Therefore, it is important to address whether transfer of immature recipient’s DC loaded with donor antigens could also induce anti-allotolerance or even working better than directly transfer donor’s DC. In this study, we generated recipient’s immature DC by adenoviral infection with a kinase-defective dominant negative form of IKK2 (IKK2dn), then loaded with donor antigens and tested their ability to induce anti-allotolerance in an allo-kidney graft rat model. Our results indicated that IKK2dn-transfected DC are capable of inducing tolerance and significantly Lorlatinib in vitro prolonged transplanted allograft survival by reducing B7-1 and B7-2 expression,
increasing IL-10 and decrease IFN-γ production. Animals and reagents. Male Lewis (LEW/CrlBR), Brown Norway (BN/CrlBR), and Wistar (Crl:(WI)BR) rats, 8–10 weeks, body weight around 180–200 g, were purchased from Charles River (Vital River, Beijing, China) and maintained in the university animal facility. Procedures involving animals and their care were conducted FK228 in vitro in accordance with the institutional guidelines that are in compliance with national and international
laws and policies. The recombinant rrGM-CSF and rrIL-4 were purchased from Peprotech (Rocky Hill, CT, USA). FITC or PE-conjugated mouse anti-Rat CD80, CD86, and MHC-II antibodies were purchased from Serotec (Oxford, UK); IL-2, IL-10, IFN-γ ELISA kits are from R&D (Minneapolis, MN, USA). All other reagents are from Sigma (St. Louis, MO, USA). The replication-deficient adenovirus encoding a kinase-defective dominant negative form of human IKK2 plasmid, pACCMVpLpASR(+)-IKK2dn, was a kind gift from Dr Rain D Martin (University of Vienna, Vienna, Austria). pAdxsi-GFP-IKK2dn and pAdxsi-GFP-0 were constructed by SinoGenoMax Company, Beijing. DC preparation
and gene transfer. Bone marrow cells were obtained from the tibias and femurs of Lewis rats. Bone marrow cells (2 × 106/ml) were cultured in 6-well plates (Becton Dickinson, Heidelberg, Germany) with recombinant rat granulocyte macrophage-colony Anacetrapib stimulating factor (GM-CSF) (5 ng/ml), in combination with recombinant rat IL-4 (5 ng/ml). On day 3 and every other day after, half of the medium containing the appropriate cytokines was replaced. Recombinant, replication-deficient adenoviral vectors encoding IKK2dn and Adv-0 were constructed as follows: IKK2dn cDNA was cloned into adenovirus transfer vector pShuttle-CMV-GFP(−)TEMP (Sinogenomax, Beijing, China) and analysed by restriction endonuclease KpnI & HindIII digestion. Then, the obtained plasmid, pShuttle-CMV-GFP(−)TEMP-IKK2dn was transferred into pAdxsi vector to construct pAdxsi-GFP-IKK2dn plasmid and was identified by XhoI digestion. The correct adenoviral recombinant was then cleaved with Pac I and transfected into 293 cells to produce and purify viral particles.