Direct evidence for native CD4 oligomers in lymphoid and monocytoid cells

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Abstract
CD4 is expressed by T lymphocytes and monocytes and is generally considered a monomer even though its structure was originally modelled on the REI Bence-Jones homodimer. However, native CD4 was demonstrated as both monomer and dimers of 55 and 110 kDa in lymphoid and monocytoid cells by immunoprecipitation and immunoblotting after solubilization with alkylating (iodoacetamide) or reducing (dithiothreitol, 2-mercaptoethanol) reagents. Full reduction yielded only the 55-kDa monomeric form. Purified CD4 oligomers from CEM-T4 cells were also resolved as homodimers by MALDI-Tof mass fingerprinting after tryptic digestion. Cell treatment with the membrane impermeable, free-thiol reactive, 5,5′-dithiobis-2-nitrobenzoic acid enhanced cell surface CD4 dimers and tetramers. The interaction sites producing dimerization were probably in the D4 domain as OKT4 inhibited self association of recombinant CD4 (rCD4). Oligomerization of rCD4 by glutathione and thioredoxin indicates that thiol exchange interactions were responsible. Enhanced CD4 dimer expression was also observed after PMA (20 ng/ml) activation of THP-1 cells. These findings demonstrate that different quaternary forms of CD4 such as monomers, homodimers and tetramers are expressed by T lymphocytes and monocytes/macrophages.