1% saponin, 0.2% NaN3), followed by staining with αIL-7-biotin and streptavidin-APC.

Samples were measured and analyzed as described in “Antibodies and flow cytometry”. Single-cell suspensions of naïve CD45.1+ splenocytes were prepared, and erythrocytes were removed. Half of the cells were pulsed with gp33 (10−6 M) at 37°C for 90 min. Then, the cells were washed twice with PBS, adjusted learn more to 2×106 cells/mL, and labeled with CFSE (Molecular Probes, Eugene, OR, USA) at either a final concentration of 5 μM (gp33-pulsed splenocytes, CFSE high) or of 0.1 μM (unpulsed splenocytes, CFSE low) for 10 min at 37°C. After labeling, FCS was added up to a final concentration of 10%, and cells were washed with PBS at 4°C. Briefly, 3×107 CFSE-labeled, gp33-pulsed and 3×107 CFSE-labeled, unpulsed CD45.1+ splenocytes were buy Dorsomorphin injected i.v. into H8-CML mice, αCD8-treated H8-CML mice, naïve C57BL/6 and LCMV-immune mice which had been infected i.v. with 200 pfu LCMV-WE 8 wk previously. After 8, 24 and 48 h, blood was collected, and the reduction of the CFSE high population normalized to the CFSE

low population was calculated by flow cytometry analysis. P14×CD45.1 T cells were isolated and purified by MACS (Miltenyi Biotec) for CD8+Va2+ T cells. In total, 2.5−4×106 CD8+Va2+CD45.1+ cells were injected i.v. into H8-CML mice, H8×IL-7−/−-CML mice, naïve C57BL/6 control mice and C57BL/6 mice chronically infected with 107 pfu LCMV Docile (all recipient mice were CD45.1−). CML disease progression and expansion of transferred CD8+Va2+ T cells were monitored Resveratrol by FACS analysis of blood and spleen. For isolation of total spleen mRNA, 30 mg of tissue were frozen in liquid nitrogen and homogenized using a stainless steel bead and tissue lyser (Qiagen, Hombrechtikon, Switzerland), followed by RNA extraction (RNeasy

mini kit, Qiagen). For isolation of granulocyte mRNA, single-cell suspensions of naïve C57BL/6 or CML spleens were sorted for 1.5×106 granulocytes or GFP+ granulocytes, respectively, into RNAprotect® cell reagent (Qiagen) on a FACS Aria unit (BD Biosciences). RNA was extracted and its concentration was determined by spectrophotometry (Nanodrop ND-1000, Witec AG, Littau, Switzerland). Reverse transcription was performed using 0.25–1 μg of mRNA, random oligonucleotides and AMV-RT (Roche, Basel, Switzerland). For conventional RT-PCR, we used Taq-Polymerase (Roche) and the following primers: β-actin sense 5′-TGGAATCCTGTGGCATCCATGAAA-3′, β-actin antisense 5′-TAAAACGCAGTCCAGTAACAGTCCG-3′, IL-7 sense 5′-GGAATTCCTCCACTGATCCT-3′, IL-7 antisense 5′-CTCTCAGTAGTCTCTTTAGG-3′ (Microsynth, Balgach, Switzerland). For quantitative real-time RT-PCR, we used 10 ng of cDNA per well, TaqMan® Universal PCR Master Mix and TaqMan® Gene Expression Assays for IL-7 (Mm00434291_m1) and the four housekeeping genes GAPDH (Mm99999915_g1), β-actin (Mm00607939_s1), β-Glucuronidase (Mm00446957_m1) and Transferrin-Receptor (Mm00441941_m1) (Applied Biosystems, Rotkreuz, Switzerland).

Modifying the classical suppression Ibrutinib assay to measure cytokine production by the responder (CD4+CD25–) T cells activated has revealed that there may be a hierarchy of suppression, with down-regulation of IFN-γ mRNA occurring earlier than suppression of Th2 cytokine production [79]. A similar study examining the transcriptional profile of T cells activated in the presence or absence of Tregs revealed down-regulation of factors promoting both Th1 and Th2 development [IL-12Rα, IL-12Rβ2 and Irf-4 as well as T-bet and GATA binding

protein 3 (GATA-3)] in ‘suppressed’ T cells [80]. Notably, expression of IL-21, a Th17-associated cytokine, was also suppressed upon co-culture, suggesting that Tregs can down-regulate at least one element of Th17 effector function. Sakaguchi

et al. reported that mice lacking CD25+ T cells develop exacerbated responses to non-self antigens and eventually develop various autoimmune NVP-AUY922 pathologies [13]. This seminal observation implicated Tregs in governing the magnitude of immune responses and setting the threshold for the development of clinical autoimmune disease. If Tregs are particularly important in restraining one type of effector T cell response, this might be revealed by looking at what type of pathology is most prevalent in the absence of Tregs or when their regulatory function is impaired. ifoxetine Several mouse models have been utilized to investigate defects in FoxP3 function with varied degrees of severity, from global impairment [18,81,82] and inducible ablation [34], to attenuated expression of FoxP3 [35], to Treg specific-disruption of selected suppressive mechanisms [30,31] or homing mechanisms [29]. T cells from scurfy mice are hyperproliferative to TCR ligation [17] and produce higher levels of cytokines than wild-type littermates [83]. Heightened production of IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IFN-γ and TNF-α in scurfy mice

indicated that components of both Th1 and Th2 responses are exacerbated in the absence of functional Tregs, while pathology results from an excessive ‘non-polarized’ response. Because IL-17 was not recognized as an important proinflammatory product of T cells at the time the scurfy mouse was characterized, levels of IL-17 were not determined. Mice with a targeted disruption of FoxP3 recapitulated the phenotype of scurfy mice displaying allergic airway inflammation and hyperproduction of immunoglobulin (Ig)E, indicative of overactive Th2 responses. However, both Th1 and Th2 cytokines were overproduced in FoxP3 knock-out mice, suggesting a non-selective dysregulation of both Th1 and Th2 responses [82].

In this study, we analyzed the impact of IL-7/IL-7R signaling components on the generation, composition and function of circulating Treg. We hypothesized that an impairment of this CHIR-99021 concentration pathway might add to the aberrant T-cell homeostasis and Treg dysfunction associated with MS. Most resting lymphocytes express the IL-7 receptor, which is composed of the IL-7Rα-chain and the common cytokine γ-chain. Basal responsiveness of naïve subsets to IL-7 is important for their sustained survival and facilitates homeostatic cycling and differentiation of RTEs 10, 22. In consistence

with an essential role of IL-7/IL-7R signaling for the maintenance of naïve T cells, we provide evidence that the expression level of the IL-7Rα chain on Tconv is closely linked to the percentage of RTEs defined as CD31-coexpressing naïve T cells within the peripheral T-cell pool. This applies not only to conventional CD4+ T cells as described earlier 11, 12, but also to the Treg subset despite the distinctively lower overall levels of CD127 expressed on Treg, which together with intracellular FOXP3 expression and high surface expression of CD25 defines the Treg phenotype in humans 23–25. The reciprocal relation between IL-7Rα-MFIs on Tconv and plasma concentrations of

IL-7 detectable in our study underlines the tight balance between the components of this signaling pathway. Here, we show that the amount of IL-7Rα expressed on the surface of Tconv and Selleckchem Mitomycin C Tconv subsets is significantly decreased in patients with MS. As an important finding, reduced IL-7Rα-MFIs in MS-derived Tconv strongly correlated with both reduced frequencies of RTE-Tconv and RTE-Treg and with reduced Treg-mediated inhibitory activities. Therefore, by determining the prevalences of circulating RTE-Treg, IL-7Rα expression appears to affect the suppressive capacity of total Treg, which is in line with previous studies demonstrating that proportions of RTE-Treg are critical for the function of total Treg 2, 3. A decline of IL-7Rα-MFIs was detectable

in approximately two-thirds of patients with MS, whereas 30% of patients showed HC-like levels of IL-7Rα together with normal RTE-frequencies and normal Treg functions. This observation is consistent with earlier findings of a minority of MS patients 5-Fluoracil mouse exhibiting normal Treg homeostasis and suppressive properties 26. Of note, IL-7Rα expression on Tconv and RTE-Treg were decreased in HC donors of older age whereas age related effects were abolished in MS patients. This substantiates the assumption that immunosenescence might play a role in the development of this disorder 27. As a typical phenotypic feature of the Treg subset IL-7Rα expression is downregulated on circulating Treg 23–25. As expected, we found low levels of IL-7Rα on Treg and Treg subsets in all blood samples analyzed (data not shown). Thus, the MS-related reduction of IL-7Rα-MFIs on Tconv was not detectable in Treg.

In each of the outbreaks there was high sequence identity between the strains isolated within each individual outbreak. SB525334 mw The strain causing the outbreak in November of the same year had the closest

sequence identity to the Gulu 2000 outbreak strain [20]. The first recorded outbreak caused by BDBV, representing the species Bundibugyo ebolavirus, occurred in Uganda in 2007 [7] (Table 3). The virus was found again in a 2012 outbreak in Isiro in the DRC: this was the first identification of BDBV in the DRC. The BDBV isolate showed 98.6% full genome sequence identity with the prototype BDBV isolated in the 2007 outbreak in Bundibugyo, Uganda [20]. While FHF outbreaks have been reported in few countries in Africa (Fig. 1, Tables 2

and 3), the geographical distribution of filoviruses may be wider than previously thought. A feature of recent outbreaks is new strains/species in new locations, as has been the case with the MVD outbreak in Angola, the discovery of BDBV in Uganda and the DRC, and the current EBOV infection in West Africa [7, 20, 29, 35]. Using ecological niche modeling, filovirus distribution was generally predicted to occur across the Afro-tropics, with ebolaviruses occurring in the central and western African rain forests learn more and marburgviruses in the drier and less forested central and eastern Africa [3]. Countries like Tanzania, Mozambique, Madagascar and Mauritania have had no reported outbreaks of filovirus infections, but do fall within the ecological niche of

this virus and its reservoir(s). It is possible that there have been misdiagnosed and undiagnosed cases in countries with no FHF outbreak history. In some areas with no recorded outbreaks of EVD, EBOV seroprevalence in humans and some species of nonhuman primates has been found to be unexpectedly high [32, 36]. This suggests either MRIP the presence of non-pathogenic variants of EBOV or unknown filoviruses antigenically similar to EBOV, but with lower pathogenicity, causing high seropositivity [32, 37-39]. This also implies high exposure of these populations to the virus [36]. Wider filovirus distribution, even into the Eurasian continent, has been suggested by recent studies that have reported the discovery of RESTV in domestic pigs in China [40]; identification of a new filovirus, LLOV in Spain [41] and detection of antibodies to filoviruses or unknown filovirus-related viruses in Indonesian orangutans [42] and fruit bats in Bangladesh [43]. Apart from R. aegyptiacus, the only bat species from which infectious marburgviruses have been isolated, other bat species in which filovirus genome RNAs have been detected are Epomops franqueti, Hypsignathus monstrosus and Myonycteris torquata for EBOV [44]; Miniopterus inflatus and Rhinolophus eloquens for MARV [45], and Miniopterus schreibersii for LLOV [41]. Many more bat species have been found to have antibodies to various filoviruses [46].

The specificity of MICA upregulation was reflected by a higher cytolytic activity of an NK cell line (NK92MI) against C. trachomatis-infected cells compared with uninfected control cells. Significantly, data also indicated that NK cells exerted a partial, but incomplete sterilizing effect on C. trachomatis as shown by the reduction in recoverable inclusion forming units (IFU)

when cocultured with C. trachomatis-infected cells. Taken together, our data suggest that NK cells may play a significant role in the ability buy Cilomilast of the host to counter C. trachomatis infection. Genital infections with Chlamydia trachomatis serovars D-K are the most prevalent sexually transmitted bacterial infection (CDC, 2010). The propensity for these intracellular infections to remain relatively asymptomatic in women, combined with the ability of C. trachomatis to survive for extended periods in the genital tract,

make this pathogen a major public health challenge. Although the microorganism is susceptible to antibiotics, asymptomatic patients typically go untreated. Infection that ascends into the upper tract can cause pelvic inflammatory disease that can eventually lead to tubal infertility, ectopic pregnancy, and chronic pelvic pain (Brunham & Rey-Ladino, 2005). Chlamydia trachomatis infection also enhances human immunodeficiency virus acquisition and shedding (Plummer et al., 1991; Ghys et al., 1997) and has been implicated as a cofactor in HPV-induced cervical Buspirone HCl neoplasia (reviewed in Paavonen, 2011] and possibly preterm Selleckchem PLX4032 labor (Baud et al., 2008). Co-evolution of C. trachomatis with its human host has driven the acquisition of several immune evasion strategies that likely contribute to the above and promote continued spread of disease (Brunham & Rey-Ladino, 2005). Chlamydia trachomatis is an obligate intracellular pathogen and genital serovars have a tropism for columnar epithelial cells of the female and male genital tracts. When C. trachomatis is recognized by the host immune system, innate [natural killer (NK) cells (Tseng & Rank,

1998; Hook et al., 2004, 2005)]; innate-like [NK T (NKT) cells (Yang, 2007)] and adaptive [CD4+ (Ficarra et al., 2008) and CD8+ T cells (Igietseme et al., 1994; Roan & Starnbach, 2006; Ficarra et al., 2008; Igietseme et al., 2009)] immune constituents contribute to host cellular immune defense and/or host immune pathogenesis. To avert detection by CD8+ and CD4+ cells, genital serovars of C. trachomatis decrease epithelial cell surface expression of major histocompatibility (MHC) class I and class II antigen presenting molecules through the secretion of C. trachomatis Protease-like Activity Factor (CPAF), a chlamydia-encoded protein (Zhong et al., 1999, 2000, 2001; Shaw et al., 2002). CPAF is also involved in the degradation of CD1d, the host cell ligand for NKT cells, in penile genital epithelial cells (Kawana et al., 2007, 2008). While most experiments are conducted using supraphysiologic C.

In total,

we obtained 52 find more and 30 Equ c 1143–160-specific TCLs from allergic and non-allergic subjects, respectively (Fig. 1). When the number of Equ c 1143–160-specific TCLs was analysed per person, it was found to be similar between the subject groups [3·7 ± 0·6 (mean ± SEM) and 3·3 ± 1·1 TCLs, respectively; P > 0·05, Fisher's exact test]. However, when the Equ c 1143–160-specific TCLs that were also specific to the Equ c 1 protein (protein-specific TCLs) were analysed (30 lines from allergic and 12 from non-allergic subjects) the number of TCLs showed some tendency for difference between the groups (2·1 ± 0·6 and 1·3 ± 0·9 TCLs per person, P = 0·19; Fig. 1: black columns). When one non-allergic subject out of nine (subject Q, Fig. 1; Grubb’s test for outliers P < 0·01 = significant outlier) with an exceptionally high number of protein-specific TCLs (eight; the next largest number for a non-allergic individual was two, Fig. 1) was excluded from the analysis, the difference was statistically highly significant (0·5 ± 0·3 TCLs per non-allergic person, P < 0·001). Therefore, this finding suggests that the recognition of the naturally processed epitope from Equ c 1 by CD4+ T cells may be a distinguishing factor between the allergic patients and most of the healthy subjects. The frequency of Equ c 1143–160-specific

CD4+ T cells was estimated by the number of positive wells in the split-well cultures on 96-well plates. When a total of six million PBMCs GW-572016 mouse were seeded per person (30 wells, 2 × 105 PBMCs per well), assuming that each positive well represents a monoclonal T-cell growth, the mean Alanine-glyoxylate transaminase frequency of Equ c 1143–160-specific T cells of allergic subjects was 0·63 per 106 and that of non-allergic subjects

was 0·56 per 106 PBMCs. Presuming that a person’s PBMCs contain 30% of CD4+ T cells it can be estimated that there are approximately 2·10 per 106 and 1·85 per 106 Equ c 1143–160-specific CD4+ cells in the circulating CD4+ T-cell pool of allergic and non-allergic subjects, respectively. Extending the estimation to the CD4+ cells that were Equ c 1 protein-specific as well, the frequencies of specific cells were even lower, around 1·18 per 106 CD4+ cells for an allergic and 0·74 per 106 for a non-allergic subject. Again, if the eight protein-specific lines obtained from the non-allergic subject Q were excluded, the protein-specific CD4+ T cells were detected extremely rarely in most non-allergic subjects (0·28 per 106). We have previously observed that although T-cell responses to lipocalin allergens are weak in general,[11, 15, 17] allergen-specific TCLs from allergic subjects have stronger proliferative capacity than TCLs from non-allergic subjects.

64 Amongst these cytokines, IL-6, IL-21 and IL-23 all signal through STAT3, and not surprisingly, STAT3 is essential for Th17 development. Indeed, disrupted STAT3 expression in T cells blocks Th17 differentiation,65 and confers resistance to experimental autoimmune Dinaciclib encephalomyelitis (EAE) and colitis.66,67 STAT3 controls the expression

of several key Th17 genes such as il17a, il17f, rora, il6r and il2167–69 but also promotes RORγt while repressing Foxp3 expression,65 so STAT3 is key at all stages of Th17 commitment (Fig. 4). Interestingly, the activation of STAT5 by IL-2 is required for optimal differentiation of Th1, Th2 and Foxp3+ Treg cells, but inhibits the development of Th17 cells.70 Indeed, STAT5 binds several sites on the il17 promoter and directly antagonizes STAT3 transcriptional activity,71 showing that STAT3 and STAT5 exert polar opposite effects on IL-17 expression in the context of Th17 differentiation (Fig. 4). This suggests that STAT5 is an essential regulator of CD4+ T-cell plasticity because IL-2 promotes Th1 and Th2 responses, whereas the absence of IL-2 favours the emergence of Th17 cells, as summarized in Table 1. The SOCS3 protein is a well known inhibitor of STAT3 activation in various cell types, and in particular inhibits IL-6 and IL-23 signalling in CD4+ T cells60–62 (Fig. 4). As might have been expected, SOCS3 deletion in T cells favours IL-17

secretion in vitro62 and in vivo,72 whereas enforced expression of SOCS3 Ferroptosis inhibitor review inhibits polarization towards Th17 and delays the onset

of EAE.61 Moreover, mutation of the SOCS3 binding site on gp130 results in increased IL-17 secretion60 and spontaneous arthritis.73 Finally, it has been proposed that TGF-β inhibits SOCS3 expression, and subsequently prolongs STAT3 activation, which perhaps explains how TGF-β enhances Th17 differentiation.74 Therefore, SOCS3 clearly inhibits the development of Th17 cells, but SOCS1 and SOCS2 appear to have the opposite effect. Indeed, disruption of SOCS1 expression in T cells strongly inhibits Th17 differentiation and diminishes disease in EAE models.61 This is associated with increased IFN-γ-mediated STAT1 activation, enhanced SOCS3 levels, attenuated STAT3 phosphorylation and reduced TGF-β transcriptional activity. These observations indicate that SOCS1 Endonuclease promotes Th17 differentiation possibly by modulating TGF-β signalling, but also indirectly by preventing Th1 lineage polarization and by regulating SOCS3 levels. Interestingly, SOCS2-deficient CD4+ T cells also have impaired IL-17 secretion, consistent with reduced STAT3 activation and elevated SOCS3 levels.59 Therefore the positive effect of SOCS1 and SOCS2 on Th17 differentiation might well be simply the consequence of increased SOCS3 levels, which confirms that the regulation of STAT3 activation by SOCS3 is an essential mechanism to limit Th17 development.

Mechanistically, our data show that the type I IFN response to PbA is essential for CXCL9 and CXCL10 expression that govern pathogenic T-cell recruitment to the brain, and ECM pathology (Fig. 7). Indeed, the increased

survival, reduced neurological signs, ischemia and microvascular pathology, and brain morphologic changes seen by MRI/MRA in the absence of type I IFN signaling were associated with a lower T-cell response in the brain. We documented earlier the parallel between flow cytometry analysis of brain CD8+ T-cell number and activation and the expression of T-cell response markers such as IFN-γ measured by qPCR [8]. Here, ECM protection was concurrent with decreased AZD9668 in vitro brain levels of CD3ε, CD8α, Granzyme B, IFN-γ, and IL-12Rβ2 expression, although these decreases were less prominent than in ECM resistant IFN-γR1−/− mice. The reduced Granzyme B expression in ECM-protected IFNR-deficient mice was in line with the reported essential role of CD8+ T-cell Granzyme B expression Regorafenib for ECM development [38].

Reduced brain T-cell sequestration and decrease in IFN-γ expression, essential for ECM development [11, 12], might explain the ECM protection seen in IFNAR1−/− mice. The reduced brain sequestration of activated effector CD8+ and CD4+ T lymphocytes upon PbA infection in IFNAR1-deficient mice was associated with a reduced membrane expression of CXCR3, a chemokine receptor associated with murine ECM [45]. T-cell chemoattractants, CXCR3 ligands CXCL9, CXCL10, and CXCL11 expression were strongly reduced in IFNAR1−/− mice and almost abrogated

in IFN-γR1−/− mice. Both CXCL9 and CXCL10 were shown to be essential for CD8+ T-cell trafficking to the brain and ECM development [39, 40]. They are the initial chemokines induced in the brain during ECM onset, 6 days post PbA infection, at a time when IFN-γ, CCL5, CCL3, or CCL2 are still low, thus likely induced by the innate immune response [39]. CXCL9 and CXCL10 induction was reported to be MyD88-dependent [46], attributed to TLR responses to PbA [39]. But IFNs are also strong inducers of CXCL9 and CXCL10. AT-rich Plasmodium DNA induced IFN-β via a pathway involving STING, TBK1, and IRF3/IRF7 signaling [42]. Early splenic release of IFN-α was reported 1–2 days post-PbA infection in mice [21]. Microglia respond to IFN-β 3-mercaptopyruvate sulfurtransferase by increasing chemokines and cytokines, and most prominently CXCR3 ligands CXCL9, CXCL10, and CXCL11 [47]. CXCL9 is further expressed by brain endothelial cells and astrocytes in response to IFN-γ, while CXCL10 is expressed by endothelial cells, neurons, astrocytes, and microglial cells in response to either type I IFNs or IFN-γ [39, 47, 48]. Thus, we propose that type I IFNs might be a missing link between innate and adaptive response to PbA, central for chemokines expression and pathogenic T-cell recruitment to the brain and ECM development.

5% BSA and 0.05% Tween20). Blots were washed repeatedly in washing check details buffer (15 mM NaCl, 50 mM Tris-HCl, 0.05% Tween20; pH 7.6) and incubated for 1 h at room temperature with 0.1 μg/mL peroxidase-conjugated donkey anti-mouse IgG in blocking buffer. Peroxidase activity was detected using chemiluminescence substrate (Pierce) and recorded with a chemiluminescence detector

(Vilber Lourmat). Mouse anti-MEK1/2 (phosphorylated and non-phosphorylated), mouse anti-JNK (phosphorylated and non-phosphorylated) and mouse anti-p38 (phosphorylated and non-phosphorylated) were obtained from Cell Signaling Technology, Danvers, MA, USA For TransAm analysis, primary human keratinocytes were stimulated for 2 h with recombinant cytokines. Nuclear

extracts were generated with the Nuclear Extract Kit (Active Motif) and analyzed for activated transcription factors using TransAm Kits (Active Motif) according to the manufacturer’s protocols. For dual luciferase assays, primary human keratinocytes were grown to 70% confluence and transfected with two plasmids, MLN2238 ic50 one containing the “Firefly Luciferase” under control of an AP-1-dependent promoter and a control plasmid expressing the “Renilla Luciferase” under the CMV promoter. The transfection was performed in presence of DMRIE-C (1, 2 -Dimyristyloxypropy l-3 – Dimethyl – Hydroxy – Ethyl–Ammoniumbromide plus Cholesterol) (Dual-Luciferase-Reporter Assay System, Promega). Eighteen hours after transfection, keratinocytes were stimulated for 48 h with recombinant cytokines. Concentration of CXCL-10, CXCL-11 and HBD-2 in cell-free supernatant of primary

human keratinocytes stimulated with 50 ng/mL IL-22, 50 ng/mL TNF-α or a combination of both were measured using commercially available sandwich ELISA kit according to the manufacturer’s instructions (CXCL-10, CXCL-11: R&D Systems, HBD-2: Phoenix Pharmaceuticals). C. albicans wild-type strain SC5314 was used for the infection of human oral keratinocytes (TR146, buccal carcinoma cell line) as described previously 33. C. albicans was grown on Sabouraud’s Grape seed extract dextrose agar (Difco) followed by two pre-cultures in 10 mL YPG (1% yeast extract, 2% peptone, 2% glucose) medium (Difco), first for 16 h at 25°C and then for 24 h at 37°C through orbital shaking. Human oral keratinocytes were cultured in DMEM medium supplemented with 10% FCS and 0.1% gentamicin solution (50 mg/mL) at 37°C and 5% CO2. For two-dimensional skin infection models, 30 000 human oral keratinocytes (TR146) were plated per well in 96-well plates in antibiotic and antimycotic free culture medium. Twenty-four hours after plating, cells were treated with 50 ng/mL TNF-α and IL-22 or Th22 supernatant. Each treatment was performed in triplicate. Keratinocytes were infected 30 min after treatment with a total amount of 3000 yeast cells (MOI 0.1).

In contrast, in Mycobacterium leprae-infected humans, T cells using the Vβ6-, Vβ12-, Vβ14- and Vβ19-encoded TCRs are overrepresented in lesions when compared to blood (50). Similarly, Vβ3, Vβ6 and Vβ7 are dominant in the lesions of 50% of patients with Leishmania braziliensis infection

(50), and the Vβ14 and Vβ24 gene families are overrepresented in lesions caused by Wuchereria bancrofti (21). These differences may be because of the divergent access of blood supply Selleckchem GW572016 to lesions and the liver. Indeed, in other diseases, parallels in the Vβ expression have been detected in sites of disease pathogenesis and peripheral blood. For example, there is selective expansion of TCR Vβ6 in tonsillar and peripheral blood T cells in patients with IgA nephropathy (51), and another study (52) demonstrated identical β cell-specific CD8+ T cell clonotypes in both peripheral blood and pancreatic islets of individual non-obese diabetic mice. The ability to detect CD8+

TEM cells in the blood of mice immunized with Pbγ-spz indicates that it will be highly relevant to assess in clinical trials the peripheral blood of human volunteers immunized with attenuated sporozoites. By analysing TCR Vβ expression in blood, we were able to follow the expansion of CD8+ TEM cells in Stem Cell Compound Library clinical trial individual mice. The expansion pattern observed after immunization did not change with challenge and remained the same for 8 weeks thereafter. In a similar

fashion, Walker et al. (53) monitored the expression of Vα8 on Ag-selected CD8+Vβ10+ cells in response to an immune-dominant epitope expressed on P815-CW3-transfected cells. While there was substantial variation among responder mice in Vα8 usage, the repertoires of individual animals remained relatively stable over long periods of time (<1 year). Analysis of C57BL/6 mice infected with influenza virus demonstrated the persistence of CD8+Vβ7+ PA-specific T cells 200 days after infection (54). In recent years, there has been renewed interest in the use of a whole parasite vaccine strategy and there are now intense efforts under way to prepare and formulate attenuated IKBKE sporozoites that could be cryopreserved and then inoculated by syringe (55). This interest is fuelled mainly by the ability of the whole parasite to successfully induce long-term protection. Although the single recombinant protein vaccine, RTS,S, induces protective immunity in nonexposed adults and children residing in malaria endemic areas, the protection is short-lived, and CD8+ T cell responses are not detected (56). However, little is known about the nature, source and long-term maintenance of CD8+ T cell memory induced by attenuated parasite vaccination. It is likely that the induction and maintenance CD8+ T cell immune response generated to a whole parasite is different than that seen in response to a single protein, such as in a subunit vaccine.