Cancer Immunol Immunother (2002) 51: 130–138 DOI 10.1007/s00262-002-0264-8
O R I GI N A L A R T IC L E
Gonzalo Rubio Æ Xabier Ferez Æ Ana Mora Jesus Galvez Æ Antonio Chicano Pilar Garcia-Pen˜arrubia
b1 Integrin triggering affects leukemic cell line sensitivity to natural killer cells Received: 15 July 2001 / Accepted: 20 December 2001 / Published online: 28 February 2002 Springer-Verlag 2002
Abstract The role of b1 (CD29) integrins in natural killer (NK) cell–target cell conjugation and cytotoxicity has not been clearly established. Ligation of b1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the b1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of b1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-b1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207–218 of the b1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from b1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-b1 epitope A1 mAb. Importantly,
G. Rubio Division of Immunology, Miguel Hernandez University, 03550 San Juan de Alicante, Spain X. Ferez Æ A. Chicano Æ P. Garcia-Pen˜arrubia (&) Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, 30100 Murcia, Spain E-mail:
[email protected] Tel.: +34-968-364673 Fax: +34-968-364150 A. Mora Department of Allergy, Morales Meseguer Hospital, 30007 Murcia, Spain J. Galvez Department of Physical Chemistry, University of Murcia, 30100 Murcia, Spain
pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-b1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-b1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through b1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity. Keywords b1 integrin Æ Binding isotherm Æ Cytotoxicity Æ Leukemia cell line Æ NK cell
Introduction Natural killer (NK) cell cytotoxic activity against tumor cells is a process that results from a complex still unsettled counterbalance between the action of inhibitory and triggering receptors, in which adhesion molecules participate [15]. In humans, inhibitory receptors recognize HLA class I molecules on target cells and transduce inhibition signals that protect normal autologous cells from killing. In opposition, an array of molecules that is displayed on all or on subsets of NK cells triggers NK cytotoxicity upon their occupation by ligands [4, 16, 19]. As the missing self hypothesis postulates, a pathological reduction of HLA class I molecules on target cells limits the inhibition and NK cells lyse the target cells [15]. In addition to inhibitory and triggering receptors, NK-cell mediated lysis depends on adhesion molecules to enable the formation of conjugates with target cells. Conjugation occurs through b2 integrins (leukocyte associated molecule-1; LFA-1; CD11a/CD18) and CD2 expressed on NK cells, and their ligands on target cells, i.e. intercellular adhesion molecule-1 (ICAM-1; CD54), ICAM-2 (CD102) and LFA-3 (CD58), as suggested by the partial inhibition exerted by specific monoclonal antibodies (mAb) [13, 32, 35]. b1 [very late activation antigens; VLA (CD29)] integrins are also involved in
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cell–cell adhesion, although their role in NK cell cytotoxicity still remains controversial. b1 integrins operate either as receptors for extracellular matrix proteins (e.g. a6b1; VLA-6 for laminin) or as receptors for both extracellular ligands and cell-surface molecules [11]. VLA-4 molecule is the b1 integrin that is mostly implicated in cell–cell interactions. Notably, occupation of either the b1 or a4 subunit by selected mAb is able to trigger myeloid and B lymphocyte cell line homotypic adhesion, as well as to enhance adhesion of bone marrow mononuclear cells to stroma, the process being dependent on VLA molecules themselves [1, 8, 21]. Previous data have indicated that b1 integrins are implicated in cytolytic processes. Recently, it has been shown that engagement of VLA-4 and VLA-5 inhibits CD16-triggered NK granule exocytosis [18, 30]. However, other studies have reported that that b1 integrin engagement with mAb or natural ligands mobilizes Ca2+, activates cytokine production, and stimulates NK cytotoxic activity [17, 22]. Moreover, it has been shown that increased expression of VLA molecules on melanoma target cells improves their susceptibility to cytotoxic T lymphocytes [3]. Taken together, the above observations suggest that VLA molecules expressed by potential target cells may be involved in the interaction with NK cells, that in turn can modulate their cytolytic activity either positively or negatively upon occupancy of their VLA counter-receptor molecules. In the present study, using a model developed by our group the conjugation of NK cells to leukemia cell lines treated with anti-b1 integrin mAb was analyzed. Conjugation was measured by flow cytometry in normalized assays and adhesion-related binding parameters were calculated by binding isotherms [6, 12, 28]. Further, 51Cr release assays were performed to determine the involvement of VLA molecules in the cytotoxicity process.
Materials and methods Antibodies The anti-b1 integrin mAb used were Lia 1/2, Alex 1/4 [8] (kindly provided by F. Sanchez-Madrid, Hospital de la Princesa, Madrid, Spain), TS2/16 [14] (ATCC, Manassas, Va.), 4B4 [20] and K20 [2] (Beckman Coulter, Fullerton, Calif.). F(ab’)2 fragments of mAb Alex 1/4 were prepared as previously described [27]. mAb TS1/11 (anti-CD11a/CD18), TP1/36 (anti-CD43), RP2/21 (anti-CD45) and HP2/19 (anti-ICAM-3, CD50) have been previously described and were obtained from F. Sanchez-Madrid [7, 24, 29]. mAb HP-1F7 (anti-HLA class I) [10] was a gift from M. Lopez-Botet (Hospital de la Princesa), and mAb W6/32 (anti-HLA class I) was obtained from ATCC. mAb anti-CD3-FITC, anti-CD16-PE and anti-CD56PE used for the phenotypic analysis as well as isotype-matched controls were purchased from Caltag (Burlingame, Calif.). Immunofluorescence staining methods have been described previously [28]. A FACSort flow cytometer (Becton Dickinson, San Jose, Calif.) was used throughout the study. Cells and tissue culture reagents Purified fresh NK cells as well as the NKL cell line were used as effector cells. Highly enriched populations of NK cells were purified
by negative selection from the heparinized blood of healthy donors, as previously described [28]. Obtained cells were routinely >90% CD3– CD56/CD16+ and
