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ENHANCEMENT OF HUMAN KIDNEY ALLOGRAFTS BY COLD B-LYMPHOCYTE CYTOTOXINS
1228 ENHANCEMENT OF HUMAN KIDNEY ALLOGRAFTS BY COLD B-LYMPHOCYTE CYTOTOXINS YUICHI IWAKI MIN SIK PARK
PAUL I. TERASAKI RONALD BILLING
Department of Surgery, U.C.L.A. School of Medicine, University of California, Los Angeles, California 90024, U.S.A.
The sera of 233 kidney transplant patients before transplantation were tested by cytotoxicity against a panel of B and T lymphocytes at 5°C and 37°C. The results divided the patients into four groups: those whose sera reacted with B lymphocytes at 5°C only; those reacting with B lymphocytes at 5°C and 37°C; those reacting with T lymphocytes at 37°C; and those with no antibodies. The patients with pre-transplant antibodies reactive with B lymphocytes at 5°C had a significantly higher kidney-transplant survival rate at 6 months (70%) and 1 year (65%) than patients who had no antibodies (47% and 46%, respectively). Patients with antibodies reactive at 37°C had a 6-month survival-rate of 38% when reactive against B cells and 43% when reactive against T lymphocytes. The cold cytotoxins were IgM.
Summary
Introduction ALTHOUGH allograft enhancement in animals is well established,1 its role in human transplantation is unclear. Leucocytes from the kidney donor were injected into 4 graft recipients in 1973 by Newton and Anderson2 but none showed evidence of immunisation. If we use "enhancement" in its broadest sense-i.e., induction of lower responsiveness by pretreatment with allogeneic cells-blood-transfusions may be thought to induce enhancement. Without blood-transfusions and with immunosuppressive treatment, approximately 30% of kidney allografts survive a year after transplantation,3 but this can be improved to 50-70% by pre-transplant transfusion of allogeneic blood. If this is considered to be enhancement, it has been achieved since human kidney transplants began. In the early days of the operation most patients were given numerous transfusions before transplantation,4 and the survival rates of recent years may be lower because transfusions have been less fre-
grafts
at
twenty-seven
transplant
centres
between
ranged from 3 months to 2 years. Methods
Lymphocytotoxicity B and T lymphocytes were isolated and tested by the lymphocyte microcytotoxicity test as previously described.6 A serum was tested against B and T lymphocytes from 17-30 normal individuals. Cells from
one
person
were
as B-warm-reactive. Sera classified as T-warm-reactive were those which killed T lymphocytes from >15% of the panel at 37°C whether or not they had cold or warm reactivity or killed T lymphocytes at 5°C. Fig. 1 shows examples of these three patterns of reaction. It should be noted that there are fifteen different patterns of reaction possible by testing sera against B and T cells at 5°C and 37°C. However, the B-cold, B-warm, and T-warm reactive patterns and the absence of lymphocyte reactivity were the most common.
Serum Fractionation 2 ml of B-cold-reactive serum was fractionated by gel filtration on ’Biogel 5M’. 2 ml fractions were eluted in 0-15 mol/1 sodium chloride, 10 mmol/l phosphate buffer (pH 7.2). The fractions under peaks with optical density 280 nm were pooled, concentrated to 2 ml, and tested for IgM or IgG by Ouchterlony immunodiffusion using goat antibodies against human IgG and IgM. There was a clear separation of the IgG and IgM fractions. Each fraction was tested for cold anti-Bcell activity against at least three different B-lymphocyte preparations at 5 IC and 37°C.
Results Of the 233 pretransplant sera tested, 139 (60%) did not react against T or B lymphocytes at 3°C or 37°C. 94 sera had anti-lymphocyte activity, 40 with the
If the state of enhancement could be detected by invitro tests, patients who were properly "prepared" by transfusions and pregnancies could be identified. We describe here a test which may detect enhancing antibodies.
Patients
patients
were
233
recipients of cadaver kidney allo-
Harland, P. S. E. G. in The Child in the African Environment (edited by R. Owor, V. L. Ongom, and B. G. Kirya); p. 349. Kampala, 1975. 22. Kirchner, H., Rühl, H. Exp. Cell. Res. 1970, 61, 229. 23. Rühl, H., Kirchner, H., Bochert, G. Proc. Soc. exp. Biol. Med. 1971, 137, 21.
1089. 24. Williams, R. O., Loeb, L. A. J. Cell Biol. 1973, 58, 594. 25. Chesters, J. K. Biochem. J. 1975, 150, 211 26. Phillips, J. L., Azari, P. Cell. Immun. 1974, 10, 31. 27. Chandra, R. K. J. Pediat. 1972, 81, 1194. 28. Harland, P. S. E. G. Lancet, 1965, ii, 719.
tested separ-
ately against: B lymphocytes at 5°C; B lymphocytes at 37°C; T lymphocytes at 5°C; and T lymphocytes at 37°C. Incubation with complement in all cultures was at 25 °C. Incubationtimes were 1 h with serum and 2 h with complement. Classification of Sera When a serum killed B lymphocytes from > 15 % of the panel at 5°C and all other reactions were negative, it was classified as B-cold-reactive. If it killed B lymphocytes from > 15 % of the panel at 37°C (whether or not it had B-cold reactivity) and killed T lymphocytes from <15% of the panel, it was classified
quent.5
The
September,
1975, and November, 1977. Serum samples were all taken before transplantation. 183 patients had first allografts, 38 had second, and 12 had third allografts. The follow-up period
Fig.
1-Patterns of
serum
reactivity.
1229 anbibodies. These anti-B lymphocyte antibodies are not harmful-as others have claimed anti-B-lymphocyte antibodies to be9,10—but are beneficial when they react only at 5 oc and not at both 5 °C and 37°C. B-cold lymphocytotoxins are not directed to any of the four HLA loci. This does not argue against the conclusion that these are the antibodies with enhancing activity; Fabre and Morrist6 and Fabre and Batchelor17 have shown in rats that enhancing antibodies are not as
Fig. 2-Actuarial graft survival for cadaver donor transplants, according to type of pre-transplant cytotoxins.
B-cold pattern (17% of total), 26 with the B-warm pat(11%), and 28with the T-warm pattern (12%). The B-cold group contained twice as many males as females while in the B-warm and T-warm groups females predominated, but this difference was not significant. Significantly more kidney transplants survived in patients with the B-cold pattern than in those without antibodies (fig. 2). At 3 months 80% of grafts survived in the B-cold group, compared with 56% in patients without antibodies (P<0002); at 6 months, the survival rates were 70% and 47%, respectively (P<0-01), and at In comone year 65% and 46%, respectively (P<0.05). parison, the patients with the B-warm or T-warm patterns had poor survival rates (38% and 43%, respectively, at 6 months). When B-cold-reactive serum was fractionated, cold activity against B cells was detected only in the IgM fraction.
tern
Discussion The presence of lymphocytotoxins in graft recipients before transplantation and its association with transplant failure was previously taken to indicate that the patient was presensitised.8 More recent studies have emphasised the importance of distinguishing lymphocytotoxins acting solely on B lymphocytes, which have no deleterious effect on transplants even when a positive crossmatch is obtained with the B cells of the donor.9 10 Such antibodies, for the most part, detect HLA-D antigens analogous to Ia antigens in mice. They could be enhancing antibodies, as suggested by experiments with rat heart" and rat kidney grafts. 12 Lymphocytotoxins not directed at HLA specificities and more reactive in the cold" also appear in the sera of potential transplant recipients. Klouda and Jeannet noted that 14 of 15 grafts in patients with pre-transplant cytotoxins reactive at 13°C against unfractionated lymphocytes survived at 1 year, compared with 3 out of 10 who had cytotoxins reactive at 25OC,14 In another study grafts in all 4 patients with cold cytotoxins (13°C) before transplantation survived for 1 year, while 6 out of 10 grafts survived in patients with antibodies reactive at37°C. We have shown that cold cytotoxins to B lymphocytes distinguish patients in whom the rate of transplant survival is higher than in patients with no pre-transplant
specific as previously supposed. Experimental evidence suggests that after the induction of active enhancement by injection of allogeneic cells, the timing of the graft is critical ; 18 the IgM response peaks 1 week after injection, when enhancement of rat kidney allografts is most effective," and these IgM antibodies disappear from the circulation and are not found in rats with long-surviving allografts.t9 Our results-although the number of B-cold-reactive patients was limited and no crossmatching with the kidney donor was done-are consistent with these findings: the presence of cold-reactive antibodies to B lymphocytes before transplantation was associated with a higher survival rate, and cold antibodies were often absent after a successful graft. It is a puzzle why human kidney transplants function in the face of the worst possible types of HLA incompatibility : of 495 kidney transplants done with four mismatched HLA antigens, 42% survived for one year.20 Enhancing antibodies in the recipient before transplantation may account for the high graft-survival rate in these patients, and in patients with intermediate grades of incompatibility. From a practical point of view, if the B-cold lympho-
cytotoxins
are
enhancing antibodies, they
can serve to
monitor the effect of transfusions before transplantation, and help to calculate the proper number of transfusions, the dosage, and the most effective time for
transplantation. We thank Mark Hermes, Roswitha Heintz, and Emi Yoshihara for technical assistance. This study was supported by grant AM 02375 from the National Institute of Arthritis and Metabolic Diseases and contract N01 CM 02092 from the National Cancer Institute.
Requests for reprints
should be addressed
to
Y. I., 1000 Veteran
Avenue, Los Angeles, California 90024, U.S.A. REFERENCES
1. Batchelor, J. R., Welsh, K. I. Br. med. Bull. 1976, 32, 113. 2. Newton, W. T., Anderson, C. B. Surgery, 1973, 74, 430. 3. Opelz, G., Terasaki, P. I. Transplant. Proc. 1977, 9, 121. 4. Dossetor, J. B., MacKinron, K. J., Gault, M. H., MacLean, L. D. Transplantation, 1967, 5, 844. 5. Terasaki, P. I., Opelz, G., Mickey, M. R. Transplant. Proc. 1976, 8, 139. 6. Terasaki, P. I., Bernoco, D., Park, M. S., Ozturk, G., Iwaki, Y. Am. J. clin. Path. 1978, 69, 103. 7. Hannon, R., Haire, M., Wisdon, G. B., Neil, D. W. J. immun. Methods,
1975, 8, 29. Terasaki, P. I., Kreisler, M. Mickey, M. R. Postgrad. med. J 1971, 47, 89. Ettenger, R. B., Terasaki, P. I., Opelz, G., Malekzadeh, M., Pennisi, A., Uittenbogaart, C., Fine, R. Lancet, 1976, ii, 56. 10. Morris, P. J., Ting, A., Oliver, D. O., Bishop, M., Williams, K., Dunhill, M. S. ibid. 1977, i, 1288. 11. Davies, D. A. L., Alkins, B. J. Nature, 1974, 247, 294 12. Soulillou, J. P., Carpenter, C. B., d’Apice, A. J. F., Strom, T. B. J. exp. Med. 1976, 143, 405. 13. Park, M. S., Terasaki, P. I., Byrns, G., Ayoub, G. Transplant. Proc. 1977, 9, 1701. 14. Klouda, P. T., Jeannet, M. Lancet, 1976, i, 876. 15. Roy, R., Robert, M. Union méd. Canada, 1977, 106, 297. 16. Fabre, J. W., Morris, P. J. Transplantation, 1974, 18, 436. 17. Fabre, J. W., Batchelor, J. R. ibid. 1975, 20, 269. 18. Carpenter, C. B., d’Apice, A. J., Abbas, A. K. Adv. Immun. 1976, 22, 1. 19. Desai, S B. R., Ruszkiewicz, M. Transplantation, 1977, 23, 230. 20. Opelz, G., Mickey, M R., Terasaki, P I ibid. 1977, 23, 490. 8. 9.
PAUL I. TERASAKI RONALD BILLING
Department of Surgery, U.C.L.A. School of Medicine, University of California, Los Angeles, California 90024, U.S.A.
The sera of 233 kidney transplant patients before transplantation were tested by cytotoxicity against a panel of B and T lymphocytes at 5°C and 37°C. The results divided the patients into four groups: those whose sera reacted with B lymphocytes at 5°C only; those reacting with B lymphocytes at 5°C and 37°C; those reacting with T lymphocytes at 37°C; and those with no antibodies. The patients with pre-transplant antibodies reactive with B lymphocytes at 5°C had a significantly higher kidney-transplant survival rate at 6 months (70%) and 1 year (65%) than patients who had no antibodies (47% and 46%, respectively). Patients with antibodies reactive at 37°C had a 6-month survival-rate of 38% when reactive against B cells and 43% when reactive against T lymphocytes. The cold cytotoxins were IgM.
Summary
Introduction ALTHOUGH allograft enhancement in animals is well established,1 its role in human transplantation is unclear. Leucocytes from the kidney donor were injected into 4 graft recipients in 1973 by Newton and Anderson2 but none showed evidence of immunisation. If we use "enhancement" in its broadest sense-i.e., induction of lower responsiveness by pretreatment with allogeneic cells-blood-transfusions may be thought to induce enhancement. Without blood-transfusions and with immunosuppressive treatment, approximately 30% of kidney allografts survive a year after transplantation,3 but this can be improved to 50-70% by pre-transplant transfusion of allogeneic blood. If this is considered to be enhancement, it has been achieved since human kidney transplants began. In the early days of the operation most patients were given numerous transfusions before transplantation,4 and the survival rates of recent years may be lower because transfusions have been less fre-
grafts
at
twenty-seven
transplant
centres
between
ranged from 3 months to 2 years. Methods
Lymphocytotoxicity B and T lymphocytes were isolated and tested by the lymphocyte microcytotoxicity test as previously described.6 A serum was tested against B and T lymphocytes from 17-30 normal individuals. Cells from
one
person
were
as B-warm-reactive. Sera classified as T-warm-reactive were those which killed T lymphocytes from >15% of the panel at 37°C whether or not they had cold or warm reactivity or killed T lymphocytes at 5°C. Fig. 1 shows examples of these three patterns of reaction. It should be noted that there are fifteen different patterns of reaction possible by testing sera against B and T cells at 5°C and 37°C. However, the B-cold, B-warm, and T-warm reactive patterns and the absence of lymphocyte reactivity were the most common.
Serum Fractionation 2 ml of B-cold-reactive serum was fractionated by gel filtration on ’Biogel 5M’. 2 ml fractions were eluted in 0-15 mol/1 sodium chloride, 10 mmol/l phosphate buffer (pH 7.2). The fractions under peaks with optical density 280 nm were pooled, concentrated to 2 ml, and tested for IgM or IgG by Ouchterlony immunodiffusion using goat antibodies against human IgG and IgM. There was a clear separation of the IgG and IgM fractions. Each fraction was tested for cold anti-Bcell activity against at least three different B-lymphocyte preparations at 5 IC and 37°C.
Results Of the 233 pretransplant sera tested, 139 (60%) did not react against T or B lymphocytes at 3°C or 37°C. 94 sera had anti-lymphocyte activity, 40 with the
If the state of enhancement could be detected by invitro tests, patients who were properly "prepared" by transfusions and pregnancies could be identified. We describe here a test which may detect enhancing antibodies.
Patients
patients
were
233
recipients of cadaver kidney allo-
Harland, P. S. E. G. in The Child in the African Environment (edited by R. Owor, V. L. Ongom, and B. G. Kirya); p. 349. Kampala, 1975. 22. Kirchner, H., Rühl, H. Exp. Cell. Res. 1970, 61, 229. 23. Rühl, H., Kirchner, H., Bochert, G. Proc. Soc. exp. Biol. Med. 1971, 137, 21.
1089. 24. Williams, R. O., Loeb, L. A. J. Cell Biol. 1973, 58, 594. 25. Chesters, J. K. Biochem. J. 1975, 150, 211 26. Phillips, J. L., Azari, P. Cell. Immun. 1974, 10, 31. 27. Chandra, R. K. J. Pediat. 1972, 81, 1194. 28. Harland, P. S. E. G. Lancet, 1965, ii, 719.
tested separ-
ately against: B lymphocytes at 5°C; B lymphocytes at 37°C; T lymphocytes at 5°C; and T lymphocytes at 37°C. Incubation with complement in all cultures was at 25 °C. Incubationtimes were 1 h with serum and 2 h with complement. Classification of Sera When a serum killed B lymphocytes from > 15 % of the panel at 5°C and all other reactions were negative, it was classified as B-cold-reactive. If it killed B lymphocytes from > 15 % of the panel at 37°C (whether or not it had B-cold reactivity) and killed T lymphocytes from <15% of the panel, it was classified
quent.5
The
September,
1975, and November, 1977. Serum samples were all taken before transplantation. 183 patients had first allografts, 38 had second, and 12 had third allografts. The follow-up period
Fig.
1-Patterns of
serum
reactivity.
1229 anbibodies. These anti-B lymphocyte antibodies are not harmful-as others have claimed anti-B-lymphocyte antibodies to be9,10—but are beneficial when they react only at 5 oc and not at both 5 °C and 37°C. B-cold lymphocytotoxins are not directed to any of the four HLA loci. This does not argue against the conclusion that these are the antibodies with enhancing activity; Fabre and Morrist6 and Fabre and Batchelor17 have shown in rats that enhancing antibodies are not as
Fig. 2-Actuarial graft survival for cadaver donor transplants, according to type of pre-transplant cytotoxins.
B-cold pattern (17% of total), 26 with the B-warm pat(11%), and 28with the T-warm pattern (12%). The B-cold group contained twice as many males as females while in the B-warm and T-warm groups females predominated, but this difference was not significant. Significantly more kidney transplants survived in patients with the B-cold pattern than in those without antibodies (fig. 2). At 3 months 80% of grafts survived in the B-cold group, compared with 56% in patients without antibodies (P<0002); at 6 months, the survival rates were 70% and 47%, respectively (P<0-01), and at In comone year 65% and 46%, respectively (P<0.05). parison, the patients with the B-warm or T-warm patterns had poor survival rates (38% and 43%, respectively, at 6 months). When B-cold-reactive serum was fractionated, cold activity against B cells was detected only in the IgM fraction.
tern
Discussion The presence of lymphocytotoxins in graft recipients before transplantation and its association with transplant failure was previously taken to indicate that the patient was presensitised.8 More recent studies have emphasised the importance of distinguishing lymphocytotoxins acting solely on B lymphocytes, which have no deleterious effect on transplants even when a positive crossmatch is obtained with the B cells of the donor.9 10 Such antibodies, for the most part, detect HLA-D antigens analogous to Ia antigens in mice. They could be enhancing antibodies, as suggested by experiments with rat heart" and rat kidney grafts. 12 Lymphocytotoxins not directed at HLA specificities and more reactive in the cold" also appear in the sera of potential transplant recipients. Klouda and Jeannet noted that 14 of 15 grafts in patients with pre-transplant cytotoxins reactive at 13°C against unfractionated lymphocytes survived at 1 year, compared with 3 out of 10 who had cytotoxins reactive at 25OC,14 In another study grafts in all 4 patients with cold cytotoxins (13°C) before transplantation survived for 1 year, while 6 out of 10 grafts survived in patients with antibodies reactive at37°C. We have shown that cold cytotoxins to B lymphocytes distinguish patients in whom the rate of transplant survival is higher than in patients with no pre-transplant
specific as previously supposed. Experimental evidence suggests that after the induction of active enhancement by injection of allogeneic cells, the timing of the graft is critical ; 18 the IgM response peaks 1 week after injection, when enhancement of rat kidney allografts is most effective," and these IgM antibodies disappear from the circulation and are not found in rats with long-surviving allografts.t9 Our results-although the number of B-cold-reactive patients was limited and no crossmatching with the kidney donor was done-are consistent with these findings: the presence of cold-reactive antibodies to B lymphocytes before transplantation was associated with a higher survival rate, and cold antibodies were often absent after a successful graft. It is a puzzle why human kidney transplants function in the face of the worst possible types of HLA incompatibility : of 495 kidney transplants done with four mismatched HLA antigens, 42% survived for one year.20 Enhancing antibodies in the recipient before transplantation may account for the high graft-survival rate in these patients, and in patients with intermediate grades of incompatibility. From a practical point of view, if the B-cold lympho-
cytotoxins
are
enhancing antibodies, they
can serve to
monitor the effect of transfusions before transplantation, and help to calculate the proper number of transfusions, the dosage, and the most effective time for
transplantation. We thank Mark Hermes, Roswitha Heintz, and Emi Yoshihara for technical assistance. This study was supported by grant AM 02375 from the National Institute of Arthritis and Metabolic Diseases and contract N01 CM 02092 from the National Cancer Institute.
Requests for reprints
should be addressed
to
Y. I., 1000 Veteran
Avenue, Los Angeles, California 90024, U.S.A. REFERENCES
1. Batchelor, J. R., Welsh, K. I. Br. med. Bull. 1976, 32, 113. 2. Newton, W. T., Anderson, C. B. Surgery, 1973, 74, 430. 3. Opelz, G., Terasaki, P. I. Transplant. Proc. 1977, 9, 121. 4. Dossetor, J. B., MacKinron, K. J., Gault, M. H., MacLean, L. D. Transplantation, 1967, 5, 844. 5. Terasaki, P. I., Opelz, G., Mickey, M. R. Transplant. Proc. 1976, 8, 139. 6. Terasaki, P. I., Bernoco, D., Park, M. S., Ozturk, G., Iwaki, Y. Am. J. clin. Path. 1978, 69, 103. 7. Hannon, R., Haire, M., Wisdon, G. B., Neil, D. W. J. immun. Methods,
1975, 8, 29. Terasaki, P. I., Kreisler, M. Mickey, M. R. Postgrad. med. J 1971, 47, 89. Ettenger, R. B., Terasaki, P. I., Opelz, G., Malekzadeh, M., Pennisi, A., Uittenbogaart, C., Fine, R. Lancet, 1976, ii, 56. 10. Morris, P. J., Ting, A., Oliver, D. O., Bishop, M., Williams, K., Dunhill, M. S. ibid. 1977, i, 1288. 11. Davies, D. A. L., Alkins, B. J. Nature, 1974, 247, 294 12. Soulillou, J. P., Carpenter, C. B., d’Apice, A. J. F., Strom, T. B. J. exp. Med. 1976, 143, 405. 13. Park, M. S., Terasaki, P. I., Byrns, G., Ayoub, G. Transplant. Proc. 1977, 9, 1701. 14. Klouda, P. T., Jeannet, M. Lancet, 1976, i, 876. 15. Roy, R., Robert, M. Union méd. Canada, 1977, 106, 297. 16. Fabre, J. W., Morris, P. J. Transplantation, 1974, 18, 436. 17. Fabre, J. W., Batchelor, J. R. ibid. 1975, 20, 269. 18. Carpenter, C. B., d’Apice, A. J., Abbas, A. K. Adv. Immun. 1976, 22, 1. 19. Desai, S B. R., Ruszkiewicz, M. Transplantation, 1977, 23, 230. 20. Opelz, G., Mickey, M R., Terasaki, P I ibid. 1977, 23, 490. 8. 9.