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Evaluation of whole blood as effector for antibody-dependent cellular cytotoxicity against autologous targets
Journal of Immunological Methods, 35 (1980) 225--231
225
© Elsevier/North-Holland Biomedical Press
E V A L U A T I O N OF WHOLE BLOOD AS EFFECTOR FOR A N T I B O D Y - D E P E N D E N T CELLULAR CYTOTOXICITY A G A I N S T AUTOLOGOUS TARGETS i
E. DUPONT 2
Departments of Nephrology and Immunology, Cliniques Universitaires de Bruxelles, Hdpital Erasme, Brussels, Belgium (Received 3 December 1979, accepted 20 February 1980)
An assay for antibody-dependent cell-mediated cytotoxicity which uses whole blood as effector and autologous lymphocytes sensitized with rabbit anti-human lymphocyte serum as targets is assessed. Comparison with performances of purified lymphocytes shows that whole blood yields approximately the same cytotoxicity levels as the lymphocytes which can be separated from the corresponding volume of blood. This equivalence is not seen at the early stage of cytolysis, whole blood reaching its plateau stage 1 h later. A contribution of phagocytic cells to lysis of the target cells is ruled out. The presence of plasma does not modify the cytolytic capacity of the blood of healthy subjects. The test may be performed with less than 1 ml of blood and is suitable for repeated evaluation of the immunologic status of patients, for evaluation of plasma factors in diseases and of the effects of agents inducing lymphocyte redistribution.
INTRODUCTION
Whole blood has been used for the study of cellular immunity in mitogeninduced proliferation (Park and Good, 1972) and more recently of cellmediated cytotoxicity (Gale and MacLennan, 1976). Apart from practical advantages such as the requirement for only small volumes and avoidance of isolation procedures, the main rationale is to maintain in vitro conditions existing in vivo such as humoral factors (e.g. immune complexes) and proportions of cell subpopulations which are otherwise likely to be modified during isolation procedures. We investigated various parameters of antibody-dependent cell-mediated cytotoxicity (ADCC) effected by whole blood against autologous lymphocytes sensitized with rabbit anti-human lymphocyte serum. As these target
1 Supported by the Fonds de la Recherche Scientifique Mddicale (contract 3.4526.78) and by the Fondation Universitaire David et Alice van Buuren. 2 Reprint requests: Dr. E. Dupont, H6pital Erasme, 808 route de Lennick, 1070 Brussels, Belgium.
226 cells are lysed by a restricted population of null cells (Brier et al., 1975), they are appropriate for evaluation of K cell activity. MATERIAL AND METHODS Blood was taken in preservative-free heparin from healthy subjects. Decreasing volumes (see below) were added to tubes. An aliquot was submitted to Ficoll-Hypaque density centrifugation (BSyum, 1968). The mononuclear cells recovered, containing 85--90% l y m p h o c y t e s and 5--10% m o n o c y t e s , with granulocyte contamination less than 5% were then used b o t h as targets and effectors. In one experiment, polymorphonuclear (PMN) cells were isolated from the pellet containing both PMN and red blood cells by sedimentation for 1 h at r o o m temperature in 1 vol of 5% dextran (Povite; MW = 200,000). After lysis of the remaining red cells with 0.83% NH4C1, the cell preparation contained 90% PMN, 7% m o n o c y t e s and 3% lymphocytes.
Target cells 1 X 106 isolated mononuclear cells used were labeled for 60 min with 100 pCi [S'Cr]sodium chromate (specific activity, 200--400 mCi/mg of Cr) in PBS containing 10% heat-activated fetal calf serum. After washing, the cells were incubated for 45 min with 0.2 ml heat-inactivated rabbit antihuman l y m p h o c y t e serum (ALS) at 10 -3 dilution in HBSS. The ALS was obtained by immunization of a rabbit with pooled lymphocytes from 30 different donors. After incubation, u n b o u n d antibody was removed by 3 washings and the cell concentration readjusted to 1 X 106/ml in McCoy's 5A medium (Flow Laboratories) supplemented with 2% fetal calf serum, L-glutamine, Hepes buffer and antibiotics.
The effector cell preparation Effectors consisted of decreasing volumes of whole blood (100, 50, 25, and 10 pl) added in triplicate to Beckman 0.4 ml microtubes. The plasma was removed by 3 washings in HBSS, and 200 pl of McCoy's medium was added on t o p of the red cell layer covered by the b u f f y coat. In some experiments, the plasma was left and its volume measured by hematocrit. Medium was added to a final volume (plasma + medium) of 200 pl. Separated lymphocytes from the same individuals were tested in parallel in 200 pl volumes at the following concentrations: 2 × 106/ml, 1 × 106/ml, 0.5 X 10~/ml and 0.25 X 106/ml. In 3 experiments, phagocytic cells were removed from whole blood b y incubation at 37°C for 60 min with carbonyl iron powder and constant agitation b y a magnetic stirrer. In one experiment, the cytolytic capacity of isolated PMN was compared with that of the l y m p h o c y t e s of the same donor at the same cell concentrations.
227
Cytotoxic assay Of sensitized target cells (5 X 103 l y m p h o c y t e s / t u b e ) 5 pl were added to the effector dell preparation (whole blood, isolated lymphocytes or PMN), each test being performed in triplicate. Effector and target cell mixtures were then centrifuged for 1 min at 700 X g on a Beckman microfuge and incubated at 37°C for 4 h (except for kinetic studies). The tubes were then thoroughly shaken, centrifuged at 1000 X g and 100 #l of the supernate transferred to another tube. The radioactivity was counted in a gamma counter. Each experiment included minimum or spontaneous release controls (SlCr release from antibody-coated targets in 0.2 ml medium) and m a x i m u m release b y lysis of target cells in distilled water. The following formulae were used for calculation: %SlCr experimental release = 2 X [(cpm in supernate)/(cpm in supernate + cpm in residue)] X 100. %SlCr specific release = [(exp. r e l e a s e - min. release)/(max, r e l e a s e - min. release)] X 100. RESULTS Fig. 1 compares the performance of isolated lymphocytes from 5 different donors with that of whole blood from the same individuals. The two curves have similar shapes. At the concentrations of cells used in these experiments, isolated l y m p h o c y t e s yielded somewhat higher c y t o t o x i c i t y values. Blood aliquots greater than 100 pl were n o t tested. Approximately 50% cytolysis was obtained with 50 pl of whole blood and with 10 X 104 lymphocytes. The latter represents approximately the number of lymphocytes present in 50 pl of blood, since the mean l y m p h o c y t e count of our normal donors was 2200 + 400/pl. Thus the use of whole blood was n o t associated with significant loss of c y t o t o x i c activity. Kinetic studies (Figs. 2 and 3) showed that equivalence is reached only at a later stage of cytolysis b u t this disappears if only random cell contact is taking place by spontaneous sedimentation. Centrifugation leads to an 'explosive' c y t o t o x i c i t y as early as 10 min after initiation of incubation with isolated lymphocytes, while lysis effected by corresponding volumes of whole blood is more gradual, probably reflecting hindrance b y platelets, granulocytes and red cells to early effective contact between Fc receptors of K cells and Fc portions of IgG coating the target. The potential c y t o t o x i c activity of phagocytic cells was assessed in t w o ways: absorption of whole blood with carbonyl iron powder (Fig. 3) and use of isolated PMN as effectors (Fig. 4). Both show that phagocytic cells do not contribute to the lysis of ALS-coated lymphocytes. Removal o f plasma by 3 extensive washings (Fig. 5 ) d i d not m o d i f y cyto-
228 BLOOD
WHOLE
806040-
5
t
N
20-
,-,, 0¢:
0
,J bJ O_ U%
/~
80--
ISOLATED LYMPHOCYTES
,3 80
an
60
~
6o~0-
40,~++ sE~, ,,:s
20-
10 20 10:1 20:1 40~
o~
20-
4o.~o ~ 80:1
Lymphoc. 5
_____---o b
Hin.
E/T Ratio/tube
Fig. 1. C o m p a r i s o n of p e r f o r m a n c e s o f w h o l e b l o o d ( t o p ) a n d of isolated l y m p h o c y t e s ( b o t t o m ) f r o m 5 n o r m a l d o n o r s . R e s u l t s are e x p r e s s e d as t h e m e a n ± S.E.M. o f t i t r a t i o n c u r v e s o b t a i n e d w i t h v a r i o u s v o l u m e s o f b l o o d a n d c o n c e n t r a t i o n s o f l y m p h o c y t e s as m e n t i o n e d . E f f e c t o r c e l l / t a r g e t cell ( E / T ) r a t i o s are i n d i c a t e d . Fig. 2. K i n e t i c s of 10 × 10 a p u r i f i e d l y m p h o c y t e s (A A) a n d o f 50 pl o f w h o l e b l o o d (m --) f r o m o n e d o n o r , a i n d i c a t e s m a x i m u m a n d b m i n i m u m release. A f t e r s t a r t i n g t h e i n c u b a t i o n a t 37~C, c y t o l y s i s was s t o p p e d a f t e r t h e various t i m e s i n d i c a t e d by rapid harvesting of the supernates. A A a n d [] [], results w i t h o u t c e n t r i f u g a t i o n b e f o r e i n c u b a t i o n a t 37°C.
•
absorbed
x
non absorbed
100 -
_i
80
dIJJ
60
,2,
4o
~
20
W
O_
N=3
1;
5'0
16o
Fig. 3. E f f e c t of a b s o r b i n g w h o l e b l o o d w i t h c a r b o n y l i r o n p o w d e r .
229 X Washed whole blood • Unwashed whole blood
80-
80.J
bJ
60t-Y
4020-
o~
~:
60
bJ o. cn
L,O
~n
20
N:3
OI
5
I
10
I
20
I
~,010/+
i
i
10 25
i
50
I
lOOpt
Fig. 4. Comparisons of cytolysis effected by similar concentrations of lymphocytes (x X) and granulocytes (A A) against autologous sensitized lymphocytes. Results with unsensitized autologous lymphocytes (o . . . . . . o) or lymphocytes (o o) obtained from an unrelated donor are shown. Fig. 5. Effects of plasma removal by extensive washing.
toxicity, ruling out the role of factors inhibiting or enhancing K cell activity in the plasma of normal persons. DISCUSSION
Depending on the target system used, ADCC can be performed by a variety of lymphoid cells carrying an Fc surface receptor: K cells (MacLennan, 1972; Perlmann et al., 1972), monocytes and macrophages (MacDonald et al., 1975) and neutrophils (Gale and Zighelboim, 1975). K cells share surface properties responsible for spontaneous cytotoxicity with NK cells but not sensitivity to corticosteroids (Parillo and Fauci, 1978). This draws attention to the importance of using a target cell which is destroyed exclusively by K cells. Chang cells (MacLennan et al., 1976) or lymphoblastoid cell lines (Trinchieri et al., 1975) are targets currently used for ADCC which show lysis without sensitizing antibody. Whole blood is a complex reagent in which the influence of the number of lymphocytes, plasma factors, the role of phagocytic cells and of interactions between cell subpopulations might affect ADCC. A lack of correlation between lymphocytosis and cytotoxic level has been previously shown (Gale and Zighelboim, 1975; Sasaki et al., 1980). We have shown above that plasma factors present in normal individuals do not modify cytotoxicity. Participation of human complement present in plasma has also been shown to be insignificant (Sasaki et al., 1980). These findings favor use of this method for the study of plasma factors in disease. The present findings rule out the participation of phagocytic cells, both on the results of absorption studies and
230
b y testing the capacity of isolated granulocytes to lyse the target cells. Large numbers of granulocytes if present in whole blood could potentially lyse target cells sensitized with rabbit antiserum (Gale and Zighelboim, 1975). More extensive depletion experiments which have been performed with isolated l y m p h o c y t e s (Brier et al., 1975) on the same ADCC system are not feasible on whole blood for technical reasons. Two methodological aspects may also be mentioned. The use of fresh autologous l y m p h o c y t e s is suitable for studies in which the c y t o t o x i c capacity of a patient is compared in the same experiment before and after a given treatment. For repeated evaluations of many patients with different diseases, use of one or several stored standardized target cells is probably preferable (Ting and Terasaki, 1974; D u p o n t et al., 1977; Sasaki et al., 1979). The presence of variable volumes of red cells at the b o t t o m of the tubes (in patients with different hematocrits or in titration experiments) influences the volume of medium into which SlCr is released during the c y t o t o x i c reaction. When plasma is left in place the amount of supplementary medium which must be added to plasma may be calculated from the hematocrit value. This is unnecessary if plasma is removed b y washing, as a constant volume of medium may be added. Practical aspects of this test for frequent monitoring of transplant and cancer patients has been emphasized (Sasaki et al., 1979). Recent unpublished findings indicate that the test may be used to study plasma factors (by comparison of the c y t o t o x i c level and without washing) and agents such as corticosteroids which modify cellular traffic, and induce an increase of c y t o t o x i c activity in isolated l y m p h o c y t e s as compared with a decrease of c y t o t o x i c i t y of whole blood (Parillo and Fauci, 1978). The true redistribution of cell subpopulations occurring in vitro is thus assessed. Discrepancies between performances of whole blood and of purified l y m p h o c y t e s have already provided information a b o u t the effects of immunosuppression on T l y m p h o c y t e s (Lopez et al., 1975). REFERENCES BSyum, A., 1968, Scand. J. Clin. Lab. Invest 21 (Suppl. 97), 1. Brier, A.M., L. Chess and F.S. Schlossman, 1975, J. Clin. Invest. 56, 1580. Dupont, E., G. Opelz, L.A. Gustafsson, S.M. Mikulski and P.I. Terasaki, 1977, Transplantation 23,165. Gale, D. and I.C.M. MacLennan, 1976, Clin. Exp. Immunol. 23,252. Gale, R.P. and J. Zighelboim, 1975, J. Immunol. 114, 1047. Lopez, C., R.L. Simmons, J.L. Touraine, B.H. Park, D.F. Kiszkiss, J.S. Najarian and R.A. Good, 1975, Clin. Immunol. Immunopathol. 4, 135. MacDonald, H.R., A.D. Bonnard, S. Sordat and S.H. Zawodnik, 1975, Scand. J. Immunol. 4,437. MacLennan, I.C.M., 1972, Transplant. Rev. 13, 67. MacLennan, I.C.M., A.C. Campbell and D.G.L. Gale, 1976, in: In Vitro Methods in Cell-
231 mediated and Tumor Immunity, eds. B.R. Bloom and J.R. David (Academic Press, New York) p. 511. Parillo, J.E. and A.S. Fauci, 1978, Scand. J. Immunol. 8, 99. Park, B.H. and R.A. Good, 1972, Proc. Natl. Acad. Sci. U.S.A. 69, 371. Perlmann, P., H. Perlmann and H. Wigzell, 1972, Transplant. Rev. 13, 91. Sasaki, M., P.I. Terasaki, H. Silberman and T. Berne, 1979, Transplantation 27,246. Sasaki, M., B. Looman and P.I. Terasaki, 1980, Tissue Antigen 15, 225. Ting, A. and P.I. Terasaki, 1974, Cancer Res. 34, 2694. Trinchieri, G., P. Baumann, M. Demarchi and Z. Tokes, 1975, J. Immunol. 115,249.
225
© Elsevier/North-Holland Biomedical Press
E V A L U A T I O N OF WHOLE BLOOD AS EFFECTOR FOR A N T I B O D Y - D E P E N D E N T CELLULAR CYTOTOXICITY A G A I N S T AUTOLOGOUS TARGETS i
E. DUPONT 2
Departments of Nephrology and Immunology, Cliniques Universitaires de Bruxelles, Hdpital Erasme, Brussels, Belgium (Received 3 December 1979, accepted 20 February 1980)
An assay for antibody-dependent cell-mediated cytotoxicity which uses whole blood as effector and autologous lymphocytes sensitized with rabbit anti-human lymphocyte serum as targets is assessed. Comparison with performances of purified lymphocytes shows that whole blood yields approximately the same cytotoxicity levels as the lymphocytes which can be separated from the corresponding volume of blood. This equivalence is not seen at the early stage of cytolysis, whole blood reaching its plateau stage 1 h later. A contribution of phagocytic cells to lysis of the target cells is ruled out. The presence of plasma does not modify the cytolytic capacity of the blood of healthy subjects. The test may be performed with less than 1 ml of blood and is suitable for repeated evaluation of the immunologic status of patients, for evaluation of plasma factors in diseases and of the effects of agents inducing lymphocyte redistribution.
INTRODUCTION
Whole blood has been used for the study of cellular immunity in mitogeninduced proliferation (Park and Good, 1972) and more recently of cellmediated cytotoxicity (Gale and MacLennan, 1976). Apart from practical advantages such as the requirement for only small volumes and avoidance of isolation procedures, the main rationale is to maintain in vitro conditions existing in vivo such as humoral factors (e.g. immune complexes) and proportions of cell subpopulations which are otherwise likely to be modified during isolation procedures. We investigated various parameters of antibody-dependent cell-mediated cytotoxicity (ADCC) effected by whole blood against autologous lymphocytes sensitized with rabbit anti-human lymphocyte serum. As these target
1 Supported by the Fonds de la Recherche Scientifique Mddicale (contract 3.4526.78) and by the Fondation Universitaire David et Alice van Buuren. 2 Reprint requests: Dr. E. Dupont, H6pital Erasme, 808 route de Lennick, 1070 Brussels, Belgium.
226 cells are lysed by a restricted population of null cells (Brier et al., 1975), they are appropriate for evaluation of K cell activity. MATERIAL AND METHODS Blood was taken in preservative-free heparin from healthy subjects. Decreasing volumes (see below) were added to tubes. An aliquot was submitted to Ficoll-Hypaque density centrifugation (BSyum, 1968). The mononuclear cells recovered, containing 85--90% l y m p h o c y t e s and 5--10% m o n o c y t e s , with granulocyte contamination less than 5% were then used b o t h as targets and effectors. In one experiment, polymorphonuclear (PMN) cells were isolated from the pellet containing both PMN and red blood cells by sedimentation for 1 h at r o o m temperature in 1 vol of 5% dextran (Povite; MW = 200,000). After lysis of the remaining red cells with 0.83% NH4C1, the cell preparation contained 90% PMN, 7% m o n o c y t e s and 3% lymphocytes.
Target cells 1 X 106 isolated mononuclear cells used were labeled for 60 min with 100 pCi [S'Cr]sodium chromate (specific activity, 200--400 mCi/mg of Cr) in PBS containing 10% heat-activated fetal calf serum. After washing, the cells were incubated for 45 min with 0.2 ml heat-inactivated rabbit antihuman l y m p h o c y t e serum (ALS) at 10 -3 dilution in HBSS. The ALS was obtained by immunization of a rabbit with pooled lymphocytes from 30 different donors. After incubation, u n b o u n d antibody was removed by 3 washings and the cell concentration readjusted to 1 X 106/ml in McCoy's 5A medium (Flow Laboratories) supplemented with 2% fetal calf serum, L-glutamine, Hepes buffer and antibiotics.
The effector cell preparation Effectors consisted of decreasing volumes of whole blood (100, 50, 25, and 10 pl) added in triplicate to Beckman 0.4 ml microtubes. The plasma was removed by 3 washings in HBSS, and 200 pl of McCoy's medium was added on t o p of the red cell layer covered by the b u f f y coat. In some experiments, the plasma was left and its volume measured by hematocrit. Medium was added to a final volume (plasma + medium) of 200 pl. Separated lymphocytes from the same individuals were tested in parallel in 200 pl volumes at the following concentrations: 2 × 106/ml, 1 × 106/ml, 0.5 X 10~/ml and 0.25 X 106/ml. In 3 experiments, phagocytic cells were removed from whole blood b y incubation at 37°C for 60 min with carbonyl iron powder and constant agitation b y a magnetic stirrer. In one experiment, the cytolytic capacity of isolated PMN was compared with that of the l y m p h o c y t e s of the same donor at the same cell concentrations.
227
Cytotoxic assay Of sensitized target cells (5 X 103 l y m p h o c y t e s / t u b e ) 5 pl were added to the effector dell preparation (whole blood, isolated lymphocytes or PMN), each test being performed in triplicate. Effector and target cell mixtures were then centrifuged for 1 min at 700 X g on a Beckman microfuge and incubated at 37°C for 4 h (except for kinetic studies). The tubes were then thoroughly shaken, centrifuged at 1000 X g and 100 #l of the supernate transferred to another tube. The radioactivity was counted in a gamma counter. Each experiment included minimum or spontaneous release controls (SlCr release from antibody-coated targets in 0.2 ml medium) and m a x i m u m release b y lysis of target cells in distilled water. The following formulae were used for calculation: %SlCr experimental release = 2 X [(cpm in supernate)/(cpm in supernate + cpm in residue)] X 100. %SlCr specific release = [(exp. r e l e a s e - min. release)/(max, r e l e a s e - min. release)] X 100. RESULTS Fig. 1 compares the performance of isolated lymphocytes from 5 different donors with that of whole blood from the same individuals. The two curves have similar shapes. At the concentrations of cells used in these experiments, isolated l y m p h o c y t e s yielded somewhat higher c y t o t o x i c i t y values. Blood aliquots greater than 100 pl were n o t tested. Approximately 50% cytolysis was obtained with 50 pl of whole blood and with 10 X 104 lymphocytes. The latter represents approximately the number of lymphocytes present in 50 pl of blood, since the mean l y m p h o c y t e count of our normal donors was 2200 + 400/pl. Thus the use of whole blood was n o t associated with significant loss of c y t o t o x i c activity. Kinetic studies (Figs. 2 and 3) showed that equivalence is reached only at a later stage of cytolysis b u t this disappears if only random cell contact is taking place by spontaneous sedimentation. Centrifugation leads to an 'explosive' c y t o t o x i c i t y as early as 10 min after initiation of incubation with isolated lymphocytes, while lysis effected by corresponding volumes of whole blood is more gradual, probably reflecting hindrance b y platelets, granulocytes and red cells to early effective contact between Fc receptors of K cells and Fc portions of IgG coating the target. The potential c y t o t o x i c activity of phagocytic cells was assessed in t w o ways: absorption of whole blood with carbonyl iron powder (Fig. 3) and use of isolated PMN as effectors (Fig. 4). Both show that phagocytic cells do not contribute to the lysis of ALS-coated lymphocytes. Removal o f plasma by 3 extensive washings (Fig. 5 ) d i d not m o d i f y cyto-
228 BLOOD
WHOLE
806040-
5
t
N
20-
,-,, 0¢:
0
,J bJ O_ U%
/~
80--
ISOLATED LYMPHOCYTES
,3 80
an
60
~
6o~0-
40,~++ sE~, ,,:s
20-
10 20 10:1 20:1 40~
o~
20-
4o.~o ~ 80:1
Lymphoc. 5
_____---o b
Hin.
E/T Ratio/tube
Fig. 1. C o m p a r i s o n of p e r f o r m a n c e s o f w h o l e b l o o d ( t o p ) a n d of isolated l y m p h o c y t e s ( b o t t o m ) f r o m 5 n o r m a l d o n o r s . R e s u l t s are e x p r e s s e d as t h e m e a n ± S.E.M. o f t i t r a t i o n c u r v e s o b t a i n e d w i t h v a r i o u s v o l u m e s o f b l o o d a n d c o n c e n t r a t i o n s o f l y m p h o c y t e s as m e n t i o n e d . E f f e c t o r c e l l / t a r g e t cell ( E / T ) r a t i o s are i n d i c a t e d . Fig. 2. K i n e t i c s of 10 × 10 a p u r i f i e d l y m p h o c y t e s (A A) a n d o f 50 pl o f w h o l e b l o o d (m --) f r o m o n e d o n o r , a i n d i c a t e s m a x i m u m a n d b m i n i m u m release. A f t e r s t a r t i n g t h e i n c u b a t i o n a t 37~C, c y t o l y s i s was s t o p p e d a f t e r t h e various t i m e s i n d i c a t e d by rapid harvesting of the supernates. A A a n d [] [], results w i t h o u t c e n t r i f u g a t i o n b e f o r e i n c u b a t i o n a t 37°C.
•
absorbed
x
non absorbed
100 -
_i
80
dIJJ
60
,2,
4o
~
20
W
O_
N=3
1;
5'0
16o
Fig. 3. E f f e c t of a b s o r b i n g w h o l e b l o o d w i t h c a r b o n y l i r o n p o w d e r .
229 X Washed whole blood • Unwashed whole blood
80-
80.J
bJ
60t-Y
4020-
o~
~:
60
bJ o. cn
L,O
~n
20
N:3
OI
5
I
10
I
20
I
~,010/+
i
i
10 25
i
50
I
lOOpt
Fig. 4. Comparisons of cytolysis effected by similar concentrations of lymphocytes (x X) and granulocytes (A A) against autologous sensitized lymphocytes. Results with unsensitized autologous lymphocytes (o . . . . . . o) or lymphocytes (o o) obtained from an unrelated donor are shown. Fig. 5. Effects of plasma removal by extensive washing.
toxicity, ruling out the role of factors inhibiting or enhancing K cell activity in the plasma of normal persons. DISCUSSION
Depending on the target system used, ADCC can be performed by a variety of lymphoid cells carrying an Fc surface receptor: K cells (MacLennan, 1972; Perlmann et al., 1972), monocytes and macrophages (MacDonald et al., 1975) and neutrophils (Gale and Zighelboim, 1975). K cells share surface properties responsible for spontaneous cytotoxicity with NK cells but not sensitivity to corticosteroids (Parillo and Fauci, 1978). This draws attention to the importance of using a target cell which is destroyed exclusively by K cells. Chang cells (MacLennan et al., 1976) or lymphoblastoid cell lines (Trinchieri et al., 1975) are targets currently used for ADCC which show lysis without sensitizing antibody. Whole blood is a complex reagent in which the influence of the number of lymphocytes, plasma factors, the role of phagocytic cells and of interactions between cell subpopulations might affect ADCC. A lack of correlation between lymphocytosis and cytotoxic level has been previously shown (Gale and Zighelboim, 1975; Sasaki et al., 1980). We have shown above that plasma factors present in normal individuals do not modify cytotoxicity. Participation of human complement present in plasma has also been shown to be insignificant (Sasaki et al., 1980). These findings favor use of this method for the study of plasma factors in disease. The present findings rule out the participation of phagocytic cells, both on the results of absorption studies and
230
b y testing the capacity of isolated granulocytes to lyse the target cells. Large numbers of granulocytes if present in whole blood could potentially lyse target cells sensitized with rabbit antiserum (Gale and Zighelboim, 1975). More extensive depletion experiments which have been performed with isolated l y m p h o c y t e s (Brier et al., 1975) on the same ADCC system are not feasible on whole blood for technical reasons. Two methodological aspects may also be mentioned. The use of fresh autologous l y m p h o c y t e s is suitable for studies in which the c y t o t o x i c capacity of a patient is compared in the same experiment before and after a given treatment. For repeated evaluations of many patients with different diseases, use of one or several stored standardized target cells is probably preferable (Ting and Terasaki, 1974; D u p o n t et al., 1977; Sasaki et al., 1979). The presence of variable volumes of red cells at the b o t t o m of the tubes (in patients with different hematocrits or in titration experiments) influences the volume of medium into which SlCr is released during the c y t o t o x i c reaction. When plasma is left in place the amount of supplementary medium which must be added to plasma may be calculated from the hematocrit value. This is unnecessary if plasma is removed b y washing, as a constant volume of medium may be added. Practical aspects of this test for frequent monitoring of transplant and cancer patients has been emphasized (Sasaki et al., 1979). Recent unpublished findings indicate that the test may be used to study plasma factors (by comparison of the c y t o t o x i c level and without washing) and agents such as corticosteroids which modify cellular traffic, and induce an increase of c y t o t o x i c activity in isolated l y m p h o c y t e s as compared with a decrease of c y t o t o x i c i t y of whole blood (Parillo and Fauci, 1978). The true redistribution of cell subpopulations occurring in vitro is thus assessed. Discrepancies between performances of whole blood and of purified l y m p h o c y t e s have already provided information a b o u t the effects of immunosuppression on T l y m p h o c y t e s (Lopez et al., 1975). REFERENCES BSyum, A., 1968, Scand. J. Clin. Lab. Invest 21 (Suppl. 97), 1. Brier, A.M., L. Chess and F.S. Schlossman, 1975, J. Clin. Invest. 56, 1580. Dupont, E., G. Opelz, L.A. Gustafsson, S.M. Mikulski and P.I. Terasaki, 1977, Transplantation 23,165. Gale, D. and I.C.M. MacLennan, 1976, Clin. Exp. Immunol. 23,252. Gale, R.P. and J. Zighelboim, 1975, J. Immunol. 114, 1047. Lopez, C., R.L. Simmons, J.L. Touraine, B.H. Park, D.F. Kiszkiss, J.S. Najarian and R.A. Good, 1975, Clin. Immunol. Immunopathol. 4, 135. MacDonald, H.R., A.D. Bonnard, S. Sordat and S.H. Zawodnik, 1975, Scand. J. Immunol. 4,437. MacLennan, I.C.M., 1972, Transplant. Rev. 13, 67. MacLennan, I.C.M., A.C. Campbell and D.G.L. Gale, 1976, in: In Vitro Methods in Cell-
231 mediated and Tumor Immunity, eds. B.R. Bloom and J.R. David (Academic Press, New York) p. 511. Parillo, J.E. and A.S. Fauci, 1978, Scand. J. Immunol. 8, 99. Park, B.H. and R.A. Good, 1972, Proc. Natl. Acad. Sci. U.S.A. 69, 371. Perlmann, P., H. Perlmann and H. Wigzell, 1972, Transplant. Rev. 13, 91. Sasaki, M., P.I. Terasaki, H. Silberman and T. Berne, 1979, Transplantation 27,246. Sasaki, M., B. Looman and P.I. Terasaki, 1980, Tissue Antigen 15, 225. Ting, A. and P.I. Terasaki, 1974, Cancer Res. 34, 2694. Trinchieri, G., P. Baumann, M. Demarchi and Z. Tokes, 1975, J. Immunol. 115,249.