title: Polymorphisms of CD3epsilon in cynomolgus and rhesus monkeys and their relevance to anti-CD3 antibodies and immunotoxins.
abstract: The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T-cell-depleting reagent when conjugated to diphtheria toxin that was mutated to prevent binding to non-targeted cells. The antibody recognizes a conformational epitope on the ectodomain of monkey CD3epsilon and displays a range of binding activity to the T cells from different rhesus and cynomolgus monkeys. Our quantitative fluorescence-activated cell sorting analysis of the FN18 reactivity to T cells from different rhesus and cynomolgus monkeys showed that there are at least three levels of FN18 reactivity in the monkeys tested: high, moderate and low. On the basis of available DNA sequence information, we determined the gene structure of rhesus CD3epsilon chain and designed primers that can be used to amplify and quickly sequence the ectodomain of monkey CD3epsilon. Our sequence analysis revealed that the extent of nucleotide sequence variation in this area is greater than that previously reported. In addition to the amino acids at positions 45 and 50, we demonstrated that position 35 of CD3epsilon was also important and substitution of amino acid A for V at this position greatly reduced T-cell reactivity to FN18. We found that T cells from monkeys with high FN18 reactivity all had V, E and R at positions 35, 45 and 50 in CD3epsilon, respectively; those having low FN18 reactivity were homozygous in CD3epsilon with at least one of the changes: V35 to A, E45 to G and R to 50Q, whereas members in the moderate group are heterozygous, having both V and A, E and G, R and Q at these locations. A cytotoxicity assay revealed that T cells from a heterozygous rhesus monkey with moderate FN18 reactivity were much (about 40 times) less sensitive to a FN18-derived immunotoxin than those from a homozygous rhesus monkey having high FN18 reactivity.
authors.affiliation: Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892, USA. yyliu@mail.nih.gov
authors.forename: Yuan Yi
authors.initials: YY
authors.lastname: Liu
authors.forename: Zhirui
authors.initials: Z
authors.lastname: Wang
authors.forename: Judith
authors.initials: J
authors.lastname: Thomas
authors.forename: K Jeanine
authors.initials: KJ
authors.lastname: Goodwin
authors.forename: Scott
authors.initials: S
authors.lastname: Stavrou
authors.forename: David M
authors.initials: DM
authors.lastname: Neville
mesh_terms: D000409:Alanine; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D017252:CD3 Complex / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D007135:Immunoglobulin Variable Region / Q000378:metabolism; D000922:Immunotoxins / Q000276:immunology*; D008252:Macaca fascicularis / Q000235:genetics*; D008253:Macaca mulatta / Q000235:genetics*; D008297:Male; D050505:Mutant Proteins / Q000276:immunology; D011110:Polymorphism, Genetic; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D014633:Valine
chemical_list: D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D050505:Mutant Proteins; D014633:Valine; D000409:Alanine