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Analysis of chicken CD4 by monoclonal antibodies indicates evolutionary conservation between avian and mammalian
M Luhtala1, J Salomonsen, Y Hirota
1Department of Medical Microbiology, Turku University, Finland.
We have created a panel of mouse monoclonal antibodies detecting different epitopes on avian CD4 molecule. Two-color immunofluorescence analysis shows that chicken peripheral alpha beta T cells are either CD4 or CD8 single positive whereas most gamma delta 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 (M(r) 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 M(r) 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.
We have created a panel of mouse monoclonal antibodies detecting different epitopes on avian CD4 molecule. Two-color immunofluorescence analysis shows that chicken peripheral alpha beta T cells are either CD4 or CD8 single positive whereas most gamma delta 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 (M(r) 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 M(r) 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.