We recently reported that blockade of the CD40CCD154 ligand interaction with

We recently reported that blockade of the CD40CCD154 ligand interaction with the cross-reacting mouse anti-human CD154 antibody, 5c8, together with donor-specific transfusion led to enhanced but not completely successful engraftment in a canine model of DLA-identical marrow transplantation after 100cGy total body irradiation (TBI). 1997; Hogan et al., 2003). However, A-443654 when TBI conditioning was decreased to 1 1 Gy, all dogs eventually rejected their grafts. Extended and sustained engraftment was accomplished in most but not all dogs when 1 Gy TBI was preceded by intravenous injections of both peripheral blood mononuclear cells (PBMC) from the marrow donor and the T-cell costimulatory blockers recombinant human (rh) CTLA4-Ig or cross-reacting mouse anti-human CD154 antibody 5c8 (Storb et Rabbit Polyclonal to CLIC6. al., 1999; Jochum et al., 2007). One possible explanation for the lack of uniform success might be reduced affinity of these cross-reacting anti-human products for canine cell surface determinants. A-443654 Therefore, we focused on developing a canine specific reagent to block the CD40CCD154 interaction. Instead of generating an anti-CD154 monoclonal antibody, we developed a canine specific fusion protein, CD40-Ig. In other similar studies, CD40-Ig has been shown to be active with human (McLellan et al., 1996) cells and in rodent models of liver (Nomura et al., 2002), heart (Guillot et al., 2002), and other organ transplantation models (Jin and Xie, 2003; Kanaya et al., 2003; Yamashita et al., 2003). 2. Materials and Methods 2.1. Experimental animals and blood cell preparations Beagles, mini-mongrel, basenji, and golden retriever crossbreeds used for all experiments were raised at the Fred Hutchinson Cancer Research Center (Seattle, WA, USA) or purchased from commercial kennels. PBMC were isolated on Ficoll-Hypaque (density 1.074). Lymph node and A-443654 tonsil cells were obtained from dogs, which were euthanized for other reasons. 2.2. Cloning of the extra cellular domain of canine CD40 Oligonucleotides were custom-made by Invitrogen (Carlsbad, CA, USA). Total RNA was isolated from the lymph node, tonsil, and thymus using TRIzol reagent (Invitrogen). cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen) and oligo (dT) primer (Promega, Madison, WI, USA). The cDNA of CD40 was synthesized by RT-PCR using Platinum PCR Supermix (Invitrogen) and a forward primer (CGGGAATATTACGGGGAACT) and a reverse primer (CCACTGAATCACAAACAATGCC) based on the GenBank sequence (“type”:”entrez-nucleotide”,”attrs”:”text”:”AY333789″,”term_id”:”32700004″,”term_text”:”AY333789″AY333789) of CD40 mRNA. The PCR product was isolated from an agarose gel using QIAquick Gel Extraction kit (Qiagen, Valencia, CA) and ligated into the pGEM-T Easy vector (Promega, Madison, WI) for sequencing. DNA sequencing was performed with an automated sequencer by PCR amplification using BigDye terminator v3.1 reagents (Applied Biosystems, Foster City, CA) and T7 and SP6 promoter primers (Promega)E 2.3. Cloning of murine IgG2a The cDNA of murine IgG2a was isolated from the IgG2a-secreting mouse myeloma cell line RPC5.4 (ATCC, Manassas, VA) by RT-PCR using Platinum PCR Supermix and a forward primer (TAAAGAGCCCAGAGGGCCCACAATCAA) and a reverse primer (TCATTTACCCGGAGTCCGGGAGAA) based on the GenBank sequence (“type”:”entrez-nucleotide”,”attrs”:”text”:”V00798″,”term_id”:”51835″,”term_text”:”V00798″V00798) of mouse gamma 2a immunoglobulin heavy chain. The PCR product was isolated and ligated into the pGEM-T Easy vector (Promega, Madison, WI) for sequencing as outlined above. 2.4. Assembly of canine CD40 murine Ig fusion vector A-443654 An AflII and HindIII restricted PCR product of the signal peptide and extracellular domain of CD40 was generated from CD40 cDNA using forward (CATTAGCTTAAGATGGTTCTCCTGCCTCTGCGC) and reverse (TCCGGGAAGCTT-GGCTCTTAACCGAGGCTGGGG) primers. A HindIII restriction site and a Gly4Ser linker were added at the 5 end of the hinge region and a NotI restriction site was added at the 3 end of the CH3 region of murine IgG2a using forward (ATAATTAAGCTTGGAGG-TGGAGGTAGTGAGCCCAGAGGGCCCACATC) and reverse (CCATTATAGCGGCCG-CTCATTTACCCGGAGTCCGGGA) primers, respectively (Figure 2). Following gel purification, PCR products were digested with the appropriate restriction enzymes and ligated into AflII and NotI digested pcDNA3.1 (+) (Invitrogen). Plasmids from DH5 (Invitrogen) transformants were sequenced with T7 forward and BGH reverse primers. Figure 2 Schematic diagram of CD40-Ig expression vector containing the leader and extracellular domain of canine CD40 fused to a Gly4Ser linker and the hinge A-443654 through CH3 regions of murine IgG2a. 2.5. Cell culture and protein production CHO cells deficient in the gene (CRL-9096; ATCC) were co-transfected with linearized canine CD40/murine Ig2a/pcDNA3.1 and.

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