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Enhancement of human natural killer activity by the monoclonal Leu-11 antibodies
CELLULAR
104,386-399 (1987)
IMMUNOLOGY
Enhancement of Human Natural Killer Activity by the Monoclonal Leu-1 1 Antibodies SHUN-ENEGAWA,TORU ABO,*,’ AND NOBUOHIWATASHI Third Department oflnternal Medicine, Tohoku University School OfMedicine, and *Department of Microbiology, Tohoku University School ofDentistry, Sendai 980, Japan Received June 27,1986; accepted October 21,1986 The Fc receptor for IgG on human natural loller (NK) cells can be identified by a series of murine monoclonal antibodies. When these antibodies with IgGi &type (e.g., Leu-1 la and Leu-1 lc), but not with IgM (e.g., Leu- 11b and VEPl3), were added to NK assay culture against K562 targets of 4-hr analysis, a considerable enhancement of NK activity was induced. An enhancement was shown at a concentration of up to lo-’ mg/ml of Leu-1 la. Furthermore, the present experiments demonstrated that many established cell lines with diverse cell origins expressed the Leu- 11 antigens, and enhancement of NK activity by Leu- 11 was induced when these Leu-1 I+ cell lines were used as targets in NK assays. The results that Leu-1 la caused an increase in effector-to-target cell conjugates, and that F(ab’)* of Leu-1 la was effective in enhancement of NK activity, but the Fab was not, indicated that Leu- 11might become a linkage between the effector and target cells. Significance of this phenomenon was discussedregarding practical application of L.eu-11 antibodies in laboratory experiments and in clinical studies. 8 1987 Academic
F’rm.
Inc.
INTRODUCTION Natural killer (NK)* cells are a subpopulation of lymphoid cells that have several unique characteristics in terms of their morphology, phenotype, NK function, and cytochemical distribution pattern of cytoplasmic enzymes, and are therefore distinguishable from classicahy identified T and B lymphocytes (l-5). Cumulative evidence has suggestedthat NK cells may exert their cytotoxicity to aberrant cells yielded in the host (e.g., rapidly dividing normal and malignant cells and virally infected cells) and, in the long run, may play an important role in immunoregulatory functions and host defense mechanisms against malignancy and infections (3,4). The recent availability of monoclonal antibodies enables us to intensively characterize the phenotype of human NK cells. By using monoclonal antibodies directed to human NK cells, some subpopulations of NK cells (or granular lymphocytes) have been identified (2, 5). A major population of NK cells with prominent NK activity expressesFc receptor (FcR) defined by a series of monoclonal Leu-1 1 antibodies as ’ To whom correspondence should be addressed. ’ Abbreviations used: NK, natural killer; FcR, Fc receptor; MNC, mononuclear cells; IFN, interferon; IL2, interleukin 2; FITC, fluorescein isothiocyanate; PE, phycoerythrin; ADCC, antibody-dependent cellmediated cytotoxicity. 386 0008-8749187$3.00 Cawisht 0 1987 by Academic Fws, Inc. AU right.5 of reproduction in my form revved.
ENHANCEMENT
OF NK ACTIVITY
BY Lea-1 1
387
well as other myeloid antigens (e.g., OKM 1, Leu-M4, and Leu- 15) (6-8). In addition to myeloid antigen expression, we have recently demonstrated that this NK population is capable of selective phagocytosis and the subsequent production of interleukin 1, both of these functions being similar to those of myelomonocytes (9). Studies using monoclonal antibodies also have revealed another unique lymphoid population with a morphology of granular lymphocyte that has minimal NK activity and expresses T-cell-associated antigens instead of the Leu- 11 antigens ( 10, 11). As it lacks many features ascribed to classically identified T cells, the relationship of this population to NK cells or T cells remains to be elucidated. In the present study, we investigated an interaction between monoclonal Leu-ll antibodies and FcR for IgG on human NK cells. Thus, when the Leu-ll antibodies with IgG, isotype were added to NK assay culture, a considerable enhancement of NK activity was induced. This enhancement was produced when target cells used in NK assaysexpressed the Leu-I 1 antigens (FcR). Surprisingly, many target cells of established cultured cell lines commonly used for human NK assays,including K562 targets with the highest susceptibility to human NK cells, were demonstrated to express FcR. The present experiments also indicated a possibility that Leu- 11-induced enhancement of NK activity might be mediated by a receptor distinguishable from that of original spontaneous NK cytotoxicity. In this study, we analyzed the precise phenomenon and mechanism of this cytotoxicity and discussed what possible significance the phenomenon might have regarding practical application of Leu- 11 antibodies in laboratory experiments and in clinical studies. MATERIALS AND METHODS Cell preparations. Heparinized blood was obtained from healthy Japanese adult donors. Mononuclear cells (MNC) were separated by Ficoll-Isopaque density gradient centrifugation (12). Nonadherent cells were prepared from MNC by depleting monocytes on a nylon-wool column (12). The prepared cells were suspended in RPM1 1640 medium with 10%heat-inactivated fetal calf serum (FCS). Cultured cell lines. Human cultured cell lines, which were used as targets in NK assays,were myelomonocytoid lines K562, THP- 1, and U-937, and lymphoid lines Jurkat, JM, BALL 1, BJAB, and Raji. All cell lines were cultured in RPM1 1640 medium with 10%heat-inactivated FCS in our laboratory. Antibodiex Monoclonal antibodies used in this study included Leu-l la (IgG,), Leu-1 lb (IgM), and Leu-1 lc (or B73.1) (IgGr), all of which reacted with the FcR on NK cells and granulocytes (Beckton-Dickinson Co.) (13, 14). Another monoclonal antibody, VEP13 (IgM) reacting to the same FcR, was kindly provided by Dr. H. Rumpold (University of Vienna, Austria) (15). HNK- 1 (or Leu-7) (IgM) and ML2 (IgG,) monoclonal antibodies which reacted with a subpopulation of granular lymphocytes were prepared in this laboratory (9, 12). Monoclonal N901 antibody (IgG,) which reacted with NK cells was provided from Dr. S. F. Schlossman (Harvard Medical School) (16). Monoclonal anti-HLA-A, B, C common determinant antibody (PA2.6) (IgG,) and anti-HLA-DR antibody (MHM4) (IgG,) were gifts from Dr. A. J. McMichael (Oxford University, England) ( 17). Monoclonal anti-HLA-DQ antibody (Leu 10) (IgG,), fluorescein isothiocyanate (FITC)-conjugated Leu-l la, and phycoerythrin (PE)-conjugated Leu-l lc also were obtained from Beckton-Dickinson Co. The purified IgG fraction with high protein concentration (10 mg/ml) of Leu- 11a was
388
EGAWA,
ABO, AND
HIWATASHI
provided from Dr. L. L. Lamer (Beckton-Dickinson Co.). The IgM monoclonal LeuMl (MMA) antibody (Be&ton-Dickinson Co.) was used to estimate the proportion of monocytes in the purified cell fractions (18). Polyclonal anti-mouse IgG antibody, derived from goats, was obtained from Cappel Laboratories, Inc. NK assays. NK function was examined by a specific 5’Cr-releaseassaywith the use of various cultured cell lines. The method and formula for calculating specific cytolysis have been described ( 12). NK activity in this report was represented asthe percentage of cytotoxicity at a 10:1 effector-to-target cell (E:T) ratio, unless otherwise stated, by 4-hr incubation analysis. Lytic units (LU) per lo6 MNC were calculated as the number of cells required to lyse 30% of the target cells as described ( 19). Conjugate assay. Conjugates were formed by mixing equal numbers of MNC and K562 targets together in small tubes as described (20). The mixture was centrifuged, kept on ice for 30 min, and then gently resuspended for counting. The percentage of target-to&e&or cell conjugates was determined by counting the percentage of MNC binding target cells. Lymphokine assays. Interferon (IFN) titer of culture supernatants was determined by antiviral activity on human amnion (FL) cells infected with Sindbis virus and by the 50% plaque-reduction method. The method and formula for calculating IFN titer have been described (2 1). Standard IFN used in this study was human recombinant IFN-7 (Shionogi Pharmaceutical Co., Japan). Our murine NK cloned cells (NK-7) maintained with recombinant human interleukin 2 (IL-2) (Shionogi Pharmaceutical Co.) were used for the assayof IL-2 activity (22). Briefly, lo4 NK-7 cells/well in 0.1 ml medium were cultured with twofold 0. lml serially diluted samples. [3H]Thymidine uptake was measured after 48 hr of culture; 16 hr before terminating the culture, cells were pulsed with 0.5 &i/well of [3H]thymidine. Immunofluorescence tests. Expression of surface antigens on MNC or cultured cell lines was identified by monoclonal antibodies in conjunction with direct or indirect immunofluorescence tests (12). The FcR on MNC or cultured cell lines were detected by using PE-Leu- 11c or FITC-Leu- 11a, whereas the I-INK- 1 antigens on MNC were stained with FITC-Leu-7. The monoclonal Leu-Ml antibody was developed by FITC-conjugated F(ab’)* fragment of goat anti-mouse p-chain antibody (2). Fluorescent-positive cells were counted under a fluorescence microscope with phase-contrast equipment. F(aH)* and Fab preparations of murine immunoglobulins. The purified murine immunoglobulins of monoclonal Leu- 11a antibody were fractionated to F(ab’)* or Fab fragment by enzyme digestion methods. The F(ab’)Z and Fc fragments were separated by S-200 column chromatography after pepsin digestion as described (23). In this separation, gel filtration pattern indicated that a peak of undigestive molecule of Leu- 11a after pepsin treatment was very small and there was no overlapping between peaks of the intact molecule and F(ab’)* fragment of Leu- 11a. The Fab fragment was collected through S-200 column elution after papain digestion as specifically described for murine immunoglobulins (24). All preparations were dialyzed against phosphate-buffered saline (0.15 M) and sterilized by Millipore (Millipore Products Division). The staining titer of fragments F(ab’)Z and Fab of Leu- 11a in immunofluorescencetests was almost identical to that of the intact molecule. Trypsin treatment of cells. To eliminate NK functional capability of cells, trypsin treatment of cells was carried out (25,26). Ten million cells suspended in 1 ml of the
ENHANCEMENT a. Donor
Leull’. 0’
’ 20:1
1
Leul
9.4% I 10:1
I 5:l
b. Donor
OF NK ACTIVITY 2
1 +, 11 .Q%
c. Donor
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,
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to Target
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BY Leu-11
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FIG. 1. Enhancement of NK activity by Leu- 1la. Blood MNC from five different donors were incubated with K562 targets at effector-to-target cell ratios of 20: 1, 10:1, and 5: 1, in the presence of Leu- 11a, 0.25 &ml (a), or in the absence of Leu-1 la (0). Leu-1 la was added to assay culture of 4-hr analysis. The percentagesof Leu- 1l+ cells were enumerated by immunothtorescence tests with the use of FITC-Leu- 11a.
medium were incubated with 0.25% trypsin and 15 pg/ml deoxyribonuclease I (Sigma Chemical Co.) at 37°C for 30 min. After incubation, the cells were washed three times and then suspended in the medium. The viability was more than 95% as measured by the trypan blue dye exclusion test. RESULTS
Enhancement of NK activity by Leu-Ila. When blood MNC from five different donors were incubatated with K562 targets at the indicated ratios in the presence or absence of Leu- 11a (0.25 &ml), a marked enhancement of NK activity by Leu- 11a was observed in the samples taken from four donors (Donors 1 to 4, but not Donor 5) (Fig. 1). In these experiments, Leu- 1la was added to assay cuhure and thus was present throughout the 4 hr of NK assay.If we represent the results in Fig. 1 as lytic units per 1O6cells, the lytic units in MNC assayedwith or without Leu- 11a are 121.O rt 47.2 and 5 1.7 & 3 1.4 (LU/ lo6 ceils), respectively (P C 0.05, n = 5). The percentagesof Leu- 1l+ cells in MNC were estimated by FITC-Leu- 11a in parallel with NK assay in Donors 1,2, and 5 (Fig. 1). All of the donors had a significant level of Leu1la+ cells and of spontaneous NK activity against K562 targets. However, the enhancement of NK activity by Leu- 11a in Donor 5 was minimal. When we further examined this enhancement of NK activity by Leu- 11a in 278 healthy Japaneseadults at a 10:1 e&&or-to-target (K562) cell ratio, only 12 persons were demonstrated to be nonresponders (4.3%); they showed lessthan 10%enhancement in the percentage of cytotoxicity at the effector-to-target cell ratio used. These nonresponders, however, did not necessarily always lack either Leu- 11+ cells or NK functional activity against K562 targets (data not shown).
EGAWA, ABO, AND HIWATASHI
390 Lou-11
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0(-)
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Addition
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FIG. 2. Isotype (IgGr)-related enhancement of NK activity by the monoclonal Leu-1 I antibodies. A possible enhancing capability of NK activity by monoclonal antibodies cross-reactive with NK cells or granular lymphocytes was examined: the monoclonal antibodies used here included antibodies against FcR (Leu- 1la, Leu- 11b, Leu-1 Ic, and VEPl3), antibodies against granular lymphocytes or NK cells (Leu7, N90 1, and ML-2), and antibodies against HLA antigens (anti-HLA-A,B,C, anti-DR, and anti-DQ). All of these antibodies were added to assayculture at a final concentration of 0.1 pg/ml.
Isotype-relatedenhancementof NK activity by monoclonal Leu-I 1 antibodies. Several monoclonal antibodies directed to FcR on NK cells have been established in addition to Leu- 11a. The other monoclonal antibodies against FcR, including Leu11b, Leu- 1lc (B73. l), and VEP13, also were examined as to their enhancement capacity (Fig. 2). The monoclonal Leu-7, N901, and ML-2 antibodies reacted with molecules other than FcR on NK cells, and the monoclonal anti-HLA-A,B,C, anti-DR, and anti-DQ antibodies were used in parallel. All of these antibodies were added to assayculture at a final concentration of 0.1 &ml. As shown in the figure, both Leu1lc and Leu-1 la induced enhancement. Among the antibodies directed to FcR on NK cells, Leu- 11a and Leu- 1lc are IgG, , whereas Leu- 11b and VEP 13 are IgM. It is conceivable that the antibodies with IgG, isotype reacting with FcR on NK cells may induce enhancement of NK activity. The antibodies directed to molecules other than FcR did not induce prominent enhancement irrespective of their isotypes. Titration of Leu-1 la antibody-concentrationfor enhancement.The concentration of Leu- 11a effective for enhancement was titrated, using two different means of antibody exposure to cells (Fig. 3). When Leu- 11a was added to NK assayculture of 4hr analysis, a significant enhancement of NK activity was shown at a concentration of up to 1Op5mg/ml. On the other hand, pretreatment of cells with Leu- 11a-induced enhancement at a concentration of more than lop4 mg/ml. Here, pellets of lo6 MNC were mixed with 10 ~1 of Leu-l la at the indicated concentrations and incubated at 4°C for 30 min. These pretreated cells were washed three times and then assayedon
ENHANCEMENT
OF NK ACTIVITY
lo-6
10-7
Concentration
10-6
of
10-5
Leu-11
BY Leu-1 1
10-4
10-3
391
10-z
a (mg/ml)
FIG. 3. Titration of Leu-1 la antibody concentration for killing enhancement. The concentrations of Leu-1 la effective for enhancement were titrated, by using two different means of antibody-exposure to cells: Leu-1 la was added at the indicated concentrations to assayculture (0) or cells were pretreated with Leu- 11a at 4°C for 30 min (0).
their NK function. All of the pretreated cells with Leu-1 1 antibodies were equally prepared in the present study. Adherent monocyteswere not involved in enhancement.Involvement of adherent monocytes in enhancement was investigated, since monocytes are known to have FcR for IgG and may interact with the Leu- 11a molecule which binds to FcR on NK cells (Table 1). Nonadherent cells were prepared from three donors by passing MNC through a nylon-wool column. Enhancement was induced in all preparations depleted of monocytes by adding Leu- 11a to assayculture. The nonadherent cells used lacked any detectable levels of contaminating monocytes (<0.2%) defined by using the monoclonal Leu-M 1 antibody in conjunction with immunofluorescence tests. Eflect of anti-mouse IgG on enhancedNK activity by Leu-1 la. In this experiment, we investigated whether a secondary antibody directed to Leu-1 la inhibited or furTABLE 1 Leu- 11a-Induced Enhancement of NK Activity in Nonadherent Cells Depleted of Monocytes Addition of Leu- 1la Nonadherent cells”
(-)
1
19.56
2 3
15.5 46.5
(+) 60.5 64.2 71.4
’ Nonadherent cells were prepared from MNC of three different donors by depleting monocytes on a nylon-wool column. ’ % cytotoxicity.
EGAWA, ABO, AND HIWATASHI
392
% Cytotoxioity Supematant
from : -
cell8 3TC.
without 4houl-8
FIG. 4. Enhancing capability for NK activity in the culture supematants of cells pretreated with LeuI la. Culture supematants were harvested after 4-hr incubation from cells pretreated with or without Leu1la and then added to the NK assay system at a concentration of 50%. Data were represented as means f SE of the percentage of cytotoxicity (E:T = 10:1) from four experiments.
ther augmented the enhanced NK activity by Leu-1 la. MNC pretreated with 10 ~1 of Leu-1 la (10 &nI) were assayedwith or without anti-mouse IgG (50 &ml) for their NK function of 4-hr analysis. All of enhancement was completely eliminated by the addition of anti-mouse IgG. However, original spontaneous NK activity was not affected by the treatment of anti-mouse IgG (data not shown). Analysis of the culture supernatantsof cellspretreated with Leu-I la. In these experiments, the culture supernatants of MNC pretreated with Leu- 11a were investigated for their NK enhancing activity. MNC were pretreated with 10 &ml of Leu- 1la ( 10 &ml) at 4°C for 30 min, washed three times, and then incubated in the medium at 37 or 4°C. The culture supematants were harvested after a 4-hr incubation and then added to NK assay system at a concentration of 50% (Fig. 4). Although the control supematant from cells untreated with Leu- 11a did not show any effects,the supematant of MNC pretreated with Leu- 11a showed a potent enhancing activity. The cells incubated at 4°C also produced a considerable enhancing activity in the supematant. In recent studies, NK cells have been demonstrated to produce several lymphokines, including IFN and IL-2. It is well known that IFN and IL-2 are potent enhancers of NK activity. The possibility that these lymphokines could be produced from MNC pretreated with Leu- 11a was examined. Despite a potent enhancing activity in the supematant from ceils pretreated with Leu- 11a, no activity of IFN (< 1 IU/ml) or IL-2 (< 1 U/ml) was detectable, nor was activity detectable in the control supematant (data not shown). This result also was confirmed by experiments with the use of excessamounts of the monoclonal antibodies against IFN-y and IL2 (Shionogi Pharmaceutical Co.). When either anti-IFN-y or anti-IL2 was added to assayculture, no reduction of killing enhancement by Leu- 11a was observed (data not shown). In the next experiment, we investigated the possibility that MNC pretreated with Leu- 11a released a significant amount of Leu- 11a itself from their cell surface into the supematant during the culture. The culture supematant with enhancing activity was incubated with anti-mouse IgG (50 pg/ml) and then added to the NK assaysys-
ENHANCEMENT
OF NK ACTIVITY THP-
Jurkat
K 562
393
BY Leu-11 BALL-
1
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0%
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0%
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FIG. 5. Enhancing effects of Leu- 1la and Leu- 1lc on NK activity against various target cells. Enhancing effects of NK activity by Lcu- 1I monoclonal antibodies, Lcu- 11a and Leu- 11c, were investigated by using various target cell lines. The antibody was added to assay culture of 4-hr analysis at a concentration of 0.25 &ml. The Leu- 11 antigen expression on target ceils was examined by using PE-Leu-1 lc. Significant proportions of cell lines K562, Jurkat, THP-1, and BALL1 were demonstrated to express the Leu-1 lc antigens on their cell surface.
tern. The enhancing activity was completely abrogated by this treatment (data not shown). Enhancing eflects of Leu-lla and Leu-llc on NK activity against various target cells. Enhancing effect of NK activity by Leu-1 1 antibodies was further confirmed by using various target cell lines with diverse cell origins (Fig. 5). Interestingly, the vulnerability of half of the cell lines used, K562, Jurkat, THP-I and BALL-l, was augmented by adding both Leu- 11a and Leu- 11c in assayculture. Cytotoxicity of the other target cells, BJAB, U-937, JM, and Raji, was not changed by the additions. The cell origins of target cells did not seemto be related to the enhancement. In the following experiment, the Leu- 11 antigen expression on target cells was examined by using PE-Leu- 1lc. Significant proportions of cell lines K562, Jurkat, THP- 1 and BALL 1 were demonstrated to express the Leu-1 1 antigens on their cell surface (Fig. 5). In contrast with the strong reactivity of both Leu- 11a and Leu- 11c to NK cells in blood, these cell lines were reactive with Leu- 11c, but to a lesser extent Leu- 11a (data not shown). It is obvious that the enhancing effect of Leu-1 1 antibodies may be induced only when target cells that expressFcR identified by Leu- 11 antibodies are utilized. Enhancement of target-to-eflector cell conjugates in the presence of Leu-I Ic. Evidence for the existence of FcR on some target cells suggeststo us a possible mecha-
394
EGAWA, ABO, AND HIWATASHI TABLE 2
Enhancement of Target-to-Effector Cell Conjugates after Pretreatment of Effector Cells with Leu- 11a Addition of Leu- I la Donor”
C-1
(+)
1 2 3
12.ob 13.7 18.4
21.6 21.2 22.7
’ MNC from three different donors were analyzed on their target (K562)-to-effector cell conjugates after pretreatment with Lxx- 11a. In order to clearly represent the effect of Leu- 11a, MNC were prepared from the donors with relatively high levels of Leu- 11a+ cells in blood. The percentagesof Leu- 11a+ cells in MNC from Donors 1,2, and 3 were 22.6,21.5, and 23.4, respectively. ’ % MNC binding K562 targets.
nism for enhancement of NK activity by Leu-1 1 antibodies, explained as follows: Leu- 11 antibodies react with FcR on both effector and target cells and then accelerate their bindings in the initial phase of their interaction. This possibility was examined by conjugate assayusing K562 targets (Table 2). As expected, a sign&ant increase of the target-to-effecter cell conjugates was observed in the presence of Leu-1 lc (IgG,). We also demonstrated that Leu-1 lb (IgM) induced an increase of the target-toeffector K562 cell conjugates at the comparable level of that of Leu-1 la (data not shown). Nevertheless, Leu- 1lb did not induce any NK augmentation as already shown in Fig. 2. A control antibody anti-HLA-A,B,C did not induce any increase of the target-to-effector cell conjugates when K562 and Jurkat cells were used as target cells. Here, Jurkat cells all expressed human class I MHC antigens HLA-A,B,C on their surface, whereas K562 cells lacked class I MHC antigens (data not shown). Eflects of F(ab’)* and Fab fragments of Leu-1 la on NK activity. We examined the effects of F(ab’h and Fab of Leu-1 la on the enhancement capacity of NK activity (Fig. 6). It was clearly demonstrated that F(ab’)*, but not Fab, of Leu- 1la was effective in enhancing killing capacity. It is presumed that a bivalent activity of the antibody may be essential to induce the enhancement. Diflerential eflects oftrypsin treatment of eflector cells on Leu-I l-induced cytotoxicity and original spontaneous NK activity. It has been established that NK activity is abrogated by trypsin treatment of MNC. NK functional receptor seemsto be labile to trypsin. Indeed, when effector cells were pretreated with trypsin (0.25%) at 37°C for 30 min, almost all NK activity was eliminated (Fig. 7). The NK activity of these trypsin-treated MNC no longer could be enhanced when these cells were exposed to human recombinant EN-y (500 u/ml) at 37°C for 2 hr (Fig. 7b). This incubation was enough to enhance NK activity for the trypsin-untreated effector cells. On the other hand, this mild trypsin treatment of cells did not deteriorate the FcR expression on MNC. More than 80% activity of antibody-dependent cell-mediated cytotoxicity (ADCC; Raji cells and anti-Raji antibody system) was retained in the trypsin-treated MNC (data not shown). Leu-1 lc still induced a significant cytotoxicity in these trypsin-treated cells (Fig. 7a). The results showed a possibility that a functional receptor of Leu-1 l-induced cytotoxicity might be distinguishable from that of spontaneous NK activity.
ENHANCEMENT
OF NK ACTIVITY
a 60
395
BY Leu-ll
b
I
-
I
I
I-1
(+I
01 Ftab’lp
of Leu-11
t-1 Fab
a
(+I of
Leu-lla
FIG. 6. F(ab’)*, but not Fab, of Leu- 1la was effective for enhancement. MNC from three different donors were assayed on their NK function (E:T = 10: 1) with or without F(ab’)z of Leu-1 la (a) and the Fab of Leu1la (b). Each fragment was added to assayculture of 4-hr analysis at a concentration of 0.1 &ml.
DISCUSSlON In recent studies, a series of murine monoclonal antibodies directed to the FcR for IgG on human NK cells and granulocytes have been established (13- 15). In the present study, we demonstrated that among these monoclonal antibodies, the antibodies
40
* .t .u :: z‘:
,-
a
b
30
20
0 8 10
/ 0
t-1
(+I Leu-llc
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7. Differential effectsof trypsin treatment of effector cells on Leu- 1l-induced cytotoxicity and original spontaneous NK activity MNC were pretreated with trypsin (0.25%) at 37°C for 30 min (0) or without trypsin (0). The NK activity of these trypsin-treated or untreated effector cells were assayedin the presence of Leu- 1 lc (0.25 &ml) (a) or alter again exposing them with recombinant IFNr (500 U/ml)(b). Although trypsin-treated cells showed decreased NK activity and no longer could be augmented on their NK activity by recombinant IFN-y, these cells were still induced a significant cytotoxicity by Leu- 1lc. A representative result from three experiments was depicted. FIG.
396
EGAWA, ABO, AND HIWATASHI
with IgGl isotype (e.g., Leu-l la and Leu- 1lc) e5ciently enhanced NK activity against several target cells, including a commonly used target cell line K562 with the highest susceptibility to human NK cells. This phenomenon was observed only when MNC, including NK cells, were used as effector cells. Granulocytes originally lacked any binding (<5%) and killing activities against K562, and this result was not changed even in the presence of Leu- 11a (up to 10e3mg/ml in NK assayculture). To our knowledge, there has been no report on enhancement of NK activity by Leu-1 1 monoclonal antibodies. In a study using the IgM monoclonal antibody VEP13, Rumpold et al. demonstrated that the NK activity of cells stained with VEP13, but unsorted, was unchanged compared with that of untreated cells (15). These results were consistent with ours. In earlier studies, Perussia et al. investigated the effects of B73.1 (or Leu-1 lc) monoclonal antibody on antibody-dependent cytotoxicity and spontaneous cytotoxicity of NK cells (26,27). In their reports, the B73.1 antibody did not induce the enhancement of NK activity against K562 targets. Our findings do not agree with these results. To clear up the discrepancies between these findings, we first examined individual variations of enhancing NK activity mediated by monoclonal Leu-1 1 antibodies. Although MNC from a few individuals tested (4.3%) did not show any significant enhancement of NK activity by Leu-1 la, MNC taken from more than 95% of 278 healthy Japaneseadults showed that Leu-1 la induced enhancement. Ethnic differences in the enhancement of NK activity by Leu11 antibodies remain to be tested. Earlier, we reported ethnic differences in the functional expression of FcR for murine IgG, immunoglobulins on human monocytes (28). Another possibility to explain the discrepancies was that the K562 targets Perussia et al. used expressed Leu- 11 antigens to a lesserextent on the cell surface than the K562 targets we used. Magnitude of killing enhancement by Leu- 11 antibodies was proportional to Leu-1 1 antigen expression of the target cells used. Enhancement of NK activity by Leu- 11 monoclonal antibodies was produced only when Leu- 11+ cell lines were used as target cells in assay.It is, therefore, conceivable that Leu-1 1 antibodies may become a linkage between effector and target cells. A similar crosslinking of effector cells to targets using monoclonal antibody has been described in the T-cell system (29). This conception was supported by the result that Leu- 1la actually increased the number of target-to-effector cell conjugates. The effectivenessof F(ab’h but not of the Fab fragment of Leu- 11a further supported this conception. This result also indicated that a mechanism of ADCC might not be, if any, a major part of the enhancement by Leu-1 1 antibodies in this study. Furthermore, it was reported that the ADCC of human NK cells preferentially mediated with IgG,, but not IgG, of anti-target cell murine monoclonal antibodies (30). Although Leu-1 lb (IgM) also induced a significant level of the increase of conjugation, there was no induction of the NK augmentation at all. This result suggestedthat a crosslinking between NK cells and targets was not su5cient enough to induce the NK augmentation. Even after crosslinking, isotype-specific molecular interaction (IgGi but not IgM) with cells might be required. Enhancement of NK activity by Leu-1 1 antibodies was restricted to the IgGl monoclonal antibodies. In cross-competition experiments using ‘251-labeledLeu- 11 antibodies, Peru&a et al. have demonstrated that Leu- 1la (IgGI) and Leu-1 1b (IgM), but not Leu-1 lc (B73.1) (IgGI), recognize a same epitope on NK cells (3 1). It is clear, therefore, that only the isotope of Leu- 11 monoclonal antibody is important for killing enhancement, whereas the epitope recognized by the antibody is not. Effects of
ENHANCEMENT
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BY La-1 1
397
the other FcR monoclonal antibodies with IgGh, IgGlb, or IgG3 isotype remain to be tested, because these antibodies are not available yet. We do not know why only IgG, , but not IgM, of Leu-1 1 monoclonal antibodies is effective to the NK augmentation. However, similar isotype-restricted phenomena of monoclonal antibodies have been reported. For example, murine IgGza is effective in a certain human ADCC system (30), whereas murine IgG, and IgGz, of anti-CD3 antibody are effective to CD3-induced human T-cell proliferation (28). Murine IgM antibodies are not working at all in both systems. In the present experiments, we also examined the effect of monoclonal antibodies reacting with molecules other than FcR on enhancement of NK activity; the antibodies used were Leu-7 (IgM), N901 (IgGr), and MG2 (IgG,). Although all of these monoclonal antibodies reacted with a considerable proportion of human NK cells, any significant enhancement of NK activity was not produced. The monoclonal antiHLA-A,B,C, anti-DR, and anti-DQ antibodies induced a minimal augmentation of NK activity. In the murine system, antibody-induced augmentation of NK activity was demonstrated by using monoclonal antibodies, such as anti-H-2, anti-Ia, and anti-Thy- 1.2 (32,33). When MNC pretreated with Leu-1 1 monoclonal antibody were incubated overnight at 37°C enhancing activity was detectable in the culture supernatants. However, the supematants did not contain any activities of either IFN or IG2. As polyclonal anti-mouse IgG completely neutralized enhancing activity in the supematants, Leu-1 1 antibody itself might be released from the cell surface during incubation. It was reported that the Leu- 11 antibody that bound to cells was rapidly shedded from the cell surface when cells were incubated at 37°C (26). It has been revealed that a series of the Leu-1 1 monoclonal antibodies recognize the FcR on NK cells and granulocytes but not on monocytes and B cells in normal human leukocytes (13- 15). The present results, however, demonstrated that many established cell lines with diverse cell origins, including myelomonocytoid, T-, and B-cell lines, expressed the antigens identified by Leu-1 lc. The observed staining of Leu-1 lc on K562 was unlikely to be nonspecific binding of murine IgG, immunoglobulins to their FcR, because the other IgG, monoclonal antibodies (e.g., antiHLA-A,B,C and anti-N901) were not reactive with K562 (data not shown). In contrast with the strong reactivity of both Leu- 11a and Leu- 11c to NK cells in blood, these cell lines were reactive with Leu- 11c but to a lesser extent with Leu- 11a. It has been established that the epitope on FcR recognized by Leu-1 la (and Leu-1 lb) is distinct from that recognized by Leu-1 lc (3 1). Despite these staining variations, Leu1la and Leu-1 lc were both functionally effective for enhancement. Here, the pretreatment of target cells with Lcu- 11 antibodies Leu- 11a and Leu- 1lc, was found to be always less efficient for enhancement than the effector cell pretreatment (data not shown). It is presumed that the low efficiency of target cell pretreatment may be due to the relatively lower expression of Leu- 11 antigens on established cultured cell lines than that of fresh MNC. There have been several reports that FcR on NK cells is relatively resistant to trypsin treatment, whereas the NK functional receptor is sensitive to the same treatment (25,26). Indeed, we were able to eliminate almost all spontaneous cytotoxicity of NK cells by this treatment. The NK activity of these trypsin-treated cells could not be augmented by pretreatment with recombinant IFN-7. Of particular interest was that the Leu-1 1 antibody still induced a significant enhancement of NK activity in the
398
EGAWA, ABO, AND HIWATASHI
trypsin-treated cells. These results indicated a possibility that a functional receptor of Leu- 11-induced cytotoxicity might be distinguishable from that of spontaneous NK cytotoxicity. Namely, enhancement of NK activity by the Leu- 11 antibody was felt not to be a mere augmentation of spontaneous NK activity, but was felt to be the cytotoxicity mediated via the distinct functional receptor, probably a linkage of an FcR (on targets)-Ieu- 11-FcR (on effectors) complex. Similar cytotoxicity mediated by receptors other than NK functional receptors is the lectin-induced cell-mediated cytotoxicity (LICC), where a linkage of lectin and lectin receptors on both target and effector cells may be functional receptors for LICC (34). The lytic machinery itself seems,however, to be common to all of these cytotoxicities. In the present study, we demonstrated the enhancement of human NK activity by the Leu- 11 monoclonal antibodies. This phenomenon occurred when target cells in NK assaysexpressed the Leu-1 1 antigens on their cell surface. Surprisingly, many NK target cells of established cultured cell tines, including K562 targets, were demonstrated to expressthe Leu- 11 antigens. For practical application of Leu- 11 antibodies, attention should be paid to the isotypes of Leu- 11 antibodies in conjunction with the nature of target cells in NK assays.For example, when the target cells used in a given performing system is Leu-1 l+, the Leu-1 1 antibodies with IgM isotype rather than IgG, should be used (e.g., for cell sorting experiments) to avoid variations of spontaneous NK cytotoxicity. Finally, we observed only a few nonresponders to killing enhancement by Leu- 11 antibodies among normal healthy adult donors. In the preliminary experiments, we demonstrated that many pregnant women and some patients with autoimmune diseases(e.g., Crohn’s disease)are nonresponders to the enhancement, despite their normal levels of Leu- 11+ cells in blood. Experiments are currently in progress to investigate whether defective responsesto Leu- 11 antibodies observed in pregnant women and some patients were due to the paralysis of NK cells by some serum components via their FcR. The Leu- 11-mediated cytotoxicity presented here may contain some clinical values to help us understand and evaluate the clinical status of diseased persons. ACKNOWLEDGMENTS We are grateful to Drs. Hiroshi Itoh and Katsuo Kumagai for helpful discussion, and Ms. Junko Hirata and Ms. Hiroko Akaishi for their expert editorial assistance. Note added in proof: After completing this manuscript, M. Jondal et al. have reported the enhancement of NK activity by moncclonal antibodies against NK cell FcR (35). They have also observed the isotype (IgG,)-specific function in this phenomenon. Our manuscript includes several experiments to analyze the basis of the mechanism.
REFERENCES 1. Timonen, T., Ortaldo, J. R., and Herberman, R. B., J. Exp. Med. 153,569,198 1. 2. Abo, T., Miller, C. A., and Balch, C. M., Eur. J. Immunol. 14,616, 1984. 3. Ferrarini, M., and Grossi, C. E., Seminars Hematol. 21,270, 1984. 4. Trinchieti, G., and Perussia, B., Lab. Invest. 50,489, 1984. 5. Lanier, L. L., Le, A. M., Phillips, J. H., Warner, N. L., and Babcock, G. F., .I. Zmmunol. 131, 1789, 1983. 6. Zarling, J. M., Clause, K. A., Biddison, W. E., and Kung, P. C., J. Immunol. 127,2575, 1981. 7. Schlesinger, M., Lew, F., and Bekesi, J. G., J. C/in. Lab. Immunol. 16, 13, 1985.
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OF NK ACTIVITY
BY Leu-ll
399
8. Landay, A., Gartland, G. L., and Clement, L. T., J. Immunol. 131,2X’, 1983. 9. Abe, T., Sugawara, S., Amenomori, A., Itoh, H., Rikiishi, H., Moro, I., and Kumagai, K., J. Immunol. 136,3189,1986.
10. Abo, T., Cooper, M. D., and Balch, C. M., J. ZmmunoZ.129,1752, 1982. 11. Abo, T., and Balch, C. M., J. Zmmunol. 129,1758,1982. 12. Abo, T., and Balch, C. M., J. Zmmunol. 127, 1024, 1981. 13. Phillips, J. H., and Babcock, G. F., Zmmunol. Lett. 6, 143, 1983. 14. Pen&a, B., Starr, S., Abraham, S., Fanning, V., and Trinchieri, G., J. Immunol. 130,2133, 1983. 15. Rumpold, H., Krafi, D., Obexer, G., Bock, G., and Gebhart, W., J. Immunol. 129, 1458, 1982. 16. Griffin, J. D., Hercend, T., Beveridge, R., and Schlossman, S. F., J. Zmmunol. 130,2947, 1983. 17. Wallace, L. E., Moss, D. L., Rickinson, A. B., McMichael, A. J., and Epstein, M. A., Eur. J. Zmmunol. 11,694, 1981.
18. Hanjan, S. N. S., Keamey, J. F., and Cooper, M. D., Clin. Immunol. Immunopathol. 23, 172, 1982. 19. Abo, T., Miller, C. A., Gartland, G. L., and Balch, C. M., J. Exp. Med. 157,273, 1983. 20. Rubin, P., Pross, H. F., and Roder, J. C., J. Immunol. 128,2553, 1982. 2 1. Itoh, K., Shiiba, K., Shimizu, Y., Suzuki, R., and Kumagai, K., J. Zmmunol. 134,3124, 1985. 22. Suzuki, R., Suzuki, S., Ebina, N., and Kumagai, K., J. Zmmuno!. 124,2139,198X 23. Mandy, W. J., and Nisonoff, A., .I. Biol. Chem. 23&206,1963. 24. Fahey, J. L., and Askonas, B. A., J. Exp. Med. 115,623, 1961. 25. Koide, K., and Takasugi, M., J. Natl. Cancer Inst. 59,1099, 1977. 26. Perussia, B., Acute, O., Terhorst, C., Faust, J., Lazarus, R., Fanning, V., and Trinchieri, G., J. Immunol. 130,2142,1983.
27. Perussia, B., and Trinchieri, G., J. Immunol. 132, 1419, 1984. 28. Abo, T., Tilden, A. B., Balch, C. M., Kumagai, K., Troup, F. M., and Cooper, M. D., J. Exp. Med. 160,303, 1984.
29. Staerz, U. D., Kanagawa, O., and Bevan, M. J., Nature (London) 314,628, 1985. 30. Lanier, L. L., Kipps, T. J., and Phillips, J. H., J. Exp. Med. 162,2089, 1982. 3 1. Peru&a, B., Trinchieri, G., Jackson, A., Warner, N. L., Faust, J., Rumpold, H., Kraft, D., and Lanier, L. L., J. Zmmunol. 133,180, 1984. 32. Saxena, R. K., and Adler, W. H., J. Zmmunol. 123,846, 1979. 33. Brunda, M. J., Herberman, R. B., and Holden, H. T., Znt. J. Cancer 27,205, 198I. 34. Brunda, M. J., Varesio, L., Herberman, R. B., and Holden, H. T., Znt. J. Cancer 29,299, 1982. 35. Jondal, M., Kullman, C., Alter, M.-B., and Ljunggren, K., Cell. Zmmunof. 100, 158, 1986.
104,386-399 (1987)
IMMUNOLOGY
Enhancement of Human Natural Killer Activity by the Monoclonal Leu-1 1 Antibodies SHUN-ENEGAWA,TORU ABO,*,’ AND NOBUOHIWATASHI Third Department oflnternal Medicine, Tohoku University School OfMedicine, and *Department of Microbiology, Tohoku University School ofDentistry, Sendai 980, Japan Received June 27,1986; accepted October 21,1986 The Fc receptor for IgG on human natural loller (NK) cells can be identified by a series of murine monoclonal antibodies. When these antibodies with IgGi &type (e.g., Leu-1 la and Leu-1 lc), but not with IgM (e.g., Leu- 11b and VEPl3), were added to NK assay culture against K562 targets of 4-hr analysis, a considerable enhancement of NK activity was induced. An enhancement was shown at a concentration of up to lo-’ mg/ml of Leu-1 la. Furthermore, the present experiments demonstrated that many established cell lines with diverse cell origins expressed the Leu- 11 antigens, and enhancement of NK activity by Leu- 11 was induced when these Leu-1 I+ cell lines were used as targets in NK assays. The results that Leu-1 la caused an increase in effector-to-target cell conjugates, and that F(ab’)* of Leu-1 la was effective in enhancement of NK activity, but the Fab was not, indicated that Leu- 11might become a linkage between the effector and target cells. Significance of this phenomenon was discussedregarding practical application of L.eu-11 antibodies in laboratory experiments and in clinical studies. 8 1987 Academic
F’rm.
Inc.
INTRODUCTION Natural killer (NK)* cells are a subpopulation of lymphoid cells that have several unique characteristics in terms of their morphology, phenotype, NK function, and cytochemical distribution pattern of cytoplasmic enzymes, and are therefore distinguishable from classicahy identified T and B lymphocytes (l-5). Cumulative evidence has suggestedthat NK cells may exert their cytotoxicity to aberrant cells yielded in the host (e.g., rapidly dividing normal and malignant cells and virally infected cells) and, in the long run, may play an important role in immunoregulatory functions and host defense mechanisms against malignancy and infections (3,4). The recent availability of monoclonal antibodies enables us to intensively characterize the phenotype of human NK cells. By using monoclonal antibodies directed to human NK cells, some subpopulations of NK cells (or granular lymphocytes) have been identified (2, 5). A major population of NK cells with prominent NK activity expressesFc receptor (FcR) defined by a series of monoclonal Leu-1 1 antibodies as ’ To whom correspondence should be addressed. ’ Abbreviations used: NK, natural killer; FcR, Fc receptor; MNC, mononuclear cells; IFN, interferon; IL2, interleukin 2; FITC, fluorescein isothiocyanate; PE, phycoerythrin; ADCC, antibody-dependent cellmediated cytotoxicity. 386 0008-8749187$3.00 Cawisht 0 1987 by Academic Fws, Inc. AU right.5 of reproduction in my form revved.
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well as other myeloid antigens (e.g., OKM 1, Leu-M4, and Leu- 15) (6-8). In addition to myeloid antigen expression, we have recently demonstrated that this NK population is capable of selective phagocytosis and the subsequent production of interleukin 1, both of these functions being similar to those of myelomonocytes (9). Studies using monoclonal antibodies also have revealed another unique lymphoid population with a morphology of granular lymphocyte that has minimal NK activity and expresses T-cell-associated antigens instead of the Leu- 11 antigens ( 10, 11). As it lacks many features ascribed to classically identified T cells, the relationship of this population to NK cells or T cells remains to be elucidated. In the present study, we investigated an interaction between monoclonal Leu-ll antibodies and FcR for IgG on human NK cells. Thus, when the Leu-ll antibodies with IgG, isotype were added to NK assay culture, a considerable enhancement of NK activity was induced. This enhancement was produced when target cells used in NK assaysexpressed the Leu-I 1 antigens (FcR). Surprisingly, many target cells of established cultured cell lines commonly used for human NK assays,including K562 targets with the highest susceptibility to human NK cells, were demonstrated to express FcR. The present experiments also indicated a possibility that Leu- 11-induced enhancement of NK activity might be mediated by a receptor distinguishable from that of original spontaneous NK cytotoxicity. In this study, we analyzed the precise phenomenon and mechanism of this cytotoxicity and discussed what possible significance the phenomenon might have regarding practical application of Leu- 11 antibodies in laboratory experiments and in clinical studies. MATERIALS AND METHODS Cell preparations. Heparinized blood was obtained from healthy Japanese adult donors. Mononuclear cells (MNC) were separated by Ficoll-Isopaque density gradient centrifugation (12). Nonadherent cells were prepared from MNC by depleting monocytes on a nylon-wool column (12). The prepared cells were suspended in RPM1 1640 medium with 10%heat-inactivated fetal calf serum (FCS). Cultured cell lines. Human cultured cell lines, which were used as targets in NK assays,were myelomonocytoid lines K562, THP- 1, and U-937, and lymphoid lines Jurkat, JM, BALL 1, BJAB, and Raji. All cell lines were cultured in RPM1 1640 medium with 10%heat-inactivated FCS in our laboratory. Antibodiex Monoclonal antibodies used in this study included Leu-l la (IgG,), Leu-1 lb (IgM), and Leu-1 lc (or B73.1) (IgGr), all of which reacted with the FcR on NK cells and granulocytes (Beckton-Dickinson Co.) (13, 14). Another monoclonal antibody, VEP13 (IgM) reacting to the same FcR, was kindly provided by Dr. H. Rumpold (University of Vienna, Austria) (15). HNK- 1 (or Leu-7) (IgM) and ML2 (IgG,) monoclonal antibodies which reacted with a subpopulation of granular lymphocytes were prepared in this laboratory (9, 12). Monoclonal N901 antibody (IgG,) which reacted with NK cells was provided from Dr. S. F. Schlossman (Harvard Medical School) (16). Monoclonal anti-HLA-A, B, C common determinant antibody (PA2.6) (IgG,) and anti-HLA-DR antibody (MHM4) (IgG,) were gifts from Dr. A. J. McMichael (Oxford University, England) ( 17). Monoclonal anti-HLA-DQ antibody (Leu 10) (IgG,), fluorescein isothiocyanate (FITC)-conjugated Leu-l la, and phycoerythrin (PE)-conjugated Leu-l lc also were obtained from Beckton-Dickinson Co. The purified IgG fraction with high protein concentration (10 mg/ml) of Leu- 11a was
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provided from Dr. L. L. Lamer (Beckton-Dickinson Co.). The IgM monoclonal LeuMl (MMA) antibody (Be&ton-Dickinson Co.) was used to estimate the proportion of monocytes in the purified cell fractions (18). Polyclonal anti-mouse IgG antibody, derived from goats, was obtained from Cappel Laboratories, Inc. NK assays. NK function was examined by a specific 5’Cr-releaseassaywith the use of various cultured cell lines. The method and formula for calculating specific cytolysis have been described ( 12). NK activity in this report was represented asthe percentage of cytotoxicity at a 10:1 effector-to-target cell (E:T) ratio, unless otherwise stated, by 4-hr incubation analysis. Lytic units (LU) per lo6 MNC were calculated as the number of cells required to lyse 30% of the target cells as described ( 19). Conjugate assay. Conjugates were formed by mixing equal numbers of MNC and K562 targets together in small tubes as described (20). The mixture was centrifuged, kept on ice for 30 min, and then gently resuspended for counting. The percentage of target-to&e&or cell conjugates was determined by counting the percentage of MNC binding target cells. Lymphokine assays. Interferon (IFN) titer of culture supernatants was determined by antiviral activity on human amnion (FL) cells infected with Sindbis virus and by the 50% plaque-reduction method. The method and formula for calculating IFN titer have been described (2 1). Standard IFN used in this study was human recombinant IFN-7 (Shionogi Pharmaceutical Co., Japan). Our murine NK cloned cells (NK-7) maintained with recombinant human interleukin 2 (IL-2) (Shionogi Pharmaceutical Co.) were used for the assayof IL-2 activity (22). Briefly, lo4 NK-7 cells/well in 0.1 ml medium were cultured with twofold 0. lml serially diluted samples. [3H]Thymidine uptake was measured after 48 hr of culture; 16 hr before terminating the culture, cells were pulsed with 0.5 &i/well of [3H]thymidine. Immunofluorescence tests. Expression of surface antigens on MNC or cultured cell lines was identified by monoclonal antibodies in conjunction with direct or indirect immunofluorescence tests (12). The FcR on MNC or cultured cell lines were detected by using PE-Leu- 11c or FITC-Leu- 11a, whereas the I-INK- 1 antigens on MNC were stained with FITC-Leu-7. The monoclonal Leu-Ml antibody was developed by FITC-conjugated F(ab’)* fragment of goat anti-mouse p-chain antibody (2). Fluorescent-positive cells were counted under a fluorescence microscope with phase-contrast equipment. F(aH)* and Fab preparations of murine immunoglobulins. The purified murine immunoglobulins of monoclonal Leu- 11a antibody were fractionated to F(ab’)* or Fab fragment by enzyme digestion methods. The F(ab’)Z and Fc fragments were separated by S-200 column chromatography after pepsin digestion as described (23). In this separation, gel filtration pattern indicated that a peak of undigestive molecule of Leu- 11a after pepsin treatment was very small and there was no overlapping between peaks of the intact molecule and F(ab’)* fragment of Leu- 11a. The Fab fragment was collected through S-200 column elution after papain digestion as specifically described for murine immunoglobulins (24). All preparations were dialyzed against phosphate-buffered saline (0.15 M) and sterilized by Millipore (Millipore Products Division). The staining titer of fragments F(ab’)Z and Fab of Leu- 11a in immunofluorescencetests was almost identical to that of the intact molecule. Trypsin treatment of cells. To eliminate NK functional capability of cells, trypsin treatment of cells was carried out (25,26). Ten million cells suspended in 1 ml of the
ENHANCEMENT a. Donor
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FIG. 1. Enhancement of NK activity by Leu- 1la. Blood MNC from five different donors were incubated with K562 targets at effector-to-target cell ratios of 20: 1, 10:1, and 5: 1, in the presence of Leu- 11a, 0.25 &ml (a), or in the absence of Leu-1 la (0). Leu-1 la was added to assay culture of 4-hr analysis. The percentagesof Leu- 1l+ cells were enumerated by immunothtorescence tests with the use of FITC-Leu- 11a.
medium were incubated with 0.25% trypsin and 15 pg/ml deoxyribonuclease I (Sigma Chemical Co.) at 37°C for 30 min. After incubation, the cells were washed three times and then suspended in the medium. The viability was more than 95% as measured by the trypan blue dye exclusion test. RESULTS
Enhancement of NK activity by Leu-Ila. When blood MNC from five different donors were incubatated with K562 targets at the indicated ratios in the presence or absence of Leu- 11a (0.25 &ml), a marked enhancement of NK activity by Leu- 11a was observed in the samples taken from four donors (Donors 1 to 4, but not Donor 5) (Fig. 1). In these experiments, Leu- 1la was added to assay cuhure and thus was present throughout the 4 hr of NK assay.If we represent the results in Fig. 1 as lytic units per 1O6cells, the lytic units in MNC assayedwith or without Leu- 11a are 121.O rt 47.2 and 5 1.7 & 3 1.4 (LU/ lo6 ceils), respectively (P C 0.05, n = 5). The percentagesof Leu- 1l+ cells in MNC were estimated by FITC-Leu- 11a in parallel with NK assay in Donors 1,2, and 5 (Fig. 1). All of the donors had a significant level of Leu1la+ cells and of spontaneous NK activity against K562 targets. However, the enhancement of NK activity by Leu- 11a in Donor 5 was minimal. When we further examined this enhancement of NK activity by Leu- 11a in 278 healthy Japaneseadults at a 10:1 e&&or-to-target (K562) cell ratio, only 12 persons were demonstrated to be nonresponders (4.3%); they showed lessthan 10%enhancement in the percentage of cytotoxicity at the effector-to-target cell ratio used. These nonresponders, however, did not necessarily always lack either Leu- 11+ cells or NK functional activity against K562 targets (data not shown).
EGAWA, ABO, AND HIWATASHI
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FIG. 2. Isotype (IgGr)-related enhancement of NK activity by the monoclonal Leu-1 I antibodies. A possible enhancing capability of NK activity by monoclonal antibodies cross-reactive with NK cells or granular lymphocytes was examined: the monoclonal antibodies used here included antibodies against FcR (Leu- 1la, Leu- 11b, Leu-1 Ic, and VEPl3), antibodies against granular lymphocytes or NK cells (Leu7, N90 1, and ML-2), and antibodies against HLA antigens (anti-HLA-A,B,C, anti-DR, and anti-DQ). All of these antibodies were added to assayculture at a final concentration of 0.1 pg/ml.
Isotype-relatedenhancementof NK activity by monoclonal Leu-I 1 antibodies. Several monoclonal antibodies directed to FcR on NK cells have been established in addition to Leu- 11a. The other monoclonal antibodies against FcR, including Leu11b, Leu- 1lc (B73. l), and VEP13, also were examined as to their enhancement capacity (Fig. 2). The monoclonal Leu-7, N901, and ML-2 antibodies reacted with molecules other than FcR on NK cells, and the monoclonal anti-HLA-A,B,C, anti-DR, and anti-DQ antibodies were used in parallel. All of these antibodies were added to assayculture at a final concentration of 0.1 &ml. As shown in the figure, both Leu1lc and Leu-1 la induced enhancement. Among the antibodies directed to FcR on NK cells, Leu- 11a and Leu- 1lc are IgG, , whereas Leu- 11b and VEP 13 are IgM. It is conceivable that the antibodies with IgG, isotype reacting with FcR on NK cells may induce enhancement of NK activity. The antibodies directed to molecules other than FcR did not induce prominent enhancement irrespective of their isotypes. Titration of Leu-1 la antibody-concentrationfor enhancement.The concentration of Leu- 11a effective for enhancement was titrated, using two different means of antibody exposure to cells (Fig. 3). When Leu- 11a was added to NK assayculture of 4hr analysis, a significant enhancement of NK activity was shown at a concentration of up to 1Op5mg/ml. On the other hand, pretreatment of cells with Leu- 11a-induced enhancement at a concentration of more than lop4 mg/ml. Here, pellets of lo6 MNC were mixed with 10 ~1 of Leu-l la at the indicated concentrations and incubated at 4°C for 30 min. These pretreated cells were washed three times and then assayedon
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Concentration
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391
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a (mg/ml)
FIG. 3. Titration of Leu-1 la antibody concentration for killing enhancement. The concentrations of Leu-1 la effective for enhancement were titrated, by using two different means of antibody-exposure to cells: Leu-1 la was added at the indicated concentrations to assayculture (0) or cells were pretreated with Leu- 11a at 4°C for 30 min (0).
their NK function. All of the pretreated cells with Leu-1 1 antibodies were equally prepared in the present study. Adherent monocyteswere not involved in enhancement.Involvement of adherent monocytes in enhancement was investigated, since monocytes are known to have FcR for IgG and may interact with the Leu- 11a molecule which binds to FcR on NK cells (Table 1). Nonadherent cells were prepared from three donors by passing MNC through a nylon-wool column. Enhancement was induced in all preparations depleted of monocytes by adding Leu- 11a to assayculture. The nonadherent cells used lacked any detectable levels of contaminating monocytes (<0.2%) defined by using the monoclonal Leu-M 1 antibody in conjunction with immunofluorescence tests. Eflect of anti-mouse IgG on enhancedNK activity by Leu-1 la. In this experiment, we investigated whether a secondary antibody directed to Leu-1 la inhibited or furTABLE 1 Leu- 11a-Induced Enhancement of NK Activity in Nonadherent Cells Depleted of Monocytes Addition of Leu- 1la Nonadherent cells”
(-)
1
19.56
2 3
15.5 46.5
(+) 60.5 64.2 71.4
’ Nonadherent cells were prepared from MNC of three different donors by depleting monocytes on a nylon-wool column. ’ % cytotoxicity.
EGAWA, ABO, AND HIWATASHI
392
% Cytotoxioity Supematant
from : -
cell8 3TC.
without 4houl-8
FIG. 4. Enhancing capability for NK activity in the culture supematants of cells pretreated with LeuI la. Culture supematants were harvested after 4-hr incubation from cells pretreated with or without Leu1la and then added to the NK assay system at a concentration of 50%. Data were represented as means f SE of the percentage of cytotoxicity (E:T = 10:1) from four experiments.
ther augmented the enhanced NK activity by Leu-1 la. MNC pretreated with 10 ~1 of Leu-1 la (10 &nI) were assayedwith or without anti-mouse IgG (50 &ml) for their NK function of 4-hr analysis. All of enhancement was completely eliminated by the addition of anti-mouse IgG. However, original spontaneous NK activity was not affected by the treatment of anti-mouse IgG (data not shown). Analysis of the culture supernatantsof cellspretreated with Leu-I la. In these experiments, the culture supernatants of MNC pretreated with Leu- 11a were investigated for their NK enhancing activity. MNC were pretreated with 10 &ml of Leu- 1la ( 10 &ml) at 4°C for 30 min, washed three times, and then incubated in the medium at 37 or 4°C. The culture supematants were harvested after a 4-hr incubation and then added to NK assay system at a concentration of 50% (Fig. 4). Although the control supematant from cells untreated with Leu- 11a did not show any effects,the supematant of MNC pretreated with Leu- 11a showed a potent enhancing activity. The cells incubated at 4°C also produced a considerable enhancing activity in the supematant. In recent studies, NK cells have been demonstrated to produce several lymphokines, including IFN and IL-2. It is well known that IFN and IL-2 are potent enhancers of NK activity. The possibility that these lymphokines could be produced from MNC pretreated with Leu- 11a was examined. Despite a potent enhancing activity in the supematant from ceils pretreated with Leu- 11a, no activity of IFN (< 1 IU/ml) or IL-2 (< 1 U/ml) was detectable, nor was activity detectable in the control supematant (data not shown). This result also was confirmed by experiments with the use of excessamounts of the monoclonal antibodies against IFN-y and IL2 (Shionogi Pharmaceutical Co.). When either anti-IFN-y or anti-IL2 was added to assayculture, no reduction of killing enhancement by Leu- 11a was observed (data not shown). In the next experiment, we investigated the possibility that MNC pretreated with Leu- 11a released a significant amount of Leu- 11a itself from their cell surface into the supematant during the culture. The culture supematant with enhancing activity was incubated with anti-mouse IgG (50 pg/ml) and then added to the NK assaysys-
ENHANCEMENT
OF NK ACTIVITY THP-
Jurkat
K 562
393
BY Leu-11 BALL-
1
1
Leul1+,7.6% 50 40 30
LL
20 ~~ 10 0 It-1
WH
L.u-31.
ct)
(-I
L.u-,,c
BJAB Leu11+,
u-937 Ld1+,
0%
JM Leu11+,
0%
l+1H
(+I
Raji Leu11+,
0%
0%
I aI
50
Y !i
40
xO
30
0 x
2o 10
2
)c h
O
t-1
w-1
Lou-1
I*
(+I L.u-,
,c
k
i (-I L.u-(1.
WH
t (i+
L.u-8,s
\% (4
L (+I(-)
L.“-lt.L.“-IlC
f+)
(4 L.“-1,.
(+I(-)
fii Lw-ttc
FIG. 5. Enhancing effects of Leu- 1la and Leu- 1lc on NK activity against various target cells. Enhancing effects of NK activity by Lcu- 1I monoclonal antibodies, Lcu- 11a and Leu- 11c, were investigated by using various target cell lines. The antibody was added to assay culture of 4-hr analysis at a concentration of 0.25 &ml. The Leu- 11 antigen expression on target ceils was examined by using PE-Leu-1 lc. Significant proportions of cell lines K562, Jurkat, THP-1, and BALL1 were demonstrated to express the Leu-1 lc antigens on their cell surface.
tern. The enhancing activity was completely abrogated by this treatment (data not shown). Enhancing eflects of Leu-lla and Leu-llc on NK activity against various target cells. Enhancing effect of NK activity by Leu-1 1 antibodies was further confirmed by using various target cell lines with diverse cell origins (Fig. 5). Interestingly, the vulnerability of half of the cell lines used, K562, Jurkat, THP-I and BALL-l, was augmented by adding both Leu- 11a and Leu- 11c in assayculture. Cytotoxicity of the other target cells, BJAB, U-937, JM, and Raji, was not changed by the additions. The cell origins of target cells did not seemto be related to the enhancement. In the following experiment, the Leu- 11 antigen expression on target cells was examined by using PE-Leu- 1lc. Significant proportions of cell lines K562, Jurkat, THP- 1 and BALL 1 were demonstrated to express the Leu-1 1 antigens on their cell surface (Fig. 5). In contrast with the strong reactivity of both Leu- 11a and Leu- 11c to NK cells in blood, these cell lines were reactive with Leu- 11c, but to a lesser extent Leu- 11a (data not shown). It is obvious that the enhancing effect of Leu-1 1 antibodies may be induced only when target cells that expressFcR identified by Leu- 11 antibodies are utilized. Enhancement of target-to-eflector cell conjugates in the presence of Leu-I Ic. Evidence for the existence of FcR on some target cells suggeststo us a possible mecha-
394
EGAWA, ABO, AND HIWATASHI TABLE 2
Enhancement of Target-to-Effector Cell Conjugates after Pretreatment of Effector Cells with Leu- 11a Addition of Leu- I la Donor”
C-1
(+)
1 2 3
12.ob 13.7 18.4
21.6 21.2 22.7
’ MNC from three different donors were analyzed on their target (K562)-to-effector cell conjugates after pretreatment with Lxx- 11a. In order to clearly represent the effect of Leu- 11a, MNC were prepared from the donors with relatively high levels of Leu- 11a+ cells in blood. The percentagesof Leu- 11a+ cells in MNC from Donors 1,2, and 3 were 22.6,21.5, and 23.4, respectively. ’ % MNC binding K562 targets.
nism for enhancement of NK activity by Leu-1 1 antibodies, explained as follows: Leu- 11 antibodies react with FcR on both effector and target cells and then accelerate their bindings in the initial phase of their interaction. This possibility was examined by conjugate assayusing K562 targets (Table 2). As expected, a sign&ant increase of the target-to-effecter cell conjugates was observed in the presence of Leu-1 lc (IgG,). We also demonstrated that Leu-1 lb (IgM) induced an increase of the target-toeffector K562 cell conjugates at the comparable level of that of Leu-1 la (data not shown). Nevertheless, Leu- 1lb did not induce any NK augmentation as already shown in Fig. 2. A control antibody anti-HLA-A,B,C did not induce any increase of the target-to-effector cell conjugates when K562 and Jurkat cells were used as target cells. Here, Jurkat cells all expressed human class I MHC antigens HLA-A,B,C on their surface, whereas K562 cells lacked class I MHC antigens (data not shown). Eflects of F(ab’)* and Fab fragments of Leu-1 la on NK activity. We examined the effects of F(ab’h and Fab of Leu-1 la on the enhancement capacity of NK activity (Fig. 6). It was clearly demonstrated that F(ab’)*, but not Fab, of Leu- 1la was effective in enhancing killing capacity. It is presumed that a bivalent activity of the antibody may be essential to induce the enhancement. Diflerential eflects oftrypsin treatment of eflector cells on Leu-I l-induced cytotoxicity and original spontaneous NK activity. It has been established that NK activity is abrogated by trypsin treatment of MNC. NK functional receptor seemsto be labile to trypsin. Indeed, when effector cells were pretreated with trypsin (0.25%) at 37°C for 30 min, almost all NK activity was eliminated (Fig. 7). The NK activity of these trypsin-treated MNC no longer could be enhanced when these cells were exposed to human recombinant EN-y (500 u/ml) at 37°C for 2 hr (Fig. 7b). This incubation was enough to enhance NK activity for the trypsin-untreated effector cells. On the other hand, this mild trypsin treatment of cells did not deteriorate the FcR expression on MNC. More than 80% activity of antibody-dependent cell-mediated cytotoxicity (ADCC; Raji cells and anti-Raji antibody system) was retained in the trypsin-treated MNC (data not shown). Leu-1 lc still induced a significant cytotoxicity in these trypsin-treated cells (Fig. 7a). The results showed a possibility that a functional receptor of Leu-1 l-induced cytotoxicity might be distinguishable from that of spontaneous NK activity.
ENHANCEMENT
OF NK ACTIVITY
a 60
395
BY Leu-ll
b
I
-
I
I
I-1
(+I
01 Ftab’lp
of Leu-11
t-1 Fab
a
(+I of
Leu-lla
FIG. 6. F(ab’)*, but not Fab, of Leu- 1la was effective for enhancement. MNC from three different donors were assayed on their NK function (E:T = 10: 1) with or without F(ab’)z of Leu-1 la (a) and the Fab of Leu1la (b). Each fragment was added to assayculture of 4-hr analysis at a concentration of 0.1 &ml.
DISCUSSlON In recent studies, a series of murine monoclonal antibodies directed to the FcR for IgG on human NK cells and granulocytes have been established (13- 15). In the present study, we demonstrated that among these monoclonal antibodies, the antibodies
40
* .t .u :: z‘:
,-
a
b
30
20
0 8 10
/ 0
t-1
(+I Leu-llc
t-1
(+I IFN
7. Differential effectsof trypsin treatment of effector cells on Leu- 1l-induced cytotoxicity and original spontaneous NK activity MNC were pretreated with trypsin (0.25%) at 37°C for 30 min (0) or without trypsin (0). The NK activity of these trypsin-treated or untreated effector cells were assayedin the presence of Leu- 1 lc (0.25 &ml) (a) or alter again exposing them with recombinant IFNr (500 U/ml)(b). Although trypsin-treated cells showed decreased NK activity and no longer could be augmented on their NK activity by recombinant IFN-y, these cells were still induced a significant cytotoxicity by Leu- 1lc. A representative result from three experiments was depicted. FIG.
396
EGAWA, ABO, AND HIWATASHI
with IgGl isotype (e.g., Leu-l la and Leu- 1lc) e5ciently enhanced NK activity against several target cells, including a commonly used target cell line K562 with the highest susceptibility to human NK cells. This phenomenon was observed only when MNC, including NK cells, were used as effector cells. Granulocytes originally lacked any binding (<5%) and killing activities against K562, and this result was not changed even in the presence of Leu- 11a (up to 10e3mg/ml in NK assayculture). To our knowledge, there has been no report on enhancement of NK activity by Leu-1 1 monoclonal antibodies. In a study using the IgM monoclonal antibody VEP13, Rumpold et al. demonstrated that the NK activity of cells stained with VEP13, but unsorted, was unchanged compared with that of untreated cells (15). These results were consistent with ours. In earlier studies, Perussia et al. investigated the effects of B73.1 (or Leu-1 lc) monoclonal antibody on antibody-dependent cytotoxicity and spontaneous cytotoxicity of NK cells (26,27). In their reports, the B73.1 antibody did not induce the enhancement of NK activity against K562 targets. Our findings do not agree with these results. To clear up the discrepancies between these findings, we first examined individual variations of enhancing NK activity mediated by monoclonal Leu-1 1 antibodies. Although MNC from a few individuals tested (4.3%) did not show any significant enhancement of NK activity by Leu-1 la, MNC taken from more than 95% of 278 healthy Japaneseadults showed that Leu-1 la induced enhancement. Ethnic differences in the enhancement of NK activity by Leu11 antibodies remain to be tested. Earlier, we reported ethnic differences in the functional expression of FcR for murine IgG, immunoglobulins on human monocytes (28). Another possibility to explain the discrepancies was that the K562 targets Perussia et al. used expressed Leu- 11 antigens to a lesserextent on the cell surface than the K562 targets we used. Magnitude of killing enhancement by Leu- 11 antibodies was proportional to Leu-1 1 antigen expression of the target cells used. Enhancement of NK activity by Leu- 11 monoclonal antibodies was produced only when Leu- 11+ cell lines were used as target cells in assay.It is, therefore, conceivable that Leu-1 1 antibodies may become a linkage between effector and target cells. A similar crosslinking of effector cells to targets using monoclonal antibody has been described in the T-cell system (29). This conception was supported by the result that Leu- 1la actually increased the number of target-to-effector cell conjugates. The effectivenessof F(ab’h but not of the Fab fragment of Leu- 11a further supported this conception. This result also indicated that a mechanism of ADCC might not be, if any, a major part of the enhancement by Leu-1 1 antibodies in this study. Furthermore, it was reported that the ADCC of human NK cells preferentially mediated with IgG,, but not IgG, of anti-target cell murine monoclonal antibodies (30). Although Leu-1 lb (IgM) also induced a significant level of the increase of conjugation, there was no induction of the NK augmentation at all. This result suggestedthat a crosslinking between NK cells and targets was not su5cient enough to induce the NK augmentation. Even after crosslinking, isotype-specific molecular interaction (IgGi but not IgM) with cells might be required. Enhancement of NK activity by Leu-1 1 antibodies was restricted to the IgGl monoclonal antibodies. In cross-competition experiments using ‘251-labeledLeu- 11 antibodies, Peru&a et al. have demonstrated that Leu- 1la (IgGI) and Leu-1 1b (IgM), but not Leu-1 lc (B73.1) (IgGI), recognize a same epitope on NK cells (3 1). It is clear, therefore, that only the isotope of Leu- 11 monoclonal antibody is important for killing enhancement, whereas the epitope recognized by the antibody is not. Effects of
ENHANCEMENT
OF NK ACTIVITY
BY La-1 1
397
the other FcR monoclonal antibodies with IgGh, IgGlb, or IgG3 isotype remain to be tested, because these antibodies are not available yet. We do not know why only IgG, , but not IgM, of Leu-1 1 monoclonal antibodies is effective to the NK augmentation. However, similar isotype-restricted phenomena of monoclonal antibodies have been reported. For example, murine IgGza is effective in a certain human ADCC system (30), whereas murine IgG, and IgGz, of anti-CD3 antibody are effective to CD3-induced human T-cell proliferation (28). Murine IgM antibodies are not working at all in both systems. In the present experiments, we also examined the effect of monoclonal antibodies reacting with molecules other than FcR on enhancement of NK activity; the antibodies used were Leu-7 (IgM), N901 (IgGr), and MG2 (IgG,). Although all of these monoclonal antibodies reacted with a considerable proportion of human NK cells, any significant enhancement of NK activity was not produced. The monoclonal antiHLA-A,B,C, anti-DR, and anti-DQ antibodies induced a minimal augmentation of NK activity. In the murine system, antibody-induced augmentation of NK activity was demonstrated by using monoclonal antibodies, such as anti-H-2, anti-Ia, and anti-Thy- 1.2 (32,33). When MNC pretreated with Leu-1 1 monoclonal antibody were incubated overnight at 37°C enhancing activity was detectable in the culture supernatants. However, the supematants did not contain any activities of either IFN or IG2. As polyclonal anti-mouse IgG completely neutralized enhancing activity in the supematants, Leu-1 1 antibody itself might be released from the cell surface during incubation. It was reported that the Leu- 11 antibody that bound to cells was rapidly shedded from the cell surface when cells were incubated at 37°C (26). It has been revealed that a series of the Leu-1 1 monoclonal antibodies recognize the FcR on NK cells and granulocytes but not on monocytes and B cells in normal human leukocytes (13- 15). The present results, however, demonstrated that many established cell lines with diverse cell origins, including myelomonocytoid, T-, and B-cell lines, expressed the antigens identified by Leu-1 lc. The observed staining of Leu-1 lc on K562 was unlikely to be nonspecific binding of murine IgG, immunoglobulins to their FcR, because the other IgG, monoclonal antibodies (e.g., antiHLA-A,B,C and anti-N901) were not reactive with K562 (data not shown). In contrast with the strong reactivity of both Leu- 11a and Leu- 11c to NK cells in blood, these cell lines were reactive with Leu- 11c but to a lesser extent with Leu- 11a. It has been established that the epitope on FcR recognized by Leu-1 la (and Leu-1 lb) is distinct from that recognized by Leu-1 lc (3 1). Despite these staining variations, Leu1la and Leu-1 lc were both functionally effective for enhancement. Here, the pretreatment of target cells with Lcu- 11 antibodies Leu- 11a and Leu- 1lc, was found to be always less efficient for enhancement than the effector cell pretreatment (data not shown). It is presumed that the low efficiency of target cell pretreatment may be due to the relatively lower expression of Leu- 11 antigens on established cultured cell lines than that of fresh MNC. There have been several reports that FcR on NK cells is relatively resistant to trypsin treatment, whereas the NK functional receptor is sensitive to the same treatment (25,26). Indeed, we were able to eliminate almost all spontaneous cytotoxicity of NK cells by this treatment. The NK activity of these trypsin-treated cells could not be augmented by pretreatment with recombinant IFN-7. Of particular interest was that the Leu-1 1 antibody still induced a significant enhancement of NK activity in the
398
EGAWA, ABO, AND HIWATASHI
trypsin-treated cells. These results indicated a possibility that a functional receptor of Leu- 11-induced cytotoxicity might be distinguishable from that of spontaneous NK cytotoxicity. Namely, enhancement of NK activity by the Leu- 11 antibody was felt not to be a mere augmentation of spontaneous NK activity, but was felt to be the cytotoxicity mediated via the distinct functional receptor, probably a linkage of an FcR (on targets)-Ieu- 11-FcR (on effectors) complex. Similar cytotoxicity mediated by receptors other than NK functional receptors is the lectin-induced cell-mediated cytotoxicity (LICC), where a linkage of lectin and lectin receptors on both target and effector cells may be functional receptors for LICC (34). The lytic machinery itself seems,however, to be common to all of these cytotoxicities. In the present study, we demonstrated the enhancement of human NK activity by the Leu- 11 monoclonal antibodies. This phenomenon occurred when target cells in NK assaysexpressed the Leu-1 1 antigens on their cell surface. Surprisingly, many NK target cells of established cultured cell tines, including K562 targets, were demonstrated to expressthe Leu- 11 antigens. For practical application of Leu- 11 antibodies, attention should be paid to the isotypes of Leu- 11 antibodies in conjunction with the nature of target cells in NK assays.For example, when the target cells used in a given performing system is Leu-1 l+, the Leu-1 1 antibodies with IgM isotype rather than IgG, should be used (e.g., for cell sorting experiments) to avoid variations of spontaneous NK cytotoxicity. Finally, we observed only a few nonresponders to killing enhancement by Leu- 11 antibodies among normal healthy adult donors. In the preliminary experiments, we demonstrated that many pregnant women and some patients with autoimmune diseases(e.g., Crohn’s disease)are nonresponders to the enhancement, despite their normal levels of Leu- 11+ cells in blood. Experiments are currently in progress to investigate whether defective responsesto Leu- 11 antibodies observed in pregnant women and some patients were due to the paralysis of NK cells by some serum components via their FcR. The Leu- 11-mediated cytotoxicity presented here may contain some clinical values to help us understand and evaluate the clinical status of diseased persons. ACKNOWLEDGMENTS We are grateful to Drs. Hiroshi Itoh and Katsuo Kumagai for helpful discussion, and Ms. Junko Hirata and Ms. Hiroko Akaishi for their expert editorial assistance. Note added in proof: After completing this manuscript, M. Jondal et al. have reported the enhancement of NK activity by moncclonal antibodies against NK cell FcR (35). They have also observed the isotype (IgG,)-specific function in this phenomenon. Our manuscript includes several experiments to analyze the basis of the mechanism.
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ENHANCEMENT
OF NK ACTIVITY
BY Leu-ll
399
8. Landay, A., Gartland, G. L., and Clement, L. T., J. Immunol. 131,2X’, 1983. 9. Abe, T., Sugawara, S., Amenomori, A., Itoh, H., Rikiishi, H., Moro, I., and Kumagai, K., J. Immunol. 136,3189,1986.
10. Abo, T., Cooper, M. D., and Balch, C. M., J. ZmmunoZ.129,1752, 1982. 11. Abo, T., and Balch, C. M., J. Zmmunol. 129,1758,1982. 12. Abo, T., and Balch, C. M., J. Zmmunol. 127, 1024, 1981. 13. Phillips, J. H., and Babcock, G. F., Zmmunol. Lett. 6, 143, 1983. 14. Pen&a, B., Starr, S., Abraham, S., Fanning, V., and Trinchieri, G., J. Immunol. 130,2133, 1983. 15. Rumpold, H., Krafi, D., Obexer, G., Bock, G., and Gebhart, W., J. Immunol. 129, 1458, 1982. 16. Griffin, J. D., Hercend, T., Beveridge, R., and Schlossman, S. F., J. Zmmunol. 130,2947, 1983. 17. Wallace, L. E., Moss, D. L., Rickinson, A. B., McMichael, A. J., and Epstein, M. A., Eur. J. Zmmunol. 11,694, 1981.
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27. Perussia, B., and Trinchieri, G., J. Immunol. 132, 1419, 1984. 28. Abo, T., Tilden, A. B., Balch, C. M., Kumagai, K., Troup, F. M., and Cooper, M. D., J. Exp. Med. 160,303, 1984.
29. Staerz, U. D., Kanagawa, O., and Bevan, M. J., Nature (London) 314,628, 1985. 30. Lanier, L. L., Kipps, T. J., and Phillips, J. H., J. Exp. Med. 162,2089, 1982. 3 1. Peru&a, B., Trinchieri, G., Jackson, A., Warner, N. L., Faust, J., Rumpold, H., Kraft, D., and Lanier, L. L., J. Zmmunol. 133,180, 1984. 32. Saxena, R. K., and Adler, W. H., J. Zmmunol. 123,846, 1979. 33. Brunda, M. J., Herberman, R. B., and Holden, H. T., Znt. J. Cancer 27,205, 198I. 34. Brunda, M. J., Varesio, L., Herberman, R. B., and Holden, H. T., Znt. J. Cancer 29,299, 1982. 35. Jondal, M., Kullman, C., Alter, M.-B., and Ljunggren, K., Cell. Zmmunof. 100, 158, 1986.