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Production of a monoclonal antibody inhibiting the killer activity
Immunology Letters, 36 (1993) 125-130 0165 - 2478 / 93 / $ 6.00 © 1993 Elsevier Science Publishers B.V. All rights reserved IMLET 01949
Production of a monoclonal antibody inhibiting the killer activity Y a s u m o t o S u z u k i a, M i t s u r u F u n a b a s h i a, R y o j i S u z u k i a, K u n i h i r o N o t a k e b and Takashi Yokochi b aLaboratory of Bacteriology, Aichi Prefectural Institute of Public Health, Nagoya, Aichi 462, Japan; and bDepartment of Microbiology, Aichi Medical University, School of Medicine, Nagakute, Aichi 480-11, Japan (Received 7 May 1992; revision received 17 February 1993; accepted 5 March 1993)
I.
Summary
We established the hybridoma producing the monoclonal antibody (mAb) 3C3 by immunizing rats with mouse natural killer (NK)-like cells. The 3C3 mAb seemed to react mainly with T cells and T-lineage cell lines. The 3C3 antigen also seemed to be coincidentally expressed on a part of asialo GM 1 ÷ cells from nude mice, suggesting its expression on N K cells. Treatment of effector cells with 3C3 mAb markedly inhibited the killer activity against RL~-I cells, but less so against YAC-1 cells, in vitro. It is suggested that the cell surface molecule defined by 3C3 mAb was closely associated with the killer activity of T cells and N K cells.
main enigmatic [3-5]. The study of N K cells has been difficult because they account for only a small percentage of the peripheral blood and splenic lymphocytes. A number of monoclonal antibodies defining N K cells have been reported [611]. However, the role of cell surface molecules on the killer activity of NK cells is still unclear. Previously, we established NK-like hybridoma cells by fusing spleen cells from BALB/c nude mice and mouse myeloma line (NS-1) [12]. In the present study, we tried to produce a mAb which inhibited the killer activity of N K cells. Here we describe the establishment of a mAb which is reactive with T cells and N K cells and can inhibit their killer activity. 3.
2.
Materials and Methods
Introduction 3.1.
Natural killer (NK) cells represent a population of lymphoid cells morphologically and functionally distinct from conventional T and B lymphocytes [1,2]. Despite accumulating evidence that N K cells play significant roles in certain aspects of host defense mechanisms, they largely reKey words: Monoclonal antibody; Killer activity; NK cell; T cell
Correspondence to: Dr. Yasumoto Suzuki, Laboratory of Bacteriology, Aichi Prefectural Institute of Public Health, Kita-ku, Nagoya, Aichi 462, Japan.
Cell lines
A variety of cell lines, RL3-1, YAC-1, NS-I, and 4D1D4, were maintained as described previously [12]. The 4DID4 cells are NK-like hybridoma cells which were obtained from a fusion between spleen cells from BALB/c nude mice and NS-1 cells [12]. 3.2.
Production of mAb
F344/NSIC rats were intraperitoneally injected twice over an interval of two weeks with 1 × 107 4D1D4 cells. Three days after the second injec125
tion, the spleens were removed, and spleen cells were fused with the murine myeloma NS-1 as described previously [12]. The supernatants of clones were tested for their reactivity with 4D 1D4 cells and NS'-I cells by an immunofluorescence method. Subsequently, the positive clones were examined for inhibitory action on the N K activity of 4D1D4 cells against RL~-I cells. Finally, the hybridoma, 3C3, was selected from 117 hybrid clones.
3.3.
The laser flow cytometry
Cells were treated with the culture supernatant of 3C3 mAb or rabbit anti-asialo GM1 serum for 30 min at 4°C and then stained with fluoresceinconjugated goat anti-rat or rabbit Ig serum (1:40). Immunofluorescence-positive cells were analyzed with the aid of a laser flow cytometer (FACS 440, Becton Dickinson, USA). The intensity of fluorescence is expressed on a log scale.
3.4.
NK assay
The N K activity of 4D1D4 cells or BALB/c nude mouse spleen cells was measured by the carboxyfluorescein diacetate (CFDA) method [13].
In an inhibition experiment, spleen cells of BALB/c nude mice were preincubated with serially diluted 3C3 mAb for 60 min at 37°C and then mixed with CFDA-labeled target cells in 96-well plates. Spontaneous release was determined by incubating labeled targets alone, and the maximal release was determined by the addition of 0.5% NP-40. The target lysis was calculated by use of the following equation: % specific lysis = (experimental release (R) - spontaneous R/maximal R - spontaneous R) x 100. All samples were assayed in triplicate, and the values were presented as the mean + one standard deviation of triplicate determinations. 4.
4.1.
Results
Reactivity of 3C3 mAb against normal lymphocytes and cell lines
The reactivity of 3C3 mAb against normal lymphocytes and cell lines was examined with laser flow cytometry (Fig. 1). The histogram pattern of all thymocytes and a large proportion of lymph node cells and spleen cells from normal mice was widely shifted to the right compared with the control curve (Fig. 1A). Only a small population of
A
a
o°
., .
=
/4"X,
o
B
.
.
.
@ >
"~
a
b
c
d
~2
J\\
L Fluorescence intensity
Fig. 1. The laser flow cytometric analysis of normal lymphocytes (A) and cell lines (B) stained with 3C3 mAb. The intensity of tuorescence is expressed on a log scale. Dotted lines indicate the histogram of the negative control treated with FITC-conjugated :econd antibody alone. A: (a) thymocytes, (b) lymph node cells, (c) spleen cells, (d) spleen cells from nude mice; B: (a) RLS'-I, (b) YAC-I, (c) 4D1D4, (d) NS-1.
126
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.........&f, ir ,~.-:~-:.
o
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,':'t
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...... :
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i i!!i!iiiii!!!! :-.~i !!~!!:?:i~::, ........
,,::..;a'
.
...
.:~:.
~:.I...............,
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.
3C3-FITC
Fig. 2. Two-color analysis of spleen cells from nude mice, double-labeled with rabbit anti-asialo GM 1 antiserum plus phycoerythrin (PE)-conjugated anti-rabbit lgG, and 3C3 mAb plus FITC-conjugated anti-rat IgM, respectively. Note the coexpression of 3C3 antigen and asialo GM 1 antigen.
spleen cells f r o m n u d e mice was m a r g i n a l l y positive. T h e R L ~ - I a n d Y A C - 1 T l y m p h o m a cell lines were s t r o n g l y positive, a n d m o s t o f the 4 D 1 D 4 N K - l i k e cells were also positive (Fig. 1B). In c o n t r a s t , NS-1 m y e l o m a cells were negative.
4.2. Coexpression between asialo GM1 antigen and 3C3 antigen on spleen cells of nude mice T o e x a m i n e the r e l a t i o n s h i p between the expression o f asialo G M 1 a n t i g e n a n d 3C3 antigen,
spleen cells o f n u d e mice were d o u b l e - s t a i n e d with a n t i - a s i a l o G M 1 a n t i s e r u m a n d 3C3 m A b . Fig. 2 d i s p l a y s the d u a l p a r a m e t e r F A C S profile for 3C3 antigen (x-axis) a n d asialo G M 1 a n t i g e n (y-axis). T h e 3C3 antigen + a n d asialo G M 1 antigen + cells a n d the 3C3 antigen + a n d asialo G M 1 a n t i g e n - cells were a p p r o x i m a t e l y 10% each, suggesting t h a t h a l f o f the 3C3 a n t i g e n + cells coexpressed asialo G M I antigen. T h e 3C3 antigen was also c o e x p r e s s e d with C D 4 , C D 8 , o r N K cell-associated G A - 1 antigen, b u t with neither
TABLE 1 Inhibitory action of 3C3 mAb on the NK activity of spleen cells from nude mice against RLS'-I and YAC-1. Dilution of 3C3 mAb
% Specific lysis (% inhibition) RL~-I
1:4 1:40 1:400 (-)
YAC-1
20a
40
80
40
2.6+ 1.6 (79) 8.3 -I-2.2 (34) 9.3+6.4 (26) 12.5+4.4
2.6+2.7 (87) 10.6 _ 3.5 (49) 19.0-t- 1.8 (8) 20.6+3.3
9.0___2.7 (61) 11.6+6.3 (50) 18.0+2.1 (21) 22.8-1-2.8
37.1 +0.2 (19) 44.5___8.5 (3) not tested 46.1 +3.3
aE/T ratio. 127
B220 nor IgM (data not shown).
4.3.
Inhibition of the NK activity of spleen cells and 4DID4 cells by 3C3 mAb
The effect of 3C3 mAb on the NK activity of spleen cells and 4D1D4 NK-like cells was examined (Table 1). The addition of the 3C3 culture supernatant markedly inhibited the N K activity of spleen cells against RL3-1. The inhibition was much lower in the case of YAC-I cells. The inhibition against RL3-1 cells was roughly dose-dependent. The inhibitory action could not be removed by washing out extra antibodies from mAb-treated effector cells. In a typical experiment, the NK activity of 3C3 mAb-treated effector cells against RL~-I cells was less than 1% when the untreated control showed 12.2% killing. Similarly, mAb-treated 4D1D4 cells showed negligible NK activity at a 5/1 E/T ratio compared with 15.1% killing in the untreated control. The 3C3 mAb also blocked the binding of 4D1D4 cells with RL~-I target cells. In addition, we studied the effect of 3C3 mAb on the cytotoxicity of Lyt-2 ÷ T cell clone (K7L) [14] against L1210 leukemia cells. The addition of 3C3 mAb partially inhibited the killer function of K7L clone cells (30% inhibition).
5.
Discussion
The present study demonstrated that 3C3 mAb inhibited the killer activity of mouse spleen cells or 4D1D4 NK-like cells in a dose-dependent manner (Table 1) and that the inhibition was due to the binding of 3C3 mAb with effector cells. The 3C3 mAb markedly inhibited the killer activity against syngenic RL3'-I cells but less against allogenic YAC-1 cells. This might be related to the fact that the 4D1D4 cells used for the immunization could kill RL~-I cells, but not YAC-1 cells [12]. NK-like 4DID4 cells did not express either CD4 or CD8 and showed no allospecific or MHC-restricted cytotoxicity [12]. It is, therefore, very likely that 3C3 mAb may inhibit the killer activity of N K cells. Furthermore, the killer function of tumor-specific cytotoxic T lymphocytes also seemed to be inhibited by 3C3 mAb. There are several reports on mAb capable of 128
inhibiting the NK activity. It has been reported that anti-CD2 and anti-LFA-1 mAb could inhibit NK cytotoxicity in humans [6,7], but other reports did not support this notion in mice [15]. Anti-Ly-5 and nonpolymorphic anti-T200 antibodies also blocked the NK activity in the absence of complement [10]. However, 3C3 mAb did not recognize Ly-5 antigen and a polymorphic determinant of the T200 molecule expressed on all hematopoietic cells, including N K cells, T cells, and B cells, because the 3C3 antigen is selectively expressed on T-lineage cell lines and asialo GM1 ÷ cells. On the other hand, anti-Thy-1 and anti-asialo GM1 antibody cannot inhibit the NK activity without the complement [16], and other NK-specific mAb, such as anti-NK-l.1, anti-LGL-1, and anti-Qa-5 mAb, cannot block the NK activity without complement either [8,9]. The LAA antigen defined by the KBA mAb is expressed at low levels on tnymocytes, spleen cells, and lymph node cells, and the KBA mAb could inhibit the NK activity and LAK activity without the complement [11]. However, 3C3 mAb reacted strongly with some of those lymphoid cells. Therefore, the reactivity of 3C3 mAb definitely differed from that of NK cell-related mAb which can inhibit the NK activity. The experiment on coexpression between asialo G M I antigen and 3C3 antigen showed the existence of 3C3 antigen ÷ and asialo GM1 antigencells in the spleen cells from nude mice. Those cells might be asialo GM1 antigen- immature NK cells or some T-lineage cells. Our 3C3 mAb might be useful for the study of the killer function of T cells and N K cells. The biochemical nature of the 3C3 antigen has not yet been clarified because of the difficulty caused by the IgM class.
Acknowledgements We wish to thank Dr. S. Migita, Kanazawa University, and Dr. R. Ueda, Aichi Cancer Center Institute, for the gift of YAC-1, RL~-I, and NS-1 cell lines, and Dr. K. Yoshikawa, Aichi Medical University, for his comments and discussion. We are grateful to Dr. K. Okumura, Juntendo University, for the analysis for cell surface markers.
References [1] Lanier, L.L., Phillips, J.H., Hackett Jr., J., Tutt, M. and Kumar, V. (1986) J. lmmunol. 137, 2735. [2] Ortaldo, J.R. and Reynolds, C.W. (1987)J. Immunol. 138, 4545. [3] Arai, S., Yamamoto, H., Itoh, K. and Kumagai, K. (1983) J. lmmunol. 131, 651. [4] Kiessling, R., Klein, E. and Wigzell, H. (1975) Eur. J. lmmunol. 5, 117. [5] Walker, C.M., Rawls, W.E. and Rosenthal, K.L. (1984) J. Immunol. 132, 469. [6] Hart, M.K., Kornbluth, J., Main, E.K., Spear, B.T., Taylor, J. and Wilson, D.B. (1987) Cell. lmmunol. 109, 306. [7] Siliciano, R.F., Pratt, J.C., Schmidt, R.E., Ritz, J. and Reinherz, E.L. (1985) Nature 317, 428. [8] Mason, L.H., Mathieson, B.J. and Ortaldo, J.R. (1990) J. lmmunol. 145, 751. [9] Hammerling, G.J., Hammerling, U. and Flaherty, L.
(1979) J. Exp. Med. 150, 108. [10] Seaman, W.E., Talal, N., Herzenberg, L.A., Herzenberg, L.A. and Ledbetter, J.A. (1981) J. Immunol. 127, 982. [11] Nishimura, T., Yagi, H., Yagita, H., Uchiyama, Y. and Hashimoto, Y. (1985) Cell. Immunol. 94, 122. [12] Suzuki, Y., Funabashi, M., lsomura, S., Yoshikawa, K., Notake, K. and Yokochi, T. (1992) Microbiol. Immunol. 36, 401. [13] Suzuki, Y., Yoshikawa, K. and Yokochi, T. (1991) J. Immunoassay 12, 145. [14] Nagase, F., Ueda, K., Nakashima, I., Kawashima, K., lsobe, K., Nagura, E., Yamada, K., Yokochi, T., Hasegawa, Y., Yoshida, T. and Ando, K. (1986) Int. J. Cancer 38, 907. [15] Nakamura, T., Takahashi, K., Fukazawa, T., Koyanagi, M., Yokoyama, A., Kato, H., Yagita, H. and Okumura, K. (1990) J. immunol. 145, 3628. [16] Kumagai, K., ltoh, K., Suzuki, R., Hinuma, S. and Saitoh, F. (1982)J. lmmunol. 129, 388.
129
Production of a monoclonal antibody inhibiting the killer activity Y a s u m o t o S u z u k i a, M i t s u r u F u n a b a s h i a, R y o j i S u z u k i a, K u n i h i r o N o t a k e b and Takashi Yokochi b aLaboratory of Bacteriology, Aichi Prefectural Institute of Public Health, Nagoya, Aichi 462, Japan; and bDepartment of Microbiology, Aichi Medical University, School of Medicine, Nagakute, Aichi 480-11, Japan (Received 7 May 1992; revision received 17 February 1993; accepted 5 March 1993)
I.
Summary
We established the hybridoma producing the monoclonal antibody (mAb) 3C3 by immunizing rats with mouse natural killer (NK)-like cells. The 3C3 mAb seemed to react mainly with T cells and T-lineage cell lines. The 3C3 antigen also seemed to be coincidentally expressed on a part of asialo GM 1 ÷ cells from nude mice, suggesting its expression on N K cells. Treatment of effector cells with 3C3 mAb markedly inhibited the killer activity against RL~-I cells, but less so against YAC-1 cells, in vitro. It is suggested that the cell surface molecule defined by 3C3 mAb was closely associated with the killer activity of T cells and N K cells.
main enigmatic [3-5]. The study of N K cells has been difficult because they account for only a small percentage of the peripheral blood and splenic lymphocytes. A number of monoclonal antibodies defining N K cells have been reported [611]. However, the role of cell surface molecules on the killer activity of NK cells is still unclear. Previously, we established NK-like hybridoma cells by fusing spleen cells from BALB/c nude mice and mouse myeloma line (NS-1) [12]. In the present study, we tried to produce a mAb which inhibited the killer activity of N K cells. Here we describe the establishment of a mAb which is reactive with T cells and N K cells and can inhibit their killer activity. 3.
2.
Materials and Methods
Introduction 3.1.
Natural killer (NK) cells represent a population of lymphoid cells morphologically and functionally distinct from conventional T and B lymphocytes [1,2]. Despite accumulating evidence that N K cells play significant roles in certain aspects of host defense mechanisms, they largely reKey words: Monoclonal antibody; Killer activity; NK cell; T cell
Correspondence to: Dr. Yasumoto Suzuki, Laboratory of Bacteriology, Aichi Prefectural Institute of Public Health, Kita-ku, Nagoya, Aichi 462, Japan.
Cell lines
A variety of cell lines, RL3-1, YAC-1, NS-I, and 4D1D4, were maintained as described previously [12]. The 4DID4 cells are NK-like hybridoma cells which were obtained from a fusion between spleen cells from BALB/c nude mice and NS-1 cells [12]. 3.2.
Production of mAb
F344/NSIC rats were intraperitoneally injected twice over an interval of two weeks with 1 × 107 4D1D4 cells. Three days after the second injec125
tion, the spleens were removed, and spleen cells were fused with the murine myeloma NS-1 as described previously [12]. The supernatants of clones were tested for their reactivity with 4D 1D4 cells and NS'-I cells by an immunofluorescence method. Subsequently, the positive clones were examined for inhibitory action on the N K activity of 4D1D4 cells against RL~-I cells. Finally, the hybridoma, 3C3, was selected from 117 hybrid clones.
3.3.
The laser flow cytometry
Cells were treated with the culture supernatant of 3C3 mAb or rabbit anti-asialo GM1 serum for 30 min at 4°C and then stained with fluoresceinconjugated goat anti-rat or rabbit Ig serum (1:40). Immunofluorescence-positive cells were analyzed with the aid of a laser flow cytometer (FACS 440, Becton Dickinson, USA). The intensity of fluorescence is expressed on a log scale.
3.4.
NK assay
The N K activity of 4D1D4 cells or BALB/c nude mouse spleen cells was measured by the carboxyfluorescein diacetate (CFDA) method [13].
In an inhibition experiment, spleen cells of BALB/c nude mice were preincubated with serially diluted 3C3 mAb for 60 min at 37°C and then mixed with CFDA-labeled target cells in 96-well plates. Spontaneous release was determined by incubating labeled targets alone, and the maximal release was determined by the addition of 0.5% NP-40. The target lysis was calculated by use of the following equation: % specific lysis = (experimental release (R) - spontaneous R/maximal R - spontaneous R) x 100. All samples were assayed in triplicate, and the values were presented as the mean + one standard deviation of triplicate determinations. 4.
4.1.
Results
Reactivity of 3C3 mAb against normal lymphocytes and cell lines
The reactivity of 3C3 mAb against normal lymphocytes and cell lines was examined with laser flow cytometry (Fig. 1). The histogram pattern of all thymocytes and a large proportion of lymph node cells and spleen cells from normal mice was widely shifted to the right compared with the control curve (Fig. 1A). Only a small population of
A
a
o°
., .
=
/4"X,
o
B
.
.
.
@ >
"~
a
b
c
d
~2
J\\
L Fluorescence intensity
Fig. 1. The laser flow cytometric analysis of normal lymphocytes (A) and cell lines (B) stained with 3C3 mAb. The intensity of tuorescence is expressed on a log scale. Dotted lines indicate the histogram of the negative control treated with FITC-conjugated :econd antibody alone. A: (a) thymocytes, (b) lymph node cells, (c) spleen cells, (d) spleen cells from nude mice; B: (a) RLS'-I, (b) YAC-I, (c) 4D1D4, (d) NS-1.
126
t,IJ
.:3
!
":!:"
.-."
..":3-~ .
.........&f, ir ,~.-:~-:.
o
+~., .2..~
~:"~
°~
,'-~¢'':'..:..
.......
" .:i::--'~ •
,':'t
.
.:::.
...... :
.
< ,~...;':::: ~ :~,
/ .[~ ::::
i i!!i!iiiii!!!! :-.~i !!~!!:?:i~::, ........
,,::..;a'
.
...
.:~:.
~:.I...............,
:':
.
3C3-FITC
Fig. 2. Two-color analysis of spleen cells from nude mice, double-labeled with rabbit anti-asialo GM 1 antiserum plus phycoerythrin (PE)-conjugated anti-rabbit lgG, and 3C3 mAb plus FITC-conjugated anti-rat IgM, respectively. Note the coexpression of 3C3 antigen and asialo GM 1 antigen.
spleen cells f r o m n u d e mice was m a r g i n a l l y positive. T h e R L ~ - I a n d Y A C - 1 T l y m p h o m a cell lines were s t r o n g l y positive, a n d m o s t o f the 4 D 1 D 4 N K - l i k e cells were also positive (Fig. 1B). In c o n t r a s t , NS-1 m y e l o m a cells were negative.
4.2. Coexpression between asialo GM1 antigen and 3C3 antigen on spleen cells of nude mice T o e x a m i n e the r e l a t i o n s h i p between the expression o f asialo G M 1 a n t i g e n a n d 3C3 antigen,
spleen cells o f n u d e mice were d o u b l e - s t a i n e d with a n t i - a s i a l o G M 1 a n t i s e r u m a n d 3C3 m A b . Fig. 2 d i s p l a y s the d u a l p a r a m e t e r F A C S profile for 3C3 antigen (x-axis) a n d asialo G M 1 a n t i g e n (y-axis). T h e 3C3 antigen + a n d asialo G M 1 antigen + cells a n d the 3C3 antigen + a n d asialo G M 1 a n t i g e n - cells were a p p r o x i m a t e l y 10% each, suggesting t h a t h a l f o f the 3C3 a n t i g e n + cells coexpressed asialo G M I antigen. T h e 3C3 antigen was also c o e x p r e s s e d with C D 4 , C D 8 , o r N K cell-associated G A - 1 antigen, b u t with neither
TABLE 1 Inhibitory action of 3C3 mAb on the NK activity of spleen cells from nude mice against RLS'-I and YAC-1. Dilution of 3C3 mAb
% Specific lysis (% inhibition) RL~-I
1:4 1:40 1:400 (-)
YAC-1
20a
40
80
40
2.6+ 1.6 (79) 8.3 -I-2.2 (34) 9.3+6.4 (26) 12.5+4.4
2.6+2.7 (87) 10.6 _ 3.5 (49) 19.0-t- 1.8 (8) 20.6+3.3
9.0___2.7 (61) 11.6+6.3 (50) 18.0+2.1 (21) 22.8-1-2.8
37.1 +0.2 (19) 44.5___8.5 (3) not tested 46.1 +3.3
aE/T ratio. 127
B220 nor IgM (data not shown).
4.3.
Inhibition of the NK activity of spleen cells and 4DID4 cells by 3C3 mAb
The effect of 3C3 mAb on the NK activity of spleen cells and 4D1D4 NK-like cells was examined (Table 1). The addition of the 3C3 culture supernatant markedly inhibited the N K activity of spleen cells against RL3-1. The inhibition was much lower in the case of YAC-I cells. The inhibition against RL3-1 cells was roughly dose-dependent. The inhibitory action could not be removed by washing out extra antibodies from mAb-treated effector cells. In a typical experiment, the NK activity of 3C3 mAb-treated effector cells against RL~-I cells was less than 1% when the untreated control showed 12.2% killing. Similarly, mAb-treated 4D1D4 cells showed negligible NK activity at a 5/1 E/T ratio compared with 15.1% killing in the untreated control. The 3C3 mAb also blocked the binding of 4D1D4 cells with RL~-I target cells. In addition, we studied the effect of 3C3 mAb on the cytotoxicity of Lyt-2 ÷ T cell clone (K7L) [14] against L1210 leukemia cells. The addition of 3C3 mAb partially inhibited the killer function of K7L clone cells (30% inhibition).
5.
Discussion
The present study demonstrated that 3C3 mAb inhibited the killer activity of mouse spleen cells or 4D1D4 NK-like cells in a dose-dependent manner (Table 1) and that the inhibition was due to the binding of 3C3 mAb with effector cells. The 3C3 mAb markedly inhibited the killer activity against syngenic RL3'-I cells but less against allogenic YAC-1 cells. This might be related to the fact that the 4D1D4 cells used for the immunization could kill RL~-I cells, but not YAC-1 cells [12]. NK-like 4DID4 cells did not express either CD4 or CD8 and showed no allospecific or MHC-restricted cytotoxicity [12]. It is, therefore, very likely that 3C3 mAb may inhibit the killer activity of N K cells. Furthermore, the killer function of tumor-specific cytotoxic T lymphocytes also seemed to be inhibited by 3C3 mAb. There are several reports on mAb capable of 128
inhibiting the NK activity. It has been reported that anti-CD2 and anti-LFA-1 mAb could inhibit NK cytotoxicity in humans [6,7], but other reports did not support this notion in mice [15]. Anti-Ly-5 and nonpolymorphic anti-T200 antibodies also blocked the NK activity in the absence of complement [10]. However, 3C3 mAb did not recognize Ly-5 antigen and a polymorphic determinant of the T200 molecule expressed on all hematopoietic cells, including N K cells, T cells, and B cells, because the 3C3 antigen is selectively expressed on T-lineage cell lines and asialo GM1 ÷ cells. On the other hand, anti-Thy-1 and anti-asialo GM1 antibody cannot inhibit the NK activity without the complement [16], and other NK-specific mAb, such as anti-NK-l.1, anti-LGL-1, and anti-Qa-5 mAb, cannot block the NK activity without complement either [8,9]. The LAA antigen defined by the KBA mAb is expressed at low levels on tnymocytes, spleen cells, and lymph node cells, and the KBA mAb could inhibit the NK activity and LAK activity without the complement [11]. However, 3C3 mAb reacted strongly with some of those lymphoid cells. Therefore, the reactivity of 3C3 mAb definitely differed from that of NK cell-related mAb which can inhibit the NK activity. The experiment on coexpression between asialo G M I antigen and 3C3 antigen showed the existence of 3C3 antigen ÷ and asialo GM1 antigencells in the spleen cells from nude mice. Those cells might be asialo GM1 antigen- immature NK cells or some T-lineage cells. Our 3C3 mAb might be useful for the study of the killer function of T cells and N K cells. The biochemical nature of the 3C3 antigen has not yet been clarified because of the difficulty caused by the IgM class.
Acknowledgements We wish to thank Dr. S. Migita, Kanazawa University, and Dr. R. Ueda, Aichi Cancer Center Institute, for the gift of YAC-1, RL~-I, and NS-1 cell lines, and Dr. K. Yoshikawa, Aichi Medical University, for his comments and discussion. We are grateful to Dr. K. Okumura, Juntendo University, for the analysis for cell surface markers.
References [1] Lanier, L.L., Phillips, J.H., Hackett Jr., J., Tutt, M. and Kumar, V. (1986) J. lmmunol. 137, 2735. [2] Ortaldo, J.R. and Reynolds, C.W. (1987)J. Immunol. 138, 4545. [3] Arai, S., Yamamoto, H., Itoh, K. and Kumagai, K. (1983) J. lmmunol. 131, 651. [4] Kiessling, R., Klein, E. and Wigzell, H. (1975) Eur. J. lmmunol. 5, 117. [5] Walker, C.M., Rawls, W.E. and Rosenthal, K.L. (1984) J. Immunol. 132, 469. [6] Hart, M.K., Kornbluth, J., Main, E.K., Spear, B.T., Taylor, J. and Wilson, D.B. (1987) Cell. lmmunol. 109, 306. [7] Siliciano, R.F., Pratt, J.C., Schmidt, R.E., Ritz, J. and Reinherz, E.L. (1985) Nature 317, 428. [8] Mason, L.H., Mathieson, B.J. and Ortaldo, J.R. (1990) J. lmmunol. 145, 751. [9] Hammerling, G.J., Hammerling, U. and Flaherty, L.
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