Inhibition of PHA-induced cell proliferation by polyclonal CD4 antibodies generated by DNA immunization

Although the role of CD4 molecule as associative binding element to MHC class II is well documented, their role in T cell activation is unclear. In the present report we used DNA immunization, which is currently shown to induce potent immune responses, to produce the polyclonal antibodies specific for the CD4 molecule and used the generated antibodies to characterize the CD4 function. A rabbit was pre-treated with bupivacaine hydrochloride for 24 h which was followed by intramuscular injection of DNA encoding CD4 protein (CD4-DNA) at weekly interval. By this procedure, CD4 antibodies were detected in the immunized serum after two DNA inoculations. The CD4 antibodies titer was up to 1:800 after five DNA inoculations. The rabbit polyclonal CD4 antibodies recognized both recombinant CD4 protein expressed on CD4-DNA transfected COS cells and native CD4 protein presented on peripheral lymphocytes and CD4+ cell lines. These generated CD4 antibodies could block the binding of standard CD4 mAb, Leu3a and 13B8.2, to the CD4 molecule. To characterize the function of CD4 molecule, PBMC were cultured in the presence of sub-optimal dose of PHA and the produced polyclonal CD4 antibodies. We found that the polyclonal CD4 antibodies strongly suppressed PHA induced cell proliferation. The inhibitory effect of CD4 antibodies may be due to their steric inhibition of the CD4-TCR/CD3 association or may interfere with the binding of CD4 to its ligand IL-16, resulting in the reduction of signal transduction and subsequent cellular responses. Our results indicate the possibility of utilizing DNA immunization to produce polyclonal antibodies against cell surface molecule.