It is, however, unclear whether these Abs have any impact on virus elimination. In the current study, we have addressed this https://www.selleckchem.com/products/apo866-fk866.html question by infecting B-cell-sufficient mice with an impaired ability to produce antigen-specific Abs with low doses of LCMV strains that

differ in their replication speed. The results revealed that the requirement for adaptive humoral immunity to control the infection is dependent on the replicative capacity of the viral strains used. Ab transfer experiments further demonstrated that nonneutralizing NP-specific IgG Abs were capable of accelerating virus elimination in vivo. Surprisingly, these Abs functioned in an Fcγ receptor (FcγR) and C3 complement-independent manner. To overcome the caveats of mice lacking B cells, B-cell-sufficient MD4 mice were used. MD4 mice express a transgenic B-cell receptor specific for hen egg lysozyme and due to allelic exclusion, their B-cell repertoire is compromised [15]. For our experiments, we used the LCMV strains Armstrong, WE, and Docile, which differ in their replication speed (Docile > WE > Armstrong) [16]. MD4 mice were first infected with the slowly replicating LCMV strain Armstrong using a low virus infection dose (200 PFU). This induced a strong GP33- and NP396-specific

CD8+ T-cell response and marked upregulation of the effector cell marker killer lectin-like receptor G1 (KLRG1) on CD8+ T cells similar as in B6 wild-type mice (Fig. 1A). As in wild-type mice, virus was completely cleared in spleen, liver, and lungs of MD4 mice at day 8 postinfection (p.i.) (Fig. 1B). Selleckchem Cabozantinib The same result was obtained with IgMi mice, which are severely impaired in the production of soluble Abs due to a mutated IgH gene locus [17] (Supporting Information Fig. 1). These data demonstrate that MD4 and IgMi mice were not inherently impaired in mounting a potent LCMV-specific CD8+ T-cell response and that an adaptive Ab response was not required to control LCMV Armstrong infection. When the faster replicating LCMV strain WE was used, we observed a decrease in KLRG1 induction

and fewer GP33-specific CD8+ T cells in MD4 compared with B6 wild-type mice at day 14 p.i. (Fig. 1C). Virus elimination selleck inhibitor in the spleen was delayed, nevertheless, virus was cleared in these mice as well (Fig. 1D, left). Similar to MD4 mice, virus clearance was also delayed in IgMi mice (Fig. 1D, right). Thus, after LCMV WE infection, the virus-specific CD8+ T-cell response and virus elimination were delayed in the absence of an Ab response. Most strikingly, infection of MD4 mice with the fast replicating LCMV strain Docile led to classical signs of CD8+ T-cell exhaustion indicated by low KLRG1 expression, strongly decreased IFN-γ production and significant expression of the exhaustion markers, PD-1 and 2B4 (Fig. 2A and B). LCMV Docile infected B6 wild-type mice mounted a vigorous CD8+ T-cell response characterized by high-KLRG1 expression and potent IFN-γ production.

, 2006a; Bragonzi et al., 2009; Hoboth et al., 2009; Rau et al., 2010). Thus, the propensity for genetic change appears to be important for the adaptation of P. aeruginosa Navitoclax concentration isolates for chronic infection. We have previously shown that clinical isolates of P. aeruginosa indeed generate higher morphotypic and phenotypic diversity when grown as biofilms than does the laboratory strain of P. aeruginosa PAO1 (Kirov et al., 2007; data not shown). We now report that variants derived from in vitro grown biofilms have regained hallmarks of acute infection isolates, suggesting a mechanism by which biofilm growth may contribute to acute exacerbations associated with chronic

infection in the CF airway. We compared the dispersal response of a panel of clinical isolates from patients with CF and showed that all strains exhibited cell death and seeding dispersal from biofilms, high morphotypic diversity and the production of superinfective phage during dispersal (Kirov et al., 2007). Pseudomonas aeruginosa strain 18A was selected from that panel of clinical isolates as a representative strain for further study here. The phenotypes tested in this study included metabolic capacity, virulence factor production and colonisation traits. In comparison with strain PAO1, functional diversification was greatest in the dispersal progeny

of the chronic infection CF isolate, strain 18A. For both strains, the development of stable genetic variants was a feature of biofilm dispersal and was not observed in planktonic cultures. selleck inhibitor The diversification in metabolic capacity may play a crucial role in the establishment of chronic, persistent pulmonary infections of P. aeruginosa in patients with CF. For example, the ability of P. aeruginosa to catabolise alanine is known to provide a competitive advantage over other bacterial strains in vivo (Boulette ROS1 et al.,

2009) and could therefore explain why the clinical strain 18A is able to utilise alanine while the laboratory strain PAO1 cannot. Additionally, Hoboth et al. (2009) reported that clinical CF P. aeruginosa isolates that are hypermutators have increased amino acid uptake. These authors further suggested that ornithine metabolism may play a pivotal role in adaptation within the patient’s lungs. Hence, the higher mutation frequency of strain 18A compared to strain PAO1 may be linked to the increased substrate utilisation by the clinical strain and its biofilm variants. The ability to grow on d-alanine, l-alaninamide and l-ornithine was consistently lost in the dispersal population of the clinical isolate strain 18A. This may be a consequence of biofilm development on a glucose medium in contrast to sputum that contains a range of amino acids, including ornithine and alanine (Palmer et al., 2005, 2007).

The study was approved by the local ethics committee (journal number H-C-2007–0123) and all subjects included gave written informed consent before enrolment. All subjects were sensitized with DPCP in acetone on buttock skin. Petrolatum-backed 11-mm filter disks were soaked in 50 µl

of 0·0625% DPCP in acetone (25 µg/50 µl). Each filter disc was mounted inside a 12-mm aluminium Finn chamber® and taped to the skin (Scanpor®; Epitest Oy, Tuusula, Finland). The disks were left for 48 h. Challenges were carried out on the upper inner arm 3 weeks after sensitization, using four concentrations BIBW2992 datasheet of DPCP in acetone (0·39, 0·78, 1·56, 3·125 µg/15 µl) and one acetone control on 7-mm filter discs in 8-mm Finn chambers®. The discs were left for 6 h. The challenge sites were all marked with a surgical skin marker for evaluation 48 h later. Sensitization as well as challenge was performed on healthy skin. The elicitation

responses were assessed using a visual score, as suggested by Cooper and co-workers [10]: 1 = no reaction, 2 = mild, macular erythema, 3 = moderate erythema, occasionally with population, 4 = strong erythematous reaction (including vesicular changes) and 5 = extreme or spreading reaction (including bullous or ulcerative reaction). Palbociclib The sum increase in clinical score was calculated as the sum of values at the five challenge sites. Increase in dermal thickness, measured using the ultrasound technique, has been shown to correlate well with the strength of an elicitation reaction [11]. In addition to visual scoring, dermal thickness of each elicitation site was determined using a high-frequency ultrasound scanner (Dermascan, Cortex Technology,

Horsens, Denmark). Twelve-mm scanned images were recorded pre- and post-challenge. Five dermal thickness measurements were taken from each pre- and post-challenge scan at fixed distances of 2, 4, 6, 8 and 10 mm along the horizontal length 4-Aminobutyrate aminotransferase of the scanned image. A mean percentage increase in dermal thickness was calculated for each elicitation site. Two 4-mm punch biopsies were taken from each patient, one from the challenge area where the highest dose of DPCP (3·125 µg/15 µl) had been applied and one from the area where acetone had been applied. The biopsies were taken 48 h after challenge. Biopsies were taken from 29 individuals; 11 were used for immunohistochemistry and 17 were used for the microarray study. Biopsies taken from 11 individuals, six patients with psoriasis, two of whom had a positive elicitation reaction and five healthy controls, three of whom had a positive elicitation reaction, were prepared for immunohistochemistry. These skin biopsies were embedded in Tissue Tek octreotide (OCT) compound (Sakura Finetek, Zoeterwoude, the Netherlands), frozen instantly in liquid nitrogen and stored at −80°C until use.

The amplified DNA fragments were ligated to pGEM-T Easy vector DNA, yielding recombinant plasmids pGEM-T/Rv3874, pGEM-T/Rv3875 and pGEM-T/Rv3619c, respectively. The DNA fragments corresponding to rv3874, rv3875 and rv3619c genes from recombinant pGEM-T were subcloned in the expression vector pGES-TH-1,

and their identity was confirmed by DNA sequencing (data not shown). E. coli IWR 1 BL-21 cells were transformed with recombinant pGES-TH-1, and SDS–PAGE analysis of cell lysates from transformed E. coli showed the expression of proteins that corresponded to the size of GST/Rv3874 (Fig. 2, panel A, lane 3), GST/Rv3875 (Fig. 2, panel A, lane 4) and GST/Rv3619c (Fig. 2, panel B, lane 3). The E. coli cells carrying the parent plasmid (pGES-TH-I) also expressed free GST that migrated to its expected position (30 kDa) in the gel (Fig. 2, panel A and B, lane 2). The absence of major protein bands at these positions with the parent E. coli cells (Fig. 2, panel A and B, lane 1) implied that the major protein bands in transformed E. coli cells were as a result of the expression of additional proteins from the parent or recombinant

plasmids. The identity of the expressed fusion proteins was established by Western immunoblotting with anti-GST and anti-penta His antibodies. There was no reaction with any cellular protein from the negative control (parent E. coli BL-21 cells) (Fig. 2, panel C, D, E, F; lane 1), while Ivacaftor mw the GST protein alone, expressed from the parent plasmid (pGES-TH-l), reacted with the anti-GST antibodies and anti-penta His antibodies, as expected (Fig. 2, panel C, D, E, F; lane 2). A major band of reactivity was obtained with anti-GST antibodies for GST-Rv3874, GST-Rv3875 (Fig. 2C; lane 3, 4, respectively), and GST-Rv3619c (Fig. 2E, lane

3), and with anti-penta His antibodies for GST-Rv3874, GST-Rv3875 (Fig. 2D; lane 3, 4, respectively), and GST-Rv3619c fusion proteins (Fig. 2F, lane 3), which corresponded with the major protein band in Coomassie blue-stained gels and to the expected migration positions of the three fusion proteins. The SDS–PAGE analysis of cell-free extracts and pellets of sonicates Meloxicam of induced E. coli cells containing pGES-TH/Rv3874, pGES-TH/Rv3875 and pGES-TH/Rv3619c showed that GST-Rv3874 and GST-Rv3875 proteins were present in the soluble fraction (Fig. 3A, B, lane 1), whereas GST-Rv3619c was present in the pellet, which solublized best in 4 m urea (Fig. 3C, lane 1). To purify the recombinant mycobacterial proteins, the soluble/solublized fractions were loaded on to glutathione-Sepharose affinity matrix and the GST-free mycobacterial proteins were released from the fusion proteins bound to the column matrix by cleavage with thrombin protease. The analysis of eluted fractions by SDS–PAGE showed that the recombinant Rv3874 and Rv3875 proteins were contaminated with another protein of nearly 70 kDa (Fig.

A CLP polymicrobial sepsis model was applied to the rats. All groups were killed 16 h later, and lung, kidney and blood samples were analysed histopathologically and biochemically. Sildenafil increased glutathione (GSH) and decreased the activation of myeloperoxidase (MPO) and of lipid peroxidase (LPO) and levels of superoxide dismutase (SOD) in the septic rats. We observed a significant decrease in LPO and MPO and a decrease in SOD activity in the buy Silmitasertib sildenafil-treated CLP rats compared with the sham group. In addition, 20 mg/kg sildenafil treatment in

the sham-operated rats improved the biochemical status of lungs and kidneys. Histopathological analysis revealed significant differences MLN8237 in vivo in inflammation scores between the sepsis group and the other groups, except the CLP + sildenafil 10 mg/kg group. The CLP + sildenafil 20 mg/kg group had the lowest inflammation score. Sildenafil treatment decreased the serum tumour necrosis factor (TNF)-α

level when compared to the CLP group. Our results indicate that sildenafil is a highly protective agent in preventing lung and kidney damage caused by CLP-induced sepsis via maintenance of the oxidant–anti-oxidant status and decrease in the level of TNF-α. Sepsis is a systemic inflammatory response to infection and a major cause of morbidity and mortality worldwide. Sepsis may result in hypotension and organ dysfunction called septic shock [1]. Sepsis/septic shock is characterized by profound hypotension, progressive metabolic acidosis, systemic inflammatory response syndrome (SIRS), tissue damage and multiple Calpain organ dysfunction syndrome (MODS), acute respiratory distress syndrome (ARDS) and/or acute lung injury (ALI), or even death. Although its pathophysiology is not well defined, monocytes orchestrate the innate immunity response to Gram-positive and Gram-negative bacteria by expressing a variety of inflammatory cytokines, including tumour necrosis factor (TNF)-α and interleukin (IL)-6, which are considered to play an essential role in the pathogenesis

of sepsis [2–6]. These mediators extend the inflammatory response and can lead to multiple organ dysfunction syndrome [7] and, ultimately, death [8]. Some of these oxidants are known to modulate the expression of various genes that are involved in immune and inflammatory responses [9]. Sepsis and endotoxaemia lead to the production of reactive oxygen species (ROS) [10,11], which have been assumed to play a role in the induction of many proinflammatory cytokines and mediators important in producing the acute inflammatory responses associated with sepsis [12]. Endotoxaemia and sepsis are associated with a reduced endogenous antioxidant capacity, and may therefore result in an oxidant–anti-oxidant imbalance [13].

Some examples of these are, but are not limited to, T-bet, GATA-3, interferon regulatory factor family and Foxp3.90,91 These transcription factors play an important role in the differentiation of T cells, but

are beyond the scope of this review. So far, we have reviewed the transcription factors that are activated downstream Palbociclib in vitro of TCR signalling and how components of the immunological synapse activate them. T cells can differentiate to perform various effector functions, be tolerized or be deleted. All these processes require engagement of TCRs by peptide–MHC complexes and happen over days. Tolerance induction can occur when TCR signals are delivered in the absence of co-stimulatory signals, whereas deletion can occur when high-affinity self-peptide Etoposide mouse interactions occur in the periphery.21 Effector T-cell differentiation occurs as a result of co-operation between TCR, co-stimulatory and cytokine signals.92,93 Differentiation is also accompanied by epigenetic changes occurring at specific promoters, particularly

in the promoters of cytokine genes.9,94 Antigen dose and affinity, however, also play an important role in determining the differentiation state of effector T cells in the absence of polarizing cytokines. O’Garra and colleagues demonstrated that increasing antigen dose led to more IFN-γ production by T cells whereas very low or very high antigen doses caused cells to produce

IL-4.95 Another study, from Bottomly and colleagues, showed that a high dose led to IFN-γ-producing cells whereas stimulation with a lower antigen Doxacurium chloride dose caused cells to produce IL-4.96 A requirement for co-stimulation through CD28 was demonstrated in this system for Th2 responses by way of weak TCR signals.97 Although peptide dose and affinity do show an impact on Th1 versus Th2 choices, Croft and colleagues demonstrated that the time of differentiation also played an important role in determining whether cells produced IL-4 or IFN-γ.98 Bottomly and colleagues also demonstrated that antigen dose affected the balance of NFATp versus NFATc DNA-binding activity, with lower potency ligands favouring higher levels of nuclear NFATc and lower levels of NFATp conducive for IL-4 transcription.99 More recently, Paul and colleagues have explored the mechanism by which high and low doses of peptide induce Th1 versus Th2 responses. They report that T cells stimulated by low peptide concentrations result in IL-2-dependent signal transducer and activator or transcription 5 (STAT5) phosphorylation, TCR-induced IL-4-independent early GATA-3 expression and IL-4 production. Stimulation with a higher concentration of peptide caused, by way of the ERK pathway, abrogation of GATA-3 expression and IL-2-dependent STAT5 phosphorylation and IL-4 production.

These findings demonstrate that the poxviral protein can negatively affect signalling from a mammalian counterpart. Given that viral Pellino can functionally antagonise its mammalian

counterparts and the latter has been demonstrated to participate in multiprotein signalling complexes 14, 27, we next examined the functional regulation of other TLR signalling molecules by viral Pellino. Components of the TLR/NF-κB pathway were expressed at levels sufficient to induce NF-κB activation. Co-expression of viral Pellino led to a substantial inhibition of reporter gene activity mediated by the TIR-containing adaptor proteins MyD88 and Mal (Fig. 8A), whereas TRIF- and TRAM-mediated Everolimus nmr activation of NF-κB was less sensitive to viral Pellino (data not shown). The poxviral protein also displayed inhibitory activity towards NF-κB activation by downstream TLR signalling pathway components IRAK-1, TRAF6 and

IKKβ but not p65 C646 solubility dmso (Fig. 8A). The lack of effect of viral Pellino on p65 suggests specificity of action for viral Pellino, albeit with multiple targets. The regulation of a number of these signalling molecules by viral Pellino is consistent with its functional antagonism of mammalian Pellinos. Since Pellinos interact with IRAK-1 and TRAF-6 and promote polyubiquitination of IRAK-1 that subsequently recruits IKK-containing complexes, it is not surprising that viral Pellino-induced degradation of mammalian Pellinos negatively regulates IRAK-1, TRAF6 and IKKβ. However, viral Pellino also showed inhibitory potential upstream of IRAK-1 in functional assays, suggesting that viral Pellino targets signalling molecules beyond IRAK-1. Indeed, this is further corroborated by our earlier findings demonstrating that truncation mutants of viral

Pellino, lacking a FHA domain, fail to interact with IRAK-1 and yet partially retain inhibitory effects on TLR signalling. We thus next investigated other potential targets for viral Pellino and more specifically probed whether it could also interact with the TIR adaptor Levetiracetam proteins, MyD88 and Mal, given their sensitivity to viral Pellino. Co-immunoprecipitation studies demonstrated that viral Pellino can associate with MyD88 (Fig. 8B, upper panel) and Mal (Fig. 8C, upper panel). Interestingly, in the case of both adaptors, interaction with viral Pellino led to reduced levels of adaptor protein (Fig. 8B and C, second panels). Such effects on the expression levels of the adaptor proteins were observed reproducibly and appear to represent some degree of specificity, given that viral Pellino fails to affect the expression levels of co-expressed IRAK-1 (Fig. 4A and B). The lack of an intact RING domain eliminates the possibility that viral Pellino itself can directly induce polyubiquitination and subsequent degradation of TLR signalling components, suggesting that it may recruit an intermediary protein capable of such regulation.

To date, there have been no large reports of their success in the anatomical region with the highest free flap failure rate, the lower extremity. Methods: A retrospective review of 67 consecutive patients who underwent lower extremity microvascular reconstruction performed from August 2003 to September 2010 was performed. Patient charts were reviewed for age, sex, medical comorbidities, HDAC inhibitor mechanism etiology of defect, location of defect, flap type, anastomotic technique, complications, flap survival, and limb salvage outcome. Results:

No patients returned to the operating room to have an arterial or venous anastomosis revised. Despite 100% vascular anastomosis patency rates in 67 consecutive lower extremity free flaps, flap survival rate was 95.5%. Total complication rate (13.4%) was due to two partial and one complete flap loss, three infections, two skin graft loses, and one hematoma. There were no intraoperative or perioperative complications involving the use of a microvascular anastomotic coupling device itself. Thirty-day and long term limb salvage rate was 97% and 92.5%, respectively. Conclusion: Microvascular anastomotic coupling devices create

effective venous anastomoses in lower extremity microvascular reconstruction. Thus, it presents an important tool in the armamentarium for lower extremity microsurgical reconstruction. Selleck DAPT © 2011 Wiley-Liss, Inc. Microsurgery 2011. “
“Defects sustained at the distal forearm are common and pedicled perforator flaps have unique advantages in resurfacing it. The purpose of this study is to reappraise the anatomy of the perforator in the posterolateral aspect of the mid-forearm and present our clinical experience on using perforator flaps based on it for reconstruction of defects in the distal forearm. Methods: This study was divided into anatomical study and clinical application. In the anatomical study, 30 preserved upper limbs were used. Clinically, 11 patients with defects

at the forearm underwent reconstruction with the posterolateral mid-forearm perforator flaps. The defects, ranging from 4.5 × 2.5 cm to 10.5 × 4.5 cm, were located at the dorsal aspect of the distal forearm Reverse transcriptase in 6 cases and at the volar aspect of the distal forearm in 5 cases. Three patterns of the perforator were observed in the posterolateral aspect of the mid-forearm, which originated from the posterior interosseous artery, the proximal segment of the radial artery or the radial recurrent artery, and the middle segment of the radial artery, respectively. The perforator was located 11.8 ± 0.2 cm to 15.8 ± 0.4 cm inferior to the lateral humeral epicondyle. Clinically, flaps in 8 cases survived uneventfully, while the other 3 cases suffered mild marginal epidermal necrosis, which was cured with continuous dress changing.

trachomatis infection and in the development of disease. Therefore, while our data indicate that C. trachomatis infection may generally induce susceptibility to NK cell activity,

we hypothesize that an individual’s NK2GD and MICA allelic composition may modify the degree of protection conferred by NK cells. Thus, in some individuals, find more a specific NKG2D and MICA allelic composition may facilitate C. trachomatis’ escape from the NK cell-mediated immune response more efficiently than other alleles. Such possibilities may explain why C. trachomatis infection remains an endocervical infection is some women but establishes acute ascending infection in others. They may also provide insight into why infection may be spontaneously cleared in several weeks or months in some individuals but remain for highly extended periods of time in others (Morre et al., 2002; Molano et al., 2005; Brunham & Rekart, 2008). This work was supported by NIH grants U19AI061972 and AI095859 and by the Louisiana Vaccine Center and the South Louisiana Institute for Infectious Disease Research

click here sponsored by the Louisiana Board of Regents. We thank Connie Porretta for technical assistance with flow cytometric experiments and Dr. Tim Foster for insightful comments with respect to data presentation. tetracosactide “
“Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USAFax: +1-617-525-5566 Intracellular pathogen-specific antibodies (Abs) can contribute to host protection by a number of different mechanisms. Ab opsonization of pathogens residing outside a host cell can prevent infection of

target cells either via neutralization of the critical surface epitopes required for host cell entry, complement-mediated degradation, or via subsequent intracellular degradation. In the case of intracellular localization, Abs can bind to infected cells and thus mark them for destruction by Fc receptor (FcR)-bearing effector cells. This review focuses on the protective role of Abs against intracellular bacteria and parasites involving FcR interactions that modulate the intracellular trafficking of the pathogen, the ability of FcRs to interfere with the establishment of an intracellular replicative niche and the involvement of FcRs to modulate pathogen-specific T-cell responses. Antibodies (Abs) have been implied in protection against all types of pathogenic organisms, i.e. viruses, bacteria, fungi, and multicellular parasites. In order to fulfill their action against this multitude of pathogens, Abs mediate their protective effects through a wide panel of direct and indirect effector mechanisms.

Thus, a more detailed understanding of the mechanism by which TNFR2 affects the survival of CD8+ T cells

is useful for devising more effective therapies against cancer and autoimmune diseases. B6 and B6.TNFR2−/− mice were obtained from The Jackson Laboratory. Mice of 6–10 weeks of age were used for the experiments. Animal studies were performed according to guidelines established by the Canadian Council of Animal Care and approved by our institutional review board. CD8+ T cells from the lymph nodes of WT and TNFR2−/− mice were purified using miniMACS microbeads selleck (Miltenyi Biotec) according to the manufacturer’s protocol. After purification the cells were stained with anti-CD8 conjugated FITC (eBioscience). FACS analysis of the purified cells indicated that the purified cells were>95% CD8+ (Supporting Information Fig. 1). The purified CD8+ T cells

were cultured at 37°C and 5% CO2 in Iscove’s DMEM (Invitrogen Life Technologies) supplemented with 10% FBS (Invitrogen Life Technologies), 5×10−5 M 2-mercaptoethanol (Sigma), and antibiotics (Invitrogen Life Technologies). Purified CD8+ T cells were cultured with 10 μg/mL RAD001 concentration plate-bound anti-CD3 (2C11) and 20 U/mL IL-2 for 48 h in 96-well flat-bottom plates. Purified CD8+ T cells were incubated with 10 μg/mL plate-bound anti-CD3 and 20 U/mL IL-2 in a 96-well flat-bottom plate for 48 h. The cells were then restimulated with 10 μg/mL anti-CD3 and 20 U/mL IL-2 for another 24 h. In some experiments, anti-TNF-α (R&D Systems), anti-TNFR2 (Biolegend) or control antibodies (purified Armenian hamster IgG from eBioscience) were added during the 24 h restimulation period. At the end of this SPTLC1 24-h culture period, the cells were harvested and stained with 7-AAD (Invitrogen Life Technologies) and annexin V (BD Biosciences Pharmingen) following the manufacturer’s protocols and subsequently analyzed by FACS.

Proliferation assay was performed by incubating 5×105 purified CD8+ T cells with 10 μg/mL plate-bound anti-CD3. Cells were cultured in triplicate in a volume of 0.2 mL in 96-well flat-bottom plate, and 1 μCi [3H]-thymidine (PerkinElmer) was added for the last 8 h of the 48-h culture period. In some experiments anti-TNF-α or anti-TNFR2 antibodies were added to the cultures. Purified CD8+ T cells were cultured with 10 μg/mL plate-bound anti-CD3 and 20 U/mL IL-2 for 48 h. The activated CD8+ T cells were then stimulated with 10 ng/mL TNF-α (R&D Systems) for the indicated time period. Cell lysates were prepared with lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% TritonX-100) supplemented with protease inhibitors (Roche Diagnostics) for 30 min on ice. Protein quantification was determined by DC protein assay (Bio-Rad Laboratories). Thirty microgram of total cell lysates were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocking the filters with TBS containing 0.