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<strong>2. MATERIALS ANDMETHODS</strong> <p><strong>2.1. DC and T lymphocytes preparation</strong></p> <p><strong>2.1.1. Donors</strong></p> <p>Blood was collected from allergic patients sensitive to house dust mite (specific IgE antibodies; positive skin prick tests towards Dermatophagoides pteronyssinus (Dpt) (RAST class > 3); total serum IgE > 250 kU/mL) and from healthy donors (total IgE level </p> <p><strong>2.1.2. Cell preparation</strong></p> <p>Monocyte-derived DC were generated from blood monocytes purified by positive selection using monoclonal anti- CD14 antibodies coupled to magnetic microbeads (Miltenyi Biotech, Bergsch Gladbach, Germany) as described [20]. Cells were cultured at 1 × 106 cells/ml for 5-6 days in complete medium containing 25 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF) (Preprotech, London, UK) and 10 ng/ml IL-4 (R&D system, Oxon, UK) to obtain immature DC. At the end of the culture, 95% of the population is CD1a+HLA-DR+CD80/C86low.</p> <p>Naive CD4+ T cells were isolated from the eluted CD14− cell fraction by negative selection using a CD4+CD45RA+ T cell isolation kit (Miltenyi Biotech, Germany) (purity > 95%) and frozen until use.</p> <p><strong>2.2. Caco2-DC Transwell coculture system</strong></p> <p>To mimic the intestinal barrier, we used an in vitro Transwell coculture system as described [10]. Caco2 cell line was grown in Dulbecco’s MEM (Cambrex Bio Sciences, Verviers, Belgium) supplemented with 10% heat-inactivated fetal calf serum(Invitrogen, Paisley, UK), 1% nonessential amino acid (Gibco BRL, UK), and antibiotics. Cells were seeded on the upper face of 6.5mm filters (3 μm pore Transwell filters, Corning Incorporated, Acton, MA) for 15 days until a transepithelial resistance (TER) of ∼ 300Ω/cm2 was achieved. Transwell filters were turned upside-down, and immature DC (1 × 106) were added for 3 hr on the filter facing the basolateral membrane of the epithelium to allow cell attachment. Filters were replaced into 24-well plates. TER was checked to be unchanged during the coculture with DC and after L. casei stimulation (Grangette personal observation) as described [21].</p> <p><strong>2.3. Preparation of bacteria</strong></p> <p>L. casei ATCC 393 was prepared as previously described [20]. Briefly, the bacteria were cultured overnight in MRS broth medium (Difco, Detroit, MI), at 37◦C. The bacterial suspension was diluted at 1 : 20 in fresh medium and further cultured until exponential phase. After washing, bacteria were resuspended in PBS containing 20% of glycerol and stored at −80◦C before using. The bacteria concentration was determined as described [20].</p> <p>As live and killed LAB were not different in their capacity to regulate peripheral blood mononuclear cells (PBMC) or DC stimulation ([19, 20], Grangette personal observation), we used killed LAB obtained after fixation by 45min incubation in endotoxin-free PBS 4% paraformaldehyde (PFA).</p> <p>These killed LAB were stored at +4◦C until use.</p> <p><strong>2.4. Uptake analysis</strong></p> <p><strong>2.4.1. FITC-labelled bacteria</strong></p> <p>Fixed bacteria were resuspended in RPMI (1 ml) and incubated with ethanol (2ml, 70%) for 1 h at room temperature.</p> <p>After centrifugation (2500 g, 15 min), bacteria were suspended in carbonate/bicarbonate (pH = 9.7) buffer and incubated with 0.1 mg/ml fluorescein isothiocyanate (FITC) (Sigma, Germany) for 1 h, with constant stirring. After washing in PBS 0.1% gelatine, bacteria were stored in RPMI at −20◦C before use. FITC-labelled bacteria were added on the apical side for 24 or 48 h at the dose of DC/bacteria: 1/10, 1/100. TER were checked to be unchanged during this time. </p> <p><strong>2.4.2. Flow cytometer analysis</strong></p> <p>Monocyte-derived DC from allergic (n=2) or healthy (n = 2) donors, adherent or not to the filter, were collected and immediately analyzed by FACScalibur (Becton Dickinson, San Diego, CA).</p> <p><strong>2.4.3. Cell staining for confocal microscopy</strong></p> <p>After 48 h of culture, Transwell filters were washed and fixed 30 min in PFA (4%). After 15 min treatment with PBS 0.5% Triton (Hopkin & Williams) and 30min saturation with human serum (2% in PBS), filters were incubated either with monoclonal anti-human CD11c antibody coupled to Phycoerythrin (PE) or with an irrelevant antibody (Becton Dickinson) for 30 min. After washing, filters mounted with Fluoprep (Biom´erieux SA, France) were analyzed with confocal laser scanning microscope (SP2 AOBS, LCS software, Leica, Wetzlar, Germany).</p> <p>The dose of 100 bacteria for 1 DC, allowing the highest uptake intensity in preliminary studies, was chosen for all the experiments with the epithelium (data not shown).</p> <p><strong>2.5. DC activation</strong></p> <p>After 48 h of culture with bacteria, monocyte-derived DC from allergic (n = 6) or from healthy (n = 7) donors were harvested for phenotype analysis and their supernatants collected. Only one test per individual was performed in each group.</p> <p><strong>2.5.1. DC surfacemarker analysis</strong></p> <p>Monocyte-derived DC adherent or nonadherent to the filter were washed in PBS and incubated for 30min at 4◦C with different monoclonal antibodies (mAbs) : FITC-conjugated anti-CD86, anti-HLA-DR, and PE-conjugated anti-CD80, anti-CD54 or irrelevant mAbs (Becton Dickinson). Cells were fixed in PBS 1% PFA and analyzed using a FACScalibur (Becton Dickinson). Variations in DC phenotype were expressed as the difference betweenmean fluorescence intensity (MFI) minus the isotype control MFI (ΔMFI).</p> <p><strong>2.5.2. Cytokine production by DC</strong></p> <p>DC supernatants were harvested from the lower chamber of the Transwell 48 h following stimulation and assayed for the presence of IL-10 or IL-12 (IL-12 p70) by specific ELISA (Diaclone, France). The sensitivity of both assays was 5 pg/ml.</p> <p><strong>2.6. T cell activation</strong></p> <p><strong>2.6.1. DC-T coculture</strong></p> <p>After 24 hours of culture with bacteria, monocyte-derived DC (allergic: n = 5; healthy: n = 5) activated in the Transwell system and adherent or not to the filter were harvested from the lower chamber (Figure 1(a)). After washing, DC were resuspended in complete RPMImedium (105/ml) and cultured with autologous CD4+CD45RA+ T cells (106/ml) (ratio: 1 DC/10 T lymphocytes) for 5 days. Only one test per individual was performed in each group.</p> <p><strong>2.6.2. Cytokine production by T cells</strong></p> <p>Supernatants were collected and assayed for IL-5 (PharMingen), IL-10, and IFN-γ (Diaclone) by specific ELISA (sensitivity = 5pg/ml). </p> <p><strong>2.7. Statistical analysis</strong></p> <p>Nonparametric statistical analyses were performed with paired samples and the permutation tests (STATEXACT, Cytel Software,MA). P values of .05 or less were considered statistically significant.</p>
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