Aur-A and overexpression rates of Aur-A mRNA were observed in breast cancer, gastric cancer, hepatocellular carcinoma, and ovarian cancer. We found that aggressive head and neck cancers exhibited overexpression and stabilization of Aur-A protein without gene amplification or mRNA overexpression. Here we tested the hypothesis that aberration of the protein destruction system induces accumulation and consequently overexpression of Aur-A in cancer.The serine/threonine kinase Aurora-A (Aur-A) is a proto-oncoprotein overexpressed in a wide range of human cancers. Overexpression of Aur-A is thought to be caused by gene amplification or mRNA overexpression. However, recent evidence revealed that the discrepancies between amplification of Cdh1 and consequently degraded when cells exited mitosis, and phosphorylation of Aur-A on Ser51 was observed during mitosis. Phosphorylation of Aur-A on Ser51 inhibited its APCCdh1-mediated ubiquitylation and consequent degradation. Interestingly, constitutive phosphorylation on Ser51 was observed in head and neck cancer cells with protein overexpression and stabilization. Indeed, phosphorylation on Ser51 was observed in head and neck cancer tissues with Aur-A protein overexpression. Moreover, an Aur-A Ser51 phospho-mimetic mutant displayed stabilization of protein during cell cycle progression and enhanced ability to cell transformation.Aur-A protein was ubiquitinylated by APCBroadly, this study identifies a new mode of Aur-A overexpression in cancer through phosphorylation-dependent inhibition of its proteolysis in addition to gene amplification and mRNA overexpression. We suggest that the inhibition of Aur-A phosphorylation can represent a novel way to decrease Aur-A levels in cancer therapy. Aur-A/STK15 is located at chromosome 20q13.2, which is commonly amplified in various cancers, including breast, colon, bladder, ovarian, pancreatic and head and neck cancers A series of periodic kinase reactions by cyclin-dependent kinases (CDKs) promote the progression of cell cycle Xenopus Aur-A XenopusIt is well established that most cell cycle regulators are degraded by the ubiquitin-proteasome system (UPS) Aur-A gene Cdh1-mediated ubiquitylation and sequential degradation of Aur-A.Previous studies have indicated that the level of Aur-A protein in tumors does not always correlate with amplification of the Overexpression of Aur-A is shown in a wide range of human cancers. By immunohistochemistry, head and neck cancer cells expressed Aur-A at higher levels, in comparison with normal oral epithelial cells . ImportaCdh1-mediated Aur-A degradation. Aur-A has four putative D-Box and one KEN box motifs, which could potentially be recognized by the APCCdh1 ubiquitin ligase complex. In Xenopus, the N-terminus A-box and C-terminus D-box of Aur-A are essential for its degradation Xenopus, the A-box and D-box motifs are essential for the degradation of human Aur-A protein  and then transfected in cells with or without Cdh1 , a paralogue of Aur-A, differs in localization and timing of activation during cell cycle from Aur-A, despite the ∼60% sequence identity between them. Comparison of the schematic structure between Aur-A and -B is shown in out Cdh1 . The schNext, we examined if phosphorylation on Ser51 was involved in regulation of Aur-A expression during cell cycle progression. We raised a phospho-specific antibody against a synthetic peptide that spans the phosphorylated Ser51 residue of Aur-A. This antibody specifically recognized wild type and S51D mutant, but not S51A mutant , indicatIn consideration of the above findings, we hypothesized that Aur-A overexpression in head and neck cancer cells may be caused by stabilization of Aur-A protein through a constitutive phosphorylation on Ser51. Therefore, we examined the status of phosphorylation on Ser51 in head and neck cancer cell lines. Phosphorylation on Ser51 was detected in HSC2, HSC3 and Ho-1-U-1 cells . Interesc 3T3 A31-1-1 cells (Aur-A and G12V-HRAS (T24-ras), and observed that Aur-A potentiated the frequency of G12V-HRAS-induced transformation. Interestingly, a much larger number of foci were found using the S51D mutant, suggesting that constitutive phosphorylation on Ser51 has enhanced oncogenic potentials.In order to further assess the tumorigenesis induced by overexpression of Aur-A protein due to phosphorylation on Ser51, we performed the stability of S51D mutant and cell transformation in comparison with wild type. While the expression of the wild type protein and the S51D mutant is virtually identical in mitotic cells, the S51D mutant was not degraded when cells exited mitosis . In addi-1 cells . We co-tCdh1 at the exit of mitosis Cdh1 ubiquitin ligase complex recognizes proteins containing either D-Box or KEN-box motifs 47RxLxPSN52) were essential for the degradation of human Aur-A, in similar to previous reports Xenopus Ser53 within the A-box is phosphorylated during mitosis and that phosphorylated Ser53 (or 51 in human) is essential for mitotic specific stabilization Cdh1-mediated degradation. As shown in Cdh1Cdh1 . Unfortunately, we could not find the possible correlation between PP2A and Aur-A Ser51 phosphorylation status in cancer. To demonstrate the correlation between PP2A and Aur-A Ser51 phosphorylation status needs further experiments.Aur-A kinase is associated with the centrosome from the time of centrosome duplication through to mitotic exit, and is also associated with regions of microtubules proximal to centrosomes in mitosis dh1Cdh1 , strongldh1Cdh1 . Ser51 pdh1Cdh1 . Therefodh1Cdh1 . MoreoveCdh1. In the case of Aur-A, Ser51 is located far from the D-box, but Ser51 is located in the A-box, which is also essential for ubiquitylation. However, S51D Aur-A mutant as well as wt and S51D mutant can bind to Cdh1  was obtained from Peptide institute inc. . Cycloheximide (CHX), nocodazole (Noc) and okadaic acid (OA) were obtained from Sigma. The Aur-A phospho-Ser51-specific antibody was generated by immunizing rabbits with the synthetic peptide P*SNSSQRIPC, corresponding to amino acids 50–59 of human Aur-A sequence with a phospho-Serine at position 51 (*S). The antibody was purified from serum by two rounds of affinity chromatography on a phospho-Ser51 peptide column followed by a non-phosphopeptide column. The polyclonal antibody to cyclin A has been described previously Tissue samples of head and neck cancer were retrieved from the Surgical Pathology Registry of Hiroshima University Hospital from 1998 to 2004 after their written informed consent. 10% buffered-formalin fixed and paraffin embedded tissues were used for immunohistochemical examination. The histological grade and stage of tumor were classified according to the criteria of the Japan Society for Head and Neck Cancer. For Western blot analysis, 9 head and neck cancer tissues and 1 normal oral mucosal tissue were obtained from patients underwent surgery at Dental hospital, Peradeniya, Sri Lanka after their written informed consent. These tissue specimens were immediately frozen and stored in −80°C. Informed consent was obtained from all patients for this study. Our work was approved by the Ethical Committee of Hiroshima University and Peradeniya University.Immunohistochemical detection of Periostin in head and neck cancer cases was performed on 4.5 µm sections mounted on silicon-coated glass slides, using a streptavidin-biotin peroxidase technique as described previously Aur-A cDNA was isolated from the HeLa cDNA library by RT–PCR using sense and antisense primers. Aur-A cDNA was then subcloned by insertion into the KpnI/XbaI restriction site of pcDNA3 with N-terminal FLAG tagging Human 2 in air at 37°C. HeLa and 293T cells were routinely maintained in Dulbecco's Modified Eagle Medium  supplemented with 10% heat-inactivated fetal bovine serum (Boehringer Mannheim) and 100 U/ml penicillin-streptomycin (Gibco) under conditions of 5% CO2 in air at 37°C. For experiments, they were grown to subconfluence in this medium.Six OSCC cell lines , HeLa and 293T cells were used. All cell lines were provided by Japanese Cancer Research Resources Bank. OSCC cell lines were routinely maintained in RPMI-1640  supplemented with 10% heat-inactivated fetal bovine serum (Boehringer Mannheim) and 100 U/ml penicillin-streptomycin (Gibco) under conditions of 5% CO293T cells and HeLa were transfected with vectors with the use of FuGENE6 (Roche). Cell lysis and immunoprecipitation were performed as described 35S] methionine-labeled human Aur-A protein and were prepared by coupled transcription-translation reactions in rabbit reticulocyte lysate (Promega). Cold in vitro-translated human Cdh1 protein was also used. The extracts from HeLa cells were immunoprecipitated with anti-Cdc27 antibody (Sigma). Immunoprecipitants were incubated with Cdh1 in reaction mixtures contained the following in a volume of 10 µl: 40 mM Tris-HCl (pH 7.6), 1 mg/ml carboxymethyl bovine serum albumin, 1 mM DTT, 5 mM MgCl2, 10 mM phosphocreatine, 50 µg/ml creatine phosphokinase, 0.5 mM ATP, 50 µM ubiquitin, 1 µM ubiquitin aldehyde, 1 pmol of E1, 5 pmol of E2-C, 1 µM okadaic acid, 1-2 pmol of [35S] methionine-labeled human Aur-A protein. Following incubation at 30°C for 1 h, samples were subjected to electrophoresis on a 10% polyacrylamide-SDS gel.[c 3T3 A31-1-1 cells were used in this study. For detection of oncogenes, exponentially growing cells (105) were seeded in 60-mm dishes (3 dishes per experiment), after which the cells were transfected with each expression plasmid using Lipofectamine (Invitrogen). The expression plasmids for FLAG-tagged wt and mutants (S51A and S51D) were used. For the positive control, H-Ras (G12V)-induced transformation, 100 ng of mutated H-Ras plasmid  The transformation target BALB/Table S1Summary of Aur-A expression in head and neck cancer.(0.09 MB TIF)Click here for additional data file.Figure S1Cell cycle-dependent regulation of Aur-A protein by APCCdh1. A: T98G cells were released from serum starvation and collected at the indicated times. Samples were analyzed by SDS-PAGE followed by Western blotting with Aur-A, Aur-B, Cyclin A, Cyclin B, p27, phospho-histone H3 (Ser10) and Cul1 antibodies. Anti-cyclin B antibody was purchased from Transduction Laboratories. B: HeLa cells were released from nocodazole-induced prometaphase arrest and collected at the indicated times. Samples were analyzed by SDS-PAGE followed by Western blotting with Aur-A, Aur-B, Cyclin A, Cyclin B, p27, phospho-histone H3 (Ser10) and Cul1 antibodies. C: Cells at M phase (noc) and G1 phase (noc+7h) were treated with or without proteasome inhibitor, ZLLL/MG132. Samples were analyzed by SDS-PAGE followed by Western blotting with Aur-A, Aur-B, Cyclin A, phospho-histone H3 (Ser10) and Cul1 antibodies.(0.15 MB TIF)Click here for additional data file.Figure S2Correlation between the expression of PP2A and Aur-A Ser51 phosphorylation status. Expression of PP2A  is examined by Western blot analysis. We used an anti-PP2A  monoclonal antibody (Transduction Laboratories). Ser51 phosphorylated Aur-A protein is examined by immunoprecipitation (IP) with a phosopho-specific antibody against Ser51 of Aur-A followed by immunoblottoing (IB) analysis with a monoclonal antibody to Aur-A in head and neck cancer cells. Gene amplification and mRNA expression were previously examined (0.08 MB TIF)Click here for additional data file.Figure S3Aur-A binds to Cdh1 and APC component, cdc27 at M phase. A: Wild type and mutant (S51A and S51D) Aur-A bind to Cdh1. Cdh1 was co-transfected with or without Aur-A wt and two S51 mutants in 293T cell, and then ZLLL was added for 6h before the cells were collected. Cell extracts were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted with anti-Cdh1 antibody. Cul1 was used as a loading control. Aur-A wt, S51A and S51D bind to Cdh1. B: Aur-A binds to Cdh1 at M phase. After noc treatment, cell extracts were immunoprecipitated (IP) with anti-Cdh1 antibody and immunoblotted with anti-Aur-A antibody in HeLa cells. We confirmed the expression of Aur-A, Cdh1, phospho-histon H3 (P-HH3) and Cul1 in lysates. Endogenous Aur-A binds to Cdh1 at M phase. C: Aur-A binds to Cdc27 and Cdh1 at M phase. In HeLa cells at 0h and 9h after nocodazole (noc) treatment, cell extracts were immunoprecipitated (IP) with anti-Cdc27 and anti-Cdh1 antibody and immunoblotted with anti-Aur-A antibody in HeLa cells. Endogenous Aur-A binds to Cdc27 and Cdh1 at M phase. At G1 phase (noc+9h), these bindings were not observed.(0.10 MB TIF)Click here for additional data file.


The insulin-like growth factor 1 receptor (IGF-1R) plays numerous crucial roles in cancer biology. The majority of knowledge on IGF-1R signaling is concerned with its role in the activation of the canonical phosphatidyl inositol-3 kinase (PI3K)/Akt and MAPK/ERK pathways. However, the role of IGF-1R ubiquitination in modulating IGF-1R function is an area of current research. In light of this we sought to determine the relationship between IGF-1R phosphorylation, ubiquitination, and modulation of growth signals.Wild type and mutant constructs of IGF-1R were transfected into IGF-1R null fibroblasts. IGF-1R autophosphorylation and ubiquitination were determined by immunoprecipitation and western blotting. IGF-1R degradation and stability was determined by cyclohexamide-chase assay in combination with lysosome and proteasome inhibitors.IGF-1R autophosphorylation was found to be an absolute requirement for receptor ubiquitination. Deletion of C-terminal domain had minimal effect on IGF-1 induced receptor autophosphorylation, however, ubiquitination and ERK activation were completely abolished. Cells expressing kinase impaired IGF-1R, exhibited both receptor ubiquitination and ERK phosphorylation, however failed to activate Akt. While IGF-1R mutants with impaired PI3K/Akt signaling were degraded mainly by the proteasomes, the C-terminal truncated one was exclusively degraded through the lysosomal pathway.Our data suggest important roles of ubiquitination in mediating IGF-1R signaling and degradation. Ubiquitination of IGF-1R requires receptor tyrosine kinase activity, but is not involved in Akt activation. In addition we show that the C-terminal domain of IGF-1R is a necessary requisite for ubiquitination and ERK phosphorylation as well as for proteasomal degradation of the receptor. In the past few years, the insulin-like growth factor 1 receptor (IGF-1R) has emerged as a receptor tyrosine kinase (RTK) with important roles in cancer biology. The physiological responses to IGF-1R tyrosine kinase activation are diverse and include differentiation, proliferation, protection from apoptosis, cellular transformation, and cancer progression The IGF-1R is a tetrameric receptor tyrosine kinase consisting of two ligand-binding extracellular α-subunits and two β-subunits composing a transmembrane domain, an intracellular tyrosine kinase domain and a C-terminal domain Recent data has shown that IGF-1R is a substrate for ubiquitination, however, the role is unclearIn general, activated receptors must be cleared from the cell surface in order to desensitize the cell to mitogenic signals The fact that IGF-1R is ubiquitinated makes it as a possible substrate for proteasomal degradation. However, several studies have demonstrated that degradation of epidermal growth factor receptor (EGFR), being the most investigated RTK in this respect, is mediated by lysosomal proteases Using different mutated constructs we aimed to identify functional sites and domains of IGF-1R necessary for receptor ubiquitination and to address whether ubiquitination is involved in control of signaling and degradation.The β-subunit of the IGF-I receptor spans the membrane and contains the TK domain, responsible for the overall kinase activity of the receptor . The lysIn order to study these constructs in cell systems we used R- cells (IGF-1R knockout) stably transfected with wild type IGF-1R (here referred to as wt), Y1136F, Δ1245 and Y950F+Δ1245 as well as transiently transfected with K1003R. All constructs are of human origin.The expression level of the above IGF-1R variants under basal conditions is demonstrated in Upon ligand (IGF-1) stimulation the IGF-1R is rapidly autophosphorylated and, as recently demonstrated, ubiquitinated Next we sought to investigate the potential requirement of specific sites/domains of the IGF-1R for receptor ubiquitination. In this respect, we used cells stably transfected with the 4 aforementioned IGF-1R mutants . As showSince we now have identified an IGF-1R construct with deficient ubiquitination capacity we sought to investigate whether ubiquitination influences the signaling responses mediated by IGF-1R. We stimulated serum starved wt, K1003R, Y1136F and Δ1245 cells with IGF-1 for different time points (0–20 min). Phosphorylation of ERK1/2 was assessed as a measure of MAPK activation and phosphorylation of Akt as a measure of PI3K activation. The levels of expression of phospho-ERK1/2 and phospho-(S473) Akt were investigated by immunoblot analysis . Akt phode novo protein synthesis as described elsewhere We investigated degradation of the different IGF-1R constructs using cyclohexamide (CHX) to block P<0.05), stabilizing effect on IGF-1R. Wt, K1003R and Δ1245 cells were pre-treated with epoxomicin or chloroquine before treatment with CHX and the levels of IGF-1R expression were analyzed by western blotting  and quanUnexpectedly, we observed that the mutant receptors in Y1136F and Y950F cells, which both exhibited IGF-1R ubiquitination , are maiIn this study we sought to shed light on regulation of and role of IGF-1R ubiquitination. The results presented here show that phosphorylation of the receptor is necessary for its ubiquitination. Furthermore, we can state that the C-terminal domain of the receptor is needed in this context. In wild type IGF-1R the profile of ubiquitination matches its phosphorylation status. However, C-tail truncated IGF-1R cannot be ubiquitinated in spite of its normal kinase activity. Since, C-terminal truncation of IGF-1R inhibits receptor ubiquitination, one could speculate that the lysine(s) required for ubiquitination are located in the C-tail. However, out of the 29 lysine residues in the β-subunit of the IGF-1R only three are in the C-tail domain. Although these three lysines might function as ubiquitin acceptors, another possibility is that the C-terminal domain functions as an E3 ligase binding site. Further studies are, however, required to understand the distinct mechanism by which C-terminal truncation inhibits ubiquitination of IGF-1R. Importantly, when ubiquitination of IGF-1R is prevented by C-tail truncation the receptor is degraded explicitly through the lysosomes, suggesting a role of IGF-1 induced ubiquitination in proteasomal degradation of the receptor.Intriguingly, we observed that the IGF-1R mutant Y1136F is mainly degraded via the proteasomes. Since this mutant did not exhibit any Akt phosphorylation, but intact phosphorylation of ERK1/2, it is possible that the PI3K/Akt pathway has a positive regulatory influence on lysosomal degradation. A similar predominance of proteasomal degradation occurred in case of the Y950F mutant, which has an impaired IRS-1 binding In summery we have identified a receptor domain critical for the IGF-1R ubiquitination and show that this post-modification is dependant of phosphorylation of the receptor. Furthermore, we show the co-existence of both proteasomal and lysosomal pathway for IGF-1R degradation, in which PI3K/Akt signaling may be important for the lysosomal pathway and ubiquitination for the proteasomal one. Also, IGF-1R activated MAPK/ERK signaling may be controlled by ubiquitination of the receptor.Polyclonal IGF-1R antibodies , monoclonal antibody to phosphotyrosine (PY99) and monoclonal antibody to ubiquitin (p4D1) were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Anti-pErk1/2, anti-Erk1/2, anti-pAkt (serin 473), and anti-Akt antibodies were purchased from Cell Signaling Technology, Danvers, MA. Protein G Sepharose from Amersham Pharmacia Biotech . All other reagents unless stated otherwise were from Sigma .Cell lines used are derived from 3T3-like fibroblasts isolated from mouse embryos with a targeted disruption of the IGF-IR gene (R-cells) R- cells were seeded to 90% confluence in 10-cm dishes  with plasmids using Lipofectamine 2000 , according to the manufacturer's instructions. 24 h after transfection cells were splited into 6 well plates and cultured for an additional 24 h in the presence of 0.6 mg/ml G418. During the last 24 h, cells were starved and then stimulated for indicated times with 50 ng/ml IGF-1. Cells were lyzed in lysis buffer , phosphatase inhibitor 1 and 2, and 10 mM N-Ethylmaleimide). Protein extracts were prepared for immunoprecipitation or western blot analyses.Cell lysate were distracted as described above. Protein samples were dissolved in a sample buffer containing 0.0625 M Tris·HCl (pH 6.8), 20% glycerol, 2% SDS, bromophenol blue, and 2β-mercaptoetanol. Samples corresponding to 50–100 µg of cell protein were separated by 7.5% or 4–12% gradient sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Molecular weight markers (Bio-Rad) were run simultaneously. After SDS/PAGE the proteins were transferred onto nitrocellulose membranes  and blotted with the indicated antibodies. This was followed by washes and incubation with a HRP conjugated secondary antibody , and detected with .Cells were cultured to subconfluency in 6-cm plates. The cells were serum-depleted for 24 h, and then stimulated by IGF-1 (50 ng/ml) at indicated time points. For determination of IGF-1R phosphorylation and ubiquitination, cell lysates were extracted with lysis buffer as described above. Twenty µl of protein G Sepharose and 1 µg of antibody were added to 1 mg of protein material. After overnight incubation at 4°C on a rocker platform, the immunoprecipitates were collected by centrifugation in a microcentrifuge at 2,500 rpm for 2 min. The supernatant was discarded, whereupon the pellet was washed 2 times with lysis buffer and 1 time with PBS and then dissolved in a sample buffer for SDS/PAGE and further was analyzed by western blotting.Protein degradation was assessed by cycloheximide-chase assay as to previously described


The intercellular adhesion molecule-1 (ICAM-1) gene is located on chromosome 19p13, which is linked to Type 1 diabetes (T1D). ICAM-1 expression is related to development of T1D and diabetic nephropathy. The present study aims to evaluate the genetic influence of ICAM-1 gene polymorphisms on the development of T1D and diabetic nephropathy.Five valid single nucleotide polymorphisms (SNPs) were genotyped in 432 T1D patients (196 patients had diabetic nephropathy) and 187 non-diabetic control subjects by using dynamic allele-specific hybridization (DASH) and pyrosequencing.P = 0.026, OR = 1.644 (95% CI 1.138–2.376) and P < 0.001, OR = 2.456 (1.588–3.8)]. Frequencies of the C allele in SNP rs281432(C/G) and the A allele in SNP rs5498 E469K(A/G) increased stepwise from non-diabetic control subjects to T1D patients without diabetic nephropathy and T1D patients with diabetic nephropathy. Further analysis for these two SNPs indicated that T1D patients had increased frequency of the common haplotype C-A, in comparison with non-diabetic control subjects .SNPs rs281432(C/G) and rs5498 E469K(A/G) had high heterozygous indexes. They were significantly associated with T1D [The present study provided evidence that SNPs rs281432(C/G) and rs5498 E469K(A/G) in the ICAM-1 gene confer susceptibility to the development of T1D and might also be associated with diabetic nephropathy in Swedish Caucasians. Type 1 diabetes mellitus  is an autoimmune disease. The destruction of insulin-secreting β-cells of the islets of Langerhans leads to hyperglycaemia, which is a key factor in the development of diabetic microvascular complications such as retinopathy, nephropathy and neuropathy. The development of T1D and diabetic complications is strongly modulated by genetic and environmental factors . In SNP rs5498 E469(A/G), similar results were found, suggesting that the SNP was associated with T1D . There was no significant difference in genotype distributions of SNPs rs5491(A/T), rs5492(C/G) and rs1799969 R241G(A/G) between the case and control groups studied.The allele frequencies of five SNPs in the ICAM-1 gene are given in subjects . The comWe found that SNP rs281432(C/G) and rs5498 E469K(A/G) had high heterozygous indexes, although the genotype distributions of SNPs studied in non-diabetic individuals were in the Hardy–Weinberg equilibrium. We thus performed and confirmed the genotyping experiments by using DASH and pyrosequencing. Data using both techniques were matched, and no indication of genotyping error was found. To ascertain whether ‘duplicons’, which might cause high heterozygous indexes, were involved in these two SNPs, direct sequencing analysis by using forward and reverse primers was performed, but no ‘duplicons’ were found.We attempted to detect possible association of SNPs rs281432(C/G) and rs5498 E469K(A/G) with diabetic nephropathy, although no significant difference of genotype distribution in these two SNPs between the groups of T1D patients with diabetic nephropathy and the patients without diabetic nephropathy was found . We dissD′| = ∼0.7) appeared to extend over the region between SNPs rs281432(C/G) and rs5498 E469K(A/G). Frequencies of haplotypes constructed by these two SNPs in T1D patients and non-diabetic control subjects are summarized in P = 0.035).To further search for evidence that ICAM-1 gene polymorphisms influence the development of T1D, the degree of LD for all SNPs was examined. Data indicated that the ICAM-1 gene had a low LD  and the We have carried out a genetic association study of five SNPs in the ICAM-1 gene in Swedish T1D patients and non-diabetic control subjects with a high-throughput SNP scoring technique called DASH. The results indicated that SNPs rs281432(C/G) and rs5498 E469K(A/G) were significantly associated with the development of T1D. Interestingly, these two SNPs had a high heterozygous index in the genotype distribution. We thus confirmed the genotyping experiments by using pyrosequencing. Segmental duplications (duplicons) with > 90% similarity between copies comprise at least 5% of the human genome, which may cause specific allelic and genotypic diversities, such as a high heterozygous index in complex diseases ,25. In oRs5498 E469K(A/G) is a non-synonymous SNP and resides in the fifth immunoglobulin-like domain of ICAM-1. To date, no report indicates that this domain is involved in ligand binding of ICAM-1. However, this domain may play a role in an immunodominant epitope of B lymphocytes and dendritic cells . A recenet al. found that SNP rs1799969 R241G(A/G) had an increased transmission rate in T1D patients [Single marker analysis is a useful tool for genetic association studies. Multiplex marker analysis, however, may be more powerful for detecting an association in complex diseases. All previous genetic association reports of the ICAM-1 gene in T1D studied only one or two SNPs, which may reduce the possibility of detecting an association. In the present study, we genotyped five SNPs along the ICAM-1 gene and performed a haplotype analysis based upon population-specific LD. We found that SNP rs281432(C/G) and rs5498 E469K(A/G) might exist in an LD block. The common haplotype C-A of these two SNPs was found to be significantly associated with T1D in Swedish Caucasians. Previously, Nejentsev patients . This noBiological experiments suggest that genetic variation of the ICAM-1 gene might be involved in the development of diabetic nephropathy ,11. To tIn conclusion, the C allele of SNP rs281432(C/G) and the A allele of SNP rs5498 E469K(A/G) in the ICAM-1 gene are significantly associated with T1D in Swedish Caucasians. These two alleles confer an increased risk for the development of T1D and might also be associated with susceptibility to diabetic nephropathy.None declared.


The pulmonary surfactant protein (SP)-A has potent immunomodulatory activities but its role and regulation during allergic airway inflammation is unknown.Aspergillus fumigatus (Af) challenge of sensitized animals.We studied changes in SP-A expression in the bronchoalveolar lavage (BAL) using a murine model of single Af provocation of sensitized mice. Decrease of SP-A was associated with influx of inflammatory cells and increase of IL-4 and IL-5 mRNA and protein levels. Since levels of SP-A showed a significant negative correlation with these BAL cytokines (but not with IFN-γ), we hypothesized that SP-A exerts an inhibitory effect on Th2-type immune responses. To study this hypothesis, we used an in vitro Af-rechallenge model. Af-induced lymphocyte proliferation of cells isolated from sensitized mice was inhibited in a dose-dependent manner by addition of purified human SP-A (0.1–10 μg/ml). Flow cytometric studies on Af-stimulated lymphocytes indicated that the numbers of CD4+ (but not CD8+) T cells were significantly increased in the parental population and decreased in the third and fourth generation in the presence of SP-A. Further, addition of SP-A to the tissue culture inhibited Af-induced IL-4 and IL-5 production suggesting that SP-A directly suppressed allergen-stimulated CD4+ T cell function.SP-A protein levels in the BAL fluid showed a rapid, transient decline that reached the lowest values (25% of controls) 12 h after intranasal We speculate that a transient lack of this lung collectin following allergen exposure of the airways may significantly contribute to the development of a T-cell dependent allergic immune response. Cells were then harvested on a PHD Harvester . [3H] thymidine incorporation was determined via liquid scintillation counting . Samples were plated in triplicates and the experiments were repeated n = 4 times unless otherwise stated.Human peripheral blood mononuclear cells were isolated from healthy volunteers using a standard protocol approved by the IRB of the University of Pennsylvania.2b isotype control  and Allophycocyanin conjugated anti-human CD8 . Data were acquired on a four-color, dual laser FACSCalibur . For FACS analysis the gated cell populations were divided into four different generations  according to their CFSE content. Isotype controls were used to distinguish positive and negative populations. The SP-A-treated samples were compared with, and data were expressed as the percentage of the non-treated samples.The effects of SP-A on lymphocyte proliferation was further assessed using CFSE labeling as previously described . SurfaceA relative quantitation of lung IL-4, IL-5, and IFN-γ mRNA expression was performed by real time PCR using TaqMan(R) Gene Expression Assay and TaqMan(R) Universal PCR Master Mix: Mm00445259, Mm00439646, Mm00434204, and Mm00801778, respectively, (Applied Biosystems) according to the manufactures instructions. In brief three RNA samples isolated from the lungs per time points, were reverse transcribed using Superscript(TM) III First Strand Synthesis System (Invitrogen). Approximately 100 ng of cDNA was used per singleplex PCR reaction in a total volume of 25 μl. Cycling was performed on an ABI SDS-7000 and ABI SDS-7500 respectively with an initial denaturation step at 95°C for 10 min and 40 cycles of 15 sec. at 95°C and 1 min. at 60°C. Every sample was run in triplicate and β-2-microglobin (Mm00437762) was used as endogenous control. The Comparative Ct Method was used to analyze the results using ABI PRISM(R) 7900 SDS software. Results are expressed as fold difference  relative to naïve animals.Splenic lymphocytes were cultured for 48 h as described. The supernatant was then removed and frozen at -80°C pending analysis. OptEIA mouse IL-4, IL-5 and IFN-γ ELISA kits (Pharmingen) were used.Data were expressed as mean ± SEM. Time courses and dose responses were compared using ANOVA. To test differences between individual groups Student's t test assuming equal variances were performed. Correlations were investigated by regression analysis. A p value of < 0.05 was considered as significant. Data were analyzed with the Sigma Stat standard statistical package (Jandel Scientific).Af challenge. Recovery was observed 72 h later (135 ± 40 %)  were compared with mice that received vehicle (glycerol) treatment instead of antigen  Fig.  and 1B. vs. 106 ± 16 p < 0.05, n = 5, Fig Af challenge.SP-A protein decreases were not associated with commensurate mRNA changes. Northern blot analysis on RNA extracted from the lung tissue of each group of sensitized mice showed that SP-A mRNA actually increased 12 h after allergen challenge in comparison with the 0 h controls  in comparison with glycerol challenge . The glycerol challenge controls were not different from mice that received no sensitization or challenge  and eosinophils  in comparison with glycerol challenged controls. These cell numbers were also reduced  by the 72 h time point.In order to examine the proinflammatory changes that were linked with the greatest fall in SP-A concentrations, we chose to study the 12 h time point. Following allergen provocation there were significant Penh increases 12 h after enge Fig . Levels Af-induced models of allergic airway inflammation [Af challenge induced a local production of predominantly Th2-type cytokines. In comparison with glycerol challenged controls there was a significant release of IL-4 (559 pg/ml vs. 3 pg/ml p < 0.01), IL-5 . There was no correlation between SP-A and IFN-γ (not shown). Further, by the 72 h time point after allergen challenge IL-4 (8.7 ± 2.7 pg/ml), IL-5 (10.3 ± 7.3 pg/ml) and Eotaxin (9.72 ± 3.5 pg/ml) returned to close to baseline. To verify that the amount of airway SP-A is negatively associated with Th2 cytokine production, we also performed quantitative real-time PCR analysis of IL-4, IL-5 and IFN-γ mRNA. These results showed that mRNA levels for the Th2 cytokines increased approximately 35 fold in comparison with naïve controls 12 h after allergen challenge and returned to control levels by 72 h . The background cpm throughout our experiments ranged between 0 and 16. The SP-A preparation had < 0.07 endotoxin units/ml. This level is consistent with other cell culture SP-A preparations [Af were markedly inhibited by SP-A in a dose-dependent manner  and ionomycin (500 ng/ml) were inhibited by SP-A (10 μg/ml) to a similar extent as the Af-stimulated lymphocytes by 66 ± 4% , G2, G3 and G4 population according to decreasing levels of CFSE expression. Cells incubated in the presence of 10 μg/ml of SP-A were compared with equivalent populations of cells stimulated in the absence of SP-A and the results are expressed as a percentage of the SP-A-free (100%) control. In comparison with medium treatment, (represented by the middle line) addition of SP-A increased the proportion of cells that remained in the G1 population in the samples stimulated by Af .To further confirm the effects of SP-A on the proliferating cells, we added an intracellular fluorescent dye 5,6-carboxy- fluorescein diacetate succinimidyl ester (CFSE) to the cell cultures. The amount of this dye is halved as the f Figure . The proAf-induced  to sensitized lymphocytes significantly inhibited IL-4 and IL-5 production both in the d Figure  and PMA/in vitro.Emerging evidence suggests that collectins provide a link between innate and adaptive immunity. The relationship of SP-A production to inflammatory changes in the lung however is poorly defined. This study reports that a transient decrease in airway SP-A levels is associated with mRNA transcription and release of Th2-type cytokines following allergen provocation. The results also demonstrate that this lung collectin has a direct negative regulatory effect on allergen-induced lymphocyte activation Af challenge but completely recovered 72 h later. The exact mechanisms that regulate the changes of SP-A concentrations in the airways are uncertain. Since reduction in the absolute amounts of SP-A was only 42%, the effect of dilution by the extravasated plasma proteins 12 h after allergen challenge could have significantly added to the fall in the fractional SP-A concentrations. Nevertheless, while further studies are needed to unravel its intricate regulation [Studies investigating allergen-induced airway inflammation previously described either significant increases ,23 or degulation , it appein vitro model where SP-A mRNA but not protein expression was induced by dexamethasone, cyclic AMP and IL-4 [SP-A changes were comparable to that of SP-B and SP-C, described previously in a similar model . Interesand IL-4 . In addiand IL-4 . Taken tAf [The inverse association we observed between levels of SP-A and the inflammatory changes 12 h after allergen provocation was used to generate our hypothesis that SP-A negatively regulates Th2 cytokine production. This hypothesis was then tested by directly adding SP-A to allergen-stimulated T cells. These results are in support of previous reports showing that administration of SP-A alleviated allergic inflammatory changes in mice (reviewed in ), howeveAf ,29 and hAf  may be oAf ,31. FurtAf  and T ceAf -12. SP-Ain vitro. Here we present for the first time the inhibitory effects of SP-A on generational progression of sensitized lymphocytes to Af re-challenge in vitro. We show that this inhibition was dose-dependent. Further, inhibition of lymphocyte activation by SP-A was not due to toxic effects since baseline proliferation of the lymphocytes was not affected. In addition, Figure Af concentrations, confirming that the antigen-responsive, stimulated T helper cell population is the target of SP-A.The mechanism of T cell susceptibility to inhibition by SP-A needs further clarifications. While the inhibitory effects of SP-A have been demonstrated in antigen-stimulated human PBMC  and moreex vivo [in vivo and that there was a significant, negative correlation between SP-A and the amount of IL-4, IL-5 and eotaxin supporting the hypothesis that this collectin exerts an inhibitory effect on Th2 processes. Indeed addition of SP-A abolished production of the Th2 cytokines IL-4 and IL-5 by Af-stimulated lymphocytes. Thus, SP-A may act as a natural immunosuppressant in the lung with direct inhibitory actions on allergen-stimulated CD4+ Th2 lymphocytes.The effects of SP-A treatment on inhibiting Th2 cytokines were previously studied ex vivo . Here weA transient decrease in airway levels of SP-A was associated with transcription of mRNA and release of the Th2-type cytokines IL-4 and IL-5 but not IFN-γ. On the other hand, addition of SP-A to lymphocyte cultures from sensitized mice suppressed allergen-induced Th2-type immune response. We speculate that this lung collectin may play a protective role to prevent allergen-inhalation provoked immune reactions and propose the novel concept that a transient SP-A deficiency contributes to the pathogenesis of the allergic airway response.Af: Aspergillus fumigatusAHR: Airway hyperresponsivenessBAL: Bronchoalveolar lavageCFSE: 5,6-carboxyfluorescein diacetate succinimidyl estercpm: Count per minuteELISA: Enzyme linked immunosorbent assayFACS: Fluorescence activated cell sorterFITC: Fluorescein isothiocyanateIg: ImmunoglobulinIL: Interleukini.n.: Intranasali.p.: IntraperitonealLPS: LipopolysaccharideMBL: Mannose binding lectinSP: Surfactant proteinPAP: Pulmonary Alveolar ProteinosisPI: PMA and ionomycinPMA: Phorbol 12-myristate 13-acetatePBMC: Peripheral Blood Mononuclear CellsTh: T helperSeth Thomas Scanlon carried out the surfactant protein analysis and the protein and phospholipids measurements. Tatyana Milovanova carried out the CFSE studies and FACS analysis. Sonja Kierstein performed the cytokine mRNA analysis. Yang Cao participated in the surfactant protein analysis and in the animal experiments. Elena N. Atochina analyzed the SP-A Western and Northern blots. Yaniv Tomer carried out the animal experiments. Scott J. Russo performed the mRNA extraction and Northern blots. Michael F. Beers participated in the design of the study and contributed to the manuscript writing with comments. Angela Haczku conceived of, designed and coordinated the study, and participated in its writing. All authors approved the manuscript.


A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein). Stable expression of both viral GAAP (v-GAAP) and human GAAP (h-GAAP), which is expressed in all human tissues tested, inhibited apoptosis induced by intrinsic and extrinsic apoptotic stimuli. Conversely, knockout of h-GAAP by siRNA induced cell death by apoptosis. v-GAAP and h-GAAP display overlapping functions as shown by the ability of v-GAAP to complement for the loss of h-GAAP. Lastly, deletion of the v-GAAP gene from vaccinia virus did not affect virus replication in cell culture, but affected virus virulence in a murine infection model. This study identifies a new regulator of cell death that is highly conserved in evolution from plants to insects, amphibians, mammals, and poxviruses. Apoptosis is a conserved and strictly regulated process of cell suicide that, among other things, can remove virus-infected cells. In turn, many viruses, including poxviruses, have evolved strategies to block apoptosis to keep cells alive until virus replication is completed. Here, a novel viral anti-apoptotic protein from vaccinia virus and camelpox virus, viral Golgi anti-apoptotic protein (v-GAAP), and its human counterpart, h-GAAP, are described. Evolutionarily, GAAPs are extremely well conserved with closely related proteins in plants, insects, amphibia, and mammals, the viral and human counterparts sharing a striking 73% sequence identity. GAAPs are resident in the Golgi and inhibit apoptosis induced by a wide range of apoptotic stimuli. Knockout of h-GAAP, which is expressed in every tissue tested, induced cell death by apoptosis. The close relationship between the viral and the human proteins was confirmed in that v-GAAP could complement for the loss of h-GAAP and promote cell survival. Deletion of the v-GAAP gene from vaccinia virus affected virus virulence. Thus, this study identifies a new regulator of cell death that is highly conserved in evolution and has been hijacked by poxviruses. These data support a role for the Golgi complex in sensing pro-apoptotic stimuli. Cell death is the result of either necrosis or apoptosis. Necrosis is the outcome of severe and acute injury, whereas apoptosis is an evolutionarily conserved, strictly regulated, energy-dependent process of cell suicide responsible for the ordered removal of superfluous, aged, or damaged cells, including virus-infected cells . The sigOrthopoxvirus genus is the most extensively characterised genus of the Chordopoxvirinae [, the cause of smallpox, vaccinia virus (VACV), the vaccine used to eradicate smallpox, and camelpox virus (CMLV), which is closely related genetically to variola virus [Poxviruses are a family of large DNA viruses that replicate in the cytoplasm . The Ortxvirinae  and inclla virus . The ortla virus . Proteinla virus . Alternala virus ).This study describes the identification and characterisation of a new poxvirus anti-apoptotic protein that is located in the Golgi and is called viral Golgi anti-apoptotic protein (v-GAAP). v-GAAP is non-essential for virus replication in cell culture but affects virus virulence in a murine model of infection. Strikingly, v-GAAP shows very high conservation with a hitherto uncharacterised human protein (h-GAAP) that is expressed in all human tissues tested, is essential for cell viability, is anti-apoptotic, and is conserved in plants, insects, amphibia, and mammals.6L encodes a 237–amino acid (aa) protein with a putative molecular mass of 26.5 kDa [Xenopus laevis (69% identity), and zebrafish (65% identity). In addition, there are related proteins of similar size predicted to be encoded by Arabidopsis thaliana (38% identity) and Drosophila melanogaster (34% identity) (CMLV gene 26.5 kDa . Databasdentity) A.Comparison of the sequence of all putative GAAPs showed that, evolutionarily, GAAPs are extremely well conserved in length (235–239 aa) and sequence identity. The close relationship between GAAPs is supported further by their conserved hydrophobicity profile, which predicted a six or seven transmembrane topology , an inhibitor of DNA replication and late gene expression. Reverse transcription (RT)–PCR detected v-GAAP messenger RNA (mRNA) from 2 h post infection (p.i.) onward and at 22 h p.i. in the presence of AraC. In contrast, the CMLV late gene gene D8L , was onlhttp://www.poxvirus.org) showed that the majority of orthopoxviruses do not encode an orthologue of CMLV gene 6L, but an exception was VACV Lister [6L yielded a 714–base pair (bp) fragment, the same length as the CMVL 6L gene (unpublished data). DNA sequencing confirmed that these viruses encoded proteins of 237 aa with 94%–95% identity with CMVL 6L.There is no established small animal model for studying the contribution of specific genes to CMLV pathogenesis, so to investigate the role of v-GAAP in virus virulence, we searched other orthopoxviruses for orthologues of CMLV 6L. Bioinformatic analysis of poxvirus genomes (V Lister . PCR ampv-GAAP gene was deleted (v-ΔGAAP) and re-inserted (v-GAAP Rev) into the deletion mutant. We also constructed a virus in which v-GAAP contained a C-terminal hemagglutinin (HA) tag (vGAAP RevHA). PCR and Southern blot analysis confirmed that the genomic structures of all viruses were as predicted. Comparisons of the growth of the recombinant viruses in cell culture showed there were no differences in plaque phenotype or yield of intracellular virus (unpublished data). Immunoblotting of extracts from v-GAAP RevHA–infected cells with anti-HA antibody (Ab) detected v-GAAP as a 23-kDa protein that was present early and throughout infection  were constructed in which the To study the intracellular location of GAAPs, an Ab was raised against a GAAP peptide, SSIEDDFNYGSSV (h-GAAP aa 11–23 and v-GAAP aa 10–22). Cells were infected with v-GAAP WT, v-ΔGAAP, and v-GAAP Rev and fixed for immunofluorescence analysis 3, 6, or 24 h p.i.  A and 3B n-A gold B or immun-A gold A, but eln-A gold B. Moreovn-A gold .Therefore, both human and viral GAAPs are distributed throughout the Golgi stacks, and when they are overexpressed, glycoprotein trafficking to the plasma membrane is normal.To study h-GAAP function, three small interfering RNAs (GAAP siRNAs 1–3) were designed to down-regulate h-GAAP expression. RT-PCR showed that GAAP siRNAs 1 and 3 down-regulated h-GAAP mRNA substantially by 36 h post transfection, whereas GAAP siRNA2 and a control siRNA against GFP did not A. Striki2-ceramide) [The similarity of GAAPs to anti-apoptotic proteins (lifeguard and Bax inhibitor-1) and the observation that knock down of h-GAAP expression induced apoptosis, suggest that GAAPs regulate apoptosis. Accordingly, we tested whether h-GAAP and v-GAAP affected the induction of apoptosis by five different pro-apoptotic stimuli that act through the intrinsic pathway  –24. To tApoptosis was then induced using staurosporine, a broad-spectrum kinase inhibitor. Staurosporine (3 μM) induced apoptosis in only 28% and 46% U2OS-v-GAAP and U2OS-h-GAAP cells, respectively, when compared to U2OS-neo cells B. To ass2-ceramide (10 μM). This treatment induced apoptosis in 35% of U2OS-neo cells, whereas apoptosis in U2OS v-GAAP and U2OS-h-GAAP cells was inhibited completely and remained similar to untreated cells , compared to control cells treated with anti-Fas antibody or TNF-α (designated as 100%), apoptosis was reduced to 46.3% and 62.5% in U2OS h-GAAP cells and to 28.2% and 29.6% in U2-OS v-GAAP cells, respectively  or human tumour necrosis factor (TNF)–α. Treatment with anti-Fas Ab and cycloheximide (CHX) caused apoptosis in 22% of U2OS-neo cells A compareectively B.To define the site of action of h-GAAP, we treated U2OS-neo and U2OS-h-GAAP cells with TNF-α and CHX and examined cleavage of caspase-8. There was no gross difference in proteolytic cleavage of caspase-8 into its active p43/p41 and p18 fragments between both cell lines after 2, 4, and 6 h C. In conHuman and viral GAAP RNA sequences share 77% nucleotide identity, so the use of specific siRNAs enables differential degradation of viral and/or human mRNA. GAAP siRNA1 was designed to down-regulate h-GAAP and v-GAAP, whereas GAAP siRNAs 2 and 3 were specific for h-GAAP. Accordingly, we tested GAAP siRNAs 1–3 for their ability to down-regulate v-GAAP in the U2OS human cell line that we had engineered to stably express v-GAAP, U2OS-v-GAAP. Immunofluorescence analysis showed that 36 h post transfection, GAAP siRNA1 down-regulated h-GAAP and v-GAAP, whereas siRNAs 2 and 3 did not affect v-GAAP expression, and siRNA2 affected neither h-GAAP nor v-GAAP A. Just aTherefore, downregulation of h-GAAP causes cell death by apoptosis, and v-GAAP can complement for loss of h-GAAP to promote cell survival.7 plaque-forming units (pfu) of v-GAAP WT, v-ΔGAAP, or v-GAAP Rev, and the weight change and signs of illness , there was no significant difference in weight loss when comparing mice infected with v-ΔGAAP to those infected with v-GAAP WT and v-GAAP Rev  was observed in v-ΔGAAP compared to both control groups on days 5 and 8 p.i. (6 pfu (unpublished data): mice infected with v-ΔGAAP showed increased signs of illness compared to mice infected with v-GAAP WT and v-GAAP Rev.The role of v-GAAP protein in virus virulence was investigated using a murine intranasal model ,26. Miceneumonia ) were asGAAP Rev A. Howeved 8 p.i. B. Simila6 pfu/gm of tissue by 3 d p.i., and there were no statistically significant differences between virus titres of the different groups detected on this day or later (unpublished data). Next, the inflammatory infiltrate in the lungs of infected mice was examined. In repeated experiments (n = 3), at days 6 and 8 p.i., significantly more cells were recovered from bronchoalveolar lavages (BALs) of mice infected with v-ΔGAAP than from mice infected with v-GAAP WT and v-GAAP Rev . As reported previously after VACV infection [To assess why infection with v-ΔGAAP resulted in more severe signs of illness, the virus titres in lungs were determined at different times p.i. Each virus replicated well in lung tissue to reach titres >10nfection , there wThese results show that loss of v-GAAP altered the virulence of VACV in an intranasal murine model of infection.A new regulator of apoptosis is described that is encoded by VACV, CMLV, and many other eukaryotes. This protein is called Golgi-anti-apoptotic protein because of its function and subcellular location. Overexpression of h-GAAP or v-GAAP rendered cells resistant to induction of apoptosis, while knock down of expression of the human gene by siRNA caused cells to die by apoptosis. Deletion of v-GAAP from VACV did not affect virus replication in cell culture, but affected virulence in a murine model of infection. v-GAAP therefore represents a new virus-encoded apoptosis inhibitor. v-GAAP is remarkable in that it is very closely related to a previously uncharacterised human protein (h-GAAP) that is conserved in a wide range of eukaryotes, is expressed in all 16 human tissues tested, and is essential for cell viability.VACV and other poxviruses express a wide range of proteins that interfere with aspects of the host response to infection . TheFirst, v-GAAP (and h-GAAP) is present in the Golgi, a compartment that until recently was not thought to have an important role in the regulation of apoptosis ,30. ThisArabidopsis GAAPs share 38% aa identity and differ in length by only 4 aa. Hitherto, the function of the protein we have called GAAP had not been determined in any species, although the human gene was one of several reported to be up-regulated in some breast tumours [Second, v-GAAP is very closely related to a human protein that shows remarkable conservation throughout eukaryotes. The virus and human protein have 73% aa identity, a similar hydropathy profile, and a very similar length. Moreover, the genomes of many eukaryotes  encode a closely related protein. For instance, the human and  tumours . Data pr tumours , interfe tumours , and 3β- tumours  from VACThird, v-GAAP (and h-GAAP) inhibits apoptosis induced by an unusually broad range of pro-apoptotic stimuli from both the extrinsic and intrinsic pathways. Instrinsic pro-apoptotic stimuli inhibited by GAAPs include bax, staurosporine, doxorybicin, cisplatin, and ceramide. Extrinsic stimuli inhibited include Fas and TNF. The mechanism by which GAAPs inhibit apoptosis remains to be determined. The ability of GAAPs to inhibit cell death induced by various instrinsic apoptotic stimuli suggests strongly that it acts at a step in the mitochondrial pathway. The finding that h-GAAP suppressed TNF-induced apoptosis at a step downstream of caspase-8 activation is consistent with this idea.Homo sapiens, is highly conserved in eukaryotes, and is another example of a host gene acquired during virus evolution to combat the host response to infection. The human protein is essential for cell viability, and in its absence cells die by apoptosis. Both human and viral GAAPs inhibit an unusually wide range of pro-apoptotic stimuli. The viral protein affects virus virulence and can also substitute for the h-GAAP to maintain cell viability.In summary, a new regulator of apoptosis has been discovered by the study of poxviruses. The protein, called v-GAAP in VACV and h-GAAP in −143, U2OS , and Saos2  cells were grown in Dublbecco's modified Eagle's medium  with 10% (v/v) heat-inactivated fetal bovine serum. The source of VACV strain Evans and CMLV strain CM-S were described previously [Monkey BS-C-1 and human HeLa, TKeviously ,27.http://www.sigmaaldrich.com/Brands/Sigma_Genosys.html). The specificity of the Ab was confirmed by immunofluorescence using U2OS cells stably expressing HA-tagged human or viral GAAPs showing that the signal obtained is identical to the signal given by the anti-HA monoclonal antibody (mAb).The peptide sequence SSIEDDFNYGSSV (h-GAAP aa 11–23 and v-GAAP aa 10–22) was conjugated to keyhole limpet hemocyanin via a C-terminal cysteine residue and used to immunize rabbits  and semi-quantitative RT-PCR with Platinum PCR SuperMix . cDNAs were amplified with h-GAAP specific primers. To define the optimal number of PCR cycles for linear amplification, an aliquot from each of the samples was removed at 20 cycles, and thereafter at every fifth additional cycle.The presence of h-GAAP mRNA in different tissues was analyzed using Human Multiple Tissue cDNA panels I and II . Oligonucleotides were identical for both viral proteins: v-GAAP exp1 (5′-GCCCCGAATTCCGCCGCCATGGCAATGCCG) and v-GAAP exp2-HA (5′-GCCCCTCGAGTAAagcgtaatcaggcacgtcgtaagggtaTTTCTTATTAGATACTTCCAAAAGC). Bold, underlined, or lower case nucleotides represent the translation initiation and termination codons, restriction sites, and HA tag, respectively.CMLV strain CM-S and VACV strain Evans genomic DNA were used for PCR amplification of the h-GAAP gene, total RNA was isolated from HeLa cells and h-GAAP cDNA was cloned by RT-PCR into pCDNA3.1 using the ThermoScript RT-PCR System (Invitrogen) and oligonucleotides h-GAAPexp1 (5′-GCCCCGAATTCTGCCATCATGGCTGACCCCG) and h-GAAPexp2HA (5′- GCCCCTCGAGTTAagcgtaatcaggcacgtcgtaagggtaCTTTTTATTAACTGCTTCCAGAAACC). Bold, underlined, or lower case nucleotides represent the translation initiation and termination codons, restriction sites, and HA tag, respectively. The fidelity of all DNA sequences was confirmed by sequencing. For transient transfection experiments, human and viral GAAPs cDNA were subcloned into pCI .For the GAAP gene was constructed using a plasmid assembled by splicing by overlap extension (SOE)-PCR [GAAP gene, using oligonucleotides ΔGAAP-1 (5′-GCCCCGTCGACGATGTGATAAATACAGCGATACC) and ΔGAAP-2 (5′-CCAATCCATGTTTTGACCTATATATTAATACTC) or oligonucleotides ΔGAAP-3 (5′-TAATATATAGGTCAAAACATGGATTGGAAAC) and ΔGAAP-4 (CGGGGAATTCGTAGCGTGGCAATGACAG), respectively. Annealing regions are shown in bold and restriction sites are underlined. The 5′ and 3′ GAAP deletion fragments were joined by SOE-PCR using oligonucleotides ΔGAAP-1 and ΔGAAP-4, and the product was cloned into pSJH7 [A VACV mutant lacking the SOE)-PCR . Two DNAto pSJH7 , formingGAAP gene was amplified by PCR using oligonucleotides ΔGAAP-1 and ΔGAAP-4 and VACV Evans genomic DNA as template. This product was cloned into pSJH7, forming pSJH7-GAAP-Rev. For the construction of a VACV expressing v-GAAP with a C-terminal HA tag, plasmid pSJH7-GAAPRevHA was constructed by SOE-PCR as for pSJH7-ΔGAAP using oligonucleotides ΔGAAP-1, ΔGAAP-2RevHA (5′-AGCGTAATCAGGCACGTCGTAAGGGTATTTCTTATTAGATACTTCCAAAAGC), ΔGAAP-3RevHA (5′- TACGACGTGCCTGATTACGCTTAAAGTTTAAAATAGAATTAATAAAAACATATAGG), and ΔGAAP-4 and VACV genomic DNA as template.To make a revertant virus, a wild-type copy of the Escherichia coli guanine xanthine phosphoribosyltransferase (Ecogpt) gene as the selectable marker [A41L gene [Recombinant viruses were constructed by transient dominant selection  using the marker . Briefly41L gene . A rever41L gene .U2OS cells were transfected with either the pCDNA3.1- or pCDNA3.1-encoding human or viral GAAPs-HA using Lipofectamine (Invitrogen); 48 h post-transfection, cells were treated with 500 μg/ml of G418. After 1 mo, isolated colonies were cloned and the expression of transfected sequences was determined by immunofluorescence and immunoblotting. Staurosporine- and Fas-induced apoptosis assays (see apoptosis assay section below) were carried out with two independent U2OS-v-GAAP clones or three independent U2OS-h-GAAP clones. Each clone gave similar results to those shown for the two clones described throughout the paper.6) were seeded in 10-cm diameter plates; after 24 h, cells were transfected with plasmids expressing CD20 (2 μg), Bax (5 μg), and 15 μg of pCI or pCI-v-GAAP or pCI-h-GAAP-HA as indicated. Then, 36 h after transfection, both attached and floating cells were harvested and analyzed as described [Saos-2 cells , or 10 ng/ml of human TNF-α . Fas and TNF-α were used in combination with 10 μg/ml of CHX (Sigma). For induction of apoptosis by C2-ceramide , cells were preincubated in modified KRB buffer  supplemented with 1 mM CaCl2 for 4 h and treated with 10 μM C2-ceramide in the same medium. At the indicated time post all treatments, both attached and floating cells were harvested using 4 mM EDTA/PBS. The DNA content of all the cells was analyzed using flow cytometry as described above.Cells were seeded into 6-well dishes at 4–6 × 10http://www.zeiss.com). Primary antibodies used were mouse α-HA mAb , rat high affinity α-HA mAb , rabbit α-GAAP (1:50), mouse α-GM130 , or mouse α-A36R mAb .http://www.fei.com) using a MegaView III CCD camera  operating under analySIS Docu software.For conventional electron microscopy and for cryo-immunocytochemistry, cells were treated as described . All imahttp://www.cellsignal.com).Cells were lysed in RIPA or LDL buffer and between 50 and 200 μg of protein was analyzed by immunoblotting as described . Primaryhttp://www.ambion.com). GAAP siRNA1: 5′- CGAUCGAGGACGACUUCAACU-3′ (sense) and 5′- UUGAAGUCGUCCUCGAUCGAG-3′ (antisense); GAAP siRNA2: 5′- CUGUACGGACAUUUGUACAUG-3′ (sense) and 5′- UGUACAAAUGUCCGUACAGAC-3′ (antisense); GAAP siRNA3: 5′- CGGAUCUCUGGGUUUGAUUUU-3′ (sense) and 5′- AAUCAAACCCAGAGAUCCGAG-3′ (antisense). As a control, siRNA to GFP  was used. Cells were grown to 50% confluency in 6-well plates and transfected with 1 μg of siRNAs using siFECTamine  [siRNA sequences specific to h-GAAP were designed using “siDIRECT” ,45 and wKingdom) . The traFor semi-quantitative RT-PCR after siRNA transfection, total RNA was extracted from HeLa cells using TRIzol reagent (Invitrogen) and treated with Amplification grade DNAseI (Invitrogen) before semi-quantitative RT-PCR analysis was performed using the SuperScriptTM III One-Step RT-PCR System (Invitrogen). Each reaction contained 0.2 μg of total RNA and the optimal number of cycles for linear amplification for tissue expression analysis. Primers were h-GAAP forward: 5′-GCCCCGAATTCTGCCATCATGGCTGACCCCG-3′ and reverse: 5′-GCCCCTCGAGTTACTTTTTATTAACTGCTTCCAGAAACC-3′; and actin forward: 5′-GTGGGGCGCCCCAGGCACCA-3′ and reverse 5′-CTCCTTAATGTCACGCACGATTTC-3′.−143 cells were infected with 5 pfu/cell of CMLV with or without 40 μg/ml AraC. After 2, 5, or 22 h, total RNA was extracted from cells using the RNEasy Midi kit  treated with Amplification Grade DNAseI (Invitrogen). Semi-quntitative RT-PCR analysis was performed as above. Primers were v-GAAP exp1, v-GAAP exp2, D8L-1 (5′-GTCTCTCTCAAATCGGAC), and D8L-2 (5′-GCAAAAAAAACACGATGA).To determine the time of expression of VACV GAAP during infection, TKhttp://probes.invitrogen.com). Cells were loaded with TMRE by incubating cells in medium containing 0.2 μM TMRE for 30 min at 37 °C. TMRE fluorescence was acquired using flow cytometry through the FL-2 channel.Mitochondrial membrane potential was quantified by staining with TMRE  for 5 min at RT. Cells were washed with RPMI 1640 medium (Gibco) and cell viability was assessed using trypan blue exclusion.For intranasal infections, groups of BALB/c mice (6–8 wk old) were inoculated under general anaesthesia with virus in 20 μl PBS. Each day, mice were weighed individually and monitored for signs of illness , and thot-test (two-tailed) was used to test the significance of the results.The Student's Figure S1U2OS-neo, U2OS-v-GAAP, or U2OS-h-GAAP cells were transfected with a plasmid encoding vesicular stomatitis virus glycoprotein G fused to GFP and were fixed for analysis by fluorescence microscopy 4, 7, and 24 h post transfection.Scale bars, 20 μm.(584 KB PPT)Click here for additional data file.Figure S2HeLa cells, mock-transfected or transfected with h-GAAP siRNAs 1–3 for 56 h, were loaded with the mitochondrial membrane potential sensor TMRE. Loss of mitochondrial membrane potential was measured as a decrease in TMRE fluorescence by two-color flow cytometry (cell population labeled M1).(76 KB PPT)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the proteins discussed in this paper are A. thaliana GAAP (NP_193209), CMLV strain CM-S GAAP (AAG37461), dog GAAP (XP531662), D. melanogaster GAAP (Q9V6H6); h-GAAP , mouse GAAP (Q9DA39), orangutan GAAP (Q5R8M0), VACV Evans GAAP (AY690669), VACV Lister GAAP (AY699305), VACV USSR GAAP (AY699306), X. laevis GAAP (AAH90219), and zebrafish GAAP (Q5TZH2).The Genbank (


The generation and use of MHC class II tetramers to study antigen-specific CD4+ T cells appears less straightforward. Most MHC class II tetramers are produced with a homogeneously built-in peptide, reducing greatly their flexibility of use. We attempted the generation of "empty" functional HLA-DR*1101 tetramers, receptive for loading with synthetic peptides by incubation. No such reagent is in fact available for this HLA-DR allele, one of the most frequent in the Caucasian population.MHC class I-peptide tetramers are currently utilised to characterize CD8+ T cells. Both staining temperature and activation state of CD4+ T cells are critical for the binding of peptide-pulsed HLA-DR*1101-Bir to the cognate TCR.We compared soluble MHC class II-immunoglobulin fusion proteins (HLA-DR*1101-Ig) with soluble MHC class II protein fused with an optimised Bir site for enzymatic biotynilation (HLA-DR*1101-Bir), both produced in insect cells. The molecules were multimerised by binding fluorochrome-protein A or fluorochrome-streptavidin, respectively. We find that HLA-DR*1101-Bir molecules are superior to the HLA-DR*1101-Ig ones both in biochemical and functional terms. HLA-DR*1101-Bir molecules can be pulsed with at least three different promiscuous peptide epitopes, derived from Tetanus Toxoid, influenza HA and the tumour associated antigen MAGE-3 respectively, to stain specific CD4+ T cells. As shown for other HLA-DR alleles, we confirm that not all the strategies to produce soluble HLA-DR*1101 multimers are equivalent.It is therefore possible to generate a soluble recombinant HLA-DR*1101 backbone that is receptive for loading with different peptides to stain specific CD4 The antigen specificity of T cells is determined by the highly specific interaction between their TCR and the cognate MHC-peptide complex. The low affinity and fast off-rate of this interaction, however, precludes its exploitation to identify specific T cells [+ T cells in many basic or clinical settings [+ T cell responses, seems somewhat less straightforward. Structural differences between MHC class I and class II molecules might account for the greater difficulties to standardise the generation and production of MHC class II tetramers. In MHC class I molecules, the α chain alone contains the complete peptide-binding groove and it is stable in the soluble form when associated with the β2-microglobulin chain [k have been successfully produced in bacteria [E. coli as denatured proteins, which are allowed to refold correctly as αβ heterodimer in the presence of a single peptide. Once refolded, however, the MHC class II heterodimer cannot exchange the peptide any longer. Another way to produce functional class II tetramers relies on the generation of soluble MHC class II αβ heterodimers engineered with a covalently linked peptide at the N-terminus of either chain, via a flexible amino acid linker of various lengths. This approach results in a homogeneous binding of the target peptides to the soluble MHC class II heterodimer [The direct  T cells -5. Tetra T cells ,7. MHC csettings -13. By cin chain . In contin chain ,16. A vabacteria -19. In aerodimer -23. Thisin vitro incubation.The generation of soluble recombinant MHC class II molecules without covalently linked peptide has been successfully used with HLA-DR*0401 and HLA-DR0*404 molecules -26. ThisMoreover, no univocal strategy to multimerise soluble recombinant MHC class II monomers has been yet devised. For this purpose, in fact, two main modifications are currently introduced at the C-terminus of the MHC class II monomer. In the first approach, the extracellular domains of either MHC class II α or β chain are linked to the Ig constant region. Chimeric MHC-class II-Ig molecules are generally multimerised by using protein A, which is bivalent, thus generating a tetrameric (dimer of dimers) peptide/MHC complex ,22,27,28+ T cell responses to several peptide epitopes derived from either environmental or tumour-associated antigens. This molecule represents one of the most frequent HLA-DR allele in Caucasians, being expressed in up to 20% of the population, we therefore decided to attempt the production of HLA-DR*1101 heterodimers without covalently linked built-in peptide, receptive for loading the desired peptide by incubation. Since no information is currently available on functional empty tetramers made of this HLA-DR allele, and given the premises outlined above, we compared two strategies for the production of soluble HLA-DR*1101 molecules in insect cells: the first generating soluble chimeric HLA-DR*1101-Ig molecules, the second HLA-DR*1101-Bir ones. We show here that it is indeed possible to produce functional HLA-DR*1101 tetramers, which can be loaded with the desired peptide after the purification of the protein. For this purpose, HLA-DR*1101-Bir molecules resulted superior to the HLA-DR*1101-Ig chimeric constructs.We were interested in generating HLA-DR*1101 tetramers to characterise CD4To produce HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules, the cDNAs coding the extracellular domains of the HLA-DR α and the HLA-DR*1101 β chain were fused in frame at the 5' end with the sequence encoding for the Drosophila leader peptide Bip, and at the 3' end with the acidic (AZ) and basic (BZ) leucin zipper sequences, respectively Figure . A sequeTo generate a chimeric HLA-DR*1101-Ig molecules, the DR α chain was linked at the 3' end with a cDNA encoding for the hinge and Fc fragment of human IgG1 Figure . To alloDrosophila melanogaster cells, that were initially grown in selective medium as bulk culture. To stabilise and optimise the production of both recombinant soluble HLA-DR*1101 molecules, bulk cultures of transfected S2 cells were cloned by limiting dilution, and clones producing highest quantity of soluble molecules were selected and expanded.Each construct was stably transfected into S2 The HLA-DR*1101-Ig recombinant molecule was secreted in the culture supernatant as dimers of αβ heterodimers, owing to the presence of an inter-chain disulfide bond between two Ig hinge regions, and was purified using an affinity chromatography with protein A-sepharose Figure . The forThe HLA-DR*1101-Bir recombinant molecule was secreted as αβ heterodimer by S2 cells and purified by immunoaffinity chromatography with the human HLA-DRα chain-specific mAb L243 Figure . NoteworThe immunoprecipitation of HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules with the mAb L243 shows that only a little fraction of HLA-DR*1101-Ig molecule can be bound by the mAb, and most of the protein remains in the culture supernatant, suggesting that the secreted HLA-DR*1101-Ig molecules have lost the native conformation recognised by L243 Figure . A seconTo further verify the biochemical properties and stability of the two types HLA-DR*1101 molecules, we performed analytical size-exclusion chromatography on the purified proteins. Figure Altogether these data indicate that the HLA-DR*1101-Bir exhibited superior biochemical quality to HLA-DR*1101-Ig molecule both in terms of tertiary/quaternary structure and stability.830–845 (p2), a very well characterized promiscuous peptide binding with high affinity to different HLA-DR alleles [The relative disadvantage in the production of HLA-DR*1101-Ig molecules could be compensated by possible peculiar physical properties displayed by this molecule in comparison with HLA-DR*1101-Bir molecules. We speculated in fact that, once pulsed with peptide and tetramerised, the presence of the flexible Ig hinge region in the HLA-DR*1101-Ig construct could represent an advantage and facilitate the binding of cognate TCRs, in comparison with the more rigid HLA-DR*1101-Bir molecules. For this reason, we investigated the functional properties of both HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules. The purified soluble HLA-DR*1101 molecules were loaded with the Tetanus Toxoid-derived peptide TT alleles . Both pu alleles . The hig alleles . As showin vitro to activate p2-specific CD4+ T cells. Since the assay was performed in the absence of antigen presenting cells, a sub-optimal dose of phorbol myristate acetate (PMA) was added as costimulatory signal for T cells. As shown in Figure 830–845-loaded HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules were able to induce IFN-γ production by p2-specific CD4+ T cells, but not by CD4+ T cells specific for an irrelevant peptide or restricted for another HLA-DR allele. These results indicated that both HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules could be loaded in vitro with a peptide of interest.To characterise further the loading of both soluble recombinant HLA-DR*1101 molecules with the exogenous peptides, we relied on an indirect functional assay, in which p2-loaded HLA-DR*1101 molecules were utilised + T cell clones displaying low and high affinity for the peptide respectively, as shown in Figure We finally compared the staining capacity of both soluble HLA-DR*1101 molecules, loaded with different CD4 peptide epitopes. We selected for the staining experiment two p2-specific CD4+ T cells, and required an optimal temperature of staining of 37°C. By contrast, HLA-DR*1101-Ig could not stain peptide-specific T cells, despite their capacity to be loaded with peptides.As shown in Figure + T cells by peptide-pulsed HLA-DR*1101-Bir molecules was consistently higher when T cells had been recently restimulated with either cognate peptide or polyclonal stimuli in the presence of PBMCs as APCs. This observation prompted a more systematic investigation of the phenomenon. This is in fact a relevant issue when considering the use of HLA-DR*1101 tetramers for ex vivo staining, since primary CD4+ T cells might display a whole range of different activation states, which in turn could affect the "detectability" of the whole peptide-specific T cell repertoire by the HLA-DR*1101 tetramers. We therefore compared the capacity of HLA-DR*1101-Bir tetramer to stain the high affinity p2-specific CD4+ T cell clone 162 at different time points from restimulation. As shown in Figure + T cells influences the binding of HLA-DR*1101-Bir tetramers to the TCR.In an independent series of experiments, we noticed that the staining level of CD4+ T cells. Two different helper epitopes from proteins expressed by environmental pathogens were selected: TT-p2 and Influenza Hemagglutinin HA306–318 (HA) [ex vivo or at different time points after in vitro stimulation with the corresponding peptide. In line with published data [+ T cells could be hardly detected ex vivo by HLA-DR*1101-Bir tetramers pulsed with either peptides (data not shown), with frequencies of tetramer positive cells in the range of those obtained with peptide-unloaded (empty) HLA-DR*1101 tetramer . After 4 days of stimulation in vitro with the specific peptide, however, a sizeable fraction of CD4+ T cells could be clearly detected with either peptide-pulsed-HLA-DR*1101-Bir tetramer  , anotherhed data ,26,36, pr Figure , with a t Figure . Again, + T cells specific for the naturally processed, promiscuous p39 peptide derived from the MAGE-3 TAA [+ T cell clone 2C3.35 could be stained with p39-pulsed HLA-DR*1101-Bir tetramers. The affinity of this CD4+ T cell clone for the p39 peptide, as shown in a dose response experiment of IFN-γ production  in frame with the The extracellular region of the HLA-DRα chain, deprived of the leader sequence, was cloned by RT-PCR using the following primers: oligo-1-Up DRα (BglII) 5'GGGAGATCTATCAAAGAAGAACATGTGATCATCCAG3', oligo-2-Dw DRα (BamHI) 5'GGATCCTCCACCTCCGTTCTCTGTAGTCTCTGGG3'. The PCR product was cloned into the PCR2.1 vector (Invitrogen) and sequenced.To generate the HLA-DRα-Bir chain, a cassette containing the sequence for the Acidic Leucine Zipper-Bir A target peptide was generated by fusion PCR with the following primers, as illustrated in Figure Oligo-3-Up AZ (BamHI) 5'GGAGGATCCACTACAGCTCCATCAGCTCAG3', Oligo-4-Dw AZ (Bir) 5'ACCACCACCGTCCCCACCCTGAGCCAGTTC3', Oligo-5-Up Bir (AZ) 5'GGTGGGAGCGGTGGTGGTCTGAACGATATTTTTG3' and Oligo-6-Dw Bir (NotI) 5'CTAGCGGCCGCTATTCATGCCATTCGAT3'. The PCR product was cloned into the PCR2.1 vector and sequenced, then subcloned in frame with the HLA-DRα fragment separated by a five-aminoacid linker (GGGGS). The resulting sequence encoding the HLA-DRα-AZ-Bir fusion protein was excised with BglII and NotI restriction enzymes, and cloned into the Drosophila melanogaster expression vector pMT/Bip/V5/His.To generate the HLA-DRα-Ig chain, a cassette containing the sequence for the Acidic Leucine Zipper-hIgG1 constant region was generated by fusion PCR as illustrated in Figure oligo-3-upAZ(BamHI) described above, oligo-9-Dw AZ (Ig) AGATTTGGGCTCAGATGCTGCCTGAGCCAGTTC, oligo-10-Up Ig (AZ) GCAGCATCTGACGCCCAAARCTTGTGACAAAACTCAC and oligo-11-Dw IgG1 (NotI) 5'TGGCGGCCGCCGCACTCATTTACCCGGAGA. The PCR product was cloned into the PCR2.1 vector, sequenced and then subcloned in frame with the HLA-DR*1101α chain fragment. The resulting sequence encoding for the HLA-DR*1101α-AZ-IgG1 fusion protein was excised with BglII and NotI, and cloned in the Drosophila melanogaster expression vector pMT/Bip/V5/His.The HLA-DR*1101β chain, shared by both type of soluble recombinant HLA-DR*1101 molecules, was generated as follows. The extracellular region of the HLA-DR*1101β, chain deprived of its leader sequence, was cloned by RT-PCR using the following primers:oligo-7-Up DRβ (BglII) 5'GGGAGATCTGGGGACACCAGACCACGTTTC3', oligo-8-Dw DRβ (BamHI) 5'GTGGATCCTCCACCTCCCTTGCTCTGTGCAGATTC3'. The PCR product was cloned into the PCR2.1 vector and sequenced. The HLA-DR*1101β cDNA was subcloned in frame with a Basic Leucine Zipper-His tag cassette derived from the pCO268 plasmid spaced by a five-aminoacid linker (GGGGS) . The res6 cells/ml. The α- and β-chain expressing vectors (15 μg each) were co-transfected with 0,5 μg of the selection plasmid pCoHYGRO using a calcium phosphate transfection kit (Invitrogen). Three days after the transfection, selection medium containing 300 μg/ml of Hygromicin B  was added to cells. Transfected cells were cultured in SFX medium supplemented with 2% of foetal calf serum and 300 μg/ml of Hygromicin B. Cells were kept in the exponential growth phase (7–20 × 106 cells/ml) and the protein production was induced by the addition of 1 mM of CuSO4 when the cells were at a density of 107/ml. Protein production was monitored by dot blot analysis with either mouse anti-human HLA-DR α monoclonal antibody L243  or rabbit anti-His tag polyclonal antibody His-probe , followed by an HRP-labelled goat anti mouse or anti rabbit antibody . The assay was developed with ECL . To improve the production of recombinant proteins, transfected cells were cloned by limiting dilution. S2 cells expressing the desired HLA-DR*1101 molecule were diluted to 2.5 cell/ml in a suspension of irradiated (8000 rad) S2 wild type cells at the concentration of 3 × 105 cells/ml in medium+Hygromicin B, and plated in flat bottomed 96 w plates. The best producer clones were selected by dot blot analysis on the culture supernatant as described above, upon induction with 1 mM of CuSO4 of the same number of cells, and used for routine protein production.S2 cells were grown in SFX medium  supplemented with 10% heat inactivated foetal calf serum , 50 u/ml penicillin, 25 μg/ml streptomycin, 25 μg/ml Kanamycin (Gibco) and 1 μg/ml Amphotericin B  at 27°C to a density of 2–4 × 10Both HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules were purified from the S2 cells supernatant by immunoaffinity chromatography using ProtA-Sepharose (Amersham Bioscience) and HLA-DRα-specific L243 mAb-Sepharose, respectively. The HLA-DR*1101-Ig molecules were stored at -80°C at a concentration of 1 mg/ml in PBS buffer. HLA-DR*1101-Bir molecules were dialyzed against 10 mM Tris buffer, pH 8.0, and concentrated up to 3 mg/ml on Vivaspin2 concentrator . The protein was biotinylated using the BirA enzyme according to the manufacturer's instructions , then was diluted to 1 mg/ml and stored at -80°C. The formation of molecular aggregates was assessed by size exclusion chromatography on a Superdex 200 HR 10/30 column using a ÅKTA FPLC chromatography system (Amersham Bioscience). The calibration of the Superdex 200 column was performed with the Gel Filtration Calibration Kit high molecular weights (Amersham Bioscience), following the manufacturer indications. Partition coefficients were calculated using the relation Kd = (Ve-Vo)/(Vc-Vo), where Ve, Vc and Vo are the elution, column and void volumes, respectively.Both HLA-DR*1101-Ig and HLA-DR*1101-Bir were loaded with synthetic peptides by incubation at 37°C for 72 hours with 50 fold molar excess of either Tetanus Toxoid peptide residues 829–844 (p2), Hemagglutinin peptide residues 306–318 (HA) or MAGE-3 tumour associated antigen peptide residues 191–205 (p39) in 10 mM Tris, 50 mM Glycine pH 5.0 with 0.2% of n-octyl-β-D-glucopyranoside .+CD4+ viable cells.Tetramerization of HLA-DR*1101-Ig and HLA-DR*1101-Bir were achieved by adding FITC-labelled protein A  at 5:1 molar ratio, or PE-labelled streptavidin (Molecular Probes) at 5:1 molar ratio, respectively. Tetramer staining was performed for 2 hours at 37°C in complete medium. The cells are then washed twice, transferred on ice and stained for the other surface markers. Immediately before the analysis TOPRO-3 (Molecular Probes) is added following the manufacturer's instructions to exclude dead cells. The analysis is performed on CD36 cells/ml of a mixture of two different allogeneic PBMCs supplemented with 1 μg/ml of PHA-L (Roche) and 200 u/ml of human recombinant IL-2 (Roche). The cloning mix is dispensed in 25 μl in flat-bottomed Terasaki plates and incubated at 37°C for 10–20 days. Growing cells are then tested for the recognition of peptide pulsed HLA matched EBV cell lines.20 million of freshly purified PBMCs from a HLA-DR*1101/1104 healthy donor were cultured for 7 days in RPMI 1640 (Gibco) supplemented with 10% of human serum, 2 mM Glutamax I (Gibco), 100 u/ml penicillin and 50 μg/ml streptomycin (Gibco), in the presence of the specific peptide at a concentration ranging from 1 to 10 μg/ml. After seven days, blasts were separated from small T cells on a 30–70% discontinuous Percoll gradient , and put4 T cells were plated in U bottom 96 well plates in RPMI 1640 supplemented with 10% of human serum, 2 mM Glutamax I, 100 u/ml penicillin, 50 μg/ml streptomycin, together with 5 × 104 irradiated (6000 rad) HLA-DR*1101 LCL cells as antigen presenting cells, and the specific peptide at the concentrations detailed in the figure legends. IFN-γ production in the supernatant was measured by ELISA after 48 hours of incubation (IFN-γ detection kit-Endogen).To test IFN-γ production, 10i. without peptide; ii. with 5 μg/ml of peptide; and iii. with 50 ng/ml of PMA (Sigma) plus 500 ng/ml of Ionomycin (Sigma). After the first hour of stimulation, Brefeldin A was added at the final concentration of 10 μg/ml. At the end of the stimulation, the cells were harvested, fixed with PFA1%, permeabilized with 0,5% saponin and stained for intracellular cytokine expression.For the intracellular analysis of IFN-γ production, T cells were stimulated for 6 hours with HLA-DR*1101 LCL of at 1:5 ratio in three conditions: 4 specific T cells were added to the wells and incubated for 48 hours at 37°C in the presence of 1 ng/ml of PMA. Cells incubated either with PMA alone or in the presence of 1 μg of plate coated TR66 anti-CD3 mAb are used as negative and positive control, respectively. IFN-γ production in the supernatant is measured by ELISA  after 48 hours of culture.For the T cell activation with peptide-loaded soluble recombinant HLA-DR molecules, 1 μg of peptide loaded HLA-DR*1101-Ig or HLA-DR11-Bir molecules in 50 μl of PBS were incubated in U bottomed 96 well plates for 3 hours at 37°C. After washing, 2 × 10The following antibodies have been used: FITC- and APC-labelled mouse anti human CD3 , Quantum Red-conjugated mouse anti-human CD4 (SIGMA), PE-labelled rat anti-human IFN-γ (Becton Dickinson). Data were collected using a Becton Dickinson FACScalibur flow cytometer and analysed using the Cell Quest software (Becton Dickinson)M.M, P.D., G.C designed experiments. M.M, V.C., C.M., E.D. produced and characterised the tetramers. B.G., M.D. contributed to the purification of the HLA-DR monomers. N.G., M.P.P contributed reagents and expertises. M.M, V.C., P.D., G.C. analysed data. M.M, P.D., G.C. wrote the paper. All authors read and approved the final manuscript.