genes in the HLA region on chromosome 6 - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5688-5692, August 1986 Medical Sciences

Natural killer susceptibility of human cells may be regulated by genes in the HLA region on chromosome 6 (natural killer cells/natural killer targets/mutant cell lines)

A. HAREL-BELLAN*, A. QUILLET*, C. MARCHIOL*, R. DEMARSt, T. TURSZ*, AND D. FRADELIZI* *Laboratoire d'Immunologie, Batiment de Recherches, Institut Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif, France; and tLaboratory of Genetics, Genetics Building, University of Wisconsin, Madison, WI 53706

Communicated by Jean Dausset, April 2, 1986

ing either to different subsets of NK cells or to a polyspecific NK cell population. One attractive hypothesis would be that the susceptible cells either are completely lacking major histocompatibility complex (MHC) antigen expression or are expressing abnormal MHC antigens on their surface. This hypothesis is sustained by several lines ofevidence (10, 11). We report here results about variants of a human B-lymphoblastoid cell line in which the loss of histocompatibility leukocyte antigen (HLA) expression at the cell surface was accompanied by the acquisition of NK susceptibility.

ABSTRACT Natural killer (NK) cells exist in each individual in the absence of any intentional immunization. They are able to kill a wide range of targets from tumoral as well as from normal origin. However, their exact physiologic role is not clearly understood. In this study we report results about a human Epstein-Barr virus-transformed B-cell line from which variants perturbed in the expression of HLA molecules have been derived. Our results indicate that in these cell lines an inverse relationship exists between expression of HLA antigens and susceptibility to NK lysis. The original cell line is highly resistant to NK lysis. On the contrary, the variant perturbed in class I antigen expression is highly susceptible. Variant perturbed in class II antigen expression is intermediate in susceptibility. Interferon, which induces HLA class I expression and NK resistance in the unrelated classical K-562 target cells, does not change either HLA expression or NK susceptibility in the variant cell lines. The difference between the original cell line and the variants does not reside in the ability to be bound by NK effectors. Our results suggest a different role for HLA molecules. By some unknown mechanism discussed here, the presence of HLA molecules at the surface of a cell would prevent this cell from being killed by NK cells. The loss of this "good health" signal would lead to the elimination of the cell through NK lysis.

MATERIAL AND METHODS Cell Lines. LCL 721 is an Epstein-Barr virus (EBV)transformed human B-cell line that expresses the following haplotypes: HLA-DP4; DQ2; DR3; Al; B8 and HLA-DP2; DQ1; DRI; A2; BS (12). We studied variants 721.84.5 and 721.134 (referred to as .84 and .134 below), both ofwhich had been derived from an intermediate variant clone, .45, from which the entire HLA-DP4; DQ2; DR3; Al; B8 haplotype had been physically deleted (12). Mutant .84 does not express DR and DQ antigens (13, 14) because it has homologous deletions of DR and DQ genes (15) but it does express DP2 as well as A2 and B5 antigens. Mutant .134 has normal expression of the class II antigens (15) but has greatly reduced expressions of the HLA-A2 and -B5 antigens (16). Residual binding of monoclonal antibodies BB 1.2 (anti-A2) and BB M.1 (anti,82-microglobulin) indicates significant expression of HLAA2 and possibly other non-HLA-B class I antigens on .134. The class I deficient phenotype of .134 and several similar mutants derived from LCL 721 (16) results from a defect in a posttranscriptional aspect of class I antigen expression that may result from a homozygous mutation in the MHC interval between the DP and complement genes (17, 18). Priess is a human EBV-transformed B-lymphoblastoid cell line. K-562 is a human erythroblastic cell line. Fluorescence-Activated Cell Sorter (FACS) Analysis. Cells from the different cell lines were labeled for FACS analysis by using different antibodies. M18 is an anti-,32-microglobulin antibody (kind gift from G. Kalil); W6/32 (13) and B12-32 (kind gift from J. Colombani) are two monomorphic antiHLA class I antibodies; L 112 (kind gift from G. Kalil) is a monomorphic anti-HLA class II antibody. All antibodies were spun (Beckman Airfuge) 30 min at 30 psi (1 psi = 6.89 kPa) before use. Cells (106) were incubated at 4°C in 50 ,ul of phosphate-buffered saline (PBS, 0.15 M sodium chloride/10 mM sodium phosphate, pH 7) supplemented with 1% fetal calf serum containing the optimal concentration of antibodies

One intriguing problem in immunology is the role played by natural killer (NK) cells in vivo. NK cells are able to kill in vitro targets cells that are mainly of tumoral but also of normal origin (1, 2). They exist in each individual in the absence of intentional immunization. The cells responsible for NK activity have been characterized as large granular lymphocytes (LGL) that possibly are of the T-cell lineage, although this remains controversial. Two principal physiological roles for NK lysis have been proposed: immune surveillance against the growth of tumors or metastasis (3) and/or surveillance of hematopoiesis (4), being responsible in both cases for the destruction of cells expressing aberrant non-self antigens. A decisive advance in the understanding of the function of NK cells would be the identification of the exact characteristics of the target cells. In fact, if NK lysis is not considered specific, it is mainly because its specificity is unknown even though the susceptibility to NK lysis is restricted to certain targets. The nature of the molecule(s) responsible for (i) the binding of effector to target cells and (it) susceptibility to NK lysis is still controversial. They have been identified alternatively as sugars (5), asialo-GM2 (6), differentiation antigens (3), immature structures (7), viral structures (8), receptor for transferrin (9), or proteins of different molecular weight (3). The current hypothesis is that different target molecules exist, correspond-

Abbreviations: FACS, fluorescence-activated cell sorter; HLA,

histocompatibility leukocyte antigen; MHC, major histocompatibility complex; NK, natural killer; PBL, peripheral blood lymphocyte(s); LGL, large granular lymphocyte(s); FITC, fluorescein isothiocyanate.

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Proc. Natl. Acad. Sci. USA 83 (1986)

Medical Sciences: Harel-Bellan et al. in microtiter plates. After 45 min, cells were washed three times and incubated under the same conditions with the appropriate (M18, W6/32, and B12-32 are IgG; L 112 is an IgM) goat anti-mouse Ig coupled with fluorescein isothiocyanate (FITC). After 45 min, cells were washed in PBS and analyzed on a FACS 440 (Becton Dickinson). Chromium-Release Cytolysis Assay. For NK cytolysis assays, peripheral blood lymphocytes (PBL) from normal human volunteers (from the Centre de Transfusion Sanguine, Hopital St-Louis, Paris) were isolated on Ficoll/Hypaque (MSL, Eurobio, Paris), washed, and depleted of adherent cells by 1 hr of incubation at 370C in plastic Petri dishes. For lymphokine-activated killer cytolysis assays, PBL from human volunteers were cultured for 2 days at 1 x 106 cells per ml in RPMI medium supplemented with 10% fetal calf serum and human crude supernatant containing interleukin 2 at 25 units/ml [Biological Response Modifier Program (BRMP) units, National Cancer Institute, Bethesda, MD] and washed before use. Serial dilutions of effector cells were distributed in round-bottom microtiter plates in RPMI medium supplemented with 10% fetal calf serum (each dilution was performed in duplicate). Target cells (107 in 300 /l) were labeled with 51Cr [sodium chromate, CEA, Saclay, France; 200 ,Ci (1 Ci = 37 GBq) in a total volume of 200 /4 of RPMI medium] for 1 hr and washed three times. 51Cr-labeled targets (104) were added to each well (total volume, 200,ul). Spontaneous release and total release were measured in quadruplicate in wells receiving no PBL and medium or 1 M HCl, respectively. Plates were incubated 16-18 hr at 37TC. Supernatants were collected by using a Skatron device (Skatron, Lier, Norway) and released 51Cr was measured by a Kontron -counter. Percentage of specific lysis was calculated as follows: % specific lysis = [(sample release - spontaneous release)/(total release - spontaneous release)] x 100. Unlabeled Target Inhibition Experiments. Unlabeled target inhibition experiments were performed as cytotoxicity assays except that graded doses of unlabeled inhibitors were added as indicated. The effector-to-target ratio was 40:1. b

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Target Binding Assay. PBL depleted of adherent cells were further fractionated on a Percoll density gradient (Pharmacia). Fraction 3 (bottom of the 34% layer), which was enriched in LGL as assessed by Giemsa staining and which contained most of the NK activity, was used as the effector for the target binding assay. Effectors and targets were mixed in a total volume of 0.2 ml (effector/target ratio, 5:1), spun down 15 sec at 600 x g, and incubated at room temperature. After 30 min, cells were gently resuspended and the percentage of conjugated LGL (LGL in conjugates/total LGL) was estimated by using an hemocytometer. Treatment with Interferon. Cells (1-2 x 105 per ml in RPMI medium with 10% fetal calf serum) were incubated 4 days with 1000 units of recombinant interferon (Biogen, Geneva) per ml in RPMI medium supplemented with 10% fetal calf serum and washed.

RESULTS Expression of HLA Class I Antigens: Study by FACS Analysis. Expression of HLA antigens by these three cell lines was restudied by FACS analysis using monomorphic anti-HLA monoclonal antibodies (Fig. 1). LCL 721, .84, and .134 cells were incubated with antibodies recognizing 32microglobulin (M18 antibodies), with antibodies recognizing class I molecules [B12-32 and W6/32 (19)], and with antibodies recognizing class II molecules (DR molecules) [L 112 (20)]. Cells from LCL 721, bound all of these antibodies (line 1). Variant .84 (line 3) bound anti-B2-microglobulin and anti-A/B antibodies only, indicating that this cell line lacks the expression of DR molecules and expresses A/B and P2-microglobulin. Mutant .134 (line 2) bound anti-f32-microglobulin and anti-DR antibodies, indicating that this cell line expresses DR molecules normally. It was positively stained by W6/32 antibodies but only very faintly by B12-32, indicating that this line lacks expression of some, but not all, HLA-A and -B molecules, confirming previous studies (16, 18). K-562 cells (line 4), the classical line used in NK assays,

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Fluorescence intensity FIG. 1. Expression of HLA antigens on the different lines used in this study assessed by FACS. Cells from LCL 721, from its variants .134 and .84, and from K-562 were labeled by using different anti-HLA antibodies and FITC-conjugated goat anti-mouse Ig antibodies and then submitted to FACS analysis. Abscissa, fluorescence intensity (arbitrary units, logarithm scale); ordinate, number of cells. Column a, negative control, goat anti-mouse IgG-FITC alone; column b, B12-32 antibodies; column c, W6/32 antibodies; column d, M18 antibodies; column e, negative control, goat anti-mouse IgM-FITC alone; column f, L 112 antibodies. Line 1, 721 cells; line 2, .134 cells; line 3, .84 cells; line 4, K-562 cells.

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Table 1. Target binding assay using Percoll gradient-purified LGL and the different cell lines % of conjugate Donor 2 Donor 1 Target 23 18 K-562 20.9 21 721 23 34 .134 37 .84 17 5.8 7 CTL-L2* LGL from PBL depleted of adherent cells were purified on Percoll and used as effectors for the target binding assay. The % of conjugated LGL (LGL in conjugates/total LGL) was estimated by using a hemocytometer. *CTL-L2 is a mouse interleukin 2-dependent T-cell line used here as a negative control.

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ciently lysed by lymphokine-activated killer cells (PBL that have been activated by lymphokine in vitro) or by in vitro induced HLA-specific cytolytic T lymphocytes cells (data not shown). Target Binding Assay Using the Sensitive and the Resistant Cell Lines. To test if the different susceptibilities to NK lysis were associated with significant differences in the ability of target cells to bind effector cells, LGL were purified from PBL on a Percoll density gradient and incubated with target cells. The conjugation of the LGLs to the targets was then assessed microscopically. Table 1 shows that NK-resistant LCL 721 bound LGL as efficiently as the NK-sensitive K-562 positive control, showing that this cell line, although resistant to lysis, expresses the molecule(s) necessary for the binding of LGL. Unlabeled Target Inhibition Assay Using the Sensitive and Resistant Cell Lines. Once established that resistance of LCL 721 to NK lysis does not appear to result from general resistance to cell-mediated cytolysis or from failure to bind NK cells, we used unlabeled target inhibition experiments to determine if the target molecule for NK cells could be detected indirectly on the cell lines. Graded numbers of unlabeled K-562, LCL 721, .84, .134, and Priess cells were added during the cytolysis tests to fixed numbers of PBL and 51Cr-labeled K-562, .84, or .134. As shown in Fig. 3C, only unlabeled K-562 was inhibitory for the lysis of labeled K-562. The lysis of .134 (in which expression of HLA-A and -B is

5 10 20 Effector/target ratio

FIG. 2. NK susceptibility of the different cell lines studied. Cells from K-562 (positive control; c), Priess (negative control; o), 721 (m), .134 (A), and .84 (A) were used as target cells in a NK cytolysis assay using PBL from normal human volunteers depleted of adherent cells as effector cells.

was very faintly stained by anti-class I antibodies and not by anti-class II. Susceptibility of the Different Cell Lines to NK Lysis. The susceptibility of these cell lines to lysis by NK cells in a long-term (18 hr) 51Cr-release assay was measured and results of a typical experiment (1 of 10 experiments) are shown in Fig. 2. Lysis of LCL 721 by normal human PBL was not significantly above the background. The variant lacking HLA-DR and DQ expression (.84) was significantly but slightly lysed by the same effector cells. The variant showing perturbation in the expression of class I antigen (.134), on the contrary, was highly susceptible to NK lysis. Positive (K-562) and negative (Priess) controls are also shown. Absence of LCL 721 cells lysis by NK cells is not due to general resistance to lysis since these cells were very effi-

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Competitor/target ratio FIG. 3. Unlabeled target inhibition study. Cytotoxicity assay was as described in the legend to Fig. 2 except that graded doses of unlabeled inhibitors were added as indicated. Effector-to-target ratio, 40:1. 51Cr-labeled targets: .134 (a), .845 (b), K-562 (c). Inhibitor: K-562 (c), Priess (o), 721 (C), .134 (A), .845 (A).

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Fluorescence intensity FIG. 4. Effect of pretreatment with interferon on HLA antigen expression by K-562 and .134 cells. Cells (1-2 x 105 per ml in RPMI medium with 10%6 fetal calf serum) were incubated 4 days with 1000 units of recombinant interferon (Biogen) per ml. Cells were then washed, labeled with anti-HLA monoclonal antibodies as described in the legend to Fig. 1, and analyzed with the FACS 440. Line 1, .134 cells; line 2, K-562 cells. Column a, negative control, goat anti-mouse IgG antibodies alone; column b, W6/32 antibodies; column c, B12-32 antibodies. Solid lines, untreated controls; broken lines, interferontreated cells.

reduced) was inhibited by the addition of unlabeled .134 and also by unlabeled K-562 (Fig. 3a). The lysis of .84 (lacking HLA-DR and -DQ molecules) was inhibited by the addition of unlabeled .84, unlabeled K-562, and unlabeled .134 (Fig. 3b). In the three systems, unlabeled LCL 721 cells were not able to inhibit the lysis of the susceptible targets, indicating that the 721 cells do not express the target molecule(s) present on the surface of .84, .134, and K-562 cells. Competition results can be interpreted in two ways. Different target molecules (three or more) could be involved in the lysis of K-562, .84, and .134. K-562 would express all of these targets, .134 would express only two of them, and .84 would express only one of them. Alternatively, the same target molecule(s) could be involved in the three systems. K-562 would express more of these molecules than .134, which, in turn, would express more molecules than .84.

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Effect of Treatment with Interferon on Sensitive Cell Lines. Treatment with interferon has been shown to protect target cells against NK lysis (21) and simultaneously to enhance the expression of HLA molecules at the cell surface (22). We therefore tested the effect of interferon on the susceptibility of .134 to NK lysis. Cells were treated with interferon (1000 units/ml) during 4 days and then submitted to FACS analysis and to NK lysis. Results of one of four experiments are shown in Figs. 4 and 5. Without any treatment, K-562 expressed very low levels of HLA class I and class II molecules. As described (23), interferon enhanced HLA-A/B expression on K-562 cells (Fig. 4, line 2). Coordinately, interferon induced protection of these cells against NK lysis (Fig. 5a). However, interferon neither modified the level of expression of HLA class I molecules on the variant (Fig. 4, line 1) nor its susceptibility to NK lysis (Fig. 5b). This cell line is, to our knowledge, the only one in which interferon does not induce protection from NK lysis. It also differs strikingly from the other cell lines studied by the fact that interferon does not induce the expression of HLA class I. A similar study with .84 cells gave identical results (data not shown).

DISCUSSION A role for murine class I molecules in NK susceptibility has been discussed previously. Decreased expression of H-2 was accompanied by increased NK susceptibility in variants of mouse lymphoma RBL5 (10). Furthermore, treatment of target fibroblasts with interferon resulted in a loss of sensitivity to NK lysis (24) and enhanced expression of MHC antigens (25). However, opposite results have been described in the mouse system. Variants from the highly susceptible mouse YAC-1 cell line, selected for reduced expression of H-2 molecules, were relatively insensitive to lysis in general and to NK lysis in particular (26). Loss of H-2 expression also resulted in decreased susceptibility to activated killer cells in a variant thymoma cell line (27). In the human cell system described here, susceptibility to NK lysis seems to be related to reduced expression of HLA antigens. K-562 expresses trace amounts of class I antigens and no class II HLA molecules and is highly susceptible to NK lysis. After interferon treatment, K-562 is less susceptible to NK lysis and expresses increased amounts of class I antigens, whereas class II molecules remain unchanged (23). LCL 721 expresses high levels of class I and class II antigens and is resistant to lysis. Mutant .84 does not express the DR b

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FIG. 5. Effect of pretreatment with interferon on the NK susceptibility of the K-562 and .134 cells. Untreated controls (o) and interferon-treated cells (m) (see legend to Fig. 4) were 51Cr labeled and submitted to NK cytolysis as described in the legend to Fig. 2. (a) K-562 cells. (b) .134 cells.

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and DQ class II antigens and is relatively resistant to NK lysis. In contrast, the reduced expression of class I antigens in mutant .134 is associated with distinct susceptibility to lysis by NK cells. Furthermore, when these cells are pretreated with interferon, the lack of resistance to NK lysis is related to the absence ofinduction of HLA class I expression. Thus, an inverse relation between class I expression and sensitivity to NK cells is suggested. It remains possible, however, that susceptibility to NK lysis is caused by altered expression of a non-class I molecule as a direct result of mutation in the MHC or as an indirect effect of reduced class I antigen expression. Although the involvement of mutations outside the MHC cannot be totally excluded, the chance of such involvement is remote, since the induced rate of single mutations in these experiments was 4 x 10-5, the rate of two coincidental non-MH'C mutations being 10-9 (18). The mechanism by which reduction in class I antigen expression would result in NK lysis susceptibility is not understood. Target binding assays indicate that LCL 721 expresses the molecule necessary to bind effector cells. However, results from unlabeled target inhibition experiments indicate that the expression of the lytic target molecule could not be detected on 721 cells, whereas it was present on the two variants. We propose one model to explain these two results. The various target cells bind the effector cells to the same extent by means of binding structures. Once bound, interaction between the effector and a molecule on the target cell (possibly HLA molecules) would decide whether the target cell is lysed or not. If HLA molecules are intact on the surface, the target would not be lysed and the effector cells would be recycled. If class I antigen expression is sufficiently reduced or modified, the targets would be lysed. This mechanism could be ancestral to that of the cytolytic T lymphocyte system, in which the induction of specific cytolytic T cells results from their recognition of allogeneic class I amino acid sequence differences'or of autologous class I antigens that are modified as a result of viral infection. The molecule responsible for this inhibition of NK target expression could act in several ways: it could mask the NK target antigen at the cell surface, it could transform the target molecule to an inactive form, or it could suppress the biosynthesis of this molecule at the level of transcription or translation. If the inverse relationship between the presence of HLA molecules at the cell surface and susceptibility to NK lysis is confirmed in the human system, it could help us to understand the exact role played by class I HLA antigens, on one hand, and NK cells, on the other. In fact, HLA molecules could be a sign of cellular integrity, a "good health" signal for the cells. When a cell loses this HLA, it becomes susceptible to NK lysis and is eliminated. We acknowledge volunteer donors from Banque du Sang, Hopital St Louis and Dr. J. Reviron, Dr. G. Kalil, and Dr. J. Colombani for the kind gift of antibodies. We are indebted to Dr. Z. Mishall for the help in FACS analysis and to Dr. D. Grausz for helpful discussion. This work was supported by grants from Institut Gustave Roussy,

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