Generation of anti-human CD8β-specific antibodies using transfectants expressing mixed-species CD8 heterodimers

Abstract

The CD8 glycoprotein is a lymphocyte differentiation antigen comprised of two distinct polypeptide chains, α and β, which have the capacity to form homodimeric (CD8 α/α) or heterodimeric (CD8 α/β) cell surface complexes. The majority of monoclonal antibodies which recognize the human CD8 antigen react with the CD8α chain, while a single mAb, referred to as T8/2T8-5H7 (or 2ST8-5H7), has been identified which binds to the CD8α/β heterodimer. In order to generate antibodies specific for CD8β, murine fibroblast transfectants were constructed which express the human CD8β chain in combination with either the human CD8α chain or the murine CD8α homologue, the Lyt-2 molecule. These transfectants were used to raise polyclonal heteroantisera in mice. Transfectants expressing human CD8α/β heterodimers induced moderate anti-CD8α titers, but were weakly effective in generating anti-CD8β titers, despite high level cell surface expression of this protein. In contrast, transfectants expressing mixed-species CD8 heterodimers (murine CD8α and human CD8β) induced high anti-CD8β 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 CD8β chain. Unlike T8/2T8-5H7, the mAb 5F2 can bind the CD8β chain irrespective of its pairing partner, and can immunoprecipitate the CD8β protein from cells transfected with the CD8β gene in the absence of the human or mouse CD8α gene product. Anti-CD8β antibodies should help elucidate the extent of biochemical heterogeneity of the CD8β protein, and will also be useful indefining the role of the CD8β protein in thymocyte and lymphocyte development, recognition and activation.