Tissue Antigens (1975),5, 262-265 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)
MLC Results among Unrelated can be Predicted F. JBRGENSEN, L. u. LAMMAND F. KISSMEYER-NIELSEN Tissue Typing Laboratory, Arhus Kommune Hospital, Arhus, Denmark
Typing for LD determinants which has been practicable for some time is shown to be very predictive for the MLC response between unrelated, since LD identical individuals - selected and paired solely on the basis of LD typing - mutually show very low or no MLC reaction.
Received f o r publication 13 January, accepted 28 January 1975
Activation in the mixed leucocyte culture whereas the high responders do not possess (MLC) in man is controlled by the major the determinant. This paper presents a study of the prehistocompatibility complex ( M H C ) (Amos dictive value of LD typing for the outcome & Bach 1968). A strong MLC activating unit has been documented which is outside of MLC tests between unrelated people sethe LA-FOUR stretch (Yunis & Amos lected and paired solely on the basis of 1971) and linked to the FOUR-end (Du- their response against LD-homozygous pont et al. 1971). Furthermore, a weakly typing cells. activating unit may be found in the viciniMethods ty of the LA-end (Eijsvoogel et al. 1972). The terms SD (serological defined) and The methods used are described in detail LD (lymphocyte defined) antigens or de- elsewhere (Jerrgensen & Lamm 1974) as terminants have been introduced (Bach are the bulk of the population studies dur1973), covering the classic HL-A antigens ing which HL-A unidentical but “LDand the “MLC-antigens”, respectively. The identical” unrelated individuals were found concept of typing for LD determinants by (Jerrgensen et al. 1974). I n short, 5X 104 an LD homozygous cell (a “typing cell”), responder cells and 5 X 104 irradiated stime.g. A/A, was introduced by Mernpel et al. ulator cells (5000 Rads from CoGO) are (1973a,b) and simultaneously confirmed by cultured in 0.15 rnl of medium RPMI 1640 Dupont et al. (1973b) and Jerrgensen et al. supplemented with 25 % pooled, heat(1973). Responders in the MLC test can inactivated human serum plus standard be divided in low-responders and high- antibiotics. The cells used have been either responders to a “typing cell”, the interpre- freshly collected or frozen samples stored tation being that the former have the LD in liquid nitrogen. The experiments were determinant in either heterozygous or ho- performed in round-bottomed Linbro mimozygous dose (A/- or A/A) or at least a crotiter plates (IS-MRC-TC96) which determinant which is “typing-cell-like”, were incubated at 37’C in a humidified
263
M I X RESULTS AMONG UNRELATED CAN BE PREDICTED
atmosphere containing 5 (@ COP. After 4 days of culture, thymidine-2-Cl4 (0.04 pC, spec. act. 60 mC/mM) was added and after a further 24 h incubation the cultures were processed for scintillation counting on filters by a methodology similar to that described by Hartzman et al. (1972). Each combination is performed in triplicate and the median of the replicates is used in the evaluation. The raw data are transformed to “relative responses”.
-
ses - expressed as relative response against three MLC typing cells J, L and S, each describing an MLC allele, j , 1 and s, respectively (Jsrgensen et al. 1974). There are five “low-responders” against J, 10 “low-responders” against L and five against S. Three persons are low-responders against both J and L, thus having the “genotype” ill, and three show low response against L and S giving the genotype Ils; finally, none 284
4 Materials and Results Nineteen persons were included in the present experiments. Fig. 1 shows their respon-
%
150
70
150
*. 0
+-
f w
z
v,
2 v, C w T -
+
100 b
5
0.
c= Y
50
I
2
1
:
3
J
L
I
I
S
Figure 1 . Plot of the “relative response” elicited by the typing cells, J, L and S , in the 19 persons involved. Relative response is the counts obtained in a given combination expressed as a percentage of the “typical” response shown by the actual responder. The “typical” response is defined as the median of the responses against a t least three different stimulators unrelated to the responder; (for details see Jorgensen & Lamm 1974).
1
0
Figure 2. Relative response in 57 allogeneic MLC combinations involving six persons as responders sorted out in groups according to sharing of two, one or zero LD alleles. In the group of combinations sharing one allele there appear two related combinations (open symbols), involving different responders; one where the stimulator is the mother of the responder and another where the stimulator is the daughter of the responder. T o the right of each cluster the mean and standard deviation of the group is given. The arrows indicate the mean of the responses of related combinations with equivalent compatibility, i.e. (from left to right) HL-A identical siblings, one haplotype differing and two haplotypes differing combinations within families.
264
JORCENSEN ET AL.
of the persons give low response against all three typing cells. Within each set of fully
genotyped individuals, combinatorial one way MLC testing was performed including stimulation with cells carrying the one or the other or none of the alleles in question. Thus, each responder was stimulated by cells sharing either two, one or zero LDdeterminants with the responder. The experiment with the ill set included two related combinations (see legend to Fig. Z), but for these two, all the other combinations were unrelated. I n Fig. 2, the results have been sorted out according to the LD compatibility as defined by LD typing. I t appears that there is a striking correlation between LD compatibility and MLC response. I n fact, the picture has a close resemblance to the finding within families, when LD compatibility is inferred indirectly from the HL-A typing (SD-types). Thus LD typing as a basis for matching of unrelated individuals has a substantial predictive value. Most pertinent here is the high frequency of low- to non-reacting combinations found, when the MLC partners are “LD-identical”. This frequency is much higher than would be expected by pairing unrelated randomly and also higher than the 10 % or so found between HL-A identical (SD identical) unrelated individuals (Mempel et al. 1973a). Discussion The present material extends and generalizes the initial findings (Dupont et al. 1973b, Jerrgensen & Lamm 1974), and documents that LD typing is the method, which has, so far, shown the highest predictive value for the outcome of the MLC between unrelated individuals. In most cases there is a distinct, but weak, reaction between the “LD identical” test persons. This is easily explained by the fact that ‘‘likeness’’ of two “unknown”
compared to the same reference does, of course, not mean identity between the unknown. The only statement we give about a person giving “typing-response” against a typing cell is that the person has a strong LD-determinant on his lymphocytes, which is like the strong LD-determinant carried by the typing cell. The typing response is generally not zero (see Fig. l ) , this can, among other things, be explained by the “mu1tiple-determinants-on-one-haplotypeconcept” (Dupont et al. 1973a), which covers heterogeneity within the major determinant as well as differences in minor determinants within the HL-A region, pertinent here especially is the weak MLC activating entity, apparently linked to the LA end of the region suggested by Eijsvoogel et al. (1972). The implication of these above possibilities will be as written in the first part of this paragraph. The basic logic in the currently used approach to LD typing resembles the phenomenon of the “serum-control’’ (confirmatory test) in ABO blood grouping and carries with it the same inherited danger (negative observation as a basis for positive conclusion), viz. the occasional discrepancy between the presence of ABO antigens on the red cells and the isoantibody in the serum. The fact that LD typing has predictive value for the outcome of the MLC test supports the common interpretation of a typing response, i.e. the presence of a structure - an “antigen” - on the cells being typed. Thus, although the formal logic behind the approach to MLC typing is wrong - or at least equivocal - the procedure apparently leads to the right conclusion. The structural concept is further underlined by the finding of a dominant or codominant inheritance of the “LD-determinants” in families unrelated to the typingcell donor (Jsrgensen et al. 1973 and other unpublished results), and finally by the
MLC RESULTS AMONG UNRELATED CAN BE PREDICTED
fact that serological techniques able to detect “LD-determinants” directly are now emerging (Kovithavongs et al. 1975, van Rood et al. 1975). Nevertheless, the MLC test as such and also “LD typing” will still be an important tool in immunogenetic and clinical work. Acknowledgements Mrs. Sussi Ipsen and Mrs. Helen Jargensen are cordially thanked for their technical assistance and Mrs. EIly Andersen and Miss Hanne Pedersen for their secretarial help. This work was aided by grants from the Danish Medical Research Council. References Amos, D. B. & Bach, F. H. (1968) Phenotypic expressions of the major histocompatibility locus in man (HL-A), leucocyte antigens and mixed leucocyte culture reactivity. J . exp. Med. 128, 623-637. Bach, F. H. (1973) The major histocompatibility complex in transplantation immunology. Transplant. Proc. 5, 23-29. Dupont, B., Nielsen, L. S. & Svejgaard, A. (1971) Relative importance of FOUR and LA loci in determining mixed lymphocyte reaction. Lancet ii, 1336-1340. Dupont, B., Jersild, C., Hansen, G. S., Nielsen, L. S., Thomsen, M. & Svejgaard, A. (1973a) Multiple MLC (LD) determinants on the same HL-A haplotype. Transplant. Proc. 5, 1481-1487. Dupont, B., Jersild, C., Hansen, G. S., Nielsen, L. S., Thomsen, M. & Svejgaard, A. (1973b) Typing for MLC determinants by means of LD-homozygous and LD-heterozygous test cells. Transplant. Proc. 5, 1543-1549. Eijsvoogel, V. P., van Rood, J. J., du Toit, E. P. & Schellekens, P. Th. A. (1972) Position of a locus determining mixed lymphocyte reaction distinct from the known HL-A loci. Eur. J . Immunol. 2,413-418.
265
Hartzmann, R. J., Bach, M. L., Bach, F. H., Thurman, G. B. & Sell, K. W. (1972) Precipitation of radioactively labeled samples : A semiautomatic multiple-sample processer. Cell. Zmmunol. 4, 182-186. Jerrgensen, F., Lamm, L. U. & Kissmeyer-Nielsen, F. (1973) Mixed lymphocyte cultures with inbred individuals: An approach to MLC typing. Tissue Antigens 3, 323-339. Jergensen, F., Lamm, L. U. & Kissmeyer-Nielsen, F. (1974) Three LD (MLC) determinants. A Danish population study. Tissue Antigens 4,419-428. Jerrgensen, F. & Lamm, L. U. (1974) MLC a micro-modification of the mixed lymphocyte culture technique. Tissue Antigens 4, 482494. Kovithavongs, T., Hyshka, L., McConnachie, P. R . & Dossetor, J. B. (1975) Serotyping for MLC gene products: I Presumptive evidence that ABCIL may detect MLC factors. Tissue Antigens 5, 165-172. Mempel, W., Grosse-Wilde, H., Baumann, P., Netzel, B., Steinbauer-Rosenthal, I., Scholtz, S., Bertrams, J. & Albert, E. (1973a) Population genetics of the MLC response: Typing for MLC determinants using homozygous and heterozygous reference cells. Transplant. Proc. b, 1529-1534. Mempel, W., Grosse-Wilde, H., Albert, E. & Thierfelder, S. (1973b) Atypical MLC reactions in HL-A typed related and unrelated pairs. Transplant. Proc. 5,401-408. van Rood, J. J., van Leeuwen, A., Keuning, J. J. & BlussC van Oud Albas, A. (1975) The serological recognition of the human MLC determinants using a modified cytotoxicity technique. Tissue Antigens 5, 73-79. Yunis, E. J. & Amos, D. B. (1971) Three closely linked genetic systems relevant to transplantation. Proc. nut. Acad. Sci. (Wash.) 68, 3031-3035.
Address: Fritz Jsrgensen Tissue Typing Laboratory The University Hospital DK-8000 Arhus C Denmark
MLC Results among Unrelated can be Predicted F. JBRGENSEN, L. u. LAMMAND F. KISSMEYER-NIELSEN Tissue Typing Laboratory, Arhus Kommune Hospital, Arhus, Denmark
Typing for LD determinants which has been practicable for some time is shown to be very predictive for the MLC response between unrelated, since LD identical individuals - selected and paired solely on the basis of LD typing - mutually show very low or no MLC reaction.
Received f o r publication 13 January, accepted 28 January 1975
Activation in the mixed leucocyte culture whereas the high responders do not possess (MLC) in man is controlled by the major the determinant. This paper presents a study of the prehistocompatibility complex ( M H C ) (Amos dictive value of LD typing for the outcome & Bach 1968). A strong MLC activating unit has been documented which is outside of MLC tests between unrelated people sethe LA-FOUR stretch (Yunis & Amos lected and paired solely on the basis of 1971) and linked to the FOUR-end (Du- their response against LD-homozygous pont et al. 1971). Furthermore, a weakly typing cells. activating unit may be found in the viciniMethods ty of the LA-end (Eijsvoogel et al. 1972). The terms SD (serological defined) and The methods used are described in detail LD (lymphocyte defined) antigens or de- elsewhere (Jerrgensen & Lamm 1974) as terminants have been introduced (Bach are the bulk of the population studies dur1973), covering the classic HL-A antigens ing which HL-A unidentical but “LDand the “MLC-antigens”, respectively. The identical” unrelated individuals were found concept of typing for LD determinants by (Jerrgensen et al. 1974). I n short, 5X 104 an LD homozygous cell (a “typing cell”), responder cells and 5 X 104 irradiated stime.g. A/A, was introduced by Mernpel et al. ulator cells (5000 Rads from CoGO) are (1973a,b) and simultaneously confirmed by cultured in 0.15 rnl of medium RPMI 1640 Dupont et al. (1973b) and Jerrgensen et al. supplemented with 25 % pooled, heat(1973). Responders in the MLC test can inactivated human serum plus standard be divided in low-responders and high- antibiotics. The cells used have been either responders to a “typing cell”, the interpre- freshly collected or frozen samples stored tation being that the former have the LD in liquid nitrogen. The experiments were determinant in either heterozygous or ho- performed in round-bottomed Linbro mimozygous dose (A/- or A/A) or at least a crotiter plates (IS-MRC-TC96) which determinant which is “typing-cell-like”, were incubated at 37’C in a humidified
263
M I X RESULTS AMONG UNRELATED CAN BE PREDICTED
atmosphere containing 5 (@ COP. After 4 days of culture, thymidine-2-Cl4 (0.04 pC, spec. act. 60 mC/mM) was added and after a further 24 h incubation the cultures were processed for scintillation counting on filters by a methodology similar to that described by Hartzman et al. (1972). Each combination is performed in triplicate and the median of the replicates is used in the evaluation. The raw data are transformed to “relative responses”.
-
ses - expressed as relative response against three MLC typing cells J, L and S, each describing an MLC allele, j , 1 and s, respectively (Jsrgensen et al. 1974). There are five “low-responders” against J, 10 “low-responders” against L and five against S. Three persons are low-responders against both J and L, thus having the “genotype” ill, and three show low response against L and S giving the genotype Ils; finally, none 284
4 Materials and Results Nineteen persons were included in the present experiments. Fig. 1 shows their respon-
%
150
70
150
*. 0
+-
f w
z
v,
2 v, C w T -
+
100 b
5
0.
c= Y
50
I
2
1
:
3
J
L
I
I
S
Figure 1 . Plot of the “relative response” elicited by the typing cells, J, L and S , in the 19 persons involved. Relative response is the counts obtained in a given combination expressed as a percentage of the “typical” response shown by the actual responder. The “typical” response is defined as the median of the responses against a t least three different stimulators unrelated to the responder; (for details see Jorgensen & Lamm 1974).
1
0
Figure 2. Relative response in 57 allogeneic MLC combinations involving six persons as responders sorted out in groups according to sharing of two, one or zero LD alleles. In the group of combinations sharing one allele there appear two related combinations (open symbols), involving different responders; one where the stimulator is the mother of the responder and another where the stimulator is the daughter of the responder. T o the right of each cluster the mean and standard deviation of the group is given. The arrows indicate the mean of the responses of related combinations with equivalent compatibility, i.e. (from left to right) HL-A identical siblings, one haplotype differing and two haplotypes differing combinations within families.
264
JORCENSEN ET AL.
of the persons give low response against all three typing cells. Within each set of fully
genotyped individuals, combinatorial one way MLC testing was performed including stimulation with cells carrying the one or the other or none of the alleles in question. Thus, each responder was stimulated by cells sharing either two, one or zero LDdeterminants with the responder. The experiment with the ill set included two related combinations (see legend to Fig. Z), but for these two, all the other combinations were unrelated. I n Fig. 2, the results have been sorted out according to the LD compatibility as defined by LD typing. I t appears that there is a striking correlation between LD compatibility and MLC response. I n fact, the picture has a close resemblance to the finding within families, when LD compatibility is inferred indirectly from the HL-A typing (SD-types). Thus LD typing as a basis for matching of unrelated individuals has a substantial predictive value. Most pertinent here is the high frequency of low- to non-reacting combinations found, when the MLC partners are “LD-identical”. This frequency is much higher than would be expected by pairing unrelated randomly and also higher than the 10 % or so found between HL-A identical (SD identical) unrelated individuals (Mempel et al. 1973a). Discussion The present material extends and generalizes the initial findings (Dupont et al. 1973b, Jerrgensen & Lamm 1974), and documents that LD typing is the method, which has, so far, shown the highest predictive value for the outcome of the MLC between unrelated individuals. In most cases there is a distinct, but weak, reaction between the “LD identical” test persons. This is easily explained by the fact that ‘‘likeness’’ of two “unknown”
compared to the same reference does, of course, not mean identity between the unknown. The only statement we give about a person giving “typing-response” against a typing cell is that the person has a strong LD-determinant on his lymphocytes, which is like the strong LD-determinant carried by the typing cell. The typing response is generally not zero (see Fig. l ) , this can, among other things, be explained by the “mu1tiple-determinants-on-one-haplotypeconcept” (Dupont et al. 1973a), which covers heterogeneity within the major determinant as well as differences in minor determinants within the HL-A region, pertinent here especially is the weak MLC activating entity, apparently linked to the LA end of the region suggested by Eijsvoogel et al. (1972). The implication of these above possibilities will be as written in the first part of this paragraph. The basic logic in the currently used approach to LD typing resembles the phenomenon of the “serum-control’’ (confirmatory test) in ABO blood grouping and carries with it the same inherited danger (negative observation as a basis for positive conclusion), viz. the occasional discrepancy between the presence of ABO antigens on the red cells and the isoantibody in the serum. The fact that LD typing has predictive value for the outcome of the MLC test supports the common interpretation of a typing response, i.e. the presence of a structure - an “antigen” - on the cells being typed. Thus, although the formal logic behind the approach to MLC typing is wrong - or at least equivocal - the procedure apparently leads to the right conclusion. The structural concept is further underlined by the finding of a dominant or codominant inheritance of the “LD-determinants” in families unrelated to the typingcell donor (Jsrgensen et al. 1973 and other unpublished results), and finally by the
MLC RESULTS AMONG UNRELATED CAN BE PREDICTED
fact that serological techniques able to detect “LD-determinants” directly are now emerging (Kovithavongs et al. 1975, van Rood et al. 1975). Nevertheless, the MLC test as such and also “LD typing” will still be an important tool in immunogenetic and clinical work. Acknowledgements Mrs. Sussi Ipsen and Mrs. Helen Jargensen are cordially thanked for their technical assistance and Mrs. EIly Andersen and Miss Hanne Pedersen for their secretarial help. This work was aided by grants from the Danish Medical Research Council. References Amos, D. B. & Bach, F. H. (1968) Phenotypic expressions of the major histocompatibility locus in man (HL-A), leucocyte antigens and mixed leucocyte culture reactivity. J . exp. Med. 128, 623-637. Bach, F. H. (1973) The major histocompatibility complex in transplantation immunology. Transplant. Proc. 5, 23-29. Dupont, B., Nielsen, L. S. & Svejgaard, A. (1971) Relative importance of FOUR and LA loci in determining mixed lymphocyte reaction. Lancet ii, 1336-1340. Dupont, B., Jersild, C., Hansen, G. S., Nielsen, L. S., Thomsen, M. & Svejgaard, A. (1973a) Multiple MLC (LD) determinants on the same HL-A haplotype. Transplant. Proc. 5, 1481-1487. Dupont, B., Jersild, C., Hansen, G. S., Nielsen, L. S., Thomsen, M. & Svejgaard, A. (1973b) Typing for MLC determinants by means of LD-homozygous and LD-heterozygous test cells. Transplant. Proc. 5, 1543-1549. Eijsvoogel, V. P., van Rood, J. J., du Toit, E. P. & Schellekens, P. Th. A. (1972) Position of a locus determining mixed lymphocyte reaction distinct from the known HL-A loci. Eur. J . Immunol. 2,413-418.
265
Hartzmann, R. J., Bach, M. L., Bach, F. H., Thurman, G. B. & Sell, K. W. (1972) Precipitation of radioactively labeled samples : A semiautomatic multiple-sample processer. Cell. Zmmunol. 4, 182-186. Jerrgensen, F., Lamm, L. U. & Kissmeyer-Nielsen, F. (1973) Mixed lymphocyte cultures with inbred individuals: An approach to MLC typing. Tissue Antigens 3, 323-339. Jergensen, F., Lamm, L. U. & Kissmeyer-Nielsen, F. (1974) Three LD (MLC) determinants. A Danish population study. Tissue Antigens 4,419-428. Jerrgensen, F. & Lamm, L. U. (1974) MLC a micro-modification of the mixed lymphocyte culture technique. Tissue Antigens 4, 482494. Kovithavongs, T., Hyshka, L., McConnachie, P. R . & Dossetor, J. B. (1975) Serotyping for MLC gene products: I Presumptive evidence that ABCIL may detect MLC factors. Tissue Antigens 5, 165-172. Mempel, W., Grosse-Wilde, H., Baumann, P., Netzel, B., Steinbauer-Rosenthal, I., Scholtz, S., Bertrams, J. & Albert, E. (1973a) Population genetics of the MLC response: Typing for MLC determinants using homozygous and heterozygous reference cells. Transplant. Proc. b, 1529-1534. Mempel, W., Grosse-Wilde, H., Albert, E. & Thierfelder, S. (1973b) Atypical MLC reactions in HL-A typed related and unrelated pairs. Transplant. Proc. 5,401-408. van Rood, J. J., van Leeuwen, A., Keuning, J. J. & BlussC van Oud Albas, A. (1975) The serological recognition of the human MLC determinants using a modified cytotoxicity technique. Tissue Antigens 5, 73-79. Yunis, E. J. & Amos, D. B. (1971) Three closely linked genetic systems relevant to transplantation. Proc. nut. Acad. Sci. (Wash.) 68, 3031-3035.
Address: Fritz Jsrgensen Tissue Typing Laboratory The University Hospital DK-8000 Arhus C Denmark
