After 48 h, cultures were pulsed with 0.4 μCi [3H]thymidine (Amersham Biosciences, Braunschweig, Germany),

and incubated for another 24 h. After harvesting, incorporated DNA was measured in a β-counter (Perkin Elmer, Rodgau, Germany). Cytotoxicity of freshly sorted splenic CXCR3− and CXCR3+ NK cells EX 527 (5×105/mL) against YAC-1 target cells was assessed by standard 4 h chromium release assay. Target cells were labeled with 3 MBq Na51CrO4 (Hartmann Analytic, Braunschweig, Germany), incubated for 1 h at 37°C, washed two times and used for the assay within 1 h. Cells were plated in V-bottom 96-well plates. Background values were determined by incubating target cells without effector cells. Maximal values were obtained by lysing target cells with 1% Triton X-100 (Sigma-Aldrich).

After 4 h, cells were pelleted and 100 μL supernatant of each well was used for measurement of 51Cr release in a γ counter (MicroBeta/PerkinElmer, Waltham, MA, USA) in triplicates with E:T ratios of 10:1, 5:1, 2.5:1 and 1.25:1. Specific lysis was calculated by: [(experimental release–spontaneous release)/(maximum release–spontaneous release)] ×100. Lysosomal granule exocytosis was determined by CD107a expression. For this experiment, lymphocytes (E:T ratio 10:1) or sorted CXCR3− and CXCR3+ NK cells (E:T ratio 2:1) were incubated at 37°C in 5% CO2 together click here with YAC-1 cells for 4 h. Anti-CD107a mAb was added directly Interleukin-3 receptor to the cell suspensions at a final concentration of 0.01 mg/mL. After 1 h of incubation, Monensin (BD Biosciences) was added as a golgi block at a final concentration of 5 μg/mL and incubation was continued for additional 3 h. In case of subsequent intracellular cytokine staining, brefeldin A (Sigma-Aldrich) was added at a final concentration of 2 μg/mL for the last 3 h of incubation time. Samples were finally surface-stained and analyzed via multicolor flow cytometry. In order to determine the IFN-γ production, sorted

CXCR3− and CXCR3+ NK cells were cultured in 96-well round-bottom culture plates (Greiner, Frickenhausen, Germany) in the presence of rIL-2 (100 U/mL), rIL-12 (10 ng/mL) and rIL-18 (5 ng/mL) for 15–17 h. Optimal cytokine concentrations were determined by earlier dose titrations. Brefeldin A (Sigma-Aldrich) was added at a final concentration of 2 μg/mL for the last 2 h of incubation time. Analysis of intracellular IFN-γ was preceded by surface staining at 4°C. After 30 min, cells were washed twice and resuspended in PBS containing 3% FCS. After fixation with 4% paraformaldehyde (Merck) for 10 min, cells were perforated with 0.1% saponin buffer (PBS supplemented with 0.1% saponin (Riedel-de Haën, Seelze, Germany) and 0.01 M HEPES (Roth, Karlsruhe, Germany)) and anti-IFN-γ mAb was added. After 30 min of incubation and three washes, cells were analyzed as described above.

“
“Pathological heterogeneity of Aβ deposition in senile plaques (SP) and cerebral amyloid angiopathy (CAA) in Alzheimer’s disease (AD) has been long noted. The aim of this study was to classify cases of AD according to their pattern of Aβ deposition, and to seek factors which might Daporinad cost predict, or predispose towards, this heterogeneity. The form, distribution

and severity of Aβ deposition (as SP and/or CAA) was assessed semiquantitatively in immunostained sections of frontal, temporal and occipital cortex from 134 pathologically confirmed cases of AD. Four patterns of Aβ deposition were defined. Type 1 describes cases predominantly with SP, with or without CAA within leptomeningeal vessels alone. Type 2 describes cases where, along with many SP, CAA is present in both leptomeningeal and deeper penetrating arteries. Type 3 describes cases where capillary CAA is SRT1720 present along with SP and arterial CAA. Type 4 describes a

predominantly vascular phenotype, where Aβ deposition is much more prevalent in and around blood vessels, than as SP. As would be anticipated from the group definitions, there were significant differences in the distribution and degree of CAA across the phenotype groups, although Aβ deposition as SP did not vary. There were no significant differences between phenotype groups with regard to age of onset, age at death, disease duration and brain weight, or disease presentation. Women were over-represented in the type 1 phenotype and men in type 2. Genetically, type 3 (capillary subtype) cases were strongly associated with possession of the APOE ε4 allele. This study offers an alternative method of pathologically classifying cases of AD. Further studies may derive additional genetic, environmental

or clinical factors which associate with, or may be responsible for, these varying pathological presentations of AD. Classically, Alzheimer’s disease (AD) can be defined as a progressive neurodegenerative disorder Vitamin B12 which presents as a disturbance of memory and cognition and is characterized histopathologically by the presence of numerous senile plaques (SP) and neurofibrillary tangles (NFT) within neocortical and certain subcortical regions, accompanied in most cases by a deposition of amyloid β protein (Aβ) in the walls of leptomeningeal and intracortical (parenchymal) arteries, arterioles, capillaries and veins, and known as cerebral amyloid angiopathy (CAA). The same Aβ protein deposited in blood vessel walls is also present in the brain parenchyma within the SP, although this is mostly composed of the longer peptide, Aβx-42, whereas CAA Aβ protein is mostly composed of the shorter peptide, Aβx-40 [1]. Nonetheless, the origins of CAA are still poorly understood. Various mechanisms have been proposed, which include a derivation from blood and or cerebrospinal fluid [2], local production by smooth muscle cells and/or pericytes [3] or through secretion from neurones and perivascular drainage [4].

01 EU/μg pDNA by the Triton X-114 extraction. For polyI:C and imiquimod, polymyxin B, which binds to LPS, was added to cells at a final concentration of 5 μg/mL. ODNs, nucleotides and nucleosides were used as obtained without further purification or addition of polymyxin B. TLR9 KO mice were purchased from the Oriental Yeast Company (Tokyo, Japan). C57BL/6 WT mice SAHA HDAC and Institute for Cancer Research (ICR)

mice were purchased from Japan SLC (Shizuoka, Japan) and maintained on a standard food and water diet under conventional housing conditions. All animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for animal experiments were approved by the Institutional Animal Experimentation Committee of the Graduate School of Pharmaceutical Sciences, Kyoto University. In the experiment of subcutaneous injection

of ODN into mouse footpad, 3 nmol of ODN1668 in 20 μL PBS were subcutaneously injected into the footpad of the right hind leg of male ICR mice with or without 10 nmol DNase I-treated or untreated ODN1720. Before and 24 h after injection of ODN, the thickness of footpad was measured using a micrometer caliper with a minimum scale of 10 μm (Mitutoyo, Kawasaki, Japan). Separately, the footpad was removed at 24 h after injection and submerged into

4% paraformaldehyde in PBS for 24 h at 4°C. The fixed footpad tissues BTK inhibitor price were decalcified and embedded in paraffin and sectioned into 3-μm slices. The paraffin sections were stained with hematoxylin and eosin to evaluate the infiltration of blood cells. The number of mononuclear cells and neutrophils infiltrating into the injection site in 25 mm2 was counted. Splenic macrophages were collected as previously described 16 and cultured on 96-well culture plates at a density of 3×105 cells/well in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/mL), streptomycin (100 μg/mL), L-glutamine (292 μg/mL) and 2-mercaptoethanol (10−5 M). They were used for the cytokine release experiment soon after isolation. The murine macrophage-like cell line, RAW264.7 cells, was cultured Branched chain aminotransferase on 96-well culture plates at a density of 5×104 cells/well in RPMI-1640 supplemented with 10% FBS, penicillin G (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (292 μg/mL). They were cultured for 24 h prior to use. The human leukemic plasmacytoid DC line, PMDC05 cells 17, was cultured on 96-well culture plates at a density of 4×105 cells/well in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, penicillin G (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (292 μg/mL). They were plated before the cytokine release experiment. RAW264.

In another set of experiments, CFSE-labelled allogeneic naive and memory CD3+ T cells were added to PDC, and T cell proliferation was determined by flow cytometric measurement of CFSE dilution. The supernatants of the stimulated PDC were analysed

for IFN-α, interleukin-6 MK-1775 mouse and TNF-α concentrations by standard ELISA, according to the manufacturer’s instructions. The IFN-α ELISA detects the main subtypes IFN-α2a, IFN-α2b and IFN-α2c. The supernatants of T cells co-cultured with allogeneic PDC were analysed for IFN-γ, IL-10, IL-4, IL-17 and CXCL-10 also by standard ELISA, according to the manufacturer’s instructions. In other cases the cytokine production of LOX-PDC stimulated T cells was assessed by restimulating the see more T cells with PMA (40 ng/ml) and ionomycin (1 ug/ml) for 6 h. During the last 5 h of restimulation brefeldin A (5 ug/ml) was added to inhibit protein transport processes. Intracellular IFN-γ, IL-17 and IL-10 expression was determined by using Fix&perm cell permeabilization kit, according to the manufacturer’s instructions. To assess the suppressive capacity of CD8+CD38+LAG3+ regulatory T cells generated during co-cultures with allogeneic PDC, CD8+CD38+LAG3+ T cells were purified

from cultured cells by flow cytometric sorting using a FacsAria Cell Sorter (Becton Liothyronine Sodium Dickinson), and added in graded doses to cultures of CD3+ T cells (1 × 105/200 μl) that were stimulated with allogeneic irradiated (3000 rad) donor-specific

MoDC (1·5 × 104) in round-bottomed wells. In these experiments Mo-DC and PDC were derived from the same donor. After 5 days, proliferation was assessed by determination of [3H]-thymidine incorporation for 18 h. All experiments were performed n times, as indicated in the figure legends, with cells from different individuals, and mean values ± standard error of the mean (s.e.m.) were calculated. Significance of differences between paired observations was tested in the paired t-test using Microsoft Excel 2003 software. A P-value of less than 0·05 was considered significant. The effects of rapamycin were studied using purified human PDC stimulated with TLR-9 ligand CpG-A-ODN 2336 or TLR-7 ligand loxoribine, in the presence of IL-3 as essential survival factor. To determine whether a clinically relevant concentration of 20 ng/ml rapamycin, which is similar to the blood peak level reached during rapamycin treatment (Rapamune summary of product characteristics; Wyeth-Ayerst Pharmaceuticals Inc., Philadelphia, PA, USA), inhibits mTOR-signalling in PDC, we measured phosphorylation of the 40S ribosomal protein S6, which is a downstream phosphorylation target of mTOR [22].

One of the advanced lipid-based delivery systems is the solid–lipid nanoparticles (SLNs), which can be one of the alternative delivery system to electroporation. SLNs are basically composed of high-melting-point lipids that act as a solid core, covered by surfactants. The use of materials that are generally recognized as safe (i.e. triglycerides, partial glycerides, fatty acids, steroids) [35] leads to an advantageous toxicity profile [36].The SLN production by hot high-pressure homogenization is easy, and no organic solvents are required [37]. Scaling-up is standardized up to 50-kg batches [38], and steam sterilization is possible [39]. The excellent activity and superiority of DOTAP–cetyl palmitate–SLN were reproducible.

The positively charged SLN would bind to polyanionic DNA via electrostatic

force leading selleck products to SLN–DNA complex that will protect DNA from interaction with small molecules in the environment and will be taken into cell by an endocytosis process [40]. An additional advantage of delivering vaccine candidates by nanoparticles is the potential to enhance their stability during transport, and this is critical in areas that lack reliable cold storage chain (2–8°C) [41]. Our previous results revealed that stable formulation of cSLN was able to protect pDNA in DNase I challenge assay and deliver it to the right immune cells for the proper immune response induction [22]. In this study, we generated a DNA vaccine encoding A2–CPA–CPB−CTE as a trifusion gene and compared the impact of DNA vaccine delivery to immune cells (e.g. physical/electroporation vs. chemical/cSLN formulation) on the development of protective immune response against an infectious VX-809 concentration L. infantum challenge. The pcDNA–A2–CPA–CPB−CTE was formulated into cationic

lipid particles with nanometre triclocarban range (~240–250 nm). In our experimental system, the administration of pcDNA–A2–CPA–CPB−CTE in BALB/c mice elicited the induction of specific Th1 and Th2 clones, indicating a mixed immune response and the production of IFN-γ and IL-10, although IFN-γ was much higher than IL-10, especially in G2 using the cSLN formulation. However, a higher amount of IFN-γ was obtained in G1 immunized via electroporation in response to both rA2–rCPA–rCPB and F/T L. infantum antigens at 4 and 8 weeks after challenge. Although IFN-γ secretion at 8 weeks after challenge in G1 was higher than in G2, there were no significant differences in IFN-γ: IL-10 ratio between these two groups. Also, at 8 weeks after challenge, the IFN-γ: IL-10 ratio in splenocytes from mice immunized with pcDNA–A2–CPA–CPB−CTE (G1 and G2) stimulated with rA2–rCPA–rCPB was significantly higher than G3 (~28·25- and 26·5-fold; P < 0·01) and G4 (~8·69- and 8·154-fold; P < 0·01). The same result was obtained with splenocytes stimulated by F/T L. infantum antigen. So, we can conclude that these two delivery strategies elicit the same immune responses with efficient protection.

These results suggest that the immune system exploits the different CTLA-4 isoforms for either intrinsic or extrinsic regulation of T-cell activity. CTLA-4

is an important regulator of T-cell responses [1-4]. Its critical role is highlighted by CTLA-4 knockout mice, which develop a fatal lymphoproliferative disorder soon after birth, arising from a profound failure of T-cell homeostasis [5, 6]. Despite these potent effects, the activities of CTLA-4 are only partially understood. CTLA-4 shares sequence homology and B7 ligands (CD80/CD86) with the costimulatory molecule, CD28, but differs by delivering inhibitory, rather than activating, signals to the T cells on which it is expressed as a receptor [7, 8]. Upregulation of CTLA-4 on activated T cells provides a mechanism for negative feedback www.selleckchem.com/products/AG-014699.html to control their responses. However, not all its regulatory effects are explained by inhibitory costimulation, since CTLA-4 can also suppress activated effector T-cell populations without the need for them to express it [9, 10]. This latter, cell-extrinsic mechanism has

been largely attributed to CD4+ regulatory T (Treg)-cell subsets, which constitutively express high levels of CTLA-4, Liproxstatin-1 molecular weight and require it for their regulatory function [11-16]. How Treg cells might use CTLA-4 to regulate effector T-cell responses remains controversial. It has been suggested that CTLA-4 on Treg cells binds B7 and thus blocks CD28-mediated effector T-cell costimulation, or that it induces inhibitory mechanisms CYTH4 in the APC such as the IDO tryptophan catabolic enzyme cascade [17], or the FoxO3 transcription factor that controls inflammatory cytokine production [18]. Recently, a direct role for CTLA-4 in mediating cell-extrinsic activity has been supported by the observation that CTLA-4 is a component of a transendocytosis process to remove CD80/CD86 from APCs, an inhibitory mechanism that suppresses costimulation of activated effector T-cell populations

[19]. However, it remains unclear whether any of these mechanisms fully explains the regulatory properties of CTLA-4. A paradox arising from the competing models of CTLA-4 activity is that the same T-cell surface molecule can apparently mediate not only cell-intrinsic negative costimulation, but also extrinsic regulation of other cells. This might be resolved if CTLA-4 had functions other than as a receptor. It has been widely assumed that all the activities of CTLA-4 are exclusive to the full-length membrane-bound receptor isoform (mCTLA-4), encoded in humans by exons 1–4 on chromosome 2, but other alternatively spliced mRNA transcripts have been detected, including one that generates a secretable soluble form, sCTLA-4 [20, 21].

These polyclonal autoantibodies to foreign antigens might cross-react with self-antigens and, in the case of a normally developed immune systems, this type of immune reaction is self-limiting [21]. Meanwhile, these antibodies may develop as a result of ‘molecular mimicry’ wherein an epitope on the surface of foreign infectious antigen stimulation. Those

produced antibodies are also considered to be polyclonal and are present relatively long period (month or year) [26]. The aetiology of KS remains unknown, although infectious agents are suspected and being discussed even now. Hence, it is conceivable that the possibility of infectious antigens induced these autoimmune GDC0068 phenomena. Various drugs are also thought to be associated with neutropenia [27]. These mechanisms include immune-mediated destruction of granulocytes or granulocytic precursors, dose-dependent inhibition of granulopoiesis and direct toxic effect on myeloid precursors or the marrow microenvironment [28, 29]. In this case, the DLST of PAPM/BP was positive, suggesting that it may be one of the causes of immune-mediated

this website neutropenia. The antibiotics might function as a hapten and recognize antigens on the neutrophil membrane, resulting in the production of neutrophil-specific autoantibody. However, when the drug acts as a hapten, the ANC should also improve within 1–2 weeks after cessation of drug administration [26]. In addition, potential role of IVIG-induced neutropenia also should be considered. IVIG-induced neutrophil apoptosis in KS had been suggested by the rapid occurrence after IVIG administration and was experimentally demonstrated in circulating neutrophils in patients after IVIG administration [7, 30]. The more commonly suggested mechanisms Oxymatrine are the presence of anti-neutrophil antibodies in preparing immunoglobulin, and we examined and confirmed the absence of antibodies to neutrophils in the same lots of immunoglobulin used for IVIG treatment. These mechanisms, therefore, did not

explain the disease course of the present case. Thus, autoantibodies to immature myeloid cells and neutrophils might be developed as part of a polyclonal activation of B cells and cause transient neutropenia. In conclusion, an autoantibody to a novel antigen on immature myeloid cells or neutrophils was produced and was revealed as a possible cause of severe neutropenia in a patient with KS. Our findings provide further insight into the potential mechanisms of antibody-induced neutropenia associated with KS. The authors are especially thankful to Dr Takashi Satoh, Associate Professor, Department of Pediatrics, Hiroshima University School of Medicine, Hiroshima, Japan, for technical support. “
“Macrophages orchestrate the immune response via the polarization of CD4+ T helper cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described.

In this study, we investigated the role of SQSTM1 in host responses to Legionella pneumophila, an intra-cellular pathogen that infects macrophages, in both an SQSTM1-deficient

(SQSTM1−/−) mouse model and macrophages from these mice. Compared with wild-type (WT) macrophages, the production and secretion of the proinflammatory cytokine IL-1β was Autophagy inhibitors library significantly enhanced in SQSTM1−/− macrophages after infection with L. pneumophila. Inflammasome activity, indicated by the level of IL-18 and caspase-1 activity, was also elevated in SQSTM1−/− macrophages after infection with L. pneumophila. SQSTM1 may interact with nucleotide-binding oligomerization domain-like receptor family, caspase Selleck Palbociclib recruitment domain-containing 4 and nucleotide-binding oligomerization domain like receptor family, pyrin domain containing 3 proteins to inhibit their self-dimerization. Acute pulmonary inflammation induced by L. pneumophila and silica was enhanced in SQSTM1−/− mice with an increase in IL-1β levels in the bronchoalveolar lavage fluids. These findings suggest

that SQSTM1 is a negative regulator of acute pulmonary inflammation, possibly by regulating inflammasome activity and subsequent proinflammatory cytokine production. “
“Common variable immunodeficiency disorders (CVID) are a group of heterogeneous L-NAME HCl conditions that

have in common primary failure of B cell function, although numerous T cell abnormalities have been described, including reduced proliferative response and reduced regulatory T cells. This study compared the T cell phenotype of CVID patients subdivided into clinical phenotypes as well as patients with partial antibody deficiencies [immunoglobulin (Ig)G subclass deficiency and selective IgA deficiency], X-linked agammaglobulinaemia (XLA) and healthy and disease controls. Absolute numbers of T cell subpopulations were measured by four-colour flow cytometry: naive T cells, central and effector memory and terminally differentiated (TEM) T cells, using CD45RA and CCR7 expression. Early, intermediate and late differentiation status of T cells was measured by CD27/CD28 expression. Putative follicular T cells, recent thymic emigrants and regulatory T cells were also assessed. Significant reduction in naive CD4 T cells, with reduced total CD4 and recent thymic emigrant numbers, was observed in CVID patients, most pronounced in those with autoimmune cytopenias or polyclonal lymphoproliferation. These findings suggest a lack of replenishment by new thymically derived cells. CD8 naive T cells were reduced in CVID patients, most significantly in the autoimmune cytopenia subgroup.

The causative association of allergen-specific Immunglobulin E (IgE), the high-affinity IgE receptor (FcεRI), and mast cells for immediate type allergy and anaphylaxis has been studied for decades [3-5]. However, since the discovery of anaphylaxis in IgE-deficient mice [6] and more recently studies on basophil biology, a number of publications

have focused on the contribution of alternative pathways to anaphylaxis [7-9]. It has become evident that the isotype, quantity, and quality of the sensitizing antibodies are important parameters for anaphylaxis [9]. In summary, at least two mutually nonexclusive pathways exist that employ allergen-mediated cross-linking of either receptor bound IgE and/or receptor bound IgG and PF-562271 order lead to activation of mast cells and/or basophils leading to release of inflammatory substances, e.g. histamine or platelet activating factor [7, 10]. Nevertheless, experiments to examine the role of the active polyclonal antibody response in anaphylaxis are hampered by the low expression of IgE and a low frequency of IgE expressing B cells in WT mice [11, 12]. In order to circumvent this problem, we generated an IgE knock-in mouse strain to

study the role of IgE regulation in vivo. We created a high-IgE expressing mouse model this website for allergy research based on work by Rajewsky et al. [13], who showed that the replacement of the murine IgG1 heavy chain locus by human IgG1 leads to humanized antibody production in vivo. We adapted this approach and replaced the exons encoding the soluble part of the constant region of murine IgG1 with the murine IgE counterpart. The advantage of this approach over conventional selleck screening library IgE transgenic mice is twofold. First, it is possible to study the regulatory influences of the genetic region in a defined way, excluding positional effects of the classic transgenic approach. Second, it allows the natural usage of the endogenous variable, diversity, and joining segment of the antibody gene region and, therefore, the generation of polyclonal

IgE antibody responses against any given antigen and not only the monoclonal IgE production against a single model antigen [13, 14]. Indeed, both IgE and IgG1 are dependent on Th-2 type T-cell and cytokine signals, e.g. CD40–CD40L interaction and IL-4. However, a number of studies suggest that the developmental switch to IgE has unique features as it can occur outside secondary lymphoid structures [15] or initiate in germinal centers (GCs) and rapidly progresses to IgE+ plasma cells located outside the GC [16]. Recently, membrane IgE GFP-reporter mouse strains suggested a scenario where IgE+ B cells develop with similar kinetics compared with those of IgG1+ B cells, but without an IgG1+ intermediate stage.

Figure 5a shows that opsonized C. neoformans drastically inhibited the production of H2O2 by GM-CSF-stimulated eosinophils (P < 0·03; eosinophils plus opsonized C. neoformans versus eosinophils in medium alone). This phenomenon was exclusively dependent on FcγRII, because, in the presence of a blocking antibody, opsonized C. neoformans were unable to suppress H2O2 production. To a lesser extent, opsonized C. neoformans also inhibited NO production by GM-CSF-stimulated eosinophils (Fig. 5b; P < 0·05; eosinophils plus opsonized C. neoformans versus eosinophils in medium alone) through FcγRII interactions.

Similarly, in the absence of GM-CSF, opsonized C. neoformans also inhibited the basal production of H2O2 or NO by eosinophils (data not shown). Experiments were see more then performed in order to evaluate the ability of eosinophils to present fungal antigens. Taking into account that the expression of MHC class II was significantly higher on eosinophils cultured with C. neoformans in the presence of GM-CSF than in its absence (Fig. 2b), eosinophils were pulsed with opsonized C. neoformans in the presence of GM-CSF for 24 hr before being fixed with paraformaldehyde.

Then, they were cultured with MSCs or purified T lymphocytes Dasatinib purchase (CD4+ and CD8+) obtained from untreated rats (naive lymphocytes) or from rats infected with 107 yeasts 7 days previously (C. neoformans-primed lymphocytes). Seven days after culture, the lymphoproliferation was measured by thymidine incorporation. The results showed that C. neoformans-primed lymphocytes (MSCs or purified CD4+ plus CD8+ T cells), but not naive lymphocytes, proliferated significantly in the presence of C. neoformans-pulsed eosinophils, compared with MSCs or T cells cultured in medium alone, or with Casein kinase 1 fixed C. neoformans yeasts or unpulsed eosinophils (Fig. 6a,b). Moreover, in the absence of eosinophils, neither MSCs nor T cells proliferated, even when incubated with C. neoformans alone, discounting any possible effect of APC contamination

within the eosinophil preparation or among the purified T cells. In addition, Fig. 6b shows that C. neoformans-pulsed peritoneal Mφ did not stimulate T-cell proliferation. In this regard, it has been previously demonstrated that monocytes pretreated with encapsulated cryptococci have little or no ability to stimulate T-cell proliferation.30 To evaluate if C. neoformans-primed CD4+ or CD8+ T cells were responsible for the lymphoproliferation observed in Fig. 6b, the CD4+ and CD8+ T-cell proliferations were measured separately in the presence of C. neoformans-pulsed eosinophils. Figure 6c shows that both CD4+ and CD8+ T cells proliferated in the presence of C. neoformans-pulsed eosinophils compared with CD4+ and CD8+ T cells cultured in medium alone. However, CD4+ T cells were the main population sensitive to the stimulation of C.