Expression and characterization of recombinant soluble monkey CD3 molecules: mapping the FN18 polymorphic epitope

Area of Science:

• Immunology
• Molecular Biology
• Primate Genetics

Background:

• The monoclonal antibody FN18 serves as a marker for T cells in non-human primates and can be used for T cell depletion.
• FN18 shares characteristics with human anti-CD3 antibodies, precipitating CD3 chains from detergent extracts.
• CD3 polymorphisms in rhesus and cynomolgus monkeys lead to FN18-negative phenotypes, unlike in humans.

Purpose of the Study:

• To investigate the molecular basis of FN18 binding to primate CD3.
• To characterize the CD3 polymorphisms responsible for FN18-negative phenotypes in monkeys.
• To identify the specific CD3 chain composition recognized by the FN18 antibody.

Main Methods:

• Expression and refolding of cynomolgus CD3-epsilon, CD3-gamma, and CD3-delta ectodomains in E. coli.
• Fractionation of CD3 homo- and heterodimers using anion exchange chromatography.
• Enzyme-linked immunosorbent assay (ELISA) to determine FN18 epitope specificity.

Main Results:

• The FN18 epitope is exclusively located on the CD3-epsilon gamma ectodomain heterodimer.
• CD3-epsilon delta and CD3-epsilon epsilon heterodimers do not react with FN18.
• Amino acid polymorphisms in the CD3-epsilon chain abolish the bioactivity of CD3-epsilon gamma towards FN18.

Conclusions:

• FN18 specifically recognizes the CD3-epsilon gamma heterodimer in cynomolgus monkeys.
• CD3 polymorphisms, particularly in the CD3-epsilon chain, are responsible for the FN18-negative phenotype.
• Understanding these molecular interactions is crucial for using FN18 in immunological studies and therapies involving non-human primates.