These studies identify bacterial cag pathogenicity island and the cooperative interaction among host innate receptors TLR2, NOD2, and NLRP3 as important regulators of IL-1β production in H. pylori infected DCs. “
“Although it is widely believed that interleukin (IL)-27 is anti-inflammatory, its role in

controlling human immune responses is not fully established. In particular, its interactions with T helper type 17 (Th)17 cytokines are unclear. Our aims were to establish the relationships between IL-27 and proinflammatory cytokines, including IL-17A, in human sera and cultures of peripheral blood mononuclear cells. Plasma IL-27 levels in 879 healthy humans from 163 families varied widely, but with relatively low heritability (19%).

Despite IL-27 including a subunit encoded by Epstein–Barr virus-induced gene 3 (EBI3), there was mTOR inhibitor no correlation of levels with serological evidence of infection with the virus. Although IL-27 has been reported to inhibit IL-17A production, we demonstrated a strong positive correlation in sera, but lower correlations of IL-27 with other proinflammatory cytokines. We verified that IL-27 inhibited IL-17A production by human peripheral blood T cells in vitro, but not that it stimulated IL-10 secretion. Importantly, Transferase inhibitor addition of IL-17A decreased IL-27 production by stimulated T cells but had the opposite effect on resting T cells. Together, these data suggest a model whereby IL-27 and IL-17A exerts complex reciprocal effects Ketotifen to boost inflammatory responses, but restrain resting cells to prevent inappropriate activation. “
“In this study, mice were vaccinated intranasally with recombinant N. caninum protein disulphide isomerase (NcPDI) emulsified in cholera toxin (CT) or cholera toxin subunit B (CTB) from Vibrio cholerae.

The effects of vaccination were assessed in the murine nonpregnant model and the foetal infection model, respectively. In the nonpregnant mice, previous results were confirmed, in that intranasal vaccination with recNcPDI in CT was highly protective, and low cerebral parasite loads were noted upon real-time PCR analysis. Protection was accompanied by an IgG1-biased anti-NcPDI response upon infection and significantly increased expression of Th2 (IL-4/IL-10) and IL-17 transcripts in spleen compared with corresponding values in mice treated with CT only. However, vaccination with recNcPDI in CT did not induce significant protection in dams and their offspring. In the dams, increased splenic Th1 (IFN-γ/IL-12) and Th17 mRNA expressions was detected. No protection was noted in the groups vaccinated with recNcPDI emulsified in CTB. Thus, vaccination with recNcPDI in CT in nonpregnant mice followed by challenge infection induced a protective Th2-biased immune response, while in the pregnant mouse model, the same vaccine formulation resulted in a Th1-biased inflammatory response and failed to protect dams and their progeny.

04 or 0.08 μM of each primer, a DNA template, and 1× multiplex PCR mixture (Qiagen KK, Tokyo, Japan). The PCR conditions were as follows: find more an initial denaturation step at 95°C for 15 min; 35 cycles of denaturation at 95°C for 20 sec, annealing at 60°C for 90 sec, and extension at 72°C for 60 sec; and the final extension step at 72°C for 10 min. The PCR products were diluted and separated with an ABI 3130 genetic analyzer, using GeneScan LIZ 600 (Applied Biosystems) as

the size standard. The size of each PCR product was converted to a repeat copy number by using the Gene Mapper software (Applied Biosystems). The data were incorporated into the BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) and analyzed as previously described (7). Repeat copy number for the null allele, namely, when no PCR product was obtained, was designated as JNK signaling pathway inhibitor −2. Simpson’s index of diversity (D) and 95% CI were calculated according

to formulas described in a previous report (12). The number of alleles indicates the number of variations detected in the repeat copy numbers at a locus and is hereafter referred to as the ‘allele number’. PFGE was carried out according to the PulseNet protocol developed at the Centers for Disease Control and Prevention by using Salmonella enterica serovar Braenderup H9812 strain as a standard for normalization (4, 13). DNA was digested with XbaI and separated using a CHEF DR III apparatus (Bio-Rad Laboratories, Hercules, CA) under the following conditions: switching time from 2.2 to 54.2 sec at 6 V/cm for 21 hr at 14°C. After the gels were stained with ethidium bromide, they were imaged using Gel Doc EQ and Quality One System (Bio-Rad Laboratories). Cluster analysis was carried out using the BioNumerics software as previously described (14). Our initial analysis of the genome sequences of the O26 and O111 strains (8) revealed that for among the nine loci that are routinely used for analyzing O157 (O157-3, O157-9, O157-10, O157-17, O157-19, O157-25, O157-34, O157-36, and O157-37), five and four loci are not present in the O26 and O111 strains, respectively (Table 1). This finding indicates that

additional genomic loci are required for MLVA of the O26 and O111 strains. Therefore, we selected nine additional loci on the basis of the results obtained after analyzing the genome sequences of the O26 and O111 strains and comparing their genome sequence to that of O157; moreover, we developed a system by which these 18 loci can be simultaneously analyzed, as described previously (Table 1). By using this system and the 469 representative EHEC isolates (153 O157, 219 O26, and 97 O111 isolates), we examined whether these 18 loci can be used for MLVA of the O26 and O111 isolates, as well as the O157 isolates (Fig. 1). Of the nine loci that are currently used for analyzing the O157 isolates, four (O157-3, O157-10, O157-17, and O157-36) were not detected in any of the O26 or O111 isolates.

2a), while caspase-3 activity was significantly higher after 8 and 24 h (Fig. 2b,c). With LPS, AUY-922 neutrophils experienced a decrease in caspase-3 and caspase-8 activity at 8 h

(P < 0·05) (Fig. 2b), while a fivefold increase of caspase-3 was observed at 24 h compared to control cells (P < 0·05) (Fig. 2c). Hypoxia did not alter the apoptosis rate in tracheobronchial epithelial cells within 24 h of exposure to 5% oxygen (Fig. 3a–c), while stimulation with LPS increased caspase-3 activity by 129% and caspase-9 activity by 80% at 4 h of incubation (P < 0·05) (Fig. 3a). After 8 h of LPS stimulation, a 79% increase of caspase-3 activity was observed, while caspase-9 was twofold higher compared to the control group (P < 0·05) (Fig. 3b). At Selleck Midostaurin 24 h, caspase-3 activity reached 206% and caspase-9 95% compared to the adequate control group with 100% expression (P < 0·05) (Fig. 3c). Alveolar epithelial cells as possible target cells showed a different apoptosis pattern as tracheobronchial epithelial cells. Hypoxia did not

induce changes in the apoptosis rate in alveolar epithelial cells, while LPS increased caspase-3 activity by 56%, 78% and 70% after 4, 8 and 24 h, respectively (all P-values <0·05) (Fig. 4a–c). No changes of caspase-8 and -9 activity were observed upon LPS injury for all time-points (Fig. 4a–c). As the increase of caspase activities might not necessarily correlate with the process of apoptosis, neutrophils were analysed assessing apoptosis-induced cellular changes. Flow cytometric measurements of annexin V staining showed that changes of caspases reflect the process of apoptosis (Fig. 5a,b). many At 4 h of injury, apoptosis rate decreased by 19% (range 35%) under hypoxia and by 32% (range 39%) with LPS, respectively (P < 0·05). In tracheobronchial

epithelial cells, apoptosis increased upon 24 h of LPS stimulation, as shown previously with the help of a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining [10]. Numerous studies have been conducted to understand ALI/ARDS more clearly. Cell death has been demonstrated to play a key role in the lung during the pathogenesis of ALI/ARDS. In this study we focused on different cell types of the respiratory compartment, and determined apoptosis in vitro in the model of hypoxia- or endotoxin-induced injury. Alveolar macrophages, tracheobronchial cells as well as alveolar epithelial cells showed a similar apoptosis response pattern to injuries, such as hypoxia or LPS: (i) no increased apoptosis rate was observed under hypoxia at early time-points; (ii) for all three cell types, LPS induced apoptosis at any time-point. In alveolar macrophages, LPS stimulation activated caspase-3, caspase-8 and caspase-9, while in tracheobronchial epithelial expression of caspase-9 and caspase-3 was increased.

5a). These results showed that the presence of MyD88 is not essential

for the signalling initiated by zymosan. While the deletion of MyD88 was partial in these animals, they showed reduced neutrophil recruitment to LPS, confirming the role of the TLR4–MyD88 pathway in detecting LPS and also validating that the deletion was sufficient to impair responses (Fig. 5b). In contrast, tamoxifen treatment of wild-type mice did not impair responses (data not shown). On the other hand, when cKO mice when PLX4032 datasheet treated with tamoxifen from Day 0 of birth, these mice exhibited reduced neutrophil recruitment to zymosan as compared with untreated mice (Fig. 5c). These results supported our hypothesis OSI-906 molecular weight that for inflammatory ligands like zymosan, MyD88 is required during the pre-challenge phase for activation of immune cells but is dispensable during the actual inflammatory

challenge. One of the major findings of this study is that for neutrophil-mediated acute inflammation to several pro-inflammatory agents, the immune system needs to be previously stimulated by intestinal flora in a MyD88-dependent fashion. This stimulation enables the host to mount a neutrophil response to future inflammatory insults. We have shown that germ-free and flora-deficient mice are defective in neutrophil migration to a number of different microbial and sterile inflammatory ligands. This defect can be corrected by supplementing the drinking water with LPS, a TLR4–MyD88 agonist, before challenge with the inflammatory agent. Furthermore, pre-treatment of flora-deficient MyD88 knockout mice with LPS failed to restore neutrophilic infiltration, showing that LPS specifically acts through MyD88 to prime the immune system. Presumably other PAMPs that stimulate MyD88–TLRs would have similar effects, Etofibrate although this has not yet been tested. There is some evidence that PAMPs derived

from intestinal flora are present systemically in the mammalian body under physiological conditions.[29, 30] These ligands presumably translocate into the circulation via the intestinal epithelium. In a similar fashion, we hypothesize that ligands derived from gut flora, such as LPS (TLR4–MyD88), bacterial DNA (TLR9–MyD88), peptidoglycan (TLR2–MyD88) as well as others, activate MyD88 signalling that then enables systemic neutrophilic inflammatory responses. A previous report published by our laboratory had shown that MyD88 knockout mice do not show a defect in zymosan-induced neutrophil migration.[31] The basis for this discrepancy is unclear. It is possible that this difference was the result of the extent of backcrossing of the MyD88-deficient mice; the mice in the present study were fully backcrossed onto the B6 background whereas those in the earlier study were not.

The use of bisphosphonates in renal transplant

recipients is yet to be supported by large randomized controlled trials. In the non-transplant population concerns exist regarding the association between atypical fractures and bisphosphonates caused by reduced bone remodelling. Nevertheless, the absolute risk of atypical fractures with bisphosphonate use may be small compared with the beneficial effects of the drug.1 A randomized, prospective, controlled trial in 72 new renal transplant patients was performed with prophylactic pamidronate at months 0, 1, 2, 3 and 6.2 A subgroup of patients had bone biopsies. Pamidronate preserved vertebral, but not hip BMD during treatment and for 6 months after cessation. Fifty per cent of all patients had low bone turnover disease at baseline selleck and all pamidronate-treated patients had adynamic bone disease at 6 months. The study was not powered to examine fracture rates and did not determine whether improved BMD with adynamic bone disease is ultimately beneficial or harmful. Dual energy X-ray absorptiometry of the hip region has been shown to predict fractures in renal transplant Torin 1 purchase recipients in 238 patients investigated between 1995 and 2007 in a single-centre study.3 Bisphosphonates had been prescribed

in 12.8% and 13% had undergone parathyroidectomy. Osteoporosis was present in 13.9% and osteopaenia in 46% of hips studied. Forty-six of the 238 patients suffered any fracture after DEXA. Osteopaenia and osteoporosis were independent risk factors for fracture,

with a relative risk of 2.7 and 3.5 respectively. Hip BMD was found to be a better predictor of future fractures compared with lumbar BMD possibly because of aortic calcification or undiagnosed lumbar spine fractures. Hyperparathyroidism post-kidney transplantation may be caused by secondary hyperparathyroidism and hyperplastic parathyroid glands or tertiary hyperparathyroidism with autonomous functioning of monoclonal parathyroid cells. Common practice is to delay parathyroidectomy for at least 6 months from the time of transplantation Carbohydrate as involution of the parathyroid glands may obviate the need for surgery. Kidney Disease: Improving Global Outcomes (KDIGO) has no specific guidelines advising on post-transplantation parathyroidectomy.4 A single-centre retrospective analysis between 1983 and 1995 examined 37 kidney transplant patients who underwent parathyroidectomy and were followed for up to 13 years.5 Parathyroidectomy was performed after an average of 36.7 months post-transplantation. Of this cohort, 13 patients experienced rejection and became dialysis-dependent, 24 had persistent good renal function, 7 died and 4 developed hypoparathyroidism. Fifty-six per cent of patients still required parathyroidectomy after more than 1 year post-transplantation and the authors therefore advocated early surgery after transplantation.

ILCs lack an antigen receptor or other linage markers, and ILC subsets that express the transcriptional factor RORγt have been found to secrete IL-17. Evidence is emerging that these newly

recognised sources of IL-17 play both pathological and protective roles in inflammatory diseases as discussed in this article. Although early studies suggested that IL-17 was produced primarily by αβ T cells [1, 2], it has recently been found that various “innate” subsets of lymphoid cells can produce this cytokine [3-6]. Indeed the term Th17 cell, which refers to IL-17-secreting CD4+ T cells, does not include CD8+ T cells and γδ T cells, which have been revealed to be high producers of this cytokine [7]. γδ T cells, together with natural killer (NK) cells, VX-770 chemical structure NKT cells, and several populations of innate lymphoid cells (ILCs), belong to a family of IL-17-secreting lymphocytes that fits more closely with the innate rather than the adaptive immune system. The discovery of these innate sources of IL-17 has led to a re-examination of the roles played by effector and pathogenic cells in diseases where IL-17 is implicated, such as bacterial and fungal www.selleckchem.com/products/3-methyladenine.html infection and cancer,

as well as in gut homeostasis. In addition, these innate IL-17 producers have been shown to participate in the initiation of autoimmune diseases including experimental autoimmune encephalomyelitis (EAE), arthritis, and colitis [6, 8, 9]. While much of the work identifying and characterizing Succinyl-CoA the function of IL-17-producing γδ T cells and ILCs discussed in this review is based on the studies from mouse models, these cells have also been identified in humans. While there are some differences in repertoire and phenotype of the human IL-17-producing γδ T cells and ILCs as compared with those in the mouse, evidence to

date suggests that both cell populations perform the same functions. γδ T cells account for approximately 3–5% of all lymphoid cells found in the secondary lymphoid tissues and the blood. These cells are the first immune cells found in the fetus and provide immunity to newborns prior to activation of the adaptive immune system [10]. γδ T cells are much more prevalent at mucosal and epithelial sites, especially the gut, where they can account for up to 50% of the total intraepithelial lymphocyte population. Although γδ T cells express a TCR, this TCR does not engage MHC-antigen complexes in the same manner as αβ T cells [11]. Instead, it appears to act more like pattern recognition receptors, recognizing conserved phosphoantigens of bacterial metabolic pathways, as well as products of cell damage [12]. Activation via the γδ TCR in the thymus has, however, been shown to determine the cytokine profile of γδ T cells following their departure from the thymus.

Cells were analyzed on a FACScan flow cytometer (BD Biosciences). Cytokines (IL-4,

IL-10, and IFN-γ) were determined by ELISA using commercially available kits, according to manufacturer’s instructions (BD Biosciences). The sensitivity limits of the assays were 7 pg/mL for IL-4 and 30 pg/mL for IL-10 and ABT 888 IFN-γ. CD4+CD25− and CD4+CD25+ T cells were isolated from pooled draining LN cells of L. major infected mice or from spleens of normal mice (n = 4) using a mouse TREG-cell isolation kit (Miltenyi Biotec, Bergish Gladcach, Germany) according to the manufacturer’s instructions. The suppressive capacity of TREG cells was studied in co-culture suppression assays, which were set up in 96-well plates

in RPMI 1640 (Gibco, Z-IETD-FMK molecular weight CA, USA) supplemented with 10% heat-inactivated fetal bovine serum Gibco). Proliferation was assessed by (3H)-thymidine incorporation. Briefly, CD4+CD25− (TEFF) cells isolated from draining LNs of infected WT mice (or Lgals3−/− mice, when indicated) were seeded at 5 × 104 cells per well and restimulated with 20 μg/mL of L. major antigen. Then, CD4+CD25+ TREG cells or CD4+CD25− T (TEFF) cells from either WT- or Lgals3−/−-infected mice were incorporated to cultures at different ratios. At day 5, proliferation was measured by adding 0.5 μCi (3H)-thymidine (Amersham Biosciences, Piscataway, NJ, USA) to each well. After 12 h, radioactivity was measured using a β-plate counter (Packard, Canberra, Australia). Culture supernatants were collected for cytokine measurement by ELISA. Tests were set up in triplicate. For differentiation of naïve CD4+CD25− T cells into a TREG-cell phenotype, CD4+CD25− T cells were enriched from total spleen cells of WT or Lgals3−/− mice by negative selection. CD4+CD25− T cells were resuspended at 1 × 105

cells per well in RPMI 1640 medium plus 5% fetal bovine serum, seeded in a 96-well plate coated with anti-CD3 mAb (BD Biosciences) at Tenoxicam the indicated concentrations, and stimulated with soluble TGF-β1 (3 ng/mL), IL-2 (20 ng/mL), and anti-CD28 mAb (at the indicated concentrations) (all from BD Biosciences). In some experiments, cells were cultured in the presence of different concentrations of DAPT(1–10 μM, Sigma-Aldrich). After 5 days of culture, cells were harvested and analyzed for CD25 and Foxp3 by flow cytometry as described above. Cytokines were measured in culture supernatants by ELISA. Footpad tissue from infected WT and Lgals3−/− mice was frozen in Tissue Tek (Qiagen, CA, USA) medium and cut into 8–10 μm sections.

[11] Clearance of infectious pathogens is also dependent on the action of cytokines secreted by Teff. Critical T-cell–DC interactions occur at sites of inflammation in lymph nodes and thereby control susceptibility to the development of an autoimmune disease. Therefore, it is crucial to understand how the dynamics of T-cell recirculation, localization and interaction in vivo within tissues such as lymph nodes contribute to effective immune responses

that either promote or prevent inflammation and autoimmune disease. Recent application of intravital imaging technology, which uses two-photon (2P) microscopy to detect the location, behaviour, movement and interactions of viable cells in vivo, has significantly advanced our understanding of several factors that mediate T-cell–DC see more and T-cell–B-cell interactions.[50-54] We have learned how such cells behave in resting tissue, how they interact with one another, exchange information, respond to pathogenic stimuli, and mediate various functions. This technique has also been informative about disease processes that occur in cells by defining the impact of specific changes in real-time. Visualization and quantification of these cellular dynamics in vivo relies on the ability to fluorescently tag different cell types under analysis.

For example, the FDA approved Drug Library solubility dmso use of ‘photoswitchable’ fluorescent proteins that transition from green to red can track individual cells as they move between blood vessels and tissues in the body. Currently,

most studies are limited to a tissue depth of about 300–400 mm. Major conclusions reached so far using 2P microscopy of fluorescently tagged cells are summarized in Table 3. Another conclusion of particular interest is that the duration of T-cell contact with APCs may vary from being long-lived if MG-132 in vitro they occur during an immune response to short-lived while they are in a state of peripheral tolerance. Conceivably, this difference in duration of T-cell–APC contact could be diagnostic of the capacity of various agents administered in vivo to treat a given disease to induce (pre-disease onset) or restore (post-disease onset) immune tolerance. In this regard, imaging studies have reported that the inhibitory receptors cytotoxic T-lymphocyte antigen-4 and programmed death-1 on Teff or Treg cells may suppress immune responses by limiting the duration of T-cell interaction with antigen-bearing DCs.[55-57] While intriguing, these results on duration of T-cell–APC contacts remain controversial and may vary depending on the specific experimental systems used.[58-60] It is also controversial as to whether brief contacts between T-cell effectors (e.g. cytokines) and APCs deliver a sufficient quantity of effector molecules to elicit chronic inflammation.

CDPs further differentiate into classical DCs (cDCs) and plasmacytoid DCs (pDCs). Here, we studied the impact of histone acetylation Sotrastaurin in vitro on DC development in C57BL/6 mice by interfering with histone acetylation and deacetylation, employing histone deacetylase (HDAC) inhibitors. We observed that commitment of MPPs into CDPs was attenuated by HDAC inhibition and that pDC development was specifically blocked. Gene expression profiling revealed that HDAC inhibition prevents establishment of a DC-specific gene expression repertoire. Importantly,

protein levels of the core DC transcription factor PU.1 were reduced in HDAC inhibitor-treated cells and consequently PU.1 recruitment at PU.1 target genes Fms-like tyrosine kinase 3 (Flt3), interferon regulatory factor 8 (IRF8),

and PU.1 itself was impaired. Thus, our results demonstrate that attenuation of PU.1 expression by HDAC inhibition causes reduced expression of key DC regulators, which results in attenuation of DC development. We propose that chromatin modifiers, such as HDACs, are required for establishing GSK2118436 purchase a DC gene network, where Flt3/STAT3 signaling drives PU.1 and IRF8 expression and DC development. Taken together, our study identifies HDACs as critical regulators of DC lineage commitment and development. “
“Neutrophils are the primary cells contributing to initial defense against Niclosamide mycobacteria. Yet, little is known about the potential of various mycobacterial strains to stimulate neutrophils. This study was focused to compare the differential capacity of vaccine strains, Mycobacterium bovis bacillus Calmette–Guerin (BCG) and

Mycobacterium indicus pranii (Mw), and laboratory strain H37Rv to activate and enhance neutrophil functions. The expression of phenotypic markers like Fcγ receptor, toll-like receptor (TLR), and chemokine receptor; secretion of pro-inflammatory cytokines; and the rate of apoptosis were studied in infected neutrophils. Increased expression of CD32, CD64, TLR4, and CXCR3; increased TNF-α secretion; and downregulation of early apoptosis were observed in H37Rv-infected neutrophils. Among the vaccine strains, BCG increased the expression of only CD32 on neutrophils, while Mw was comparatively ineffective. To understand the paracrine role of neutrophils, the supernatants from infected neutrophils were used to stimulate monocytes and T helper cells. The secretory molecules from all infected neutrophils increased the expression of CCR5 on monocytes, whereas only H37Rv-infected supernatant increased the expression of CCR7 on monocytes and CD69 on T cells. Thus, H37Rv was more effective in activating neutrophils and in turn stimulating monocytes and T cells. By comparison, vaccine strains were less effective in modulating neutrophil functions.

However, it is also notable that the inhibitory effect of DN T cells in an antigen-specific setting is superior to non-specific inhibition. As a result of the vigorous HBeAg-specific proliferative

property of the DN T-cell population during in vitro culture, it is possible AZD8055 ic50 that the DN cells are derived from HBeAg-specific CD4+, CD8+ or from an independent DN progenitor population. To determine the origin of the DN T-cell population, depletion of T-cell subpopulations from total spleen of HBeAg × 7/16-5 dbl-Tg mice was performed and the remaining cells were cultured in vitro with p120–140 for 4 days and compared with total spleen cells. As shown in Fig. 6, CD4+ and CD8+ T-cell depletion from total spleen did not affect the generation of the DN T-cell population in the culture (i.e. 40–48%). However, negative depletion of DN T cells before culture prevented the generation of the HBeAg-specific DN T-cell population in the 4-day culture (i.e. 8%). Hence, HBeAg-specific DN T cells exist in the periphery and are not generated from CD4+ or CD8+ T cells in the periphery. It is notable that without DN T cells, CD4+ T cells from HBeAg × 7/16-5 dbl-Tg mice demonstrate robust proliferation selleck kinase inhibitor and cytokine

production in vitro (data not shown). The frequency of Vβ11+ DN T cells in thymus and spleen ex vivo was measured. The Vβ11+ DN T cells in thymus of 7/16-5 × HBeAg dbl-Tg mice were present at a slightly higher frequency (5% higher) than in the thymus of 7/16-5 × HBcAg dbl-Tg mice. There was also a 20% higher frequency of Vβ11+ DN T cells in

the ex vivo spleens of 7/16-5 × HBeAg compared with the spleens of 7/16-5 single TCR-Tg mice (data not shown). However, in absolute terms DN Vβ11+ T cells are present in low frequency in situ in HBeAg × 7/16-5 dbl-Tg mice (i.e. 3–5%) and require antigen stimulation for expansion. It is not clear if DN T cells can proliferate and be activated in vivo. To determine the capability of DN T cells to expand in vivo, we injected HBeAg-derived p120–140 (250 μg) into 7/16-5 × HBeAg-dbl Tg mice. As shown in Fig. 7, at least a twofold increase in the DN T-cell frequency in vivo was observed 1 and 2 weeks after injection, whereas in control, 7/16-5 mice no evidence of expansion of DN T cells occurred. Although HBeAg-specific Treg cells appear quiescent in vivo Methamphetamine in HBeAg × 7/16-5 dbl-Tg mice, these cells are capable of being activated in vivo, in this case by exogenous antigen. The ability to activate DN T cells in vivo will permit further studies of their in vivo function. To further pursue the origins of DN T cells, we bred 7/16-5 × HBeAg dbl-Tg mice onto MHC class I KO, and TCR α-chain KO backgrounds. Because the 7/16-5 TCR is surprisingly expressed on CD8+ as well as CD4+ T cells in the thymus and the periphery, it was important to determine if expression of CD8 was necessary for selection of the DN T-cell population.