Blocking IDO reduced the immunosuppressive effect of cytokine-treated ASC to levels found in control ASC, but did not abolish the immunosuppressive capacity completely. This shows that IDO is important for the induced immunosuppressive capacity

of ASC treated with cytokines, but less so for the basic immunosuppressive capacity of ASC. As a consequence, other factors must play a role in the immunosuppressive function of ASC, of which several have been reported in the literature, such as HGF, HLA-G and nitric oxide (NO) [5,19,20]. We found high expression of Apoptosis inhibitor HLA-G, TGF-β1 and COX-2, which have been reported to be involved in the immunosuppressive effect of ASC [5,18,19]. In MLR-cultured ASC we found strong up-regulation of COX-2, which could indicate that prostaglandin E2 is responsible for some of the enhanced immunosuppressive capacity selleck compound of these cells. Culture under inflammatory conditions not only changed the expression of anti-inflammatory factors by ASC, but also

increased the expression of HLA class I. The expression of HLA class II was increased predominantly by proinflammatory cytokines, whereas culture of ASC with MLR had less effect. Up-regulation of HLA makes ASC potentially more immunogenic. This could have consequences for clinical application of ASC of allogeneic origin. Inflammatory conditions also increased the expression of proinflammatory factors and chemokines. The type of proinflammatory factors and chemokines produced by ASC depended upon the inflammatory condition. Whereas ASC cultured with MLR showed up-regulation of chemokines for neutrophils,

monocytes and macrophages, in particular, culture of ASC with proinflammatory cytokines resulted in the up-regulation of chemokines for T lymphocytes. The relevance of the chemoattraction of the different immune cells by ASC is not clear, but could lead to binding of activated immune cells to ASC [23]. Close contact of activate selleck inhibitor immune cells and ASC may increase the efficacy of the immunomodulatory function of ASC [20,35]. These results indicate that ASC can exhibit diverse immunomodulatory effects. The local inflammatory milieu is of crucial importance for the balance between the pro- and anti-inflammatory effects of ASC. Furthermore, it determines the mechanisms that ASC employ to execute their immunomodulatory function. Apart from their immunomodulatory properties, ASC have potential to support tissue regeneration. While this is mediated partially via their differentiation in other cell types [2], there is now increasing evidence that the regenerative effect of ASC is also the result of the production of trophic factors, which stimulate resident progenitor cells [4]. Under inflammatory conditions, ASC maintained the capacity to differentiate in adipogenic and osteogenic lineages.

The first four stages are approximately 5 days each in duration whereas Stage V lasts for 69 days.

Stage VI duration is indeterminate and can last for many years until immunological control fails (Fig. 1). The temporal appearance of functional responses in relation to viral dynamics provides important clues about the mechanisms of immunological control. In this regard, it is also possible to discriminate between recent and chronic infections in Fiebig Stage VI using a sensitive/less-sensitive algorithm that employs a standard HIV ELISA (sensitive) and a ‘detuned HIV ELISA’ (less sensitive) that detects increasing antibody titres that emerge early after infection.[30] Hence, the detuned ELISA can discriminate individuals in the early part of Fiebig Stage VI who were recently infected versus those who are chronically infected. More recent studies show that increased levels of acute-phase proteins, such as selleck serum amyloid precursor A, are elevated as early as the eclipse phase but wane around day 20 post-T0.[31] A cytokine storm follows beginning 6 days after T0 in Fiebig

Stage II, waning around day 20 post-T0.[32] Immune complexes of HIV with either IgM or IgG appear at day 8 post-T0 and become undetectable around day 20 post-T0. Free IgG non-neutralizing antibodies to gp41 appear 13 days after T0, early in Fiebig Stage IV.[29] Free IgG non-neutralizing antibodies appear 28 days after T0, midway in Fiebig Stage IV.[29] Autologous neutralizing antibodies appear approximately at day Protease Inhibitor Library 82 post-T0, late in Fiebig Stage V, followed by neutralization insensitive viral variants around 10 days later, apparently selected by neutralization pressure (reviewed in ref. [21]).

These antibodies are narrowly specific for autologous virus with neutralization breadth increasing slowly over time thereafter.[33] Hence, there is a 55-day window between the appearance of the first free IgG antibodies that bind to gp41 or gp120 and the emergence of narrowly specific neutralizing antibodies.[21] By contrast, the first CD8+ cytotoxic T-lymphocyte (CTL) responses appear at the beginning of Fiebig Stage III, around day 20, followed by the emergence of CTL escape viruses 10 days later at Amisulpride the beginning of Fiebig Stage V, suggesting that these responses exert immunological pressure on the virus (reviewed in ref. [21]). Because there is a 60-day lag between the CD8+ CTL response and neutralizing antibody response, it has been widely accepted that post-infection control of viraemia is largely due to CTLs. This conclusion is also supported by CD8 depletion studies in NHPs.[34, 35] By contrast, in acutely HIV-infected individuals, there is evidence that antibody-mediated cellular cytotoxicity (ADCC) responses appear around day 36 post-T0, at the beginning of Fiebig Stage V, and that these responses correlate inversely with viral load.

W.), the Collaborative Research Project (2012–2209)

of the Brain Research Institute, Niigata University (F.M.), Grants-in Aid from the Research Committee for Ataxic Disease, the Ministry of Health, Labour and Welfare, Japan (K.W.), and the Intramural Research Grant (24-5) for Neurological and Psychiatric Disorders of NCNP (K.W.). The authors wish to express their gratitude to M. Nakata for her technical assistance. “
“The role of nonclassical human leukocyte antigens G and E (HLA-G and HLA-E) was originally thought to be restricted to the protection of the fetus from a maternal allorecognition. Now it is known that HLA-G and HLA-E exert multiple immunoregulatory functions. A prognostic significance of the expression of HLA-G and HLA-E by

neoplastic BMN 673 cells in glioblastoma is not well characterized. In this study, we evaluated the expression of HLA-G and HLA-E by neoplastic cells in 39 cases of glioblastoma. We found the production of HLA-G and HLA in a majority of cases. There was an unexpected positive correlation between the expression of HLA-E and length of survival. We speculate that the expression of this molecule by neoplastic cells may represent a coincidental selective pro-host advantage related to better response to subsequent therapeutic modalities. Mechanisms of glioblastoma cell pathophysiology and mechanisms of responses to therapeutic interventions in respect to the expression MG-132 cell line of these molecules deserves further study. “
“Focal cerebral ischemia induces cellular responses that may result in secondary tissue damage. We recently demonstrated multi-facetted spatial and temporal

patterns of neuroinflammation by multimodal imaging. In the present study, we especially focus on the separation of vital and necrotic tissue, which enabled us to define a demarcation zone. Focal cerebral ischemia was induced via macrosphere embolization of the middle cerebral artery in Wistar rats. Subsequent cellular processes were investigated immunohistochemically from 3 to 56 days after onset of ischemia. We detected several infarct subareas: a necrotic infarct core and its margin adjacent to a nerve/glial antigen 2 (NG2)+ zone delineating it from a vital peri-infarct zone. Initially transition from science necrotic to vital tissue was gradual; later on necrosis was precisely separated from vital tissue by a narrow NG2+ belt that was devoid of astrocytes, oligodendrocytes or neurons. Within this demarcation zone NG2+ cells associate with ionized calcium binding adaptor molecule 1 (Iba1) but not with GFAP, neuronal nuclear antigen (NeuN) or 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase). During further infarct maturation NG2 seemed to be positioned in the extracellular matrix (ECM) of the demarcation zone, whereas Iba1+ cells invaded the necrotic infarct core and GFAP+ cells built a gliotic containing belt between the lesion and NeuN+ unaffected tissue.

IgG derived

from a SS patient positive for antibodies to the see more third extracellular loop had no effect on (Ca2+)I, as well as IgG derived from an anti-M3R antibody-negative SS patient (Figs 3e and 4). Recently, anti-M3R antibodies have been the focus of interest in rheumatology because of their potential pathogenic role, use as diagnostic markers and being therapeutic targets in patients with SS [1]. Several methods have been used to detect anti-M3R antibodies in SS patients [1]. In functional assays using smooth muscles, IgG fractions from patients with SS (SS-IgG) inhibited carbachol-evoked or nerve-evoked bladder or colon contractions [8,9]. In salivary gland cells, SS-IgG inhibited the rise in (Ca2+)i induced by carbachol, and also inhibited pilocarpine-induced AQP5 trafficking to the apical membrane from the cytoplasm [2]. The inhibitory actions of SS-IgG on

the rise in (Ca2+)i was acutely reversible [10]. Anti-M3R antibodies from SS patients can be detected by immunofluorescent analysis using rat lacrimal glands [11], and by flow cytometry using the M3R-transfected Chinese hamster ovary (CHO) cell line [12]. Moreover, anti-M3R antibodies in sera of SS patients were detected by ELISA using synthetic peptides or recombinant proteins of the second extracellular loop of M3R [13]. We have reported previously the presence of anti-M3R antibodies in a group of patients with SS, which recognized the second extracellular loop by ELISA using synthetic buy Torin 1 peptides [4,5]. In the present study, we established a standard method to detect anti-M3R antibodies that can be used for screening large patient populations. Functional assays and flow cytometry are too laborious for routine use. Although ELISA is easy, the results from some ELISA systems used for screening anti-M3R antibodies differ Mannose-binding protein-associated serine protease widely with regard to the prevalence of anti-M3R antibodies (from 11 to 90%) [4,14]. Furthermore, Cavill et al.[15]

reported failure to detect anti-M3R antibodies by ELISA using synthetic peptides. In the present study, we reported higher frequencies and titres of anti-M3R antibodies against all extracellular domains in SS patients than the control. The prevalence of anti-M3R antibodies against the second extracellular loop in SS (55%) determined in the present study was much higher than that reported in our previous study (11%) [4]. The reason for this difference is probably related to the change in the methodology, such as increased sensitivity resulting from purity of the synthetic peptides, modification of the washing procedure or other factors introduced in the modified ELISA system. In the present study, we also determined the precise B cell epitopes of M3R molecules.

Urinary cytology, nucleic acid testing of urine and/or plasma, and viral-specific staining of biopsy specimens are necessary for diagnosis. Infected tubular cells show intranuclear inclusions, lysis or necrosis, and shedding into the tubular lumen. But such light microscopy findings are quite focally observed in many cases, and varying degrees of tubulointerstitial inflammation mimicking T-cell-mediated

acute rejection make accurate diagnosis difficult. There is a histological classification of BKVN originally reported by the University of Maryland in 2001, and modified by American Society of Transplantation Infectious Disease Community of Practice, which focuses on interstitial inflammation and fibrosis. Another see more classification was proposed by the Banff Working Group in 2009 (Banff Working Proposal), which focuses

on acute tubular injury instead of interstitial inflammation. The usefulness of the Banff Working Proposal is now under consideration with a multicenter study being conducted, but it has not yet reached a clear conclusion. In this review, the current screening strategies for the replication of BK virus, difficulties with diagnosis, histopathological classifications, treatments, and prognostic factors of BKVN are discussed. Polyomavirus BK (BKV) is an important pathogen in organ transplant patients. BKV was first isolated from mTOR inhibitor urine and ureteral epithelial cells of a kidney transplant patient,[1] and is known

to cause ureteral stenosis and hemorrhagic cystitis in kidney and hematopoietic stem cell transplant patients. The first case of tissue destructive nephropathy, called polyomavirus BK nephropathy (BKVN), in a kidney allograft was reported in 1995,[2] and numerous studies on various aspects of the causative virus and the disease have been published. Vasopressin Receptor BKV is ubiquitously present in the general population, and 90% or more of tested individuals may be seropositive.[3, 4] It is demonstrated that BKV is transmitted to the patient through the donor kidney with a latent infection,[5] and is reactivated with immunosuppressive treatment. Urinary shedding of the virus, called viruria, is the first step of viral reactivation, followed by viraemia, and nephropathy after the 6–12-week window period.[6] Progression of BKVN is associated with interstitial fibrosis, and subsequent acute rejection followed by the reduction of immunosuppression also induces allograft injury. Since graft survival in patients with BKVN is much poorer than those without the disease,[7] current clinical practice focuses on the early detection of viral replication and pre-emptive reduction of immunosuppression.[8-10] The management of BKV infection appeared in Kidney Disease Improving Global Outcome (KDIGO) guidelines in 2009,[8] and the American Society of Transplantation (AST) Infectious Disease Community of Practice also published guidelines.

After 2 h of incubation at room temperature, bound biotinylated peptide-MHC complexes were detected colorimetrically, as indicated

previously 31. TEPITOPE is a T-cell epitope prediction software that enables the identification of ligands binding in a promiscuous or allele-specific mode to HLA-DR molecules 32. We set the TEPITOPE prediction threshold to 10% to select all potential peptide binders to HLA-DR*0401, DR*0404, DR*0101 (which includes weak putative binders and may yield false positives, i.e. peptides not able to bind to these alleles; see 32). HLA-DR*0401-Tg mice were previously described 33 and were purchased from Taconics Farm (Germantown, NY, USA). Mice BI 2536 mw were bred and kept under specific pathogen-free conditions. Mice were immunized subcutaneously with 3 nmole hnRNP-A2 peptide emulsified in CFA containing H37Ra Mycobacteria (Difco, Becton Dickinson). Eight days later, cells from the draining lymph nodes were cultured (6×105 cells/well) in 96-well culture plates (Costar) with 30 μM of C646 hnRNP-A2 peptides in synthetic HL-1 medium (BioWhittaker) supplemented with 2 mM L-glutamine and 50 μg/mL gentamicin (Sigma). PPD was used

as positive control for each culture at a final concentration of 10 μg/mL. Cultures were incubated at 37°C and supernatants were harvested after 20 h (for the detection of IL-2) or 60 h (for the detection of IFN-γ). All animal procedures were performed according to Austrian laws (BGBI. I Nr 162/2005) and approved by the local ethical committee. IFN-γ and

IL-2 were detected by ELISA as previously described 34, using for capture anti-IFN-γ mAb Suplatast tosilate Ph551216 (PharMingen) or anti-IL-2 mAb JES6-1A12 (PharMingen), and for detection biotin-conjugated AN 18.17.24 mAb (kindly provided by Dr. Edgar Schmidt, Mainz) or biotinylated anti-IL-2 mAb JES6-5H4. The cytokine detection limit was 30 pg/mL. ELISPOT plates (Millipore Multiscreen-HA MAIP), pre-incubated with 70% ethanol and washed with distilled water, were coated overnight at 4°C with 10 μg/mL capture mAb (anti-IFN-γ mAb, BMS228ESCA/1, Bender Med Systems, Vienna, Austria) dissolved in 0.1 M NaHCO3 pH 9.5. The plates were then washed once with 200 μL sterile PBS and blocked with 200 μL/well complete RPMI medium (RPMI 1640, 1 mM sodium pyruvate, 200 μM L glutamine, MEM with nonessential amino acids, 10% heat-inactivated AB human serum, all purchased from PAA GmbH, and β2-mercaptoethanol from Gibco) for 2 h at 37°C. The blocking medium was removed and freshly isolated human PBMC (8×105 cells) were cultured with recombinant hnRNP-A2 protein (10 μg/mL), hnRNP-A2 peptides (10 μM), PPD of tuberculin (10 μg/mL), TT (10 μg/mL), or PHA (1/50), in a final volume of 200 μL complete RPMI medium. Control wells contained PBMC with medium alone. After 18- to 24-h incubation at 37°C, cells were removed, plates were washed with PBS/0.

The level of AOPP was independently associated with IHD only in HD patients. “
“Adriamycin nephropathy (AN) is a rodent model of chronic kidney disease that has been studied extensively and has enabled a greater understanding of the processes underlying the progression of chronic proteinuric renal disease. AN is characterized by podocyte injury followed by glomerulosclerosis, tubulointerstitial inflammation and fibrosis. Genetic studies have demonstrated a number of loci that alter both risk and severity of renal injury induced by Adriamycin. Adriamycin-induced renal injury has been shown in numerous studies to be modulated by both non-immune and immune factors, and has facilitated further study of mechanisms

of tubulointerstitial injury. This review will outline the pharmacological behaviour of Selleck Seliciclib Adriamycin, and describe in H 89 ic50 detail the model of AN, including its key structural characteristics, genetic susceptibility and pathogenesis. Most types of chronic kidney disease (CKD) are characterized by the development of glomerulosclerosis, tubulointerstitial inflammation and fibrosis. Adriamycin® (Pfizer, Sydney, Australia) (doxorubicin) is a well-known inducer of renal injury in rodents, which mirrors that seen in human CKD due to primary focal segmental glomerulosclerosis.

The first published record of anthracyclines causing renal injury was in 1970 by Sternberg.1 The first description of Adriamycin inducing renal injury was in 1976 in rats,2 and 1998 in mice.3 In 1977, Burke and colleagues4 described a case of a 78-year-old man developing renal failure after the administration of doxorubicin. Since then, Adriamycin nephropathy (AN) in rodents has been extensively studied and

has enabled a greater understanding of the processes underlying the progression of renal injury. Adriamycin nephropathy has several strengths as an experimental model of kidney disease. It is a highly reproducible model of renal injury. It is also a ‘robust’ model in that the degree of tissue injury is severe while associated with acceptable mortality (<5%) and morbidity (weight loss). Because the model is characterized by the induction of renal injury within a few days of drug administration, the timing of injury is consistent and predictable. The severity and timing of renal injury means that it is a model mafosfamide suitable for testing interventions that either worsen or protect against renal injury. The type of structural and functional injury is very similar to that of chronic proteinuric renal disease in humans (see below). Last but not least, this model is similar in rats and mice. Rodent models are extremely useful in the study of disease. Rodents are characterized by their short reproduction period, easy (and cheap) availability of animals and reagents, and amenability to genetic manipulation.5 There are also limitations in the use of AN as an experimental model.

We considered that this problem could be overcome by the eventual demise

of plasma cells, alone or in combination with B cell depletion. However, plasma cells have very long half-lives, measured in months or even years [11]. Finally, in this study we show that anti-mCD20 mAb depletes B cells efficiently and that, although therapeutically less effective, B cell depletion by this agent is highly efficient for preventing development of experimental Graves’ hyperthyroidism. Our results indicate that B cells are critical not only as antibody-producing cells but also as antigen presenting/immune-modulatory cells in the early phase of the disease pathogenesis. Further studies are necessary to find efficient means to suppress the pathogenic autoantibody production therapeutically as novel therapeutic modalities GSK3235025 ic50 for Graves’ disease and also other autoantibody mediated autoimmune diseases. We thank buy IWR-1 Drs R. Dunn and M. Kehry at Biogen Idec, San Diego, CA, for kind gifts of monoclonal anti-mCD20 (18B12) or control (2B8) antibodies, and Professors Sandra M. McLachlan and Basil Rapoport, at Autoimmune Disease Unit, Cedars-Sinai Medical Center and University of California Los Angeles, CA, for critical reading of the manuscript. The authors have nothing to disclose. “
“Because

Helicobacter pylori has a role in the pathogenesis of gastric cancer, chronic gastritis and peptic ulcer disease, detection of its viable form is very important. The objective of this study

was to optimize a PCR method using ethidium monoazide (EMA) or propidium monoazide (PMA) for selective detection of viable H. pylori cells in mixed samples of viable and dead bacteria. Before conducting the real-time PCR using SodB primers of H. pylori, EMA or PMA was added to suspensions of viable and/or dead H. pylori cells at concentrations between 1 and 100 μM. PMA at a concentration of 50 μM induced the highest DNA loss in dead cells with little loss of genomic DNA in viable cells. In addition, selective detection of viable cells in the oxyclozanide mixtures of viable and dead cells at various ratios was possible with the combined use of PMA and real-time PCR. In contrast, EMA penetrated the membranes of both viable and dead cells and induced degradation of their genomic DNA. The findings of this study suggest that PMA, but not EMA, can be used effectively to differentiate viable H. pylori from its dead form. Helicobacter pylori, a Gram-negative and microaerophilic bacterium that infects human gastrointestinal organs such as the stomach, exhibits various shapes during colonization, including spiral, U-shaped, and coccoid forms (1, 2). H. pylori has a role in the pathogenesis of gastric cancer, chronic gastritis, and peptic ulcer disease (2–5). Social and economic underdevelopment associated with inadequate hygiene practices, consumption of unhealthy food, and paucity of pure drinking water are the main risk factors for the development of H. pylori infection (6).

Normal interleukin (IL)-7, IL-12 and IL-15 plasma levels were found. In one of the patients sporadic NK T cells were detected at the tumour site. α-Galactosylceramide (αGalCer) stimulation of peripheral blood mononuclear cells or isolated NK T cell lines from both patients induced IFN-γ, but no IL-4 and no response towards autologous tumour LY2835219 cell line cells or lysates. The clinical course of disease in both patients was not exceptional with regard to histological subtype and extent of metastatic disease. Therefore, despite a constitutive high peripheral frequency

and in vitroαGalCer responsiveness, the NK T cells in the two RCC patients did not show anti-tumour responsiveness. Invariant NK T cells are a distinct set of T cells characterized by

expression of an invariant T cell receptor (TCR) Vα14-Jα18 chain, coupled preferentially to Vβ8·2,7 or -2 in mice or TCR Vα24-Jα18 and Vβ11 in humans [1]. NK T cells recognize glycolipids, rather than peptide antigens, presented by the major histocompatibility complex class I-like molecule CD1d. This results in rapid release of large amounts of T helper type 1 (Th1) [interferon (IFN)-γ] or Th2 [interleukin (IL)-4] cytokines, which in turn can activate dendritic cells, NK cells and B cells as well as conventional Poziotinib concentration CD4+ and CD8+ T cells [2,3]. Thereby, NK T cells play a pivotal role as intermediates between the innate and the adaptive immune system and have the capacity to enhance host immunity to microbial infections and cancer as well as prevent autoimmunity [4–6]. In healthy individuals, the frequency of NK T cells in the peripheral blood is relatively low and ranges between 0·01% to 0·2% of total lymphocytes [7–9]. In cancer patients, NK T cell counts are reduced further compared to age- and gender-matched healthy controls [7,8] and usually defective in IFN-γ production upon stimulation [10,11]. Low circulating NK T cell numbers were found to predict poor clinical outcome in patients with Farnesyltransferase head and neck cancer [12]. Attempts have been made

to stimulate NK T cell expansion with the glycolipid α-galactosylceramide (αGalCer) in order to stimulate anti-tumour responses in cancer patients [13–18]. In 10 of 17 non-small cell lung cancer patients this resulted in prolonged median survival time [19]. In an IFN-α trial of patients with metastatic renal cell carcinoma (RCC), a disease that has not been associated with high NK T cell numbers previously, we detected unusually high levels of circulating NK T cells in two of 14 patients. This prompted us to characterize these cells further to elucidate whether they were related to the therapy and had anti-tumour effectivity. All patients had primary metastatic RCC, patient B2 had clear cell RCC with sarcomatoid component and patient B7 had papillary RCC.

As a general observation, the iIEL compartment showed substantially higher basal [Ca2+]i levels than systemic T cells (Fig. 1B). The systemic populations had equal basal [Ca2+]i levels, though 50% less in relation to iIEL populations (Fig. 1B). In spite of these differences, all five T-cell populations showed robust ionomycin-induced Ca2+-fluxes (Fig. 1C). However, Ca2+ response amplitudes were higher in CD8+ p-αβ and CD8− p-γδ representing systemic T cells. Next, we studied the Ca2+-flux of isolated iIEL or systemic T cells from γδ reporter mice after TCR-clustering with antibodies. For this, we applied an anti-γδ TCR mAb clone (GL3) and an anti-CD3ε clone (145-2C11, here 2C11) and subsequently clustered

them on the cell surface with secondary goat anti-hamster antibody. This procedure induced robust anti-CD3-induced Ca2+-fluxes in the systemic populations CD8+ p-αβ and CD8− p-γδ (Fig. 1D). Similarly, clustering BIBW2992 ic50 with anti-γδ TCR mAb specifically induced

Ca2+-flux of systemic CD8− p-γδ cells (Fig. 1D). However, in the iIEL compartment, we observed discrete Ca2+-fluxes in response to anti-CD3 or anti-γδ TCR mAb only in CD8− i-γδ but not in CD8+ i-γδ (Fig. 1E). This suggested that high basal [Ca2+]i levels in γδCD8αα+iIEL correlated with TCR-unresponsiveness. Taken together, we found that systemic αβ and γδ T cells showed comparable Ca2+-flux responses to TCR ligation, whereas https://www.selleckchem.com/products/ensartinib-x-396.html CD8αα+ αβ and γδ iIEL were presumably pre-activated and thus refractory to further stimulation of the TCR complex and displayed high intrinsic [Ca2+]i levels. These results suggest a chronic stimulation of CD8α+ iIEL in vivo. Next, we sought to investigate the outcome of αβ- and γδ-specific TCR stimulation on isolated iIEL in ex vivo stimulation assays. Since systemic γδ T cells in lymph nodes, spleen and circulation 19, 21, 34 as well as intraepithelial γδ T cells in the skin 35 have been described to be biased to produce IL-17A, we tested whether this pro-inflammatory cytokine was produced by intestinal γδ Fludarabine cell line iIEL. We found that, irrespective of CD8α expression,

γδ iIEL did not produce IL-17A upon stimulation with anti-TCR mAb or PMA/ionomycin (Fig. 2). This is in accordance with a recent report showing that intestinal γδ IEL are not ‘pre-wired’ toward a specific lineage 36. Therefore, we focused in this study on the well-established γδ IEL effector molecules CC chemokine ligand 4 (CCL4) and IFN-γ. Chemokine and cytokine production of αβ, γδ and total iIEL from WT mice was monitored by stimulation with plate-bound anti-γδ TCR (GL3 and GL4), anti-αβ TCR (H57-597, called H57) and anti-CD3 (2C11), respectively, followed by cytokine measurement in the supernatants. Here, αβ or γδ TCR triggering induced similar concentrations of CCL4 (Fig. 3A, upper panel), whereas higher amounts of IFN-γ were produced through anti-αβ TCR stimulation (Fig. 3A, lower panel).