B-lymphoid cells with attributes of dendritic cells regulate T cells via ...

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Jun 8, 2010 - tinctive B-lymphoid cell type with quintessential T-cell regulatory attributes ..... (2003) CD19 regulates innate immunity by the toll-like receptor.
B-lymphoid cells with attributes of dendritic cells regulate T cells via indoleamine 2,3-dioxygenase Burles A. Johnson IIIa, David J. Kahlera,1, Babak Babana,b, Phillip R. Chandlera,c, Baolin Kanga, Michiko Shimodaa,b, Pandelakis A. Konia,c, Jeanene Pihkalad, Bojan Vilagose, Meinrad Busslingere, David H. Munna,f, and Andrew L. Mellora,c,2 a

Immunotherapy and Cancer Centers, Departments of bPathology and cMedicine, and dFlow Cytometry Core Facility, Medical College of Georgia, Augusta, GA 30912; eResearch Institute of Molecular Pathology, A-1030 Vienna, Austria; and fDepartment of Pediatrics, Medical College of Georgia, Augusta, GA 30912

Edited* by Rafi Ahmed, Emory University, Atlanta, GA, and approved April 27, 2010 (received for review December 14, 2009)

A discrete population of splenocytes with attributes of dendritic cells (DCs) and coexpressing the B-cell marker CD19 is uniquely competent to express the T-cell regulatory enzyme indoleamine 2,3-dioxygenase (IDO) in mice treated with TLR9 ligands (CpGs). Here we show that IDO-competent cells express the B-lineage commitment factor Pax5 and surface immunoglobulins. CD19 ablation abrogated IDO-dependent T-cell suppression by DCs, even though cells with phenotypic attributes matching IDO-competent cells developed normally and expressed IDO in response to interferon γ. Consequently, DCs and regulatory T cells (Tregs) did not acquire T-cell regulatory functions after TLR9 ligation, providing an alternative perspective on the known T-cell regulatory defects of CD19-deficient mice. DCs from B-cell–deficient mice expressed IDO and mediated T-cell suppression after TLR9 ligation, indicating that B-cell attributes were not essential for B-lymphoid IDO-competent cells to regulate T cells. Thus, IDO-competent cells constitute a distinctive B-lymphoid cell type with quintessential T-cell regulatory attributes and phenotypic features of both B cells and DCs. B cells

| T-cell regulation | CD19

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ndoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme expressed in response to interferons (IFNs) at sites of inflammation. IDO inhibitors exacerbated T-cell-mediated pathology in models of tumor growth and autoimmune, infectious, and allergic diseases, and are currently under evaluation as potential tumor vaccine adjuvants in clinical settings (1, 2). By analogy to the tumor-protective effects of IDO, some pathogens may exploit IDO to facilitate persistence and IDO may attenuate vaccine-induced immunity (3–6). Consistent with this paradigm, artificially enhanced IDO activity attenuated graft versus host disease after bone marrow engraftment and prolonged rat lung allograft survival (7– 9). Thus, IDO-dependent T-cell regulation occurs in multiple settings of chronic inflammation relevant to clinical syndromes. Despite the potential benefits of manipulating IDO activity to improve therapy in chronic inflammatory syndromes, little is known about the cellular basis of IDO-mediated T-cell regulation in physiologic settings. Antigen presenting cells (APCs) expressing IDO, such as dendritic cells (DCs), activate regulatory T cells (Tregs) and directly suppress effector T cells at sites of tissue inflammation (10, 11). Cells with functional and phenotypic attributes of DCs in mice and humans are competent to express IDO and regulate T-cell responses (11–15). In mice, splenocytes uniquely competent to mediate T-cell suppression via IDO after in vivo treatment with TLR9 ligands (CpGs) were a rare cell type located in splenic red pulp with phenotypic attributes of DCs (CD11c, CD8α, CD80/86, MHCII). IDO-competent cells also expressed B220 and the B-cell lineage marker CD19, but not the plasmacytoid DC (pDC) marker PDCA/120G8 (13). After CTLA4-Ig and CpG treatment, IDO-competent cells produced IFNα (15, 16), a functional attribute of pDCs and some B cells. Here we show that IDO-competent DCs exhibit phenotypic attributes of B-lineage cells, including Pax5 and surface Ig (sIg) expression. Moreover, cells with phenotypic attributes of IDO-competent cells developed in CD19-deficient mice but were not competent to express IDO, 10644–10648 | PNAS | June 8, 2010 | vol. 107 | no. 23

whereas IDO-competent cells with attributes of DCs but not B cells were present in B-cell–deficient mice. Results IDO-Competent Cells Express the B-Lineage Commitment Factor Pax5.

As reported previously (13), systemic CpG treatment (100 μg, i.v.) induced rapid and selective IDO expression by rare CD19+ cells in red pulp of mouse spleen (Fig. 1). CD19+ B cells located in lymphoid follicles did not express IDO (Fig. 1). Thus, two distinct subtypes of CD19+ cells reside in mouse spleen. CD19 is a downstream target of the B-lineage commitment factor Pax5 (17), and CD19 expression in B-lymphoid cells is dependent on Pax5 function (18). We tested whether IDO-competent cells expressed Pax5 by evaluating Pax5 expression of reporter genes encoding human CD2 (hCD2) and GFP by splenocytes from Pax5ihCD2/ihCD2 and Pax5iGFP/iGFP knockin mice (19). We used gating criteria (Fig. 2A) described previously (13) to distinguish IDO-competent cells—defined phenotypically as CD11chighCD19+ cells (CD19+ DCs)—from myeloid DCs (mDCs), CD11chighCD19neg and B cells (CD11cnegCD19+). CD19+ DCs and B cells from Pax5ihCD2/ihCD2 knockin mice expressed hCD2 at comparable (high) levels; as expected, mDCs from Pax5ihCD2/ihCD2 mice did not express hCD2 (Fig. 2A). Similar results were obtained when GFP expression was analyzed in spleen of Pax5iGFP/iGFP knockin mice (Fig. S1). As Pax5 is a definitive marker of B cells and their progenitors (20), these findings identify IDO-competent cells as a distinct B-lymphoid subtype with attributes of DCs. We hypothesized that Pax5 marks the rare DC subset uniquely competent to mediate T-cell suppression after CpG treatment. We treated Pax5iGFP/iGFP knockin mice with CpGs (100 μg, i.v.) to induce IDO, and 24 h later measured the T-cell stimulatory properties of flow-sorted Pax5+ (GFP+) and Pax5neg (GFPneg) CD11c+ DCs by culturing sorted DCs with responder (H-2Kbspecific) T cells from BM3 TCR transgenic mice with or without the IDO inhibitor 1-methyl-[D]-tryptophan (D-1MT) (13). Sorted Pax5+ (GFP+) DCs did not stimulate T-cell proliferation unless IDO inhibitor was present (Fig. 2B). In the absence of IDO inhibitor, sorted Pax5neg (GFPneg) DCs stimulated robust T-cell proliferation, which was not enhanced by adding IDO inhibitor (Fig. 2B). Thus, IDO-competent cells express Pax5.

Author contributions: B.A.J., M.B., D.H.M., and A.L.M. designed research; B.A.J., D.J.K., B.B., P.R.C., B.K., and B.V. performed research; B.B., M.S., P.A.K., J.P., B.V., M.B., D.H.M., and A.L.M. contributed new reagents/analytic tools; B.A.J., D.J.K., B.B., P.R.C., B.K., B.V., M.B., D.H.M., and A.L.M. analyzed data; and B.A.J. and A.L.M. wrote the paper. Conflict of interest statement: D.H.M. and A.L.M. receive consulting income and research support from NewLink Genetics Inc., which has licensed patent rights based on intellectual property emanating from their laboratories. *This Direct Submission article had a prearranged editor. 1

Present address: The New York Stem Cell Foundation, New York, NY 10023.

2

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.0914347107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0914347107

Fig. 1. IDO-competent cells are a distinct subset of CD19+ splenocytes. B6 mice were treated with CpGs (100 μg, i.v.) for 24 h and spleen sections were stained with anti-IDO Ab (A) or to detect cells expressing IDO and CD19 (B–D). Stained cells were visualized using immunohistochemical (A, red) and immunofluorescence (B–D) methods to detect IDO (B, green), CD19 (C, red), and IDO/CD19 (D, merged images). Original magnifications, 25× (A), 400× (B–D). f, follicles; r, red pulp.

Next, we examined the phenotypic attributes of IDO-competent cells. Resting B cells (CD11cnegCD19+; Fig. 3B) and IDO-competent cells (CD11chighCD19+; Fig. 3C) exhibited radically different light-scattering properties, indicating that IDO-competent cells were larger and more granular than B cells. Like B cells, IDO-competent cells expressed uniformly high levels of immunoglobulin (IgM, Igκ, IgD), the complement receptor CD21, and the IgE receptor CD23 (Fig. 3D). As expected, mDCs (CD11chighCD19neg) did not express these markers. Unlike B cells, IDO-competent cells and mDCs expressed CD4, CD5, CD44, CD8α, CD11b, and Flt3 at comparable levels (Fig. 3E and Fig. S2 A, B, and E). Sorted IDO-competent cells from CpGtreated mice also expressed IFNα, whereas mDCs did not (Fig. S2C). Sorted B cells and IDO-competent cells expressed low levels of the helix-loop-helix transcription factor E2-2 (Fig. S2D), which is expressed at much higher levels by PDCA+ pDCs and is essential for pDC development (21). Selective ablation of the Tcf4 gene encoding E2-2 in CD23+ B-lymphoid cells had no effect on development of IDO-competent cells (Fig. S2 F and G). As the Cd23-Cre transgene is first expressed by immature splenic B-lymphoid cells (22), this confirms that IDO-competent cells develop from immature B cells and are unrelated to conventional pDCs. Few cells with phenotypic attributes of IDO-competent cells were present in bone marrow (Fig. S2H). However, IDOexpressing cells in inflamed tumor-draining lymph nodes (LNs), and LNs draining skin exposed to tumor promoters, closely resembled splenic IDO-competent cells (23, 24). We compared the T-cell stimulatory (APC) properties of IDO-competent cells and B cells from untreated B6 mice. Sorted IDO-competent cells stimulated robust BM3 T-cell proliferation, whereas resting B cells did not (Fig. 3F). Thus, like DCs—but unlike resting B cells—IDO-competent cells were potent T-cell APCs when not induced to express IDO, confirming that IDOcompetent cells are a distinct cell subtype with attributes of B cells and DCs. Intact CD19 Is Required for DCs to Express IDO in Response to CpGs.

CD19 amplifies B cell receptor (BCR) signaling and promotes Bcell responses to membrane-bound antigens (25–28). We hypothesized that CD19 is required for IDO induction in DCs. Johnson et al.

Fig. 2. IDO-competent cells express Pax5. (A) Flow cytometric analyses of hCD2 reporter gene expression by splenic B cells and CD11c+ DC subsets from Pax5ihCD2/ihCD2 knockin mice gated as indicated in the dot plot. Numbers are the mean fluorescence intensities of hCD2+ cells in each gated subset. (B) FACS-sorted GFP+ and GFPneg DCs from CpG-treated Pax5iGFP2/iGFP knockin mice were cultured with responder BM3 T cells (±D-1MT), and T-cell proliferation was assessed after 96 h. Data are representative of at least two experiments.

Consistent with this hypothesis, splenic DCs from CpG-treated CD19 knockout (KO) mice stimulated robust T-cell responses ex vivo which were not enhanced by IDO inhibitor (Fig. 4A). Moreover, no IDO-expressing cells were detected in spleens of CpG-treated CD19-KO mice (Fig. S3A). CD19 ablation did not compromise development of cells with phenotypic attributes of IDO-competent cells, as CD11chighB220+ cells—a population encompassing IDO-competent cells (29)—developed normally and expressed CD4 and CD5 in CD19-KO and B6 mice (Fig. S3 B and C). Moreover, sorted CD11c+B220+ cells from CD19-KO and B6 mice expressed IDO when incubated with IFNγ, whereas sorted CD11c+B220neg cells did not express IDO (Fig. 4B). Thus, intact CD19 was essential for TLR9-mediated IDO induction, but CD19 ablation did not impair development of B220+ DCs competent to express IDO in response to IFNγ. Systemic CpG treatment induced rapid Treg activation in IDO-sufficient mice (30). To test whether CD19 ablation compromised Treg activation via IDO, we added Tregs from CpGtreated mice to cultures containing responder CD4+ (H-Yspecific) T cells from A1 TCR transgenic mice, APCs from CBA mice, and H-Y peptide; in this assay system, Tregs (H-2b) are MHC-mismatched with responder T cells and APCs (H-2k) so that Tregs are not activated during culture and suppression must be generated in vivo (30). Up to 10,000 Tregs from CpG-treated CD19-KO mice failed to suppress T-cell proliferation, whereas PNAS | June 8, 2010 | vol. 107 | no. 23 | 10645

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IDO-Competent Cells Exhibit Attributes of B Cells as Well as DCs.

Fig. 4. Defective IDO induction in CD19-deficient mice. (A) Magnetic-activated cell sorted (MACS)-enriched splenic DCs from CpG-treated B6 and CD19-KO mice were cultured with BM3 T cells ± D-1MT as before. (B) Sorted B220+ and B220neg DCs were incubated with IFNγ (200 U/mL, 24 h) and cytospins were stained to detect IDO; original magnifications, 1,000×. (C) Graded numbers of MACS-enriched Tregs from CpG-treated B6 (closed symbols) and CD19-KO (open symbols) mice were cultured with responder T cells and stimulator APCs (72 h) before assessing T-cell proliferation. Data are representative of two or more experiments.

Fig. 3. Phenotypic and functional analyses of IDO-competent cells. (A–E) Splenocytes from untreated B6 mice were stained with CD11c and CD19 (A) and B-cell (D) and non-B-cell markers (E). (A) B cells (CD11cnegCD19+), IDO-competent cells (CD19+CD11chigh; shown in red), and mDCs (CD11chighCD19neg) were gated as indicated. (B and C) Forward and side light scatter (FSC, SSC) properties of gated B cells (B) and IDO-competent cells (C). (D and E) Histograms of markers expressed by gated cells. (F) Graded numbers of sorted B cells and IDO-competent cells from untreated mice were cultured with BM3 responder T cells (96 h) and T-cell proliferation was assessed. Data are representative of two or more experiments.

as few as 1,250 Tregs from CpG-treated B6 mice blocked T-cell proliferation completely (Fig. 4C). Thus, CD19 is essential for splenic DCs to become competent to express IDO and mediate T-cell suppression after TLR9 ligation. IDO-Competent Cells Are Present in Spleens of B-Cell–Deficient Mice.

We tested whether IDO-competent cells were present in B-cell– deficient mice. For this approach, we used B-cell–deficient μMTKO and JH-KO mice (31, 32), in which B-cell development is arrested at the pro-B-cell stage due to defective BCR expression (Fig. 5A). As expected, no splenocytes from JH-KO mice, including gated B220+ DCs, expressed IgM (Fig. S4A). Despite the loss of IgM expression, DCs from CpG-treated μMT-KO and JHKO mice mediated potent IDO-dependent T-cell suppression (Fig. 5 B and C), and IDO-expressing cells from JH-KO mice were indistinguishable from their counterparts in B6 mice (Fig. 5D). Induced IDO1 gene expression in CpG-treated μMT-KO mice segregated with (CD11c+) DCs (Fig. S4B), and induced IDO protein expression segregated with CD8α+ DCs in B6 and μMT10646 | www.pnas.org/cgi/doi/10.1073/pnas.0914347107

KO mice (Fig. S4C). However, B220+ DCs from B6 and JH-KO mice exhibited comparable CD8α staining profiles, indicating that blockade of B-cell development had little impact on development of cells with DC attributes matching those of IDO-competent cells in wild-type mice (Fig. S4A). Thus, IDO-competent cells with the same DC attributes as IDO-competent cells in wild-type mice developed in μMT-KO and JH-KO mice. Thus, attributes critical for B-cell development and function such as surface BCR expression were dispensable for development of IDO-competent cells. Because CD19 is essential for TLR9-mediated IDO induction, we analyzed CD19 expression by splenocytes from CpG-treated μMT-KO mice. No cells expressed CD19 at levels detected in wild-type mice, and CD19 expression was abrogated completely on CD11cneg cells (Fig. S4D). However, CD11c+ cells expressed low levels of CD19, and CD8α expression profiles on CD19lowCD11chigh cells from μMTKO mice were comparable to IDO-competent cells from wild-type mice (Fig. S4D). Discussion In this study, we show that cells competent to express IDO after systemic TLR9 ligand treatment are a unique cell subtype with phenotypic attributes of B cells and DCs. Like B cells, IDOcompetent cells expressed Pax5, IgM/D/κ, CD21, and CD23. Like mDCs, IDO-competent cells expressed CD4, CD5, CD44, CD8α, CD11b, and Flt3 and stimulated robust T-cell responses when not induced to express IDO. Like pDCs, IDO-competent cells expressed IFNα after TLR9 ligation but, unlike pDCs, IDOcompetent cells did not require E2-2 for development. The most striking attribute of IDO-competent cells is their unique ability (among DCs) to suppress T cells and activate Tregs when induced to express IDO (13, 16, 30). Thus, IDO-competent cells are pivotal regulators of T-cell responses that help create and maintain immune privilege at local sites of inflammation (1). IDO-competent cells are easy to overlook, due to their paucity (∼105/spleen), unusual light-scattering properties, and unconventional array of phenotypic markers, making them prone to Johnson et al.

inadvertent inclusion (or exclusion) from populations of B cells, pDCs, and DCs. Moreover, small cohorts of IDO-expressing cells mediate potent T-cell regulation that may predominate over a large excess of T-cell stimulatory DCs. Splenic IDO-competent cells closely resemble IDO-expressing cells in inflamed LNdraining sites of tumor growth and topical tumor promoter application, and these cells have been implicated in tumor progression and resistance to antitumor immunity (23, 24, 33). Pax5 is a definitive B-lineage marker and commitment factor, and Pax5 expression by IDO-competent cells formally identifies them as B-lymphoid cells (20). Selective Tcf4 gene ablation in IDO-competent cells and B cells from mice harboring a Cd23-Cre transgene confirmed that both cell types develop from immature B-cell progenitors (22). Cells with classic features of B cells and DCs can develop from B-cell progenitors (34). Plasmablasts in splenic extrafollicular regions that promote T-independent humoral responses also expressed CD11c, albeit at lower levels than IDO-competent cells (35). However, it is unclear whether CD11c+ B cells were IDO-competent. Recently, Vinay and colleagues described a discrete B-cell subtype expressing the pDC marker PDCA that expressed IFNα and IDO after ex vivo CpG treatment (36). Unlike IDO-competent cells, PDCA+ B cells did not express CD19 uniformly, and it is unclear whether PDCA+ B cells are located in lymphoid follicles or in splenic red pulp, where IDO-competent DCs are found (Fig. 1). Moreover, the ability of PDCA+ B cells to suppress T cells and activate Tregs—quintessential functional attributes of IDO-competent cells—was not evaluated. Previously, we showed that rare CD19+ cells coexpressing CD11c, B220, and CD8α were the only splenocytes competent to regulate T cells when induced to express IDO; in contrast, sorted PDCA+ (120G8+) pDCs were not IDO-competent (13). Moreover, unlike conventional pDCs that express PDCA as an archetypal marker, E2-2 is not required for development of IDO-competent cells (Fig. S2F). Thus, CD19 and CD11c are excellent markers for rare IDOcompetent cells, whereas PDCA is a poor marker for these key functional attributes. IDO-competent cells were not present in CD19-KO mice, although B220+ DCs were present in comparable proportions and had comparable phenotypes in spleens of CD19-KO and wildJohnson et al.

Materials and Methods Mice. Pax5iGFP/iGFP, Pax5ihCD2/ihCD2 (19), CD19cre/cre (38), μMT-KO (31), and JHKO (32) mice were described previously. All procedures were approved by the Medical College of Georgia Institutional Animal Care and Use Committee, and mice were housed in specific pathogen-free facilities. Antibodies and Reagents. 1-Methyl-[D]-tryptophan (Aldrich) was used at a final concentration of 200 μM. Antibodies (BD Pharmingen) for flow cytometry were: IgMb-PE (AF6-78, 553521), Igκ-FITC (550003), IgD-FITC (11-26c.2a, 553439), CD23-PE (B3B4, 553139), CD4-PE (GK1.5), CD5-PE (53-7.3, 553023), CD44-FITC (S7; eBioscience; 11-0441-82), CD16/CD32 (2.4G2, 553142), CD11cAPC (HL3, 550261), CD19-PerCP-Cy5.5 (1D3, 551001), and CD8α (53-6.7, 553033). CD21-PE (7G6) was a gift from J. Kearney (Birmingham, AL). CpG Treatment. CpG oligonucleotides (CpG B 1826 with a fully phosphorothioate backbone) (Coley Pharmaceuticals) were injected (i.v., 100 μg) 24 h before harvesting spleens (13). Analytical Flow Cytometry. Fluorescence-activated cell sorter (FACS) analyses were performed as described (15). Erythrocyte-free cell suspensions were treated with normal mouse serum and rat anti-mouse CD16/CD32 before mAb addition. Cells were analyzed on a FACSCanto system (Becton Dickinson). Splenic DC and T-Cell Isolation. DCs and CD8α+ T cells were prepared using magnetic beads (Miltenyi Biotec) as described (16). T-Cell Proliferation Assay. Mixed lymphocyte cultures (96-h) contained MACSenriched CD11c+ DCs (2.5 × 104) and CD8α+ T cells (5 × 104) from BM3 TCR transgenic mice ± D-1MT as described (16). Thymidine incorporation was measured using a Betaplate scintillation counter (Wallac). Treg Suppression Assay. Treg suppression was assessed as described (30, 33). MACS-enriched Tregs were isolated 24 h after CpG treatment, and graded numbers were added to cultures containing responder A1 (H-Y-specific) T cells, stimulators (splenic APCs) from CBA mice, and 100 nM H-Y peptide. Immunohistochemistry. Spleen sections were stained as described (13, 16).

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Fig. 5. IDO-competent cells develop in B-cell-deficient mice. (A) B-cell development is blocked after the pro-B-cell stage in μMT-KO and JH-KO mice due to defective BCR expression. (B and C) MACS-enriched splenic DCs from CpG-treated mice were cultured with BM3 T cells (96 h, ± D-1MT). Data are representative of two or more experiments. (D) IDO-expressing cells (stained red) in spleen of CpG-treated mice (24 h). Original magnifications, 200× (Top and Middle) and 600× (Bottom).

type mice. The defect in IDO induction caused by CD19 ablation was selective for signaling downstream of TLR9, because signaling via IFNγ—which induces IDO via the Jak/Stat pathway— induced B220+ DCs from CD19-KO mice to express IDO. It is unclear why CD19 is required for IDO induction after TLR9 ligation in vivo. CD19 regulates intracellular TLR signaling in B cells, lowers B-cell activation thresholds, and is required for optimal B-cell responses to membrane-bound antigens (25–28). Thus, CD19 may transduce intracellular signals from TLR9 in IDO-competent cells. CD19 ablation had little impact on conventional B-cell (B2) development, but CD19 ablation impaired regulatory (B-1a) and marginal zone B-cell development (37–40). Consequently, CD19-KO mice exhibited immune regulatory defects, including TH1 hyperresponsiveness leading to contact hypersensitivity, and more severe forms of experimental autoimmune encephalomyelitis (41, 42). The finding that CD19 ablation abrogated TLR9induced suppression by DCs and Tregs provides a unique perspective on the T-cell regulatory defects of CD19-KO mice. IDO-competent cells with DC attributes were present in B-celldeficient μMT-KO and JH-KO mice despite the absence of IgM, indicating that BCR expression—the archetypal feature of mature B cells—was not essential for development of cells competent to express IDO and regulate T cells. Thus, IDO-competent cells develop from immature B-lymphoid progenitors, but BCR is a residual attribute. However, IDO-competent cells may develop from other, non-B-lymphoid progenitors when B-cell development is blocked. The requirement for CD19 is consistent with an essential role for CD19 in intracellular TLR signal transduction in B cells (25). Thus, residual CD19 expressed by IDO-competent cells in B-cell-deficient mice may suffice for intracellular signaling required to induce IDO after TLR9 ligation.

Statistical Methods. Student’s t test was used to evaluate significance (P < 0.05) of differences in mean values of triplicate readouts from suppression assays and quantitative RT-PCR analyses. ACKNOWLEDGMENTS. We thank Doris McCool for providing mice and Doris Cawley for preparing tissue sections. We thank Art Krieg (Coley Pharmaceut-

icals) and NewLink Genetics for gifts of CpGs and D-1MT, respectively. We thank Dan Homberg (Umeå University, Sweden) for providing the conditional Tcf4 (E2-2) mouse. D.H.M. and A.L.M. receive consulting income and research support from NewLink Genetics. This work was supported by National Institutes of Health grants to A.L.M. (AI063402, AI075165) and D.H.M. (CA103320, CA096651, and CA112431) and by Boehringer-Ingelheim (B.V. and M.B.).

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