15 Administration of interleukin (IL)-10-treated DCs markedly suppressed the development of AHR, inflammation, and Th2 cytokine production.16 Similarly, activation of DCs with antibodies directed

to a member of the family of B7 costimulatory molecules PD-1 costimulatory molecule ligand ex vivo before adoptive transfer into pre-sensitized mice Vemurafenib was shown to be sufficient to protect animals from inflammatory lung disease induced by subsequent repeated airway exposure to the offending antigen.17 In this study, we investigated whether transfer of histamine-treated allergen-pulsed DCs changed the course of the allergic response, in a well-defined model of OVA-induced allergic airway inflammation.18 All experiments were carried out using 2-month-old virgin female BALB/c mice raised at the National Academy of Medicine, Buenos Aires, Argentina. Mice were housed six per cage and

kept at 20 ± 2° under an automatic 12 hr light/dark schedule. Animal care was in accordance with institutional guidelines. Mice were sensitized using a standard protocol, as described previously.18 Briefly, mice were injected intraperitoneally (i.p.) with 20 μg of OVA (grade V; Sigma-Aldrich, Sigma, San Louis, MO) in 2 mg of aluminium hydroxide (alum) at days 0 and 7. Control mice received a saline injection instead of OVA/alum solution. On day 14, sensitized mice were challenged intranasally with 50 μl of phosphate-buffered learn more saline (PBS) containing 3% OVA for 5 days. Control mice were instillated with PBS. The procedure used to obtain DCs was as described by Inaba et al.,19 with minor modifications.20 Florfenicol Briefly, bone marrow was flushed from the long bones of the limbs using 2 ml of RPMI-1640 (Invitrogen, Carlsbad, CA) with a syringe and 25-gauge needle. Red cells were lysed with ammonium chloride. After washing, cells were suspended at a concentration of 1·5 × 106 cells/ml in 70% RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 5·5 × 10−5 mercaptoethanol (Sigma, San Louis, MO) (complete medium) and 30% J588-GM cell line supernatant. The cultures were fed every 2 days by gently swirling the plates, aspirating 50% of the medium,

and adding fresh medium with J588-GM cell line supernatant. At day 9 of the culture, > 90% of the harvested cells expressed MHC class II, CD40 and CD11c, but not Gr-1 (not shown). DCs obtained from bone marrow precursors were incubated in the absence or presence of histamine (1 μm) (DCs and DCHISs, respectively) for 30 min at 37°. Cells were then incubated for 3 hr at 37° in the presence or absence of OVA (100 μg/ml). Finally, DCs were washed and injected intratracheally (i.t.) into BALB/C mice after intranasal challenge of sensitized mice with OVA. For this purpose, mice were anaesthetized with embuthal (2% v/v in PBS), and 100 μl of PBS, DCs or DCHISs (5 × 105 cells) was injected. Lungs were cut into small pieces and treated with Type I collagenase (250 U/ml) (Roche; Bs.As.

Therefore, shrimp antiviral immunity combines aspects of the insect selleck compound antiviral RNAi pathway with aspects of the mammalian dsRNA response. Whether this is also the case for other arthropods or other organisms thought to exclusively rely on antiviral silencing, remains unclear. Of note, while there is no specific therapeutic against WSSV, genetic selection for shrimps that are resistant to infection by WSSV or that do not develop the pathological consequences of infection (white spot disease) has led to the development of three selected lines of Litopenaeus vannamei. While there was still some mortality post WSSV challenge, all

infection survivors were qPCR negative for WSSV [37] but whether this is due to an increase in the efficacy of antiviral silencing is unknown. Nevertheless, harnessing this cocktail of antiviral responses may one day be used to protect marine animals and valuable food sources from viral pathogens. Moreover,

an understanding of the antiviral pathways conserved between shrimp and insects, such as mosquitoes, may aid in efforts to develop immune-based therapies against human arboviruses. This work was supported by grants from the National Institutes of Health (R01AI074951, U54AI057168, and R01AI095500) to S.C. L.R.S. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2016-12). S.C. is a recipient of the Burroughs Wellcome Investigators in the Pathogenesis

of Infectious Disease Award. The authors declare no financial or commercial conflicts of interest. “
“Eosinophils not only have multiple functions as ABT263 effector cells of the innate immune system but also as modulators of immune responses. As producers of cytokines required for plasma cell survival, they are essential for the long-term maintenance of plasma cells in the BM. Here we show that the activation of eosinophils both in vitro and in vivo enhances the expression of the plasma cell survival factors APRIL, IL-6, IL-4, IL-10 and TNF-α. The in vivo activation of eosinophils was independent of the type of adjuvant used for primary immunization. Although eosinophils were activated by adjuvant itself, a stable activation and a constant increase Loperamide in BM eosinophils were observed only in the presence of antigen. Thus, the numbers and the quality of eosinophils were dependent on priming the adaptive immune system. With secondary immunization and re-activation of antigen-dependent memory cells, the ability of eosinophils to promote plasma cell survival was further increased. These findings suggest that in T-cell-dependent immune responses eosinophils are conditioned to support the long-term survival of plasma cells in the BM, and furthermore imply that through accelerated numbers of eosinophils, stable plasma cell survival niches are established and the long-term survival of plasma cells is ensured.

Thus, in Australia and New Zealand in 2005, live donor transplants accounted for 41% of the total transplants performed.

In comparison, although the number of deceased donor transplants performed was similar 10 years earlier in 1995 (348 in Australia and 70 in New Zealand), fewer live donor transplants were performed (94 in Australia and 24 in New Zealand), thus in 1995, live donor transplants accounted for only 22% of the total transplants performed.1 This progressive increase in the number of live donor transplants performed is indicative of the overall success of kidney transplantation as well as the increased confidence in using live donors. However, it also reflects the continued shortage of deceased donor organs. Since 2000, 12-month primary BYL719 deceased donor recipient

survival in Australia and New Zealand has been approximately 96%, and 12-month primary deceased donor graft survival has been approximately 92%.1 In comparison, 12-month primary Alectinib manufacturer live donor recipient survival has been approximately 99%, and 12-month primary live donor graft survival has been approximately 96%.1 Examining longer term results: recent 5-year primary deceased donor recipient survival has been approximately 87%, with 5-year primary deceased donor graft survival being approximately 80%. In comparison, 5-year live donor recipient survival has been approximately 94%, with 5-year live donor graft survival being approximately 86%. These recipient and graft survival outcomes for both deceased and live donation are excellent. Unadjusted figures show superior outcomes for live donor transplantation relative to deceased donor transplantation. Various studies have assessed the success of live donor kidney transplantation relative to the donor source (e.g. related, unrelated, spousal). In general, graft survival is excellent and equivalent regardless of whether the donor is related or

unrelated.2–5 Decitabine Unmatched, unrelated live donor transplants show similar or superior results compared with deceased donor transplants.2–5 Gjertson and Cecka analyzed United Network for Organ Sharing (UNOS) Registry data and found that 5-year graft survival rates for spousal, living unrelated and parental donation were all similar (75%, 72% and 74%, respectively).5 Graft half-lives were 14, 13 and 12 years, respectively.5 Mandal et al. analyzed USRDS data and compared primary deceased donor versus primary live donor transplantation for different age groups.6 The outcomes for recipients aged over 60 years (n = 5,142) demonstrated that live donation was always associated with a better outcome. Comparing deceased donor with live donor renal transplant in this older age group, the relative risk of death was 1.72 and the relative risk of graft failure was 1.64. Living donor renal transplantation for recipients aged 18–59 years was also generally associated with better outcomes compared with deceased donor renal transplantation.

Recombinant HSP20 was amplified from E. multilocularis cDNA by PCR (primer set: 5′-CAGTGGATCCTTGATTTTCCCTGTTCGC-3′ and, 5′-CAGTAAGCTTTCATTTAAAGAGAGGTGCCT-3′). The fusion protein was expressed in Escherichia coli (strain SG130009). The recombinant protein (HSP20), which contains an N-terminal His-tag, was purified on a Ni-NTA agarose column, according to the methodology provided by the supplier (Qiagen, Hilden, Germany). The recombinant protein was used as

Adriamycin datasheet antigen in SDS-PAGE and immunoblotting. HSP20 in 10% SDS-PAGE in reducing and nonreducing conditions showed two bands at 34 and 50 kDa (Fig. 1c). Mass spectrometry analysis revealed that the spectra obtained after tryptic digestion of the bands at 34 and 50 kDa both corresponded to the same protein (Fig. 1d). We identified, using an IB assay, IgG against the 34 kDa subunit of HSP20 in sera from 61/95 (64%) patients with CE, but not in sera from age-matched healthy subjects. Conversely, all sera from CE patients and from healthy subjects recognised the 50 kDa subunit of HSP20. As the subunit at 34 kDa appears the most specific, in this study, we evaluated exclusively antibodies specific to it (Fig. 1e). The pre-absorption with HSP20 of the sera from two patients with CE completely inhibited the antibody reactivity confirming the specificity MK-2206 cost of IB. Dividing

the 95 patients with CE accordingly to the state of the disease (active or inactive), we observed that serum antibodies to HSP20 were present in sera from 54/66 (81%) patients with active disease (CE1-CE2 cysts), and from 7/29 (24%) patients with inactive disease (CE4-CE5 cysts) (P = 10−4 with Fisher test). To highlight the usefulness of the protein for monitoring disease progression, we tested by IB, in a long-term follow-up, sera from 20 patients Rucaparib nmr with CE surgically and/or pharmacologically treated (Table 1). IB analysis revealed HSP20-specific IgG in sera from 10 of the 13 patients (78%) with cured disease in the active phase of the disease (T0) and no reaction at the end of follow-up (T1). Conversely, the IB pattern of anti-HSP20 antibodies unchanged during follow-up in sera from six of the seven

(86%) with progressive disease (P = 0·017 with Fisher test). To note, IB analysis revealed that antibodies specific for a partially purified fraction of hydatid fluid and for antigen B unchanged in all patient’ sera during follow-up (data not shown). In the present study, using a proteomic strategy, we identified HSP20 as a new antigenic target of IgG in patients with CE. As HSP20-IB detected specific antibodies in an elevated percentage (81%) of patients with active disease, this new antigen might be a marker of disease status. Confirming these results, in long-time follow-up, serum antibodies specific for HSP20 markedly decreased over the course of treatment in patients with cured disease relative to patients with progressive disease.

0001) (Fig. 2C). The establishment of functional T-cell memory is vital for the success of an immunization protocol. To assess if functional CTL responses could be generated by a single immunization or if a prime boost regime JQ1 concentration were required, C57BL/6 mice were given single or multiple immunizations with TRP2/HepB human IgG1 DNA. No epitope-specific responses were detectable 20 days after a single immunization with TRP2/HepB human IgG1 DNA, but high-frequency responses were detectable after two immunizations (p=0.026) which increased further

with another immunization (p<0.0001) (Fig. 2D). The avidity of responses after two or three immunizations was analyzed. The responses induced in mice receiving two or three DNA immunizations were of high avidity (1.4×10−12 M and 1.8×10−12 M,

respectively). There is no significant difference in avidity between these two groups (p=0.89) (Fig. 2E). As both the frequency and avidity of the CTL response appear enhanced, the question “was avidity related to frequency?” arose. Over 80 mice were immunized with TRP2/HepB human IgG1 DNA and the frequency and avidity of responses measured. The avidity of the TRP2-specific responses ranged from 5×10−8 M to 5×10−13 M peptide. No significant correlation learn more between avidity and frequency of TRP2 peptide-specific responses was identified, suggesting they are independent events (Fig. 3A). It is possible that xenogeneic human Fc influences the frequency and avidity of responses induced. Comparison of responses from immunization with human IgG1 or an equivalent murine IgG2a construct reveals similar frequency and avidity (Fig. 3B), suggesting that the xenogeneic human Fc was not influencing the response. Synthetic peptides have short half lives in vivo and are poor immunogens as they

have no ability to specifically target professional Ag presenting cells such as DC. Current therapies are showing DC pulsed with peptide induce an efficient immune response. TRP2/HepB human IgG1 DNA immunization was compared to DC pulsed with HepB/TRP-2 linked peptide. TRP2/HepB human IgG1 DNA demonstrated similar frequency responses compared to those Celastrol elicited by peptide-pulsed DC, both of which were superior to peptide immunization (p=0.0051 and p=0.0053) (Fig. 4A). Analysis of the avidity of responses reveals that the avidity in TRP2/HepB human IgG1 DNA immunized mice is 10-fold higher than with peptide-pulsed DC (p=0.01) (Fig. 4B). The TRP2 specific responses were analyzed for ability to kill the B16F10 melanoma cell line in vitro. Figure 4C shows that although responses from peptide and peptide-pulsed DC immunized mice demonstrate a good peptide-specific lysis, mice immunized with TRP2/HepB human IgG1 DNA showed better killing of the B16 melanoma cells (p=0.003). The enhancement of avidity could be related to direct presentation of the epitopes by the Ab–DNA vaccine and similar responses may be elicited by a DNA vaccine incorporating the native TRP2 Ag.

Briefly, the DE52-purified parasites were resuspended in

Balts-buffer (50 mM sodium phosphate buffer, pH 5.5) and incubation on ice for 30 min followed by a 5-min incubation at 37°C. The solution was subsequently centrifuged (1400 rpm, 7 min, 4°C) and the supernatant treated with benzonase (VWR) to remove potential DNA/RNA contamination Pictilisib solubility dmso (as described by the supplier). The supernatant was dialyzed against 10 mM Tris, pH 7.4, and the sVSG was purified using ion-exchange chromatography and gel filtration as described previously 79, 80. mfVSG was prepared as described previously 81. Prior to performing a size exclusion chromatography (equilibrated against 10 mM Tris, pH 7.4, containing 0.02% N-octylglucoside, Sigma-Aldrich), the mfVSG was treated with benzonase (similar as for sVSG) to remove potential nucleic acid contamination. The protein concentration of both VSGs was estimated spectrofotometrically by a detergent-compatible protein assay kit (Bio-Rad) using BSA as a standard. The purity of both sVSG and mfVSG was checked in SDS-PAGE and found to be >95%. In addition, Western blot analysis, using rabbit polyclonal anti-VSG and anti-cross-reacting determinant Abs confirmed the presence of the GPI anchor on mfVSG 82. Finally, the endotoxin levels were determined using the Limulus amebocyte lysate (LAL) test (Cambrex) according to the manufacturers’ instructions and found to be <0.5 pg/μg VSG. BM-DCs were generated as

described previously 83.

Briefly, BM-precursor cells were isolated from the hind limbs and seeded out in petri dishes (10 cm, Greiner) at 3×106 cells per dish. For microarray analysis, BM-precursor AZD0530 supplier cells were depleted of B and T cells by using anti-CD19 and anti-CD90 magnetic beads (Miltenyi Biotec), respectively. Cells were cultured in RPMI 1640 (PAA) supplemented with 10% heat-inactivated fetal calf serum (FCS, second PAA), penicillin (100 U/mL; PAA), streptomycin (100 mg/mL; PAA), L-glutamine (2 mM; PAA) and β-mercaptoethanol (50 mM; Sigma-Aldrich). Culture medium was additionally supplemented with 10% supernatant from a GM-CSF-transfected cell line 84. At d7 or d8, BM-derived DCs were harvested and replated at a density of 106 cells/mL in a 24-well plate (nontissue culture treated; Greiner). For maturation analysis of cytokine production and surface marker expression, BM-DCs were cultured for 20–24 h in the presence of TNF (500 U/mL; PeproTech), LPS (Escherichia coli 0127:B8 0.1 μg/mL; Sigma-Aldrich), sVSG or mfVSG from clone AnTat1.1 (2 μg/mL), or sVSG MiTat1.5 (2 μg/mL). For in vivo polarization assays, BM-DCs were seeded at a density of up to 5×106 cells/mL, matured for 4 h only with different maturation stimuli and additionally loaded with 40 μg/mL MOG35–55-peptide (synthesized and HPLC purified by R. Volkmer, Charité, Berlin, Germany), 10 μM OVA-peptide327–339 (Activotec) or 50–100 μg/mL OVA protein (endotoxin-free; Hyglos) as indicated.

In this study, the activation of other TLRs such as TLR4 and TLR5 had no effect on Treg generation, supporting our results for TLR4 activation. In our study, TLR7 and TLR9 ligands triggered stronger IL-6 and IL-12 responses in DC–T-cell cocultures than TLR4 ligand LPS.

The defect in stable Foxp3 expression caused by addition of TLR7 ligands to the coculture LDE225 purchase could be mimicked by supernatants of TLR7-stimulated DCs, but not by supernatants of unstimulated DCs or TLR7 ligand-stimulated DCs, which had been pretreated with neutralizing antibody against IL-6. These results suggest that IL-6 produced by splenic DCs early during the coculture in response to TLR7 ligand is largely responsible for the observed loss of Foxp3 expression after transient induction. The addition of neutralizing antibodies to the DC–T-cell cocultures confirmed the major Proteases inhibitor role of IL-6 and additionally revealed a minor role for IFN-γ and IL-4 in inhibiting Treg generation in the presence of TLR7

ligand, which is in accordance with a recent report describing the influence of Th1/Th2-polarizing cytokines on Treg differentiation 22. In the study by Hall et al. using lamina propria DCs stimulated with TLR9 ligand CpG, the inhibitory effects of IL-4 and IFN-γ prevailed over the inhibitory effect of IL-6 on Treg generation. Thus, IL-6 appears to play a less prominent

role for inhibiting Foxp3 expression in the context of lamina propria DCs stimulated with TLR9 ligand than in our study using splenic DCs stimulated with TLR7 ligand 27. It has been previously shown that IL-6 PRKD3 inhibits conversion of naïve T cells into Tregs and supports Th17 differentiation 28, 29. In fact, we also observed higher concentrations of IL-17 in cocultures stimulated with TLR7 and TLR9 ligands correlating with reduced numbers of Tregs. Expression of RORγτ and IL-17 mRNA in Foxp3+ T cells generated in the presence of TLR7 ligand (Supporting Information Fig. S3B) suggests that this population contains cells which are in transition to Th17 cells resembling the recently described proinflammatory “ex Foxp3” cells 26. LPS induced even higher IL-17 production disproportionate to the low amounts of IL-6 induced by LPS compared with TLR7 and TLR9 stimulation. These results support the finding that Th17 induction can also occur independently of IL-6 29. IL-23 did not play a role in our experimental system since it was not induced in DC–T-cell cocultures stimulated with TLR7 or TLR9 ligands. We can exclude that the lower Treg numbers generated in DC–T-cell cocultures in the presence of TLR7 ligands are due to a proliferation or survival advantage of Foxp3− T cells, which could have outgrown Foxp3-expressing Tregs.

Two micrograms of RNA was then reverse transcribed with High Capacity RNA-to-cDNA kit following manufacturers’ instructions (Applied Biosystems, Foster City, CA, USA). Complementary DNA samples (cDNA) were then diluted 1 : 5 in RNAse-free water and stored at −20°C for further use. The expression level of IL-4, IL-10 and IFN-γ was determined by relative quantification using Taqman Q-RT-PCR. Hypoxanthine phosphoribosyl transferase (HPRT) was included as a housekeeping gene and custom-designed by

Applied Biosystems based on sequences obtained from Genbank for IL-4, IFN-γ and HPRT (Accession numbers AF169170, D84216 and M31642, respectively), while for rabbit IL-10, a predesigned assay from Applied Biosystems was used (Oc03396942_m1). EX 527 in vivo Primer-probe pairs sequence for the three cytokines, and the house keeping gene are reported in Pathak et al. (28). Reactions Epigenetics inhibitor were performed in MicroAmp® Optical 96-well plates using 1× Taqman Gene Expression Master Mix, 1× expression assay and 100 ng

cDNA in a 25 μL reaction. PCRs were performed on a 7500 Real Time PCR system using the default cycling conditions: 50°C for 2 min, 95°C for 10 min, 95°C for 15 s for 40 cycles, 60°C for 1 min (Applied Biosystems). Real-time data were expressed as Ct (cycle threshold) values. Ct values for IL-4, IL-10 and IFN-γ were normalized to the HPRT to control for variability in cDNA amount and reaction efficiencies. To quantify local (mucus) and systemic (serum) changes in the IgA and IgG response to the establishment

(L3) and survival (adults) of both nematodes, an enzyme-linked immunosorbent assay (ELISA) was performed. As a source of antigen, we used L3 larvae extracted from a culture of faeces harvested from rabbits infected with the same batch of nematode larvae used in these experiments, while adult nematodes were collected from our wild rabbit population. Nematodes from wild rabbits showed less antibody background noise at the ELISA than the adults extracted from the laboratory infected rabbits (results not showed). Nematodes were washed in PBS and protease inhibitors and subsequently homogenized in a Hybaid ribolyser (2 mm steel balls, twelve 30 s pulses). The extract was spun at 13 000 rpm for 5 min, ID-8 the soluble extract removed, and the protein concentration determined using the Bradford assay (Sigma, Dorset, UK) and then stored at −20°C. The ELISA design was similar for serum and mucus samples of both infections. Antigen concentrations and antibody dilutions were optimized using a checkerboard titration and the optimal dilutions selected at the inflection point from the resulting dilution curves. The dilutions established for the antigen, mucus and secondary antibodies to T. retortaeformis and G. strigosum are reported in Table 1.

[7] The role of intestinal flora in preventing enteric infections was initially attributed to its ability to prevent invasion and colonization by opportunist pathogens in selleck inhibitor the intestinal niche. However, in recent years it has become increasingly apparent that the host microbiota plays a more active role in the development and functioning of the immune system in the gastrointestinal system. Germ-free mice have anatomical defects in the gut-associated lymphoid tissue, including poorly developed Peyer’s patches and isolated lymphoid follicles, fewer plasma cells and fewer intraepithelial lymphocytes.[8-11] These animals also produce lower

levels of antimicrobial peptides and immunoglobulin A in their gastrointestinal tract.[10, find more 12] Certain species of the microbiota, namely segmented filamentous bacteria, have been shown to induce the

production of T helper type 17 cells in the small intestinal lamina propria.[13] Likewise, the gut organism Bacteroides fragilis facilitates the production of inducible regulatory T cells in the gut.[14] Hence, commensal microbiota are pivotal for the development of gut-associated immunity. Recent studies have demonstrated that gut flora have more far-reaching effects on host adaptive systemic immunity. Germ-free mice have a systemic defect in the proliferation of effector CD4+ T cell numbers and exhibit a T helper type 1/type 2 imbalance.[15] Mazmanian et al. showed that in the absence of intestinal flora, splenic CD4+ T

cells made more interleukin-4 (IL-4) and low levels of interferon-γ, which was characteristic of a T helper type 2 response. Urocanase There is much less information available as to how the gut flora influences innate immunity at sites outside the gastrointestinal tract, although commensal flora has been shown to influence bone marrow and blood neutrophils in ways that promote their phagocytosis of Streptococcus pneumoniae and Staphylococcus aureus.[16] In this study, we sought to determine the contribution of intestinal flora in regulating acute neutrophilic inflammatory responses. In acute inflammatory responses there is a rapid recruitment of neutrophils from the blood to the affected tissue site. Diverse agents including invading pathogens, injured or dead cells and other irritants like crystals may stimulate this response. These pro-inflammatory agents are sensed by tissue-resident cells like macrophages, dendritic cells and mast cells. The latter, once activated, release inflammatory mediators like histamines, prostaglandins and cytokines like interferon-γ, macrophage inflammatory protein-2 (MIP-2), tumour necrosis factor-α and IL-1. These mediators promote vasodilatation and also activate the endothelium, facilitating the transmigration of leucocytes into the affected tissue.

7A–C). In addition, whereas stressed mice demonstrated a significant increase in the frequency of splenic CD4+CD25+ T cells as compared with nonstressed mice (17.3 and 14.7%, respectively, p < 0.05; Fig. 7D and E), the fraction of CD127− cells among CD4+CD25+ T cells was significantly lower in stressed than in nonstressed mice in the spleen (76 and 82%, respectively, p < 0.05; Fig. 7D and E) and in the blood (65.6 and 77%, respectively, p < Selleckchem HSP inhibitor 0.01; Supporting Information Fig. 5A and B). Comparing the frequency of cells expressing CD127+ and CD127+ within splenic (Fig. 7D and F) and blood-derived (Supporting Information Fig.

5A and C) CD4+ T cells revealed a significant decrease in the CD127+/CD127+ ratio in stressed mice compared with nonstressed mice. This was evident primarily within the CD4+CD25high subpopulation Proteasome inhibitor and to a lesser extent within the CD25low population, but was not evident in the CD25+ subpopulation. Notably, the frequency of CD25+CD127+, but not CD25+CD127+, within splenic (Fig. 7G) and blood-derived (Supporting Information Fig. 5D) CD4+ T cells was significantly higher in stressed than in nonstressed mice. This indicates that the increased Teff/Treg ratio in stressed mice resulted from an increase in the effector T-cell population with no change in the Treg-cell population. The frequency of Foxp3+ cells and the CD127−/CD127+ ratio among CD4+CD25+

T cells were then examined following EAE induction. As shown in Figure 7H, whereas the frequency of splenic Foxp3 Treg cells among CD4+ T cells was generally reduced in stressed mice prior to EAE induction, no difference was observed between stressed and nonstressed mice following EAE. Similarly, no difference was observed in the CD127+/CD127+ ratio among blood-derived CD4+CD25+ T cells between stressed and nonstressed mice

following EAE induction or remission (Supporting Information Fig. 5E). Notably, both the frequency of Foxp3+ cells (Fig. 7H) and the CD127+/CD127+ ratio among CD4+CD25+ T cells (Supporting Information Fig. 5E) were reduced at EAE onset and gradually recovered toward disease remission. The present study aimed to test the effects of chronic variable stress on immunoregulatory processes involved in autoimmune diseases. Although stress has been traditionally considered to suppress the immune system and shift it toward an antiinflammatory Bcl-w response through the secretion of CORT [3, 13], our results show that prolonged stress exposure exacerbates, rather than ameliorates, EAE in female C57BL/6 mice; this phenomenon, however, could be prevented by blocking CORT signaling throughout the stress exposure period. We also show that CORT levels under basal conditions are significantly lower in male than in female mice, which is associated with exacerbated EAE symptoms. Finally, we show that stress decreases the Treg/Teff ratio, and increases the Th1-Th17/Th2 ratio, within the Teff-cell subsets.