We observed the preferential presence of certain HLA class II DR molecules in our responding patients, HLA-DR15 and HLA-DR7 in 50% of the responding women and DR11 in 30%. No such an association between HLA class II molecules, T anti-HPV T cell responses and classic VIN has been described previously. A significantly high frequency of DRB1* 0901 or DQB1*03032 was observed in HPV-16-positive CIN3/invasive MAPK inhibitor cervical carcinoma patients in Japan and China [51–53]. An increased risk of CIN3 has been associated with DRB1*1501 or DQA1*0102 in New Mexico [54]. Conversely, DRB1*1501 and DQB1*0602 haplotypes were shown

recently to be protective against CIN2+, especially in individuals infected with oncogenic HPV in Canada [55]. In CIN1, DRB1*1301 was associated with an increased probability of regression [56] and DR B1*11, DR B1*15, DR B1*3 with persistence [57]. By studying the immunodominant E6 and E7 large peptides in HLA-DR-specific binding assays, we observed that E6/2 14–34 and E/4 45–68 peptides bound HLA-DR7, 11 and 15 (molecules shared by our patients) and to other HLA-DR such as DR1, DR3, DR4, DRB5. Nevertheless, it remains to be proven that HLA-DR molecules are the restricting element for proliferative CD4+ T cells. Indeed, HLA-DQ and -DP were described recently as proliferative response-restricting elements during BIBW2992 HPV-16 infection [58,59]. The present

study shows that following the disappearance of the lesions, either spontaneously or after destructive treatment, proliferative responses can persist at least for 1 year with a broadening of peptide recognition concomitant with a loss Benzatropine of some specificities and acquisition of others. This observation can be related to an immunospreading of the cellular immune response following deliverance of new HPV antigens in the blood after destruction of the lesions or to

recirculation of effector T lymphocytes from the epithelium to the blood. Using ELISPOT–IFN-γ assay, ex-vivo frequencies of specific anti-E6 or E7 peptides T lymphocytes were stronger in the present study in the two patients with large clinical lesions of classic VIN compared to the patients with smaller or no detectable lesions who had low blood T cell responses. In a previous study, six of nine patients with classic VIN had ex-vivo frequencies of specific anti-E6 or E7 peptides; CD8+ T lymphocytes comprised between 21 and 1360 SFC/106 CD4-depleted T lymphocytes [60]. However, no clinical correlation was reported in the latter study. Our results may be the consequence of better contact between T lymphocytes and a large area of HPV-16-infected keratinocytes, generating better ex-vivo T cell responses. After treatment and disappearance of the lesions in our patients, ex-vivo T cell responses became undetectable by ELIPSPOT–IFN-γ assay. In conclusion, we have defined two immunodominant regions in HPV-16 E6 protein.

There was a trend, albeit not significant, toward a decrease in Treg-cell function after OK-432 administration (Fig. 4C). In contrast, we did not observe any differences in frequency and function of Treg cells in PBMCs before

and after OK-432 administration (data not shown). These data propose that in vivo injection of OK-432 decreases the local Treg-cell accumulation and function. To further explore the effect of OK-432 on the inhibition of in vivo Treg-cell activity, we also examined the potential of OK-432 as an adjuvant in a cancer vaccine. We have reported that high-avidity NY-ESO-1–specific CD4+ T-cell Selleckchem Atezolizumab precursors are present in naive CD45RA+ populations and that their activation is rigorously suppressed by CD4+CD25+ Treg cells [20, 21]. We also found that synthetic peptide vaccination with incomplete Freund’s adjuvant induces only peptide-specific CD4+ T cells with low-avidity TCRs (recognition of >1 μM peptide but not naturally processed NY-ESO-1 protein), but not high-avidity CD4+ T cells (recognition of naturally processed NY-ESO-1 protein or <0.1 μM peptide) that are susceptible to Treg-cell suppression [21]. Together, BI 6727 mouse these data highlight the importance of blocking Treg-cell activity to allow activation/expansion of high-avidity NY-ESO-1–specific CD4+ T-cell precursors. For this reason, we investigated whether

high-avidity NY-ESO-1–specific CD4+ T-cell precursors were activated by NY-ESO-1 protein vaccination with OK-432 as an adjuvant and were present in memory CD45RO+ populations. Samples from two patients who received vaccination with cholesteryl hydrophobized pullulan (CHP)-HER2 and NY-ESO-1 with OK-432 (Supporting Information Fig. 1) were available for this analysis. Whole CD4+ T cells or CD4+CD25−CD45RO+ (effector/memory) T cells before and after vaccination Buspirone HCl were presensitized with NY-ESO-1–overlapping peptides covering the entire sequence of NY-ESO-1 and specific CD4+ T-cell induction was analyzed with ELISPOT assays. As the sample size was not sufficient to analyze specific CD4+ T-cell induction within CD4+CD25−CD45RA+

(naive) T cells, we analyzed whether NY-ESO-1–specific high-avidity CD4+ T cells were induced from the CD4+CD25−CD45RO+ (effector/memory) T-cell population after vaccination in Pt #1 (HLA-DR 4, 12 and HLA -DQ 4, 8) and #2 (HLA-DR 9, 15 and HLA-DQ 6, 9). Pt #1 exhibited spontaneously induced CD4+ T-cell responses against NY-ESO-191–110 before vaccination and the responses were maintained after extensive vaccination (Fig. 5A). These spontaneously induced NY-ESO-191–110–specific CD4+ T cells were detected in the CD4+CD25−CD45RO+ (effector/memory) T-cell population before and after vaccination. Following vaccination with NY-ESO-1 protein in the presence of OK-432, CD4+ T-cell immune responses against NY-ESO-1111–130 were newly elicited (Fig. 5A).

While an animal model mimicking the entire complexity of AD is currently lacking, certain aspects of typical pathophysiological alterations can be modelled by using transgenic mice expressing mutant forms

of AD-related proteins Depsipeptide cell line (see, e.g. [12-15]). Aged triple-transgenic (3xTg) mice which harbour mutated amyloid precursor protein (APP) and tau as well as knocked-in human presenilin-1, display both β-amyloidosis and tau hyperphosphorylation [16-19], although their causal relationship remains controversial. However, details regarding the third hallmark of AD – that is, the degeneration of cholinergic projection neurones known to contribute significantly to cognitive decline in AD patients [20] – have often been neglected in animal models of AD. On a descriptive level, two studies have recently addressed cholinergic alterations in 3xTg mice [21, 22], which resulted in only marginal changes and conflicting data concerning their age-related starting time point. In detail, Girão da Cruz et al. [21, 23] reported a reduction in the number of cholinergic neurones in the medial septum/vertical limb of the diagonal band (MS/DB) complex,

comparing 4- and 12-month old 3xTg Selleckchem LEE011 and control mice. In contrast, Perez et al. [22] described a 23% reduction in the number of cholinergic neurones in the MS/DB of 3xTg mice compared to controls, but this effect failed to reach statistical significance until an age of 18–20 months. Beyond this descriptive perspective, a method to experimentally induce cholinergic degeneration in a widely accepted animal model of AD might be useful to more reliably capture the complexity of AD, and therefore, to further

explore interrelations between the cholinergic system and Aβ accumulation as well as tau hyperphosphorylation. To address this, we introduce an extended model in which mice with genetically induced age-dependent β-amyloidosis and tauopathy undergo selective loss of CPN in the basal forebrain. For this purpose, an immunolesioning technique was applied for CPN degeneration, Akt inhibitor based on a selective immunotoxin containing the ribosome-inactivating saporin from soapwort Saponaria officinalis. This method of ‘molecular surgery’ [24] was originally described by Wiley and co-workers [25, 26] and briefly acts in the following way: After intracerebroventricular (icv) application, saporin-conjugated antibodies directed against extracellular epitopes of the low-affinity neurotrophin receptor p75 (in the forebrain exclusively on CPN) are first bound by the receptor located on cortical terminals, subsequently internalized as anti-p75-saporin/p75 complexes and then retrogradely transported to the perikarya where saporin inactivates ribosomes causing selective death of CPN.

Accordingly, a rare IL-23R polymorphism in humans protects against the development of Crohn’s disease 35, likely due to reduced Th17-cell responses. In contrast, our data predict that humans with IL-23R variants, although protected against Sotrastaurin concentration autoimmune diseases, may not generate effective BCG vaccine-induced Th1-cell immunity, potentially resulting in poor protection outcomes upon M. tuberculosis challenge. Furthermore, since recombinant BCG strains are a

likely choice for priming or boosters in future TB vaccine strategies against TB 36, the findings presented here suggest that including IL-23-promoting factors into recombinant BCG vaccines may be one approach to promote Th17-cell responses and improve upon current levels of Th1-cell-induced protection against TB. In contrast, identifying and eliminating IL-10-inducing factors in BCG may directly increase

Th1-cell responses and generate better efficacy against M. tuberculosis challenge as seen in the il10−/− BCG-vaccinated mice. Our data also GSK2118436 mouse suggest that eliminating PGE2-inducing factors in BCG may eliminate IL-10 production and directly induce Th1-cell responses without dependence on IL-17. Therefore, our study defines several molecular mechanisms that can be exploited to improve upon current vaccine strategies against TB. In summary, we propose that some intracellular bacteria such as BCG avoid direct induction of Th1-cell responses by producing PGE2 and IL-10. The fact that BCG-induced IL-10 inhibits IL-12 production and limits IFN-γ production has been demonstrated previously 27. However, our study extends these findings and shows that il-10−/− BCG-vaccinated mice have better vaccine-induced protection outcomes. Moreover inhibitory effects of IL-10 are not limited to attenuated strains of mycobacteria, since even in models of virulent M. tuberculosis infection,

il10−/− mice exhibit enhanced IFN-γ production and reduced lung bacterial loads during chronic stages of infection 28. Furthermore, novel data presented here show that pathogen-induced PGE2 has dual functions to play in host immunity, apart from its role in driving IL-10 production, PGE2 is also required to drive IL-23 responses in DCs and subsequent IL-17 production in T cells. IL-17 then overcomes IL-10-mediated inhibition of Niclosamide Th1-cell induction by downregulating IL-10 and upregulating IL-12 production in DCs, thereby allowing for the generation of an effective IFN-γ response. The broader understanding of the specific host factors required to induce an optimal Th1-cell immune response against intracellular bacteria will allow us to exploit this knowledge in design of better vaccine strategies against infections. C57BL/6 (B6), OT-II αβ TCR Transgenic (Tg) mice (OT-II) which are MHC class II I-Ab restricted and specific for OVA323–339 and il-10−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

HRP-conjugated goat antirabbit IgG (Dingguo Biotechnology, Beijing, China) diluted by 1:10000 was added and incubated for 1 hr at 37°C. The plates were washed four times with PBS before adding diaminobenzidine substrate (Dingguo Biotechnology), 20 M H2SO4 was added to cease the reaction and the OD490nm was measured. A positive control, a negative control and a blank control were always included on each plate. Six BALB/c mice (6–8 weeks of age) were immunized with the purified recombinant protein. For primary immunization, each mouse was s.c.

injected with 50 μg of antigen (recombinant click here 56-kDa protein) emulsified in Freund’s complete adjuvant. Ten days later, they were given an i.p. booster injection of GSK126 mouse 50 μg antigen emulsified in Freund’s incomplete adjuvant. Control mice were injected similarly with PBS emulsified in Freund’s complete adjuvant or incomplete adjuvant. After that, mice were bled and sera were obtained and stored at −20°C. The animal use was reviewed and approved by the Beijing Administrative Committee for Laboratory Animals and the animal care met the standard of the committee. Bleeding of the mice was performed by tail clip after primary immunization and cardiac puncture after booster immunization. To determine

IgG titers of the sera, an IFA test with antigen slides of O. tsutsugamushi Karp was carried out with fluorescein isothiocyanate-conjugated goat antimouse IgG (Kierkegaard & Perry Laboratories, Gaithersburg, MD,

USA). Meanwhile, an ELISA test was also performed as described above. A fragment of 1107 bp that would yield a 46-kDa His-tagged protein with a deletion of 99 amino acid residues at the N terminal and 64 amino acid residues at the C terminal was amplified by PCR and the product was cloned into pET30a. The resulting recombinant plasmid, designated pET30a-Ot56, was detected by both PCR and restriction enzyme digestion (Fig. 1) and was verified by direct DNA sequencing. Analysis by SDS-PAGE showed that a band approximately at 46 kDa, the expected size Carnitine palmitoyltransferase II for the truncated protein, was observed in E. coli Rossetta cells transformed with pET30a-Ot56 (Fig. 2a). The purified protein appeared as a single band corresponding to the molecular mass of the recombinant protein on SDS-PAGE (Fig. 2b). The amount of protein after purification was 0.7 mg/mL. Immunoblot assay showed that the protein was recognized by O. tsutsugamushi Karp-immunized rabbit serum (Fig. 2c). The recombinant protein was also validated by MALDI-TOF-MS, which revealed that it had 100% identity to 56-kDa protein of O. tsutsugamushi (Fig. 3). Enzyme-linked immunoassay was performed to assess the extent of cross-reactivity of the recombinant protein with the rabbit polyclonal sera described above. All of the sera detected, except sera against O. tsutsugamushi strains TA763, TH1817, Kato, B quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis were negative (Tables 1,2).

Patients were randomly assigned to the treatment group (750 mg/day probucol combined with 160 mg/day valsartan) or the control group (160 mg/day valsartan alone). Initially, GS-1101 patients were followed up once every 4 weeks. When the target blood pressure (BP) of 130/80 mmHg was not achieved, a β-adrenergic antagonist was administered; if blood pressure was still not controlled, a α-adrenergic antagonist was added. Diuretics and calcium antagonists were used only temporarily if necessary.

Mild dietary sodium restriction limited to 90 mmol/day was advised. At study entry, complete medical histories were taken and physical examinations were performed for all patients. Initial clinical and laboratory results were sent to the coordinating centre. Follow-up

patient examinations and measurements of blood pressure (BP), serum creatinine (Scr); blood urea nitrogen (BUN); 24-h urinary protein excretion, estimated glomerular filtration rate (eGFR; estimated with the MDRD (Modification of Diet in Renal Disease) equation), haemoglobin (HGB); total cholesterol (CHOL), and low-density lipoprotein cholesterol (LDL-C); triglycerides (TG); serum albumin (ALB); and electrocardiogram (ECG) were scheduled at 2-month intervals. The results of echocardiography examination were obtained at admission and at the end of the study. Also, first morning urinalysis, liver function, including total protein (TP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), direct bilirubin (DBIL) and serum potassium were Ensartinib mw Amobarbital collected and analyzed at the local centre at each scheduled visit. All clinical and laboratory results were recorded on case report forms, forwarded to the coordinating centre, and entered for data processing. Proteinuria, serum creatinine and eGFR are the key indicators for evaluating the risk for rapid disease progression. In the present study, these indicators are chosen to evaluate

the efficacy of probucol combined with valsartan treatment. The primary endpoint of the study was time to doubling serum creatinine as compared with the baseline or the development of end-stage renal disease that required renal replacement therapy or death during the study period. The secondary endpoint was reduction of 24-h urinary protein by 50% or more or rate of eGFR decrease relative to the baseline. Results are expressed as mean ± scanning electron microscopy (SEM) for continuous data and as percentages for categorical variables. Statistical analysis was performed using the statistical package SPSS for Windows Ver. 19.0 (SPSS, Chicago, IL, USA). Descriptive analysis was used for evaluation of the general characteristics of patients and a χ2 test or a rank sum test was used to compare baseline parameters of the two groups. A repeated-measure analysis of variance (anova), student’s t-test or the rank sum test was used to compare parameters of the two groups was used to compare parameters before and after treatment.

In contrast, circulating IgA levels in control wool lambs remained low and stable following initial larval exposure, and breed differences in circulating IgA were thus larger in control lambs. Greater responses in circulating IgA in response to transient exposure to parasite larvae in control hair lambs suggests a more robust response to this ‘natural’ vaccination protocol. IgA concentrations in hair sheep were somewhat higher than those reported for wool sheep in previous studies. Larval antigen-specific IgA production has been reported to peak between 1 and 2 weeks p.i. (42,44). However, total IgA in serum in our infected lambs continued to increase through 3 weeks

p.i. in both breeds. Previous studies have not observed higher serum IgA concentrations in resistant breeds including the Gulf Ulixertinib mw coast native (40), Santa Ines (17) and Barbados Blackbelly (34) compared with susceptible wool breeds, which may indicate that St. Croix hair sheep have a novel resistance mechanism that is absent in other resistant breeds. Globule leucocytes are described as partially

de-granulated intraepithelial mast cells (45) and have been suggested to be responsible for larvae damage and expulsion within the first few days of infection. Unlike eosinophils (46,47), mast cells bind IgE (41), leading to similar co-dependency to that suggested for eosinophils buy Sirolimus and IgA. Breed differences in abomasal globule leucocytes were not significant in this study, but levels tended to be greater in hair sheep at 27 days p.i. This result is less striking than the 15- to 40-fold increase in globule leucocytes of younger infected hair compared with wool lambs reported by Gamble and Zajac (18). However, breed differences in concentration PRKACG of globule leucocytes have been reported to be minimal by 1 year of age (3) and hence age differences probably contribute to apparent inconsistencies among studies. Associations of increased globule leucocytes with lower FEC, lower worm numbers and decreased female worm length are present in young animals (11,15,48), suggesting a role for these cells in resistance and are consistent with our favourable association of globule

leucocyte numbers with IgE in the lymph nodes and PCV at 21 days p.i. Measurement of sheep IgE was first reported by Shaw et al. (49) and Kooyman et al. (8) and several studies have shown that sheep infected with GIN have increased total and worm-antigen-specific IgE (8,12,13,50,51). Mean serum IgE levels in our lambs exceeded 60 ng/mL through 16 days p.i. in infected lambs of both hair and wool types. IgE levels were similarly elevated through the first 16 days following exposure and subsequent de-worming in control hair lambs, but were only transiently elevated in control wool lambs over the same period. These patterns suggest a similar change in circulating IgE following infection in the two breeds with a potentially more robust vaccination response in hair lambs, similar to that observed for circulating IgA.

4A and Supporting Information Fig. 2F–J), consistent with a first-order kinetics of irreversible dissociation of a single monomeric bond with a single state selleck [39]. Using this model, the off-rate is evaluated from the negative slope of the linear regression of the lifetime distribution data. The off-rates of pMHC dissociating from the individual TCRs in the panel are summarized in Fig. 4C. As the off-rates of some

TCRs (W2C8, L2G2, and K4H5) are too fast to be determined by SPR [36] and because the pMHC tetramer only stained the two highest affinity TCRs when expressed in the CD8− hybridoma (Supporting Information Fig. 1C and D), the 2D data obtained here show that the thermal fluctuation assay has a higher sensitivity and temporal resolution than SPR or tetramer staining and allows us to obtain kinetic parameters for low-affinity fast dissociating TCRs that are otherwise unobtainable. The effective 2D on-rates were then calculated based on Ackon = AcKa × koff (Supporting Information Fig. 2K). We observed no correlation between 2D and 3D on-rates (R2 = 0.13; p = 0.55, Supporting Information Fig. 3B). The 2D off-rates for the individual TCRs (Fig. 4C) are at least 15-fold faster than their 3D counterparts (Supporting Information Fig. 3C). The TCR with slowest 3D off-rate (19LF6; ∼0.012/s) [36] has the fastest 2D off-rate (∼11.4/s), amounting to a three orders of magnitude difference. KU-60019 in vitro Thus, for the panel

of human TCRs interacting with a single pMHC, the 2D measurements substantially differ from the 3D measurements in both on- and off-rates and in affinity, similar

to previous observations obtained when analyzing a single mouse TCR interacting with a panel of pMHCs [27, 28, 33]. All of the TCRs studied here (except for 19LF6) rely on the co-receptor CD8 for their functional activities (Fig. 1C and Supporting Information Fig. 1A), yet, tetramer staining of TCR+CD8+ hybridoma cells yielded only insignificant correlation with the TCR functional outcome (Fig. 2D). Therefore, we asked whether 2D kinetic analysis of pMHC binding to these cells would better predict their T-cell responses. To dissect how CD8 contributes to 2D binding of pMHC to TCR+CD8+ cells, we first measured the HLA-A2–CD8 interaction kinetics Oxymatrine in 2D. Micropipette adhesion frequency revealed fast kinetics of the HLA-A2–CD8 interaction on a TCR−/CD8+ cell line (Fig. 3B). The off-rate measured by the thermal fluctuation assay was 17.4/s (Fig. 4B and C). The effective 2D affinity was 1.3 × 10−6 μm4 (Fig. 3C). This is the first 2D kinetics measurement for human CD8 (hCD8) interacting with HLA-A2. In comparison, mouse CD8 (mCD8) has 2D affinities of 5.8 × 10−6 μm4 and 7.8 × 10−7 μm4 for H2-Kb and H2-Db, respectively [40]. The hCD8 2D affinity is more than two orders of magnitude lower than the affinities for the panel of TCRs (Fig. 3C, except for the weakest TCR, W2C8 with an affinity of 5.

2 ml normal saline. After 10 days of challenge the organs (liver and spleen) were aseptically removed and homogenized with a tissue homogenizer (Potter-Elvehjem homogenizer) using 5 ml of saline. The blood (100 μl) was collected from the orbital plexus for HT. The tissue suspensions were transferred to petri-dishes containing the mixture (Sabouraud’s broth + yeast extract + chloramphenicol) and maintained at room temperature for 48 h. The colonies were counted and the number of CFU per organ was determined. Under these conditions the culture gave a yield of approximately 20 × 106 cells per ml and the organisms grew as an essentially pure yeast phase population. The cells were washed twice,

suspended in saline to get desired concentrations and BGB324 research buy the viability was checked by methylene blue staining [12]. Statistical analysis.  The statistical analysis of data was done using analysis of variance

(ANOVA) with post-hoc analysis. The Tukey–Kramer post-hoc test was BAY 57-1293 nmr applied to analyze significant changes among groups. The significance of results was ascertained at P < 0.05. All the data are presented as means ± standard error (SE) of the means. GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis. Deltamethrin (18 mg/kg) induced suppression in humoral immunity showing reduction in both parameters, PFC (Fig. 1) and antibody titre (Table 1). Significant (P < 0.001) reduction in PFC was observed in animals treated with deltamethrin alone when compared with control animals (group I). In case of pre and post exposure of fungal infection with deltamethrin the PFC response was significantly (P < 0.001) reduced when compared with the control group.

Infections with C. albicans alone also showed significant (P < 0.001) decrease in PFC response. In case of titre value, there was remarkable decrease in animals treated with deltamethrin when compared with the control group. The titre value in control was 1:682, whereas in DM-treated mice it was 1:64. In animals, treated with fungal infection alone antibody titre was 1:48, whereas it was 1:16 in pre and post exposure of C. albicans with deltamethrin group of animals. Liver of control animals and those treated with deltamethrin Fenbendazole alone showed almost no CFU. Animals treated with deltamethrin before as well as after candida infection showed significantly increased number of CFU (Fig. 2). When data of two candida + deltamethrin groups were compared it was observed that animals which were given deltamethrin exposure after C. albicans exposure showed a significantly high number of CFU (P < 0.01). Similar observation was made in case of CFU in spleen (Fig. 3). Deltamethrin appears to be the best compromise between the effectiveness and disadvantages of insecticides, being widely used to control a large variety of agricultural pests and to protect stored products [8, 16, 17].

Finally, we analysed the observed frequencies of cytokine-producing CD4+ T cells by scoring the results as negative (responses <0.01%) versus positive and compared the 3+ CD4+ T cells statistically in the different groups of individuals. As summarized in INCB024360 Table 1, the highest proportion of positive responses was found among patients with active TB, followed by those patients with cured TB (at the end of anti-mycobacterial treatment). Lower proportions of 3+ CD4+ T cells positive responses were found in individuals with LTBI, whereas all of the controls were negative (data not shown). Pair-wise comparisons of the positivity

PI3K inhibitor rates for 3+ CD4+ T cells in the four groups of individuals are summarized in

Table 1: the proportion of positive responses among active TB-infected patients was significantly higher than that recorded among patients with cured TB, individuals with LTBI and control subjects. Taken together, these data suggest that 3+ CD4+ T cells simultaneously secreting IFN-γ, IL-2 and TNF-α to three antigens of M. tuberculosis, Ag85B, ESAT-6 and the 16-kDa antigen, are more frequently found in patients with current or historic TB disease compared with LTBI which are able to control M. tuberculosis replication. This study provides a detailed analysis of the frequency and quality of cytokine-producing CD4+ T cells in patients with active TB disease, cured TB and in subjects with LTBI. Importantly, we show here that the frequency of CD4+ T lymphocytes that produce multiple cytokines (IFN-γ, IL-2 Branched chain aminotransferase and TNF-α)

is significantly higher in subjects with active TB disease, not supporting current beliefs that such responses may be associated with protection. In contrast, CD4+ T cells that produced IL-2 and IFN-γ, or IFN-γ alone, were lower in active TB-infected patients compared with cured TB patients or individuals who controlled infection naturally (LTBI). Lending further support to our results is the observation that this pattern of distribution of cytokine-producing CD4+ T cells was consistently observed in response to three different M. tuberculosis antigens, Ag85B, ESAT-6 and 16 kDa antigen. Data from HIV and other chronic viral infections have associated CD4+ and/or CD8+ T cells that simultaneously produce the three cytokines IFN-γ, IL-2 and TNF-α, with non-disease progression and efficient control of infection 20, 22, 23. Such “multifunctional” cell profiles have subsequently also been used to define correlates of vaccine-mediated protection against Leishmania11 and M. tuberculosis12, 24, 25 in mouse models of vaccination.