Subsequent allergen contact leads to a boost of allergen-specific IgE and T cell responses as well as to allergic inflammation (28). sensitized mice. However, co-stimulation blockade had no influence on established IgE responses in sensitized mice. Immediate type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by co-stimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and secondary allergen-specific antibody responses. Allergen-specific T cell activation was suppressed in mice by early as well as late co-stimulation blockade suggesting that IgE-responses Rabbit Polyclonal to TIMP1 in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in allergy. Y1090 were grown overnight in LB medium containing 0.4 % w/v maltose and 50 g/ml ampicillin, harvested by centrifugation and resuspended in 10 mM MgSO4. Cells were dissolved in 0.6 % w/v agarose and plated onto LB plates containing 50 mg/L ampicillin. Two l aliquots of phage lysates containing 105 Pfu were dotted onto the plates. Plates were incubated at 43C until plaques became visible and protein synthesis was induced by overlay with nitrocellulose filters (Schleicher & Schull, Dassel, Germany) soaked with 10 mM IPTG for 4 h at 37C. Filters were cut into stripes. Stripes, containing Leucyl-alanine the expressed allergen fragments from clones 11,14, 21,26, 47, 50, 57, 59, 68, 81, 117, and 120, and the phage gt11 as negative control, or 1 g rPhl p 5 as positive control, were incubated with mouse sera diluted 1:1000 overnight, a monoclonal rat anti-mouse IgG1 antibody (Pharmingen, San Diego, USA) diluted 1:1000 for 5 h, and a 125I-labelled goat anti-rat IgG antibody (Sigma-Aldrich, St.Louis, MO, USA) diluted 1:2000 for 2 h. Leucyl-alanine Reactivity with the allergen fragments was detected by autoradiography. The intensities of the signals where determined by densitometry using the AlphaEaseFC? ChemiImager 4400 software. ELISA experiments To measure antigen-specific antibodies in the sera of immunized mice an ELISA was performed as described earlier (14, 26). Plates were coated with rPhl p 5 (5g/ml), sera were diluted 1:10 for IgE, 1:100 for IgM, IgA, and IgG2a, and 1:1000 for IgG1 and bound antibodies where detected with monoclonal rat anti-mouse IgM, IgG1, IgE, IgA, and IgG2A antibodies (Pharmingen, San Diego, USA) diluted 1:1000 and a HRP-coupled goat anti-rat antiserum (Amersham, Biosciences, U.K.) diluted 1:2000. T cell proliferation assay Spleens were removed under aseptic conditions (day 100) and Leucyl-alanine homogenized. After lysis of erythrocytes, cells were washed and re-suspended in complete medium (RPMI, 10% fetal calf serum, 0.1 mg/ml gentamicin, 2mM glutamine). Single cell suspensions were plated into 96-well round-bottom plates at a concentration of 2 105 cells / well (200 l) in triplicates and stimulated with or without concavalin A (0.5g/well), rPhl p 2 (3g/well), and rPhl p 5 (3g/well) for 4 days. The cultures were pulsed with 0.5 Ci / well tritiated thymidine for 16 h and harvested. The proliferation responses were measured by scintillation counting. The ratio of the mean proliferation after antigen stimulation and medium control values, i.e. the Leucyl-alanine stimulation index (SI), was calculated. RBL assay For the quantification of IgE antibody-mediated immediate type reactions, rat basophil leukemia (RBL) cell mediator release assays were performed as previously described (27). RBL-2H3 cells were cultivated in 96 well tissue culture plates (4104 cells/well) for 24 h at 37 C using 7% CO2. Passive sensitization was performed by incubation with 1:30 diluted murine sera for 2 h. Cells were washed twice with Tyrode’s buffer (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 0.4 mMNaH2PO4, 5.6 mM D-glucose, 12 mM NaHCO3, 10 mM HEPES, and 0.1% w/v BSA, pH 7.2) to remove unbound antibodies. Leucyl-alanine Degranulation of RBL cells was induced by adding 0.3 g/ml rPhl p 5. After 30 min, ?-hexosaminidase release was analysed. Results are expressed as percentages of total ?-hexosaminidase released after addition of 1% Triton X-100 and represent mean of triplicate determinations. Mixed lymphocyte reaction (MLR) As a positive control for the biological activity of CTLA4Ig, an MLR was performed in the presence of 100 g/ml CTLA4Ig. Spleen cells were washed with PBS and resuspended in MLR-Medium (42.5 ml RPMI 1640 (Bio-Whittaker), 7.5 ml CPSR-2 (Sigma), 0.5 ml HEPES buffer (ICN, Biomedica, Vienna Austria), 1.55 ml Nutrient.