Tissue Antigens (1975), 5, 165-172 Published by Munksgaard, Copenhagen, Denmark
No part may be reproduced by any process without written permission from the author(s)
Serotyping for MLC Gene Products: I. Presumptive Evidence that ABCIL" may Detect MLC Factors THAVISAKDI KOVITHAVONGS, LINDAHYSHKA,PETERR. MCCONNACHIE AND JOHN
B. DOSSETOR
MRC Transplantation Group, University of Alberta and Department of Clinical Pathology, University of Alberta Hospital, Edmonton, Alberta, Canada
Serum from a grand multiparous Inuit (Eskimo) woman, who is HL-A identical but stimulatory in MLC with cells of her husband, contains strong ABCIL reactivity. This serum is operationally monospecific against a cell membrane determinant on lymphocytes, but not platelets, of some unrelated persons. Cells of those who are ABCIL positive with this serum always give a lower MLC stimulation index compared with their MLC responsiveness to cells that are ABCIL negative. This serum also contains good MLC inhibitory activity but only in those one way MLC reactions in which it reacts, by ABCIL, with the stimulating cells. I t is postulated that the ABCIL activity is directed against a common MLC factor on lymphocytes. Received for publication 19 August, accepted 25 October 1974
This report represents part of a present study in this laboratory to attempt to establish a serological method for LD typing. The method used is the antibody-mediated, cell-dependent immune lympholysis or the ABCIL system which is complementindependent. This method is simple, fast, accurate and sensitive and does not require any costly laboratory instruments except
* The editorial board
for a gamma spectrometer for the 51Cr release assay. Earlier reports from this laboratory have shown that the ABCIL antibody is different from the complementdependent cytotoxic antibody (Kovithavongs & Dossetor 1973) that is frequently detected both in parous women and in multi-transfused hemodialysis patients in whom cytotoxic antibody may not be de-
is rather unhappy to see a new terminology used for an old phenomenon, but will have t o accept individuality. ABCIL is equivalent to LDA as used by Perlmann, AIC as used by MGller, ADCC as used by Fahey and LALI, as used by Ceppellini.
166
KOVITHAVONGS ET AL.
tectable (Kovithavongs et al. 1974a,b) and that the specificity of ABCIL is not defined by the restrictions of the HL-A system (Kovithavongs et al. 1974a). In one parous woman's serum, ABCIL reactivity and inhibitory activity in one way mixed lymphocyte cultures (UMLC) were correlated in the sense that they were both widespread and not restricted to the HL-A system. Using this and another serum in a study in a large family, we were able to identify individuals who were HL-A haploidentical yet did not stimulate in MLC (Kovithavongs et al. 1974c). We are now extending that line of research to unrelated individuals by ABCIL screening individuals, who were HL-A typed using a panel of ABCIL reactive sera most of which also have known cytotoxic specificity. The finding that one unique serum was able to predict the response in MLC by cells that were grouped according to ABCIL reactions to this serum is reported here. The correlation of ABCIL reactivity and UMLC inhibitory activity by this serum will also be presented. Our findings confirm our earlier report that certain ABCIL sera can identify LD factors and suggest that the ABCIL technique may provide a rapid serological approach for L D typing in unrelated individuals. 51Cr release
(%)
=
Materials and M e t h o d s ABCZL T e s t As part of the screening study, the ABCIL procedure, as used in this study, was a semi-micro modification of our original technique (Kovithavongs et al. 1974a). The present technique has the advantage of consuming only (onequarter of the original amount of serum required at appropriate dilutions, yet preserving the accuracy of the test. The preparation of lymphocytes and the 5Wr labelling of the target cells were as previously described. The effector cells were finally resuspended in medium 199 with 10 % fetal calf serum (FCS) at a concentration of 5 X 106 cells per ml. Sufficient numbers of 51Cr labelled target cells (5X lOS/ml) were added to the effector suspension to give an effector to target cell ratio of 20:l. This effectortarget cell mixture was then instilled to 1 0 x 7 5 mm glass tubes which were preloaded with 0.025 ml antiserum, at 0.2 ml mixture per tube. After mixing, the tubes were incubated at 37' C in 5 % CO, atmosphere for 3 h, then 2 ml cold Hanks BSS was added and the supernatants were separated from the cell pellets after centrifugation. 51Cr release was calculated according to the formula:
cpm in supernatant cpm in supernatant cpm in cell pellet
+
x
100
and specific 51Cr release
(%)
=
experimental 51Cr release - backgnound 51Cr release maximal 5lCr release -background 51Cr release
Experimental release twice the background release or greater was considered to be significantly positive.
A n t iser u m The results of only one of many sera
x
100
studied will be reported. This serum, designated as STIN serum, was obtained from an Inuit (Eskimo) woman 10 years after her last (twelth) pregnancy. As reported previously, this woman is HL-A identical with her husband, but they did
Cells
Racea
Sexb
HL-A
167
:I
SEROTYPING FOR MLC GENE PRODUCTS
1
% specific 51Cr releasee Background
Experimental
a C = Caucasoid, 0 = Oriental.
F = female, M = male. after 3 h incubation at 37" C in 5% CO, atmosphere. The first six are ABCIL negative, the rest are ABCIL positive.
stimulate each other in MLC (Dossetor et al. 1973). As expected, her serum did not contain any cytotoxin yet it had good ABCIL activity against her husband and some children (Kovithavongs & Dossetor 1973). This serum was kept frozen a t - 20" C until the time of study when it was thawed and heat inactivated. Part of it was used unabsorbed in both ABCIL and UMLC reactions and part was used in ABCIL after appropriate absorption with platelets and lymphocytes described below.
Target Cells Since this is only part of a present study, only 12 cells are shown. Six of them were ABCIL positive and the other six negative with STIN serum. As presented in Table 1, the race, sex and HL-A profile did not differ significantly between the two groups. S.I. =
R R
Effector Cells Our earlier experience indicates that some effector cells have better activity than others (Kovithavongs et al. 1974d). Only cells with good effector cell activity were used in this study. Mixed Lymphocyte Culture Test This was done in triplicate according to a modification of the method of Bach et al. ( 1970) : 105 responding cells, purified by the Ficoll-Isopaque separation technique and 105 mitomycin treated stimulating cells were cultured together for five days in 1 ml of medium 199 with 15% normal AB serum, tritiated thymidine was added to the cultures 4 h before they were processed for counting in a liquid scintillation (Packard) counter. The results are expressed as stimulation indices (S.I.) calculated according to:
+ Sm or cpm of responding cells + stimulating cells (mitomycin treated) + Rm cpm of responding cells 4-responding cells (mitomycin treated)
168
KOVITHAVONGS ET AL.
In experiments in which the inhibitory effect of antiserum was investigated, cultures of responding and stimulating cells as described above were made with and with-
"/o change in S.I. by STIN serum =
R
+ SM + serumJR + Rm + serum R + S d R + Rm
Results As shown in Table 1, cells A, B, C, D, E and F are ABCIL negative with STIN serum; cells G, H, I, J, K and L are positive. I n order to explore whether or not STIN serum was directed against a similar membrane bound determinant on different cells and whether or not this determinant is present on platelets, an absorption study was carried out. Lymphocytes and platelets from individual G at a dosage similar to that adopted by van Leeuwen et al. (1973) were used. The absorbed sera were then used to react with cells G, H, I, J, and K. It is clear from this experiment that the ABCIL activity of STIN serum cannot be removed by platelets from G (Fig. 1) . O n the other hand, G lymphocytes cannot only remove the ABCIL activity against G but also the activity against H, I, J, and K to a comparable extent. This suggests that the ABCIL activity of STIN serum is monospecific and directed against a common 30
20 SPECIFIC
51Cr
-
determinant on G, H, I, J, and K lymphocytes, but not on their platelets. The effects of STIN serum on one way MLC are shown in Table 2. I t is remarkable that all responding cells exhibited striking differences to stimulation by the ABCIL negative and the ABCIL positive cells in the presence of STIN serum. With the exception of cells C and J, all other cells showed inhibition by STIN serum to proliferate in response to stimulation by cells G, H, I and J, all ABCIL positive with STIN serum. These same cells were not inhibited in proliferation in response to the ABCIL negative cells A, B, C, and D. On the contrary, many of them showed enhanced proliferation especially cells A, C, and J, thus, taking both enhancement and inhibition into consideration, it becomes clear that all the MLC responses to ABCIL positive cells were inhibited in the presence of STIN serum, although this inhibition might be masked by the enhancing effect as in cells C and J. We conclude
0
UNABSORBED
~-E ABSORBED D ~ ~ L 0y GMPLATELETS p H o c y T E s
~
RELEASE
x
out the addition of the antiserum to a final concentration of 1.5 %. The inhibitory effect of antiserum was calculated as follows :
...: 2
lo-
; ..
01
..
:*
0,
G
H
I
-2
.'. ..
:i..
..
J
K
TARGETS Figure I. Absorption study. See text for interpretation.
169
SEROTYPING FOR M1.C GENE PRODUCTS: I
Table 2 Effect of STIN serum on MLC involving cells that were ABCIL negative (A-D) and positive (G-J). % change in S. I. by STIN seruma
Respond ing cells
Stimulating cells (mitomycin treated) Am
-
1
Bm
+lo7
1
1
Cm
+147
Dm
I
Gm
1 Hm I
mean difference
mean
Im
I
+
Jm
+136.3
+155
f 14.8
P
188.0
No part may be reproduced by any process without written permission from the author(s)
Serotyping for MLC Gene Products: I. Presumptive Evidence that ABCIL" may Detect MLC Factors THAVISAKDI KOVITHAVONGS, LINDAHYSHKA,PETERR. MCCONNACHIE AND JOHN
B. DOSSETOR
MRC Transplantation Group, University of Alberta and Department of Clinical Pathology, University of Alberta Hospital, Edmonton, Alberta, Canada
Serum from a grand multiparous Inuit (Eskimo) woman, who is HL-A identical but stimulatory in MLC with cells of her husband, contains strong ABCIL reactivity. This serum is operationally monospecific against a cell membrane determinant on lymphocytes, but not platelets, of some unrelated persons. Cells of those who are ABCIL positive with this serum always give a lower MLC stimulation index compared with their MLC responsiveness to cells that are ABCIL negative. This serum also contains good MLC inhibitory activity but only in those one way MLC reactions in which it reacts, by ABCIL, with the stimulating cells. I t is postulated that the ABCIL activity is directed against a common MLC factor on lymphocytes. Received for publication 19 August, accepted 25 October 1974
This report represents part of a present study in this laboratory to attempt to establish a serological method for LD typing. The method used is the antibody-mediated, cell-dependent immune lympholysis or the ABCIL system which is complementindependent. This method is simple, fast, accurate and sensitive and does not require any costly laboratory instruments except
* The editorial board
for a gamma spectrometer for the 51Cr release assay. Earlier reports from this laboratory have shown that the ABCIL antibody is different from the complementdependent cytotoxic antibody (Kovithavongs & Dossetor 1973) that is frequently detected both in parous women and in multi-transfused hemodialysis patients in whom cytotoxic antibody may not be de-
is rather unhappy to see a new terminology used for an old phenomenon, but will have t o accept individuality. ABCIL is equivalent to LDA as used by Perlmann, AIC as used by MGller, ADCC as used by Fahey and LALI, as used by Ceppellini.
166
KOVITHAVONGS ET AL.
tectable (Kovithavongs et al. 1974a,b) and that the specificity of ABCIL is not defined by the restrictions of the HL-A system (Kovithavongs et al. 1974a). In one parous woman's serum, ABCIL reactivity and inhibitory activity in one way mixed lymphocyte cultures (UMLC) were correlated in the sense that they were both widespread and not restricted to the HL-A system. Using this and another serum in a study in a large family, we were able to identify individuals who were HL-A haploidentical yet did not stimulate in MLC (Kovithavongs et al. 1974c). We are now extending that line of research to unrelated individuals by ABCIL screening individuals, who were HL-A typed using a panel of ABCIL reactive sera most of which also have known cytotoxic specificity. The finding that one unique serum was able to predict the response in MLC by cells that were grouped according to ABCIL reactions to this serum is reported here. The correlation of ABCIL reactivity and UMLC inhibitory activity by this serum will also be presented. Our findings confirm our earlier report that certain ABCIL sera can identify LD factors and suggest that the ABCIL technique may provide a rapid serological approach for L D typing in unrelated individuals. 51Cr release
(%)
=
Materials and M e t h o d s ABCZL T e s t As part of the screening study, the ABCIL procedure, as used in this study, was a semi-micro modification of our original technique (Kovithavongs et al. 1974a). The present technique has the advantage of consuming only (onequarter of the original amount of serum required at appropriate dilutions, yet preserving the accuracy of the test. The preparation of lymphocytes and the 5Wr labelling of the target cells were as previously described. The effector cells were finally resuspended in medium 199 with 10 % fetal calf serum (FCS) at a concentration of 5 X 106 cells per ml. Sufficient numbers of 51Cr labelled target cells (5X lOS/ml) were added to the effector suspension to give an effector to target cell ratio of 20:l. This effectortarget cell mixture was then instilled to 1 0 x 7 5 mm glass tubes which were preloaded with 0.025 ml antiserum, at 0.2 ml mixture per tube. After mixing, the tubes were incubated at 37' C in 5 % CO, atmosphere for 3 h, then 2 ml cold Hanks BSS was added and the supernatants were separated from the cell pellets after centrifugation. 51Cr release was calculated according to the formula:
cpm in supernatant cpm in supernatant cpm in cell pellet
+
x
100
and specific 51Cr release
(%)
=
experimental 51Cr release - backgnound 51Cr release maximal 5lCr release -background 51Cr release
Experimental release twice the background release or greater was considered to be significantly positive.
A n t iser u m The results of only one of many sera
x
100
studied will be reported. This serum, designated as STIN serum, was obtained from an Inuit (Eskimo) woman 10 years after her last (twelth) pregnancy. As reported previously, this woman is HL-A identical with her husband, but they did
Cells
Racea
Sexb
HL-A
167
:I
SEROTYPING FOR MLC GENE PRODUCTS
1
% specific 51Cr releasee Background
Experimental
a C = Caucasoid, 0 = Oriental.
F = female, M = male. after 3 h incubation at 37" C in 5% CO, atmosphere. The first six are ABCIL negative, the rest are ABCIL positive.
stimulate each other in MLC (Dossetor et al. 1973). As expected, her serum did not contain any cytotoxin yet it had good ABCIL activity against her husband and some children (Kovithavongs & Dossetor 1973). This serum was kept frozen a t - 20" C until the time of study when it was thawed and heat inactivated. Part of it was used unabsorbed in both ABCIL and UMLC reactions and part was used in ABCIL after appropriate absorption with platelets and lymphocytes described below.
Target Cells Since this is only part of a present study, only 12 cells are shown. Six of them were ABCIL positive and the other six negative with STIN serum. As presented in Table 1, the race, sex and HL-A profile did not differ significantly between the two groups. S.I. =
R R
Effector Cells Our earlier experience indicates that some effector cells have better activity than others (Kovithavongs et al. 1974d). Only cells with good effector cell activity were used in this study. Mixed Lymphocyte Culture Test This was done in triplicate according to a modification of the method of Bach et al. ( 1970) : 105 responding cells, purified by the Ficoll-Isopaque separation technique and 105 mitomycin treated stimulating cells were cultured together for five days in 1 ml of medium 199 with 15% normal AB serum, tritiated thymidine was added to the cultures 4 h before they were processed for counting in a liquid scintillation (Packard) counter. The results are expressed as stimulation indices (S.I.) calculated according to:
+ Sm or cpm of responding cells + stimulating cells (mitomycin treated) + Rm cpm of responding cells 4-responding cells (mitomycin treated)
168
KOVITHAVONGS ET AL.
In experiments in which the inhibitory effect of antiserum was investigated, cultures of responding and stimulating cells as described above were made with and with-
"/o change in S.I. by STIN serum =
R
+ SM + serumJR + Rm + serum R + S d R + Rm
Results As shown in Table 1, cells A, B, C, D, E and F are ABCIL negative with STIN serum; cells G, H, I, J, K and L are positive. I n order to explore whether or not STIN serum was directed against a similar membrane bound determinant on different cells and whether or not this determinant is present on platelets, an absorption study was carried out. Lymphocytes and platelets from individual G at a dosage similar to that adopted by van Leeuwen et al. (1973) were used. The absorbed sera were then used to react with cells G, H, I, J, and K. It is clear from this experiment that the ABCIL activity of STIN serum cannot be removed by platelets from G (Fig. 1) . O n the other hand, G lymphocytes cannot only remove the ABCIL activity against G but also the activity against H, I, J, and K to a comparable extent. This suggests that the ABCIL activity of STIN serum is monospecific and directed against a common 30
20 SPECIFIC
51Cr
-
determinant on G, H, I, J, and K lymphocytes, but not on their platelets. The effects of STIN serum on one way MLC are shown in Table 2. I t is remarkable that all responding cells exhibited striking differences to stimulation by the ABCIL negative and the ABCIL positive cells in the presence of STIN serum. With the exception of cells C and J, all other cells showed inhibition by STIN serum to proliferate in response to stimulation by cells G, H, I and J, all ABCIL positive with STIN serum. These same cells were not inhibited in proliferation in response to the ABCIL negative cells A, B, C, and D. On the contrary, many of them showed enhanced proliferation especially cells A, C, and J, thus, taking both enhancement and inhibition into consideration, it becomes clear that all the MLC responses to ABCIL positive cells were inhibited in the presence of STIN serum, although this inhibition might be masked by the enhancing effect as in cells C and J. We conclude
0
UNABSORBED
~-E ABSORBED D ~ ~ L 0y GMPLATELETS p H o c y T E s
~
RELEASE
x
out the addition of the antiserum to a final concentration of 1.5 %. The inhibitory effect of antiserum was calculated as follows :
...: 2
lo-
; ..
01
..
:*
0,
G
H
I
-2
.'. ..
:i..
..
J
K
TARGETS Figure I. Absorption study. See text for interpretation.
169
SEROTYPING FOR M1.C GENE PRODUCTS: I
Table 2 Effect of STIN serum on MLC involving cells that were ABCIL negative (A-D) and positive (G-J). % change in S. I. by STIN seruma
Respond ing cells
Stimulating cells (mitomycin treated) Am
-
1
Bm
+lo7
1
1
Cm
+147
Dm
I
Gm
1 Hm I
mean difference
mean
Im
I
+
Jm
+136.3
+155
f 14.8
P
188.0
