Moreover, the presence of more than one locus homologous to disruption constructs may also lead to additional ectopic integration, which may explain the occurrence of additional ectopic bands in five TmSSU1Δ mutants (data not shown). In conclusion, it is advantageous to use TMLIG4-defective cells in gene targeting experiments.

Further experiments to elucidate the NHEJ pathway in T. mentagrophytes are required. This research was partially supported by a Grant-In-Aid (19590457) from the ministry of Education, Science, Sports and Culture of Japan (KM). Table S1: Primers used in this study. “
“The scaffold protein caspase recruitment domain-containing protein 11 (CARD11) is implicated in the regulation of inflammation and autoimmunity. The present study aimed to explore the role of CARD11 in the pathogenesis of rheumatoid arthritis (RA). Mice with collagen-induced arthritis I-BET-762 solubility dmso (CIA) were treated with either CARD11-targeted interfering RNA (CARD11

siRNA) or control siRNA by intraperitoneal injection Smad inhibitor every 3 days after CIA establishment. The clinical score of arthritis was recorded every other day. Synovial inflammation and cartilage erosion were evaluated by histology and microcomputed tomography (micro-CT). Serum anti-type II collagen (anti-CII) antibodies and cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The CARD11/Bcl10 formation Nitroxoline and nuclear factor-kappa B (NF-κB) activation was assessed by immunoprecipitation and immunoblotting, and the percentage of T helper type 17 (Th17) cells was determined by flow cytometry. Systemic administration of CARD11 siRNA significantly reduced the clinical score of CIA severity. As indicated by the histology,

joint inflammation and destruction were attenuated by CARD11 siRNA treatment. Micro-CT demonstrated less severe joint destruction in CARD11 siRNA-treated mice than in control mice. CARD11 siRNA treatment resulted in inhibition of CARD11/Bcl10 formation and the subsequent NF-κB activation. In addition, treatment with CARD11 siRNA resulted in a pronounced decrease in proinflammatory cytokines interleukin (IL)-1β, IL-6 and IL-17. Serum anti-CII antibody and the percentage of Th17 cells were also significantly reduced. CARD11 is involved in the pathogenesis of CIA by formation of the CARD11/Bcl10 complex and enhancement of the Th17 cell response. Targeting CARD11 provides a novel research direction in the development of therapeutic strategies for RA. “
“To investigate the usefulness of serum cytokine levels in the diagnosis of active cystic echinococcosis, we evaluated the cytokine profile of patients with hepatic cystic echinococcosis in different cyst stages, CE 1-2 (active), CE3a-3b (transitional) and CE4-5 (inactive). Ex vivo assessment of Th1 (IL12, TNFα) and Th2 (IL4, IL10) cytokines in sera was carried out using ELISA.

Clinical and Experimental Immunology 2014, 175: 425–38. Diagnosis, pathogenesis and treatment of myositis: recent advances 2014, 175: 349–58. Neuromyelitis optica: clinical features, immunopathogenesis and treatment check details 2014, 176: 149–64. Multiple sclerosis (MS) and neuromyelitis optica (NMO) are two distinct chronic progressive inflammatory diseases of the central nervous system (CNS) with different pathophysiology and epidemiology. Both are commonly associated with disability, impairment in quality of life, decreased work capacity and high socioeconomic burden [1-4]. The pathophysiology of MS is complex and highly heterogeneous

with both inflammatory and neurodegenerative features [5], resulting in various phenotypes and disease courses. In contrast, the discovery of aquaporin-4 immunoglobulin (Ig)G as an autoantibody with pathogenetic relevance Ivacaftor supplier for NMO [6, 7] had a direct impact on therapeutic approaches. As most immunotherapies in neuroimmunology have been studied in MS [8-22] and – to a lesser extent – in NMO [23-27], this review focuses on disease-modifying drugs (DMDs) for these autoimmune CNS entities. Treatment options for other neuroimmunological diseases of the central or peripheral nervous system

and neuromuscular disorders such as neuro-sarcoidosis [28, 29], myasthenia gravis [30] or chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) [31] have been reviewed in [32, 33]. Whereas first-line

agents used in MS such as interferons and glatirameracetate exhibit moderate efficacy, we have witnessed several decades of use with highly favourable safety profiles [34]. In contrast, newer agents have surprised us with unexpected and sometimes even severe adverse drug reactions (SADR) or unanticipated high frequency of SADRs (Table 1) [35-37]. Due to the hypothesized selective mechanisms of action, fewer side effects were anticipated for different therapeutic monoclonal antibodies (mAB) coined initially as ‘magic bullets’ [38]. Rare but occasionally fatal adverse RAS p21 protein activator 1 drug reactions have evolved; however, their pathophysiology is still not well explained. Based on potential SADRs, approval for substances such as natalizumab (NAT), mitoxantrone (MX) and – at least in some countries – fingolimod (FTY) was restricted to patients refractory to first-line MS treatment options or with highly aggressive disease course; but labelling is different from the formal inclusion criteria of respective clinical trials. In addition, restriction to escalation therapy may carry the risk of omission bias, i.e. the decision not to treat patients with potential high benefit in order not to put them actively at risk for SADRs. In the face of newly introduced highly efficacious treatment options, strategies are thus needed that allow patient selection and counselling based on individualized safety and efficacy considerations.

In conclusion, age and CMV serostatus both contribute to the decrease in CD45RA+ CD27+ CD4+ T cells during ageing but the increase in CD45RA− CD27− and CD45RA+ CD27− T cells in old individuals is primarily the result of CMV infection. We next investigated whether the increase in CD45RA− CD27− and CD45RA+ CD27− CD4+ cells in CMV-seropositive donors only occurred within CMV-specific CD4+ T cells or also in those that are specific for different persistent viruses. To do this, we first identified virus-specific populations by intracellular IFN-γ staining after stimulation with lysates of virus-infected cells for

18 hr (see Supplementary Information, PD98059 mouse Fig. S1a).15 Background responses detected in unstimulated cells (negative control) were subtracted from those detected in stimulated samples. Only responses > 0·02% above background were considered positive. The IFN-γ secretion after stimulation with viral lysates was specific because no cytokine production was observed when CMV lysate was used to stimulate CD4+ T cells from CMV-seronegative donors as described previously.15 Y27632 We found that in CMV-seropositive donors, there was a significantly higher proportion of CMV-specific CD4+ T cells compared with T cells that were specific

for other persistent viruses such as VZV, HSV EBV or mycobacterial antigens (tuberculin PPD) (see Supplementary Information, Fig. S1b). We next investigated whether the increased proportion of CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells in CMV-seropositive donors (Fig. 1c) was only the result of changes

within the CMV-specific T-cell population. We found that there were significantly more CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells in CMV-seropositive donors compared with CMV-seronegative donors (Fig. 2a,b). However, although the majority of CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells in CMV-seropositive donors were Ceramide glucosyltransferase CMV-specific, there was also a higher proportion of CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells specific for the other viruses in CMV-seropositive subjects (Fig. 2b,c). Similar results were observed in both young and old donors (data not shown). This result reinforces the idea that CMV infection influences directly the composition of the CD4+ T-cell pools. Furthermore, our results indicate that CMV infection may have a global effect on driving the differentiation of other antigen-specific CD4+ T cells. This confirms our previous observations where the relative expression of CD28 and CD27 instead of CD45RA and CD27 was used to identify CD4+ T cells at different stages of differentiation.15 Several reports on CD8+ T cells suggest that the CD45RA+ CD27− subset is terminally differentiated17,22 with limited capacity for self-renewal.

The mice were used at the age of 8–10 weeks. The mice had free access to water and to standard mouse chow (Altromin®, Lage, Germany)

and were kept in a room with 12-h day/night cycle. All animal experiments were approved by the https://www.selleckchem.com/products/Maraviroc.html Danish Animal Inspectorate. CHS experiments were performed largely as described previously [17]. In brief, the mice were sensitized on day 0 by applying 20 μl 0·5% DNFB (1–fluoro-2·4-dinitrobenzene; Sigma, St Louis, MO, USA) or 100 μl 1% oxazolone (4-ethoxy-methylene-2-phenyl-3-oxazalin-5-one; Sigma), dissolved in 4:1 acetone (VWR)/olive oil (Sigma) on the shaved abdominal skin. Five (DNFB) or six (oxazolone) days later, the baseline ear thickness on the left ear was measured, after which both sides of the left ear were challenged by epicutaneous application of 20 μl 0·2% DNFB or 20 μl 0·75% oxazolone. The challenge treatment was performed under light anaesthesia with isoflurane. The ear thickness of the left ear was measured 24, 48 and 72 h after challenge with a dial thickness gauge from Mitutoyo (Mitutoyo Pocket Thickness Gages 7309; Kawasaki,

Japan). The ear swelling (ΔT) was calculated Selleck Staurosporine as ear thickness 24, 48 or 72 h after challenge minus baseline ear thickness. It is expressed as the mean ± standard error (s.e.m.) in units of 10−2 mm. In the dose-titration studies with CTLA-4-Ig (see Fig. 1) one group was sensitized with acetone/olive oil alone but challenged with DNFB or oxazolone, which induced a non-specific irritative ear-swelling before response. Another group was treated only with acetone/olive oil in both the sensitization and challenge phases, and together these two groups served as negative controls. For resensitization experiments, mice were repainted epicutaneously with 0·5% DNFB or 1% oxazolone on the shaved abdomen 3 weeks after the first sensitization. Five or 6 days later, 20 ul

of 0·2% DNFB or 20 ul 0·75% oxazolone was applied to the left ear and ear thickness was measured 24, 48 and 72 h post-challenge. All groups always comprised five animals. CTLA-4-Ig (Orencia®, Abatacept marketed by Bristol-Myers Squibb, New Hampshire, USA) was tested in doses of 1, 5, 25 or 125 mg/kg, as indicated. As controls, mice, injected with the Fc-part of a human IgG1 (BioXcell, Penzberg, Germany), in the same doses as CTLA-4-Ig, were included in all experiments. Serum levels of CTLA-4-Ig were determined by anti-human IgG1 enzyme-linked immunosorbent assay (ELISA) (Invitrogen, Carlsbad, CA, USA) 3 and 21 days after administration. To examine the activation status of T cells after sensitization, inguinal lymph node was removed 24 h post-sensitization. Single-cell suspension was prepared by transferring the lymph node through a 70-μm cell strainer and washing cells with 1 × phosphate-buffered saline (PBS) (w/o Mg2+ and Ca2+; Gibco/Invitrogen). Cells were resuspended at 10 × 106 cells/ml and 1 × 106 cells/sample were used for staining.

Animals in Group 4 and Group 5 received immunotherapy with 78 kDa and 78 kDa + MPL-A, respectively. This also consisted of two subcutaneous injections at same intervals. In Group 4, each mouse received 10 μg of 78 kDa, while in Group 5, each mice received 10 μg of 78 kDa antigen along with 40 μg of MPL-A. Animals in Group 6 serve as positive controls (infected mice only) and in group 7 as negative controls (normal mice). Normal mice include those animals which were neither infected with promastigotes of L. donovani nor given any kind of treatment, whereas infected mice were given 1 × 107 promastigotes of Panobinostat mouse L. donovani (Table 1). Six

mice from each treated and control groups were euthanized on 1 [55 days post-infection (d.p.i.), 15 (70 d.p.i.) and 30 (85 d.p.i.) post-treatment days (p.t.d.)]. Blood from different treated and control animals was collected by jugular vein incision. Then, blood was centrifuged to obtain serum, which was stored at −20°C until https://www.selleckchem.com/screening/kinase-inhibitor-library.html use. The liver and spleen of the individual animals were taken out and weighed. To quantitative levels of infection in liver and spleen, Giemsa-stained impression smears

were made and fixed in methanol. The parasite load was assessed as Leishman-Donovan units (LDU) and calculated as: Number of amastigotes/Number of cell nuclei X weight of organ in milligrams [22]. Two days prior to the day of sacrifice, 20 μL (40 μg) of leishmanin was injected subcutaneously in right footpad and PBS in the left footpad of mice. After 48 h, the thickness of the both foot pads was measured using a pair of vernier callipers. The DTH response was evaluated

in terms of percentage increase in footpad thickness according to the formula: difference between right and left footpad thickness/thickness of left footpad × 100 [23]. Conventional ELISA was used to determine the levels of serum immunoglobulin G (IgG) isotype antibody (IgG1 and IgG2a) by the method of Kaur et al. [23]. Shortly, 96-well plates were coated with 78 kDa antigen and incubated overnight at 4°C. After blocking with 4% bovine serum albumin, plates were incubated with serum samples at 37°C for 1 h followed by three washes and addition of 100 μL of anti-mouse secondary antibody conjugated with HRP in a dilution of 1 : 8400 3-oxoacyl-(acyl-carrier-protein) reductase of IgG1 (Serotec) and 1 : 2000 dilution of IgG2a (Serotec) and incubated further for 1 h at room temperature, after which the substrate and chromogen were added and absorbance read on ELISA reader (Bio-Rad, Hercules, CA, USA) at 450 nm. Lymphocytes from spleens of infected and drug-treated mice were seeded in 24-well plates in 1 mL of RPMI-1640 and incubated for 72 h at 37°C. Cells were stimulated with 50 μg/mL of the 78 kDa antigen. Supernatants of these cultures were collected and stored at −20°C. The release of cytokines (IL-2, IL-10, IL-4 and IFN-γ) was measured in the supernatants using commercial ELISA kits (BenderMed Systems, Diaclone, France) [23].

This commonly results in direct sensitization against the partner, potentially making

him an unsuitable donor. HAR may also occur in blood group incompatible transplantation without desensitization. Preformed antibodies cause rejection by binding to HLA antigens expressed on the endothelium of vessels in the transplanted kidney, resulting in activation of the complement cascade with resultant thrombosis and infarction of the graft (reviewed in2). HAR can occur immediately upon reperfusion of Ibrutinib the donor kidney. This catastrophic outcome necessitates the immediate removal of the graft. Clearly avoiding HAR is desirable and crossmatching helps predict and hence prevent this.3 In brief, a crossmatch involves placing recipient serum (potentially containing donor-specific anti-HLA antibodies) onto donor lymphocytes (containing HLA antigens). A cytotoxic reaction (deemed ‘positive’) suggests the presence of preformed DSAbs. A more detailed description is provided later in this manuscript. A 44-year-old woman with end-stage renal failure secondary to reflux nephropathy is interested in a renal transplant and her husband has offered to be a donor. They are of the same blood group but are unmatched on tissue typing (0/6 HLA matches at the HLA-A, -B and -DR loci). They have a complement-dependent cytotoxicity (CDC) crossmatch performed as part of their initial assessment, which shows a positive result for both the T- and B-cell crossmatch (Table 1). Is it safe

to Glycogen branching enzyme proceed? It is not safe to proceed in light of these crossmatch results but clarification steps are needed to better

understand Target Selective Inhibitor Library concentration the reason for the positive results. This could be a falsely positive result (technical issue) or there may be autoantibodies (against lymphocyte antigens) present in the recipient serum. Autoantibodies are generally IgM rather than IgG antibodies. To establish if autoantibodies are responsible for the result an auto-crossmatch should be performed. In this assay, recipient serum is crossmatched against recipient (rather than donor) lymphocytes. Second, the original crossmatch should be repeated with the addition of the agent Dithiothreitol (DTT). DTT reduces the disulfide bonds in IgM thereby preventing IgM antibodies from generating a positive result. IgM antibodies are generally regarded as having no pathological significance in transplantation.4–7 If a repeat crossmatch with DTT is negative then it may be safe to proceed with the transplant. An auto-crossmatch adds weight to this analysis by determining if the recipients are reacting against their own T or B cells in a similar way (Table 2). These results suggest that the reaction of the recipient to the donor is on the basis of autoantibodies. This means that the transplant could proceed using this pairing; however, before most live donor transplants and indeed cadaveric transplants more information is routinely available that aids in forming a more complete assessment of immunologic risk.

PAR-1, PAR-2 and PAR-3 were amplified with 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s). PAR-4 was amplified with 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s). Beta-actin (β-actin) was used as positive control using the following primer sequences: PD-0332991 in vitro β-actin (sense) 5′-CCAAGGCCAACCGCGAGAAGATG-3′ and β-actin (antisense) 5′-AGGGTACATGGTGGTGCCGCCAG-3′; yielding a expected PCR product of 587 bp. Beta-actin was amplified

with 35 cycles (94 °C for 60 s, 60 °C for 90 s, 72 °C for 60 s). Negative control was performed for each reaction and included the omission of the reverse transcriptase or the omission of cDNA in the PCR mix. PCR products were resolved on a 1.5% agarose gel for visualization. Flow cytometry analysis was performed of the freshly isolated naïve CD14+ monocytes and the CD14+ monocytes cultured for 24 h with experimental conditions. Briefly, the freshly isolated naïve CD14+ monocyte cell pellet was washed in PBS containing 1% BSA and 0.1% Na-azide and subsequently used for incubation with fluorochrome-labelled antibodies. The CD14+ monocytes cultured with experimental

conditions for 24 h were placed on ice for 1 h. Subsequently, medium with CD14+ monocytes was transferred to 1.5-ml tubes and centrifuged at 900 g for 5 min at room temperature. Supernatants were harvested; the remaining CD14+ cell pellet was washed in PBS containing 1% BSA and 0.1% Na-azide, and centrifuged at 900 g for 5 min at room temperature. After centrifuging, Mirabegron freshly isolated naïve CD14+ monocytes as well as cultured CD14+ monocytes 3-deazaneplanocin A nmr were incubated with APC-conjugated monoclonal mouse anti-human CD14 antibody, PE-conjugated monoclonal mouse anti-human PAR-1 (ATAP2) antibody, FITC-conjugated monoclonal mouse anti-human PAR-2 (SAM11) antibody, PE-conjugated monoclonal mouse anti-human PAR-3 (8E8) antibody, FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F)

antibody, PE-conjugated monoclonal mouse anti-human TF (HTF-1) antibody, and APC-, PE- and FITC-conjugated isotype control antibodies for 30 min at 4 °C in the dark. After a final washing and centrifuging step, cells were fixated in 2% paraformaldehyde. All cells were analysed using the FACS Calibur (BD Biosciences) and FlowJo software (Tree Star Inc., Ashland, OR, USA). For cytokine assays, naïve PBMCs and naïve CD14+ monocytes recuperated for 24 h and subsequently cultured according to the experimental conditions for 24 h were used. Supernatants were harvested, transferred to 1.5 ml tubes, centrifuged at 900 g for 5 min at room temperature and cryopreserved at −80 °C. Cytokine production (IL1-β, IL-6, IL-8, IL-10 and TNF-α) was determined in triplicate. Standard and positive control recovery for each ELISA assay was between 90–110%.

Surgical drainage (and sometimes excision) of infected lymph nodes and abscesses involving the liver, skin, rectum, kidney and brain is often necessary for healing, particularly for the visceral abscesses. Daily prophylaxis

with Bactrim and/or Itraconazole is recommended during infection-free periods. For more detailed treatment see more options, the interested reader is referred to Roos et al. [1]. One of the main reasons to make a rapid diagnosis of the severe forms of CGD is that such patients may be treated successfully with a bone marrow transplant [7-9]. A few reports suggest that gene therapy may eventually be successful both in X-linked and autosomal CGD [10, 11]. Thus, there are many reasons to identify precisely the genetic defect in patients with CGD. Patients suspected of suffering from CGD (Table 2) must be diagnosed by the inability of their blood phagocytes to generate

reactive oxygen species. This can be performed in various ways. The woman is a relative (mother, sister, daughter, maternal aunt, maternal grandmother) of a CGD patient The woman has symptoms of CGD (see Table 2a) The woman is a relative of a CGD patient R788 and has discoid lupus symptoms Carriership or occurrence of CGD should be tested functionally [NADPH oxidase activity in the neutrophils with a per-cell assay, e.g. nitro-blue tetrazolium (NBT) slide test or dihydrorhodamine-1,2,3 (DHR) assay] and genetically Cell press Usually, purified blood neutrophils [12] are used for these tests, but total leucocytes or even diluted full blood can also be used. Blood can be sent by courier to the testing laboratory, but several precautions must be taken. Ethylenediamine tetraacetic acid (EDTA) or heparin blood can be used for NADPH oxidase activity testing and for preparation of neutrophil lysate for NADPH oxidase component expression by Western blot. In the case of EDTA blood, the neutrophil fraction purified from it must

be recalcified and left for 30 min at room temperature before NADPH oxidase activity can be measured. For DNA preparation, EDTA blood is superior. The blood transport must take place in polypropylene tubes (completely filled) and at room temperature. This means, for instance, no transport in plane cargo compartments. The blood must arrive at the place of investigation within 48 h after vena puncture, preferably within 24 h. A control blood sample must be shipped together with the sample from the presumed patient and/or relative(s). All assays must be performed in parallel with the control cell preparation. The NADPH oxidase enzyme that is affected in CGD reduces molecular oxygen to the one-electron radical superoxide (O2−), which is subsequently reduced further to hydrogen peroxide (H2O2). The reducing equivalents for this reaction are derived from NADPH, which is converted into NADP+ and H+.

In conclusion, the results of the present study suggested that upregulation of IL-21 and IL-10 and downregulation Z-VAD-FMK price of IL-4

in periodontitis tissues may be collectively involved in the increased levels of salivary IgA in chronic periodontitis subjects. Since only cytokine profiles and salivary IgA level were evaluated and, no characterization of naïve B cell switch in the periodontal lesions was performed, these preliminary findings are still not enough to definitely define the mechanisms of Ig isotype switching on chronic periodontitis. However, our results may provide new insights into the possible role of Th-secreted cytokines in driving humoral immune response on periodontal tissue breakdown. The authors thank Ms Jeruza P. Bossonaro for technical assistance and São Paulo State Research Foundation (São Paulo, São Paulo, Brazil) for its financial support (# 2008/09687-0; # 2008/04280-0). “
“This chapter contains sections titled: The immune system Tissues and

cells of the immune system Activation, regulation and functions of immune responses Innate versus adaptive immunity Primary and secondary immune responses Immune cell development Mast cells and basophils Eosinophils Neutrophils Monocytes and macrophages Dendritic cells Natural killer cells CD4+ T helper cells CD8+, cytotoxic T cells B cells γδ T cells learn more Natural killer T cells Anatomy of the immune system Lymph nodes Spleen Summary “
“Although Fasudil has shown therapeutic potential in EAE mice, the mechanism of action are still not fully understood. Here, we examined the immunomodulatory effect of Fasudil on encephalitogenic mononuclear cells (MNCs), and tested the therapeutic

potential of Fasudil-treated MNCs in active EAE. Fasudil inhibited expression of CCL20 on T cells and migration of T cells, decreased CD4+IFN-γ+ and CD4+IL-17+ T cells, but increased CD4+IL-10+ and CD4+TGF-β+ PLEK2 T cells. Fasudil reduced expression of CD16/32 and IL-12, while elevating expression of CD206, CD23, and IL-10. Fasudil also decreased levels of iNOS/NO, enhanced levels of Arg-1, and inhibited the TLR-4/NF-κB signaling and TNF-α, shifting M1 macrophage to M2 phenotype. These modulatory effects of Fasudil on T cells and macrophages were not altered by adding autoantigen MOG35–55 to the culture, i.e., autoantigen-independent. Further, we observed that, in vitro, Fasudil inhibited the capacity of encephalitogenic MNCs to adoptively transfer EAE and reduced TLR-4/p-NF-κB/p65 and inflammatory cytokines in spinal cords. Importantly, Fasudil-treated encephalitogenic MNCs exhibited therapeutic potential when injected into actively induced EAE mice. Together, our results not only provide evidence that Fasudil mediates the polarization of macrophages and the regulation of T cells, but also reveal a novel strategy for cell therapy in MS.

One week after the last immunization, mice were killed, blood was taken and, following perfusion, intestinal samples were collected using the perfusion-extraction (PERFEXT) technique.20 Ovalbumin-specific IgG and IgA titres were determined by ELISA. Cabozantinib research buy Ninety-six-well plates (Greiner Bioscience, Frickenhausen, Germany) were coated with OVA (20 μg/ml)

and blocked with PBS/BSA. Serially diluted serum and intestinal samples were added followed by goat anti-mouse horseradish peroxidase-conjugated IgA or IgG (SouthernBiotech, Birmingham, AL). Plates were developed with o-phenylenediamine dihydrochloride, stopped with 0·1 m H2SO4 and absorbance was read at 490 nm. Titres of IgG and IgA were determined from the sample dilution giving an optical density value above 0·4. Data were statistically analysed in Prism (graphpad software) using the Student’s t-test, in which *P < 0·05, **P < 0·01 and ***P < 0·001. Although systemic immune compartments and skin-draining LN of CD47−/− mice have been extensively studied, the GALT has not been carefully characterized. We

therefore enumerated cells in the GALT of CD47−/− mice and revealed a 50% reduction of total cell numbers in MLN, LP and PP, compared with those in WT mice (Table 1). In contrast, the number of cells in skin-draining LN and spleen was not significantly different between WT and CD47−/− mice (Table 1). Although immunohistochemical analysis showed normal localization of T and B cells in MLN and PP of CD47−/− mice find more (see supplementary material, Fig. S1a), and both CD47−/− and WT CD4+ T cells in PP and MLN were found to express similar levels of CD44 and CD62L (data not shown), the frequency of CD4+ T cells in MLN and PP of CD47−/− mice was significantly reduced compared with that in WT mice (Fig. S1b). In contrast, the frequency of Foxp3+ CD4+ T cells in PP, but not in MLN, was significantly increased in CD47−/− compared with WT mice (Fig. S1c). Impaired DC migration from the skin and subset-specific DOK2 alterations in splenic DC at steady state have previously been

reported in CD47−/− mice13,14 therefore, we next assessed populations of antigen-presenting cells in the GALT of these mice. As the total number of cells in the GALT of CD47−/− mice was reduced by 50%, frequency rather than total number of cells within cell populations was determined. Flow cytometric analysis showed a significant reduction in the frequency of CD11c+ MHC-II+ conventional DC (cDC) in MLN, but not in LP or PP, of CD47−/− mice (Fig. 1a). In contrast, no significant change in the frequency of CD172a+ CD11clow MHC-IIlow SSClow cells was detected (Fig. 1b). Further phenotypic characterization was therefore focused on cDC and identified two populations of cDC in MLN (see supplementary material, Fig. S2a).