1. Cells and mice
Murine RAW264.7 macrophages were obtained from the German strain collection (DSMZ, Braunschweig, Germany), Human embryonic kidney (HEK293)/murine (m)TLR2 and HEK293/mTLR4-MD-2 lines were established by stable transfection of mTLR2 and mMD-2 expression plasmids obtained from H. Heine (Research Center Borstel, Germany), D. Golenbock (University of Massachusetts Medical School, USA) and K. Myake (Institute of Medical Science, Tokyo, Japan). The mTLR4 coding sequence was reamplified from a RAW264.7 cDNA library. Peritoneal and bone marrow derived primary murine macrophages, as well as splenocytes were prepared as described and cultured for analysis without or after challenge with TLR ligands . Macrophages and splenocytes were cultured in 50 μM Mercaptoethanol, 10% heat inactivated FCS, pen/strep as antibiotics at standard concentration in an incubator under regular cell culture conditions.
Adult matched C57BL/6 wild-type and TLR2-/- mice  were used for experiments. The mice experiments followed internationally recognized guidelines and were approved by the Government of Upper Bavaria.
2. αT2ib assembly
Total RNA of the hybridoma T2.5 was isolated using the RNeasy Mini Kit (Qiagen; Hilden, Germany) and the cDNA synthesized using a "First-strand cDNA synthesis kit" (GE Healthcare, England). DNA fragments encoding the variable domains of the heavy and the light chain of T2.5 were amplified using consensus primer , for the variable domain of the heavy chain VH1Back-1: 5'-CAG GTS MAR CTG CAG SAG TCW GG (S = G or C, M = A or C, R = A or G, W = A or T) and VH1FOR-2: 5'-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC. The variable domain of the light chain was amplified applying VK2Back: 5'-GAC ATT GAG CTC ACC CAG TCT CCA and MJK1FONX: 5'-CCG TTT GAT TTC CAG CTT GGT GCC. The PCR products of the heavy chain and light chain were purified from an 1% agarose gel and directly used for assembly of the variable domain of the heavy and light chain with a linker sequence that creates a 15 amino acid sequence (Gly4Ser)3 between both domains. The linker was generated from the scFv A7 isolated by phage display  by a PCR with the primer LINK BACK: 5'-GGC ACC ACG GTC ACC GTC TCC TCA and LINK FOR 5'-TGG AGA CTG AGT GAG CTC GAT GTC. The assembly PCR was performed in two steps. In the first reaction equimolar amounts of the DNA of the VH and VL domain and the synthetic linker were incubated with 25 pmol of the primer VH1 Back and MJK1FONX in a PCR of 50 μl containing 1.0 mM dNTPs, 2.5 mM MgCl2 (Qiagen), 5 μl 10 × PCR buffer (Qiagen) and 1 μl (1 U) Taq Polymerase (Qiagen). PCR amplification was performed by incubation for 20 cycles at 94°C for 1.5 min and 65°C for 3 min.
In a second step 25 pmol of the VH1Back primer containing a Sal I restriction site and 25 pmol of the MJK1FONX primer containing a Not I restriction site was added and the volume filled up with dNTPs (0.4 mM), 5 μl 10 × PCR buffer, H2O and 1 μl Taq Polymerase (1 U) to 100 μl. Incubation was performed for 30 cycles at 94°C for 1 min, 55°C for 2 min and 72°C for 2 min. This results in a complete scFv intrabody gene.
After purification of the assembled scFv from a 1% agarose gel the PCR product was cloned into linearised pCR2.1 vector containing 3' T overhangs (Invitrogen, Karlsruhe, Germany). The ligated DNA was transformed into E. coli Top 10 and after blue-white screening, positive clones were restricted with Sal I and Not I and respective inserts ligated into Sal I and Not I cleavage sites upon dephosphorylation with Shrimp alkaline phosphatase (USB Corporation, Cleveland, USA) of pCMV/myc/ER vector (Invitrogen). This vector encodes an ER signal peptide, contains a multicloning site (Pst I, Sal I, Xho I, Not I) as well as encodes a myc epitope and the ER retention signal SEKDEL. After cloning sequences encoding both a myc tag and an ER retention motif were fused 3'-terminally to the preceding anti-TLR2 scFv fragment. This plasmid drove expression of the scFv- myc -SEKDEL ER retention signal construct, which was identical to αT2ib.
3. Construction of an adenoviral vector for αT2ib transduction, production of recombinant virus, and infection in vitro and in vivo
Constructing an adenoviral vector carrying a bicystronic expression cassette driving expression of ER intrabody αT2ib and the reporter gene EGFP a strategy comprising two cloning steps was pursued. In the first step the intrabody expression cassette was cloned into the vector pGEM/IRES/EGFP  containing the IRES sequence of the poliovirus followed by the reporter gene EGFP. In a second step the bicystronic expression cassette containing the CMV promotor, the αT2ib coding sequence, the IRES sequence and the reporter gene EGFP was ligated into the adenoviral cosmid vector pAdcos45EGFPC1 .
First the plasmid pCMV/myc/ ER containing the anti-TLR2 ER intrabody gene (αT2ib) coding sequence was linearised using Eco RI. To generate a Sfi I restriction site the linearised vector was ligated with the oligonucleotid LINKER ADENO: 5'-AAT TGC GGC CGC CAT GGC C GC (Sfi I restriction site is marked bold). 5 μg of the linker containing the Sfi I restriction site was ligated with 1 μg of the linearised vector using 4.5 Units T4 DNA Ligase (Promega, Germany) overnight at 20°C in a volume of 15 μl. Excess of the fragment was removed by dialysis of the sample against 10 mM Tris-HCL, 0.1 mM EDTA and the ligated DNA was transformed into E. coli DH5α. From a positive clone (pCMV/myc/ER/αT2ib/Sfi I) the αT2ib gene with the CMV promoter was cut out by restriction with Sfi I and Xba I and ligated into dephosphorylated pGEM/IRES/EGFP restricted with Sfi I and Avr II. After transformation into E. coli DH5α positive clones (pGEM/αT2ib/IRES/EGFP/Sfi I) containing the bycistronic expression cassette of the αT2ib gene and the reporter gene EGFP were used for the second step. pGEM/αT2ib/IRES/EGFP/Sfi I was restricted with Pvu II and Xba I and the bicystronic expression cassette encompassing the CMV promotor was ligated into the adenoviral vector pAdcos45 EGFPC1 from which the EGFP gene with the CMV promoter was removed by restriction with Swa I and Xba I. The adenoviral vector pAdcos45 EGFPC1 contains the genome of a replication deficient adenovirus type 5 subgenus C in which the E1 and E3 region is deleted . The adenoviral vector contains the reporter gene EGFP. Its expression is driven by the immediate early CMV promoter.
After ligation the adenoviral cosmid DNA was packaged in vitro and transduced into E. coli. Two independent cosmid clones, namely AdVαT2ib/3 and AdVαT2ib/7 comprising identical sequences, were used for the generation of recombinant adenovirus. For production of AdVαT2ib particles HEK293 cells were transfected with 10 μg cosmid DNA in 80cm2cell culture flasks grown to 60% - 80% confluence. After 7-14 days viral plaques were visible. The recombinant adenovirus was propagated as described  and purified by CsCl gradient ultracentrifugation. For infection with the recombinant adenovirus RAW264.7 and primary peritoneal macrophages were cultured until 80% confluence was reached. Infection was carried out at a multiplicity of infection (moi) of 10 for the control virus AdVGFP which overexpressed EGFP but not an additional protein and at a moi of 100 for AdVαT2ib with infection buffer (PBS containing 2% FCS) for 1 h at room temperature since it resulted in equal EGFP expression levels. After infection MEM containing 5% FCS was added and the cells were incubated for 1 day to 3 days after which expression of the reporter EGFP was analysed by measurement of immunofluorescence and flow cytometry. Mice were infected by intravenous (i. v.) or intraperitoneal (i. p.) injection of 1 × 109 plaque forming units (pfu) of AdVαT2ib or control virus (AdVGFP) and analysed 6 days thereafter upon removal of cells by assaying ex vivo/invitro using ELISA.
4. Immunoblot and immunofluorescence analysis
Transiently intrabody expression plasmid transfected HEK293 cells and with recombinant adenovirus transduced RAW264.7 macrophages were lysed by incubation in lysis buffer (150 mM NaCl, 20 mM Tris/HCl pH7.4, 1% Triton-X-100, 10 mM EDTA, 100 μM vanadate, 1% Trasylol, 1 mM PMSF, 1 mM zinc acetate) for 20 min on ice. Lysates were centrifuged for 15 min in a table top centrifuge and a supernatant aliquot representing 5 × 105 cells was loaded on a lane of a PAA gel (12.5%) for subsequent SDS-PAGE. After blotting the membrane was blocked in 2.5% skimmed milk in PBS containing 0.05% Tween 20 at room temperature for 1 h. The intrabody band was visualized by using a mouse anti-myc antibody (Santa Cruz Biotechnology, Heidelberg, Germany) and a goat anti-mouse IgG Fcγ antibody coupled with horseradish peroxidase (Dianova, Hamburg, Germany). The blot was developed using 3,3'-diaminobenzidine tetrahydrochloride (DAB) liquid substrate (Sigma, Deisenhofen, Germany). For immunofluorescence based expression assay by microscopy or FACS the reporter gene product EGFP was visualized using UV light and a FITC filter. For analysis of intrabody expression cells were fixed with 3.7% formaldehyde (15 min at room temperature), washed 3 times with PBS and further permeabilized with 0.1% Triton X-100 for 10 min. After washing with PBS for 5 times, cells were blocked with PBS containing 3% BSA for 1 h at room temperature. Antibody incubation was performed with mouse anti- myc antibody and a TRITC labeled goat anti-mouse IgG antibody (Dianova).
5. NF-κB dependent luciferase assay in HEK293 cells overexpressing both specific TLRs and αT2ib
3 × 104 human embryonic kidney (HEK293) fibroblastoid cells were seeded per well of a 96-well cell culture plate and transfected with plasmids directing constitutive expression of mTLR2 or other TLRs, αT2ib or control intrabody (anti-VEGFR 2 intrabody scFv A7, αVR-ib , and Renilla luciferase, as well as NF-κB dependent expression of firefly luciferase. After 24 h cells were challenged with TLR agonists E. coli O111:B4 LPS (Sigma), tripalmitoylated hexapeptide (Pam3CSK4, EMC microcollections), poly I:C (Sigma), or oligodeoxynucleotide (1668, TIB Molbiol) for additional 16 h and lysed subsequently for analysis of luciferase activity.
6. Subcellular colocalisation and co-immunoprecipitation of αT2ib and mouse/human TLR2
For colocalisation HEK293 cells overexpressing mouse TLR2 were grown on sterile coverslips and transiently transfected with αT2ib expression plasmid. The cells were washed once with PBS-0.05% Tween 20, fixed for 10 minutes with 4% formaldehyd followed by permeabilisation of the cells with 0.1% Triton X-100 for 10 min at room temperature. After blocking with 3% BSA triple staining of αT2ib, mouse TLR2 and Calnexin was performed with polyclonal anti-mouse TLR2 serum , anti-calnexin antibody (Abcam, Cambridge, UK, clone AF18) and subsequent incubation with a FITC labelled goat anti-myc antibody, Cy3 labelled goat anti-mouse antibody (Dianova) and Cy5 labelled goat anti-rabbit antibody (Dianova). Incubation of antibodies was performed over a period of 1 h at room temperatur. Between incubation steps cells were washed 3 times with PBS-0.05% Tween 20. The coverslips were embedded in Moviol (Merk, Darmstadt, Germany) and analysed with a laser scanning confocal microscope (LSM 510 META, Carl Zeiss). Co-immunoprecipitation was performed as described before . Briefly, each partner of protein pairs to be analysed for their potential to interact in a cellular context were overexpressed as fusion proteins in HEK 293 cells upon transfection by Ca3(PO4)2-DNA precipitation. Specifically, human TLR2 was coupled to an N-terminal Flag-tag while intrabody constructs contained C-terminal myc-tags (see Fig. 1a exemplarily). Subsequently, cells were lysed (0.5% NP40, 150 mM NaCl and further ingredients ) upon which nuclei were removed by centrifugation. Myc-specific antibody (Sigma) and protein G beads (Santa Cruz) were added synchronously and lysates incubated on a roller at 4°C for 16 h. Upon 5 washes with lysis buffer sample buffer was added and samples were subjected to SDS-PAGE and analysed upon blotting using tag-specific antibodies (Sigma).
7. Analysis of TLR2 and TLR4 cell surface expression of transfected HEK293 cells and macrophages
αT2ib expression plasmid transfected HEK293 cells overexpressing mTLR2, as well as AdVαT2ib infected RAW264.7 and primary macrophages derived from bone marrow were stained with murine/human TLR2 specific T2.5 or mTLR4 specific UT41 to determine surface expression of specific TLRs by flow cytometry. Staining with antibodies was performed for 30 min at 4°C in a 96-well microtitre plate (Nunclon™ Surface plate, Nunc) in 100 μl PBS containing 2% FCS (Invitrogen) using a phycoerythrin-labelled anti-mouse TLR2 antibody (clone T2.5, HBT) or a phycoerythrin-labelled anti-mTLR4 antibody (clone UT41, HBT). As isotype control a phycoerythrin labelled mouse IgG1κ (clone P3, HBT) was used. Cells were washed once with PBS containing 2% FCS and resuspended in 300 μl PBS containing 2% FCS and 10 μg/ml propidiumiodide for subsequent analysis using a FACS Calibur™ (Becton Dickinson).
8. Analysis of intracellular TNFα by flow cytometry
2 × 106 RAW267.4 macrophages in one well of a 6-well microtitre plate (uninfected, infected with AdVGFP or AdVαT2ib) in 1 ml medium were stimulated with 100 ng/ml tripalmitoylated hexapeptide Pam3CSK4 (EMC microcollections) or 100 ng/ml LPS (Alexis) for 4 h at room temperature. Fixation, permeabilization, and intracellular TNFα transport inhibition were performed using cytofix/cytoperm™ plus fixation/permeabilization and golgiplug™ protein transport inhibitor (BD Biosciences Pharmingen). Challenge with TLR agonists was performed in the presence of 1 μl golgi stop ™ solution in 1 ml medium. Cells were washed once with PBS containing 2% FCS and resuspended in 250 μl fixation/permeabilization solution and incubated for 20 min at 4°C. After washing the pellet once with Perm/Wash™ buffer Fcγ-receptors were blocked by incubation with anti-CD16/CD32 antibodies (BD Bioscience Pharmingen) for 30 min at 4°C. The cells were washed and stained intracellularly as described above for cell surface staining. Antibodies used were a phycoerythrin-labelled hamster anti-mouse/rat TNFα antibody (clone TN3-19.12) and isotype control phycoerythrin-labelled hamster IgG1 antibody (clone G235-2356, both BD Biosciences, Pharmingen).
9. Analysis of TNFα and IL-6 mRNA accumulation by reverse transcription (RT) and subsequent PCR mediated amplification
After cellular challenge with 100 ng/ml Pam3CSK4 or 100 ng/ml LPS for 4 h cellular RNA was isolated (RNeasy, Qiagen) and cDNA synthesized using 1 μg RNA and a random hexamer primer according to supplier instructions (GE Healthcare). Amplification of murine TNFα and IL-6 mRNA was performed by PCR using the following primers: TNFα reverse: 5'-ATGAGCACAGAAAGCATGATC and TNFα forward: 5'-CACAGAGCAATGACTCCAAAG, as well as IL-6 reverse: 5'-ATGAAGTTCCTCTCTGCAAGA and IL-6 forward: 5'-GGTTTGCCGAGTAGATCTCAA. The PCR was carried out in 20 μl of PCR buffer (1 mMTris-Cl, 10 mM KCL, 2 mM (NH4)2SO4, 35 mM MgCl2, pH 8.0), 2.5 mM dNTPs, 0.5 U of Taq DNA polymerase (Qiagen) and 10 pmol primers (Operon). Hypoxanthine-guanine phosphoribosyl transferase (HPRT) mRNA was amplified as control (HPRT reverse: 5'-TCAACGGGGGACATAAAA, HPRT forward: 5'-ATTCAACTTGCGCTCATCTT).
TNFα and IL-6 concentrations in supernatants were analysed by application of ELISA kits according to supplier instructions (BD Biosciences). Aside of other TLR ligands (see above), a TLR7 specific compound (R848, Alexis) was applied to primary cells.