In-vitro differentiative potential of MSCs is not restricted
<

In-vitro differentiative potential of MSCs is not restricted

to mesodermal lineages, but their transdifferentiation into other lineages, such as endothelia, could be realized Doxorubicin both in vitro and in vivo [5]. In addition, MSCs exhibit immunoregulatory activities, inhibiting the function of different immune cells of innate and adaptive immunity [6], blocking the division of stimulated T cells, preventing irreversible G0/G1 phase arrest and stopping T cell division in mixed lymphocyte reactions (MLRs) [7]. However, the immunomodulatory activity of the MSCs does not rely solely upon T cells, but also upon the first step of the immune response through the inhibition of dendritic cell differentiation and maturation in antigen-presenting cells [8]. Furthermore, their regulatory activity may be amplified by modulating immune responses via the de-novo induction and expansion of CD4+CD25+forkhead box protein 3 (FoxP3)+ regulatory T cells (Tregs). Tregs play a critical role in peripheral self-tolerance, as well as in the regulation of acquired immunity, by inhibition of lymphocyte proliferation [9, 10]. As well as Tregs developing in the

thymus (natural Tregs), a Treg population can be induced from peripheral naive STA-9090 purchase T cell (inducible Tregs), and these inducible Tregs can be recruited directly by MSC from CD4+ T cells [11, 12]. In recent decades many studies have been published addressing the role of Treg number and function in human autoimmunity [13], suggesting that their possible defective function plays a role in many autoimmune diseases. On this basis, both the regenerative and the immunomodulatory properties of MSCs make them an attractive candidate Thalidomide for cellular therapy in autoimmune diseases. Systemic sclerosis (SSc) is an autoimmune disease in which alteration of cellular immunity, including T and B lymphocytes, has been largely

studied both in the skin and in internal organs [14, 15]. Furthermore, recent evidence has shown an aberrant dendritic cell function in SSc, contributing to the molecular milieu of the disease [16]. We have shown previously that MSCs obtained from SSc patients (SSc–MSC) were normal with respect to clonogenicity and differentiative capacity, although they displayed early senescence and were defective in acquiring some differentiative functions [17]. Senescent MSC generally show a flattened morphology, over-expression of senescence-associated β-galactosidase (β-Gal) activity, reduced telomerase activity and increased expression of both p53 and p21, which are negative regulators of cell proliferation [18]. At present, only few papers have investigated the immunoregulatory activity in SSc.

The significantly expressed genes were selected by a standard cut

The significantly expressed genes were selected by a standard cut-off at twofold increased expression compared with the values on day 0. These differentially expressed genes were then classified based on Gene Ontology (GO) software specifically for genes implicated in the ‘regulation of inflammatory response’ as well as the ‘cytokines and chemokines’ in the colonic epithelium of DSS-induced colitis in mice. Analysis using ICG-001 clinical trial Student’s t-test was applied to in vitro studies. Analysis between

individuals in groups in vivo was by analysis of variance followed by Student’s t-test. Results are expressed as mean ± SEM, and are representative of at least two individual experiments. P < 0·05, was considered https://www.selleckchem.com/products/PD-0325901.html significant. While it has been suggested that IL33 and ST2 are expressed in colonic tissue and in epithelial cells in clinical colitis,[20-23] the kinetics of their expression and relative expression compared with other DSS-induced

genes in inflamed colonic tissue is unknown. To understand the inflammatory process associated with the initiation of colitis, we systematically studied the early colon gene expression profile of DSS-induced colitis by analysing the publicly available microarray datasets deposited in the GEO using a meta-analysis approach.[26, 27] We specifically focused on the expression of cytokines and chemokines, and genes implicated in the regulation of inflammation using the Gene Ontology Analysis module in genespring gx11. Hierarchical clustering analysis showed that IL33 was the strongest of the 40 differentially expressed cytokine Chloroambucil and chemokine genes expressed early in the colonic tissue (see Supplementary material, Fig. S1A). Furthermore, IL33 and its receptor; the ST2 gene (IL1RL1) were the most highly induced

genes, among the 28 genes, involved in the regulation of the inflammatory response (Fig. S1B). The induced IL33 message in colonic tissue was detectable from day 4, and ST2 from day 6 after DSS administration (Fig. 1a and Fig. S1A,B). The expression levels of several other key inflammatory cytokine and chemokines, including IL-1β, IL-6, CXCL9 and CXCL10 were also significantly up-regulated (> 2-log fold) by DSS in the acute inflamed colonic tissue (Fig. 1a). However, Th2 (IL-4 and IL-5), Th1 (IFN-γ), IL-17 and the ‘alarmin’ (IL-1β and HMGB1) cytokine genes were not significantly induced (Fig. S1A,B, and data not shown). We further determined IL-33 protein levels in vitro in the cultured colonic tissue from mice that had received DSS or PBS as control as described in the Materials and methods. Consistent with the induction of IL33 message (Fig.

Optical densities were converted to IU/ml and/or ng/ml based on t

Optical densities were converted to IU/ml and/or ng/ml based on the standard curve. (1 IU/ml = 2.4 ng/ml). Statistical analysis.  Data are presented as mean ± standard deviation (SD). Comparisons between variables were performed using general linear models with IgE levels in vitro modelled using repeated measures to control for duplicate experiments and the experimental condition as the independent variable, including age, sex and number of positive SPT as covariates. Given the small sample

size, Kruskal–Wallis Lumacaftor chemical structure tests were also performed to confirm significant differences without making any assumptions about the data distribution. The results of the two analyses were similar and general linear models are presented. A two-tailed P value of < 0.05 was considered statistically significant. All statistical analyses were performed using

sas 9.2 (SAS Institute Inc, Cary, NC, USA). When PBMC from asthmatic patients were cultured for 10 days with anti-CD40 mAb and rhIL-4, high levels of IgE were detected in supernatants on day 10 (8.2 ± 4.7 IU) (Fig. 1A). MG-132 concentration IgE responses were not detected when PBMC were cultured with either anti-CD40 mAb or rhIL-4 alone (<1.0 IU/ml) (Fig. 1A). When 1, 10 or 100 ng/ml of GTE was added to cultures, IgE production was suppressed in a dose-dependent manner (89.3 ± 5.7%, 56.9 ± 8.9%, 0.2 ± 4.1%, respectively), compared with control (general linear models, P = 0.07, <0.0001, and <0.0001, respectively) (Fig. 1B). When 5 or 50 ng/ml of EGCG was added to cultures, IgE production was also suppressed in a dose-dependent manner (87.0 ± 7.0% and 72.6 ± 14.4%, respectively), compared with none(P = 0.02 and <0.0001, respectively) (Fig. 1C). However, 0.5 ng/ml of EGCG did not significantly suppress the IgE production (95.7 ± 3.8%, P = 0.90). When PBMC from asthmatic patients were cultured for 10 days with the addition of cat pelt O-methylated flavonoid antigen (1 AU/ml), high levels of IgE were also detected in supernatants on day 10 (8.5 ± 3.8 IU) (Fig. 1A). When 1, 10 or 100 ng/ml of GTE was added to cultures, IgE production was suppressed

in a dose-dependent manner (76.4 ± 13.8%, 59.5 ± 19.5%, 0.2 ± 3.3%, respectively), compared with control (general linear models, P = 0.001, <0.0001, <0.0001, respectively) (Fig. 1B). When 50 ng/ml of EGCG was added to culture, IgE production was also suppressed in a dose-dependent manner (69.2 ± 3.7%), compared with control (P = 0.002 and <0.0001, respectively) (Fig. 1C). However, 0.5 and 5 ng/ml of EGCG did not significantly suppress IgE production (94.1 ± 4.8% and 85.0 ± 3.1%, P = 0.73 and 0.06, respectively). This study demonstrates that GTE or its catechin EGCG suppresses in vitro allergen- and non-allergen-specific IgE production in human PBMC from allergic asthmatics (up to 98%). Our findings suggest that GTE or EGCG has immunoregulatory effects on human IgE responses.

In addition, Con A, complex mycobacterial antigens and peptides o

In addition, Con A, complex mycobacterial antigens and peptides of RD1 were used as controls. In a previous study, peptide pools covering the sequence of all ORFS of each RD deleted in all strains of M. bovis BCG, i.e. RD1, RD4–RD7, RD9–RD13 and RD15, have been tested in the above assays using PBMC obtained from culture-proven pulmonary TB patients (Al-Attiyah & Mustafa, 2008). The results showed differential effects of peptide pools of various RDs on the secretion of IFN-γ and IL-10 by PBMC, with low IFN-γ : IL-10 ratios (<1.0) in response

to RD12, RD13 and RD15, suggesting that these RDs may be involved in the pathogenesis of TB (Al-Attiyah & Mustafa, 2008). However, the focus of this study check details was RD15 because this region contains genes that encode Mce3 proteins, which may contribute to the pathogenesis of TB by facilitating the entry and survival of M. tuberculosis in host cells (Gioffréet al., 2005; El-Shazly et al., 2007; Senaratne et al., 2008). Therefore, analyses of the cellular immune responses to peptides of RD15 in TB patients and healthy subjects, with respect to

the target molecules recognized and the type of immune response induced, could be important to the understanding of protective and pathological immune mechanisms AZD1208 in TB. Furthermore, such analyses may also help in the identification of antigens suitable for the diagnosis and development of new vaccines against TB (Flynn, 2004; Mustafa,

2005a). To our knowledge, this is the first study to evaluate the cellular immune responses in TB patients and healthy subjects Liothyronine Sodium to the ORFs of RD15 of M. tuberculosis. Similar studies have previously been performed with peptides of RD1, which have shown that RD1 peptides are strong and moderate stimulators of cellular immune responses in TB patients and healthy subjects, respectively (Hanif et al., 2008; Mustafa et al., 2008). Therefore, RD1 peptides were included in this study as a reference with which to compare the cellular responses induced by peptides of RD15. The results showed that PBMC from both TB patients and healthy subjects mounted strong cellular immune responses to Con A and complex mycobacterial antigens, as indicated by strong lymphocyte proliferation and IFN-γ secretion by PBMC. These results indicate that both groups of subjects were immunocompetent, and therefore suitable for studying the cellular immune responses to peptides of RD15. Furthermore, RD1 peptides induced strong proliferation and IFN-γ responses in TB patients and moderate responses in healthy subjects, confirming our previous findings in different groups of donors (Hanif et al., 2008; Mustafa et al., 2008). Although RD1 is deleted in all strains of M. bovis BCG vaccines (Behr et al., 1999), the moderate responses to RD1 in M.

Thus, in primed CD8+ T cells, CD27 signaling contributes to survi

Thus, in primed CD8+ T cells, CD27 signaling contributes to survival by upregulating anti-apoptotic Bcl-2 family members as well as Pim-1, a serine/threonine kinase capable of sustaining survival of rapidly proliferating cells 4. Given the broad distribution of CD27, it cancer metabolism signaling pathway is

not surprising that CD27 is also expressed by γδ T cells. Furthermore, studies with human γδ T cells showed that expression of CD27 marks stages of cellular differentiation. Naïve and central memory cells within the Vγ9Vδ2+ subset, which is predominant in the blood, express CD27 on the cell surface, whereas effector memory cells within this subset lack CD27 expression 5; however, there has been little information about the functional role of CD27 expressed by γδ T cells. In three related studies, the research team headed by Bruno Silva-Santos now has filled much of this knowledge gap 6–8. Investigating Selleckchem Saracatinib the development of γδ T cells in mice, Ribot and colleagues found that CD27 already functions as a regulator of differentiation in the thymus 6, where it induces expression of the lymphotoxin-β receptor as well as genes associated with transconditioning and IFN-γ production. Thus, γδ TCR+ thymocytes that express CD27 develop into producers of

IFN-γ, whereas those that do not express CD27 are unable to generate IFN-γ but produce IL-17 instead 6. This complements an earlier report from Chien’s group indicating Dipeptidyl peptidase that TCR engagement determines whether γδ thymocytes develop into IFN-γ or IL-17 producers 9. Presumably, signals through the TCR and CD27 somehow synergize in determining γδ T-cell differentiation. Importantly, the correlation between expression of cytokines and CD27 was found to be stable, extending to mature γδ T cells in the periphery 6, and was maintained even during infection 7. As pointed out by the authors 6, this lack of plasticity in CD27+ cells distinguishes γδ T cells from αβ T cells and B cells, encouraging the notion of CD27+/− γδ T-cell functional subsets. Continuing their studies in mouse models, Ribot and colleagues

next examined the role of CD27 in γδ T-cell responses to infections with herpes virus and malaria 7. Here, in IFN-γ-producing CD27+ peripheral γδ T cells, CD27 costimulation was seen to synergize with the γδ TCR, providing survival and proliferative signals that determined the extent of in vivo γδ T-cell expansion in response to these infections. In sharp contrast, IL-17-producing CD27− γδ T cells during malaria infection relied on TLR/MyD88-mediated innate immune signals, revealing an entirely different TCR-independent pathway of immune engagement, at least in this γδ T-cell functional subset. Finally, in this issue of European Journal of Immunology, Silva-Santos’s group 8 examines the functional role of CD27 expressed by Vγ9Vδ2+ human peripheral blood γδ T lymphocytes.

Expanded Tregs and Teffs were thawed and incubated

in AIM

Expanded Tregs and Teffs were thawed and incubated

in AIM-V 10% HS at 37°C, 5% CO2 overnight, then resuspended at 0·5 × 105 cells/ml. Teffs were plated into 96-well U-bottomed plates at a density of 5 × 104 cells per well, while Tregs were plated into Teff-containing wells at Treg-to-Teff ratios of 1:1, 1:2, 1:4, 1:8 and 1:16. Treg/Teff cultures were stimulated with 5 μg/ml soluble anti-CD3 and 1 μg/ml soluble anti-CD28 antibodies. Unstimulated wells were included as negative controls, both from patients and interassay control healthy Teffs. IL-2 (1 U/ml) was added to all wells. Supernatants were collected after 3 days of culture and FG4592 cells were incubated with 0·2 μCi [3H]-thymidine (PerkinElmer, Waltham, MA, USA) for 18 h before harvesting. Thymidine incorporation was measured using a 1450 Wallac MicroBeta counter (PerkinElmer). C-peptide levels were measured in serum samples with a time-resolved fluoroimmunoassay (AutoDELFIATM C-peptide kit, Wallac; PerkinElmer), as described [3]. Stimulated C-peptide was measured during a mixed meal tolerance test (MMTT) in GAD-alum- (n = 21) and placebo- (n = 10) treated patients who had a maximal C-peptide response check details of >0·20 nmol/l at the 30-month follow-up. Clinical effect of treatment was defined by changes in stimulated

C-peptide measured as area under the curve (AUC) from baseline to 48 months. Statistically significant differences were determined using the Mann–Whitney two-tailed U-test for unpaired observations, as

data were determined to be significantly different from a Gaussian distribution. Wilcoxon’s signed-rank test was used to compare ever paired samples. Linear regression was used to compare slope and Y-intercept of suppression curves, and correlations were determined with Spearman’s rank correlation coefficient test. A probability level of <0·05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism software, version 5·04 (GraphPad Software, Inc., La Jolla, CA, USA). We have demonstrated previously that in-vitro stimulation with GAD65 induced CD4+CD25hi FoxP3+ cells in PBMC from GAD-alum-treated patients [9]. To determine whether this effect persisted 4 years after treatment, we analysed CD25hiCD127lo cells and used FoxP3 and CD39 as additional markers to discriminate Tregs from activated T cells more accurately. Thus, the expression of CD25, CD127, FoxP3 and CD39 on CD4+ lymphocytes was analysed in PBMC after 7 days of incubation with or without GAD65. Gates used for analysis and representative PBMC samples describing the expression of CD4, CD25 and CD127 are shown in Fig. 1a,b. The frequency of CD25hiCD127lo cells in the CD4+ population was increased significantly upon GAD65 stimulation in GAD-alum-treated patients compared to unstimulated cells (7·4% and 4·5%, respectively), but not in the placebo group (Fig. 1c).

16,17 Mice deficient in tumour necrosis factor-α (TNF-α) or lymph

16,17 Mice deficient in tumour necrosis factor-α (TNF-α) or lymphotoxins (LTs) reveal profound defects in FDC development.15,18,19 In addition, other cytokines including IL-4 and IL-6 appear to be associated with FDC development.20,21 In this report, we present evidence that IL-15 enhances the proliferation of human FDCs and regulates chemokine secretion of human FDCs. Interleukin-15 is an IL-2-like T-cell proliferation factor that is required for the generation

of cytotoxic T lymphocytes and natural killer cells.22–24 It is also important in humoral immunity.25–27 Interleukin-15 enhances the proliferation and immunoglobulin secretion of human peripheral B cells and is involved in B-cell lymphomagenesis.28–34 The heterotrimeric IL-15 receptor (IL-15R) specifically binds IL-15. The IL-15 receptor α-chain (IL-15Rα) is the distinctive component for this Tipifarnib specific binding, whereas the IL-15 receptor β-chain (IL-2Rβ)

and IL-15 receptor γ-chain (IL-2γ) chains in the receptor complex, which are shared with Cabozantinib order the IL-2 receptor, are involved in signal transduction.35 Unlike IL-2, however, IL-15 is expressed in various cell types including dendritic cells, keratinocytes,36 monocytes,37,38 thymic epithelial stromal cells,39 bone marrow stromal cells40 and fibroblasts.41 The membrane-bound form of IL-15 plays an essential role in proliferation, or apoptosis of various kinds of cells in an autocrine fashion.37,42–44 Previously, we showed that IL-15 is produced by human FDCs and presented on the surface in a membrane-bound form.13 The IL-15 enhances Olopatadine GC-B-cell proliferation rather than protecting GC-B cells from apoptosis. Furthermore, the level of IL-15 on the surface of FDCs increased following the cellular interaction with GC-B cells. However, the functional role of IL-15 in FDCs has not been investigated. In this study, we show that IL-15 augments the proliferation of human primary FDCs in vitro. The FDCs express the IL-15R complex that is functional

because anti-IL-15 or anti-IL-15R antibodies that block IL-15 signalling reduced FDC proliferation. In addition, blocking of FDC IL-15 signalling reduced FDC secretion of CCL-2, CCL-5, CXCL-5 and CXCL-8, suggesting potentially important roles for recruitment of other cellular components required for GC reaction. Because IL-15 is expressed by FDCs within the GC microenvironment and enhances the proliferation of both GC-B cells and FDCs, IL-15 may contribute to the rapid expansion and formation of the GC structure, suggesting an important role of IL-15 in the humoral immune response. Anti-IL-15 monoclonal antibodies (mAbs) [M110, M111 and M112: immunoglobulin G1 (IgG1)] were kindly provided by Dr R. Armitage (Amgen Inc., Seattle, WA). Anti-IL-2Rβ (Mik-β2) was purchased from BD Biosciences, (San Jose, CA). Mouse IgG1 (MOPC 21; used as an isotype control) was purchased from Sigma (St Louis, MO).

During spermiogenesis, round spermatids undergo a loss of cytopla

During spermiogenesis, round spermatids undergo a loss of cytoplasm, the formation of sperm tail that allows cell motility and a mid-piece containing mitochondria that provide energy for sperm motility. The acrosome is also created during

this process. This structure located over the rostral portion of the spermatozoon head is essential for successful fertilization.9 Sperm are then cast off into the seminiferous tubal lumen (spermiation). Dynamic endoplasmic specializations at the base and apex of Sertoli cells play active roles in the creation of an adluminal compartment isolated from the immune system, in the ascent of maturing germ cells with the seminiferous tubule, and their release into the tubular lumen10 (Fig. 1). The acrosome is a specialized granule, which contains a trypsin-like enzyme (acrosyn),11 the multi-functional Smad inhibitor adhesion molecule vitronectin,12 and other as yet not well-known moieties that play roles in gamete interactions that lead to fertilization.13 Selleckchem LY2606368 Sperm must first undergo a process termed capacitation within the female reproductive tract that endows them with the ability to fertilize, allowing the sperm to acrosome react.9 This process involves alteration in the sperm glycocalyx as well as loss of plasma membrane cholesterol.14 The ‘acrosome reaction’ occurs when sperm

Chlormezanone bind to a glycoprotein of the zona pellucida that surrounds the unfertilized egg.15 Following zona binding, sperm receptors are cross-linked, leading to an increase in intracellular calcium and the promotion of the ‘acrosome reaction.’16 During this process, the sperm plasma membrane fuses with the outer acrosomal membrane, creating fenestrations through which acrosomal

contents are released.9 The sperm plasma membrane and outer acrosomal membrane are lost from the rostral portion of the sperm head, and the completely acrosome-reacted sperm (now bounded by the inner acrosomal membrane) penetrates through the zona pellucida, entering the perivitelline space, and subsequently adhering to the egg surface, the oolemma.17 The egg recognizes this adherence in an as yet undefined manner, possibly through specific receptor-ligand interactions, and subsequently plays an active role in incorporating the sperm within its cortical ooplasm.18 At this time, the egg undergoes activation, with the completion of the second meiotic division, release of the second polar body, and release of cortical granules in the perivitelline space, which alter the zona pellucida, preventing the binding and penetration of secondary sperm.19 Evidence that Sertoli cells play a role in the morphologic changes sperm undergo during spermiogenesis has been provided in a series of experiments in mice, in which the adhesion molecule nectin-2 was knocked out.

Triferic maintained hemoglobin near the baseline level, while pla

Triferic maintained hemoglobin near the baseline level, while placebo resulted in a statistically significant decline from baseline. The LS mean treatment difference was 3.6 g/L from baseline to end of treatment (p < 0.001). The Triferic treatment effect was significant in all pre-defined subgroups. Triferic, via dialysate, provided sustained delivery of iron for erythropoiesis while maintaining reticulocyte

hemoglobin (CHr). Serum ferritin did not increase in the Triferic group during the study despite iron administration with each treatment. The tolerability, types and incidence of adverse and serious adverse events with Triferic were similar to placebo. With Triferic, no anaphylaxis occurred with 20,000 individual doses and no increase in intradialytic hypotension, cardiovascular events, infections, or vascular access thrombotic events relative to placebo were Selleck JAK inhibitor observed. No death was attributed to Triferic. Conclusion: In CKD-HD patients, the novel iron salt Triferic, infused via hemodialysate for up to 48 weeks, is well tolerated with a safety profile similar to placebo. Triferic is effective in maintaining hemoglobin without increasing body iron stores as indicated by stable ferritin levels. WU PING-HSUN1,4, LIN MING-YEN1,6, WANG ANGELA YEE-MOON7, LIN YI-TING2,3, KUO MEI-CHUAN1,5,

CHIU YI-WEN1,5, HWANG SHANG-JYH1,5, CHEN HUNG-CHUN1,5 1Division of Nephrology, Department of Internal Medicine, Kaohsiung Medical University RAD001 Hospital, Kaohsiung, Taiwan; 2Department of Family Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 3Department of Public Health, Kaohsiung Medical University,

Kaohsiung, Taiwan; 4Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 5Faculty of Renal Care, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 6Technology Research Center, National Applied Research Laboratories, Taiwan; 7Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong Introduction: End stage renal disease is associated with a high risk of coronary artery disease, which is one of the leading causes of death among dialysis patients. However, there is so far no randomized study Non-specific serine/threonine protein kinase comparing the effectiveness of aspirin versus thienopyridines for secondary prevention of acute coronary syndrome in dialysis patients. This study aimed to compare the efficacy of aspirin versus thienopyridines in reducing the subsequent risk of recurrent acute coronary syndrome and mortality in a national cohort of Taiwan dialysis patients who was hospitalized with acute coronary syndrome. Methods: We conducted a nationwide follow-up study, based on the Taiwan National Health Insurance Research Database. We identified incident dialysis patients who were experienced with an episode of acute coronary syndrome between during 1998 and 2006 were identified.

Where indicated, human cells were stimulated in the presence of h

Where indicated, human cells were stimulated in the presence of human IFN-α (1000 U/ml; PBL Biomedical Laboratories, Piscataway, NJ) and rhesus cells with universal type I IFN (1000 U/ml; PBL Biomedical Laboratories). To support viability in the rhesus B-cell cultures, IL-2 (100 ng/ml, PeproTech, Rocky Hill, NJ) and B-cell activation BAY 57-1293 supplier factor of the tumour necrosis factor family (BAFF; 100 ng/ml, PeproTech) were added to the rhesus cultures in the experiments where differentiation and antibody

production were measured. Human and rhesus PBMCs were labelled with 0·25 μm CFSE (Molecular Probes, Eugene, OR) for 7 min at 37° and thoroughly washed with complete medium as described elsewhere.2,3 Using the conditions described above 2 × 106 cells/ml were cultured at 37° in polystyrene round-bottom tubes in complete medium. TLR ligands were used at 1 μg/ml (Poly I:C and TLR7/8-L) and 5 μg/ml (CpG classes), optimal concentrations of each ligand that caused peak B-cell activation. Proliferation was measured by flow cytometry and data were analysed using FlowJo software. Live cells were gated on by exclusion of propidium iodide staining. B cells were gated based on expression of CD20 and CD19 for rhesus and human B cells, respectively, and lack of CD3 and CD14. Alternatively, proliferation was measured

Pictilisib cell line by thymidine incorporation where PBMCs or B cells were cultured in 96-well plates and pulsed with [3H]thymidine (1 μCi/well, Amersham Bioscience, GE Healthcare Biosciences AB, Uppsala, Sweden) for 16 hr after 4 days of culture. The level of incorporation Non-specific serine/threonine protein kinase of [3H]thymidine was measured by a 1450 MicroBeta PLUS counter (Wallac, PerkinElmer Sverige AB, Upplands Väsby, Sweden) and expressed as counts per minute (c.p.m.). Human or rhesus PBMCs at 6 × 106 cells/ml

were exposed to the TLR7/8-L (1 μg/ml) or CpG ODN class C (5 μg/ml) for 1 hr at 37° in polystyrene round-bottom tubes, followed by an additional 10 hr in the presence of the secretion inhibitor Brefeldin A (10 μg/ml; Sigma-Aldrich) and then stained as described previously.33,34 Briefly, the cells were fixed and permeabilized for 15 min using a BD Cytofix/Cytoperm kit (BD Pharmingen). The cells were then washed twice and stained with antibodies specific for IFN-α (clone MMHA-11, PBL Biomedical Laboratories), CD3, CD14, CD20, CD123, HLA-DR (antibodies as described above). The cells were analysed by flow cytometry. In addition, IFN-α levels in the supernatants of cells exposed for 24 hr to the TLR ligands were measured by ELISA (Mabtech, Stockholm, Sweden) performed according to the manufacturer’s instructions. Phenotypic differentiation of B cells was assessed for up to 6 days of culture by flow cytometry using antibodies against CD20, CD27, IgG and IgM (all BD Pharmingen). Expression of IgG and IgM was assessed by intracellular staining using the BD Cytofix/Cytoperm kit before staining.