EXISTENCE OF BOTH INHIBITORY (P58) AND ACTIVATORY (P50) RECEPTORS FORHLA-C MOLECULES IN HUMAN NATURAL-KILLER-CELLS

The natural killer (NK) cell-specific p58 molecules EB6 and GL183 have been shown to represent the putative surface receptors for two distin ct groups of human histocompatibility leukocyte antigen (HLA) C allele s. Interaction between p58 receptors and class I molecules expressed o n target cells results in inhibition of the NK-mediated cytolytic acti vity and thus in target cell protection. In the present study, we show that EB6 molecules may also act as receptors mediating NK cell trigge ring. Activatory EB6 molecules were found to be confined only to certa in donors. Moreover, in these donors, only a fraction of EB6(+) NK clo nes expressed the activatory form of EB6 molecules, while the remainin g clones expressed the conventional inhibitory form. Biochemical analy sis of the activatory EB6 molecules revealed a molecular mass of simil ar to 50 kD (p50), thus differing from the 58-kD inhibitory form. This difference was not due to differential glycosylation of the same prot ein, as revealed by deglycosylation experiments on isolated EB6 molecu les. Treatment of purified p58 or p50/EB6 molecules with proteolytic e nzymes, including V8-protease, chymotrypsin, and papain, showed only m inor differences in the resulting peptides. Treatment with pepsin foll owed by two-dimensional peptide mapping demonstrated that, although th e majority of peptides migrated in identical positions, differences be tween the two forms could be detected for at least one major peptide. Anti-EB6 monoclonal antibody (mAb)-mediated crosslinking of p50 molecu les was required to trigger the cytolytic activity and the intracellul ar calcium ([Ca++]i) increases in appropriate NK clones. Likewise, mab -mediated cross-linking of the p58 EB6 molecules was needed to inhibit the cytolytic activity; however, in this case, no [Ca++]i increases c ould be detected. In NK clones expressing the inhibitory p58 EB6 recep tors, soluble anti-EB6 mAb prevented recognition of protective Cw4 mol ecules and reconstituted target cell lysis. In contrast, in clones exp ressing the activatory p50/EB6 receptor, EB6 masking frequently result ed in partial inhibition of the cytolytic activity against Cw4(+) targ et cells. Therefore, it appears that NK clones expressing the p50/EB6 receptors are induced to lyse Cw4(+) target cells upon specific intera ction with Cw4 molecules. This concept was further substantiated by ex periments in which target cells were represented by the HLA-negative L CL721.221 cell line transfected with the Cw4 allele. Phenotypic and fu nctional analysis of a large number of NK clones showed that clones ex pressing the activatory p50/EB6 molecules consistently coexpressed inh ibitory receptors for other HLA class I alleles. Taken together, our d ata indicate that recognition of class I molecules may result either i n inhibition or in activation of NK-mediated cytolysis. However, the i nhibitory pathway appears to dominate the activatory one, thus prevent ing lysis of class I-protected autologous normal cells.