α-GalCer (Alexis Biochemicals) and β-GalCer (Galactocerobroside, Sigma) were dissolved in DMSO. The anti-CD1d mAb WTH-1 [13] was added to the cultures 30 min before the addition of any stimuli. Spots were analyzed

and enumerated using the CTLImmunoSpot S5 Versa analyzer ELISPOT reader and the ImmunoSpot 4.0 Software (both from CTL). Small spots (smaller than 0.096 mm2) obtained in cultures with medium only were considered nonspecific background and were subtracted from all the samples. Single cell suspensions prepared from spleens and livers were plated at a density of 106 cells per 1 ml of RPMI 1640 supplemented as aforementioned. Cells were cultured with 20 ng/ml α-GalCer during the first 7 days. During the second week, the cells were cultured Pirfenidone price with 10 ng/ml α-GalCer and 10% of T-cell

growth factor-containing medium (supernatant from Con A-stimulated rat splenocytes blocked with α-methylmannoside) usually adding fresh media at day 13. We would like to especially thank T. Hünig for his continuous support to this project and N. Beyersdorf for critical reading of the manuscript and helpful comments. This find more work was supported by the Deutsche Forschungsgemeinschaft Graduate College 520 Immunomodulation and HE 2346/6-1. EMC was also supported by a STIBET Doktoranden grant of the Deutsche Akademische Auslandsdienst. DBS was supported by NIH NIAID R01 AI083988 and AI059739 and by the Robert Wood Johnson Foundation (grant no. 67038) to the Child Health Institute. The authors declare no financial or commercial conflict 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 Pregnenolone (other than missing files) should be addressed to the authors. Figure S1. Figure S2. Table S1. Table S2. Table S3. “
“Dendritic cells (DC) are key factors in regulating immune responses, and they induce immune response or tolerance depends on its maturation states. Previous studies demonstrated that blocking IKK2 in bone marrow-derived dendritic cells (BMDC) by adenoviral transfection with a kinase-defective dominant negative form of IKK2 (IKK2dn) could inhibit NF-κB activation and impair DC maturation. Here, we transfected IKK2dn into recipient rat (Lewis) BMDC by adenovirus vector (Adv-IKK2dn-DC) and found that Adv-IKK2dn-DC had reduced B7-2 and B7-1 expression under alloantigen stimulation. Their ability to induce allogeneic T-cell proliferation was markedly reduced in comparison with uninfected DC. A higher IL-10 secretion and a lower IFN-γ secretion were detected in Adv-IKK2dn-DC-stimulated allogenic T cells. Furthermore, we showed that Adv-IKK2dn-DC pulsed with BN (Brown Norway rats) splenocyte lysates markedly prolonged the survival of renal allografts in an antigen-specific manner.