A mutation to Val could be tolerated as a Val can be accommodated

A mutation to Val could be tolerated as a Val can be accommodated in this region of the protein without creating severe steric clashes with the surrounding amino acids. However, the substitution creates a small cavity that could be slightly destabilizing and could explain why only half as much of this mutant is secreted compared with the WT. Once secreted, however, Microbiology inhibitor the protein is fully active both in the fluid phase and on cell surfaces. Accordingly, we found that M120V mutant was not impaired in any functional assay. On the contrary, its activity was

slightly enhanced compared with WT FI in most assays. The residue Asn133 is located in the CD5 domain, in a short α-helix, and is solvent exposed in the 3D structure of the individual domain (Fig. 8). This Asn is not glycosylated

and its substitution would seem to be tolerated in the model. However, CHIR99021 FI expression and secretion are severely impaired. Two explanations for this could be that the region around Asn133 either forms an interface with another domain of FI, or it could be important for interacting with chaperones or related proteins during its secretion and that the substitution impairs this contact. Further work will be needed to characterize this substitution at the structural level. The residue His165 is in the CD5 domain, in a loop structure and apparently fully exposed. It is partially conserved in the sequence and it could be replaced by any polar or charged side chain (Fig. 8). Its replacement with an Arg should be tolerated and our experimental data confirm this analysis since the secretion and function of FI is not affected by this mutation. On the contrary, its activity in a solution in the presence of C4BP and FH was slightly enhanced compared with WT FI. The Ala222 residue is in a loop structure and it forms a contact with Phe209. It is located next

to Cys223-Cys238 and close to the disulfide bond that links the LDLr1 domain to a short segment located prior to the FIMAC domain (Fig. 8). In this region, we have predicted a putative Ca2+-binding site, which are often present in LDLr domains. The Ala to Gly substitution Forskolin could destabilize this region of the domain and perturb the formation of the nearby disulfide bridge and/or the structure of the putative Ca2+-binding site. Such structural alterations would be consistent with the reduced secretion of this mutant that was observed experimentally and also with the observed diminished activity towards cleavage of cell bound C3b. This mutation did, however, appear to have a negligible effect on the solution-phase activity of FI. The residue Arg299 cannot be visualized in the present 3D model as it is located in a linker peptide just before the SP domain. It is possible that an Arg to Trp mutation could be tolerated fairly well in FI, as this substitution already occurs in other species.

The immunomodulatory properties of the selected Lactobacillus bac

The immunomodulatory properties of the selected Lactobacillus bacteria were assessed by measuring the induction of innate and adaptive cytokine production, proliferation and cell death of unstimulated, polyclonal stimulated and allergen-specific stimulated hPBMC. The Lactobacillus strains studied showed an overall stimulating effect on IL-10, decreased prototypical Th2 cytokines and differentially stimulated signature Th1 cytokine induction. Blood was collected from five birch pollen-allergic patients, two grass pollen-allergic patients

and one adult healthy control. All birch- and grass-allergic patients reported having rhinoconjunctivitis during the birch or grass pollen season, respectively, and had serum-specific IgE to birch

or grass pollen Caspase inhibitor of at least class 4 (except for one person who had class 3), measured by ImmunoCAP NVP-LDE225 (Phadia AB, Uppsala, Sweden). The healthy donor displayed no birch or grass pollen-specific IgE in his sera (<0.35 kU L−1/class 0). Blood was obtained outside the pollen season in September, and none of the patients showed allergic symptoms at the time of investigation. Furthermore, none of the patients had received allergen-specific immunotherapy or used antihistamines or corticosteroids in the month before the blood drawing. All participants gave their informed consent and the performed experiments were approved by the local ethical committee (Commissie Mensgebonden Onderzoek, regio Wageningen). Six Lactobacillus strains (Table 1) of the species Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus fermentum were selected from our culture collection on the basis of high survival rates under conditions of low pH and/or the presence of bile, isolation from gastrointestinal tract, or were strains from species that are among the predominant Lactobacillus populations in the human gut. Further selection, including 70 strains, was based on IL-10-inducing capacities in 24-h hPBMC cultures of a healthy donor according to standardized procedures in our laboratories. Furthermore,

a mixture of strains B2261 and B633 was included, further referred to as a mixture of B2261 and B633. The choice for this mixture was based on combining the highest IL-10-inducing strain (B633) and the highest GPX6 IL-12-inducing strain (B2261), of the 70 strains included in this initial screening. Strains were cultured for 24 h at 37 °C in Man Rogosa Sharpe (MRS) broth (Merck, Darmstadt, Germany), after which fresh broth was inoculated with 1% (v/v) overnight culture. After an additional 24 h of incubation at 37 °C, bacterial cells were harvested by centrifugation at 1000 g, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS. The bacterial cell numbers were determined by plate counting on MRS agar, and OD was measured at a wavelength of 600 nm.

4D) Conversely, the levels of perforin, IL-2, and granzyme B rem

4D). Conversely, the levels of perforin, IL-2, and granzyme B remained unchanged between Tat-POSH- and control-treated PD332991 cells (Fig. 4E–G). Disruption of the POSH/JIP-1 complex resulted in a modest (10–15%) but significant reduction in in vitro cytotoxicity that closely resembled JNK1−/− T cells (data not shown) [18]. Together, these data indicate

that the POSH/JIP-1 complex is specific for the regulation of JNK1-dependent effector function. To test the affect of disruption of the POSH/JIP-1 scaffold complex on CD8+ T-cell effector function in a more physiological setting, we investigated the ability of Tat-POSH-treated CTLs to control tumors in vivo. CD8+ OT-I T cells were stimulated for 2 days in vitro in the presence of Tat-POSH or control peptide. To directly test effector function and partially correct for the proliferation defect, equal numbers (1 × 106) of Tat-POSH and Tat-cont. CD90.1+ CTLs were transferred into B6 Rag−/− CD90.2 congenic hosts that had been subjected to subcutaneous inoculation with large doses (5 × 105 cells) of the OVAp-expressing thymoma (EG7). Tumor

size was tracked for 20 days and compared to a cohort of B6 Rag−/− hosts that received the tumor with no CTLs. The Tat-control-treated CTL group had significantly smaller tumors than the Tat-POSH-treated CTL and the no CTL control groups. Furthermore, GS-1101 datasheet there was no difference in tumor size between Tat-POSH-treated and no CTL control group (Fig. 5A). These results are consistent with loss of INF-γ-dependent tumor control by JNK1−/− [18], Eomes−/−, and Eomes−/−/T-Bet−/− CD8+ T cells [40, 41]. Interestingly, there was no difference in cell number or percentage of CTLs in the blood of mice from either group

over the first 9 days (Fig. 5B). However, when tumor-specific T-cell numbers were analyzed at day 20, there was a sizeable (>tenfold) reduction in both the number of Tat-POSH-treated CTLs in the spleen (Fig. 5C) and tumor-infiltrating lymphocytes in the Tat-POSH-treated group (Fig. 5D). Curiously, in spite of this marked loss of Tat-POSH-treated CTLs GBA3 late in the response, we did not observe significant differences in apoptosis between Tat-POSH- and control-treated cells in the blood, spleen, or tumor (data not shown). Regardless, the loss of tumor-specific CTLs along with their reduced effector function (TNF-α, FasL, and IFN-γ; Fig. 4 and [41]) provides convincing evidence that the POSH/JIP-1 complex regulates JNK1-dependent development of effector function important for tumor clearance by CD8+ T cells. Intriguingly, Tat-POSH-treated CTLs did not recover their defect even when they had been washed, adoptively transferred, and exposed to their cognate antigen (Fig. 5). This suggests that the POSH/JIP-1 complex regulates the programing of CD8+ T-cell differentiation and effector function.

The resulting Leishmania DNA copy number was then divided by the

The resulting Leishmania DNA copy number was then divided by the copy number of ß-actin DNA to obtain the relative parasite density. A total of 2 × 105 mesLN or 3 × 105 popLN cells were cultured Selleck Trichostatin A in 96-well round-bottom plates in RPMI 1640 medium

supplemented with 10% foetal calf serum, 20 mm HEPES, l-glutamine (2 mm) and gentamicin (50 μg/mL) at 37°C and 5% CO2. Cells were stimulated in triplicates for 72 h with either medium or anti-mouse CD3 (145-2C11, 1 μg/mL), S. ratti iL3 lysate (20 μg/mL) or with soluble Leishmania antigen (SLA) (three lysed parasites per cell). The supernatants were harvested for analysis of cytokine production by ELISA. Cell proliferation was measured by the uptake of 3H-thymidine for additional 18 h culture. For the detection of Strongyloides-specific Ig, Microlon ELISA plates (Greiner, Frickenhausen, Germany) were coated with 50 μL/well S. ratti antigen lysate (2·5 μg/mL) in PBS overnight at 4°C. For the detection of Leishmania-specific Ig, ELISA plates were coated with 1 × 105 live L. major, centrifuged at 1500 × g for 8 min, decanted and incubated with 50 μL/well 0·25% Glutaraldehyde/PBS for 5 min. Plates were washed 4× with PBS 0·05% Tween 20 and blocked

by incubation with 200 μL/well PBS 1% BSA for 2 h Lumacaftor price at 37°C. The sera of 1 : 200 dilutions in PBS 0·1% BSA were incubated in triplicates adding 50 μL/well and left overnight at 4°C. Plates were washed 5×, and antigen-specific Ig was detected by incubation with 50 μL/well of horseradish peroxidase conjugated anti-mouse IgG, IgM (Zymed, Karlsruhe, Germany), IgG2b, IgG3 (Southern Biotechnology, Birmingham, AL, USA) for 1 h at RT. Plates were washed 5× and developed by incubation with 100 μL/well tetramethylbenzidine 0·1 mg/mL, 0·003% H2O2 in 100 mm NaH2PO4 pH 5·5 for 2·5 min. Reaction was stopped by addition of 25 μL/well 2 m H2SO4, and optical density at 450 nm (OD450) was measured. Relative ELISA units (REU) were calculated by dividing the OD450 of each sample by the OD450 of the negative (buffer) control www.selleck.co.jp/products/sorafenib.html of each individual ELISA dish. Murine cytokines (IL-10, IL-13, and IFN-γ) were measured in the culture supernatant

of in vitro stimulated mesLN and popLN cells using DuoSet ELISA development kits (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. Statistical analysis was performed with graphpad prism software (GraphPad Software, San Diego, CA, USA) using either the two-tailed T-test or anova followed by Bonferroni’s post-test to calculate the significance of differences between multiple groups. The data are represented as means ± SEM. A value of P ≤ 0·05 was considered to be statistical significant. To understand the nature of immune response and host defence in situations of co-infection, we analysed the course of infection in mice carrying single or co-infections with the pathogenic nematode Strongyloides ratti and the flagellate Leishmania major. Mice were infected with S.

Control antibodies included Rat IgG2a isotype control mAb (eBiosc

Control antibodies included Rat IgG2a isotype control mAb (eBioscience), mouse anti-Border disease virus p125/p80 mAb VPM21 and purified rabbit immunoglobulin (Sigma-Aldrich, St. Louis, MO, USA), for rat, mouse and rabbit primary antibodies, respectively. All antibodies were diluted in PBS/T80 containing 10% NGS. Slides GPCR & G Protein inhibitor were washed twice in PBS, and the appropriate secondary antibody (peroxidase-labelled anti-mouse or anti-rabbit EnVision™+ reagent, Dako) was applied to sections for 30 min at RT. After a final PBS wash, sections were incubated with 3,3′-diaminobenzidine (DAB) for 7·5 min at RT, washed in distilled water, counterstained

with haematoxylin, dehydrated and mounted in Shandon synthetic mountant (Thermo Scientific). Each nodule was scanned under the light microscope. The initial scanning was performed with a wide-angle lens at low power (×20), and the following data were recorded: the predominant inflammatory cell type, the distribution of the cell infiltrate (diffuse or focal/multifocal) and the location of the infiltrate within the nodule (peripheral, central

or both). CD3+ and Pax5+ cells tended to occur in a focal/multifocal distribution pattern in the sections, and the foci of CD3+ and Pax5+ cells were counted in the most active ×20 field (the field with the highest number of foci). CD3+ and Pax5+ infiltrates were subjectively scored 0–3 (Table 1). MAC387+ infiltrates

were also scored 0–3; however, MAC387+ cells occurred more diffusely in sections, either evenly distributed or in patches, and therefore, the scoring system was slightly different Decitabine order (Table 2). Numbers of FoxP3+ cells were counted in 10 nonoverlapping ×400 fields (five peripheral and five central fields per oesophageal nodule using a 0·0625 mm2 graticule). In the normal oesophagus control group and lymph nodes, five nonoverlapping ×400 fields were counted. Counting was confined to CD3+ areas. Statistical analyses were performed with GraphPad Prism (GraphPad Software, Inc. CA, USA). The difference in prevalence and distribution ADAMTS5 of the different proportions of cell types was tested using the chi-square test. The differences between the scores of the different types of infiltrate were tested for significance between all groups using a Kruskal–Wallis test, followed by Dunn’s post hoc test. P values of <0·05 were considered significant. Myeloid cells predominated in 70% of cases, while T cells predominated in 23% of cases. In the remaining 7% of cases, the number of T cells and myeloid cells was approximately equal. There was no difference in the proportion of myeloid and T cells between the neoplastic and non-neoplastic groups (P = 0·27). When cells were present in normal oesophageal sections, they were diffusely scattered and myeloid and T cells tended to occur in equal proportions (Table 3).

For generation of memory T cells, mice were first immunized i p

For generation of memory T cells, mice were first immunized i.p. with 100 μL of emulsion consisting of CFA and 10 nmoles OVA protein, followed by two boosts with the same dose of Ag and Incomplete Freund Adjuvant keeping 10 day intervals. Ten days after the last injection, endogenous IL-2 responses of harvested splenocytes were analyzed by ELISPOT. An ELISPOT assay was carried out as described [45]. Cells isolated from spleens of immunized mice were suspended in

DMEM-10 culture media and restimulated with ovalbumin protein (10 μM). The cells were then cultured for 24 h Copanlisib ic50 (37°C and 5% CO2 concentration). After incubation, a plate was extensively washed and incubated with the secondary biotinylated antimouse IL-2 Ab (2 μg/mL in 1% BSA in PBS) and streptavidin-alkaline phosphatase (1:1000 in 1% BSA in PBS) followed by detection with alkaline-phosphate substrate (BCIP/NBT). Plates were precisely enumerated using an ELISPOT reader from Cellular Technology Ltd. with dedicated software. All single experiments involved 3–5 mice per group and were repeated at least three times. The data were expressed as means ± SD. Statistical analysis was performed with Student t-test using GraphPad Prism statistical software. p Values < 0.05 were considered as a significant. This work was supported

by National Institutes of Health grant nos. R01AI061077 (to W.S.), R01AI073718 Lumacaftor mw (to W.S.), and Leukemia & Lymphoma Society Scholar (W.S.) and Special 17-DMAG (Alvespimycin) HCl Fellow (D.B.G.) awards. R.J.X was supported by NIH grants DK043351 and HL088297. The authors declare no financial or commercial conflicts of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should

be addressed to the authors. Figure S1. Dlg1 is completely deleted in T-cell lineage of KO mice. Splenocytes from KO and WT mice (Vav1-Cre Dlg1flox/flox and Vav1-Cre Dlg1flox/+ respectively) were stimulated with polyclonal mitogen (ConA) overnight, subsequently harvested and lysed. Lysates were separated on 8% SDS-PAGE following by incubation with Dlg1 antibody to evaluate the expression of Dlg1 protein. Brain lysate was used as positive control whereas ERK expression was used as a loading control. Results are representative of three independent experiments. Figure S2. Dlg1 is dispensable for T-cell development in Lck-Cre and Vav1-Cre KO and WT mice. Lck-Cre and Vav1-Cre thymocytes from WT and KO were stained with indicated markers to analyze all thymocyte subsets. No differences in thymocyte subsets were found between WT and KO mice. Results are representative of n>20 mice. Figure S3. Dlg1 is dispensable for thymocyte selection in HY mice.

From the sequence-determining analysis of Vβ13+ cells, the TCR cl

From the sequence-determining analysis of Vβ13+ cells, the TCR clonality was less than 10% in the most frequently appeared clone, suggesting difficulty in showing clonality in the immunoscopic analysis by this case. The sequencing analysis showed the most frequently appeared clone to be Jβ2.1 and the immunoscope analysis of Vβ13-Jβ2.1 showed a skewed peak in CD8+ CD122+ CD49dhigh cells but the overall shape was not much different from that of Vβ13-Cβ. A limitation of this study is that we did not show a relationship between each TCR and the regulatory function of the cells; this could be investigated by establishing BGJ398 clinical trial many CD8+ CD122+ Treg cell clones, and then determining the regulatory

function of the clones that possess the preferential CDR3 sequences detected in this study. Unfortunately, we have not succeeded in establishing functional CD8+ CD122+ Treg cell clones yet because these Treg cells lose their proliferating capacity in in vitro culture (our unpublished observation). It is

difficult to determine the function of clonally expanded Treg cells obtained from wild-type mice because of the lack of methodology to purify a population with a single type of TCR. It may be necessary to make a high throughput screening assay number of lines of TCR transgenic mice to determine the function of T cells carrying one specific TCR. The interpretation of this study is limited by the lack of a conclusion as to which subset of CD8+ CD122+CD49dhigh or CD8+ CD122+ CD49dlow cells are Treg cells. The study of PD-1+ cells in the CD8+ CD122+ see more population by Dai et al.[16] and correlation of expression between PD-1 and CD49d (Fig. 1b) strongly suggests CD8+ CD122+CD49dhigh cells as Treg cells, while the possibility of CD49dlow as Treg cells still remains unknown (our unpublished observation). It has been demonstrated that memory T cells have skewed TCR diversity,[35] whereas there is little information regarding the TCR diversity of CD8+ Treg cells. In this study, we observed an increased number of identical clones of TCR Vβ CDR3 (Fig. 4) in both CD8+ CD122+ CD49dhigh and CD8+ CD122+ CD49dlow populations compared with that of

the CD8+ CD122− naive T-cell population, indicating clonal expansion of these CD122-expressing T cells. Importantly, identical clones were not shared between those obtained from the CD49dhigh population and the CD49dlow population (Figs. 4a,b). This result indicates that two fundamentally different cell populations (probably Treg cells and memory T cells) are efficiently separated into the CD8+ CD12-2+ CD49dlow population and the CD8+ CD122+ CD4-9dhigh population. Therefore, regardless of whether Treg cells are in the CD8+ CD122+ CD49dlow population or in the CD8+ CD122+ CD49dhigh population, the conclusion that CD8+ CD122+ Treg cells have skewed TCR diversity is unchanged. We thank Prof. Ken-ichi Isobe for financial help and useful discussions.

Despite being under constant barrage by the immune system, E  gra

Despite being under constant barrage by the immune system, E. granulosus is able to secrete several molecules that can directly modulate the host’s immune system, induce vigorous serological and cellular immune responses and sustain the infection for long time periods (3,4). The hydatid cyst is unilocular and filled with hydatid fluid (HF), a complex mixture of substances derived from the metabolism of the parasite. To date, the HF represents the major source of metacestode proteins for immunodiagnosis learn more or vaccine research (4,5). Although distinct antigens and IgG subclasses are good markers

for the diagnostic detection of E. granulosus infection, they demonstrate inadequate performance for the serological assessment of

active vs. inactive forms of CE (6). As the response to surgical and pharmacological treatments is unpredictable for the individual CE case, a constant medical supervision and regular monitoring of imaging findings and serological responses are entailed. In humans, ultrasound (relying on direct visualisation of the parasitic cyst) and serology (parasite-specific serum antibody detection) are the two tests conventionally employed to assess the outcome of infection after treatment (7). As antibodies Linsitinib molecular weight to most major E. granulosus antigens may persist in patients’ sera for several years after treatment, the identification of appropriate single parasite antigens that directly correlate with infection conditions seems an interesting approach for assessing whether the disease will progress or regress (8,9). Proteomic analysis linked to immunological characteristics of respective

antigens may yield improved to investigate the host–parasite relationship in view of metacestode viability or decay. Previous studies have shown that proteomic analysis can be useful for such an approach to identify proteins from hydatid fluid and protoscoleces (10,11). In this study, using an immunoproteomic analysis, we have compared sera from patients with active vs. inactive status of disease, aiming to identify new potential immunological markers involved in the development of CE. Two-dimensional gel electrophoresis (2-DE) of sheep HF (SHF), followed by immunoblot Farnesyltransferase (IB) analysis with sera from patients with distinct phases of the disease, enabled us to identify by mass spectrometry, among the proteins present in the HF, heat shock protein 20 (HSP20) as a potential marker of CE activity. We developed an IB assay to highlight the presence of IgG specific to HSP20 in serum from 95 patients with CE, grouped according to the status of disease and cyst type. Finally, we assessed by IB the IgG response to HSP20 during a long-term follow-up in 20 patients pharmacologically and surgically treated. Our observations suggested that antibodies specific to HSP20 might be a potential biomarker for monitoring therapy of CE.

Eagle Jr Eye Pathology: An Atlas and Text, 2nd Edition Wolter

Eagle Jr . Eye Pathology: An Atlas and Text, 2nd Edition . Wolters Kluwer/Lippincott Williams & Wilkins , Philadelphia , 2011 . 320 Pages (hardcover). Price

£96.90 (Amazon). ISBN- 10 1608317889 ; ISBN- 13 978-1608317882 This is the second edition of ‘Eye Pathology: An Atlas and Text’ authored by Ralph Cilomilast mw C. Eagle. I have to say I was delighted when I first stumbled across this book; it has been my impression in recent years that new textbooks of ophthalmic pathology have been rather thin on the ground. The author, Ralph C. Eagle, is one of the world’s best known ophthalmic pathologists and has taught ophthalmic pathology at the Wills Eye Institute in Philadelphia, the Armed Forces Institute of Pathology (AFIP) ophthalmic pathology small molecule library screening course and at academic institutions all over the world. This text bears testament to his wealth of experience. The colourful front cover instantly gives an

indication of the wealth of images that lie within. True to the title, the uniformly high-quality images throughout the book are complemented by text which is a well written and concise summary of modern ophthalmic pathology. A total of 16 chapters are presented in 304 pages. The book starts with an introductory chapter covering ocular anatomy and histology, while the second chapter reviews congenital and developmental anomalies. The remaining 14 chapters are dedicated to specific disease processes (inflammation, ocular trauma, glaucoma, intraocular tumours in adults, retinoblastoma

and stimulating lesions) and specific anatomical compartments (conjunctiva, cornea and sclera, the lens, retina, vitreous, the eyelid and lacrimal drainage system, orbit and optic nerve). The final chapter is dedicated to laboratory techniques, special stains and immunohistochemistry. For a relatively slender-looking book there is impressively wide-ranging and up-to-date coverage of ophthalmic disease processes. I have always been a fan of single-author texts and the consistency in writing style makes BCKDHA this an easy as well as informative read. The images are incorporated alongside the relevant text for easy reference. These include macroscopic and microscopic images as well as electron microscopy. All of the illustrations are high-quality and, to the delight of anyone who has to teach ophthalmic pathology, the images are downloadable from an image bank at the publisher’s website. Each chapter ends with a detailed bibliography for those interested in further reading. The second edition expands upon areas which the author felt were covered too superficially in the first edition.

CBA data was analysed using fcap Array software (BD Biosciences)

CBA data was analysed using fcap Array software (BD Biosciences). find more Statistical analyses were performed with GraphPad Prism software (Graphpad Software, Inc., La Jolla, CA, USA). Significance was determined using Kruskal–Wallis analysis with

Dunn’s multiple comparisons post-test and Wilcoxon tests. We analysed NKT cells isolated from fresh human thymus, spleen, cord blood and adult peripheral blood. The mean NKT cell frequency of donor tissues were similar for peripheral blood (0·1 (mean) ± 0·02 [standard error of the mean (s.e.m.)], cord blood (0·06 ± 0·01) and spleen (0·08 ± 0·03), but significantly lower in thymus (0·007 ± 0·001). Most (> 90%) thymus and cord blood NKT cells were CD4+, with CD4− NKT cells seen mainly in peripheral blood and spleen (Fig. 1). In contrast to findings in mice that blood NKT cells provide a poor measure of NKT cell frequency in spleen [18], we found that human spleen and blood had similar mean frequencies of NKT cells and of CD4+ and CD4− NKT cell subsets, although this applies

to group analysis, rather than to each individual donor. A recent publication identified diversity within CD4+, CD4− and CD8+ NKT cell subsets, but these cells had been expanded prior to analysis. We analysed cell surface antigen expression by CD4+ and CD4− NKT cell subsets without in-vitro expansion and compared blood-derived NKT cells to those from Proteasome inhibitor cord blood, thymus and spleen (Fig. 2). Many antigens were expressed differentially by the CD4+ and CD4− NKT cell subsets (Fig. 2a–j), including CD56 and CD161 (confirming these as ineffective surrogate markers for human NKT cells), with CD161 expressed more highly in peripheral blood and spleen Etofibrate than cord blood or thymus. This confirms CD161′s status as a marker of NKT cell maturity [19, 22, 23]. Interestingly, CD161 was expressed by more CD4− than CD4+ NKT cells (Fig. 2a), which supports the hypothesis that comparatively immature precursors

of CD4− NKT cells are present within the CD4+ subset [22] [19, 23]. Our analysis did not identify any preferential surface antigen expression by either of the CD4+ or CD4 NKT cell subsets. CD8, CD45RA and CD94 were expressed typically by more CD4− NKT cells (Fig. 2i,j and data not shown), whereas CD62L, CD127 and LAIR-1 (Fig. 2c,d,b) were expressed by a higher proportion of CD4+ NKT cells. CD25, CD56, CD16, CD45RO, CD84, CCR7 and signalling lymphocyte activation molecule (SLAM) were expressed differentially by both CD4+ and CD4− NKT cell subsets, but the pattern of expression was similar for each subset (Fig. 2a–j and data not shown). NKT cells from thymus, cord blood, peripheral blood and spleen expressed similar levels of most antigens, although there were exceptions: CD4 was expressed by more NKT cells in thymus and cord blood, CD161 was higher in peripheral blood, CCR7 expression was lowest in peripheral blood and CD25 was highest in cord blood.