01 M sterile PBS (pH 7.2). Cells were heat-killed at 110°C for 15 mins and stored at -20°C until use. All bacterial stocks were diluted to 5 × 108 or 1 × 108 cfu/mL for the experiments. For the in vivo experiments, the viable bacterial cell pellets were concentrated to 1 × 1010 cfu/mL in PBS containing 10% skim milk. Listeria monocytogenes

BA00092 (porcine origin; National Veterinary Research and Quarantine Service of Korea, Seoul, Korea) were grown overnight in BHI broth (BD) at 37°C and the number of live cells on the BHI plates counted (BD). Cell pellets were collected by centrifugation Pirfenidone purchase at 14,300 g for 5 mins at 4°C. The cells were then washed twice and diluted to 2 × 106 cfu/mL in PBS. Mouse peritoneal macrophages were isolated according to the method of Zhang et al. (18). Briefly, peritoneal macrophages were

collected from the peritoneal cavities of C57BL/6 mice (Nara Biotech, Seoul, Korea) 4–5 days after intra-peritoneal injection of Brewer thioglycollate medium (Sigma, St. Louis, MO, USA). The mice were killed with CO2 and their peritoneal cavities injected with 5 mL PBS. The fluid was then aspirated and centrifuged at 220 g for 8 mins at 4°C. The cell pellets were washed twice with PBS. After counting in a hematocytometer, cell viability was checked before they were diluted to 1 × 106 cells/mL in RPMI 1640 (Sigma) supplemented with 10% (v/v) FBS (Invitrogen, Grand Island, selleck inhibitor NY, USA), 100 mg/mL streptomycin and 100 U/mL penicillin Nintedanib (BIBF 1120) (Invitrogen). Peritoneal macrophages (5 × 105 cells/well) were cultured in triplicate

in 12-well tissue culture plates (BD). LAB (100 μL volume containing 5 × 107 cfu/mL or 1 × 107 cfu/mL LGG or JWS 833) were then added to the wells. PBS was added to the control wells. The LAB concentrations were such that the macrophages were exposed to either 20 or 100 LAB cells/macrophage at 37°C with 5% CO2. LPS (100 or 500 ng/mL; Sigma) was added to the positive control wells. After 24 hrs, the culture supernatants were collected and the NO and cytokines (IL-1β and TNF-α) concentrations measured. Nitric oxide concentrations were measured using Griess reagent (Promega, Madison, WI, USA). Briefly, 50 μL of culture supernatant or nitrite standard (0–100 μM sodium nitrite) was mixed (in triplicate) with an equal volume of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphylethylenediamine dihydrochloride at room temperature for 10 mins in the dark. The absorbance was then measured at 540 nm in a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The NO concentrations were then calculated from a standard curve. Interleukin-1β and TNF-α were measured using ELISA kits (BD) in accordance with the manufacturer’s instructions. Absorbance was read at 450 nm in a microplate reader. Cytokine standards (0–2000 pg/mL for IL-1β; 0–1000 pg/mL for TNF-α) and samples were assayed in triplicate.

The bone marrow (BM) and, to a lesser extent, the spleen represent the major homing sites of PCs, notably long-lived ones 4. Additionally, a substantial number of PCs can be found in the mucosa, especially in the gut 5. Antibody-secreting cells (ASCs) are also located in inflamed tissues, for instance within the nephritic kidneys of lupus mice and of SLE-patients 6–9 as well as in the synovial tissue of patients with rheumatoid arthritis 10. Cassese et al. reported that after immunization of New Zealand black/white (NZB/W) F1 lupus mice with ovalbumin (OVA), OVA-specific antibody producing cells were initially found in

the spleen 6. Within weeks, check details they disappeared from the spleen and could then be detected in the BM and also within the inflamed kidneys. Hence, inflamed tissues may synthesize chemokines such as CXCL10, which recruit migratory plasmablasts to sites of inflammation. Apart from recent reports identifying cells secreting antibodies to histone H2B 8 and dsDNA 13, respectively, little is known about the antigen-specificity of ASCs within inflamed tissues. Also, it remained elusive whether inflammatory lesions can solely harbor short-lived PCs, or if they can also support the survival of long-lived PCs. Non-dividing long-lived PCs play a critical role in maintaining protective antibody concentrations and may account for the majority of serum IgG 4. These long-lived PCs may be located in niches providing survival factors such

as APRIL or selleck chemical BAFF, stroma-derived factor-1 (SDF-1), IL-6, TNF-α, CD44 signaling, etc. to maintain continuous antibody production over time 11. Here, we further characterize the renal ASCs in the course of experimental lupus. Remarkably, we not only identified short-lived, but also long-lived, PCs within the inflamed kidneys of NZB/W F1 mice, a mouse model resembling many features of SLE 12. Moreover, we show that the frequencies of cells secreting IgG autoantibodies against dsDNA and nucleolin were significantly increased within nephritic kidneys when compared with those of the spleen Protirelin and BM. PCs can be detected within

the inflamed kidneys of SLE patients and lupus mice; however, these ASCs have not yet been thoroughly characterized. Immunohistochemical staining on paraffin-sections of perfused kidneys from nephritic NZB/W F1 mice using anti-CD138 (Supporting Information Fig. 1A and B) showed PCs located within the renal tubulointerstitial tissue of medulla as well as cortex and often formed small clusters, similar to previous observations 6, 13. Next we investigated if nephritic kidneys can harbor both short- as well as long-lived PCs. As shown in Fig. 1A, CD138+ intracellular κ and λ light chain+ PCs were detected at significantly increased numbers in aged lupus mice when compared with young, still healthy NZB/W F1 (8-wk-old) mice and >30-wk-old C57BL/6 mice. These results confirm the presence of significant numbers of PCs within the inflamed renal tissue in accordance to recently published data 8, 13.

A positive correlation was found between IgM antibodies to actin and ss-DNA. Absorption of plasma with soluble actin, myosin and lipopolysaccharides (LPS) resulted in significant

removal of antifilarial antibodies. Affinity-purified anti-ss-DNA antibodies were found to be reactive to filarial antigens and various autoantigens. Further, a positive correlation was found between polyreactive antibodies and B-1 cells in filarial-infected human subjects. After antifilarial treatment, levels of IgM antibodies to ss-DNA, actin, LPS and filarial antigen increased significantly indicating a role of polyreactive naturally occurring antibodies in filarial infection. Our findings add to the existing evidence that the B-cell defect in BALB.Xid mice account for susceptibility to murine https://www.selleckchem.com/products/gsk1120212-jtp-74057.html filarial infection and indicate an important role for these antibodies in providing host protection against filarial infection. “
“To promote an understanding of autoimmunity in BD,

we surveyed autoAgs in patients JAK2 inhibitor drug with BD and investigated the prevalence and clinical significance of the identified autoAbs. Specifically, proteins, extracted from peripheral blood mononuclear cells and separated by 2DE, were subjected to WB, using five serum samples from patients with BD. The detected candidate autoAgs were identified by mass spectrometry. As a result, 17 autoantigenic spots were detected by the 2DE-WB, out of which eight spots were identified. They are enolase-1, cofilin-1, vimentin, Rho-GDI β protein, tubulin-like protein, and actin-like proteins. The autoAbs to one of the identified proteins, cofilin-1, were investigated by WB using a recombinant protein in 30 patients with BD, 35 patients with RA, 32 patients with SLE, and 16 patients with PM/DM. The autoAbs to cofilin-1 were detected by WB in four (13.3%) of the 30 patients with BD, five (14.3%)

of the 35 patients with RA, two (6.3%) of the 32 patients with SLE, and eight (24.2%) of the 33 patients with PM/DM. Our data indicate that the generation of autoAbs to cofilin-1 may reflect common immunological disorders in BD, RA, and PM/DM. Our data would help understanding of the immunopathology NADPH-cytochrome-c2 reductase of BD. In addition, the proteomic approach would be a useful way to investigate autoAgs. BD is a chronic disease with multi-organ involvement, characterized by recurrent occurrence of oral and genital aphthae, skin lesions, and ophthalmological, neurological, or gastrointestinal manifestations. Prevalence of BD is reported to be higher in Japan than in northern Europe and the USA (1, 2). Although candidate pathogenic factors, such as genetic factors, infection, autoimmunity, and neutrophil overactivation, have been reported in BD, the pathogenesis remains to be solved.

Then the mir30 backbone containing the mature miRNA and EGFP were amplified using the primers fwd NotI mir30bb: 5´-attgcggccgcCTAGAAGCTTTATTGCGGT AGTTTATC-3´ and rev mir30bb: 5´-TCGCGGCCGCTTTAC-3´. GSI-IX concentration The

NotI-mir30bb + mir-EGFP-NotI-PCR-fragment was inserted downstream of the tet-responsive CMVmin promoter of the retroviral vector pSR-LP-TRE cloned and provided by C. Bouquet from our laboratory. This vector allows the expression of the miRNA of interest after binding of a cotransduced reverse transactivator (rtTA) in the presence of doxycycline. For the production of retroviral particles the retroviral packaging cell line PhoenixTM, eco was transfected with 2 μg endotoxin free retroviral vector plasmid mixed with 20 μg LipofectamineTM for 5.5 hours. Supernatant media containing virus particles were harvested 48 hours after transfection; 1 × 105 pre-B cells, stably transduced before with the retroviral plasmid pSR-rtTA-IRES-HISRes, were transduced (1150 g, 3.5 hours, 30°C). Twenty-four hours after transduction the cells were selected depending on the vector by addition of histidinol (1.25 mM; Sigma-Aldrich) or puromycin (1.5 μg/mL; Calbiochem). The establishment of inducible Pax5-expressing or miRNA-expressing pre-B-cell lines has been described [20]. Cell lines overexpressing

Neratinib clinical trial the miRNA of interest were established under limiting dilution conditions, and the resulting cell lines were tested for their GFP expression in vitro after 24 hours. Cell lines that expressed high GFP were tested in vivo for their migration behavior by transplantation into Rag1−/− hosts. Six- to twelve-week-old Rag1−/− (CD45.2) mice were sublethally γ-irradiated (4Gy) 24 hours before transplantation.

Pre-B cells (5 old × 106 per host), carrying the overexpression vector of interest, were injected intravenously. GFP+ cells from the BM of doxycycline-fed mice transplanted with miR-221 transduced pre-B cells were sorted 4 weeks after transplantation and differentiated in vitro by addition of αCD40, IL-4, and IL-5 together with doxycycline. After 3 and 4 days of cultivation the cells were analyzed by flow cytometry using anti-CD19 (ID3), MHC class II (TIB120), and IgM (M41) Abs. All Abs used for cell surface stainings were purchased from eBioscience, unless otherwise indicated. Fluorescence tagged Abs: phycoerythrin (PE) conjugated-anti-mouse Flt3 (A2F10), IL-7R (A7R34), CD4 (RM4-5), BST-I (BP-3), CXCR4 (2B11), CD45.1 (A20), CD138 (Syndecan-1, 281-2), Syndecan-4 (KY/8.2), and VLA4 (P/S 2.3, a kind gift of the Deutsches Rheumaforschungszentrum, Berlin, Germany); allophycocyanin conjugated-anti-mouse IgM (M41, a kind gift of Dr. Maria Leptin, Cologne University, Cologne, Germany), CD5 (53-7.3), CD8 (53-6.7) and CD45.1 (A20); PeCy7 conjugated-anti-mouse CD19 (1D3), CD25 (PC 61.

[7, 9, 10] this website The replication

of flavivirus generally occurs on virus-induced host cell membranes. DENV requires autophagy for efficient replication, with recent studies showing that DENV infection induces autophagy, and the inhibition of autophagy reduces significantly DENV replication and release of viral particles.[11-13] These structures may serve as a scaffold for anchoring the viral replication complexes, which consist of viral RNA, viral proteins and host cell factors.[14] Dengue is now considered an important neglected tropical disease. Although many studies have been carried out for almost a century, many aspects of disease remain unresolved. The great lack of knowledge on dengue pathogenesis is a major factor that contributes to a striking human and economic burden. Disease development is not fully understood, which has delayed the development of vaccines, treatments and effective methods for DENV detection.[15] After infection of an immune-susceptible host, an acute, self-limiting febrile systemic syndrome starts to develop. Resolution of infection normally occurs within 4–7 days and is associated with a robust innate and adaptive immune response. The diagnosis is largely clinical, treatment is supportive and disease control is limited to the elimination of its vectors.[1, 2] Primary infection in older children

and adults normally lead to DF, a febrile

illness accompanied by a combination GSI-IX chemical structure of non-specific symptoms that may include headache, retro-orbital pain, myalgia and occasionally haemorrhagic manifestations.[1, 16] Some patients, such as newborns and elderly people, occasionally develop DHF, the most severe form of dengue disease. The hallmark of DHF is the presence of plasma leakage and haemoconcentration, which can lead to the loss of intravascular volume and circulatory insufficiency.[16] Significant bleeding is also a clinical feature associated with severe disease. Bleeding can be observed in both DF and DHF; more severe bleeding, such as bleeding from the gastrointestinal tract, is found more frequently in DHF than in DF. Increased liver enzymes [aspartate aminotransferase/alanine aminotransferase (AST/ALT)] Dapagliflozin and thrombocytopenia (platelet count < 100 000 cells/mm3) are commonly observed in both DF and DHF patients but are more severe in DHF.[16, 17] However, haematocrit readings can be affected by factors such as fever, dehydration and haemorrhage. Patients with DHF who have narrow pulse pressure (<20 mmHg) or who show signs of shock are classified as having DSS. Other severe clinical manifestations including hepatic failure and encephalopathy have been reported in dengue patients.[16-18] Viral load is controlled by the host after a few days, when signs of systemic inflammation are still observed.

They also thank members of the Immunobiology Laboratory for advice and

discussions and Carine Joffre for her permanent support. Conflicts of Interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Many MHC class I molecules contain unpaired cysteine residues in their cytoplasmic tail domains, the function of which remains relatively uncharacterized. Recently, it has been shown that in the small secretory vesicles known as exosomes, fully folded MHC class I dimers can see more form through a disulphide bond between the cytoplasmic tail domain cysteines, RXDX-106 mouse induced by the low levels of glutathione in these extracellular vesicles. Here we address whether similar MHC class I dimers form in whole cells by alteration of the redox environment. Treatment of the HLA-B27-expressing Epstein–Barr virus-transformed B-cell line Jesthom, and the leukaemic T-cell line CEM transfected with HLA-B27 with the strong oxidant diamide, and the apoptosis-inducing

and glutathione-depleting agents hydrogen peroxide and thimerosal, induced MHC class I dimers. Furthermore, induction of apoptosis by cross-linking FasR/CD95 on CEM cells with monoclonal antibody CH-11 also induced MHC class I dimers. As with exosomal MHC class I dimers, the formation of these structures on cells is controlled by the cysteine at position 325 in the cytoplasmic tail domain of HLA-B27. Therefore, the redox

environment those of cells intimately controls induction of MHC class I dimers, the formation of which may provide novel structures for recognition by the immune system. Major histocompatibility complex (MHC) class I molecules function by presenting short peptides, normally of eight or nine amino acids in length, to T cells of the immune system.1 In this manner they provide a sensitive mechanism for the detection and elimination of pathogen-infected cells. Extensive polymorphism in the residues lining the peptide-binding groove of MHC class I molecules ensures that many different pathogenic peptides can be recognized.2 MHC class I molecules are also ligands for the extensive family of killer cell immunoglobulin-like receptors (KIR) expressed on natural killer (NK) cells.3 MHC class I molecules are composed of three main domains, with the α1 and α2 domains forming the peptide-binding groove, supported underneath by the α3 domain and the non-covalently attached β2-microglobulin.4 A transmembrane-spanning domain is then followed by a cytoplasmic tail domain, the full function(s) of which remain somewhat unclear, though roles in recycling,5 targeting for degradation by ubiquitination6 and influencing recognition by NK receptors have been demonstrated.

In response to whole bacteria, IFN-γ secretion by iNKT cells is mostly dependent on IL-12 released by DC in response to TLR stimulation, albeit with an essential role for CD1d. Interleukin-12 dependence was observed even with bacteria expressing characterized CD1d ligands such as Streptococcus pneumoniae and Sphingomonas yanoikuyae, suggesting a minimal role for CD1d presentation of foreign antigen. This relative independence of foreign antigen may be useful when the ubiquity of potential iNKT antigens

is considered,[28] whereas the possibility remains that iNKT-cell activation by foreign antigen is required for the establishment of pathogen-specific memory responses. With interest growing in designing Galunisertib research buy iNKT antigens to learn more modulate an immune response, it is important that they achieve the desired activation of iNKT cells. This in turn depends on the history of each iNKT cell and its current environment: we have seen that iNKT-cell antigens such as those in house

dust are ubiquitous, that iNKT cells can exist in a primed state, and that the activation state of APC strongly influences iNKT-cell activation. Hence, responses from cultured iNKT-cell lines may not recapitulate responses achieved with the same antigen in vivo. In some contexts, antigen is dispensable for iNKT-cell activation, which also merits consideration. Exactly when does an iNKT cell act solely to amplify an innate response? Fuller

understanding of the mechanisms controlling the down-regulation of an iNKT-cell response may also be relevant to understanding the activity of ‘designer’ antigens. It is also interesting to note how many inert CD1d ligands can be isolated. Are these acting as place-holders, sustaining CD1d trafficking through the cell in case more antigenic ligands are produced, or do they perform a necessary role, perhaps as ligands for type 2 NKT cells? Regarding N-acetylglucosamine-1-phosphate transferase β-GlcCer and its role as a key self-antigen for iNKT cells, we need to understand how alterations in β-GlcCer processing and presentation (induced by disease or by the arrival of a new iNKT-cell antigen) impact on the shape of an adaptive immune response. The author has no conflicts of interest to disclose. “
“The effects of nanogel encapsulation of recombinant NcPDI (recNcPDI) following vaccination of mice by intranasal or intraperitoneal routes and challenge infection with Neospora caninum tachyzoites were investigated. Nanogels were chitosan based, with an alginate or alginate-mannose surface. None of the mice receiving recNcPDI intraperitoneal (i.p.) (without nanogels) survived, whereas intranasal (i.n.) application protected 9 of 10 mice from disease. Association of recNcPDI with nanogels improved survival of i.p. vaccinated mice, but nanogels without recNcPDI gave similar protection levels. When nanogels were inoculated via the i.n.

Exogenous particles, as well as autoantigens, are involved in the pathogenesis of T-cell-mediated inflammation. For example, hypersensitivity pneumonitis (HP), including Farmer’s lung and summer-type HP, is a T-cell-mediated inflammation

caused by inhalation of particles, bacteria, etc. 12, 13. Repeated inhalation of organic dust can cause HP, which is characterized MK-2206 purchase by inflammatory lung disease with alveolitis and granuloma formation 13. Hyperactive pro-inflammatory Th1 cells are closely associated with the etiology of HP 14. It is thus important to assess whether Gal-9 might be involved in T-cell-mediated inflammation other than that associated with autoimmune diseases. The purpose of the study presented here RG7204 research buy is to show whether Gal-9 attenuates the severity of murine experimental HP characterized by Th1 and Th17 cell-mediated inflammation. We show that Gal-9 expands CD11b+Ly-6Chigh Mϕ that exhibit immunosuppression of T-cell proliferation and activation, thereby ameliorating Th1/Th17

cell-mediated HP. Preliminary experiments to assess the dose effects of subcutaneously injecting Gal-9 (0.3, 3, and 30 μg/mouse) revealed that 3 μg/mouse of Gal-9 was sufficient to ameliorate experimental HP, although 0.3 μg/mouse was not. Therefore, 3 μg/mouse of Gal-9 was used for further experiments. Significant weight loss was not observed during the course of experimental HP. Histological analyses on day 7 post-challenge with Trichosporon asahii revealed a marked infiltration of inflammatory cells, consisting mainly of mononuclear cells, in alveolar septal, peribronchial, and perivascular areas in PBS-treated mice (Fig. 1A). The histological scores for Gal-9-treated mice (1.68±0.09, n=10) were significantly lower Forskolin mw than those for PBS-treated mice (2.83±0.05, n=10), indicating that Gal-9 exerted a suppressive effect on experimental HP (Fig. 1A). The numbers of BALF cells from both groups of mice were counted. Total BALF cell numbers were similar in both groups until day 3 post-challenge (Fig. 1B). Gal-9 treatment resulted in a significant decrease in total cell number

on day 7 post-challenge. The numbers of specific inflammatory cell types, including Mϕ, PMN, and lymphocytes, were also counted using Giemsa staining. Infiltrated Mϕ exhibited kinetics similar to those of the total cells until day 3, while Gal-9 treatment decreased the number of PMN only in the early phase of experimental HP (6 h to day 1). Increased lymphocyte accumulation was detected in the BALF of PBS-treated mice from days 3 to 7, but this was markedly suppressed by Gal-9 treatment. BALF was obtained from each group on day 7 post-challenge to determine the concentrations of several cytokines by ELISA. As expected, Gal-9 treatment significantly decreased the levels of the pro-inflammatory cytokines IL-1β and IL-6 (Fig. 1C).

The patients underwent open colorectal surgery such as anterior rectal resection, colectomy or rectal amputation. In 44 patients, the indication for operation

was rectal cancer, and four patients were operated on owing to inflammatory bowel disease, Crohns disease or ulcerative colitis. Two patients had both inflammatory bowel disease and colorectal cancer. The patients were randomised into two different groups by the use of sealed envelopes. Both groups.  Before induction, 1 mg of midazolam (Dormicum®; Roche AB, Stockholm, Sweden) was given intravenously and an arterial line was inserted in the left radial artery for repeated blood analyses and continuous find more blood pressure monitoring. A thoracic epidural catheter was inserted in the Thoracic VII-XII interval. All patients also received 0.5 mg of atropine (Atropin Merck NM; Merck NM AB, Stockholm, Sweden) before induction of anaesthesia. Before endotracheal

intubation, fentanyl (Leptanal®; Janssen-Cilag AB, Sollentuna, Sweden) and rocuronium (Esmeron®; Organon AB, Göteborg, Sweden) were given in standard doses. A continuous epidural infusion was started during the operation with bupivacain 5 mg/ml (Marcain® adrenalin; AstraZeneca AB, Södertälje, Sweden) and adrenaline 5 μg/ml at an infusion rate Anti-infection Compound Library mouse of 4–6 ml/h. At the end of the operation, patients were given 5–10 mg of ketobemidon (Ketogan®; Pfizer AB, Sollentuna, Sweden), which is equipotent to 7–15 mg of morphine. Group TIVA.  Patients were anaesthetized with total intravenous technique; a combination of propofol (Diprivan®; AstraZeneca AB, Södertälje, Sweden) and remifentanil (Ultiva®; Glaxo Smith Kline AB, Solna, Sweden) was used. Propofol was administered intravenously

PtdIns(3,4)P2 with Target-Controlled Infusion (Alaris Diprifusor® IVAC TCI and TIVA; Alaris Medical Systems Ltd, Hampshire, UK). The target concentration during induction was 3 μg/ml. The target concentration was decreased to 2 μg/ml during the operation. Remifentanil was administered as a continuous intravenous infusion. The infusion rate at induction was 0.25 μg/kg/min. The infusion rate was then lowered to 0.15 μg/kg/min during surgery. Group INHALATION.  The patients received inhalation anaesthesia with sevoflurane/O2/air. Sevoflurane was used both as induction agent and for maintenance of anaesthesia (VIMA, Volatile Induction and Maintenance of Anaesthesia). Anaesthesia was induced by inhalation of a mixture of sevoflurane/O2/air (Sevorane®; Abbott Scandinavia AB, Solna, Sweden). For maintenance, the end-tidal sevoflurane concentration was kept at 1.4–2.8 vol%. Fentanyl, in repeated intravenous doses of 25–100 μg, was given at the discretion of the anaesthetist. Complement and cytokine measurements.  Blood samples were drawn at four times before, during and after surgery. The first sample (T0) was drawn after insertion of the arterial line before induction of anaesthesia.

2×106 COS-7 cells seeded in 100-mm plates were transfected with 5 μg p3×FlagBTN3Ax PR 171 constructs using 15 μL of FuGENE 6 Transfection Reagent (Roche). The human NK cell line, KHYG-1 is growing in RPMI 1640 medium supplemented with 20%

FCS and 450 UI/mL rIL-2 25. 5×106 KHYG-1 cells were transfected with 2 μg p3×FlagBTN3Ax constructs using the Amaxa™ Nucleofector™ Technology (Solution T, program Y-001) (Lonza Cologne AG). Public and home-made Affymetrix U133+2 data sets of purified CD4, CD8 and NK cells were collected. CD8 and CD4 data were retrieved from the public GEO data sets 26 (http://www.ncbi.nlm.nih.gov/gds), while NK sets were personal. We used Robust Multichip Average (RMA) with the non-parametric quantile algorithm as normalization parameter. RMA was applied to the raw data collected from the various series. Quantile normalization and Loess’ correction were carried out in R using Bioconductor and associated packages. The probe set corresponding to the three isoforms of BTN3A was retrieved from the normalized data sets and the corresponding log values were linearized for graphical representation. We used the respective Affymetrix AZD9668 probe sets corresponding

to BTN3A1, BTN3A2 and BTN3A3 isoforms: STP201623_s_at, 213282_at, 204171_at. Human CD4+ T cells were purified by negative selection from PBMCs using magnetic beads (Miltenyi Biotec) according to the manufacturer’s protocol. CD4+ T cells were routinely more than 97% pure. Cells were incubated 24 h in RPMI 1640 10% FBS at 37°C. CD4+ T cells were washed with PBS 1% FCS and stimulated with aAPCs at a ratio of 1:3 (cells to beads) comprised of magnetic beads (Dynabeads M-450 Epoxy, Dynal Biotech) coated with anti-CD3, anti-CD28 and/or anti-CD277 mAbs as described above. The contacts between cells (106 in 50 μL) and beads

(3×106 in 30 μL) are performed at 37°C in water bath for different times (2, 5, 10 and 30 min) in PBS 1% FCS. Phosphoflow analysis was performed by cytometry as previously described 27. Briefly, cells were fixed and permeabilized, incubated with anti-phospho-Akt ADP ribosylation factor S473 (#4058, Cell Signaling Technology) or anti-phospho-ERK-1/2 T202/Y204 (#4377, Cell Signaling Technology) antibodies and appropriate biotinylated secondary antibodies. Finally, revelation was performed using Streptavidin–phycoerythrin solution (#IM3325, Beckman Coulter). FACS data were acquired on an FACS Canto flow cytometer (BD Biosciences) using the Diva software. FACS data were analyzed using the Flowjo software (TreeStar, Ashland, OR, USA). All data were analyzed using GraphPad Prism version 5.00 for (GraphPad, San Diego, CA, USA) and Microsoft Excel (Microsoft Office). The Mann–Whitney test-matched non-parametric test was used to examine: the variations of CD277 and PD-1 expression from lymphoid tissue on living T lymphocyte subsets (in Fig. 1, Supporting Information Figs.