2b). To analyse further the MSC senescence during SSc, we assessed two specific markers associated with the senescent phenotype: p53 and p21. We observed that

both HC– and SSc–MSCs showed the same basal expression of p53 protein, without significant differences. Of note, p21 protein expression was increased strongly in MSCs isolated from SSc compared to HC cells, suggesting a sustained activation of the p53/p21 pathway during SSc. After doxorubicin, MSCs from HC and SSc showed a relevant increase in p53 protein level without differences, showing that instead of the disease, acute genotoxic stress normally induces p53 accumulation (Fig. 3a). Despite p53 activation, Wee1 inhibitor we did not find a clear increase of p21 protein level in either HC– or SSc–MSCs, although SSc cells showed a slightly increased expression of p21 after doxorubicin with respect to HC. The relative qRT–PCR confirmed the results obtained by Western blot analysis. In normal culture conditions, mRNA transcripts of p53 were no different in HC– and SSc–MSCs (HC–MSCs 0·97 ± 0·05 versus SSc–MSCs 1·04 ± 0·15 mRNA levels, P = 0·75). P21 mRNA expression was increased significantly in SSc–MSCs when compared to HC (HC–MSCs 1·07 ± 0·13 Saracatinib supplier versus SSc–MSCs 6·70 ± 3·84 mRNA levels, P = 0·01). (Fig. 3b,c). After treating MSCs with doxorubicin, we did not observe any change in

the p53 mRNA levels compared to non-treated cells, both in HC and SSc (HC–MSCs 0·86 ± 0·14 versus SSc–MSCs 0·72 ± 0·24 mRNA levels, P = 0·50). Of note, p21 mRNA levels were increased significantly in respect to HC cells (HC–MSCs 0·39 ± 0·06 mRNA levels versus SSc–MSCs 0·67 ± 0·09, P = 0·01, Fig. 3b,c). The immunosuppressive activity of MSCs, derived from both HC donors and SSc patients, was assessed by co-culture with PHA-stimulated healthy PBMCs. MSCs from HC and SSc patients suppressed PHA-induced proliferation without significant Liothyronine Sodium differences (HC PBMCs 12120 ± 1144

cpm versus HC PBMCs/HC–MSCs co-culture 5814 ± 867 cpm, P < 0·0001, and HC PBMCs 12120 ± 1144 cpm versus HC–PBMCs/SSc–MSCs co-culture 4678 ± 1283 cpm, P < 0·0001, Fig. 4a). Moreover, we assessed the capacity of MSCs to induce the regulatory phenotype (CD25brightFoxP3) in SSc lymphocytes. CD4+ T cells from healthy controls (HC–CD4+) and from SSc patients (SSc–CD4+) were co-cultured for 5 days with MSCs in both autologous and heterologous conditions. In circulating SSc–CD4+, we observed a significantly higher number of CD4+CD25brightFoxP3+ cells when compared with HC–CD4+ cells (11 216 ± 2088 versus 4547 ± 2182 cells, respectively; P = 0·02). Treg numbers, after MSC induction, increased significantly in each experimental condition without any difference between SSc patients and HC, as shown in Fig. 4b.

tuberculosis induced CD4+ T-cell-dependent responses. The key findings of the present study are that eight of a total of 157 peptides selected for HLA class I binding induce T-cell responses in PBMC from one or several PPD+ donors (Table 2). In contrast, only four of 10 donors, with low PPD reactivity in ELISPOT

assay, reacted with one or more of the eight antigenic peptides indicating the M. tuberculosis specificity of the responses observed. However, instead of being HLA class I restricted, these responses are apparently restricted Ulixertinib nmr by HLA class II molecules because the responses are all blocked by anti-HLA-DR antibody added to the ELISPOT assay culture. In addition, according to results from cell depletion and FACS analyses anti-M. tuberculosis peptide responses are mediated by CD4+ T cells. The

eight epitopes discovered are derived from five different M. tuberculosis proteins. Three of these [Rv1979, Rv3144c (PPE52) and Rv3532 (PPE61)] each express two positive CTL epitopes whereas the remaining two proteins [Rv0284 and Rv3507 (PE_PGRS)] only harbour a single epitope. Interestingly, six of the eight positive epitopes are derived from the PE/PPE gene family of conserved mycobacterial proteins (Table 2). The PE/PPE gene family is interesting from an immunological point of view because they comprise approximately Bcl-2 inhibitor 10% of the M. tuberculosis genome and may be a source of antigenic variation, which the bacterium uses to evade the host immune response.34 These proteins are surface-associated cell wall proteins and may also be accessible to antibodies.40 The B-cell and T-cell

immune responses have been reported against both PE and PPE proteins, but their immunological PR-171 concentration significance remains largely unknown.41–44 Only a few T-cell epitopes have been identified for the PE/PPE gene family. Two have been found in PE-PGRS proteins (Rv1818c and Rv3812) and one in PPE protein (Rv3018c). In the present study we report five new epitopes for the PE/PPE gene family: a single epitope for the Rv3507 (PE_PGRS) and four new epitopes for the PPE proteins [Rv3144c (PPE52) and Rv3532 (PPE61)]. Regarding the phenotype of M. tuberculosis peptide-responding T cells, our data from T-cell depletion of PBMC before ELISPOT and FACS analyses showed that the responding T cells are indeed CD4+ T cells. In our previous studies 26–28 in which we probed for specific T-cell immunity in PBMC against pox and flu virus-derived HLA-I binding peptides, respectively, HLA-I and HLA-II antibody blocking experiments and CD4+ and CD8+ T-cell depletion experiments showed that peptide reactivity was initiated by either CD4+ or CD8+ T cells but never by both subsets in the same ELISPOT culture. It is generally accepted that HLA class I binding peptides are composed of 8–11 amino acids, whereas HLA class II binding peptides consist of 15–20 amino acids being recognized by CD8+ and CD4+ T cells, respectively.

Were this so, females could have been relatively more attracted

to the novel rotation of the familiar shape than were males and thus have been more likely to divide attention between the novel rotation and its mirror Selleck KPT330 image. To investigate this possibility, 3- to 4-month-olds were given an angular discrimination task in which infants were familiarized with the number 1 (or its mirror image) at one rotation and then tested with the same shape in the familiarized rotation versus the shape in a novel rotation. Infants were provided with just a single 15-s familiarization presentation of a given angular rotation because that was the length of time infants were exposed to a given angular rotation in the familiarization portion of the mental rotation experiment in Quinn and Liben. Figure 3 depicts an example of the task used in Experiment 1. Participants were 24 3- to 4-month-olds, including 12 females, mean age = 114.75 days, SD = 10.13 days, and 12 males, mean age = 117.75 days,

SD = 8.39 days. The sex difference in age was not significant, t(20) = −0.94, p > .20, two-tailed. Data from three additional infants who were tested (one female) were excluded from analyses because they consistently (≥95%) favored one side of the display (N = 2) or failed to compare the test stimuli at all (N = 1). Most infants in Cobimetinib both Experiments 1 and 2 were Caucasian and from middle-class backgrounds. Each stimulus consisted of a black number 1 (or its mirror image) in a particular degree of rotation that was centered on a 17.7 × 17.7 cm white posterboard. The number 1 was 5.2 cm high and 3.2 cm wide at the base. The width of both the base and stem of the number 1 was 1.2 cm. Infants were tested in a visual preference apparatus, modeled after that of Fagan (1970). The apparatus has a gray display panel which includes two compartments to hold the stimuli. The stimuli

were illuminated by a fluorescent lamp Tau-protein kinase that was shielded from the infant’s view. Center-to-center distance between compartments was 30.5 cm, and on all trials, the display panel was situated approximately 30.5 cm in front of the infant. There was a 0.62 cm peephole located midway between the compartments that permitted an observer to record infant visual fixations. A second peephole, 0.90 cm in diameter, located directly below the first peephole, permitted a Pro Video CVC-120PH pinhole camera and Magnavox DVD recorder to record infant gaze duration. Familiarization consisted of a single 15-s familiarization trial, during which two identical copies of the number 1 (or its mirror image) were presented in a specific degree of rotation. There were two 10-s preference test trials, each of which paired the familiarized rotation with a novel rotation.

One week after the last immunization, mice were killed, blood was taken and, following perfusion, intestinal samples were collected using the perfusion-extraction (PERFEXT) technique.20 Ovalbumin-specific IgG and IgA titres were determined by ELISA. see more Ninety-six-well plates (Greiner Bioscience, Frickenhausen, Germany) were coated with OVA (20 μg/ml)

and blocked with PBS/BSA. Serially diluted serum and intestinal samples were added followed by goat anti-mouse horseradish peroxidase-conjugated IgA or IgG (SouthernBiotech, Birmingham, AL). Plates were developed with o-phenylenediamine dihydrochloride, stopped with 0·1 m H2SO4 and absorbance was read at 490 nm. Titres of IgG and IgA were determined from the sample dilution giving an optical density value above 0·4. Data were statistically analysed in Prism (graphpad software) using the Student’s t-test, in which *P < 0·05, **P < 0·01 and ***P < 0·001. Although systemic immune compartments and skin-draining LN of CD47−/− mice have been extensively studied, the GALT has not been carefully characterized. We

therefore enumerated cells in the GALT of CD47−/− mice and revealed a 50% reduction of total cell numbers in MLN, LP and PP, compared with those in WT mice (Table 1). In contrast, the number of cells in skin-draining LN and spleen was not significantly different between WT and CD47−/− mice (Table 1). Although immunohistochemical analysis showed normal localization of T and B cells in MLN and PP of CD47−/− mice PS-341 mouse (see supplementary material, Fig. S1a), and both CD47−/− and WT CD4+ T cells in PP and MLN were found to express similar levels of CD44 and CD62L (data not shown), the frequency of CD4+ T cells in MLN and PP of CD47−/− mice was significantly reduced compared with that in WT mice (Fig. S1b). In contrast, the frequency of Foxp3+ CD4+ T cells in PP, but not in MLN, was significantly increased in CD47−/− compared with WT mice (Fig. S1c). Impaired DC migration from the skin and subset-specific Ribonucleotide reductase alterations in splenic DC at steady state have previously been

reported in CD47−/− mice13,14 therefore, we next assessed populations of antigen-presenting cells in the GALT of these mice. As the total number of cells in the GALT of CD47−/− mice was reduced by 50%, frequency rather than total number of cells within cell populations was determined. Flow cytometric analysis showed a significant reduction in the frequency of CD11c+ MHC-II+ conventional DC (cDC) in MLN, but not in LP or PP, of CD47−/− mice (Fig. 1a). In contrast, no significant change in the frequency of CD172a+ CD11clow MHC-IIlow SSClow cells was detected (Fig. 1b). Further phenotypic characterization was therefore focused on cDC and identified two populations of cDC in MLN (see supplementary material, Fig. S2a).

The human lung is in contact with inhaled airborne IWR-1 mw pathogens and, via expression of a large panel of TLRs, the airway epithelial cells represent the first barrier against invading microbes. Several studies strongly suggest that chronic inflammation increases the risk of carcinogenesis. As lungs are frequently exposed to RNA viruses that are recognized by TLR7 and TLR8, the expression of TLR7 and TLR8 by tumor cells in human lung

cancer in situ and in cell lines was investigated. Stimulation with TLR7 or TLR8 agonists leads to atypical NF-κB activation, up-regulation of Bcl-2 expression, increases tumor cell survival, and induces chemoresistance. Altogether, these data emphasize that TLR signalling occurring during infection in lung cancer patients could directly favor tumor development. Peter Brossart (Bonn, Germany) then discussed current strategies of cancer immunotherapy, focusing on his groups’ studies using DCs presenting tumor antigens 5. DCs are the most powerful antigen presenting cells with the unique ability to initiate and maintain primary immune responses. Due to a better understanding of DC differentiation and function, and the establishment of

protocols for the generation of DC in vitro under GMP conditions, vaccination strategies were developed to treat patients with malignant diseases. Peter Brossart presented data from a recently finished clinical trial using autologous mature DCs pulsed with MUC1-derived HLA-A2 binding peptides. Hydroxychloroquine manufacturer This approach resulted in the induction of clinical and immunological responses in vaccinated patients with metastatic renal cell carcinoma. Currently, the Brossart group is characterizing novel tumor antigens and analyzing several approaches to improve the efficiency of such vaccines by utilizing in vitro transcribed RNA that code for defined tumor antigens or combinations with tyrosine kinase inhibitors. Peter Šebo (Prague, Czech Republic) delivered a rich and fascinating overview of Bordetella adenylate cyclase toxin (ACT) and suggested

Histamine H2 receptor its possible use in cellular therapies. ACT targets myeloid phagocytes bearing the αMβ2 integrin CD11b/CD18 (Mac-1 or CR3), such as neutrophils, macrophages, or dendritic cells (DC, CD11bhigh) 6. ACT penetrates across the cell membrane, promotes an influx of calcium ions, binds cytosolic calmodulin, and converts ATP to cAMP, thus causing phagocyte impotence. In DCs, partial maturation by ACT is induced that compromises their capacity to stimulate T cells. The AC domain of detoxified ACT, having the enzyme activity ablated genetically (dACT), in turn, exhibits an amazing capacity to accommodate foreign T-cell antigens and convey them into the cytosol of dendritic cells both in vitro and in vivo. This allowed the development of dACT toxoids into a particularly efficient tool for antigen delivery for cytosolic processing and MHC class I-restricted presentation to cytotoxic CD8+ T lymphocytes.

015% H2O2 as cosubstrate. Adjacent serial sections were used to directly compare pathological structures INCB018424 cost recognized by antibodies listed in Table 1. For double-label immunofluorescence, sections were blocked with 10% NGS (Sigma) in TBS for 30 min. Double-labelling experiments were conducted by combining two of the primary antibodies listed in Table 1. Bound monoclonal antibodies were detected with FIT-C or TRIT-C conjugated goat anti-mouse IgG (γ-specific) and anti-mouse IgM (μ-specific) (Jackson Immuno-Research laboratories, Bar Harbor, ME, USA). In all experiments, incubation with primary antibodies was done overnight at 4°C, followed by 2 h

at room temperature with the appropriate secondary antibodies. The sections were mounted LY2157299 in antiquenching medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA, USA). Labelled brain sections were viewed with a 40× Plan-Apochromat on a TCP-SP2 Leica (Heidelberg, Germany) laser scanning-confocal microscope. Additional high power lenses (60× and 100×) were used to critically evaluate colocalization in single optical sections. Confocal images were obtained as single sections and the stack of images was projected as individual two-dimensional extended focus images. Resulting images were analysed using the software included

with the microscope and Image J (Image Processing and Analysis in Java) software. Using the peroxidase technique, NFTs were counted in the area of interest (see Table 2). Morphometric quantification in the areas was assessed on three microscopic fields from randomly chosen regions in the area of interest. Observations were conducted by bright-field microscopy (Nikon FN1, Melville, NY, USA). Identification and counting of pathological structures why was conducted using 10× and 20× objective lenses and values expressed per mm2 as previously described [33]. Relative expression intensity was measured in neurones by using Image

J software (Image Processing and Analysis in Java). Values represent relative surface area expression. Student’s t-test was applied when counts were compared between different groups. Statistical analysis was conducted in Excel. Bar diagrams represent the experimental mean; the error bars represent the standard error. For statistical analysis we used the Student’s t-test with the significance set a P-value of 0.05. As mesocortices and the hippocampal formation are the most vulnerable brain areas to NFTs, they were the focus of this study. Mesocortices include entorhinal cortex, perirhinal cortex while the hippocampal formation contains parasubiculum, presubiculum, subiculum, CA1, CA2, CA3, CA4, and dentate gyrus. The same groups of neurones were compared with regard to morphological and cytopathological observations of NFTs for the different tau antibodies. For example, entorhinal layer II was compared in each case with all the tau antibodies. Furthermore, NFTs were compared across areas within each case.

34–36 Despite these anti-inflammatory properties of

IgA, its deposition in the skin is observed in inflammatory dermatoses such as blistering diseases and Henoch–Schönlein purpura and is associated with neutrophil infiltration and tissue injury. The IgA-induced pro-inflammatory properties include promoting the release of pro-IL-1β and FcαRI cross-linking can induce tumour necrosis factor-α and IL-6 from PBMC.37,38 Therefore, the presence of IgA in L-lep skin lesions may also promote the acute inflammation observed in patients developing ENL from L-lep. In fact, single nucleotide polymorphisms of the FcαRI promoting inflammation have been described in patients with systemic lupus erythematosus.39 The balance of anti- and GSK-3 inhibitor pro-inflammatory effects of IgA, as well as the ability to respond to IgA based on allelic differences among patients, may determine whether patients develop acute inflammatory reactions such as ENL in leprosy. The mechanisms by which B cells accumulate and differentiate in leprosy lesions are unresolved. Our data Selleck Dabrafenib suggest a role for T-cell production

of IL-5 in L-lep lesions in the presence of M. leprae to promote B-cell production of IgM. Although antibodies may be key in early responses for protection, the presence of B cells and their mediators in chronic infection may contribute to immunopathology. Insight into the mechanisms of antibody production may provide targets for monitoring and intervention in the treatment of tissue injury. We thank Dr Matthew GNA12 Schibler and the Advanced Light Microscopy core facility at the UCLA California Nanosystem Institute for use of the confocal

laser microscope and the UCLA Flow Cytometry core laboratories for use of the flow cytometer. We acknowledge the financial support received from the National Institutes of Health (AI022553 to R.L.M. and AR053104 to D.J.L.). The authors have no conflicts of interests to declare. “
“Natural killer T cells expressing an invariant T cell antigen receptor (iNKT cells) are cells of the innate immune system. After recognizing glycolipid antigens presented by CD1d molecules on antigen presenting cells (APCs), iNKT cells rapidly produce large quantities of cytokines, thereby stimulating many types of cells. Recent studies have described several mechanisms of iNKT cell activation and the contribution of these cells to antimicrobial responses. iNKT cells can be activated by endogenous antigens and/or inflammatory cytokines from APCs. However, iNKT cells also recognize certain microbial glycolipids by their invariant T cell antigen receptor (TCR), and they contribute to pathogen clearance in certain microbial infections. These findings indicate that the iNKT TCR is useful for detecting certain microbial pathogens. Moreover, recent studies suggest that iNKT cell glycolipid antigens may be useful in antimicrobial therapy and vaccines. Natural killer T cells are lymphocytes that express both αβ TCRs and NK receptors (1–4).

30 Moreover, LPS was shown to induce the up-regulation of COX2/PGE2 in RAW macrophages.31 The effects of a brief (10 min) treatment with PGE2 on CGRP release from dorsal root ganglion cultures have been reported before.32,33 We observed here that longer PGE2 treatment (24 hr) induced or enhanced LPS-stimulated CGRP release from RAW macrophages. As PGE2-induced CGRP release was blocked by the co-treatment with actinomycin-D or cycloheximide, de novo mRNA transcription and protein synthesis are most

likely involved. These findings suggest that long-term PGE2 treatment may not only increase the release of CGRP, but Natural Product Library also its transcription and synthesis in RAW macrophages. However, the PGE2 EP receptor subtype(s) involved here, as well as downstream signal transduction pathways, requires further studies. In parallel with previous reports showing that NF-κB is involved in LPS-induced production of inflammatory mediators in monocytes/macrophages,12,34 co-treatment of LPS with an inhibitor of IκB phosphorylation suppressed LPS-induced CGRP release. This finding suggests that the NFκB signalling pathway is involved in LPS-induced CGRP synthesis in RAW macrophages. Our data are comparable

to those in a previous report showing that NF-κB plays a role in IL-1β-induced CGRP secretion from human alveolar epithelial cells.16 However, how NF-κB mediates Selleckchem R428 LPS-induced synthesis of CGRP has yet to be fully established. Unexpectedly, we found that CGRP receptor accessory protein RAMP1 and NGF/trkA receptor signalling were negatively involved in LPS-induced CGRP synthesis. The CGRP receptor is a rather unique G protein-coupled receptor, because it shares a seven trans-membrane domain protein, CLR, with adrenomedullin (AM, a peptide member in the CGRP superfamily) and

also requires accessory protein RAMP1 to be functional. The RAMPs are essential accessory Hydroxychloroquine price proteins to chaperone CLR to the cell surface, which determines the receptor specificity.35 RAMP1 enables CLR to form CGRP receptor while RAMP2 and RAMP3 enable CLR to form AM1 and AM2 receptors,36 respectively. To our surprise, neutralizing antisera against either CGRP/RAMP1 or NGF/trkA receptor dramatically enhanced LPS-induced CGRP release, suggesting that RAMP1 and trkA exert negative feedback effects on the synthesis of CGRP. Neutralizing trkA or RAMP1 antiserum on their own had no effects on basal CGRP release from RAW macrophages, suggesting that the negative feedback action of trkA or RAMP1 occurs only when NGF or CGRP is up-regulated by inflammatory stimuli. Accordingly, when NGF or CGRP is increased, activation of RAMP1 or trkA receptor signalling can exert an inhibitory action on CGRP synthesis in RAW macrophages. This hypothesis is supported by a recent report showing that levels of serum CGRP in homozygous RAMP1-deficient mice were dramatically and transiently increased following peritoneal LPS challenge.

Adoptively transferred p14 CD8+ T cells coexpressed CD44, PD-1 and IL-7Rα as analyzed by FACS analysis of blood (Fig. 2G, Supporting Information Fig. 2C) and spleens (data not shown) 5 days after transfer. Thus, CML-specific CTL display an activated phenotype but retain IL-7Rα APO866 in vitro expression. The fact that specific CTL downregulate IL-7Rα expression in the presence of a chronic infection but maintain IL-7Rα expression in the presence of CML expressing the same viral antigen was surprising and led

to the question if IL-7 production is increased in CML mice. To analyze this, we compared IL-7 expression in mRNA isolated from spleen of CML and naïve C57BL/6 mice by RT-PCR. The thymus as organ with documented high IL-7 production served as a positive control. IL-7 mRNA was detectable in the spleen of CML and of naïve C57BL/6 mice (Fig. 3A and Supporting Information Fig. 3). Next, we analyzed whether IL-7 mRNA is detectable in CML granulocytes and in control granulocytes. We therefore quantitatively compared IL-7 mRNA production of sorted GFP+ granulocytes from CML mice with sorted granulocytes from C57BL/6 mice.

Surprisingly, IL-7 mRNA was detectable in both malignant and control granulocytes (Fig. 3B). Moreover, this experiment revealed that IL-7 mRNA was not differently expressed in malignant and in normal granulocytes. However, the total number of granulocytes in the spleen of mice with CML is three to four-fold higher than that found in C57BL/6 control mice (Fig. 3C). These findings were confirmed by quantification of IL-7 protein levels per MK0683 solubility dmso milligram spleen of naïve C57BL/6 mice and CML mice (Fig. 3D). Furthermore, IL-7 was detectable by intracellular staining of brefeldin-treated malignant (GFP+) and normal (GFP−) granulocytes but not in granulocytes from IL-7-deficient mice (MFI increase of IL-7 in MycoClean Mycoplasma Removal Kit GFP− granulocytes (12.4±2.9%) and GFP+ granulocytes (11.4±2.9%)

(Fig. 3E and F)). Taken together, the malignant granulocytes produce IL-7 and are increased in numbers in secondary lymphoid organs such as the spleen. To study the role of IL-7 produced by leukemic cells in more detail, H8×IL-7-deficient mice were used as bone marrow donors (H8×IL-7−/−-CML mice) to establish CML disease in C57BL/6 recipients. In this experiment, the leukemic cells will not produce IL-7. However, stromal and epithelial cells of the recipient mouse are capable of IL-7 secretion. Purified p14 CD8+ T cells (CD45.1+CD8+Vα2+) were adoptively transferred to H8×IL-7−/−-CML mice, H8-CML and naïve C57BL/6 mice. P14 CD8+ T cells expanded similarly in H8×IL-7−/−-CML mice and in H8-CML mice (Fig. 4A). However, significantly more p14 CTL survived long term in H8-CML mice than H8×IL-7−/−-CML mice (analyzed in blood: H8-CML: 8.2±3.7%; H8×IL-7−/−-CML: 1.2±0.6%; p=0.04).

Stably transfected cells were cultured in RPMI-SM + 2 μg/ml puromycin (Sigma, Munich, Germany). The complementary DNA (cDNA) coding for the scFv antibody recognizing the human CD3ε chain was kindly provided by Dr Thirion (Dr L Willems-Instituut, Diepenbeek, Belgium).42 The cDNA coding for the scFv antibody recognizing human CD19 antigen was kindly provided by Ibrutinib Professor Zola (Child Health Research Institute, Women’s and

Children’s Hospital, Adelaide, South Australia).43 Peripheral blood mononuclear cells (PBMC) used for the proliferation and cytotoxic assays were collected from healthy donors and purified as previously described.44 PBMC used for Ca2+ imaging experiments were purified from leucocyte reduction filters obtained from the local blood bank. Cells were collected by back-flushing the filter with 60 ml Hanks’ balanced salt solution (HBSS; PAA, #15-009) and the peripheral blood lymphocytes (PBL) were isolated by a density gradient centrifugation at 450 g R428 for 30 min at room temperature (Ficoll-Paque™plus; Amersham Biosciences, Freiburg, Germany; #17144002) in 50-ml Leucosep tubes (Greiner, Frickenhausen, Germany; #227290). The PBL layer was washed in HBSS. The remaining red blood cells were removed by the addition of 1 ml lysis buffer (155 mm NH4Cl, 10 mm KHCO3, 0·1 mm ethylenediaminetetraacetic acid, pH 7·3) for 1 min. After lysis, the

cells were washed with HBSS (200 g, 10 min, room temperature). For further purification, the PBL were resuspended in phosphate-buffered saline (PBS)/0·5% bovine serum albumin (BSA) and CD4+ T cells Cell press were negatively isolated using the CD4+ Negative Isolation kit (to avoid pre-stimulation) from Invitrogen (#113.17D) following the manufacturer’s instruction. After isolation, the purity of the CD4+ populations was analysed by fluorescence microscopy [anti-CD4/R-phycoerythrin (RPE) -conjugated antibody; Dako, Hamburg, Germany; #R0805]. CD4+ cells were cultured in AIMV medium (Invitrogen, #12055-091) supplemented with 10% fetal calf serum. To generate

effector cells from the primary naïve CD4+ cells, the cells were either incubated with anti-CD3/anti-CD28-coated beads or with 12 U/ml human interleukin-2 (hIL-2; Roche, Mannheim, Germany) and 3 μg/ml phytohaemagglutinin (PHA, Sigma).23 The cDNA sequences coding for the extracellular domains of CD80 and CD86 were amplified from human PBMC using standard reverse transcription–polymerase chain reaction (RT-PCR) technology as described elsewhere.44 The variable heavy chain (HC) and light chain (LC) sequences of anti-human CD33 antibodies45 were amplified by PCR using specific primers including restriction sites (NcoI–HindIII for HC, EcoRV–BamHI for LC) compatible with the pHOG expression vector and expressed as scFv fragments.