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 a gainst human IL2-activated NK cells. The HP-3E4 mAb recognized in diff erent donors variable proportions (<2-70%) of either fresh or activate d NK cells. A small population of T cells (alpha/beta and gamma/delta) appeared HP-3E4(+) in PBL. No reactivity was detected on other leukoc ytes and a panel of cell lines from different lineages. By immunohisto chemical staining of different tissues, few HP-3E4(+) cells were detec ted only in lymphoid organs. Analysis of CD56(+)CD16(+)CD3(-) clones ( n=167) from unrelated donors (n=6), showed that the Ag was stably expr essed on 8 to 70%, moreover it was detected on some gamma/delta(+) T c ell clones, whereas all CD3(+) alpha/beta(+)(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 compar ed with two mAbs (GL183 and EB6) previously reported to bind also a cl onally distributed 54- to 58-kDa Ag, the HP-3E4 mAb appeared to recogn ize a distinct epitope, thus allowing to further define NK cell subset s. Stimulation of IL2-activated NK cells with the mAb triggered TNF-al pha and IFN-gamma production, which was enhanced by using the mAb atta ched to plastic or in the presence of suboptimal concentration's of ph orbol esters. Although the HP-3E4 mAb did not significantly modify NK cell-mediated cytotoxicity against different targets, with the excepti on of the Hmy-C1R cell line, it activated BLT esterase secretion. Rema rkably, the HP-3E4 mAb triggered phosphoinositide turnover and an earl y increase of [Ca2(+)](i), as well as tyrosine phosphorylation of seve ral cellular substrates including CD3 zeta; inhibition of tyrosine kin ase activity with genistein hampered the HP-3E4-mediated stimulation o f cytokine production. Our data provide further support for the struct ural diversity of a 58-kDa surface Ag, whose expression is restricted to discrete NK and T cell subsets. Moreover, the results support the f act that the molecule plays an active role in regulating NK cell funct ions through signal transduction mechanisms comparable to those trigge red via Fc gamma RIII.