Abstract
We have created a panel of mouse monoclonal antibodies detecting different epitopes on avian CD4 molecule. Two-color immunofluorescence analysis shows that chicken peripheral aβ T cells are either CD4 or CD8 single positive whereas most Υδ T cells are CD4-negative both in the thymus and peripheral tissues. Unlabeled antibody competition analysis by flow cytometry demonstrates that several different epitopes on chicken CD4 are recognized by these antibodies. Antibodies precipitate a monomeric glycoprotein from surface-labeled chicken thymocytes and T cells with relative molecular mass (Mṟ) of 64 kd as analyzed by SDS gel electrophoresis. Removal of N-linked carbohydrates by endoglycosidase-F increases the electrophoretic mobility and reveals the core protein size with Mṟ of 45 kd. The anti-CD4 antibodies inhibit antigen-induced cellular proliferation of a keyhole limpet hemocyanin (KLH) -specific T cell line. They synergize in the blocking of T cell proliferation with anti-class II major histocompatibility complex (MHC) -specific antibodies indicating that chicken CD4 is involved in antigen recognition process by CD4+ T cells. We also show that chicken CD4 is down-modulated in a similar manner as its mammalian equivalent when thymocytes are stimulated in vitro with phorbol esters. Altogether these findings suggest functional and biochemical conservation of the CD4 molecule in evolution.
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