Scand. J. Immunol. 9, 239-245, 1979
Human Natural Cell-mediated Cytotoxicity against Fetal Fibroblasts IV. Comparison of Cytotoxic Activity with Antibody-dependent Cell-mediated Cytotoxicity T. TIMONEN Laboratory of Pathology, Departments of Obstetrics and Gynaecology, University Central Hospital, and Department of Pathology, University of Helsinki, Helsinki, Finland
Timonen. T. Human Natural Cell-mediated Cytoioxicity against Fetal Fibroblasts. IV. Comparison of Cytotoxic Activity wiih Antibody-dependent Cell-mediated Cytotoxicity. Seand, J. tmmunol. 9, 239-245. 1979The dependence of human natural killer (NK) cell activity on antibodies was investigated. Absorption of the culture medium with target cells, trypsinization of the efTector celts followed by a brief recovery, and incubation of the effector cells in scrum-free nulrient medium did not alTect natural killer cell activity against fetal fibrohlasts. No soluble mediator in the nutrient media of elTector cell-fibroblast co-cultures could be demonstrated. It was therefore concluded that antibodies are not involved in the cytolytic activity of natural killer cells. However, competition data and the activity of isolated NK cells against aniibody-coated target cells suggested overlapping between the effector cells mediating natural and antibody-dependent cell-mediated cytotoxicity, 7", Timonen. Lahoratory of Pathology. Departments of Obstetrics and Gynaecology. Universilv Central Hospital. SF-00290 Helsinki 29. I inland.
Details of the effector mechanisms involvedn in human natural killer cell (NK cell) activity are largely unknown [ 15]. On tbe basis of surface marker analysis it has been suggested that the «^«, 1^, *• «• . 11 ,-, r , populations of effector cells responsible for the antibody-dependent cell-mediated cytotoxicity .
)fcells. The results do not suggest a direct involvement of antibodies in human NK-cell activity. x, A - r c o i A r o Avrr. ^*c'rwr^^»o MATERIALS AND METHODS ^ ,
j •
T-
.
..
,
J
^
•'
Culture conditions. Target cells were cultured and
(K cells) and natural killer cell activity (NK cells) are overlapping [8, 9, 13, 15, 27]. However, the antibody dependence of NK-cell activity is controversial fl 6 24 26 271
experiments conducted in humidified air atmosphere with 5",', CO,^ at 37 C. The nutrient medium was Ham"s F 10(FlowLaboratories.Irvine,Scotlanij)supplemented '^'"^ '**"" heat-inactivated fetal calf serum (Flow),
~.
,,
1
y.'
' • , ' . ' , '
,..,,,
..
The methods for isolating human NK cells via adsorption-elution of effector cells from fetal fibroblast-coated beads have recently been developed in this laboratory [21]. In this study .Ue. ^^c^:u\^ ^,-.1 I.-L. 1• .L .• -. 1the possible r , , . role , , oi , antibodies m the activity•' o the isolated NK cells is analysed. Furthermore, antibody-dependent cell-mediated cytotoxicity (ADCC) and NK-cell activity are compared in a model assay, in which fetal fibroblast ...
,
-. . . .
,
0.29 m g / m l g l u t a m i n e , a n d antibiotics.
f^,,,,, ^,^,//,, 100 ^i venous citrate blood was coilected from healthy male volunteers, Mononuclear cells were separated with Fieolt-Isopaque gradient centrifugation [?.], washed twice in phosphate-buffered saline(PBS), suspended in 40 ml medium, and incubated .for, 40. mm « .m•I |[tre glass i nRouxflasks a , at 37 ,-.^ C to remove surface-adherent cells. Nonadherent cells were washed once in the medium and finally run through nylon wool columns[7].Theyield was 0.3-1.0 •-io« lymphocytes/m! ^^°^'^„ ., , , , ,, Tarter fW/j. Human fetal lung fibroblasts on passage
targets either as such or sensitized with anti,,^,i, 3_,5 ^ , , , ^,,j ^, ^^.g^^, i^ .^totoxicity experiHLA sera are exposed to nonimmune effector ments. The cuituring techniques of these cells as 0300-9475/79/0300-0239 $02.00 i!; 1979 Blackwell Scientific Publications 239
240
T. Timonen
standard monotayers or on Degalan beads has been described before [19]. For ADCC experiments, chromiumlabelled target cells growing on 100 ^1 beads were incubated in 1 ml serum-free Ham's F 10 medium with indicated dilutions of heat-inactivated anti-HLA serum pool (collected from three multitransfused patients, courtesy of Dr Anja Tiilikainen, the Finnish Red Cross Blood Bank, Helsinki) for 45 min and subsequently washed three times in the conventional medium. Cytoioxiciiy as.say. The technique of using anchoragedependent cell-coated beads as targets in cytotoxicity experiments has been described before [19]. 1,5 x 10" target cells growing on 200 [il of beads were labelled with 20 ^Ci of sodium chromaic-51 (Radiochemical Centre, Amersham, England) in 2 ml of medium for 8 h and subsequently washed three times in PBS. 4 [xl of beads (3 X IO* cells) were pipetted into 7 ml test tubes containing either 0.2 ml medium (spontaneous release). 0.2 ml distilled water (maximal release), or elTector cells in 0.2 ml medium in different concentrations as indicated, usually 2,5 x 10^/tube. Each combination was prepared in triplicate. After incubation for 20 h. 0,8 ml PBS was added and the tubes were centrifuged at 140 g for 10 min. 0.5 ml supernatant was removed to another test tube, and the radioactivity in all tubes was counted for 100 s in an automatic gamma counter (Wallac. Turku, Finland). The percentage of released radioactivity (PRR) was calculated for each pair of tubes as follows: PRR - {2 \ ST/BT f ST} > I(X1, in which ST - counts from 0,5 ml supernatant and BT - counts from 0.5 ml supernatant and heads. The percentage cytotoxicity was calculated from the formula Cx ("„) (T S/M-S) x 100, in which the tetters stand for average PRR values from triplicates: T tubes with effector cells, S -spontaneous release, M —maximal release. Competition experiments. HeLa cells cultured on beads were used in competition experiments to inhibit NK-cell activity against fetal fibroblasts. Unlabelled competitors were added both to the tubes with effector cells to measure the inhibition of cytotoxicity and to spontaneous release tubes to control possible changes in spontaneous release. The competitor to target cell ratio was 3:1. Isolation of natural killer celts with fetal fihroblasteoated beads. The adsorption-elution of NK cells from target-coated beads was carried out as previously described [21]. 400 \j.\ of libroblast-coated beads were incubated with 3 6 / 10* lymphocytes in 2,5 ml medium for 2 h in a test tube with a perforated bottom, after which the beads were released on the top ofa fetal calf serum layer, which separated effector cell-target cell aggregates from single cells. Target cell attached lymphocytes were released from the beads by gentle pipetting and washed once in serum-free medium. Three consecutive cycles of adsorption were performed. The yield of fetal fibroblast adhesive cells was 3-10"o of input. Effector cell trypsinization and serum-free medium incubation. The isolated NK cells were washed twice in PBS and incubated for .30 min at 37 C in PBS with 0,025",, trypsin. Subsequently the suspension was centrifuged and the pellet washed twice in medium. The effector cells were allowed to recover at 37 C from 0 to 6 h. In seruin-frce medium experiments, elTector cells were
washed twice in serum-free Ham's F 10 medium, incubated for 8-10 h in 37 C, washed further twice in serum-free medium, and finally suspended in conventional medium. Absorption of nutrient medium. In the analysis ofthe effect of absorbing nutrient medium with monolayers of target cell type, the microcytotoxicity assay was used as described before [20]. The method was chosen because it includes trypsinization of target cells before exposition to effector cells. The absorption of culture medium was conducted in three 1 h cycles, in which 10 mi medium was incubated on 75 cm" target cell monolayers. Absorption of anti-HLA .serum pool. For competition experiments, the serum pool described above was absorbed with 400 [i\ of HeLa-coated beads in 0.8 ml volumes and 1:20 dilution in serum-free medium. Three consecutive 1 h cycles of absorption were performed.
RESULTS The role of fetal calf serum derived antibodies The possible role of fetal calf setoim derived antibodies in NK-cell activity against fetal fibroblasts was investigated with tbe microcytotoxicity assay, which included trypsinization of target cells before exposure to effector cells. Cytotoxicity values obtained in experiments conducted entirely in the target-cell-absorbed medium did not differ from the values obtained from the experiments in which conventional non-absorbed medium had been used (Table I),
TABLE I. Effect of absorbing nutrient medium with target cells on natural killer cell activity against fetal fibroblasts
Cytotoxicity {" Exp. no. 1 2 3 4 5 Mean
Nonabsorbedt
Absorbed!
-14 94 42 25 16 33
—3 93 27 25 25 33
* Percentage cytotoxicity in microcytoxicity experiments, 20 h exposure, effector to target cell ratio 500:1. t Experiments conducted in conventional media containing three different batches of sera (1-2, 3-4, 5). J Target cells and effector cells suspended in a medium previously absorbed with monolayers of the target cell type.
Human Natural Cell-mediated Cytotoxicity agaitist Fetal Fibroblasts 241 AEsf
AE
NA
c
80
y
70
50
30 -
/
10 8:1
15:1
8:1 15=1 4:1 8:1 EFFECTOR:TARGET RATIO
4:1
8:1
FIG. 1. The ability of different fractions ai'ier adsorption-elution of iiattiral killer cells from fetal fibrohlast-coated beads lo mediate antibody depcndeni cell-mediated cytoioxicity. C cell-free bead absorbed, NA fibroblasl non-adhesive, and AF, - fibroblast-adliesive effector cells. All fractions incubated overnight at 37 C, AEsf in serum-free medium, ihe rest in conventional medium. The base of each bar (• ) indicates NK-cell activity, the top ADCC activity against target cells sensitized with 1:40 dilution of anti-HLA sera ( ). The dotted lines indicate ADCC activity against target cells sensitized with 1:80 dilution of the same sera. The mean values of three experiments with standard error are shown.
Trypsitiization and .serum-free medium incubationTABLE II. Effect of trypsinization on the natural killer cells against fetal fibroblasts of the isolated effector cells The effect of trypsinization of the isolated NK cells is shown in Table II. After 3 h of recovery at 37"C, NK cells had restored their activity, whereas immediately after the trypsinization a diminished cytotoxic activity could be detected. In Fig, l.theactivity of isolated NK cells incubated overnight in serum-free medium followed by careful washing is shown. No alteration in the intensity of cytotoxicity can be seen as compared to the activity of effector cells incubated in conventional serum-containing medium. In Fig. 1 the relative proportion of ADCC out of the total cytotoxicity in different fractions can also be seen. The adsorbed-eluted fractions were capable of exerting strong ADCC, indicating that NK cells overlap with K cells. However, ADCC was of the same magnitude when both control and NK cell-depleted effector cells were used, suggesting that this overlap was only partial. Competition experiments It has previously been shown that NK cells
activity of
Cytotoxicily (%)* Exp, no. 1 2 3 4
Mean
Ct
NAI
AES
AEtr'
AEtrO**
18 28 33 7
12 21 18 5
30 68 36 32
32 45 37 32
20
22
14
42
37
• Percentage cytotoxicity against chromiumlabelled fetal fibroblasts, exposure 20 h, effector to target cell ratio 8:1. t Effector cells absorbed with fibroblast-free beads. X Effector cells nonadhesive to libroblast-coaled beads, S Effector cells adsorbed and eluted from fibroblastcoated beads. • Adsorbed-eluted cells as effectors, trypsinized with 0.025" u trypsin for 30 min, recovery in conventional medium 3-6 h at 37C. *• Adsorbed-eluted ceils as effectors, no recovery after trypsinization.
cross-react against anchorage dependent target cells [20]. It was therefore of interest to study the ability of irrelevant target cells of anchorage
242
T. Timonen
dependent type to inhibit ADCC against sensitized and non-sensitized fetal fibroblasts. The anti-HLA serum pool used was absorbed with HeLa cells to produce selective antifibroblast ADCC. HeLa-celi-covered beads effectively inhibited cytotoxicity against non-sensitized fetal fibroblasts, confirming their ability to inhibit competitively the NK-cell-dependent cytotoxicity (Fig. 2). However, there v^'as no inhibition by HeLa-cell-coated beads against sensitized target cells, suggesting that NK cells have a stronger affinity against antibodysensitized target cells.
lower against the target cells from the more advanced feius, whereas ADCC is of ihe same intensity against both target cell strains.
30
1:40 (0
+^20
t
1:80 1:160 SERUM DILUTION
1:2550
FIG, 3. NK-ccll acUvily and ADCC against two fibroblast target cell strains derived from fetuses of 19 cm (circles) and 9 em (triangles) crown-rump length. Measured with two donors" (open symbols and closed symbols) effector cells; targel cells sensitized with indicated dilutions of anti-HLA serum pool. NK-cell activily is shown in front of each curve.
I
10
8:1
15:1 EFFECTOR:TARGET RATIO
30:1
FIG. 2, The elfect of competing unlabelleiJ HeLa cells on ADCC and NK-cell aclivity against labelled tibroblasls. S -anti-HLA sensitized (1:80) target cells. NS target cells with no serum treatment, CF —12 [J.1 cellfree beads as competitors, HeLa 12 ^l HeLa-cellcoated beads as competitors, x /'
Human Natural Cell-mediated Cytotoxicity against Fetal Fibroblasts IV. Comparison of Cytotoxic Activity with Antibody-dependent Cell-mediated Cytotoxicity T. TIMONEN Laboratory of Pathology, Departments of Obstetrics and Gynaecology, University Central Hospital, and Department of Pathology, University of Helsinki, Helsinki, Finland
Timonen. T. Human Natural Cell-mediated Cytoioxicity against Fetal Fibroblasts. IV. Comparison of Cytotoxic Activity wiih Antibody-dependent Cell-mediated Cytotoxicity. Seand, J. tmmunol. 9, 239-245. 1979The dependence of human natural killer (NK) cell activity on antibodies was investigated. Absorption of the culture medium with target cells, trypsinization of the efTector celts followed by a brief recovery, and incubation of the effector cells in scrum-free nulrient medium did not alTect natural killer cell activity against fetal fibrohlasts. No soluble mediator in the nutrient media of elTector cell-fibroblast co-cultures could be demonstrated. It was therefore concluded that antibodies are not involved in the cytolytic activity of natural killer cells. However, competition data and the activity of isolated NK cells against aniibody-coated target cells suggested overlapping between the effector cells mediating natural and antibody-dependent cell-mediated cytotoxicity, 7", Timonen. Lahoratory of Pathology. Departments of Obstetrics and Gynaecology. Universilv Central Hospital. SF-00290 Helsinki 29. I inland.
Details of the effector mechanisms involvedn in human natural killer cell (NK cell) activity are largely unknown [ 15]. On tbe basis of surface marker analysis it has been suggested that the «^«, 1^, *• «• . 11 ,-, r , populations of effector cells responsible for the antibody-dependent cell-mediated cytotoxicity .
)fcells. The results do not suggest a direct involvement of antibodies in human NK-cell activity. x, A - r c o i A r o Avrr. ^*c'rwr^^»o MATERIALS AND METHODS ^ ,
j •
T-
.
..
,
J
^
•'
Culture conditions. Target cells were cultured and
(K cells) and natural killer cell activity (NK cells) are overlapping [8, 9, 13, 15, 27]. However, the antibody dependence of NK-cell activity is controversial fl 6 24 26 271
experiments conducted in humidified air atmosphere with 5",', CO,^ at 37 C. The nutrient medium was Ham"s F 10(FlowLaboratories.Irvine,Scotlanij)supplemented '^'"^ '**"" heat-inactivated fetal calf serum (Flow),
~.
,,
1
y.'
' • , ' . ' , '
,..,,,
..
The methods for isolating human NK cells via adsorption-elution of effector cells from fetal fibroblast-coated beads have recently been developed in this laboratory [21]. In this study .Ue. ^^c^:u\^ ^,-.1 I.-L. 1• .L .• -. 1the possible r , , . role , , oi , antibodies m the activity•' o the isolated NK cells is analysed. Furthermore, antibody-dependent cell-mediated cytotoxicity (ADCC) and NK-cell activity are compared in a model assay, in which fetal fibroblast ...
,
-. . . .
,
0.29 m g / m l g l u t a m i n e , a n d antibiotics.
f^,,,,, ^,^,//,, 100 ^i venous citrate blood was coilected from healthy male volunteers, Mononuclear cells were separated with Fieolt-Isopaque gradient centrifugation [?.], washed twice in phosphate-buffered saline(PBS), suspended in 40 ml medium, and incubated .for, 40. mm « .m•I |[tre glass i nRouxflasks a , at 37 ,-.^ C to remove surface-adherent cells. Nonadherent cells were washed once in the medium and finally run through nylon wool columns[7].Theyield was 0.3-1.0 •-io« lymphocytes/m! ^^°^'^„ ., , , , ,, Tarter fW/j. Human fetal lung fibroblasts on passage
targets either as such or sensitized with anti,,^,i, 3_,5 ^ , , , ^,,j ^, ^^.g^^, i^ .^totoxicity experiHLA sera are exposed to nonimmune effector ments. The cuituring techniques of these cells as 0300-9475/79/0300-0239 $02.00 i!; 1979 Blackwell Scientific Publications 239
240
T. Timonen
standard monotayers or on Degalan beads has been described before [19]. For ADCC experiments, chromiumlabelled target cells growing on 100 ^1 beads were incubated in 1 ml serum-free Ham's F 10 medium with indicated dilutions of heat-inactivated anti-HLA serum pool (collected from three multitransfused patients, courtesy of Dr Anja Tiilikainen, the Finnish Red Cross Blood Bank, Helsinki) for 45 min and subsequently washed three times in the conventional medium. Cytoioxiciiy as.say. The technique of using anchoragedependent cell-coated beads as targets in cytotoxicity experiments has been described before [19]. 1,5 x 10" target cells growing on 200 [il of beads were labelled with 20 ^Ci of sodium chromaic-51 (Radiochemical Centre, Amersham, England) in 2 ml of medium for 8 h and subsequently washed three times in PBS. 4 [xl of beads (3 X IO* cells) were pipetted into 7 ml test tubes containing either 0.2 ml medium (spontaneous release). 0.2 ml distilled water (maximal release), or elTector cells in 0.2 ml medium in different concentrations as indicated, usually 2,5 x 10^/tube. Each combination was prepared in triplicate. After incubation for 20 h. 0,8 ml PBS was added and the tubes were centrifuged at 140 g for 10 min. 0.5 ml supernatant was removed to another test tube, and the radioactivity in all tubes was counted for 100 s in an automatic gamma counter (Wallac. Turku, Finland). The percentage of released radioactivity (PRR) was calculated for each pair of tubes as follows: PRR - {2 \ ST/BT f ST} > I(X1, in which ST - counts from 0,5 ml supernatant and BT - counts from 0.5 ml supernatant and heads. The percentage cytotoxicity was calculated from the formula Cx ("„) (T S/M-S) x 100, in which the tetters stand for average PRR values from triplicates: T tubes with effector cells, S -spontaneous release, M —maximal release. Competition experiments. HeLa cells cultured on beads were used in competition experiments to inhibit NK-cell activity against fetal fibroblasts. Unlabelled competitors were added both to the tubes with effector cells to measure the inhibition of cytotoxicity and to spontaneous release tubes to control possible changes in spontaneous release. The competitor to target cell ratio was 3:1. Isolation of natural killer celts with fetal fihroblasteoated beads. The adsorption-elution of NK cells from target-coated beads was carried out as previously described [21]. 400 \j.\ of libroblast-coated beads were incubated with 3 6 / 10* lymphocytes in 2,5 ml medium for 2 h in a test tube with a perforated bottom, after which the beads were released on the top ofa fetal calf serum layer, which separated effector cell-target cell aggregates from single cells. Target cell attached lymphocytes were released from the beads by gentle pipetting and washed once in serum-free medium. Three consecutive cycles of adsorption were performed. The yield of fetal fibroblast adhesive cells was 3-10"o of input. Effector cell trypsinization and serum-free medium incubation. The isolated NK cells were washed twice in PBS and incubated for .30 min at 37 C in PBS with 0,025",, trypsin. Subsequently the suspension was centrifuged and the pellet washed twice in medium. The effector cells were allowed to recover at 37 C from 0 to 6 h. In seruin-frce medium experiments, elTector cells were
washed twice in serum-free Ham's F 10 medium, incubated for 8-10 h in 37 C, washed further twice in serum-free medium, and finally suspended in conventional medium. Absorption of nutrient medium. In the analysis ofthe effect of absorbing nutrient medium with monolayers of target cell type, the microcytotoxicity assay was used as described before [20]. The method was chosen because it includes trypsinization of target cells before exposition to effector cells. The absorption of culture medium was conducted in three 1 h cycles, in which 10 mi medium was incubated on 75 cm" target cell monolayers. Absorption of anti-HLA .serum pool. For competition experiments, the serum pool described above was absorbed with 400 [i\ of HeLa-coated beads in 0.8 ml volumes and 1:20 dilution in serum-free medium. Three consecutive 1 h cycles of absorption were performed.
RESULTS The role of fetal calf serum derived antibodies The possible role of fetal calf setoim derived antibodies in NK-cell activity against fetal fibroblasts was investigated with tbe microcytotoxicity assay, which included trypsinization of target cells before exposure to effector cells. Cytotoxicity values obtained in experiments conducted entirely in the target-cell-absorbed medium did not differ from the values obtained from the experiments in which conventional non-absorbed medium had been used (Table I),
TABLE I. Effect of absorbing nutrient medium with target cells on natural killer cell activity against fetal fibroblasts
Cytotoxicity {" Exp. no. 1 2 3 4 5 Mean
Nonabsorbedt
Absorbed!
-14 94 42 25 16 33
—3 93 27 25 25 33
* Percentage cytotoxicity in microcytoxicity experiments, 20 h exposure, effector to target cell ratio 500:1. t Experiments conducted in conventional media containing three different batches of sera (1-2, 3-4, 5). J Target cells and effector cells suspended in a medium previously absorbed with monolayers of the target cell type.
Human Natural Cell-mediated Cytotoxicity agaitist Fetal Fibroblasts 241 AEsf
AE
NA
c
80
y
70
50
30 -
/
10 8:1
15:1
8:1 15=1 4:1 8:1 EFFECTOR:TARGET RATIO
4:1
8:1
FIG. 1. The ability of different fractions ai'ier adsorption-elution of iiattiral killer cells from fetal fibrohlast-coated beads lo mediate antibody depcndeni cell-mediated cytoioxicity. C cell-free bead absorbed, NA fibroblasl non-adhesive, and AF, - fibroblast-adliesive effector cells. All fractions incubated overnight at 37 C, AEsf in serum-free medium, ihe rest in conventional medium. The base of each bar (• ) indicates NK-cell activity, the top ADCC activity against target cells sensitized with 1:40 dilution of anti-HLA sera ( ). The dotted lines indicate ADCC activity against target cells sensitized with 1:80 dilution of the same sera. The mean values of three experiments with standard error are shown.
Trypsitiization and .serum-free medium incubationTABLE II. Effect of trypsinization on the natural killer cells against fetal fibroblasts of the isolated effector cells The effect of trypsinization of the isolated NK cells is shown in Table II. After 3 h of recovery at 37"C, NK cells had restored their activity, whereas immediately after the trypsinization a diminished cytotoxic activity could be detected. In Fig, l.theactivity of isolated NK cells incubated overnight in serum-free medium followed by careful washing is shown. No alteration in the intensity of cytotoxicity can be seen as compared to the activity of effector cells incubated in conventional serum-containing medium. In Fig. 1 the relative proportion of ADCC out of the total cytotoxicity in different fractions can also be seen. The adsorbed-eluted fractions were capable of exerting strong ADCC, indicating that NK cells overlap with K cells. However, ADCC was of the same magnitude when both control and NK cell-depleted effector cells were used, suggesting that this overlap was only partial. Competition experiments It has previously been shown that NK cells
activity of
Cytotoxicily (%)* Exp, no. 1 2 3 4
Mean
Ct
NAI
AES
AEtr'
AEtrO**
18 28 33 7
12 21 18 5
30 68 36 32
32 45 37 32
20
22
14
42
37
• Percentage cytotoxicity against chromiumlabelled fetal fibroblasts, exposure 20 h, effector to target cell ratio 8:1. t Effector cells absorbed with fibroblast-free beads. X Effector cells nonadhesive to libroblast-coaled beads, S Effector cells adsorbed and eluted from fibroblastcoated beads. • Adsorbed-eluted cells as effectors, trypsinized with 0.025" u trypsin for 30 min, recovery in conventional medium 3-6 h at 37C. *• Adsorbed-eluted ceils as effectors, no recovery after trypsinization.
cross-react against anchorage dependent target cells [20]. It was therefore of interest to study the ability of irrelevant target cells of anchorage
242
T. Timonen
dependent type to inhibit ADCC against sensitized and non-sensitized fetal fibroblasts. The anti-HLA serum pool used was absorbed with HeLa cells to produce selective antifibroblast ADCC. HeLa-celi-covered beads effectively inhibited cytotoxicity against non-sensitized fetal fibroblasts, confirming their ability to inhibit competitively the NK-cell-dependent cytotoxicity (Fig. 2). However, there v^'as no inhibition by HeLa-cell-coated beads against sensitized target cells, suggesting that NK cells have a stronger affinity against antibodysensitized target cells.
lower against the target cells from the more advanced feius, whereas ADCC is of ihe same intensity against both target cell strains.
30
1:40 (0
+^20
t
1:80 1:160 SERUM DILUTION
1:2550
FIG, 3. NK-ccll acUvily and ADCC against two fibroblast target cell strains derived from fetuses of 19 cm (circles) and 9 em (triangles) crown-rump length. Measured with two donors" (open symbols and closed symbols) effector cells; targel cells sensitized with indicated dilutions of anti-HLA serum pool. NK-cell activily is shown in front of each curve.
I
10
8:1
15:1 EFFECTOR:TARGET RATIO
30:1
FIG. 2, The elfect of competing unlabelleiJ HeLa cells on ADCC and NK-cell aclivity against labelled tibroblasls. S -anti-HLA sensitized (1:80) target cells. NS target cells with no serum treatment, CF —12 [J.1 cellfree beads as competitors, HeLa 12 ^l HeLa-cellcoated beads as competitors, x /'
