S2a and purity of the sorted cells shown in Supplementary Fig S2

S2a and purity of the sorted cells shown in Supplementary Fig. S2b,c). Unlike the CD11c–CD19+CD24+CD27+CD38+ cells, the CD11c–CD19+CD24+CD27–CD38– cells were unable to suppress T cell proliferation in allogeneic MLC (Fig. 1b,c). Unexpectedly, FACS-sorted CD11c–CD19+CD24+ cells exhibited statistically similar

suppressive ability as the CD19+CD24+CD27+CD38+ B cells (Fig. 1b,c). In all instances, the lower T cell frequency (Fig. 1c) in the MLC was due to decreased proliferation and absolute numbers of buy AZD2014 live CD3+ T cells (Fig. 1c,d) and not to an increase in the numbers of dead cells (including T cells) or changes in B cell frequency (Supplementary Figs S3 and S4). We hypothesized that iDC could directly affect the frequency of the suppressive CD19+CD24+CD27+CD38+ B cells and that a potentially significant increase in their number could account for the increased frequency of B220+CD11c– cells in the PBMC of iDC recipients [31]. To test this, freshly collected PBMC from healthy adults were enriched into CD19+ cells. Of these cells, 2 × 106 were then

cultured in the presence of an equal number of autologous cDC, iDC (generated from the same PBMC) or PBS vehicle for 3 days. The frequency of CD19+CD24+CD38+ cells in those co-cultures was then measured by flow cytometry. Figure  2a shows that, in the presence of iDC, the frequency of CD19+CD24+CD38+ B cells was increased significantly. Furthermore, the frequency of CD27+ cells inside the CD19+CD24+CD38+ population was increased substantially. this website This increase in frequency was due specifically to an increase in the proliferation of CD19+CD24+CD38+ cells, especially the CD27+ subpopulation (measured as the frequency and absolute number of BrdU+ cells; Fig. 2a,b). Interestingly, exposure of the CD19+ B cells to the iDC increased significantly the numbers of viable cells in general (Fig. 2a, P2 peak in the LIVE/DEAD histogram very at the top). When comparing the segregation of the individual cell surface markers used to identify

the B cells, the only discernible difference is in the generation of two peaks representing the CD19+ population in the presence of cDC or iDC (Fig. 2c). There are no other significant differences in the segregation of the other markers used (CD24, CD27, CD38; Fig. 2c). Specificity of the antibodies and non-specific antibody binding was controlled by the appropriate isotypes (Supplementary Fig. S5). Gene chip-based expression analysis of the autologous DC used in the Phase I trial [31] revealed that the rate-limiting enzyme for RA biosynthesis, ALDH1A2, was expressed in cDC and iDC generated from PBMC of normal adults (data not shown). To confirm the gene chip data and to demonstrate that cDC and iDC produce RA, we employed a reagent (Aldefluor) that reacts with RA-producing cells to identify and measure the frequency of RA-producing cells by flow cytometry. In Fig.

The plates were incubated for 1 hr at 37°C under 5% CO2 Cell Tit

The plates were incubated for 1 hr at 37°C under 5% CO2. Cell Titer 96 Aqueous One Solution Reagent (Promega, Madison, WI, USA) was added and incubated for a further 1 hr at 37°C under 5% CO2. Absorbance of each well was measured at 490 nm. The data are presented as percent viability to determine the concentration of toxin causing 50% cell death (EC50) as described previously [23]. Vero cells (3 × 107/mL) were treated with PI-PLC (0.5 U/mL; EMD Biosciences, Darmstadt, Germany) for 2 hr at 37°C in PBS and centrifuged

as described previously [24]. Aliquots of cells and supernatants were used for SDS–PAGE and toxin overlay assay. Vero cells were scraped from 25 cm2 flasks Epigenetics inhibitor with a rubber policeman and harvested by centrifugation at 1000 g for 5 min. After washing, cells were suspended in 1 mL of cold lysis buffer consisting of 10 mM Tris–HCl buffer (pH 7.0) containing 150 mM NaCl, 1% Triton X-114 (Pierce) and 0.1% protease inhibitor cocktail. After allowing them to stand for 1 hr on ice, the detergent-insoluble fractions were separated from the supernatants (the detergent-soluble fractions) by centrifugation at 15,000 g for 15 min, and finally resuspended in 1 mL of PBS. SDS–PAGE was carried

out in 5–20% gradient gels (ATTO, Tokyo, Japan). After electrophoresis, detergent-soluble and -insoluble fractions from Vero cells were blotted onto PVDF membranes. After blotting, the membranes were blocked with Baricitinib 5% skim milk in PBS for 1 hr at room temperature. After washing three times with PBS-0.01% Tween 20, the membranes were incubated for 1 hr at room temperature in the presence of 10 µg/mL wild-type or mutant alpha-toxin KU-57788 manufacturer in 0.5% skim milk. This was followed by washing and incubation for a further 1 hr at room temperature with 5 µg/mL affinity-purified rabbit anti-alpha-toxin

IgG [25] in 0.5% skim milk. The membranes were treated for 30 min at room temperature with goat anti-rabbit IgG (H + L) conjugated with peroxidase (1:3000 dilution; Cappel, West Chester, PA, USA) in 0.5% skim milk. After washing, the membranes were developed in 20 mL of PBS containing 0.05% 3′3-diaminobenzidine (Dojin Laboratories, Kumamoto, Japan) and 0.02% H2O2. Protein concentrations were determined by the method of Bradford [26] with bovine gamma globulin as a standard. To evaluate the roles of the tryptophan-rich region in the C-terminal domain in the cytotoxic effect of alpha-toxin, we constructed several mutant toxins by individually replacing tryptophan and some residues surrounding tryptophan with other amino acids (Table 1). We individually replaced tryptophan (W307, W309, and W311) with phenylalanine (W307F, W309F, and W311F), which is hydrophobic and also has an aromatic side chain. These tryptophans were also replaced with alanine to create loss of an aromatic side chain and substitution by its minimal side chain (W307A, W309A, and W311A).

aureus (Fig  5B) and influenza virus (Fig  5D), that is the only

aureus (Fig. 5B) and influenza virus (Fig. 5D), that is the only two microbes that promoted IL-2 and IFN-γ responses. In this study, we show that cord pDC promote a Th2 phenotype. However, the Th2-skewing effect of cord pDC could be omitted by enveloped viruses. This implies that virus can divert Th2-biased responses in human cord T

cells. Furthermore, we show that microbes capable of inducing IFN-α promote Th1 responses, whereas a microbe’s ability to induce IL-12 does not correlate to its ability to induce IL-2 or IFN-γ responses in vitro. The numbers of human studies of adaptive T cell responses in newborns compared with adults are limited and conflicting [37]. Yet, it is generally thought that the immune system of newborns is immature and differs from that in adults. The T cell polarization in newborns is correlated with impaired Th1 responses [38, 39]. drug discovery However, individual Th1/Th2 balance in newborns varies depending on parental and environmental

factors [40]. In this paper, we show that the baseline production of the Th2 cytokines IL-5 and IL-13 were elevated in cord CD4+ T cells compared with adult T cells. The Th2 cytokine induction observed in cord cells was not an intrinsic function of the neonatal T cells, but rather a Th2-inducing effect of cord pDC. This is in line with previous click here findings where pDC was shown to promote Th2 responses in healthy and allergic subjects [15, 19]. This is, to our knowledge, the first study to show that the levels of Th2 cytokines obtained in vitro activated T cells differs between newborns and adults. We could not detect any significant differences in Th1 cytokine synthesis (IFN-γ and IL-2) between T cells from adults and newborns, even though others have shown that cord blood DC is impaired in their capacity to induce both IFN-γ and IL-2 in responding T cells

[39]. Instead, our data imply that cord pDC were superior to both cord mDC and adult DC in promoting Th2 responses. The Th2-skewing effect of cord pDC can be blocked by viral stimuli. We found that enveloped viruses (i.e. HSV-1, coronavirus, CMV, morbillivirus 4��8C and influenza virus) blocked IL-13 secretion, while bacteria and non-enveloped viruses did not. This confirms previous findings from us and others, showing that the Th2 skewing effect of pDC in newborns and adults can be omitted by microbial stimuli [3, 19]. However, the diminished IL-13 production that was seen in virus stimulated cultures could not be correlated with Th1 polarization, that is IFN-α, IFN-γ, IL-2 or IL-12 secretion. None of the viruses tested could induce IL-12 secretion, and influenza was the only inactivated virus to evoke IFN-α, IFN-γ and IL-2 production. Still, these findings emphasize the importance of early life microbial stimuli of the innate immune system for an accurate maturation of the immune system, that is to avoid unwanted Th2 responses.

Both adaptive and innate immune effector mechanisms are believed

Both adaptive and innate immune effector mechanisms are believed to contribute to tissue disease aetiology. HLA-E is a non-classical MHC class Ib molecule that acts as the ligand for the NKG2A inhibitory receptor present on natural killer (NK) and CD8+ cells. Peptide binding and stabilization of HLA-E is often considered to signal infection or cell stress. Here we examine the up-regulation of HLA-E in MS brain tissue. Expression is significantly increased in white matter lesions in the brain of MS patients compared with PS-341 ic50 white matter of neurologically healthy controls.

Furthermore, using quantitative immunohistochemistry and confocal microscopy, we show increased HLA-E protein expression in endothelial cells of active MS lesions. Non-inflammatory chronic lesions express significantly less HLA-E protein, comparable to levels found in white matter from controls. Increased HLA-E protein levels were associated with higher scores of inflammation. These learn more results suggest the potential for an effect in central nervous system pathogenesis from HLA-E modulation in stressed tissue. Co-localization with infiltrating CD8+ cells implicates a possible role for HLA-E-restricted regulatory CD8+ cells, as has been proposed in other autoimmune diseases. “
“Perforin (P) is a prototypical cytotoxic molecule involved in cell-mediated immunity against various pathogens, alloantigens and particularly different tumours. The purpose

of this study was to determine P expression in different lymphocyte subpopulations isolated from

peripheral blood and prostate tissue of patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) and compare it with the P expression found in the control group. Twenty subjects were recruited in each of the groups. Prostate mononuclear cells of the BPH and PCa tissues were isolated Carnitine palmitoyltransferase II by enzymatic digestion and gradient density centrifugation, whereas peripheral blood mononuclear cells were isolated by gradient density centrifugation alone. Cells and tissue samples were labelled using monoclonal antibodies against P and different surface antigens (CD3, CD4, CD8 and CD56) and analysed by immunofluorescence and flow cytometry. Total P expression in peripheral blood lymphocytes did not differ significantly between BPH/PCa patients and control group, although the BPH and PCa tissue showed lower P expression level. A negative correlation between prostate-specific antigen levels and the overall percentage of P+, CD3+CD56−P+, and CD3−CD56+P+ cells in the prostate tissue was observed only in patients with PCa. Our findings indicate that the low frequency of P+ lymphocytes, including T, NKT and NK cells, in the prostate tissue of patients with BPH and, particularly, PCa could be the consequence of local tissue microenvironment and one of the mechanisms involved in the pathogenesis of prostate hyperplasia following malignant alteration.

They suggest that screening for microvascular dysfunction using a

They suggest that screening for microvascular dysfunction using a combination of the approaches described above may be advantageous for the early detection of microvascular disease, in aiding diagnosis, in monitoring disease progression and response to therapy. Furthermore, they demonstrate a co-linearity in the development

and progression of microvascular and macrovascular disease. Much effort has gone into establishing whether there is a causal effect in either direction or whether this co-linearity simply represents shared risk factors. It is most likely to be a complex combination of bidirectional interactions. While the techniques used to measure microcirculatory structure

and function Lorlatinib learn more have become more robust and better understood over the past few decades, their application to the study of large populations remains limited. Furthermore, their ability to provide a mechanistic understanding of the processes underlying the pathology of microvascular disease is restricted by the need to interrogate accessible microvascular beds influenced by a wide range of confounding factors. Also included in this volume of Microcirculation is a review article from Cheung and Daanen [2] in which they present longitudinal and laboratory studies investigating dynamic adaptation of the peripheral microvasculature to cold exposure and improved tolerance in those living in cold environments. Collectively, the clinical microcirculatory research evidenced

in these articles provide readers with a unique opportunity to gain further insight into the challenges facing Anacetrapib those working at the translational interface seeking new ways in which to interrogate the human microcirculation. “
“Microcirculation (2010) 17, 237–249. doi: 10.1111/j.1549-8719.2010.00026.x The mammalian transient receptor potential (TRP) superfamily consists of six subfamilies that are defined by structural homology: TRPC (conventional or canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin), and TRPML (mucoliptin). This review focuses on channels belonging to the vanilloid (V) and melastatin (M) TRP subfamilies. The TRPV subfamily consists of six members (TRPV1-6) and the TRPM subfamily has eight (TRPM1-8). The basic biophysical properties of these channels are briefly described. All of these channels except TRPV5, TRPV6, and TRPM1 are reportedly present in arterial smooth muscle from various segments of the vasculature. Studies demonstrating involvement of TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, and TRPM8 in regulation of arterial smooth muscle function are reviewed. The functions of TRPV3, TRPM2, TRPM3, and TRPM6 channels in arterial myocytes have not been reported.

Subsequently, maintenance therapy dose range is 0·1–0·4 g/kg of b

Subsequently, maintenance therapy dose range is 0·1–0·4 g/kg of body weight, approximately every 4 weeks (depending on the individual patient’s clinical course). IVIG effects usually last between 2 weeks and 3 months. Clinical trials: in MS, IVIG have been tested for their efficacy in (i) relapse treatment, their impact on the (ii) relapse rate and disease progression in RRMS and on (iii) disease progression in SPMS. (i)  Two studies compared

IVIG versus placebo as add-on treatment to methylprednisolone Bcr-Abl inhibitor in acute MS relapse. There was no statistically significant difference between the treatment groups [28, 29]. Thus, IVIG are currently not recommended for the treatment of acute relapses in MS. In CIDP, several short-term clinical trials showed beneficial Selleckchem Cisplatin effects of IVIG compared with placebo, plasma-exchange or steroids [33-35]. However, long-term data on the efficacy of IVIG in CIDP have emerged only recently. A recent randomized, double-blind, placebo-controlled, response-conditional cross-over trial included 117 patients with CIDP (ICE trail). The long-term

efficacy of IVIG (baseline loading dose of 2 g/kg over 2–4 days and then a maintenance dose of 1 g/kg over 1–2 days every 3 weeks for up to 24 weeks) PIK3C2G was compared with placebo [36]. IVIG or placebo was administered for up to 24 weeks in an initial treatment period; patients who did not show an improvement in INCAT disability score of ≥1 point received the alternate treatment in a cross-over treatment period. Patients who showed an improvement and completed 24 weeks of treatment were eligible to be reassigned randomly in a blinded 24-week extension phase. The primary outcome was the percentage of patients who had maintained an improvement from

baseline in adjusted INCAT disability score of 1 point or more to week 24. Secondary efficacy outcomes were (i) mean change from baseline in maximum grip strength at end-point during the initial treatment period; (ii) mean change from baseline in the compound muscle action potential amplitude after stimulation of the most severely affected motor nerve at the proximal site at end-point during the first period; and (iii) time to relapse for patients who were first-period adjusted-INCAT responders or cross-over-period adjusted-INCAT responders to IVIG and entered the extension phase. Relapse during the extension phase was defined as worsening of adjusted INCAT disability score by 1 point or more from the extension baseline value.

In autoimmunity, altered T lymphocyte responses are observed [3,4

In autoimmunity, altered T lymphocyte responses are observed [3,4]. Enhanced T cell antigen receptor

(TCR) signalling and immune complexes (ICs) contribute to the disease pathogenesis in systemic lupus erythematosus (SLE) [5]. ICs bind to its ligand, the low-affinity FcγRIIIA membrane receptor, which induces phosphorylation of the FcRγ chain, the signalling subunit for FcγRIIIA. The FcRγ chain mediates signalling via immunoreceptor tyrosine-based activation motif (ITAM), which upon phosphorylation recruits Syk in B cells and platelets. Syk-mediated signalling is an important event for B cell activation [6]. Interestingly, FcRγ chain in T cells associates with the ζ-chain, forming heterodimers in the TCR complex, and the FcRγ chain is able to support independently the development of the peripheral T cells in mice lacking endogenous TCR ζ-chain [7]. The FcRγ chain containing TCR complexes BVD-523 concentration are present in activated γδ+ T cells, natural killer (NK)-like T (NK T) cells, SLE T cells and in certain populations of human T effector cells [8–11]. An association of FcRγ chain with the TCR complex is also observed in TCRαβ+CD4–CD8– double-negative regulatory T cells (Tregs) [12]. In these cells, TCR ligation

results in the phosphorylation of both FcRγ chain and Syk, and this event is shown to be necessary for their suppressive activity [12]. TCR in CD4+ T effector cells show association of FcRγ chain with Syk [11]. Such events are also observed in antigen-induced arthritis (AIA), a chronic ABT-888 clinical trial arthritis regulated by ICs and T cells [13]. In AIA, inflammation and cartilage erosion is dependent on FcRγ chain-mediated signalling [14]. Also, for the full development

of experimental autoimmune encephalomyelitis (EAE), expression of FcRγ chain by γδ T cells in association with the TCR/CD3 complex is required [15]. Both these diseases show elevated levels of ICs. However, the ligand that triggers the Syk phosphorylation is unknown. In this report, we show that a subset of peripheral human CD4+ T cells bind to labelled aggregated human γ-globulin (AHG). SLE patients show a two–fourfold increase in this population when compared to the normal subjects. Thus, we explored whether ICs acts as a ligand for the activation of Syk signalling pathway PFKL in CD4+ T cells via engagement of low-affinity membrane Fc receptors (FcRs). The terminal complement complex (TCC), also referred to as soluble C5b-9, is a non-cytolytic by-product of the terminal complement activation pathway that triggers proinflammatory responses, cytokine release and vascular leakage [16]. We observed that, in human CD4+ T cells, in the presence of ICs, TCC synergistically enhances the phosphorylation of Syk. In addition, cells treated with TCC or non-lytic C5b-9 demonstrated aggregation of the membrane rafts (MRs) (Fig. 5). MRs are membrane structures that are crucial for lymphocyte signalling, i.e.

Mrs A pursued all active treatment options available to her and w

Mrs A pursued all active treatment options available to her and withdrew from dialysis

when it was no longer feasible. The achievement of ACP in Mrs A’s case was bringing her and her immediate family to a common understanding with nephrology staff about the seriousness of her medical conditions, her prognosis and the potential scenarios for future deterioration in health, despite a language barrier and a busy family who were not all available during office hours. Knowing that her life expectancy was limited, Mrs A identified and articulated, largely to her family, her personal goals and preferences for care. Her family were able to choose to spend time with her and support her, knowing this might be a limited opportunity. Mrs A’s case shows that these conversations can be difficult but when Endocrinology antagonist ACP is started when the patient is relatively well and out of hospital there is the opportunity to identify misunderstandings, resolve them and PI3K inhibitor move forward. Furthermore there is time for patients to reach a point of readiness to undertake

ACP and identify key decision-makers and personal priorities. Starting ACP early was key to reuniting Mrs A with her son. Mrs A’s ACP also highlights some issues to be aware of when using interpreters. Both Mrs A and her family commented to Dr Y that the skill of interpreters in translating these conversations was variable but unfortunately Dr Y could not consistently secure their preferred interpreter. The better interpreters were able to convey information better than some of Mrs A’s children felt they could. Language barriers within families can be a significant issue for

some, particularly where older patients have children who grew up in New Zealand or Australia and may be more comfortable speaking in English than their parent’s first language. Patients may wait for physicians to initiate end-of-life discussions and may feel uncomfortable asking for prognostic information.[7] Tolmetin Patients may perceive ACP as a health-care professional initiative to limit their future medical treatment, for example because of resource constraints.[3, 9] Patients may not be aware that their condition is life limiting. Family may wait for the patient to initiate end-of-life discussions.[8] Family may be unaware that the patient has a life limiting medical condition. Discussing death can be emotionally distressing for health professionals and skills and/or support for managing this distress are not currently commonly taught to nephrology trainees.[10, 11] The previous experience of emotional distress during end-of-life conversations may cause the health-care professional to avoid future discussions.[10] Lack of available time to hold ACP discussions.[10] Physician perceptions that end-of-life conversations are not valuable to the patient and/or may cause harm by diminishing patient hope.

Studies from the Hartmann laboratory [26] first suggested that ch

Studies from the Hartmann laboratory [26] first suggested that chromatin or ssRNA components of SLE immunocomplexes can activate TLR-9 in intracellular endosomes of B cells. Such nucleic acid-containing immunocomplexes were shown to activate autoreactive B cells and MK-1775 research buy autoantibody production. TLR-9-active sequence transgenic mice produce large amounts of anti-RNA, -DNA and -nucleosome antibodies of the IgG2a and IgG2b isotype that cause nephritis [27]. B cells

can promptly detect and mount responses to antigen after immunization. In the case of small soluble antigens, responses can be mounted following a simple diffusion of antigen into the lymphoid tissue; however, these encounters are usually mediated through macrophages, DCs and follicular DCs. In addition, macrophages are known to express a wide range of cell-surface receptors that could participate in the presentation of unprocessed antigen,

ALK inhibitor including complement receptors, pattern recognition receptors and/or carbohydrate-binding scavenger receptors [28]. Indeed, macrophage receptor 1 (MAC1; also known as αMβ2 integrin and CD11b–CD18 dimer), which is a receptor for complement component 3 (C3) that is expressed by macrophages, has been suggested to contribute to the retention of antigen on the cell surface [29]. Alternatively, the inhibitory low-affinity receptor for IgG (FcγRIIB) might mediate the internalization and recycling of IgG-containing immune complexes to the macrophage cell surface, as has been shown in DCs [30]. Finally, the C-type lectin DC-specific ICAM3-grabbing non-integrin (DC-sIGn; also known as CD209) could participate in the retention of glycosylated antigens, which Evodiamine is consistent with the observation that mice deficient in the mouse homologue of DC-sIGn, sIGnR1, fail to mount humoral immune responses following infection with Streptococcus pneumonia[31]. The cultured clone I3D spontaneously expresses a high level of MAC1, FcγRIIb and DC-sIGn when cultured in vitro (Fig. 6).

However, once these I3D cells were treated with MIP8a Fab more than 12 h, these expression levels of FcγRIIb and DC-sIGn but not MAC1 were decreased. The inhibitory effect of MIP8a Fab was concentration-dependent, with maximal inhibition at a Fab concentration of 10 µg/ml (Fig. 6). We believe these results could be one of the mechanisms that explain why MIP8a Fab treatment inhibits antibody deposition and subsequent complement activation. Taken together, these data suggest that the role of TLR-9 signalling in macrophages is predominant in the progression of HAF-CpG-GN and blockade of this signalling by monovalent targeting of FcαRI might inhibit disease activity. The inhibitory activity of FcαRIR209L/FcRγ ITAM (iITAM) has been associated with SHP-1 recruitment following weak activation [6,32].

In line with this hypothesis, the IgM released from CpGPTO-stimul

In line with this hypothesis, the IgM released from CpGPTO-stimulated B cells (14·6 ± 12 μg/ml) displayed unselective binding specificity, e.g. reactivity to lipopolysaccharide, pneumococcal polysaccharide, double-stranded DNA, www.selleckchem.com/products/Maraviroc.html single-stranded DNA or tetanus toxoid (Fig. 6b). To investigate

whether CpGPTO binds to autoantigens, we incubated HEp2G cells with supernatants from CpGPTO- or CD40L/rhIL-4-treated B cells or intravenous immunoglobulin G. Immunofluorescence microscopy showed binding of CpGPTO-induced immunoglobulin with a faint, mainly cytoplasmic staining pattern suggestive of low-degree autoreactivity (Fig. 6c). Hence, CpGPTO might preferentially target B cells expressing potentially polyreactive

IgM, which might belong to the IgM memory pool.[17] In B cells, internalization of antigen is mediated by the BCR. Recent studies suggested that physical linkage of a BCR antigen to a stimulatory nucleic acid represents the most efficient means to induce B-cell activation via TLR9.[9, 23, 24] This prompted us to ask whether CpGPTO trigger receptor Selleck PD332991 revision by simultaneously engaging BCR and TLR9 signalling in a B-cell subfraction. Notably, unmodified (phosphodiester) CpG ODN (CpGPO) lack mitogenicity (Fig. 7a), but the stimulatory activity of CpGPO was coupled to microspheres additionally click here carrying a BCR stimulus [anti-human immunoglobulin F(ab′)2] (Fig. 7b). However, physical linkage of ODN did not waive the requirement for the TLR9-specific CpG-motif: F(ab′)2-coupled microspheres failed to induce proliferation in the absence of CpGPO or when CpGPO was substituted by a control GpCPO or a poly(T)2o-ODN (Fig. 7c). Next, we asked whether CpGPTO use BCR-dependent signalling. To answer this question, we stimulated B cells with CpGPTO in the presence or absence of inhibitors selectively targeting tyrosine kinases typically recruited upon BCR activation. In support of our hypothesis we found that CpGPTO-triggered B-cell proliferation was partially inhibited by the syk

kinase inhibitor R406 in a concentration-dependent manner (Fig. 7d). By contrast, proliferation was enhanced by 20 ± 0·6% when B cells were pretreated with the lyn inhibitor SU6656 (Fig. 7e), a finding well compatible with hyper-responsiveness of lyn–/– B cells.[25, 26] We concluded that, first, syk and lyn kinases participate in CpGPTO-mediated B-cell activation, and, second, CpGPTO either directly stimulate the BCR or bypass BCR signalling by recruiting molecules associated with proximal BCR signalling. To further investigate this question we sought to perform CpGPTO stimulation in the absence of the BCR. To this end we used plasmacytoid dendritic cells because they are characterized by TLR9 and a BCR-like signalosome.