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Sterile sorting of Leu-lla-positive cells (NK cells) using flow cytometry and the antineoplastic effects on brain tumors
Life Sciences, Vol. 45, pp. Printed in the U.S.A.
2103-2107
Pergamon
Press
STERILE SORTING OF L E U - I I a - P O S I T I V E CELLS (NK CELLS) USING FLOW CYTOMETRY A N D THE A N T I N E O P L A S T I C EFFECTS ON BRAIN TUMORS Hiroaki
Fujiwara,
Keiji
Kawamoto,
Yoshihiro
Numa and Hiroshi
Matsumura
Department of Neurosurgery, Kansai Medical U n i v e r s i t y i, Fumizonocho, Moriguchi City 570, Japan (Received
in final
form September
22, 1989)
Summary Though we recently found some reports on sterile sorting of natural killer cells (NK cells) by flow cytometer (FCM), w h i c h m a n i f e s t e d cytotoxicity, no reports have concerned a n t i n e o p l a s t i c i t y of sterile NK cells on g l i o m a cell lines and cultured surgical m a t e r i a l s of brain tumor so far as we know. Monocytes were sampled from the peripheral b l o o d of h e a l t h y adults, stained with fluorescein isothiocyanate (FITC)-labeled antihuman m o n o c l o n a l antibodies, and sorted by an FCM, which had been s t e r i l i z e d with 0.5% Hibitane alcohol solution. Over 95% of the cells o b t a i n e d were NK cells, and their v i a b i l i t y was d i s c l o s e d to be 97% by the FDA staining. Using thus o b t a i n e d NK cells, the a n t i n e o p l a s t i c effects were evaluated in 3 kinds of glioma cell lines and 5 surgical specimens by the m i c r o c y t o t o x i c i t y test. The effects v a r i e d w i d e l y from 9 to 74% (E/T ratio 40) for glioma cell lines, and from i to 66% (E/T ratio 10-40) for surgical specimens. It is expected in the future to apply NK cells clinically using this sterile sorting technique. With the d e v e l o p m e n t of m o n o c l o n a l antibodies, it has been possible to analyze the surface antigen of various cells. Analysis of T cell subsets using m o n o c l o n a l antibodies has revealed change of T cell subsets in brain tumor patients (i). w i t h the use of m o n o c l o n a l antibody and the sorting function of a flow cytometer (FCM), lymphocytes for adoptive i m m u n o t h e r a p y can be obtained in a short period. We have established a sterile sorting technique using FCM, and applied it to obtain natural killer (NK) cells from human peripheral blood. In addition, the a n t i n e o p l a s t i c effects of these cells on brain tumors were examined. Methods i) Isolation of lymphocyte and m o n o c l o n a l antibody Lymphocytes were obtained from the p e r i p h e r a l blood of healthy adults by the F i c o l l - C o n r a y d i f f e r e n t i a l centrifugation. P r e l i m i n a r y experiments were carried out to decide the best conditions for fluorescein staining time and volume of m o n o c l o n a l antibody, and f l u o r e s c e i n - m a i n t a i n i n g period. Fluorescein isothiocyanate (FITC)-labeled antihuman Leu-lla (Becton-Dickinson Co., Ltd.) was used for the human NK cells. 2) Sterile sorting of F C M FCM (FACS III, B e c t o n - D i c k i n s o n Co., Ltd.) was sterilized immediately before sorting Leu-lla positive cells. Sterile p h y s i o l o g i c saline was used as the sheath fluid, and sample tubes were sterilized by filling the tubes w i t h o.5% chlohexizine gluconate alcohol solution (ICI Pharma Co., Ltd., Japan) for 20 minutes. Then, f l u o r e s c e i n - p o s i t i v e cells were sorted (Fig. I). 0024-3205/89 $3.00 + .00 Copyright (c) 1989 P e r g a m o n Press
plc
2104
Antineoplastic
Effect
of Sorted NK Cells
Positive
,
,
I
,
Vol.
45, No.
22, 1989
Fig. 1 Fluorescein h i s t o g r a m of FITCnegative cells as a control (left) and sorting range of positive cells showing NK cells (right).
,~
positive cells
negative control
Most part of the cells o b t a i n e d were used as the effector cell in the microc y t o t o x i c i t y test, some part of them for e v a l u a t i o n of the cell v i a b i l i t y using f l u o r e s c e i n d i a c e t a t e (FDA) staining, and some part for the microscopic study to c o n f i r m the a c c u r a c y of the sorting system. 3) E v a l u a t i o n of v i a b i l i t y by FDA Since FDA is a substance w h i c h emanates fluorescein by h y d r o l y s i s after taken in viable cells, viable cells can be d i f f e r e n t i a t e d from dead cells by c o n f i r m i n g the p r e s e n c e of fluorescein. FDA was d i s s o l v e d in acetone at the c o n c e n t r a t i o n of 5mg/ml and the r e s u l t i n g solution was stored as a stock solution, and just p r i o r to the experiment, FDA stock solution was d i l u t e d with m i n i m u m essential m e d i u m (MEM, Nissui) to the following concentration; FDA stock solution 5 ul: M E M 1 ml, and added to the sediment. Cell v i a b i l i t y was d e t e r m i n e d from the p e r c e n t a g e of f l u o r e s c e i n - p o s i t i v e cells o b t a i n e d using an FCM (2). 4) M i c r o c y t o t o x i c i t y test M i c r o c y t o t o x i c i t y test was c a r r i e d out by the m e t h o d by Takasugi and Klein (3). Target (T) cells of a c o n c e n t r a t i o n at 500 cells/10 ul were c u l t i v a t e d in wells of a m i c r o p l a t e (Falcon #3034). The m e d i u m was d i s c a r d e d after 6 hours and the m i x t u r e of target and e f f e c t o r (E) cells, w h i c h was adjusted to 10-120 E/T ratio, was d i v i d e d again in each well, followed by further cultivation for 24 hours. Then, the cells were fixed w i t h ethanol and stained w i t h Giemsa, and target cells were c a l c u l a t e d under a m i c r o s c o p e t o d e t e r m i n e %eytotoxicity using the following formula; % cytotoxicity
C - T = - C
x i00
C: control cell count T: target cell count in this experiment
As the target cells, three kinds of g l i o m a cell lines (U373MG, AJ, KMUI00) and five kinds of cultured cells d e r i v e d from surgical m a t e r i a l s were used for human NK cells. Results The v i a b i l i t y in lymphocytes was 99% before sorting and 97% after sorting, respectively, s u g g e s t i n g a slight decrease of the v i a b i l i t y but such decrease had no influence on the experiment. Of the cells o b t a i n e d from the p e r i p h e r a l blood by sorting, large g r a n u l a r lymphocytes o c c u p i e d 95% or more, and the cells o b t a i n e d by such sorting
Vol.
45, No.
technique
22, 1989
Antlneoplastic
were m i c r o s c o p i c a l l y
confirmed
Effect
of Sorted NK Cells
to be NK cells
(Fig.
2105
2).
Fig. 2 Sorted cell shows the c h a r a c t e r i s t i c s of large, granular lymphocytes which contains Azur granules in the cytoplasm. after sorting, no b a c t e r i a l c o n t a m i n a t i o n was d e t e c t e d and the cells were used for the succeeding m i c r o c y t o t o x i c i t y test. When the E/T ratio was 40, the c y t o t o x i c i t y of respective glioma cell lines of KMUI00, AJ and U 3 7 3 M G was 74, 28 and 9, showing difference in the antineoplastic effects of N K cells. In the study of cultured surgical materials, in which cells of secondary culture were used because they were considered not to greatly differ from the original tumor cells in character, sensitivities to N K cells differed according to each glioma cell. A high c y t o t o x i c i t y of 66% was seen in TC351 even when the E/T ratio was i0 (Table i). Table 1 The range of cytotoxicity.
Cytotoxicity of sorted NK cells against cultured cells (effector "NK cell)
AS for the relationship b e t w e e n the E/T ratio and c y t o t o x i c i t y in KMUI00 of a glioma cell line, showing a correlation. In TC282 of a surgical material cultivated for a short period, sensitivity to NK cells was lower than that of KMUI00 but the % c y t o t o x i c i t y was increased when the E/T ratio was increased from 80 to 120 (Fig. 3).
glioma cell line
target cells
E/T
ratio
~ cytotoxicity(%)
U373 MG
40
9
AJ KMU 100
40 40
28 74
ependymoblastoma glioblastoma glioblastoma
TC 282 TC 309 TC 343
40 20 40
13 1 24
glioblastoma glioblastoma
TC 351 TC 361
10 40
66 15
surgical material
Fig. 3 A n t i n e o p l a s t i c effects of human NK cells on glioma cell lines and cultured cells of surgical material.
Cytotoxic activity of NK cells against cultured cells 100KMU tO0 (gliorna cell line)
Discussion F C M has enabled it to reveal 9 c h a r a c t e r i s t i c s of a large amount ~ 50 of cell in a short p e r i o d and it has been used in various studies on factors responsible for b l o o d related disease or autoimmune disease, on the immunity of cancer patients (i) and on sensitivity of a n t i n e o p l a s t i c agent (4). However, in spite of 0 the sorting function of FCM, it has
T C 282 ( ependyrnoblastoma)
20
40
80
120
E / T ratio
2106
Antineoplastic
Effect
of Sorted NK Cells
Vol.
45, No.
not been well utilized, as compared with the use of FCM for a n a l y z i n g antigen of lymphocytes and FDA d i s t r i b u t i o n of cancer cells.
22, 1989
surface
It is the most important p r o b l e m w h e t h e r the cells serve our purpose, or w h e t h e r the cells remain sterile when cells are sorted by the sterile technique using FCM. F r o m our various p r e l i m i n a r y experiments, we c o n c l u d e d that when the sample tube was filled w i t h 0.5% Hibitane alcohol solution and p h y s i o l o g i c saline was used as the sheath fluid, least c o n t a m i n a t i o n was expected, and so this was the best m e t h o d for the s t e r i l i z a t i o n of FCM. At present, the most common m e t h o d for i s o l a t i o n of the T cell from p e r i p h e ral blood is the use of nylon wool columns. However, the isolation of the NK cells from a cell colony by this m e t h o d requires further p r o c e d u r e s such as the density gradient centrifugation. In the p r e s e n t study, over 95% of the sorted cells were m i c r o s c o p i c a l l y NK cells, which could be used for the s u b s e q u e n t experiment, compared with the cells isolated by the c o n v e n t i o n a l m e t h o d using nylon wool columns. A c c o r d i n g to the report on the NK cell by H e r b e r m a n (5) and K i e s s l i n g 1975, the NK cell was m e n t i o n e d as the cell to fuse tumor cells in vitro cells of mice which u n d e r w e n t no specific i m m u n o l o g i c a l treatment.
(6) in in the
Recently, the NK cells are c o n s i d e r e d to p l a y a role in the i m m u n e m o n i t o r i n g system in vivo, and various studies have been made on it (7). Among those, analysis of surface antigen of the NK cell is noted from the a s s o c i a t i o n w i t h d i f f e r e n t i a t i o n or function. Leu-7 (HNK-I) (8) and Leu-lla (NK-15) (9) are w e l l - k n o w n m o n o c l o n a l a n t i b o d i e s that react w i t h the surface antigen of NK cells. NK cells were sorted t h r o u g h an FCM by Lanier et al. in 1986 (10) using m o n o c l o n a l antibodies of CD 16 (Leu-ll) and Leu-19 (NKH-I) and Evans et al. in 1987 (ii) using m o n o c l o n a l antibody of Leu-7+ and they stated that these NK cells showed c y t o t o x i c effects on K562 cells. So far, however, we could not find any study of the c y t o t o x i c i t y of NK cells on cultured cells of brain tumor o b t a i n e d at surgery and basic e x p e r i m e n t s have not done yet, aiming at the clinical a p p l i c a t i o n in the literature. We sorted L e u - l l a - p o s i t i v e NK cells and confirmed the a n t i n e o p l a s t i c effects on brain t u m o r s by the microc y t o t o x i c i t y test. Some tumor cells from either e s t a b l i s h e d cell lines or cultured cells of surgical m a t e r i a l s showed s e n s i t i v i t y to NK cells. Thus, we u t i l i z e d the sterile sorting m e t h o d of N K cells w i t h an FCM, and firstly d e m o n s t r a t e d their a n t i n e o p l a s t i c effects on some sorts of brain tumor. Using the p r e s e n t method, a n t i n e o p l a s t i c effects for residual brain tumor can be e x p e c t e d c l i n i c a l l y by local a d m i n i s t r a t i o n of NK cells. References i. 2. 3. 4. 5. 6. 7.
K. KAWAMOTO, H. FUJIWARA, K. KAWAKAMI, H. NUMA, N. OKA and H. MATSUMURA: E x c e r p t a M e d i c a 174-183 (1986). Y. WADA, K. KAWAMOTO, S. OKA, T. YAMASHITA, T. K U M A Z A W A and H. MATSUMURA: Life Sci. 44:259-264 (1989). M. TAKASUGI and E. KLEIN: T r a n s p l a n t a t i o n 9:219-227 (1970). K. KAWAMOTO, K. KAWAKAMI, Y. KAWAMURA, H. M A T S U M U R A and A. OHYAMA: J. Neuro-Oncol. 6:361-370 (1988). R.B. HERBERMAN, M.E. NUNN, H.T. H O L D E N and D.H. LAVRIN: Int. J. Cancer 1 6 : 2 3 0 - 2 3 9 (1975). R. KIESSLING, E. KELIN and H. WIGZELL: Eur. J. Immunol. 5:112-117 (1975). T. ABO, C.A. MILLER, G.L. G A R T L A N D and C.M. BALCH: J. Exp. Med. 157:273-
Vol.
8. 9. i0. ii.
45, No.
22,
1989
Antineoplastic
Effect
of Sorted NK Cells
2107
284 (1983). T. ABO and C.M. BALCH: J. Immunol. 1 2 7 : 1 0 2 4 - 1 0 2 9 (1981). L.L. LANIER, A.M. LE, J.H. PHILLIPS, N.L. W A R N E R and G.F. BABCOCK: J. Immunol. 1 3 1 : 1 7 8 9 - 1 7 9 6 (1983). L.L. LANIER, A.M. LE, C.I. CIVIN, M.R. L O K E N and J.H. PHILLIPS: J. Immunol. 1 3 6 : 4 4 8 0 - 4 4 8 6 (1986). C.H. EVANS, J.A. H E I N B A U G H and J.H. RANSOM: L y m p h o k i n e Res. 6 : 2 7 7 - 2 9 7 (1987).
2103-2107
Pergamon
Press
STERILE SORTING OF L E U - I I a - P O S I T I V E CELLS (NK CELLS) USING FLOW CYTOMETRY A N D THE A N T I N E O P L A S T I C EFFECTS ON BRAIN TUMORS Hiroaki
Fujiwara,
Keiji
Kawamoto,
Yoshihiro
Numa and Hiroshi
Matsumura
Department of Neurosurgery, Kansai Medical U n i v e r s i t y i, Fumizonocho, Moriguchi City 570, Japan (Received
in final
form September
22, 1989)
Summary Though we recently found some reports on sterile sorting of natural killer cells (NK cells) by flow cytometer (FCM), w h i c h m a n i f e s t e d cytotoxicity, no reports have concerned a n t i n e o p l a s t i c i t y of sterile NK cells on g l i o m a cell lines and cultured surgical m a t e r i a l s of brain tumor so far as we know. Monocytes were sampled from the peripheral b l o o d of h e a l t h y adults, stained with fluorescein isothiocyanate (FITC)-labeled antihuman m o n o c l o n a l antibodies, and sorted by an FCM, which had been s t e r i l i z e d with 0.5% Hibitane alcohol solution. Over 95% of the cells o b t a i n e d were NK cells, and their v i a b i l i t y was d i s c l o s e d to be 97% by the FDA staining. Using thus o b t a i n e d NK cells, the a n t i n e o p l a s t i c effects were evaluated in 3 kinds of glioma cell lines and 5 surgical specimens by the m i c r o c y t o t o x i c i t y test. The effects v a r i e d w i d e l y from 9 to 74% (E/T ratio 40) for glioma cell lines, and from i to 66% (E/T ratio 10-40) for surgical specimens. It is expected in the future to apply NK cells clinically using this sterile sorting technique. With the d e v e l o p m e n t of m o n o c l o n a l antibodies, it has been possible to analyze the surface antigen of various cells. Analysis of T cell subsets using m o n o c l o n a l antibodies has revealed change of T cell subsets in brain tumor patients (i). w i t h the use of m o n o c l o n a l antibody and the sorting function of a flow cytometer (FCM), lymphocytes for adoptive i m m u n o t h e r a p y can be obtained in a short period. We have established a sterile sorting technique using FCM, and applied it to obtain natural killer (NK) cells from human peripheral blood. In addition, the a n t i n e o p l a s t i c effects of these cells on brain tumors were examined. Methods i) Isolation of lymphocyte and m o n o c l o n a l antibody Lymphocytes were obtained from the p e r i p h e r a l blood of healthy adults by the F i c o l l - C o n r a y d i f f e r e n t i a l centrifugation. P r e l i m i n a r y experiments were carried out to decide the best conditions for fluorescein staining time and volume of m o n o c l o n a l antibody, and f l u o r e s c e i n - m a i n t a i n i n g period. Fluorescein isothiocyanate (FITC)-labeled antihuman Leu-lla (Becton-Dickinson Co., Ltd.) was used for the human NK cells. 2) Sterile sorting of F C M FCM (FACS III, B e c t o n - D i c k i n s o n Co., Ltd.) was sterilized immediately before sorting Leu-lla positive cells. Sterile p h y s i o l o g i c saline was used as the sheath fluid, and sample tubes were sterilized by filling the tubes w i t h o.5% chlohexizine gluconate alcohol solution (ICI Pharma Co., Ltd., Japan) for 20 minutes. Then, f l u o r e s c e i n - p o s i t i v e cells were sorted (Fig. I). 0024-3205/89 $3.00 + .00 Copyright (c) 1989 P e r g a m o n Press
plc
2104
Antineoplastic
Effect
of Sorted NK Cells
Positive
,
,
I
,
Vol.
45, No.
22, 1989
Fig. 1 Fluorescein h i s t o g r a m of FITCnegative cells as a control (left) and sorting range of positive cells showing NK cells (right).
,~
positive cells
negative control
Most part of the cells o b t a i n e d were used as the effector cell in the microc y t o t o x i c i t y test, some part of them for e v a l u a t i o n of the cell v i a b i l i t y using f l u o r e s c e i n d i a c e t a t e (FDA) staining, and some part for the microscopic study to c o n f i r m the a c c u r a c y of the sorting system. 3) E v a l u a t i o n of v i a b i l i t y by FDA Since FDA is a substance w h i c h emanates fluorescein by h y d r o l y s i s after taken in viable cells, viable cells can be d i f f e r e n t i a t e d from dead cells by c o n f i r m i n g the p r e s e n c e of fluorescein. FDA was d i s s o l v e d in acetone at the c o n c e n t r a t i o n of 5mg/ml and the r e s u l t i n g solution was stored as a stock solution, and just p r i o r to the experiment, FDA stock solution was d i l u t e d with m i n i m u m essential m e d i u m (MEM, Nissui) to the following concentration; FDA stock solution 5 ul: M E M 1 ml, and added to the sediment. Cell v i a b i l i t y was d e t e r m i n e d from the p e r c e n t a g e of f l u o r e s c e i n - p o s i t i v e cells o b t a i n e d using an FCM (2). 4) M i c r o c y t o t o x i c i t y test M i c r o c y t o t o x i c i t y test was c a r r i e d out by the m e t h o d by Takasugi and Klein (3). Target (T) cells of a c o n c e n t r a t i o n at 500 cells/10 ul were c u l t i v a t e d in wells of a m i c r o p l a t e (Falcon #3034). The m e d i u m was d i s c a r d e d after 6 hours and the m i x t u r e of target and e f f e c t o r (E) cells, w h i c h was adjusted to 10-120 E/T ratio, was d i v i d e d again in each well, followed by further cultivation for 24 hours. Then, the cells were fixed w i t h ethanol and stained w i t h Giemsa, and target cells were c a l c u l a t e d under a m i c r o s c o p e t o d e t e r m i n e %eytotoxicity using the following formula; % cytotoxicity
C - T = - C
x i00
C: control cell count T: target cell count in this experiment
As the target cells, three kinds of g l i o m a cell lines (U373MG, AJ, KMUI00) and five kinds of cultured cells d e r i v e d from surgical m a t e r i a l s were used for human NK cells. Results The v i a b i l i t y in lymphocytes was 99% before sorting and 97% after sorting, respectively, s u g g e s t i n g a slight decrease of the v i a b i l i t y but such decrease had no influence on the experiment. Of the cells o b t a i n e d from the p e r i p h e r a l blood by sorting, large g r a n u l a r lymphocytes o c c u p i e d 95% or more, and the cells o b t a i n e d by such sorting
Vol.
45, No.
technique
22, 1989
Antlneoplastic
were m i c r o s c o p i c a l l y
confirmed
Effect
of Sorted NK Cells
to be NK cells
(Fig.
2105
2).
Fig. 2 Sorted cell shows the c h a r a c t e r i s t i c s of large, granular lymphocytes which contains Azur granules in the cytoplasm. after sorting, no b a c t e r i a l c o n t a m i n a t i o n was d e t e c t e d and the cells were used for the succeeding m i c r o c y t o t o x i c i t y test. When the E/T ratio was 40, the c y t o t o x i c i t y of respective glioma cell lines of KMUI00, AJ and U 3 7 3 M G was 74, 28 and 9, showing difference in the antineoplastic effects of N K cells. In the study of cultured surgical materials, in which cells of secondary culture were used because they were considered not to greatly differ from the original tumor cells in character, sensitivities to N K cells differed according to each glioma cell. A high c y t o t o x i c i t y of 66% was seen in TC351 even when the E/T ratio was i0 (Table i). Table 1 The range of cytotoxicity.
Cytotoxicity of sorted NK cells against cultured cells (effector "NK cell)
AS for the relationship b e t w e e n the E/T ratio and c y t o t o x i c i t y in KMUI00 of a glioma cell line, showing a correlation. In TC282 of a surgical material cultivated for a short period, sensitivity to NK cells was lower than that of KMUI00 but the % c y t o t o x i c i t y was increased when the E/T ratio was increased from 80 to 120 (Fig. 3).
glioma cell line
target cells
E/T
ratio
~ cytotoxicity(%)
U373 MG
40
9
AJ KMU 100
40 40
28 74
ependymoblastoma glioblastoma glioblastoma
TC 282 TC 309 TC 343
40 20 40
13 1 24
glioblastoma glioblastoma
TC 351 TC 361
10 40
66 15
surgical material
Fig. 3 A n t i n e o p l a s t i c effects of human NK cells on glioma cell lines and cultured cells of surgical material.
Cytotoxic activity of NK cells against cultured cells 100KMU tO0 (gliorna cell line)
Discussion F C M has enabled it to reveal 9 c h a r a c t e r i s t i c s of a large amount ~ 50 of cell in a short p e r i o d and it has been used in various studies on factors responsible for b l o o d related disease or autoimmune disease, on the immunity of cancer patients (i) and on sensitivity of a n t i n e o p l a s t i c agent (4). However, in spite of 0 the sorting function of FCM, it has
T C 282 ( ependyrnoblastoma)
20
40
80
120
E / T ratio
2106
Antineoplastic
Effect
of Sorted NK Cells
Vol.
45, No.
not been well utilized, as compared with the use of FCM for a n a l y z i n g antigen of lymphocytes and FDA d i s t r i b u t i o n of cancer cells.
22, 1989
surface
It is the most important p r o b l e m w h e t h e r the cells serve our purpose, or w h e t h e r the cells remain sterile when cells are sorted by the sterile technique using FCM. F r o m our various p r e l i m i n a r y experiments, we c o n c l u d e d that when the sample tube was filled w i t h 0.5% Hibitane alcohol solution and p h y s i o l o g i c saline was used as the sheath fluid, least c o n t a m i n a t i o n was expected, and so this was the best m e t h o d for the s t e r i l i z a t i o n of FCM. At present, the most common m e t h o d for i s o l a t i o n of the T cell from p e r i p h e ral blood is the use of nylon wool columns. However, the isolation of the NK cells from a cell colony by this m e t h o d requires further p r o c e d u r e s such as the density gradient centrifugation. In the p r e s e n t study, over 95% of the sorted cells were m i c r o s c o p i c a l l y NK cells, which could be used for the s u b s e q u e n t experiment, compared with the cells isolated by the c o n v e n t i o n a l m e t h o d using nylon wool columns. A c c o r d i n g to the report on the NK cell by H e r b e r m a n (5) and K i e s s l i n g 1975, the NK cell was m e n t i o n e d as the cell to fuse tumor cells in vitro cells of mice which u n d e r w e n t no specific i m m u n o l o g i c a l treatment.
(6) in in the
Recently, the NK cells are c o n s i d e r e d to p l a y a role in the i m m u n e m o n i t o r i n g system in vivo, and various studies have been made on it (7). Among those, analysis of surface antigen of the NK cell is noted from the a s s o c i a t i o n w i t h d i f f e r e n t i a t i o n or function. Leu-7 (HNK-I) (8) and Leu-lla (NK-15) (9) are w e l l - k n o w n m o n o c l o n a l a n t i b o d i e s that react w i t h the surface antigen of NK cells. NK cells were sorted t h r o u g h an FCM by Lanier et al. in 1986 (10) using m o n o c l o n a l antibodies of CD 16 (Leu-ll) and Leu-19 (NKH-I) and Evans et al. in 1987 (ii) using m o n o c l o n a l antibody of Leu-7+ and they stated that these NK cells showed c y t o t o x i c effects on K562 cells. So far, however, we could not find any study of the c y t o t o x i c i t y of NK cells on cultured cells of brain tumor o b t a i n e d at surgery and basic e x p e r i m e n t s have not done yet, aiming at the clinical a p p l i c a t i o n in the literature. We sorted L e u - l l a - p o s i t i v e NK cells and confirmed the a n t i n e o p l a s t i c effects on brain t u m o r s by the microc y t o t o x i c i t y test. Some tumor cells from either e s t a b l i s h e d cell lines or cultured cells of surgical m a t e r i a l s showed s e n s i t i v i t y to NK cells. Thus, we u t i l i z e d the sterile sorting m e t h o d of N K cells w i t h an FCM, and firstly d e m o n s t r a t e d their a n t i n e o p l a s t i c effects on some sorts of brain tumor. Using the p r e s e n t method, a n t i n e o p l a s t i c effects for residual brain tumor can be e x p e c t e d c l i n i c a l l y by local a d m i n i s t r a t i o n of NK cells. References i. 2. 3. 4. 5. 6. 7.
K. KAWAMOTO, H. FUJIWARA, K. KAWAKAMI, H. NUMA, N. OKA and H. MATSUMURA: E x c e r p t a M e d i c a 174-183 (1986). Y. WADA, K. KAWAMOTO, S. OKA, T. YAMASHITA, T. K U M A Z A W A and H. MATSUMURA: Life Sci. 44:259-264 (1989). M. TAKASUGI and E. KLEIN: T r a n s p l a n t a t i o n 9:219-227 (1970). K. KAWAMOTO, K. KAWAKAMI, Y. KAWAMURA, H. M A T S U M U R A and A. OHYAMA: J. Neuro-Oncol. 6:361-370 (1988). R.B. HERBERMAN, M.E. NUNN, H.T. H O L D E N and D.H. LAVRIN: Int. J. Cancer 1 6 : 2 3 0 - 2 3 9 (1975). R. KIESSLING, E. KELIN and H. WIGZELL: Eur. J. Immunol. 5:112-117 (1975). T. ABO, C.A. MILLER, G.L. G A R T L A N D and C.M. BALCH: J. Exp. Med. 157:273-
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