Isolation of A cDNA encoding A Putative Receptor on Macrophage for allogeneic MHC

Abstract

We reported previously that the major effector cells essential for allograft (e.g., BALB/c skin and Meth A tumor; both H-2D^d) rejection were allograft-induced macrophages (AIM; H-2D^b) and that AIM exhibited the cytotoxic activity against the allograft with MHC haplotype specificity. In the last meeting, we reported that we isolated a cDNA clone, 15-4-4 (261-bp fragment), by the expression cloning method using a monoclonal antibody, R15, specific for AIM and an H-2D^d tetramer. In the present study, we further characterized the cDNA. In order to know whether the protein encoded by 15-4-4 cDNA is functional or not, we ligated a full-length cDNA (1181-bp) with pEGFPNI vector; and the cDNA or vector alone was stably transfected into 293T cells. After selection of the transfectants by both EGFP expression and neomycin (G418) resistance, binding abilities of the expressed protein to an H-2D^d or H-2D^b tetramer were examined under a confocal microscope or by cell sorter. The analyses revealed that the 293T cells transfected with full-length 15-4-4 cDNA did react with R15 antibody and H-2D^d tetramer and that the cells transfected with vector alone did not. In contrast, H-2D^b tetramer did not react with 293T transfectants. These results suggest that the protein encoded by 15-4-4 cDNA may be a putative receptor for allogeneic MHC, H-2D^d. [Jpn J Physiol 55 Suppl:S68 (2005)]