Whether vascular calcification can be prevented or reversed with

Whether vascular calcification can be prevented or reversed with strategies INK-128 aimed at maintaining phosphate homeostasis is as yet unknown. One recent study also determined an association between serum phosphate within the normal range and vascular and valvular calcification.21 This study of 439 young and middle-age participants from the Multi-Ethnic Study of Atherosclerosis (MESA) with both normal renal function and CKD, and no known CVD, reported that after adjustment for eGFR, each 1 mg/dL increase in serum phosphate concentration was significantly associated with a 21%, 33%,

25% and 62% greater prevalence of coronary artery, thoracic, aortic valve and mitral valve calcification respectively. The CARDIA study, described earlier, also showed that phosphate levels within the reference range were significantly associated with coronary artery calcium levels in a young healthy adult population.19 Elevations in serum phosphate have been associated with structural changes and renal decline in animal models.68 In human observational studies, hyperphosphataemia is associated with progression of established CKD and the development of ESKD (end-stage Copanlisib in vivo kidney

disease)23,69–71 and studies of renal transplant recipients describe an association between higher serum phosphate and renal allograft loss.27,28 Serum phosphate levels in the upper-normal range have also recently been reported to be associated with an increased risk of developing incident CKD and ESKD.6,24 One study involving 2269 participants from the Framingham Heart Study showed that those in the highest phosphate category had an increased risk of CKD with OR 2.14 (95% CI 1.07–4.28) 4��8C when compared with the reference group with serum phosphate 2.5–3.49 mg/dL.6 The same study also analysed 13 372 participants

from the Third National Health and Nutrition Examination Survey (NHANES III) and reported that phosphate ≥4 mg/dL was associated with an increased risk of incident ESKD (RR 1.90 (95% CI 1.03–3.53)). Zoccali et al. recently evaluated the relationship between baseline serum phosphate, disease progression and response to angiotensin-converting enzyme (ACE) inhibition in 331 patients with proteinuric CKD in the prospective Ramipril Efficacy In Nephropathy (REIN) trial.72 Phosphate levels in the highest two quartiles were significantly associated with faster progression to both ESKD and to a composite end-point of doubling of serum creatinine or ESKD compared with patients with phosphate levels below the median. Therefore, with higher serum phosphate levels the renoprotective effect of ramipril decreased, despite adjustment for potential confounders such as GFR and urinary protein. This suggests that phosphate may potentially modify the protective effect of the only real therapeutic class of agents used in CKD. FGF-23 is the most potent hormone regulating phosphate homeostasis.73 In health, FGF-23 is secreted by osteocytes and osteoblasts in response to dietary phosphate intake.

Induction of in vitro Treg cells was most easily accomplished wit

Induction of in vitro Treg cells was most easily accomplished with anti-CD3 mAb mitogen-based stimulation. Therefore, to control for the use of mitogen-based stimulation, it was necessary to confirm that n-butyrate anergized mitogen-stimulated CD4+ T cells similarly to antigen-stimulated CD4+ T cells. Primary cultures of isolated C57BL/6 CD4+ T cells were stimulated with plate-bound anti-CD3 mAb and soluble

anti-CD28 mAb for 7 days in the presence or absence of n-butyrate. As seen in Fig. 1A, n-butyrate reduced proliferation of CD4+ T cells by approximately 95% in mitogen-stimulated primary cultures. To test whether n-butyrate induced unresponsiveness was retained after the removal of the HDAC inhibitor, the CD4+ T cells from the primary culture were re-stimulated in secondary cultures that did not contain n-butyrate. As shown in Fig. 1B, control CD4+ T cells JAK inhibitors in development from the

primary cultures proliferated vigorously when re-stimulated in secondary cultures. In contrast, CD4+ T cells from the n-butyrate-treated primary cultures proliferated 83–91% less than untreated CD4+ T cells. The retention of proliferative unresponsiveness in the secondary cultures demonstrated that the CD4+ T cells from the n-butyrate-treated mitogen-stimulated primary cultures were anergic. Anergy in CD4+ T cells usually involves an inability to generate IL-2 in association with proliferative unresponsiveness. Consequently, IL-2 secretion RAD001 solubility dmso by the CD4+ T cells was also examined to confirm the onset of anergy (Fig. 1C). CD4+ T cells from control primary cultures secreted IL-2 in secondary cultures stimulated with anti-CD3 mAb. In contrast, IL-2

secretion Non-specific serine/threonine protein kinase was inhibited in CD4+ T cells from the n-butyrate-treated primary cultures. The anergic CD4+ T cells did not generate any additional IL-2 beyond the detected background levels in response to anti-CD3 mAb stimulation in the secondary cultures. The decreased IL-2 concentration within the anergic CD4+ T cell culture supernatants had no bearing upon proliferation in the n-butyrate-treated CD4+ T cells as seen in Fig. 1B. Taken together, the results in Fig. 1 revealed that n-butyrate induced anergy within mitogen-stimulated CD4+ T cells as determined through significant reduction of proliferation and IL-2 secretion. To determine if n-butyrate increased the percentage of FoxP3+ Treg cells in primary or secondary cultures, CD4+ T cells from transgenic FoxP3EGFP C57BL/6 mice were stimulated in primary cultures with or without n-butyrate. Natural Treg cells as determined by the presence of FoxP3EGFP comprised approximately 8% of isolated lymphoid CD4+ T cells (data not shown). TGF-β was added to additional primary cultures to generate FoxP3+ T cells as a positive control [21]. Percentages of FoxP3+ T cells were quantified daily over the course of 5 days (Fig. 2A). The percentage of CD4+FoxP3+ T cells increased only in the primary cultures stimulated in the presence of TGF-β, as shown on Day 4 in Fig.

Clinical data from the group of patients are listed in Table 1 T

Clinical data from the group of patients are listed in Table 1. The age varied between 20 and 85 years (median 66 years). Almost all patients presented various comorbidities, mainly manifestations of the metabolic

syndrome like diabetes mellitus (40.2%), hypertension (58.7%), peripheral arterial occlusive disease (20.6%) or coronary heart disease (27.2%). 17.4% suffered from malignancies, and 19.6% showed various degrees of renal disease including end-stage renal failure reflecting the frequently observed comorbidity status of patients with invasive S. aureus infections (Laupland et al., 2003). Serum samples from specific pathogen-free (SPF) mice, juvenile mice and human sera from healthy adults and umbilical cord blood (UCB) were analyzed by Western blots for the presence of anti-Eap antibodies (Fig. 1a). Antibodies could be detected in various concentrations in all human LY2109761 concentration sera. However, SPF mice as well as juvenile mice did not show any anti-Eap antibody response. Further analysis of the human sera revealed IgM, IgG and IgA antibodies in adult samples, while in UCB, only IgG antibodies were found (Fig. 1b). For further analysis, anti-Eap antibodies were quantified by ELISA. In all blood donors, antibodies could be detected with a considerable variability in titers for IgM and IgG (Fig. 2a). No correlation was found between IgG and IgM antibody titers within individuals (correlation coefficient r2: 0.0074; Fig. 2b). this website Also, when comparing

the results for IgA and IgG from Pomalidomide purchase Western blot analysis, no correlation could be found (data not shown). All 92 patients suffering from S. aureus infections showed anti-Eap antibodies. Both IgM as well as IgG anti-Eap antibody titers were significantly higher in patients compared with healthy individuals (IgM, P=0.007; IgG, P<0.0001, Fig. 2a). However, no correlation could be established between IgM and IgG antibody titers. The avidities of anti-Eap antibodies from

healthy controls and patients were high in both groups, with patients displaying significantly higher avidity indices compared with healthy controls (patients mean 0.805, controls mean 0.696; P<0.0001, Fig. 2c). Because transcription of eap by S. aureus in deep wounds was promoted compared with the superficial wounds (Joost et al., 2009), we determined whether the extent of anti-Eap antibody response also differs as a function of infection type (Table 2). Patients with deep infections showed significantly higher anti-Eap antibody titers than those with superficial infections (P=0.001). Detailed analysis revealed significantly higher titers for patients suffering from abscesses compared with other types of infection (P<0.001). Extremely high titers were found in patients presenting with spondylodiscitis (mean 361.2), although in comparison with patients with other types of infections, these did not reach statistical significance (P=0.057), most likely due to the small number of patients (n=4).

Indeed, when PBMCs derived from IFN-β-treated patients were deple

Indeed, when PBMCs derived from IFN-β-treated patients were depleted of monocytes, the strong induction of IL-6 observed in total PBMCs was completely lost. In addition, a strong reduction of BAFF expression was observed in in vivo IFN-β-conditioned PBMCs after the depletion of monocytes. In a similar fashion, in the absence of monocytes, there was no induction of TLR7-driven IgM and IgG production, indicating that IFN-β treatment could exert its therapeutic effects this website by fine-tuning monocyte functions, in the context of TLR7 stimulation, that act through bystander mechanisms on the differentiation of

B lymphocytes. Taking into account that TLR7 is crucial for type I IFN release from pDC [41] and is, at the same time, an IFN-inducible gene [22], we can envisage the existence of a tight relation between IFN-β response and TLR7 responsiveness of MS monocytes, whose full comprehension deserves further investigation. In line with this view, recent data obtained by Molnarfi and collaborators showed that monocytes from RRMS patients exhibited a reduced ability to produce HGF, a neuroprotective and neuroinflammation-suppressive mediator, when compared with HD [42]. Treatment with IFN-β significantly enhanced

HGF PF-6463922 synthesis and secretion by blood monocytes, contributing to the clinical benefit of IFN-β in RRMS via the combined HGF-mediated neuroprotective and anti-inflammatory mechanisms. In this context, it is also important to remind that monocytes are abundant in inflammatory MS brain lesions and displayed also altered functions and an activated innate immune signature Glutamate dehydrogenase in MS patients with clinically more severe course [43]. In particular,

the type I IFN pathway is dysregulated in these monocytes, which may contribute to more active disease. In addition to that, conditional genetic knockout of IFNAR1 in monocytes, but not in T cells, B cells, or central nervous system cells, leads to enhanced disease severity in the animal model of MS [44]. All these evidences indicate that perturbations of the type I IFN signaling pathway and response in monocytes could represent crucial events in MS immunopathology and, at the same time, a key target of IFN-β therapy. On the other hand, we cannot exclude that the replenished TLR7 responsiveness in PBMCs and monocytes of IFN-β-treated MS patients could be related to the rescue or prevention of TLR7 tolerance, that is generally induced by specific ligands of this receptor and leads to a reduced cytokine and Ig production [45]. Indeed, Poovassery and Bishop [45] recently demonstrated that IFN-β controls TLR7 tolerance and activation through the PI3K/Akt/mammalian target of rapamycin signaling pathway but also enhancing TLR7 expression in human B cells.

Strains lacking either of these two mediators

have been s

Strains lacking either of these two mediators

have been shown to be more sensitive to pro-oxidants such as hydrogen peroxide, menadione and methyl viologen or paraquat (7, 9), suggesting that oxyR and rpoS are essential for survival and growth under oxidative conditions. Similar results have been found in other bacterial species and the role of OxyR in the response to oxidative stress is well established. Midostaurin datasheet For example, oxyR mutants of Pseudomonas aeruginosa are hypersensitive to pro-oxidants including H2O2 and paraquat (16) while E. coli with deletions of oxyR are hypersensitive to hydrogen peroxide and have increased rates of spontaneous mutation during aerobic growth (17). Similarly, oxyR mutants of Brucella abortus, Erwinia carotovora and Xanthomonas campestris, all show increased sensitivity to pro-oxidants (17–20). Negative regulation of oxyR by RpoS has been reported in E. coli (21). In particular the degree of β-galactosidase expression from a single-copy oxyR::lacZ fusion in a RpoS-defective strain has been shown to be higher than in its parental strain as the cells enter into, and remain in, the stationary phase growth (21). Additionally, increased expression of RpoS prevents the normal expression of oxyR (21). However, in contrast to this,

Schellhorn observed a significant reduction in oxyR expression in an E. coli rpoS::Tn10 mutant (22), a result supported by our own observations with B. pseudomallei in EPZ-6438 cost which Edoxaban low amounts of CAT activity were observed in oxyR::CAT/rpoS−, which contains a chromosomal oxyR::CAT fusion and is null for rpoS. More significantly, isogenic replacement of RpoS in strain oxyR::CAT/rpoS−/RpoS restored oxyR::CAT expression to the extent seen in the parental strain (oxyR::CAT), suggesting that RpoS acts as a positive regulator of oxyR transcription in

B. pseudomallei. Three genes have been shown to be under transcriptional control of OxyR, namely dpsA (23), katG (24) and gorA (25). The expression pattern of katG during growth of B. pseudomallei has been previously examined using a chromosomal katG::CAT fusion as a reporter. CAT activity was observed to increase during early exponential growth, reaching a maximum value in the early stationary phase growth, after which it declined in the late stationary phase growth (6). Significantly, expression was greater in an oxyR mutant strain during all phases of growth, suggesting that katG expression is negatively regulated by OxyR during normal growth, although further studies showed that katG was positively regulated by OxyR during oxidative stress (6). The negative regulation of katG by oxyR was confirmed in this study, a greater degree of CAT expression being seen in katG::CAT as compared to katG::CAT/oxyR−.

Adoptively transferred p14 CD8+ T cells coexpressed CD44, PD-1 an

Adoptively transferred p14 CD8+ T cells coexpressed CD44, PD-1 and IL-7Rα as analyzed by FACS analysis of blood (Fig. 2G, Supporting Information Fig. 2C) and spleens (data not shown) 5 days after transfer. Thus, CML-specific CTL display an activated phenotype but retain IL-7Rα Afatinib purchase expression. The fact that specific CTL downregulate IL-7Rα expression in the presence of a chronic infection but maintain IL-7Rα expression in the presence of CML expressing the same viral antigen was surprising and led

to the question if IL-7 production is increased in CML mice. To analyze this, we compared IL-7 expression in mRNA isolated from spleen of CML and naïve C57BL/6 mice by RT-PCR. The thymus as organ with documented high IL-7 production served as a positive control. IL-7 mRNA was detectable in the spleen of CML and of naïve C57BL/6 mice (Fig. 3A and Supporting Information Fig. 3). Next, we analyzed whether IL-7 mRNA is detectable in CML granulocytes and in control granulocytes. We therefore quantitatively compared IL-7 mRNA production of sorted GFP+ granulocytes from CML mice with sorted granulocytes from C57BL/6 mice.

Surprisingly, IL-7 mRNA was detectable in both malignant and control granulocytes (Fig. 3B). Moreover, this experiment revealed that IL-7 mRNA was not differently expressed in malignant and in normal granulocytes. However, the total number of granulocytes in the spleen of mice with CML is three to four-fold higher than that found in C57BL/6 control mice (Fig. 3C). These findings were confirmed by quantification of IL-7 protein levels per Metformin order milligram spleen of naïve C57BL/6 mice and CML mice (Fig. 3D). Furthermore, IL-7 was detectable by intracellular staining of brefeldin-treated malignant (GFP+) and normal (GFP−) granulocytes but not in granulocytes from IL-7-deficient mice (MFI increase of IL-7 in Cyclooxygenase (COX) GFP− granulocytes (12.4±2.9%) and GFP+ granulocytes (11.4±2.9%)

(Fig. 3E and F)). Taken together, the malignant granulocytes produce IL-7 and are increased in numbers in secondary lymphoid organs such as the spleen. To study the role of IL-7 produced by leukemic cells in more detail, H8×IL-7-deficient mice were used as bone marrow donors (H8×IL-7−/−-CML mice) to establish CML disease in C57BL/6 recipients. In this experiment, the leukemic cells will not produce IL-7. However, stromal and epithelial cells of the recipient mouse are capable of IL-7 secretion. Purified p14 CD8+ T cells (CD45.1+CD8+Vα2+) were adoptively transferred to H8×IL-7−/−-CML mice, H8-CML and naïve C57BL/6 mice. P14 CD8+ T cells expanded similarly in H8×IL-7−/−-CML mice and in H8-CML mice (Fig. 4A). However, significantly more p14 CTL survived long term in H8-CML mice than H8×IL-7−/−-CML mice (analyzed in blood: H8-CML: 8.2±3.7%; H8×IL-7−/−-CML: 1.2±0.6%; p=0.04).

As both neutrophils and monocytes

As both neutrophils and monocytes OTX015 research buy are versatile innate immune cells, DC functions may be either over- or underestimated in CD11c.DTR and CD11c.DOG mice, depending on the experimental setup. In this light, it is essential to determine whether other inducible DC-depletion models (e.g. zDC.DTR, Langerin.DTR, BDCA2.DTR, SiglecH.DTR, Clec9a.DTR, and CD205.DTR mice) also exhibit neutrophilia and monocytosis upon DT injection. Of note, zDC.DTR mice have been reported to possess increased neutrophil counts in the spleen upon DT treatment [12]. Our understanding of DC biology would greatly benefit from a mouse model that combines specific

depletion of DCs without the induction of neutrophilia and monocytosis. Work at the London Research Institute

is funded by Cancer Research UK. C.R.S. acknowledges additional support in the form of a prize from Fondation Bettencourt-Schueller and a grant from the European Research Council. J. v B. is supported by the Boehringer Ingelheim Fonds. B.U.S. was supported by an EMBO long-term Fellowship. The authors declare no financial or commercial conflict of interest. Apoptosis Compound Library
“Subunit vaccines have the potential advantage to boost Mycobacterium bovis Bacillus Calmette-Guérin (BCG)-primed immunity in adults. However, most candidates are antigens highly expressed in replicating bacilli but not in dormant or persisting bacilli, which exist during Mycobacterium tuberculosis infection. We constructed M. tuberculosis fusion protein Ag85B-Mpt64190–198-HspX (AMH) and Ag85B-Mpt64190–198-Mtb8.4 (AMM), which consist

of Ag85B, the Obeticholic Acid 190–198 peptide of Mpt64, HspX (Rv2031c) and Mtb8.4 (Rv1174c), respectively. AMH and/or AMM were mixed with adjuvants composed of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA-BCG PSN) to construct subunit vaccines. Mice were immunized thrice with Ag85B, AMH and AMM vaccines and the immunogenicity of the fusion protein vaccines was determined. Then, mice were primed with BCG and boosted twice with Ag85B, AMH, AMM and AMM + AMH vaccines, respectively, followed by challenging with M. tuberculosis virulent strain H37Rv, and the immune responses and protective effects were measured. It was found that mice immunized with AMH vaccine generated high levels of antigen-specific cell-mediated responses. Compared with the group injected only with BCG, the mice boosted with AMM, AMH and AMM + AMH produced higher levels of Ag85B-specific IgG1 and IgG2a and IFN-γ-secreting T cells upon Ag85B and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation. It is interesting that only mice boosted with AMM + AMH had significantly lower bacterial count in the lungs than those receiving BCG, whereas mice boosted with AMH or AMM did not.

Groups of mice were treated daily for 6 days with fusion protein,

Groups of mice were treated daily for 6 days with fusion protein, treated with vehicle, or untreated as indicated in the legend of Fig. 6. On day 7, the animals were killed; the omenta were removed and treated with collagenase, then stained for flow cytometry as described previously with minor modifications.32 Preliminary https://www.selleckchem.com/products/azd5363.html experiments were performed using normal omental cells, tumour cells and a reconstructed mixture of tumour cells and omental cells to establish the gates shown. Colony-forming assays were performed as described previously.33 Statistical analyses testing for significance were performed as indicated in the figure legends. We set out to create a cytokine fusion protein that could be cleaved

by a tumour cell expressed protease so that it becomes more active after cleavage. We initially tested a strategy based on steric hindrance learn more by constructing

a fusion protein consisting of IL-2 and Mip1-a separated by a PSA cleavage sequence.34 We hypothesized that both immunomodulatory proteins would be largely inactive in the fusion protein because of their close proximity, but would become more active if the fusion protein could be successfully cleaved, thereby separating the two proteins. Although the fusion protein could be expressed and cleaved by PSA, IL-2 did not appear to be attenuated in the intact fusion protein and the biological activity of the IL-2 did not increase after cleavage (data not shown). Hence, simply joining two molecules, even very closely, does

not necessarily interfere with their functional activity. However, we reasoned that if we constructed a molecule in which the putative inhibitory portion of the fusion protein bound the cytokine specifically, this would be more likely to inhibit its activity. As we describe below, we used two distinct strategies to inhibit the biological activity of IL-2. The first strategy employed a cytokine receptor whereas the second used an antibody fragment (scFv). The first strategy using specific inhibition employed IL-2 and a portion of the IL-2 receptor is illustrated schematically in Fig. 1(a). We used mouse IL-2 cDNA and took advantage of Sitaxentan the alpha chain of the IL-2 receptor (IL-2Rα) which can bind IL-2 in the absence of the other subunits (β and γ) of the high-affinity IL-2 receptor.35 In this construct, we eliminated the transmembrane and cytoplasmic region of the IL-2Rα chain, creating a soluble form of the receptor. To increase flexibility and allow the IL-2Rα portion of the molecule to fold back and inhibit IL-2, we also introduced a repeating Gly–Ser linker consisting of (GGGGS)2 (designated 2 ×), or (GGGGS)436 (designated 4 ×), and in some cases also added a 6 × His tag. These plasmids were used to construct recombinant baculoviruses to mediate expression in insect cells as described in the Materials and methods. As shown in Fig. 1(b), we examined the fusion proteins with a capture ELISA using antibodies reactive with IL-2Rα and IL-2. Also, the immunoblot analysis in Fig.

The ELISA technique required relatively large amounts of tissue e

The ELISA technique required relatively large amounts of tissue extracts, and one mouse could therefore be used only for the measurements of the two actual cytokines

as the oral mucosa, ear skin or lymph nodes cannot be dispersed in too large volume of extraction buffer to display measurable amounts of the cytokines. Counting of lymph node cells.  In a separate set of experiments (n = 2), submandibular and auricular (2 + 2) and axillary (4) lymph nodes were excised and kept in PBS. The lymph nodes were then squeezed through a nylon mesh (NYHC 150–80; Tidbecks, Ljungsarp, Sweden) to acquire single cell suspensions. The volume was adjusted to 10 ml before counting in a Bürker haemocytometer. Total cell counts for the combined submandibular/auricular and axillary lymph https://www.selleckchem.com/products/mitomycin-c.html nodes MG-132 solubility dmso were estimated. Calculations.  The oral mucosa and ear skin IL-2 and IFN-γ content was estimated per mg wet weight tissue. The total content of respective cytokine in quadruple regional (submandibular and auricular) and distant (axillary) lymph nodes was calculated. The tissue levels of IL-2 and IFN-γ differed in individual mice and not least between the different experimental series. However, the peaks of cytokine appearance/disappearance were consistently sharp but could vary from 4 to 24 h after hapten exposure in individual mice. The figures shown (Figs. 1–3)

are representatives of single experimental series, as assembling Nintedanib (BIBF 1120) the results from the three different experimental series into one curve causes considerable obscuring of the real biological response. Mice with normal oral mucosa or ear skin showed very low levels of IL-2. One exposure of the hapten to the oral mucosa or the ear skin (sensitization) resulted in increased levels of IL-2 locally,

(maximum 24-fold increase for the oral mucosa, and maximum 27-fold increase for ear skin, both n = 3) peaking 4–6 h after exposure and thereafter quickly subsiding. At 24 h, the IL-2 levels were back to baseline. After a second hapten exposure (elicitation), a similar peak of IL-2 occurred (maximum 39-fold increase for oral mucosa and 35-fold for the ear skin, both n = 3). The levels of IL-2 after elicitation were normalized by 12–24 h regardless whether oral mucosa or ear skin was examined. One exposure to the hapten resulted in only minor variations in the levels of IFN-γ in both ear skin and buccal mucosa. Increased levels of IFN-γ were found mainly after the second hapten exposure where a rapid increase in this cytokine was demonstrated. The peaks of IFN-γ were found between 8 and 24 h after re-exposure to the hapten (maximum 14-fold increase for oral mucosa and 8-fold for ear skin, n = 3) in tissue sensitized a week earlier. The levels of IFN-γ after elicitation were normalized by 24–48 h regardless whether oral mucosa or ear skin was examined.

A volume of 100 μl of cell suspension was added to 96-well plates

A volume of 100 μl of cell suspension was added to 96-well plates (Costar, Cambridge, MA, USA), and the Ag85A protein was added to the wells in triplicate at the final concentration of 5 μg/ml. After 72-h incubation, cell-free supernatants were harvested and were screened for the presence of IFN-γ and IL-4 with an ELISA detection system (Jingmei, Biotech) according to the manufacturer’s instruction. Intracellular IFN-γ measurement using flow CT99021 cell line cytometry.  Splenocytes from vaccinated

mice were cultured at 2.5 × 106/ml in 24-well tissue culture plates (Nunclon, Roskilde, Denmark) in the presence of 5 μg of Ag85A protein/ml for 3 days. Brefeldin A (Sigma) was added to the cultures for the last 5 h to prevent secretion of the intracellular cytokines.

One million cells from each group were first incubated with fluorescein isothiocyanate-conjugated ABT-737 anti-CD4 Ab (clone RM4 to 4 PharMingen, San Jose, CA, USA) or CD8 Ab for 30 min at 4 °C. Cells were then washed, fixed with 4% paraformaldehyde and permeabilized with phosphate-buffered saline containing 0.1% saponin. To label intracellular IFN-γ, cells were incubated with phycoerythrin-conjugated anti-IFN-γ Ab (clone XMG1.2; PharMingen) for 1 h at room temperature, washed and acquired on a cytofluorometer (FACSCALIBUR; BD, Mountain View, CA, USA). Lymphocytes were gated by their forward- and side light-scattering properties, and 100,000 cells were acquired in the lymphocyte gate. Analysis was performed by cell quest all software (BD, San Jose, CA, USA). Cytotoxicity assay of T cell [22].  Spleen cells adjusted to a concentration of 107/ml from in vivo-primed mice were co-cultured with mitomycin (10 μg/ml)-treated target cells (P815-Ag85A, 5 × 105/ml) in a 10-ml

cell suspension in RPMI 1640 for 5 days at 37 °C in 5% CO2. Twenty units per millilitre recombinant murine IL-2 (Biosource, Camarillo, CA, USA) was also added to the cell solution for 5 days. The P815 cell was used as a negative control. To measure the specific lysis of the target cells, we used the lactate dehydrogenase (LDH) release assay, which yields highly similar results as the standard chromium release assay, but does not require the use of radioisotopes. In 96-well round-bottom plates, effector cells were incubated with target cells at different E/T ratio for 4 h in phenol red-free RPMI 1640 containing 2% BSA, 2 mm glutamine, and 1% penicillin and streptomycin. After centrifuging the plates at 250 g for 10 min, 100 μl per well of the supernatant was then transferred to 96-well plates, and lysis was determined by measuring LDH release using cytotoxicity detection kit (Roche Molecular Biochemicals). The released LDH converted the added substrate tetrazolium salt into red formazan product, and the amount of colour is proportional to the number of lysed cells. The absorbance values from supernatants were recorded at OD 492 nm on an ELISA microplate reader.