Correlation between CgA and TNF receptor-I (TNFR-I) and TNFR-II has been evaluated in patients before the initiation of treatment with Infliximab® and compared it with the value calculated selleck compound during treatment [74]. The authors observed a high correlation between

CgA and both receptors. Moreover, they found that treatment with anti-TNF-α monoclonal antibodies (mAbs) abrogated the correlation between CgA and TNFR-I and TNFR-II, but it should be mentioned that in this study anti-TNF-α mAbs treatment did not modify the mean levels of CgA and TNFRs but led only to the abrogation of the correlation between CgA and TNFRs, implying that perhaps other indirect factors are associated in this effect. Three years later, the same group described that patients with RA have significantly higher serum levels of CgA

and TNFRs compared with controls and that the highest levels of CgA identify the population of patients with extra-articular manifestations [74]. Taken together, these results suggest that CgA might be involved in the pathogenesis of inflammatory autoimmune disease through a complex interaction with TNF-α, mediated by as yet-undefined factors. In a series of papers Metz-Boutigue’s group, who have published extensively on granins, showed a link between serum concentration of CgA and outcome in patients admitted with or without systemic PD98059 molecular weight inflammatory response syndrome. CgA concentrations were correlated positively with inflammation markers such as procalcitonin and C-reactive protein, but also with simplified acute physiological score (SAPS). A Cox Orotidine 5′-phosphate decarboxylase model confirmed that CgA and SAPS were independent predictors of outcome [75,76]. In addition, a significant association has been reported between CgA level and periodontitis, again

showing a close relationship between the level of CgA and the inflammatory process [77]. The hypothesis that Cgs-derived peptides are involved in mechanisms modulating altered colonic motility and visceral pain induced by gut inflammation was tested for the first time in 2004 using an application of acetic acid (AA) in vitro and in vivo. Using the writhing test, a model of somato-visceral pain, we have demonstrated that depending upon the Cgs-derived peptides used (bCgA 4–16, 47–66), they could display pro- and anti-nocicpetive effects [78,79]. In the context of smooth muscle contraction, Cgs-derived peptides modulate the effect of AA on human and rat smooth muscle contraction via a direct action on the calcium L-type channel or towards an indirect action through the enteric nervous system (motorneurone and type-C sensitive fibre) [80,81]. All these data provide proof of concept that Cgs and Cgs-derived peptides seem to play an important role in the development of inflammatory pathologies, and different groups have now focused their attention upon characterizing a mechanistic explanation. The studies discussed in this review provide evidence in favour of a key role of gut hormones in intestinal inflammation.

33–36 Other causes of genital inflammation also increase shedding of HIV, even in the absence of a known STI.37,38Neisseria gonorrhoeae has been shown to enhance HIV infection of CD4 cells39 and activated dendritic cells.40 Human papillomavirus (HPV) is receiving renewed attention in the mucosal immunity research. After years of being considered ‘the common cold’ of STI, the development of the HPV vaccine for the prevention of cervical

cancer has allowed for greater research in the area of genital mucosal Vemurafenib immunity. Much of this research has implications for studies involving HIV or risk of HIV. High-risk HPV reactivation has been shown to occur more commonly in HIV-infected women and is associated with an increase in genital shedding of HIV.41 HIV-positive serostatus is also associated with a delay in clearance of both high- and low-risk HPV.42 Disruption of the normal flora is well known to impact the delicate balance of the local genital immune system. Bacterial vaginosis has been associated with increased genital shedding of HIV RNA.43,44 Coleman et al.45 confirmed the importance of vaginal flora in a prospective study of vaginal health among HIV-infected Kenyan women. Antiretroviral naïve, HIV-infected women with normal CD4 counts had paired plasma and cervical wick samples collected for viral load measurement. Women with diminished Lactobacillus had a markedly

increased endocervical viral load, 15.8-fold (95% CI: EGFR inhibitor 2.0–123), compared to women with normal Lactobacillus levels (≥3+). Among women without

HIV, BV has been shown to significantly increase the risk of HIV acquisition, probably selleck chemicals as a function of disruption of natural immunity. In a large meta-analysis of 23 studies and including over 30,000 women, incident HIV was increased by BV, (relative risk = 1.6, 95% confidence interval: 1.2, 2.1).46 Other clinical characteristics that should be considered in studies of female genital tract mucosal immunity include age, body mass index, use of alcohol or substances, recent immunizations, use of systemic drugs (steroids, antiinflammatory agents, immune modulators, chemotherapy), gynecologic procedures (hysterectomy, curettage, biopsies), and vaginal practices. Vaginal practices include the very common practice internationally of vaginal douching. A prospective cohort study of female sex workers in Kenya showed that vaginal washing was associated with an increased risk of HIV acquisition, aHR, 1.47; 95% CI, 1.02–2.13.47 Clark et al.48 examined the effect of douching on vaginal health among HIV-infected women. The prevalence of detectable HIV genital shedding was overall low, 27.3%, compared to that of plasma viral load, 79.8%. While not statistically significant, only 18.9% of non-douchers had genital HIV shedding while 31.9% of women who douched had shedding. Recent intercourse must be noted and a large body of work is focusing on the impact of semen on HIV transmission.

The study was approved by the Ethic Committee of the University of Heidelberg and written informed consent was obtained from the patients. Paraffin-embedded tissue sections (4 μm) were analyzed using the avidin-biotin complex method as previously

described [5]. Prior to Ab incubation, heat pretreatment in an Ag retrieval solution (DAKO Cytomation, Hamburg, Germany; pH 9.0 for neutrophil elastase), respectively, using citrate buffer (pH 6.1 for E-cadherin) was performed. Primary antibodies included a mouse mAb to neutrophil elastase (American Diagnostics, Pfungstadt, Germany, diluted 1:25) and a mouse Ibrutinib research buy mAb to E-cadherin which detects the whole 120 kDa and the soluble ectodomain 82 kDa form (DAKO; diluted R428 mw 1:30). The immunohistochemical analysis was performed on tissue microarrays from 112 PDAC samples. E-cadherin expression was quantified, using the scoring system, previously described by Al-Aynati et al. [42], in which the distribution pattern of E-cadherin expression on tumor cells was quantified as absent (score: 0), focal (10% to <50%; score 1), or diffuse (≥ 50%; score 2). For comparison, healthy pancreas tissue of ten individuals, age and gender matched, were used. Correlation of E-cadherin expression with clinical and pathological parameters was performed

using Spearman’s-Rho analysis; correlation between E-cadherin expression was calculated using Mann–Whitney U-test. The invasion assay was calculated using ANOVA one-way test. For statistical analysis of survival, the nonparametric Log-rank test was performed. Significance levels were defined as p < 0.05. The statistical analyses were carried out with the SPSS software version 18.0 for Windows (SPSS, Chicago, USA). Graphs were made using OriginPro7.5 software (Additive Software, Friedrichsdorf, Germany). We thank Ms. Birgit Prior, Mr. Dieter Stefan, and Dr. Guido Wabnitz, Institute for Immunology, University of Heidelberg and Ms. Sarah Messnard, Institute of Pathology, University of Heidelberg for their excellent technical support. The study was funded by the University of Heidelberg Hospital. The authors declare no financial

or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Table S1. Dyhesion of T3M4 and T3M4 with downmodulated E-cadherin Cell press expression following treatment with either PMN or PMN elastase Table S2. Clinical, pathological parameters and E-cadherin expression of PDAC patients “
“Although various Toll-like receptors (TLRs) have been associated with immune response and tumorigenesis in the prostate cells, little is known about the role of TLR7. Accordingly, we examined the expression of TLR7 during tumour progression of TRMAP (transgenic mouse model for prostate cancer) mice and its role on cell growth. Toll-like receptor7 expression was examined by RT-polymerase chain reaction (PCR), Western blot, and immunohistochemistry.

The following consensus Nutlin-3a supplier guidelines regarding hypertensive donors were adopted: Patients with a BP of 140/90 by ABPM are generally not acceptable as donors. European Renal Association-European Dialysis and Transplant Association: Exclusion criteria include: ‘Reduced

GFR (in comparison to normal range for age), proteinuria of >300 mg/day, microhematuria (except when an urologic evaluation and a possible kidney biopsy are normal), . . . or hypertension without good control’.33 The Canadian Council for Donation and Transplantation:34 It would appear that BP increases by ∼5 mmHg after donating a kidney above the natural increase which occurs with normal aging. Most studies have not suggested an increased rate of hypertension following donation. To date no study using appropriate controls has examined whether donating a kidney increases the risk of premature death or cardiovascular disease over the long-term. This concern has been raised due to the observation that renal insufficiency is an independent risk factor for cardiovascular disease in the general population. Not unexpectedly, there is considerable variability

in practice particularly when it comes to accepting a potential living donor with hypertension or mildly abnormal renal function. In the case scenario involving a 50-year-old male with well-controlled hypertension on a single antihypertensive agent, 5 of 14 centres responded that they would never accept such an individual as a kidney donor. However, other centres would rarely (n = 2), sometimes (n = 5) and usually (n = 2) accept this individual as a living kidney donor.

Reference GSK2126458 cost is also made to recommendations from the Amsterdam Forum, the British Renal Association and the European Renal Association-European Dialysis and Transplant Association. 1 Further prospective studies with appropriate control groups are required in order to determine whether uninephrectomy in normotensive Rapamycin ic50 individuals increases the long-term risk of developing hypertension. Frank Ierino has received Educational Grants and fees for attendance at Conferences/Transplant Symposia from Wyeth, Roche, Janssen-Cilag and Novartis. He has also received an Unrestricted Research Grant from Roche and Novartis, has been a member of the medical advisory boards for Roche and Novartis and a member of the Drug Trial Safety Monitoring Board for Novartis. John Kanellis and Neil Boudville has no relevant financial affiliations that would cause a conflict of interest according to the conflict of interest statement set down by CARI. “
“Date written: April 2008 Final submission: August 2009 No recommendations possible based on Level I or II evidence (Suggestions are based on Level III and IV evidence) A discussion of the effect of dialysis on quality of life (QOL) should be included in the decision-making process for undertaking dialysis treatment.

Our study demonstrated that the population of MHC II+ cells changes during infection and that MHC II+CD11c− non-T, non-B cells become more numerous by approximately 10 days after CHIR-99021 infection. Although these cells are of non-lymphoid lineage, their increase in the spleen depends on the presence of lymphoid cells. These cells produce TNF-α and IL-6; however, their ability to activate specific CD4+ T cells is limited. Rag-2−/− mice were provided by Dr. Y. Yoshikai (Kyushu University, Fukuoka, Japan) [19], and OT-II transgenic mice expressing the TCR specific for OVA323–339/I-Ab by Dr. H. Kosaka (Osaka University, Osaka, Japan) [20]. These

mice were maintained in the Laboratory Animal Center for Animal Research at Nagasaki University and were used at the age of 8–14 weeks. C57BL/6 (B6) mice were purchased from SLC (Hamamatsu, Japan). All animal experiments were conducted according to the Guidelines of the Laboratory Animal Center for Biomedical Research at Nagasaki University. For adoptive transfer, Rag-2−/− mice were administered spleen cells (5 × 107) from B6.Ly5.1 mice i.v. via the tail vein. Mice were infected with P. yoelii 17XNL (P. yoelii) by i.p. injection of 1 × 104 iRBCs. The degree of parasitemia was monitored by

microscopic examination of standard blood films. Mouse spleens were cut into small fragments and incubated selleck compound with Hank’s balanced salt solution containing collagenase (400 U/mL, Wako)

for 45 min at 37°C. Bone marrow cells were collected from mouse femurs by flushing with medium. After lysing RBCs with Gey’s solution, the FcRs were blocked with anti-FcR mAb (2.4G2, 10 µg/mL) for 15 min at 4°C and the splenocytes stained with fluorochrome-conjugated mAbs specific for CD3 (145-2C11), CD19 (1D3), CD11c (N418), MHC II (M5/114), CD45R (RA3-6B2), CD45.1 (A20), CD80 (16-10A1), CD86 (GL-1), CD138 (281-2), IgM (11/41), IgD (11-26c), IgG1 (RMG1-1), IgG2a/2b (R2-40), Ly6C (AL-21), Ly6G (1A8), CD11b (M1/70), F4/80 (BM8), NK1.1 (PK136) and their isotype controls (all from e-Bioscience, San Diego, CA, USA) or with allophycocyanin-anti-PDCA-1 (Miltenyi Biotec, Gladbach, Germany). 7-AAD was used to gate out DNA Synthesis inhibitor dead cells and flow cytometry performed using FACS Canto II (BD Bioscience, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). To purify subpopulations of MHC II+ cells, FcRs were blocked with anti-FcR mAbs and splenocytes stained with PECy7-anti-CD3, PECy7-anti-CD19, PE-anti-CD11c, and FITC-anti-MHC II and biotin-anti-IgM mAbs plus APC-streptavidin, then labeled with anti-Cy7 Microbeads (Miltenyi Biotec). CD3+ and CD19+ cells were depleted using AutoMACS (Miltenyi Biotec). 7-AAD was added to exclude dead cells and MHC II+CD11chiCD3−CD19− (DCs), MHC II+CD11c−CD3−CD19−IgM+, and MHC II+CD11c−CD3−CD19−IgM− populations were sorted using a FACS Aria II (BD Biosciences).

We should point out that TSLP can also activate mast cells SAHA HDAC cell line [63]. Enterocytes also produce high amounts of TGF-β[64]. This cytokine functions by inhibiting the activity of NF-κB on the promoters of proinflammatory genes in macrophages and DCs [65]. Together with TSLP, TGF-β induces a tolerogenic phenotype in myeloid-derived

DCs in vitro[66]. TGF-β produced by DCs promotes a Th3 regulatory phenotype in some naive T cells in MLN [67]. TGF-β is also present in human milk [68], and rodent enterocytes have TGF-β receptors [69]. TGF-β is involved in suppressing inflammatory responses in the neonatal gut and in consolidating the barrier function of the intestinal mucosa [70,71]. Enterocytes also influence antibody production in the intestinal mucosa; through TSLP secretion, enterocytes promote B cell activating factor (BAFF) and APRIL (a proliferation inducing

ligand) production by adjacent DCs and class-switching of B cells towards the production of sIgA [72,73]. APRIL synthesis is initiated after bacterial stimulation of TLR-4 [74] and results in IgA2 production, an isoform of IgA which is more resistant to proteolysis [75]. After synthesis, sIgA translocates to the intestinal lumen via pIgR; once in the gut lumen, sIgA acts in favour of decreasing the antigenic pressure generated by food and microbes on the mucosa. Among intraepithelial cells, M cells and enterocytes are capable of mediating the encounter between antigens within the gut lumen and DCs. M cells are dedicated to this function, Omipalisib cell line differing from normal

enterocytes which are only secondarily involved in antigen presentation. M cells are located above Peyer’s patches (PP) in the small intestine and in close contact with luminal antigens, due to reduced glycocalyx and mucin secretion. They have a particular morphology that allows them to promote uptake and Bumetanide transport of luminal content to professional antigen-presenting cells present in Peyer’s patches and lymphoid follicles. M cells possess fewer lysosomes [76], probably indicating a low intracellular antigen degradation, and are present mainly in the small bowel, but also in the colon, rectum or respiratory tract [77]. They are very low in number, counting for only one cell for every 10 million normal enterocytes. Human and mouse M cells express important PRRs, such as TLR-4, platelet-activating factor receptor (PAFR) and α5b1 integrin [78]. These molecules, belonging to the innate immune system, recognize PAMPs and mediate translocation of bacteria across the epithelium. Jejunal M cells express major histocompatiblity complex (MHC)-II and contain acidic endosomal and prelysosomal structures, indicating that they are able of presenting endocytosed antigens to lymphocytes [79]. It is noteworthy that colonic M cells do not express MHC-II antigens, suggesting that they may not present antigen [80].

To distinguish whether BMPs inhibited differentiation or if the reduced percentages of plasmablasts mainly were a result of reduced proliferation, naive and memory B cells were labeled with CFSE prior to culturing in the presence of CD40L/IL-21 with or without various BMPs.

This experimental design made it possible to follow differentiation per cell division. Of Osimertinib mw the memory B cells that had divided four times or more, only 6.5% of the BMP-6-treated cells compared with 21% of the BMP-7 treated cells had differentiated to CD38+ cells (Fig. 3C). This shows that BMP-6, more potently than BMP-7, inhibits plasma cell differentiation. These findings were further confirmed by division slicing 37 and subsequent calculations of percentage of cells in each cell division that had differentiated to CD27+CD38+ plasmablasts. Small molecule library in vivo This approach identified BMP-6 as the most potent suppressor of CD40L/IL-21-induced plasma cell differentiation, whereas BMP-2 and -4 had intermediate suppressive effects and BMP-7 had limited effects (Fig. 3D). CFSE tracking of cell division further

showed that the BMPs inhibited cell cycle progression as the percentage of cells that had divided four times or more was reduced in the BMP-treated cells (Fig. 3C and not shown). Taken together, the data shown suggest that BMP-6 inhibits Ig production mainly by inhibiting plasma cell differentiation, but also via suppression of proliferation. To further investigate plasma cell differentiation, we sorted memory B cells by FACS and cultured them

with CD40L/IL-21 in the presence or absence of BMP-6 and BMP-7 for 5 days and then analyzed the acquisition of the Clostridium perfringens alpha toxin plasma cell markers IRF-4, CD138 and XBP-1 by immunocytochemistry. Freshly purified memory B cells had no or low expression of IRF-4 and no detectable level of XBP-1 (Fig. 4). After 5 days of culture in the presence of CD40L/IL-21, 84% of cells expressed IRF-4 and 50% of them co-expressed XBP-1 (Fig. 4 and Supporting Information Fig. 4). CD138 was not detected (not shown), indicating that the differentiated cells were plasmablasts, and not fully mature plasma cells. In contrast, fewer cells were present when they had been cultured in the presence of BMP-6 or BMP-7 (compare Hoechst staining across the different culture conditions), and 44 and 36% of them expressed IRF-4 in the presence of BMP-6 and BMP-7 respectively. These data further support the finding that BMP-6 and BMP-7 block CD40L/IL-21-induced differentiation to plasmablasts (Fig. 4). We have previously shown that BMP receptors can be detected by flow cytometry 38. To characterize BMP receptor expression in naive and memory B cells, CD19+ cells from peripheral blood were stained with anti-BMP receptor Abs combined with detection of CD27 and CD20.

15 In this study, we have shown that both CD14 and CD36 were responsible for the uptake of FSL-1 (Figs. 9 and 10), although it remains unknown how CD14 and CD36 in lipid rafts play roles in clathrin-dependent endocytosis. Therefore, studies are in progress to elucidate the detailed mechanism selleck screening library of FSL-1 uptake by CD14 and CD36. Mycoplasmas are wall-less prokaryotes characterized by small genomes, and known as the smallest self-replicating organisms.43 Lipoprotein, an integral component of mycoplasmal cell membrane, is a potent pathogenic factor in mycoplasmal infections.44–47 This study showed that the diacylated lipopeptide FSL-1, the active entity

of mycoplasmal lipopeptide, was internalized by a clathrin-dependent endocytosis. Some pathogenens, such as influenza A viruses, PD0325901 chemical structure adenoviruses and the bacterial pathogen Listeria monocytogenes, use clathrin-dependent

endocytosis as an invasion mechanism into target cells.48,49 Some mycoplasma species are also known to have invasive properties to host cells,43 but their invasion mechanism still remains unclear. For example, Mycoplasma penetrans, which is the most representative invasive mycoplasma, is known to possess a 65 000 molecular weight fibronectin-binding protein, which is considered to play an important role for its adhesion on a host cell.50 Our finding that the lipopeptide FSL-1 derived from mycoplasmal membrane protein is internalized by a clathrin-dependent endocytosis strongly suggests that membrane lipoproteins play a key role in the invasion of mycoplasmas into host cells. Studies to clarify the roles of mycoplasmal

lipoproteins in invasion into host cells are in progress. This work was supported by Grants-in-Aid for Scientific Research (B19390477 and C19592166) provided by the Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B2179178009) provided by the Ministry of Education, Culture, Sports, Science and Technology, and Grants-in-Aid provided by the Akiyama Foundation (PK430031). The authors have no financial conflict of interest. “
“Hematopoietic Ibrutinib manufacturer Stem Cell Laboratory, Lund University, Lund, Sweden Virus-like particles (VLPs) of human papillomavirus (HPV) are used as a vaccine against HPV-induced cancer, and recently we have shown that these VLPs are able to activate natural killer (NK) cells. Since NK cells collaborate with dendritic cells (DCs) to induce an immune response against viral infections and tumors, we studied the impact of this crosstalk in the context of HPV vaccination. NK cells in the presence of HPV-VLPs enhanced DC-maturation as shown by an upregulation of CD86 and HLA-DR and an increased production of IL-12p70, but not of the immunosuppressive cytokine IL-10. This activation was bidirectional.

In humans, systemic T-cell responses to allergens in healthy individuals are dominated by TGF-β and/or IL-10. Asthmatic children have reductions in the numbers of pulmonary Foxp3+ Treg cells, whereas the number of PLX4032 cell line Treg cells inside the allergen-challenged adult lung is clearly enhanced. This suggests that the function of Treg cells might be suppressed

in adults with asthma [130, 131]. TNF-α, IL-6, and TSLP are all overproduced in asthmatic airways and could be responsible for inhibiting the function of Treg cells [132]. The exact mechanism by which Treg cells are induced and recruited to the lungs of asthmatic patients and mouse models of asthma is being intensely studied. Initially, it was shown that DCs expressing ICOS-L and IL-10 were critical for inducing iTreg cells [133, 134]. It was also proposed that plasmacytoid DCs are necessary for Treg-cell

formation and/or expansion in the lungs [39, 135]. Recently, Siglec-F+ alveolar macrophages were found to be the major APC driving the differentiation of Foxp3+ Treg cells in the lungs of mice following allergen inhalation, in a process requiring TGF-β and the retinal dehydrogenases, RALDH-1 and RALDH-2) [136]. The means by which Treg cells become attracted to the allergically PXD101 in vitro inflamed lungs and LNs of mice involves the CCR4 and CCR7 receptor, respectively [137]. The main source of the CCR4 ligands, CCL17 and CCL22, is the CD103+ cDC subset of the lungs [34] and targeting antigens to these sDCs using a Ag-conjugated CD103 moAb has been shown to lead to the expansion and/or accumulation of Treg cells in the lungs [138]. The exact contribution of the intestinal (or pulmonary) microbiota to the induction of Treg cells in the gut and/or lungs is another topic of great interest. Several experiments have now shown that germ-free

mice or mice treated with broad-spectrum antibiotics at a very young age have increased features of allergic disease, including increased numbers of basophils and NKT cells [139-143]. These treatments also affect the lung microbiota, but we do not understand the full impact of this on asthma at present [144]. It is possible that the airway microbiota also regulate the threshold for epithelial Tideglusib and immune cell TLR activation, just as the gut microbiota does in colonic epithelium. Given the clear evidence for IL-4 and/or IL-13 in mouse models of allergic disease, and the presence of Th2 cytokines in patients with asthma, several clinical trials with inhibitors of these cytokines have been launched. A humanized anti-IL-4 neutralizing antibody (pascolizumab) showed promising results in human-derived cell lines and monkeys [145]. However, IL-4-specific antagonists (the IL-4 variant pitrakinra) used in clinical trials have failed to show convincing clinical results [146]. For IL-13, several neutralizing antibodies have been developed (IMA-638, AMG317 (lebrikizumab), and CAT-354), but trials are still in their infancy.

8% replicating cells in the FoxP3+ subset. In contrast, MAPK inhibitor TNF treatment resulted in replication of only 10.8% of FoxP3− cells replicating (Fig. 3A right

panels). Thus, IL-7 also enabled TNF to preferentially stimulate the proliferation of Tregs (p<0.001, Fig. 3B). We also investigated the effect of IL-7 with or without TNF on the proliferative responses of flow-sorted CD4+FoxP3/gfp+ Tregs to TCR stimulation. As shown in Fig. 3C, although IL-7 by itself only had minimal effect, a combination of TNF and IL-7 synergistically promoted the proliferation of Tregs. Next, we examined the effects of TNF/IL-7 on the expression of FoxP3 and TNFR2 on Tregs. As shown in Fig. 3D, after 3-day treatment with IL-7 alone, the proportion of FoxP3+ Tregs present in CD4+ T cells was only ∼4%, which was lower than that in freshly isolated CD4+ T cells (∼10%) or CD4+ T cells cultured with IL-2 (10 ng/mL, >10%) selleck inhibitor for 3 days. Even the higher molar concentration of IL-7 was not as effective as IL-2 in the maintenance of survival of Tregs. Nevertheless, TNF in conjunction

with IL-7 was able to increase the proportion of FoxP3+ cells (Fig. 3D), in a dose-dependent manner (Fig. 3E). Furthermore, in the presence of IL-7, TNF increased the proportion of TNFR2+ cells in the FoxP3+ subset, but not in FoxP3− cells (Fig. 3F), indicating that IL-7 could also promulgate the Treg-activating effect of TNF. To eliminate a possible effect of IL-2 released by activated FoxP3− Teffs present in the unfractionated CD4+ T cells, neutralizing anti-IL-2 Ab was used. As shown in Fig. 3G and H, in the presence of as high as 10 μg/mL of neutralizing anti-IL-2 Ab, TNF/IL-7 still Janus kinase (JAK) up-regulated TNFR2 expression on Tregs and expanded FoxP3+ cells (p<0.05). Furthermore, treatment with TNF alone for 24 h also resulted in an increase of TNFR2 expression on Tregs, which was not blocked by the neutralizing anti-IL-2 Ab (Fig. 3I and J). Thus, the effect of TNF on the proliferation of Tregs and up-regulation of TNFR2 on Tregs can occur independently

of IL-2. Next, we examined whether 4-1BB and OX40 induced on Tregs by TNF were functional. As shown in Fig. 4A and B, both agonistic anti-4-1BB and anti-OX40 Abs were able to partially overcome the anergic status of Tregs and induced proliferation of Tregs. Furthermore, the combination of TNF and anti-4-1BB Ab or anti-OX40 Ab synergistically stimulated the proliferation of Tregs (p<0.05–0.001. Fig. 4A and B). In contrast, isotype control IgGs did not have any effect (data not shown). CD4-depleted splenocytes were used as APCs in this study and they expressed OX40L and 4-1BBL (data not shown). We therefore examined the effect of blockade of OX40L and 4-1BBL on the proliferation of Tregs. As shown in Fig. 4C, TNF-induced proliferative responses of CD4+FoxP3/gfp+ Tregs to APC stimulation was partially abrogated by blocking antibodies to OX40L and to a greater extent by anti-4-1BBL Ab (p<0.05).