1. Division of Immunology & Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, 405 Life Sciences Addition, Berkeley, California 94720-3200, USA
2. Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, NRB-7, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA
3. Department of Medicine, and The Cardiovascular Research Institute, University of California, San Francisco, Box 0111, San Francisco, California 94143, USA

Correspondence to: Gregory M. Barton¹ Correspondence and requests for materials should be addressed to G.M.B. (Email: barton@berkeley.edu).

The subcellular localization of TLR9 and TLR7 is highly regulated. Although ligand recognition occurs in endolysosomes, it has been reported that most, if not all, TLR9 resides in the endoplasmic reticulum (ER) of resting cells^{5, 6}. Curiously, analyses of N-linked glycosylation on TLR9 indicates that the protein does not travel through the Golgi, even in activated cells^{5, 6}. These observations have led to a model in which TLR9 is recruited directly to endolysosomes from the ER, bypassing the classical secretory pathway⁵. However, general mechanistic details accounting for such unusual cell biology are lacking, and the specific details of how TLR9 transport is regulated between these compartments remain largely unknown.

To separate ER-resident TLR9 from receptors in the endolysosome, we purified phagosomes from cells fed latex beads. To track TLR9, we used macrophage-like RAW264 cells stably expressing TLR9 with a carboxy-terminal haemagglutinin (HA) tag (RAW-TLR9 cells). We have previously shown that this epitope tag does not affect TLR9 function⁴. As expected, phagosome preparations were enriched for the lysosomal protein LAMP1 whereas they were devoid of markers of other intracellular organelles and the cytosol (Fig. 1a). Surprisingly, we did not observe any TLR9 at the expected molecular mass (150 kDa). Instead, an approximately 80 kDa protein was highly enriched in the phagosomal preparation (Fig. 1a). The presence of ligand did not alter receptor translocation, as we could not detect full-length TLR9 in phagosomes containing latex beads coated with CpG oligonucleotides (Fig. 1b), despite the fact that these beads induced TLR9 signalling (Supplementary Fig. 1a). In contrast, the 80 kDa protein was evident in phagosomes at the earliest time point examined (10 min) and remained equally abundant over 4 h (Fig. 1b).

Figure 1: A truncated version of TLR9 is exclusively present in phagosomes.

a, Lysates or phagosome preparations from RAW-TLR9 cells fed latex beads were separated by SDS–PAGE and probed with antibodies specific for the indicated proteins. Full-length TLR9 (FL TLR9) and the truncated protein (cleaved TLR9) are indicated. b, Analysis of phagosomes from RAW-TLR9 cells fed unconjugated latex beads or beads conjugated to stimulatory CpG oligonucleotides. Lysates or purified phagosomes from the cells were separated by SDS–PAGE and probed with antibodies against the indicated proteins. c, The truncated form of TLR9 is a product of the full-length protein. Pulse–chase analysis of RAW-TLR9 cells or control cells (RAW). An asterisk indicates the high-molecular-mass full-length TLR9 band. NS indicates a nonspecific band present in both RAW-TLR9 and control RAW cells. d, Modelled structure of the truncated ectodomain (residues 477–790) of TLR9. Leucine-rich repeats (LRR) 15–26 are labelled. e, The TLR9 cleavage product is present in multiple cell types. Haematopoietic stem cells were transduced with a retrovirus encoding TLR9–HA and then differentiated into macrophages with M-CSF (MØ), dendritic cells with GM-CSF (cDC), or plasmacytoid dendritic cells with Flt3L (pDC). f, Lysates from RAW cells expressing TLR4–HA or TLR7–HA were probed with anti-HA antibody. Note that TLR4 is not cleaved. The upper band of TLR4 is due to glycosylation. Unless indicated otherwise, the data presented in each panel are representative of at least three independent experiments.

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To demonstrate directly that the 80 kDa band in our phagosome preparations represented a cleaved form of TLR9, we performed pulse–chase analysis. The 80 kDa band was first detectable after 3 h of chase, concomitant with the onset of full-length receptor degradation (Fig. 1c). The 80 kDa band continued to accumulate in parallel with the degradation of full-length TLR9, peaking at 6 h when the full-length protein was no longer detectable. These data formally demonstrate that the 80 kDa protein is a cleavage product of full-length TLR9 consisting of approximately half of the ectodomain, the transmembrane domain and the entire cytoplasmic domain. On the basis of molecular mass, we estimate that cleavage occurs within or distal to leucine-rich repeat (LRR) 14. Structural modelling of TLR9 suggests that the cleaved receptor adopts an abbreviated 'horseshoe' fold encompassing a region that corresponds to residues implicated in ligand binding by TLR3 (refs 7, 8) (Fig. 1d). A C-terminal cleavage product of TLR9 was also present in macrophage colony-stimulating factor (M-CSF)-derived macrophages, granulocyte–macrophage (GM)-CSF-derived dendritic cells, and Flt3L-derived plasmacytoid dendritic cells, indicating that similar processing events occur in many immunologically relevant cell types (Fig. 1e). We observed a similar cleavage product of TLR7, but not of TLR4, indicating that receptor processing may be a general feature shared by nucleic-acid-sensing TLRs (Fig. 1f).

We next sought to determine where within the cell TLR9 cleavage occurs. Although N-linked sugars on full-length TLR9 were sensitive to endoglycosidase H (EndoH), most of the sugars on the cleaved form of TLR9 were resistant to EndoH, indicating that the truncated form of the receptor has passed through the Golgi (Fig. 2a). A similar glycosylation pattern was observed for TLR7 (Supplementary Fig. 1b). In addition, a small fraction of full-length TLR9 consistently migrated slightly slower than most of the full-length protein (Fig. 2b, see asterisk). Notably, this high-molecular-mass band was almost entirely EndoH resistant, in contrast to the rest of the full-length TLR9 protein, indicating that this small pool of full-length protein has also passed through the Golgi. We also observed this high-molecular-mass, EndoH-resistant band in pulse–chase experiments (asterisks in Fig. 1c and Supplementary Fig. 1c). The band appeared after 1.5 h of chase, immediately preceding formation of the cleavage product. Furthermore, the intensity of the high-molecular-mass band appeared to match the intensity of the accumulated cleavage product. Collectively, these data suggest that this high-molecular-mass band represents the small fraction of TLR9 that exits the ER, passes through the Golgi, and is cleaved. In support of this model, cells treated with brefeldin A (BFA), a reagent that disrupts the Golgi, contained neither the high-molecular-mass band (1.5-h chase) nor the 80-kDa cleavage product (5-h chase) (Fig. 2c). Analysis of TLR9 localization by immunofluorescence microscopy corroborated our biochemical findings (Supplementary Figs 2 and 3).

Figure 2: TLR9 exits the ER and travels through the Golgi before cleavage.

a, Cleaved TLR9 is EndoH-resistant. Proteins from lysates or purified phagosomes from RAW-TLR9 cells were immunoprecipitated with anti-HA resin and treated with EndoH (E), PNGaseF (P), or left untreated (-). b, A longer exposure of TLR9 immunoprecipitated from RAW-TLR9 cells and subjected to analysis as described in a. An asterisk indicates the high-molecular-mass full-length TLR9 band that is EndoH-resistant. c, TLR9 cleavage requires passage through the Golgi. Pulse–chase analysis was performed, at the indicated chase time points, on RAW-TLR9 cells treated with brefeldin A (B) or vehicle (-). The data presented in each panel are representative of at least three experiments.

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To address the mechanism responsible for TLR9 sorting from the ER, we examined UNC93B1 (refs 9–11). Mice with a deficiency in UNC93B1 no longer respond to TLR3, TLR7 and TLR9 ligands¹¹, and UNC93B1 can associate with TLR3, TLR7 and TLR9 which may facilitate transport to endolysosomes^{9, 10}. We generated RAW cells in which Unc93b1 mRNA was knocked down by retrovirally encoded short hairpin RNA (shRNA) (Fig. 3a). As expected, these cells exhibited reduced TNF- production in response to CpG and R848 relative to control cells (Fig. 3b). Notably, the TLR9 cleavage product was greatly reduced in UNC93B1 knockdown cells, although full-length TLR9 was expressed at levels comparable to control cells (Fig. 3c). The high molecular mass, full-length TLR9 band was also absent in UNC93B1 knockdown cells (Fig. 3c), and TLR9 was not present in phagosomes purified from UNC93B1 knockdown cells (Fig. 3d). These data demonstrate that UNC93B1 is required for TLR9 exit from the ER. To test whether UNC93B1 is also sufficient for this exit, we overexpressed UNC93B1 in mouse embryonic fibroblasts (MEFs), which signal poorly in response to CpG oligonucleotides. Whereas we detected very little TLR9 cleavage in control cells, simply overexpressing UNC93B1 in MEFs resulted in a 75-fold increase in the 80-kDa band (Fig. 3e). The increase in cleaved TLR9 corresponded with a robust increase in responsiveness to CpG oligonucleotides (Fig. 3f). Thus, UNC93B1 seems to control the functional pool of TLR9 by regulating exit from the ER. Furthermore, the fraction of TLR9 that exits the ER and is cleaved seems to be responsible for TLR9 signalling.

Figure 3: UNC93B1 is required for TLR9 ER exit and cleavage.

a, Expression of Unc93b1 transcript was measured by quantitative RT–PCR in RAW cells transduced with a control retrovirus or a retrovirus encoding an shRNA specific for Unc93b1. b, Unc93b1 shRNA RAW cells respond poorly to TLR7 and TLR9 ligands but not TLR4 ligands. RAW cells or Unc93b1 shRNA RAW cells were stimulated with the indicated ligands and TNF- production was measured by ELISA (representative of two experiments). LPS, lipopolysaccharide; Unst., unstimulated. c, UNC93B1 is required for TLR9 cleavage. Lysates from control RAW-TLR9 cells (vector) or RAW-TLR9 cells transduced with the Unc93b1 shRNA retrovirus were probed with anti-HA antibodies or anti-tubulin antibodies. d, Lysates or purified phagosomes from the same cells described in c were analysed for the presence of TLR9 by anti-HA immunoblot. e, Overexpression of UNC93B1 in MEFs enhances TLR9 cleavage. TLR9 in lysates of MEFs stably expressing TLR9–HA or TLR9–HA with UNC93B1 was detected by anti-HA immunoblot. RAW-TLR9 cells were included as a control for TLR9 cleavage product. f, Cells described in e were stimulated with 5 M CpG or 25 ng ml^-1 recombinant mouse IL-1 or left unstimulated (US). CXCL1/KC (KC) production was measured by ELISA (representative of two experiments). Unless noted otherwise, all data presented are representative of at least three experiments. Error bars represent standard error.

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We next attempted to block TLR9 cleavage by inhibiting various cellular proteases. Bafilomycin A1, an inhibitor of the vacuolar ATPase, blocks endolysosomal acidification as well as signalling by TLR9, TLR7 and TLR3 (ref. 12). Pulse–chase analysis of RAW-TLR9 cells treated with bafilomycin A1 revealed that TLR9 cleavage was completely blocked when endolysosomal acidification was inhibited (Fig. 4a). Blocking TLR9 proteolysis with baflomycin A1 also resulted in a 20-fold accumulation of the high-molecular-mass TLR9 species, further supporting the idea that this band is the precursor to the cleavage product.

Figure 4: Processing of TLR9 is necessary to generate a functional receptor.

a, TLR9 cleavage requires the activity of acid-dependent proteases. Pulse–chase analysis was performed on RAW-TLR9 cells treated with 50 nM bafilomycin A1 (Baf) or DMSO (-) vehicle control. b, In vitro proteolysis assay performed on TLR9–HA immunoprecipitated from 293T cells. TLR9–HA was incubated with recombinant CTSK, CTSS or no cathepsin (–) and detected by anti-HA immunoblot. c, The cleaved form of TLR9 can bind ligand. Left: whole-cell lysates or lysates of purified phagosomes were separated by SDS–PAGE and immunoblotted with anti-HA antibody. Right: lysates of purified phagosomes were incubated with (+) or without (–) 0.2 M biotin-CpG followed by streptavidin precipitation. TLR9–HA was detected by anti-HA immunoblot. Data are representative of two experiments. d, Cell lysates were incubated with (+) or without (–) biotin-CpG and precipitated with streptavidin (SA) as described in c. Where indicated, unconjugated CpG was also added, before precipitation. Unmanipulated lysate (No IP) is shown as a control. e, Cleaved TLR9 recruits MyD88. RAW cells expressing TLR9–HA and Flag–MyD88 were stimulated with CpG oligonucleotides followed by lysis at the indicated time points. After lysis, MyD88 was immunoprecipitated and TLR9 or MyD88 were detected by anti-HA or anti-Flag immunoblot, respectively. f, TLR9 does not signal from the cell surface in UNC93B1–Ist2 cells. 293T cells were transiently transfected with an NF-B luciferase reporter together with the indicated expression plasmids. Luciferase production was assayed 12 h after stimulation with 3 M CpG, with or without pre-treatment with 50 M chloroquine (CHQ). g, TLR9, TLR9–Ist2 and TLR9–Ist2(18) expressed in RAW cells were analysed for sensitivity to EndoH or PNGaseF as described in Fig. 2a. h, 293T cells were transiently transfected with the indicated expression plasmids and NF-B activation was measured as described in f. i, Schematic of compartmentalized proteolysis and regulation of TLR9 and TLR7. For the indicated statistical comparisons, asterisk indicates P < 0.05; double asterisk indicates P < 0.1. Unless noted otherwise, all data presented are representative of at least three independent experiments.

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Two recent reports have shown that inhibitors of cathepsin K (CTSK) and cathepsin B (CTSB) block TLR9 signal transduction in dendritic cells and macrophages, respectively^{13, 14}. To address whether specific cathepsins may be involved in TLR9 processing, we tested whether recombinant cathepsins could cleave TLR9 in vitro. We observed that recombinant CTSK and CTSS could cleave full-length TLR9 in vitro to generate an approximately 80-kDa cleavage product (Fig. 4b). Although these experiments implicate cathepsin family members in TLR9 processing, we were unable to block processing using cathepsin inhibitors. Pulse–chase analysis showed that the cleaved form of TLR9 was still present in RAW-TLR9 cells treated with the broad spectrum inhibitors E64d, pepstatin A and leupeptin as well as with specific inhibitors of CTSK, CTSB and CTSS (Supplementary Fig. 4). With the exception of a CTSB inhibitor, these compounds did not affect TLR9 signalling (Supplementary Fig. 4 and data not shown). Finally, we observed no defect in TLR9 signalling in macrophages and dendritic cells from CTSS-, CTSS/CTSL-, CTSK- and CTSB-deficient mice (Supplementary Fig. 5). Notably, these results disagree with recent work implicating CTSK and CTSB in TLR9 function^{13, 14}, although these studies relied heavily on protease inhibitors that may have off-target effects. Instead, our data suggest that none of these proteases is solely responsible for cleaving TLR9 and are consistent with an interpretation that multiple proteases are capable of processing the receptor.

The results described above strongly implicate proteolysis as a step required for TLR9 function, but they do not directly demonstrate that processed TLR9 is a functional receptor. To address this possibility, we tested whether the cleaved form of TLR9 can bind stimulatory CpG oligonucleotides. We found that TLR9 purified from phagosomes and incubated with biotinylated-CpG oligonucleotides could be efficiently precipitated using streptavidin beads (Fig. 4c). Thus, this processed form of TLR9, which is exclusively in the phagosome, is capable of recognizing ligand. We next compared CpG binding between the full-length and cleaved forms of TLR9. The truncated receptor was pulled down more efficiently than the full-length receptor, suggesting that it may have a higher affinity for ligand (Fig. 4d). This interaction was specific, as binding was disrupted by addition of non-biotinylated CpG oligonucleotide (Fig. 4d).

Because both full-length and processed TLR9 can bind ligand, we sought to determine which form is responsible for signal transduction. The adaptor MyD88 associates with TLR9 after activation, so we examined which form of the receptor recruits MyD88 in response to CpG stimulation. Remarkably, immunoprecipitation of MyD88 in activated cells pulled down only the processed form of TLR9 (Fig. 4e). We were unable to detect any association between MyD88 and the full-length receptor, demonstrating that the processed form of TLR9 is the functional form of the receptor. We next hypothesized that requiring proteolysis may reduce the likelihood that functional receptors will leak to the cell surface and encounter self nucleic acids. To test this possibility, we used a recently reported strategy of fusing UCN93B1 to a yeast protein domain, Ist2, that directs transport of both UNC93B1 and TLR9 to the plasma membrane¹⁰. Whereas UNC93B1–Ist2 localized to the plasma membrane (Supplementary Fig. 6a), we did not observe significantly altered TLR9 processing or signalling in UNC93B1–Ist2 expressing cells (Fig. 4f and Supplementary Fig. 6b). These results suggest that a significant portion of TLR9 may avoid relocalization in UNC93B1–Ist2 cells and signal from internal compartments. Thus, we fused TLR9 directly to the Ist2 secretion sequence and examined the signalling and localization of this fusion protein. Consistent with relocalization of the receptor to the cell surface, EndoH analysis indicated that the TLR9–Ist2 fusion protein passed through the Golgi but remained unprocessed (Fig. 4g). Remarkably, TLR9–Ist2 no longer responded to CpG oligonucleotides (Fig. 4h). To ensure that this lack of signalling was not due to the Ist2 domain simply interfering with the TLR9 TIR domain, we generated a TLR9–Ist2 fusion protein in which the key amino acids necessary for plasma membrane targeting have been deleted (TLR9–Ist2(18))¹⁵. This receptor was processed (Fig. 4g) and regained the ability to respond to CpG oligonucleotides (Fig. 4h), despite the presence of the non-functional Ist2 domain at its C terminus.

Our results reveal that compartmentalized proteolysis of TLR9 represents a key step in nucleic acid recognition. Although both full-length and processed forms of the receptor can bind ligand, only the cleaved form of TLR9 is competent for signal transduction. It remains unclear why processing is necessary for TLR9 activation. In a previous study we showed that a surface-localized chimaeric receptor TLR9N4C (consisting of the TLR9 ectodomain and TLR4 transmembrane and cytosolic domains) was hyper-responsive to self DNA⁴. Unlike TLR9–Ist2, this receptor signals from the cell surface even when fused to Ist2 but is not processed (Fig. 4h right panel and ref. 4). Swapping the transmembrane and cytosolic domains of TLR9 for those of TLR4 has apparently released this receptor from the processing requirement. Recent work suggests that TLR9 undergoes a membrane-proximal conformational change on ligand binding and activation¹⁶. One possibility is that TLR9 cleavage is a prerequisite for this conformational change. This regulatory mechanism may not be required for cell surface receptors like TLR4, explaining the ability of full-length TLR9N4C to signal. Analyses comparing the structure and conformational changes of the full-length and truncated TLR9 receptors will be necessary to resolve these possibilities.

The proteolytic regulatory step described here is consistent with a model in which TLRs involved in nucleic acid sensing are translated as 'pro-receptors' in the ER and only function after being processed in the endolysosomal compartment (Fig. 4i). This scenario is conceptually analogous to the production of proteases as pro-enzymes that must be processed before they gain enzymatic activity. In this way, compartmentalized processing of TLR9 may regulate where within the cell TLR9 activation can occur. We and others have argued that restricting activation of TLR9 and TLR7 to intracellular compartments may limit responses to self nucleic acids while preserving the ability to recognize foreign nucleic acid^{4, 17, 18}. Thus, the requirement for processing of TLR9 and TLR7 may represent an evolutionary strategy to ensure proper self/non-self discrimination based on nucleic acid recognition.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

We thank members of the Barton and Vance laboratories for discussions, S. Akira for providing TLR knockout mice, and R. Vance and R. Medzhitov for advice. We acknowledge the CHPS Imaging Core and P. Herzmark for assistance with microscopy. This work was supported by a Novel Research Grant from The Lupus Research Institute (G.M.B.), the Hellman Faculty Fund (G.M.B.), and NIH grants HL67204 (H.A.C.), CA009179 (S.E.E.) and AI072429 (G.M.B.).

Author Contributions S.E.E. and G.M.B. planned experiments and wrote the manuscript. S.E.E., B.L.L., L.L. and K.E.W. performed the experiments. G.-P.S. and H.A.C. contributed reagents. All authors read and commented on the manuscript.

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