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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.


Journal article


Journal of Immunology

Publication Date





1662 - 1673