Tyrosine kinase-dependent activation of human NK cell functions upon stimulation through a 58-kDa surface antigen selectively expressed on discrete subsets of NK cells and T lymphocytes

We have raised a mAb, termed HP-3E4 (IgM), by immunizing BALB/c mice against human IL2-activated NK cells. The HP-3E4 mAb recognized in different donors variable proportions (<2-70%) of either fresh or activated NK cells. A small population of T cells (α/β and γ/δ) appeared HP-3E4+ in PBL. No reactivity was detected on other leukocytes and a panel of cell lines from different lineages. By immunohistochemical staining of different tissues, few HP-3E4+ cells were detected only in lymphoid organs. Analysis of CD56+CD16+CD3- clones (n = 167) from unrelated donors (n = 6), showed that the Ag was stably expressed on 8 to 70%, moreover it was detected on some γ/δ+ T cell clones, whereas all CD3+ α/β+(CD4+ and CD8+) clones analyzed (n = 90) were HP-3E4-. As assessed by SDS-PAGE analysis, the HP-3E4 mAb immunoprecipitated a 58-kDa surface structure. When compared with two mAbs (GL183 and EB6) previously reported to bind also a clonally distributed 54- to 58-kDa Ag, the HP-3E4 mAb appeared to recognize a distinct epitope, thus allowing to further define NK cell subsets. Stimulation of IL2-activated NK cells with the mAb triggered TNF-α and IFN-γ production, which was enhanced by using the mAb attached to plastic or in the presence of suboptimal concentrations of phorbol esters. Although the HP-3E4 mAb did not significantly modify NK cell-mediated cytotoxicity against different targets, with the exception of the Hmy-C1R cell line, it activated BLT esterase secretion. Remarkably, the HP-3E4 mAb triggered phosphoinositide turnover and an early increase of [Ca2+]i, as well as tyrosine phosphorylation of several cellular substrates including CD3ζ; inhibition of tyrosine kinase activity with genistein hampered the HP-3E4-mediated stimulation of cytokine production. Our data provide further support for the structural diversity of a 58-kDa surface Ag, whose expression is restricted to discrete NK and T cell subsets. Moreover, the results support the fact that the molecule plays an active role in regulating NK cell functions through signal transduction mechanisms comparable to those triggered via FcγRIII.