Generation of Monoclonal Antibodies to a Human Natural Killer Clone

The initial characterization of two monoclonal antibodies directed at antigens selectively expressed on large granular lymphocytes (LGL) is reported in the present paper. These two reagents, anti-natural killer (NK) HiA and anti-NKH2, were obtained following immunization of mouse spleen cells with a cloned human NK cell line termed JT3. In fresh human peripheral blood, both anti-NKHlA and anti-NKH2 selectively reacted with cells that appeared morphologically as large granular lymphocytes. However, complement lysis studies and two color fluorescence analysis demonstrated that some LGL express both antigens and other cells express only NKH1A or NKH2. Functional analysis of these subsets indicated that the population of NKH1A+ cells contains the entire pool of NK active lymphocytes, whereas expression of NKH2 antigen appeared to delineate a unique subpopulation of LGL which, in a resting state, display a low degree of spontaneous cytotoxicity. Expression of NKH1 and NKH2 was also investigated using a series of nine well characterized human NK clones. All NK clones were found to be NKH1A+ and four out of nine also expressed NKH2. These results strongly supported the view that NKH1A is a "pan-NK" associated antigen, and indicated that at least a fraction of cloned NKH2+ LGL are strongly cytotoxic. Anti-NKHlA was shown to have the same specificity as the previously described N901 antibody and was found here to precipitate a 200,000-220,000-mol wt molecule in SDSpolyacrylamide gel electrophoresis (PAGE) analysis. AntiNKH2 was specific for a structure that migrates at 60,000 mol wt in SDS-PAGE analysis under reducing conditions. Two color immunofluorescence analysis of NKH1A, NKH2, and other NK-associated antigens (Leu7 and B73.1) demonstrated variable degrees of coexpression of these antigens, which confirmed that NKHlA and NKH2 define distinct cell surface structures. Anti-NKHlA and anti-NKH2 appear to be useful reagents for characterizing LGL present in human peripheral blood and for identifying functionally relevant subsets within this heterogeneous population of cytotoxic lymphocytes.