Cloning, sequencing, and homology analysis of nonhuman primate Fas/Fas-ligand and co-stimulatory molecules

The finding that a single administration of select recombinant human cytoki nes to nonhuman primates leads to potent cytokine -neutralizing antibody re sponses in the heterologous host despite > 95% homology at the nucleotide a nd protein level prompted our laboratory to clone, sequence, and prepare re combinant nonhuman primate cytokines, chemokines, growth factors, and other immunoregulatory molecules. In the present report, we present findings on the gene sequences encoding the nonhuman primate homologues of human CD80, CD86, their ligands CD28 and CD152, CD154, CD95, and CD95-L from rhesus mac aques and for phylogenetic analysis from pig-tailed macaques, African sooty mangabey monkeys, baboons, and vervets as well as select molecules from th e New World aotus and marmoset monkeys. With the exception of CD95, the hom ology between nonhuman primate and human co-stimulatory molecules was above 95%. In contrast, CD95 was only 89.2% homologous to human CD95, but the di fferences were essentially found in the transmembrane and intracellular (de ath) domains. The extracellular portion of CD95 was more homologous which w as in accordance with approximately 98% homology between Old World monkey a nd human CD95-L. In general, sequences from the New World monkey species ap peared equidistant to sequences from Old World species and humans in terms of homology suggesting distinct evolutionary patterns. Of interest was the isolation of various splice variants of monkey CD86, CD152 (CTLA-4), CD154, and CD95 transcripts. This is also the first report documenting the occurr ence of natural CD86 variants with deleted transmembrane domains, found bot h in sooty mangabeys and baboon RNA samples. Monkey CD95 showed various del etions and addition of residues in the transmembrane and intracytoplasmic d omains compared with human CD95 and between Old and New World species. Subc loning of rhesus CD154 into an expression vector demonstrated expression of a functional protein in cell culture. The other genes are being cloned int o expression vectors for the preparation and biological characterization of the nonhuman primate molecules. These investigations will provide novel re agents for in vivo use as immunomodulatory reagents in nonhuman primates in studies which may provide a rationale for their use in humans.