These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells

These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. induce DC maturation. Natural interferon producing cells (NIPC, or plasmacytoid DCs) represent a danger-recognition system of the immune defences, and can respond to viruses not otherwise recognized as posing a danger. Indeed, MoDC did not respond to transmissible gastroenteritis virus (TGEV), whereas NIPC produced high levels of IFN- and TNF- after TGEV stimulation. Moreover, supernatants from the stimulated NIPC induced maturation in MoDCs. These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. Taken together, the present work demonstrates that maturation of MoDC not only results from TLR signalling, but can require co-operation with various cell types C principally NIPC and activated T cells C which would CL2-SN-38 reflect the particular immunological situation. 05) and T() 4 implying that the two distributions are different ( 001). An empirical determination for the T() cutoff value suitable for the present labellings was determined by measuring the variability within 10 replicates of unstimulated cultures labelled for CD80/86, MHC II and OVA-dQ. Within these labellings the T(X) values varied between 01 and 15. Based on this, a T(X) cutoff value 20 was selected for our data in order to have a stringent cutoff. T-cell stimulation assaysLymphocytes were purified by depletion of CD172a+ cells (made up of monocytes and APCs10) using the MACS system. Lymphocytes were cultured at 2 105 CL2-SN-38 cells/well. The microbial superantigen staphylococcal enterotoxin B (SEB; Alexis) was used to measure the potency of DCs at inducing T cell responses through MHC IICT-cell receptor cross linking.18,19 MoDCs were incubated CL2-SN-38 with SEB (100 ng/ml) for 1 hr at 39, washed four times and titrated in 96-well plates as described. After 4 days, 1 Ci (37 kBq) [3H]thymidine was added for another 18 hr to quantify the proliferation. The processing and presentation of antigen was assessed in tetanus toxoid (TT) antigen-specific T-cell proliferation assays. To this end, lymphocytes and MoDCs isolated from two pigs vaccinated against TT by immunizing with human tetanus vaccine (kindly provided by the Netherlands Vaccine Institute, Bilthoven, Netherlands) followed by a booster immunization after 4 weeks. Immature and matured MoDCs were cultured with CL2-SN-38 T cells (2 105 cells/well), to obtain T/DC ratios of 10 : 1, 30 : 1 and 90 : 1. TT or diphtheria toxoid (DT, used as a negative control antigen; Netherlands Vaccine Institute) were added at 12 Flocculation units/ml (Lf/ml). Lymphoproliferation was quantified in triplicate cultures after 5 days by measuring [3H]thymidine uptake as described above. Only immunized pigs responded to TT antigen in a T-cell proliferation assay. Cytokine responsesEnriched NIPC or MoDC were stimulated with TGEV or CpG oligodeoxynucleotides (CpG-ODN) (sequence D32 ggT GCG TCG ACG CAG ggg gg, 10 g/ml11). The virus was prepared from infected ST cell lysates as described10 and used a SA-2 multiplicity of contamination 2 TCID50/cell. Lysate preparations from uninfected ST cells were used as mock controls. Intracellular cytokine detection was after 6 hr, analysis of secreted cytokines in cell supernatants after 24 hr. Secreted IFN- was detected by enzyme-linked immunosorbent assay (ELISA) using with anti-porcine IFN- mAbs K9 and F17 (supplied by Dr Bernard Charley kindly, INRA, Jouy-en-Josas, France) as referred to.11 TNF- was assayed with actinomycin d-treated PK15-15 cells.20 IL-6 was analysed utilizing a duoset ELISA package from R & D systems (Oxford, UK). After staining of cell surface area substances, the cells had been set and permeabilized (Repair & Perm, Caltag, CA) and stained with anti-IFN- mAbs F17 and K9 (10 g/ml; R & D Systems) or anti-porcine TNF- mAbs (clone 4F4, Perbioscience). For movement cytometry recognition, isotype-specific FITC, R-PE (Southern Biotechnology Affiliates) and R-PE-Cy5 (Dako) conjugates had been utilized as referred to.11 Inhibition of IFN- induced by TGEV was attained by blocking anti-protein M mAb (clone 25-22), kindly supplied by Dr Bernard Charley, INRA). For neutralization of IFN-, examples had been incubated for 30 min on snow with 1 l of rabbit polyclonal antibody against porcine IFN- (as indicated from the supplier.