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Immunogenicity of allograft components
CELLULAR
IMMUNOLOGY
57, 85-91 (1981)
lmmunogenicity
of Allograft
Components
I. Assay for lmmunogenicity ANUSOOTS,
ELAINE~ARTHENAIS,'
ARTONEMLANDER,
ANDPEKKA
HAYRY
Transplantation Laboratory. Fourth Department of Helsinki, University of Helsinki, Helsinki, Finland Received March
18, 1980; accepted April
16. 1980
We describe an assay for in vivo quantitation of the “immunogenicity” of isolated cell populations. The assay is based on the observation that if an AgB-incompatible recipient rat is primed with donor strain spleen cells 72 hr prior to transplantation, heart allograft survival is reduced from 6.2 to 3.0 days. The effect is independent of the priming cell dose at levels above 3 X 10’ cells, whereas doseslower than 10’ spleen cells are unable to reduce the survival. The effect is suboptimal if the priming-transplantation interval is less than 3 days, or is prolonged to 4-10 days. The effect is immunologically specific: priming with irrelevant AgBincompatible spleen celis fails to reduce the survival. Priming with cell populations previously reported “less immunogenic,” such as ultrasonicated spleen cells, erythrocytes, spleen T cells, or spleen cells deriving from methotrexate or cyclophosphamide-treated rats, fails to reduce the survival, or reduces it only when given in IOO-fold higher numbers than the minimal dose of intact spleen cells giving maximal reduction.
INTRODUCTION “Immunogenicity” is a property of a substance to induce an immune response. Immunogenicity is usually detected by reintroducing the same antigen into the recipient, and if a secondary response is obtained, the material is said to be immunogenic. With simple protein antigens, immunogenicity depends of the “foreigness” of the material, molecular size, chemical complexity, the route of administration, and genetic constitution of the recipient (1). Histocompatibility antigens are glycoproteins regulated by several independently segregating genetic systems (2-4). In most allografts, the antigens of the major histocompatibility complex (MHC) are probably crucial in both triggering of the antiallograft response, and as targets for the host immune attack (5). “Transplant rejection” describes a complex biological phenomenon of both cellular and humoral immunity, which may be divided into the following largely overlapping components: initiation and generation of the specific immune response, amplification of the response by nonspecific inflammatory mechanisms, and destruction of the graft. The first of these events is crucial in determining the fate of the transplant, and the immunogenicity of the transplant is probably the most important single factor ’ Canadian Medical Research Council Fellow. 85 0008-8749/g 1/OlOOSS-07$02.00/O Copyright All
rights
0
1981
by Academic
of reproduction
in any
Press, form
Inc. reserved.
86
SOOTS ET AL.
regulating the size of the antigraft immune activation in an immunologically uncompromised host. It is, however, possible that all structural components of a given type of allograft are not equally immunogenic, but that the initiation of the antigraft response on several occasions may be primarily due to selected cell component(s) within the graft. The purpose of this communication is to describe a method that we believe makes it possible to numerically evaluate the “immunogenicity” of isolated cells including different isolated cell components of organ allografts. The method is based on the observation that if a recipient rat is primed with donor lymphocytes 72 hr prior to heart transplantation, the allograft survival is strongly reduced but if the time interval is prolonged to 4-10 days, the reduction in survival temporarily disappears before the true second-set rejection becomes apparent. MATERIALS
AND METHODS
Rats
The nuclei for the inbred strains DA (AgB-4), A0 (AgB-2), and HO (AgB-5) were obtained from Professor J. L. Gowans, Dunn School of Pathology, Oxford, England. The Lewis strain (AgB-1) was received from Dr. J. C. Howard, Agricultural Research Council, Babraham, England. The nucleus for the BN (AgB-3) strain was donated by Dr. J. Heron, University of Aarhus, Aarhus, Denmark, and the nucleus for the Wistar-Furth (WF, AgB-2) strains by Professor 0. Sjogren, Department of Tumor Biology, University of Lund, Lund, Sweden. All rats were bred in our own colony. Preparation
of Cells
Rat spleens were made into a single-cell suspension and red cells were lysed with 0.83% ammonium chloride. T- and B-lymphocyte-enriched populations were prepared as follows. The spleen cell suspension was incubated at 37°C for 4 hr on plastic petri dishes. After the incubation the cells were transferred to electrophoresis buffer, fractionated in free flow cell electrophoresis (Model FF4, Desaga, Heidelberg) under conditions described (6), and the fractions were collected. After appropriate “cuts” in the electrophoresis profile three final fractions were recovered. The fast-moving (T-enriched) fraction contained approximately 95% lymphocytes, more than 75% of which expressed the T-specific (7) surface Pta.A.l. antigen and less than 3% of which were surface Ia (3) positive. The slow-moving (B-enriched) fraction contained more than 60% of surface la-carrying lymphocytes, and less than 5% of them expressed the surface Pta.A.l. The middle fraction was intermediary in both respects, but was not used in priming. The electrophoresis input cells were treated prior to priming similarly with the fractionated cells with the electrophoresis buffer. In some experiments the spleen cell donor was injected iv, 6 hr prior to sacrifice, with 14 mg/kg amethopterin (Methotrexate, Lederle, Peare River, New York), or with 160 mg/kg cyclophosphamide (Sendoxan, Pharmacia, Uppsala, Sweden). Donor pretreatment with either drug is known to prolong heart allograft survival by at least 300% (8, 9). Rat red cells were obtained by centrifuging 1:10 diluted blood over Ficoll (Phar-
ASSAY
87
FOR IMMUNOGENICITY
macia Fine Chemicals, Uppsala, Sweden)-Isopaque (Nyegaard & Co., Oslo, Norway) density 1.078 g/ml. The red cells and granulocytes were recovered from the bottom, and granulocytes were removed by treatment with iron powder and magnet. The white cell contamination (mainly granulocytes) was less than 1 per 10,000 red cells. Thymuses were teased apart, the clumps were removed by sedimentation, and the single-cell suspension was washed three times before using for priming. Transplantations
The heart was grafted to the abdominal vessels of the recipient according to the microvascular technique of Ono and Lindsay (10). The transplantation was considered successful if the graft was beating the following day. The graft function was scored by manual palpation twice each day. Approximately 6-10 hr prior to complete cessation of function, the grafts went to fibrillation. The complete asystolic state was used as end point of survival (9). We have previously found that this method is as adequate as electrocardiographic monitoring of the cardiac allograft but far less complicated as it is not necessary to anesthetize the graft recipient. RESULTS I. Impact of Priming
Time and Cell Dose on Heart Allograft
Survival
A0 rats were injected iv with 3 X 106-50 X lo6 DA spleen cells 2 to 200 days prior to transplantation of a DA heart allograft, and the survival time was scored. The results are displayed in Fig. 1. Following the priming, there was a sharp reduction in allograft survival, which was maximal when the priming was done 72 hr (3 days) prior to transplantation. This effect disappeared when the priming-transplantation interval was prolonged to 5-10 days, but became apparent again if the interval was further prolonged up to 14 days or more. There was no apparent effect of the priming cell dose, within the range of 3-50 X IO6 cells.
FIG. 1, Time-sequence analysis of the impact of iv priming with donor strain spleen cells on the survival of DA cardiac allografts in A0 rats. Priming cell dose: closed circles, 5 X 10’; half-closed circles, 10’; open circles 3 X IO6 cells.
88
SOOTS ET AL.
:
5-
0
34-
0 L
2 m%c t-0 % 3% 2
0
6 21 i 104
105 CELL
106
10’
108
DOSE
FIG. 2. Impact of priming cell dose on the survival of DA heart allografts in A0 rats transplanted 72 hr after the priming.
The impact of cell dose was further analyzed by priming at 72 hr prior to transplantation. The results are displayed in Fig. 2. Doses smaller than 5 X lo4 spleen cells had no effect on the survival, whereas doses higher than 3 X lo5 cells reduced the survival maximally, i.e., from 6.2 to 3.0 days. 2. Specificity
of the Response
The immunological specificity of the response was analyzed in A0 recipients primed with DA, AO, WF, Lewis, BN, or HO spleen cells, and transplanted 72 hr later with a DA strain heart. The results are given in Table 1. Priming with as many as 3 X IO’ recipient strain spleen cells had no impact on the response.Priming with spleen cells from other (irrelevant) allogeneic strains, showed a high immu-
TABLE 1 Impact of Histoincompatibility of Priming Cells on the Survival of DA (AgB-4) Heart Allografts in A0 (AgB-2) Recipients Priming cell donor
Graft survival (days)”
Strain
A@
1 x lo6
DA A0 WF Lew BN HO
4 2 2 1 3 5
3.0 f 0.0
3 x lo6 3.0 6.0 7.6 8.0 6.3 7.6
+ 0.0 k 0.0 -+ 0.6 + 0.0 + 0.6 ic 1.1
1 x 10’
3 x 10’
1 x lo*
3.0 * 0.0
3.0 * 0.0 6.3 k 0.6’
3.0 + 0.0
’ Each figure ( f SD) represents mean value of at least three separate determinations. Normal survival time in the DA to A0 combination is 6.2 f 0.6 days (n = 5). b (Lew X DA)F, was used as graft donor.
ASSAY
89
FOR IMMUNOGENICITY
nological specificity for the phenomenon. On no occasion were we able to demonstrate (at the cell dose employed) any reduction in graft survival. In fact, when irrelevant spleen cells were used for priming, the survival was often slightly prolonged. 3. Impact of Priming
with Different
Cell Types
Finally, we analyzed the efficiency of various cell types in reducing heart allograft survival in the primed rejection assay. A0 recipients were primed with various numbers of normal or ultrasonicated DA spleen cells, with DA spleen cells enriched for T or B cells, with spleen cells derived from methotrexate- or cyclophosphamidetreated donors, or with erythrocytes. As illustrated in Table 2, nonfractionated and B-enriched spleen cells were highly efficient while T-enriched spleen cells were inefficient. Pretreatment of the spleen cell donor with methotrexate or cyclophosphamide, impaired the ability of the spleen cells to reduce the survival time. Priming with very high numbers of erythrocytes or ultrasonicated spleen cells had no reducing effect on graft survival; in fact the survival after priming with ultrasonicated spleen cells was slightly prolonged. TABLE
2
Effect of Priming with Different Types of DA Cells on DA Heart Allograft Survival in A0 Recipients Graft survival Cell type used for priming Spleen cells
cell dose)
1 x lo6
3 x lo6
I x 10’
I x IO8
3.0 + 0.0
3.0 f 0.0 (3)
3.0 + 0.0 (3)
3.0 f 0.0 (1)
(6) Ultrasonicated Electrophoresis Input
(days)” (priming
1 x IO9
10.0 f 0.0 separated 4.0 2k O.Ob
(2) T enriched B enriched Thymus cells Spleen cells after treatment of the cell donor with Methotrexate
5.6 ? 0.6 (3) 3.3 f 0.6 (3) 5.0 f 0.0
5.5 f 0.1
(2) Cyclophosphamide
5.5 + 0.5
(2) Erythrocytes
4.3 + 0.6 (3) 5.0 f 0.0 (3) 6.0 (1)
3.3 + 0.6
(2) 3.0 k 0.0
(2) 6.0 (1)
6.0 f 0.0 (3)
a *SD; number of determinations is given in parentheses. The survival of DA heart allograft in nontreated A0 recipient is 6.0 f 0.6 days (n = 5). ’ Both input and fractionated populations have been presented to electrophoresis medium of low ionic strength (6) prior to use in priming.
90
SOOTS ET AL.
DISCUSSION Transplants which survive for a particularly long time without intentional immunosuppression or other treatment of the recipient, are frequently called “less immunogenic.” Such prolongation of survival may, naturally, be facilitated also by other means, e.g., genetic nature of the recipient, condition of the graft, etc., and prolonged survival does not necessarily indicate low immunogenicity. Moreover, estimation of the immunogenicity of a transplant by graft survival is only applicable to such allografts (e.g., kidney, heart, thyroid, or bone marrow), where the survival of the transplanted tissue may be adequately monitored on the basis of the graft function. The survival assay is not usually applicable to single cells which, though able to elicit an immune response, are operationally afunctional unless they are components of a more complex organic system (e.g., kidney tubular cells, heart endothelial cells, thyroid follicular cells, etc.). We believe that our assay, provisionally called “the primed rejection assay” (PRA), is useful for the estimation of the relative immunogenicity of various types of allografts and isolated single cells, at least with regard to those antigens which are common with the priming cell(s) and the detection system. We consider it likely that the rejection is initiated by the priming, and completed upon the application of the test organ. The reduction of the survival is maximal at 72 hr after the priming, whereas if the interval is prolonged the effect is lost, indicating that reduced survival is not due to a true second-set response. Whether the loss of effect is due to redistribution of the primed cells (1 1), induction of an antiidiotypic response (12) or other similar mechanisms, cannot be determined on the basis of these data. The PRA assay is immunologically specific, since only relevant but not irrelevant allogeneic spleen cells were able to reduce transplant survival. As no unequivocally accepted test system for the quantitation of the immunogenicity of allografts and their components exists, our claim on the applicability of the PRA assay for this purpose is only indirect and circumvential. For priming we used cell populations which have previously been reported to be less immunogenic (for the primary response), and tested their ability to reduce graft survival in the PRA assay. Such populations include erythrocytes (5), sonicated spleen cells ( 13), and lymphocytes derived from animals pretreated with certain cytotoxic drugs, i.e., cyclophosphamide and methotrexate (8, 9). Although ineffective at the ordinary dose range, some of these cell populations reduced graft survival when injected at doses considerably higher than the minimum number of intact spleen cells required for maximal reduction. For example, although a maximal reduction was obtained with as few as 5 x lo5 intact spleen cells, lo9 erythrocytes and lo8 ultrasonicated spleen cells were unable to reduce survival, and 3 X lo6 spleen cells derived from methotrexate or cyclophosphamide-treated animals were equally inefficient. Compared to previous assays utilizing second-set graft rejection to demonstrate the immunogenicity of the priming procedure (14), the PRA assay operating entirely within the first set probably bypasses certain factors in the development of memory, which potentially affect on the detection of the priming. Such factors include interference with alloantibodies which probably render the second-set rejection operationally different from the first set, necessity of determining proper
ASSAY FOR IMMUNOGENICITY
91
time point to demonstrate the second-set response after priming with vastly differing doses of antigen, and possibly antigenic constitution of the recipient. In the current PRA assay, cardiac allografts are transplanted via primary vascular anastomosis to the recipient vessels. The method is technically demanding and laborious. It is, however, possible that simpler assays such as primarily nonvascularized heart allografts transplanted subcutaneously to the recipient ear-leaf, may be used with similar results. ACKNOWLEDGMENTS The authors are grateful to Dr. J. Howard, Agricultural Research Council, Babraham, England, for the Pta.A. 1, serum, and Ms. Leena Saraste for secretarial help. The study was financed by grants from Latike Pharmaceuticals, the Sigrid Juselius Foundation, the Canadian Medical Research Council, and the National Institutes of Health.
REFERENCES I, Goodman, J. W., In “Basic and Clinical Immunology” (H. H. Fudenberg, D. P. Stites, and J. L. Caldwell, Eds.), p. 39, Los Altos, Calif., 1978. 2. Humphreys, R. E., McCune, J. M., Chess, L., Herrman, H. C., Malenka, D. J., Mann, D. L., Parham, P., Schlossman, S. F., and Strominger, J. L., J. Exp. Med. 144, 98, 1976. 3. Rask, L., Lindblom, J. B., and Peterson, P. A., Eur. J. Immunol. 6, 93, 1976. 4. Hess, M., Transplant. Rev. 30, 40, 1976. 5. Bach, F. H., and van Rood, J. J., New Engl. J. Med. 295, 806; 872; 927, 1976. 6. Wigzell, H., and Andersson, B., Annu. Rev. Microbial. 25, 291, 1971. 7. Howard, J. C., and Scott, D. W., Immunology 27, 903, 1974. 8. Guttmann, R. D., and Lindquist, R. R., Transplantation 8, 490, 1969. 9. Soots, A., and Hiiyry, P., Transplantation 25, 259, 1978. 10. Ono, K., and Lindsey, E. S., Thoruc. Cardiovasc. Surgery 57, 225, 1969. 11. Hay, J. B., Cahill, R. N. P., and Trnka, Z., Cell. Immunol. 10, 145, 1974. 12. Aguet, M., Andersson, L. C., Andersson, R., Wight, E., Binz, H., and Wigzell, H., J. Exp. Med. 147, 50, 1978. 13. Hiiyry, P., Immunogenetics 3, 417, 1976. 14. Billingham, R. E., Brent, L., and Mitchison, N. A., Brit. J. Exp. Pathol. 38, 467, 1957.
IMMUNOLOGY
57, 85-91 (1981)
lmmunogenicity
of Allograft
Components
I. Assay for lmmunogenicity ANUSOOTS,
ELAINE~ARTHENAIS,'
ARTONEMLANDER,
ANDPEKKA
HAYRY
Transplantation Laboratory. Fourth Department of Helsinki, University of Helsinki, Helsinki, Finland Received March
18, 1980; accepted April
16. 1980
We describe an assay for in vivo quantitation of the “immunogenicity” of isolated cell populations. The assay is based on the observation that if an AgB-incompatible recipient rat is primed with donor strain spleen cells 72 hr prior to transplantation, heart allograft survival is reduced from 6.2 to 3.0 days. The effect is independent of the priming cell dose at levels above 3 X 10’ cells, whereas doseslower than 10’ spleen cells are unable to reduce the survival. The effect is suboptimal if the priming-transplantation interval is less than 3 days, or is prolonged to 4-10 days. The effect is immunologically specific: priming with irrelevant AgBincompatible spleen celis fails to reduce the survival. Priming with cell populations previously reported “less immunogenic,” such as ultrasonicated spleen cells, erythrocytes, spleen T cells, or spleen cells deriving from methotrexate or cyclophosphamide-treated rats, fails to reduce the survival, or reduces it only when given in IOO-fold higher numbers than the minimal dose of intact spleen cells giving maximal reduction.
INTRODUCTION “Immunogenicity” is a property of a substance to induce an immune response. Immunogenicity is usually detected by reintroducing the same antigen into the recipient, and if a secondary response is obtained, the material is said to be immunogenic. With simple protein antigens, immunogenicity depends of the “foreigness” of the material, molecular size, chemical complexity, the route of administration, and genetic constitution of the recipient (1). Histocompatibility antigens are glycoproteins regulated by several independently segregating genetic systems (2-4). In most allografts, the antigens of the major histocompatibility complex (MHC) are probably crucial in both triggering of the antiallograft response, and as targets for the host immune attack (5). “Transplant rejection” describes a complex biological phenomenon of both cellular and humoral immunity, which may be divided into the following largely overlapping components: initiation and generation of the specific immune response, amplification of the response by nonspecific inflammatory mechanisms, and destruction of the graft. The first of these events is crucial in determining the fate of the transplant, and the immunogenicity of the transplant is probably the most important single factor ’ Canadian Medical Research Council Fellow. 85 0008-8749/g 1/OlOOSS-07$02.00/O Copyright All
rights
0
1981
by Academic
of reproduction
in any
Press, form
Inc. reserved.
86
SOOTS ET AL.
regulating the size of the antigraft immune activation in an immunologically uncompromised host. It is, however, possible that all structural components of a given type of allograft are not equally immunogenic, but that the initiation of the antigraft response on several occasions may be primarily due to selected cell component(s) within the graft. The purpose of this communication is to describe a method that we believe makes it possible to numerically evaluate the “immunogenicity” of isolated cells including different isolated cell components of organ allografts. The method is based on the observation that if a recipient rat is primed with donor lymphocytes 72 hr prior to heart transplantation, the allograft survival is strongly reduced but if the time interval is prolonged to 4-10 days, the reduction in survival temporarily disappears before the true second-set rejection becomes apparent. MATERIALS
AND METHODS
Rats
The nuclei for the inbred strains DA (AgB-4), A0 (AgB-2), and HO (AgB-5) were obtained from Professor J. L. Gowans, Dunn School of Pathology, Oxford, England. The Lewis strain (AgB-1) was received from Dr. J. C. Howard, Agricultural Research Council, Babraham, England. The nucleus for the BN (AgB-3) strain was donated by Dr. J. Heron, University of Aarhus, Aarhus, Denmark, and the nucleus for the Wistar-Furth (WF, AgB-2) strains by Professor 0. Sjogren, Department of Tumor Biology, University of Lund, Lund, Sweden. All rats were bred in our own colony. Preparation
of Cells
Rat spleens were made into a single-cell suspension and red cells were lysed with 0.83% ammonium chloride. T- and B-lymphocyte-enriched populations were prepared as follows. The spleen cell suspension was incubated at 37°C for 4 hr on plastic petri dishes. After the incubation the cells were transferred to electrophoresis buffer, fractionated in free flow cell electrophoresis (Model FF4, Desaga, Heidelberg) under conditions described (6), and the fractions were collected. After appropriate “cuts” in the electrophoresis profile three final fractions were recovered. The fast-moving (T-enriched) fraction contained approximately 95% lymphocytes, more than 75% of which expressed the T-specific (7) surface Pta.A.l. antigen and less than 3% of which were surface Ia (3) positive. The slow-moving (B-enriched) fraction contained more than 60% of surface la-carrying lymphocytes, and less than 5% of them expressed the surface Pta.A.l. The middle fraction was intermediary in both respects, but was not used in priming. The electrophoresis input cells were treated prior to priming similarly with the fractionated cells with the electrophoresis buffer. In some experiments the spleen cell donor was injected iv, 6 hr prior to sacrifice, with 14 mg/kg amethopterin (Methotrexate, Lederle, Peare River, New York), or with 160 mg/kg cyclophosphamide (Sendoxan, Pharmacia, Uppsala, Sweden). Donor pretreatment with either drug is known to prolong heart allograft survival by at least 300% (8, 9). Rat red cells were obtained by centrifuging 1:10 diluted blood over Ficoll (Phar-
ASSAY
87
FOR IMMUNOGENICITY
macia Fine Chemicals, Uppsala, Sweden)-Isopaque (Nyegaard & Co., Oslo, Norway) density 1.078 g/ml. The red cells and granulocytes were recovered from the bottom, and granulocytes were removed by treatment with iron powder and magnet. The white cell contamination (mainly granulocytes) was less than 1 per 10,000 red cells. Thymuses were teased apart, the clumps were removed by sedimentation, and the single-cell suspension was washed three times before using for priming. Transplantations
The heart was grafted to the abdominal vessels of the recipient according to the microvascular technique of Ono and Lindsay (10). The transplantation was considered successful if the graft was beating the following day. The graft function was scored by manual palpation twice each day. Approximately 6-10 hr prior to complete cessation of function, the grafts went to fibrillation. The complete asystolic state was used as end point of survival (9). We have previously found that this method is as adequate as electrocardiographic monitoring of the cardiac allograft but far less complicated as it is not necessary to anesthetize the graft recipient. RESULTS I. Impact of Priming
Time and Cell Dose on Heart Allograft
Survival
A0 rats were injected iv with 3 X 106-50 X lo6 DA spleen cells 2 to 200 days prior to transplantation of a DA heart allograft, and the survival time was scored. The results are displayed in Fig. 1. Following the priming, there was a sharp reduction in allograft survival, which was maximal when the priming was done 72 hr (3 days) prior to transplantation. This effect disappeared when the priming-transplantation interval was prolonged to 5-10 days, but became apparent again if the interval was further prolonged up to 14 days or more. There was no apparent effect of the priming cell dose, within the range of 3-50 X IO6 cells.
FIG. 1, Time-sequence analysis of the impact of iv priming with donor strain spleen cells on the survival of DA cardiac allografts in A0 rats. Priming cell dose: closed circles, 5 X 10’; half-closed circles, 10’; open circles 3 X IO6 cells.
88
SOOTS ET AL.
:
5-
0
34-
0 L
2 m%c t-0 % 3% 2
0
6 21 i 104
105 CELL
106
10’
108
DOSE
FIG. 2. Impact of priming cell dose on the survival of DA heart allografts in A0 rats transplanted 72 hr after the priming.
The impact of cell dose was further analyzed by priming at 72 hr prior to transplantation. The results are displayed in Fig. 2. Doses smaller than 5 X lo4 spleen cells had no effect on the survival, whereas doses higher than 3 X lo5 cells reduced the survival maximally, i.e., from 6.2 to 3.0 days. 2. Specificity
of the Response
The immunological specificity of the response was analyzed in A0 recipients primed with DA, AO, WF, Lewis, BN, or HO spleen cells, and transplanted 72 hr later with a DA strain heart. The results are given in Table 1. Priming with as many as 3 X IO’ recipient strain spleen cells had no impact on the response.Priming with spleen cells from other (irrelevant) allogeneic strains, showed a high immu-
TABLE 1 Impact of Histoincompatibility of Priming Cells on the Survival of DA (AgB-4) Heart Allografts in A0 (AgB-2) Recipients Priming cell donor
Graft survival (days)”
Strain
A@
1 x lo6
DA A0 WF Lew BN HO
4 2 2 1 3 5
3.0 f 0.0
3 x lo6 3.0 6.0 7.6 8.0 6.3 7.6
+ 0.0 k 0.0 -+ 0.6 + 0.0 + 0.6 ic 1.1
1 x 10’
3 x 10’
1 x lo*
3.0 * 0.0
3.0 * 0.0 6.3 k 0.6’
3.0 + 0.0
’ Each figure ( f SD) represents mean value of at least three separate determinations. Normal survival time in the DA to A0 combination is 6.2 f 0.6 days (n = 5). b (Lew X DA)F, was used as graft donor.
ASSAY
89
FOR IMMUNOGENICITY
nological specificity for the phenomenon. On no occasion were we able to demonstrate (at the cell dose employed) any reduction in graft survival. In fact, when irrelevant spleen cells were used for priming, the survival was often slightly prolonged. 3. Impact of Priming
with Different
Cell Types
Finally, we analyzed the efficiency of various cell types in reducing heart allograft survival in the primed rejection assay. A0 recipients were primed with various numbers of normal or ultrasonicated DA spleen cells, with DA spleen cells enriched for T or B cells, with spleen cells derived from methotrexate- or cyclophosphamidetreated donors, or with erythrocytes. As illustrated in Table 2, nonfractionated and B-enriched spleen cells were highly efficient while T-enriched spleen cells were inefficient. Pretreatment of the spleen cell donor with methotrexate or cyclophosphamide, impaired the ability of the spleen cells to reduce the survival time. Priming with very high numbers of erythrocytes or ultrasonicated spleen cells had no reducing effect on graft survival; in fact the survival after priming with ultrasonicated spleen cells was slightly prolonged. TABLE
2
Effect of Priming with Different Types of DA Cells on DA Heart Allograft Survival in A0 Recipients Graft survival Cell type used for priming Spleen cells
cell dose)
1 x lo6
3 x lo6
I x 10’
I x IO8
3.0 + 0.0
3.0 f 0.0 (3)
3.0 + 0.0 (3)
3.0 f 0.0 (1)
(6) Ultrasonicated Electrophoresis Input
(days)” (priming
1 x IO9
10.0 f 0.0 separated 4.0 2k O.Ob
(2) T enriched B enriched Thymus cells Spleen cells after treatment of the cell donor with Methotrexate
5.6 ? 0.6 (3) 3.3 f 0.6 (3) 5.0 f 0.0
5.5 f 0.1
(2) Cyclophosphamide
5.5 + 0.5
(2) Erythrocytes
4.3 + 0.6 (3) 5.0 f 0.0 (3) 6.0 (1)
3.3 + 0.6
(2) 3.0 k 0.0
(2) 6.0 (1)
6.0 f 0.0 (3)
a *SD; number of determinations is given in parentheses. The survival of DA heart allograft in nontreated A0 recipient is 6.0 f 0.6 days (n = 5). ’ Both input and fractionated populations have been presented to electrophoresis medium of low ionic strength (6) prior to use in priming.
90
SOOTS ET AL.
DISCUSSION Transplants which survive for a particularly long time without intentional immunosuppression or other treatment of the recipient, are frequently called “less immunogenic.” Such prolongation of survival may, naturally, be facilitated also by other means, e.g., genetic nature of the recipient, condition of the graft, etc., and prolonged survival does not necessarily indicate low immunogenicity. Moreover, estimation of the immunogenicity of a transplant by graft survival is only applicable to such allografts (e.g., kidney, heart, thyroid, or bone marrow), where the survival of the transplanted tissue may be adequately monitored on the basis of the graft function. The survival assay is not usually applicable to single cells which, though able to elicit an immune response, are operationally afunctional unless they are components of a more complex organic system (e.g., kidney tubular cells, heart endothelial cells, thyroid follicular cells, etc.). We believe that our assay, provisionally called “the primed rejection assay” (PRA), is useful for the estimation of the relative immunogenicity of various types of allografts and isolated single cells, at least with regard to those antigens which are common with the priming cell(s) and the detection system. We consider it likely that the rejection is initiated by the priming, and completed upon the application of the test organ. The reduction of the survival is maximal at 72 hr after the priming, whereas if the interval is prolonged the effect is lost, indicating that reduced survival is not due to a true second-set response. Whether the loss of effect is due to redistribution of the primed cells (1 1), induction of an antiidiotypic response (12) or other similar mechanisms, cannot be determined on the basis of these data. The PRA assay is immunologically specific, since only relevant but not irrelevant allogeneic spleen cells were able to reduce transplant survival. As no unequivocally accepted test system for the quantitation of the immunogenicity of allografts and their components exists, our claim on the applicability of the PRA assay for this purpose is only indirect and circumvential. For priming we used cell populations which have previously been reported to be less immunogenic (for the primary response), and tested their ability to reduce graft survival in the PRA assay. Such populations include erythrocytes (5), sonicated spleen cells ( 13), and lymphocytes derived from animals pretreated with certain cytotoxic drugs, i.e., cyclophosphamide and methotrexate (8, 9). Although ineffective at the ordinary dose range, some of these cell populations reduced graft survival when injected at doses considerably higher than the minimum number of intact spleen cells required for maximal reduction. For example, although a maximal reduction was obtained with as few as 5 x lo5 intact spleen cells, lo9 erythrocytes and lo8 ultrasonicated spleen cells were unable to reduce survival, and 3 X lo6 spleen cells derived from methotrexate or cyclophosphamide-treated animals were equally inefficient. Compared to previous assays utilizing second-set graft rejection to demonstrate the immunogenicity of the priming procedure (14), the PRA assay operating entirely within the first set probably bypasses certain factors in the development of memory, which potentially affect on the detection of the priming. Such factors include interference with alloantibodies which probably render the second-set rejection operationally different from the first set, necessity of determining proper
ASSAY FOR IMMUNOGENICITY
91
time point to demonstrate the second-set response after priming with vastly differing doses of antigen, and possibly antigenic constitution of the recipient. In the current PRA assay, cardiac allografts are transplanted via primary vascular anastomosis to the recipient vessels. The method is technically demanding and laborious. It is, however, possible that simpler assays such as primarily nonvascularized heart allografts transplanted subcutaneously to the recipient ear-leaf, may be used with similar results. ACKNOWLEDGMENTS The authors are grateful to Dr. J. Howard, Agricultural Research Council, Babraham, England, for the Pta.A. 1, serum, and Ms. Leena Saraste for secretarial help. The study was financed by grants from Latike Pharmaceuticals, the Sigrid Juselius Foundation, the Canadian Medical Research Council, and the National Institutes of Health.
REFERENCES I, Goodman, J. W., In “Basic and Clinical Immunology” (H. H. Fudenberg, D. P. Stites, and J. L. Caldwell, Eds.), p. 39, Los Altos, Calif., 1978. 2. Humphreys, R. E., McCune, J. M., Chess, L., Herrman, H. C., Malenka, D. J., Mann, D. L., Parham, P., Schlossman, S. F., and Strominger, J. L., J. Exp. Med. 144, 98, 1976. 3. Rask, L., Lindblom, J. B., and Peterson, P. A., Eur. J. Immunol. 6, 93, 1976. 4. Hess, M., Transplant. Rev. 30, 40, 1976. 5. Bach, F. H., and van Rood, J. J., New Engl. J. Med. 295, 806; 872; 927, 1976. 6. Wigzell, H., and Andersson, B., Annu. Rev. Microbial. 25, 291, 1971. 7. Howard, J. C., and Scott, D. W., Immunology 27, 903, 1974. 8. Guttmann, R. D., and Lindquist, R. R., Transplantation 8, 490, 1969. 9. Soots, A., and Hiiyry, P., Transplantation 25, 259, 1978. 10. Ono, K., and Lindsey, E. S., Thoruc. Cardiovasc. Surgery 57, 225, 1969. 11. Hay, J. B., Cahill, R. N. P., and Trnka, Z., Cell. Immunol. 10, 145, 1974. 12. Aguet, M., Andersson, L. C., Andersson, R., Wight, E., Binz, H., and Wigzell, H., J. Exp. Med. 147, 50, 1978. 13. Hiiyry, P., Immunogenetics 3, 417, 1976. 14. Billingham, R. E., Brent, L., and Mitchison, N. A., Brit. J. Exp. Pathol. 38, 467, 1957.