Mechanisms Accounting for the Defective Natural Killer Activity in Patients With Hairy Cell Leukemia zyxw By Livio Trentin, Renato Zambello, Carlo Agostini, Achille Ambrosetti, Teodoro Chisesi, Roberto Raimondi, Pietro Bulian, Giovanni Pizzolo, and Gianpietro Semenzato Natural killer (NK) cell activity is severely impaired in untreated patients with hairy cell leukemia (HCL). In an attempt to investigate whether this impairment is related to a defect at the target cell binding and/or at the post target cell binding level, we evaluated the peripheral blood mononuclear cells (PBMC) of HCL patients for their ability to: (1) bind and kill K-562 NK-sensitive targets at the single cell binding level: (2) release the NK cytotoxic factor (NKCF) under different in vitro stimuli, including K-562 and phytohemoagglutinin: and (3) kill K-562 targets in a lectin- dependent cellular cytoxicity (LDCC) assay. This study demonstrates that untreated HCL patients' PBMC show a low ability to form conjugates with K-562 targets at the single cell binding level (5.7% zyxwvuts f 1.0%) with respect t o patients studied after treatment (9.3% k 1.3%) and con- trols (15.0% f 4.0%); P < .05 and P < .001, respectively. A decreased ability to kill the bound target was demon- strated in untreated cases (1.2% zyxwvuts f 1.1 %) versus patients studied after treatment and controls (12.3% k 1.6%. 17.0% zyxwvutsrqpon 2 3.1% respectively); P < .001 in both conditions. After activation of effector cells with interleukin-2 (IL-2) in zyxwvu AIRY CELL LEUKEMIA (HCL) is a neoplastic H disorder characterized by the proliferation of cells belonging to the B-cell lineage. The disorder is commonly characterized by a severe pancytopenia and several immuno- logic abn~rmalities.'-~ Among these, a defect of the natural killer (NK) cytotoxic function has been demonstrated in untreated patient^.^.' This in vitro function usually recovers after several months of interferon-a (IFN-a) therapy.6-* Although it has been suggested that a defective availibility of interleukin-2 (IL-2), possibly consequent to increased serum levels of soluble IL-2 receptor?'" accounts for the impair- ment of the cytotoxic cells in this disease, little is known about the mechanisms that regulate the different steps of this cell defect, either at the target binding level'' or after the target binding events. NK cells are commonly defined as CD3 negative granular lymphocytes that express CD 16 and CD56 surface markers and display a cytotoxic machinery that does not require a major histocompatibility complex (MHC) restriction. The ability of cytotoxic cells to kill the targets implies the capability of effectors to bind the targets and, as a further step, the property to kill the cells by releasing several cytotoxic factors. Although the nature of the receptor(s) used by NK cells for target cell recognition has not yet been characterized, more information is available now on the soluble molecules, which are released by effector cells at the postbinding step and are able to induce the The NK cytotoxic factor/s (NKCF) is/are one of these mole- cules. It is released by human N K cells after recognition and binding of the target and it has been proved to be selectively cytotoxic for NK-sensitive tumor cell^.'^,'^ Other molecules beside N K C F have been discovered to display a lytic activity against tumor cells, including lymphotoxin and tumor necro- sis factor-a.I4-I7 vitro, an increase in the ability of PBMC to form conjugates with K-562 targets and kill the bound target was demon- strated in each group of patients. Moreover, IL-2 was able to increase the cytotoxicity against NK-sensitive targets in all patients tested. Evaluation of NKCF production showed that untreated patients release low levels of NKCF when PBMC were incubated in the presence of K-562 stimulators (1.8% f 0.7%) with respect to patients after interferon-a (IFN-a) therapy (7.6% f 2.1%) and controls (12.9% k 2.2%); P < .02 and zyxw P < .001, respectively. When the recognition mechanisms were bypassed by triggering the cells with lectins in an LDCC assay, we demonstrated an increase of the lytic activity in both groups of patients with respect to the baseline values. However, the cytotoxic capacity observed in untreated patients was significantly lower than that observed in subjects after IFN-CY therapy and controls (P < .001). These findings suggest that the impaired NK activity observed in patients with HCL is related to defects both at the target and posttarget cell binding levels. zyxwv 0 1990 zyxwv by The American Society of Hematology. This study is designed to determine the events taking place at the cell level that might account for the impaired cytotoxic function observed in HCL patients. To this end, we evaluated the ability of peripheral blood lymphocytes, taken from both untreated HCL patients and patients treated with IFN-a, to form conjugates with NK-sensitive targets, release NKCF under different in vitro stimuli, and kill the NK-sensitive targets in a lectin-dependent cellular cytotoxicity assay (LDCC) . MATERIALS AND METHODS Patients. Nineteen patients zyxw (1 3 men and 6 women, aged from 40 to 68 years) were studied. Fourteen patients were studied at the time of diagnosis while five additional patients were evaluated after 9 to 12 months of IFN-a therapy. The diagnosis of HCL was based on clinical, morphologic, and cytochemical criteria.'.' From the Paduo University School of Medicine. Department of Clinical Medicine. Firsi Medical Clinic and Clinical Immunology Section; Hematology Section, University of Verona; and Depart- ment of Hematology, Hospital of Vicenza. Italy. Submitted June 29,1989; accepted December 8,1989. Supported in part by the Associazione Italiana per la Ricerca SUI Cancro (AIRC, Milan) and by the Schering Corp (Schering-Plough S.p.A.). L.T. and R.Z. are recipients of a fellowship from the Associazione Italiana p w la Ricerca sui Cancro (Milan). Address reprint requests to G. Semenzato, MD. Istituto di Medicina Clinica dell'Universitb. di Padova, Clinica Medica I * , Via Giustiniani 2, 35128 Padova, Italy. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate thisfact. 0 1990 by The American Society of Hematology. 0006-4971/90/7507-00~$3.00/0 Blood, Vol 75, No 7 (April 1). 1990: pp 1525-1530 1525 Downloaded from http://ashpublications.org/blood/article-pdf/75/7/1525/1641306/1525.pdf by guest on 24 October 2022