A monoclonal antibody specific for a cytochrome c T cell stimulatory peptide inhibits T cell responses and affects the way the peptide associates with antigen-presenting cells.

A monoclonal antibody (mAb) specific for the 93-104 segment of pigeon cytochrome c (cyt) was shown to block interleukin 2 production and proliferation by pigeon cyt-specific T cells in response to the pigeon cyt 81-104 peptide using either the LK35.2 B cell hybridoma or normal splenocytes as antigen-presenting cells (APC). The mAb inhibited the response to soluble peptide antigen presented by metabolically inactive paraformaldehyde-fixed APC but not the response to APC that were pre-pulsed with Ag. These results suggest that the mAb blocked the formation of peptide-major histocompatibility complex (MHC) class II molecule complexes at the cell surface but did not displace the peptide once bound to the MHC class II molecule. As determined by direct binding experiments using labeled peptide, the major means of free peptide association with live APC was fluid-phase endocytosis. No free peptide associated directly with the MHC class II molecule at the cell surface near 0 degrees C since APC pulsed with peptide on ice did not activate cyt-specific T cells. The mAb enhanced the association of the radiolabeled peptide with APC at 4 degrees C apparently by binding of the peptide-mAb complex to Fc receptors. By stripping molecules from the LK35.2 cell surface using a nonspecific protease it was shown that the peptide-mAb complexes were not internalized either at 4 degrees C or 37 degrees C. Since the mAb was found to stably bind the peptide at pH levels below that of endosomes (pH 5.5-6.2) even if the peptide-mAb complexes were taken up by fluid-phase endocytosis, it is likely that the peptide would not be able to associate with MHC class II molecules inside the APC. This mAb appears to inhibit T cell activation by blocking the formation of peptide-MHC class II molecule complexes at the cell surface and by interfering with uptake of the peptide into endosomes. Therefore, it is different from other antibodies that have been reported to block T cell receptor recognition of preformed peptide/MHC class II molecule complexes.