This regimen stimulates the development of Th1-polarized immunity

This regimen stimulates the development of Th1-polarized immunity to OVA. On day 50, the mice were anaesthetized and challenged with 100 µg of OVA in 30 µl of Palbociclib research buy PBS by footpad injection. The DTH reaction was assessed by measuring tissue swelling in the footpad after 24 h (i.e. on day 51) using a caliper; the mice were then killed. To measure sensitization, the popliteal lymph nodes were excised and single cells were prepared under aseptic conditions and suspended in Iscove’s

medium supplemented with 2 mM l-glutamine, 50 µM mercaptoethanol, 50 µg/ml gentamycin and 10% fetal calf serum (all from Sigma, Steinheim, Germany). Samples of 1 × 105 cells/well were transferred to 96-well microtitre plates and stimulated with 0·5 mg/ml OVA and incubated in 5% CO2 at 37°C. After 2 days, the supernatant was collected for cytokine analysis. After 7 days, [3H]-thymidine U0126 research buy was added and cells were harvested 10 h later. Cell proliferation was assessed by measuring [3H]-thymidine incorporation in a β-counter (Perkin Elmer, Waltham, MA, USA). Levels of IFN-γ, TNF and IL-6 in 2 days’ supernatants were assayed by cytometric bead array (CBA; BD Biosciences, San Jose, CA, USA) according to the manufacturer’s recommendations.

Samples were assayed using fluorescence activated cell sorter (FACS)Canto (BD Biosciences Pharmingen, San Jose, CA, USA) and analysed with FCAP Array Software (BD Biosciences). The limits of detection were 17·5 pg/ml for IFN-γ, 7·3 pg/ml for TNF and 5 pg/ml for IL-6. The effects of the three different diets were also evaluated in a Th2-driven airway hypersensitivity model (Fig. 1b) in a second set of animals. Mice were immunized on days 15 and 25 with intraperitoneal (i.p.) injections of 10 µg OVA mafosfamide absorbed onto 2 mg of Al(OH)3

(alum; Sigma). On day 33, the animals were anaesthetized briefly (Isofluran; Baxter Medical AB) and challenged with 100 µg of OVA in 25 µl of PBS by intranasal administration. This procedure was repeated on each of the following 4 days. Twenty-four hours after the final challenge, the mice were anaesthetized (xylazine 130 mg/kg and ketamine 670 mg/kg, i.p.). The chest was opened and blood was drawn by heart puncture. The blood sample was clotted and serum was collected after centrifugation (15 min at 3000 g). Bronchoalveolar lavage was performed by twice instilling 0·4 ml of PBS through the trachea followed by gentle aspiration. The proportion of eosinophils in the bronchoalveolar fluid was evaluated on slides prepared using a cytospin and stained with May–Grünwald/Giemsa. Sensitization was measured as OVA-specific IgE titres in the serum samples by passive cutaneous anaphylaxis [19]. Mouse sera were diluted serially with PBS and 50 µl was injected intradermally into the shaved dorsal skin of anaesthetized (8 mg/kg xylazine and 40 mg/kg ketamine i.p.) Sprague–Dawley rats (Scanbur AB).

, 2005; Jurcisek & Bakaletz, 2007; Weimer et al , 2010; Byrd et a

, 2005; Jurcisek & Bakaletz, 2007; Weimer et al., 2010; Byrd et al., 2011; Nguyen et al., 2011) and direct analysis of human clinical specimens where identification is more challenging (Hall-Stoodley et al., 2006; Bjarnsholt et al., 2009a, b; Nistico et al., 2011). This has prompted the development of proposed criteria that can be used to demonstrate biofilm in vivo along with molecular methods that can distinguish specific

microorganisms in situ ex vivo. Where in vitro biofilms are grown de novo from isolated cultures and the development and molecular components of extracellular polymeric substances (EPS) are known to be specifically of bacterial origin, host-derived components in experimental in vivo infections may be morphologically similar to microbial biofilms necessitating the distinction of microbial biofilms in complex host check details environments in an animal model. Clinical biofilm-associated infections (BAI) are even more challenging, because the infectious agents are often unknown, and pathologically significant biofilm infections need see more to be distinguished from microbial colonization with nonpathogenic organisms. A core definition of a biofilm

accommodating the diversity of BAI is needed. A biofilm is often defined as ‘an aggregate of microbial cells adherent to a living or nonliving surface, embedded within a matrix of EPS of microbial origin.’ Biofilm EPS is an amalgam of extracellular macromolecules including nucleic acids, proteins, polysaccharides, and lipids (Flemming & Wingender, 2010). Within the biofilm, microbial cells are physiologically distinct from planktonic or single, free-floating cells of the same organism; however, at present, this crucial distinction is not a simple determination that can be evaluated by the tests and examinations usually employed in medical diagnostic work-ups. Classically, bacteria exhibit recalcitrance to antibiotics when

they are in biofilms. Pseudomonas aeruginosa exhibits higher tolerance to tobramycin and colistin when it is surface-attached in vitro Fludarabine (Nickel et al., 1985; Alhede et al., 2011), compared with when it is planktonic. Although biofilms are typically described as being attached to a surface, they may also form at interfaces of spatially distinct microenvironments and as suspended aggregates. For example, an air–liquid interface can result in an aggregated mat of microbial cells just as well as those found on a solid surface-liquid interface. The notion that it is sufficient for a biofilm to be an aggregated mass of cells floating in liquid is supported by the observation that aggregates of a methicillin-sensitive strain of Staphylococcus aureus exhibit a much higher tolerance to the antibiotic oxacillin than single, planktonic, cells (Fux et al., 2004), and aggregates of P.

86, 95% CI: 1 04–3 31) and log-additive (OR: 1 35, 95% CI: 1 02–1

86, 95% CI: 1.04–3.31) and log-additive (OR: 1.35, 95% CI: 1.02–1.80) inheritance models. Akaike’s information criterion (AIC) is a measure of the goodness of fit of an estimated statistical model, and it can judge a model by how close its fitted values tend to be to the true values, in terms of a certain expected value. Because of the smaller AIC value (565.6), the log-additive model was accepted as the best fit for these data [30]. The result of association analysis for the haplotype of SNP4/SNP5/SNP6/SNP7 was consistent

with individual SNP analysis in our study (P = 0.00079). This suggests that at least one susceptibility locus for tuberculosis lies within or very close to the region that spans SNP4/SNP5/SNP6/SNP7 selleck chemical see more in ifngr1 in the Chinese Han population, because haplotype has more accuracy and statistical power than individual SNP in LD-based association studies. In addition, the haplotype of SNP4/SNP5/SNP6/SNP7 contained two alleles that are hypothesized to have lower promoter activity, SNP5 (rs1327474, G>A) and SNP4 (rs2234711, T>C), which further explained the reason for the haplotype to be associated with susceptibility to tuberculosis. It is known that patients with complete loss-of-function or TT-deletion alleles of ifngr1 primarily present DNA ligase with a clinical picture

of infection with mildly virulent mycobacteria or Bacille Calmette-Guérin, which occurs usually during early childhood or after vaccination [29, 31]. The sequence around −470delTT

(SNP7) of the ifngr1 gene is reminiscent of a signal transducer and activator of transcription 1 (STAT1) binding site (TTCCtcaAA), and the ifngr1−470delTT allele abolishes the crucial first two positions of this binding motif. In our selected population, no such mutation was found for TTdel of −470delTT. Our results in the Korea population were similar to those in Caucasians. There was also a low frequency of −470delTT in African-Americans [29, 31]. These data showed that −470delTT (SNP7) was a rare mutation and was not distributed widely in the Chinese populations. In addition, the result implied that differences in genotype frequency existed among the populations. In conclusion, we found that SNP6 (A/G) in ifngr1 or nearby genes might be implicated in predisposition to tuberculosis. In addition, the C-A-A-TT haplotype, which included the two alleles that are hypothesized to have lower promoter activity, was associated with susceptibility to tuberculosis. Further studies are warranted to confirm these findings. Investigation of these polymorphisms will be of benefit to our understanding of host and pathogen interactions.

A literature search of databases (MEDLINE, PUBMED and OVID) betwe

A literature search of databases (MEDLINE, PUBMED and OVID) between January 1998 and July 2010 was conducted using both MESH terms and the free text words ‘gene’ or ‘P2X7’ in combination with ‘tuberculosis’ in addition to manual searches of citations retrieved from relevant original studies and review articles and correspondence with researchers in this particular field of study. We corresponded with authors whether data on genotype frequencies were not available in their respective articles. For inclusion in this

analysis, respective studies had to be nonfamilial case–control studies and to provide information regarding the prevalence of P2X7 polymorphisms in tuberculosis patients and control subjects. All control subjects were ethnically matched with case groups. Another prerequisite was that sufficient data be available to calculate odds ratios (OR). HIV-positive patients were excluded

from the metaanalysis. Two investigators (J.X. and L.S) extracted data independently and reached a consensus on all conclusions. For each study, the characteristics of the individual research articles were collected, including author, year of publication, geographic location, gender distribution, mean age, type of tuberculosis, study size, diagnostic methods used to establish tuberculosis Selleckchem NU7441 infection, the techniques used for genotyping variants, DNA extraction methods, the frequency of the genotypes, consistency of genotype frequencies in Hardy–Weinberg equilibrium (HWE) in the control subjects and the source of the control subjects. We evaluated the risk-associated variant allele (1513 C) using the common allele (1513 A) as the reference and the protection-associated variant −762 C allele using the −762 T allele as the reference. Pooled ORs and their corresponding 95% confidence intervals L-gulonolactone oxidase (CI) were estimated using the fixed effects model (Mantel–Haenszel). The random effects model (DerSimonian and Laird) was performed when heterogeneity was present.

Because of the limited number of studies published to date, it was not possible to stratify and analyze data for P2X7 polymorphisms according to geographic location, ethnicity and types of tuberculosis, or to analyze publication bias using a funnel plot. We assessed HWE only in controls because cases may not be in HWE if there was an association between genotype and disease outcome. Statistical analysis was performed using revman software, version 5.0 (Cochrane). A P value <0.05 was considered statistically significant. We identified six studies published between 1998 and 2010 that fit our study criteria (Li et al., 2002; Fernando et al., 2007; Niño-Moreno et al., 2007; Mokrousov et al., 2008; Xiao et al., 2009; Sambasivan et al., 2010).

After calculations, the number of eggs eliminated by each infecte

After calculations, the number of eggs eliminated by each infected mouse was expressed as eggs/g of faeces. Given the fact that S. venezuelensis filiform larvae develop only into female worms in the small intestine of the host, fecundity rate was estimated by dividing number of eggs per number of worms recovered from the intestine of each animal. The eosinophil peroxidase (EPO) assay was used to measure eosinophil activity in the skin and lung as previously described by Strath et al.(28) and modified by Silveira et al.(16).

Briefly, 100 mg of tissue (skin or lung) was homogenized in 1·9 mL of PBS using a tissue homogenizer (Power Gen 125; Fisher Scientific, Pittsburgh, PA, USA). The homogenate was centrifuged (3000g for 10 min), red blood cells in the pellet underwent hypotonic lysis (1·5 mL of 0·2% NaCl) and the molarity was restored

with 1·5 mL of 1·6% NaCl solution containing 5% glucose. After a further centrifugation Fluorouracil cell line (3000g EGFR inhibitor review for 10 min), the pellet was resuspended in PBS (pH 7·4) containing 0·5% hexadecyltrimethylammonium bromide (PBS-HTAB). The cell solution was homogenized again and the homogenates were then freeze-thawed three times in liquid nitrogen, centrifuged for 15 min at 3000 g and the supernatant was used to measure EPO activity. For this assay, 75 μL of each experimental sample obtained from different tissues was incubated with 75 μL of substrate [1·5 mm o-phenylenediamine (OPD) in 0·075 mm Tris–HCl buffer, pH 8·0, containing 6·6 mm

of hydrogen peroxide] for 30 min at room temperature (RT) in the dark. Reaction was stopped by adding 50 μL of 1 m H2SO4. Reaction intensity was read at 492 nm on a micro-plate reader (Emax; Molecular Devices, Sunnyvale, CA, USA) and results are shown as absorbance units. The extent of neutrophil activity was indirectly estimated by myeloperoxidase (MPO) assay as previously described by Ivey et al. (29) and modified by Matos et al. (30). Tissue samples (100 mg of skin Smad inhibitor and lung) were homogenized in extraction buffer (0·1 m NaCl, 0·02 m NaPO4, 0·015 m NaEDTA; pH 4·7) and the pellet underwent hypotonic/hypertonic lysis as described in EPO assay. After further centrifugation (3000g for 10 min), the pellet was resuspended and rehomogenized in 0·05 m NaPO4 buffer, pH 5·4, containing 0·5% HTAB, followed by three freeze-thaw cycles using liquid nitrogen. The resulting solution was centrifuged for 15 min at 10 000g and the supernatant was used for the colorimetric assay. For this, 75 μL of supernatant was incubated with 75 μL of substrate (1·6 mm tetramethylbenzidine and 0·5 mm H2O2 in 0·05 m NaPO4 buffer, pH 5·4) for 30 min at room temperature in the dark. Reaction was stopped by adding 50 μL of 1 m H2SO4. Myeloperoxidase activity present in the sample was measured at 450 nm on a micro-plate reader (Emax; Molecular Devices).

Most of the

NK T cells of both patients were CD8+, with m

Most of the

NK T cells of both patients were CD8+, with minor numbers presenting as double-negative and hardly any as CD4+. This is in contrast to the NK T subsets found usually in the peripheral blood of healthy donors or cancer patients, in which CD4+ NK T cells outnumber double-negative NK T cells and few or virtually no CD8+ NK T cells are found [8,27,28]. Our RCC patient data are in line with the correlation noted in healthy individuals between high peripheral NK T cell frequency and increase in CD4-negative NK T cells [9,28], which has been described to reverse with age [29]. The aberrant CD4-negative (and CD8-positive) NK T phenotype in patients B2 and B7 suggests that progressive differentiation and selected expansion may have occurred [30]. Expression of CD69 and CD161 would suggest that these NK T cells are recently GSK126 in vivo activated buy APO866 and mature [1]. In humans, the number of peripheral CD4+ NK T cells is supported mainly by thymic output and survival and controlled by IL-7 [31], whereas CD4- NK T cells in the periphery are thought to be driven by IL-15-dependent homeostatic proliferation [30,32] Therefore, in the absence of a known antigenic trigger, the high NK T frequency in our patients can most probably be explained by homeostatic expansion, for which the normal levels of IL-15 that are detectable, may be sufficient. Homeostasis would also explain the relatively

stable NK T frequency observed in the patients. The strong drop in CD69 expression, but not in NK T cell numbers, after stopping IFN-α treatment Nintedanib solubility dmso (see Table 4), may indicate that IFN-α can influence activation, but has no direct effect on homeostasis. NK T cells have been described to activate downstream immune effector pathways, and this has prompted combination treatments aimed at activating T cell-mediated anti-tumour responses [3,33,34].

Three factors will determine the outcome of interactions between NK T cells and antigen-presenting cells: (i) frequency, strength and duration of antigenic stimulus; (ii) differentiation state of antigen-presenting cells; and (iii) presence or absence of cytokines that co-stimulate NK T cells, among which is IFN-α[35]. IFN-α treatment of ourpatients does not appear to be a trigger for high NK T frequency, as low to normal NK T cell counts were present in 12 of 14 RCC patients. Furthermore, in patient B7 the high NK T frequency could be shown to be already present before therapy. However, IFN-α was found to enhance the activation state in a co-stimulatory manner. As shown in Table 4, it increased CD69 expression of NK T cells, sometimes with a short delay. Particularly in patients B2 and B7, changes in activation of conventional T and non-T cells, parallel to NK T cells, were observed, indicating that IFN-α treatment also affected these cell types.

First, we evaluated the qualities of the BMT model Multilineage-

First, we evaluated the qualities of the BMT model. Multilineage-full

chimerism in the case of BMT using fully allogeneic BMC and multilineage-mixed chimerism in the case of BMT using mixed BMC were confirmed in the PBL of all recipients prior to tumour inoculation (Fig. 4A and data not shown). As previously reported, a small population of recipient-derived radio-resistant T cells were detected in recipients transplanted with BL6 BMC (the lower right dot plot in Fig. 4A). However, these T cells showed no alloresponses [29] and so should not affect the survival time of injected allogeneic DC. The B-cell and myeloid lineage chimerism RXDX-106 in the PBL was complete in the recipients transplanted with BL6 BMC (the upper right dot plot in Fig. 4A and data not shown). This suggests that the bone marrow haematopoietic cells had been completely replaced by donor-derived BMC. We also assessed the chimerism of the DC in the lymph nodes (inguinal and axillary) and tumours of the B/c recipients. A significant SB525334 solubility dmso percentage of recipient-derived DC was detected in the lymph nodes of recipients transplanted with BL6 BMC (Fig. 4B). More than 60% of residual B/c recipient-derived H-2Kd positive DC in the cutaneous draining lymph nodes expressed DEC205 and I-Ad high, both markers

found on Langerhans cells (data not shown) [34]. This suggests that these cells were derived from Langerhans cells repopulated from the radio-resistant progenitor cells in the cutaneous niche [35]. Because Langerhans progenitor cells in the cutaneous niche are destroyed by any contaminating donor-derived T cells

that mediate graft-versus-host disease (GVHD) [35], the presence of host-derived Langerhans cells in the draining lymph nodes in this study suggests that the T-cell depletion of the donor BMC was complete. This is supported by the fact that we observed no GVHD response in these mice; this observation was confirmed by histological analysis at autopsy (data not shown). To the contrary, almost all tumour-associated DC were positive for H-2Kb and negative for H-2Kd (Fig. 4B). This suggests that the tumour-associated DC were derived from donor BMC. Taken together, we judged that the developed BMT model was qualitatively appropriate to evaluate the three factors individually in ITADT. Surprisingly, we found that ITADT check details using BL6 DC showed a significant antitumour effect in terms of tumour growth suppression as well as ITADT using B/c DC in recipients of mixed BMC (BL6+ B/c B/c), despite the MHC incompatibility of the injected BL6 DC (Fig. 4, middle graph). However, ITADT using BL6 DC did not show any significant antitumour effect in recipients of BL6 BMC, despite the fact that these recipients were tolerant to BL6 (Fig. 4, left-hand graph), while ITADT using B/c DC did show a significant antitumour effect in recipients of BL6 BMC. In recipients of syngeneic BMC (B/c B/c), ITADT using B/c DC showed a significant antitumour effect.

3c) The results were obtained in two independent groups of BLT-N

3c). The results were obtained in two independent groups of BLT-NSG mice engrafted with HLA-A2+ thymus and liver. Together our data indicate that T cells obtained from selleck kinase inhibitor BLT-NSG mice during acute infection and in the memory phase secrete cytokines in response to stimulation with multiple DENV peptide pools as well as known HLA-A2-restricted DENV peptides. We next assessed the generation of DENV-2-specific antibodies in DENV-infected BLT-NSG mice by sandwich

ELISA. Sera from DENV-2 NGC-infected BLT-NSG mice had significantly higher IgM antibody responses against the DENV-2 envelope protein compared with responses detected in HLA-A2-transgenic NSG mice engrafted with human cord blood HSC (Fig. 4a) and previously published data in HLA-A2 cord blood Ruxolitinib mouse HSC-engrafted NSG mice.14 High IgM responses

were consistently validated in the sera of mice up to 8 weeks post-infection (Fig. 4b). Little or no DENV-specific IgG was detected even 8 weeks post-infection with DENV-2 NGC (Fig. 4b). We assessed whether multiple immunizations with DENV-2 NGC would enhance antibody responses and found a modest increase in IgM antibodies in the sera of mice that were infected more than once with DENV (Fig. 4c). No IgG responses were detected in the sera of mice immunized multiple times (data not shown). To determine whether the strain and dose of DENV influenced antibody responses, we infected mice with increasing doses of DENV-2 S16803 (a live-attenuated vaccine strain) (Fig. 4d). We found similar IgM antibody responses in the sera of mice infected with DENV-2 NGC and DENV S16803. Irrespective of the inoculation dose IgM responses were similar and in all cases we detected

low DENV-specific IgG responses. Our data indicate that IgM antibodies, which are neither viral strain-dependent nor dose-dependent, are the predominant isotype produced in response to dengue viral infection in BLT-NSG mice. Experiments were conducted next to determine whether splenic B cells from BLT-NSG mice were able to secrete DENV-specific antibodies. We used culture supernatants from stimulated splenocytes as a source of DENV-specific antibodies. We were able to detect antibodies in the supernatants of immune but not naive splenocytes from BLT-NSG mice that bound an inactivated lysate of DENV-2 and the DENV-2 Coproporphyrinogen III oxidase E protein (Fig. 5a). We next tested the neutralizing activity of DENV-2-specific antibodies generated by B cells in infected mice. We found that supernatants obtained from stimulated splenocytes of DENV-2-infected mice inhibited DENV-2 infection of Vero cells whereas supernatants obtained from stimulated naive splenocytes were unable to reduce infection (Fig. 5b). A summary of DENV-specific neutralizing activity (41–97% neutralization at 1 : 5 dilution) (n = 6) in supernatants obtained from splenocytes of infected mice is shown (Fig. 5c).

Interleukin-17 production by memory CD8+ T cells, displaying a CD

Interleukin-17 production by memory CD8+ T cells, displaying a CD27+ CD28+/− CD45RA− phenotype

in humans, was described by Kondo et al.62 CD4+ Tregs are characterized by co-expression of FoxP3 and high levels of CD25.63 We observed comparable frequencies of CD4+ (CD25high FoxP3+) Tregs in PBMCs from HD and NHPs. CD8+ Tregs (CD8+ CD25+ FoxP3+) have been described in humans,64,65 and in rhesus monkeys.66 We show that CD8+ Acalabrutinib manufacturer Tregs (CD8+ CD25interm./high FoxP3+) were present in PBMCs from NHPs in higher frequencies compared with HDs. The same was true for other T-cell subsets co-expressing FoxP3 and CD25 with putative regulatory functions, i.e. CD4+ CD25interm FoxP3+, CD4+ CD8+ CD25interm./high FoxP3+. The FoxP3 and CD25 can be induced upon T-cell activation, it is exclusively expressed by Tregs. The observation that NHPs showed a decreased number of bona fide IL-7Rα+ in CD4+ Tregs underlines the fact that differential suppressive functions may be present in NHPs compared with HDs. FoxP3 interacts with the IL-7Rα promoter and facilitates the down-regulation of IL-7Rα in CD4+ CD25bright Tregs;67 negative staining for IL-7Rα was postulated as a marker for human Tregs in concert with CD4, CD25 and FoxP3 analysis.68,69 A low percentage of human Tregs express IL-7Rα and these cells are important in diseases: a recent study showed that

human CD3+ CD4+ CD25+ Tregs, which stain positive for IL-7Rα, exhibit an aberrant functional capacity in patients with autoimmune diseases: they exhibit increased proliferation Exoribonuclease and more IFN-γ/IL-2 production compared with the same cells from healthy individuals.70 The number of Fostamatinib order IL-7Rα+ expressing CD4+ Tregs was lower in NHPs than in HDs and this may also provide the cellular basis for differential suppressive networks in NHPs. In summary, we showed, using high content flow cytometry, that the cellular immune system in humans and NHPs exhibited high level of communalities, including a unique CD4+ CD8αα/αβ+ T-cell population with cytotoxic potential. Differences

between humans and NHPs reside in immune cell subsets with long-term memory, i.e. in CD8αα+ T cells and in cells with regulatory functions. This may be biologically important in chronic disease models where inflammatory patterns contribute to immune pathology. We would like to thank Meryl Forman, Beckman Coulter (Miami, FL) for her valuable advice concerning antibody selection and the choice of fluorochromes on custom-labelled reagents. The project was funded in part by the AERAS foundation, from Karolinska Institutet, from SIDA, Vetenskaprådet and from the Söderberg Foundation, Sweden. The study was in part financed by the Aeras foundation, by a Marie-Curie Host Fellowship for Early Stage Researchers Training grant to I.M., from Cancerfonden, the Söderberg foundation, SIDA, Vetenskapsrådet and Karolinska Institutet to M.M.

A number of studies suggested that Treg exert their suppressive f

A number of studies suggested that Treg exert their suppressive function on effector T cells indirectly by modifying the function of antigen-presenting

dendritic cells. Interestingly, a recent in vitro study showed that LFA-1 is important for the formation of dendritic cell/Treg aggregates, because LFA-1−/− Treg were no longer able to inhibit the maturation of cocultured dendritic cells 20. Similar effects were also observed in a mixed human/mouse suppression system 21. We Selleck CYC202 show here that LFA-1 deficiency results in a reduced Treg/effector cell ratio in the inflamed CNS. The reduction in Treg was already established in the spleen and thymi of unimmunized LFA-1−/− mice. Hence, besides a possible functional impairment of Dorsomorphin order Treg lacking LFA-1, these results indicate a more fundamental role for LFA-1 in generation of FoxP3+ Treg in the thymus. ICAM-1, a ligand of LFA-1, is expressed on thymic stromal cells 22. Therefore, LFA-1 potentially increases the

physical contact between thymocytes and stromal cells, resulting in enhanced T-cell receptor triggering. Increased T-cell receptor signaling during thymocyte selection favors the generation of naturally occurring Treg 23, which would explain the contribution of LFA-1 to the generation of naturally occurring Treg. So far, LFA-1 has been mainly recognized as a molecule regulating the migration of lymphocytes. Generally, the migration of LFA-1-deficient T cells to the peripheral lymph nodes is impaired, resulting in significantly smaller lymph nodes 10, 14. However, upon immunization with MOG-peptide, we observed that these differences in cellularity in lymph nodes between WT and LFA-1 KO mice are more or less levelled out (data not shown). In the context of EAE and transendothelial migration, Laschinger et al. 11 demonstrated that encephalitogenic G protein-coupled receptor kinase T cells do not use LFA-1 for the initial adhesion to the endothelium of the blood/brain barrier. Instead, LFA-1 was involved in the later phases of migration into the CNS parenchyma. However, it should be noted that these results were obtained for the healthy spinal cord and that the role of LFA-1 for migration

could be different during later stages of an EAE disease, in which other integrin interactions may compensate for the lack of LFA-1. In our study, we did not directly address the question of lymphocyte migration via the blood/brain barrier. However, the observation that the frequency of MOG reactive CD4+ T cells in LFA-1−/− mice is already higher outside the CNS suggests an impaired suppression of effector T cells by Treg rather than an altered migration as cause for the higher ratio of effector versus Treg in the inflamed CNS in LFA-1−/−. Overall, the exacerbated EAE in the absence of LFA-1 seems to be due to the impaired suppression of autoantigen-specific effector T cells by Treg, which in LFA-1−/− mice show a more extensive expansion in secondary lymphoid organs upon immunization with the MOG-peptide.