiation. BANK1 is an adaptor molecule with a modular structure. It has up to 23 tyrosines susceptible of phosphorylation, and their substrate could alternate among a variety of molecules, forming specific complexes during CpG-induced activation, different from those occurring following BCR-induced ligation, for instance. One of those complexes induced by CpG could include p38. How BANK1 controls the p38/MNK1/2 pathway downstream of TLR9 is at present not known, but it is a subject of study in our laboratory.

We have described that BANK1 shows an interaction with the Src tyrosine kinase BLK (1), and that BLK serves to promote the interaction between BANK1 and phospholipase Cγ2, a key molecule in signal transduction during BCR ligation (42). This phenomenon is apparently not linked to CpG-induced signaling, as we do not observe phosphorylation of BANK1 or changes in binding of BANK1 to BLK following CpG stimulation (data not shown). Nevertheless, we provide in this study the first hints of a role for BANK1 in CpG-induced signaling in the production of IL-6, which could be highly relevant in the study of autoimmunity and inflammation.

We thank Huining Da and Farideh Movafagh for technical assistance.

There is a patent application in process for M.E.A.-R., Y.-Y.W., and R.K. The other authors have no financial conflicts of interest.