GENERATION OF ANTI-HUMAN CD8 BETA-SPECIFIC ANTIBODIES USING TRANSFECTANTS EXPRESSING MIXED-SPECIES CD8 HETERODIMERS

The CD8 glycoprotein is a lymphocyte differentiation antigen comprised of two distinct polypeptide chains, alpha and beta, which have the capacity to form homodimeric (CD8 alpha/alpha) or heterodimeric (CD8 alpha/beta) cell surface complexes. The majority of monoclonal antibodies which recognize the human CD8 antigen react with the CD8-alpha-chain, while a single mAb, referred to as T8/2T8-5H7 (or 2ST8-5H7), has been identified which binds to the CD8-alpha/beta-heterodimer. In order to generate antibodies specific for CD8-beta, murine fibroblast transfectants were constructed which express the human CD8-beta-chain in combination with either the human CD8-alpha-chain or the murine CD8-alpha-homologue, the Lyt-2 molecule. These transfectants were used to raise polyclonal heteroantisera in mice. Transfectants expressing human CD8-alpha/beta-heterodimers induced moderate anti-CD8-alpha-titers, but were weakly effective in generating anti-CD8-beta-titers, despite high level cell surface expression of this protein. In contrast, transfectants expressing mixed-species CD8 heterodimers (murine CD8-alpha and human CD8-beta) induced high anti-CD8-beta-titers in immunized mice. Following fusion of splenocytes from mice immunized with mixed-species CD8 transfectants, the mAb 5F2 was isolated which specifically recognizes the human CD8-beta-chain. Unlike T8/2T8-5H7, the mAb 5F2 can bind the CD8-beta-chain irrespective of its pairing partner, and can immunoprecipitate the CD8-beta-protein from cells transfected with the CD8-beta-gene in the absence of the human or mouse CD8-alpha-gene product. Anti-CD8-beta-antibodies should help elucidate the extent of biochemical heterogeneity of the CD8-beta-protein, and will also be useful in defining the role of the CD8-beta-protein in thymocyte and lymphocyte development, recognition and activation.