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Donor-specific antigen and cyclosporine in rat islet allografts
JOURNAL
OF SURGICAL
RESEARCH
Donor-Specific
42,494-491(1987)
Antigen and Cyclosporine
in Rat Islet Allografts
M.D., RALPH DIDLAKE, M.D., GIACOMO BASADONNA, M.D., BARRY D. KAHAN, PH.D., M.D., AND RONALD C. MERRELL, M.D.
KJZNJI KAKTZAKT, Department
of Surgery, The University
of Texas Medical School at Houston, 6431 Fannin, Houston, Texas 77030
Presented at the Annual Meeting of the Association for Academic Surgery, Washington, D.C., November 5-8, 1986 Combination therapy with one dose of 3 M KC1 extracted donor-soluble antigen (Ag) and a short course of cyclosporine (GA) has proven to prolong the survival of kidney allografts by enhancing specific T-suppressor populations. This regimen is tested in rat islet allografis in this study (Lewis to ACI). A 3-day perioperative course of 10 mg/kg/day CsA on Days - 1, 0, and 1 did not prolong graft survival (MST = 10.7 + 2.5 days vs 9.4 + 1.2 days in controls). When this course of CsA therapy was combined with a single dose of donor antigen on Day - 1, the survival time was prolonged slightly but significantly (MST = 14.0 + 5.8 days). Three cycles of a 3&y course of CsA therapy at 7-day intervals, a total of nine dosesof 10 mg/kg/day CsA, were effective in delaying rejection of islet allogratts (MST = 26.4 + 30.3). Moreover, combined therapy with donor antigen and three cycles of a 3day course of CsA prolonged the survival of islet allografts (MST = 57.7 f 5 1.4 days) with 50% of recipients still normoglycemic at 60 days after transplantation. These findings indicate that the combination therapy of donor antigen with a short course of CsA has a powerful effect to prevent the rejection of islet allografts, as shown in kidney allografts, in rats. 0 1987 Academic Press.Inc.
INTRODUCTION
It has been repeatedly demonstrated that isotransplantation of rat pancreatic islets can ameliorate pharmacologically induced diabetes mellitus [ 1, 21. However, results of allotransplantation of rat islets are currently very disappointing since the immunogenicity of isolated islets is apparently extreme. Even cyclosporine (CsA), which has proven to be a powerful immunosuppressive agent to prolong the survival of various organ allografts [3, 41, does not permit successful islet transplantation across sharp histocompatibility barriers [5, 61. A 3 M KC1 extracted donor soluble antigen (Ag) combined with a 3-day perioperative course of CsA prolongs the survival of rat renal allogratts (Buffalo, RTlb to Wistar Furth RTla), by enhancing specific suppressor T populations [7]. The effect of the combination of CsA with Ag is synergistic and donor specific [ 7-91. In this study, the combination of a short, 3-day perioperative course of CsA with Ag was tested in rat islet allografts (Lewis RT 1’ to ACI, RTl’). In addition, multiple short 0022-4804/87 %1.50 Copyright 0 1987 by Academic Press,Inc. All rights of reproduction in any form reserved.
cycles of CsA after the combined Ag-CsA regimen [ lo] have been shown to produce long-term survival of renal allografis (Buffalo, RT 1b to Wistar Furth RT 1”). Therefore, the benefit of repeated short cycles of CsA combined with one dose of Ag was also assessedin rat islet allografis. METHODS
Animals. Male AC1 rats (RT l*) were made diabetic by intravenous injection of streptozotocin (65 mg/kg). Nonfasting blood glucosewas determined on blood obtained from the tail vein using a YSI glucose analyzer. A rat was used as a recipient only if its blood glucose exceeded 350 mg/dl for more than 3 weeks. Islet isolation and transplantation. Islets were isolated from male Lewis rats (RT 1’) by the collagenase method [4] and Ficoll gradient separation [ 1I] with subsequent handpicking under a dissecting microscope. Isolated islets were maintained in tissue culture medium (RPM1 1640 with 10% calf serum) at 37°C for 5 days and then 1000 islets were transplanted into the liver via portal vein.
494
KAKIZAKI
ET AL.: DONOR-SPECIFIC ANTIGEN AND CYCLOSPORINE
Donor-soluble antigen. Donor-soluble antigen was prepared from Lewis spleen cells by the method of Reisfeld and Kahan [ 121. Spleens were minced with scissors and single-cell suspensions were obtained by pressing through fine mesh. Following removal of erythrocytes by osmotic shock, the cell pellet was resuspended in 3 M KC1 dissolved in phosphate-buffered saline (PBS) at 4°C for 16 hr. Cellular debris was removed by ultracentrifugation at 164,OOOg.The crude supernatant was concentrated by dialysis against 50% sucrose and then dialyzed against PBS. The protein concentration of extracts was estimated by the Bradford method [ 131.Rats were injected intravenously with a dose of 5 mg protein of antigen on the day prior to transplantation. Cyclosporine (GA). CsA was dissolved in olive oil at a concentration of 20 mg/ml. Rats were administered CsA 10 mg/kg/day by gavage. Experimental groups. The recipient rats were divided into the following groups. Group 1 (controls) received no treatment. Group 2 was treated with one cycle of CsA for 3 days by gavage on the day prior to (- I), on the day of (0) and on the day after (+ 1)
495
transplantation. Group 3 received 5 mg of soluble antigen intravenously on Day -1 plus CsA on Days - 1, 0, and 1. Group 4 had three cycles of intervals of 7 days (on Days -1,O,and1;7,8,and9;and14,15,and16). Group 5 received 5 mg of soluble antigen intravenously on Day - 1 plus three cycles of CsA on Days - 1, 0, and 1; 7, 8, and 9; and 14, 15, and 16. Criteria for rejection. Nonfasting blood glucose levels were determined daily and rejection was considered to have occurred when the blood glucose levels exceeded 200 mg/dl on two consecutive determinations. Gehan’s modification of the Wilcoxson rank sum test was used for data analysis. RESULTS
Eflect of cyclosporine therapy. The outcome of transplantation in each group is summarized in Table 1. Untreated AC1 hosts rejected Lewis grafts at 9.4 + 1.2 days. Administration of one cycle of CsA, on Days - 1, 0, and 1, did not affect graft survival. The mean survival time (MST) of 10.7 + 2.5 days (Group 2) was not significantly different from the untreated group (Group 1). On the other hand, hosts given three cycles of CsA at
TABLE I EFFECTSOF SOLUBLE ANTIGEN WITH CYCLICCsA THERAPYON ISLETALLOGRAFTSURVIVAL Group
Aga
CsA cyclesb
nc
1
None
None
7
Day of graft failure 6, 8, 9, 9, 9, 10, 10
MST + SDd
P value’
9.4 2 1.2
Graft function at 60 days 0
0.11 2
None
1
7
7,8,9, 11, 11, 11, 13
10.7 f 2.5
0 0.05
3
+
1
11
4
None
3
6
8, 8, 9, 10, 12, 14, 15, 15, 17, 21, 24 10, 13, 17, 18, 24, >60
14.0 ? 5.8
0
26.4 +- 30.3
16.7% 0.09
5
+
3
6
9, 15,26,>60 x 3
57.7 + 5 1.4
50%
Note. Group 1 vs 2, not significant; Group 1 vs 3, P = 0.018; Group 1 vs 4, P = 0.002; Group 1 vs 5, P = 0.007; Group 2 vs 3, P = 0.05; Group 2 vs 4, P = 0.009; Group 3 vs 5, P = 0.005; Group 3 vs 5, P = 0.002; Group 4 vs 5, not significant. ’ Rats received 5 mg donor antigen intravenously on the day prior to transplantation. b CsA was administered by gavage at 10 m&kg suspendedin olive oil either in one cycle on days - 1, 0, and + 1 or in three cycles on days -1, 0, and 1; 7, 8, and 9; 14, 15, and 16. ’ Number of animals in each group. d Mean survival time + standard deviation. eP value determined by Gehan’s modification of the Wilcoxson rank sum test.
496
JOURNAL OF SURGICAL RESEARCH: VOL. 42, NO. 5, MAY 1987
survival time of allograhed islets, but three cycles of 3-day CsA courses at intervals of 7 days (a total of nine doses of 10 mg/kg/day CsA) prolonged the graft survival to 26.4 days. In vitro culture of islets for 7- 14 days at Efect of combination of soluble antigen 37°C prior to transplantation does not prowith cyclic CsA therapy, Combined adminis- long the survival of allotransplanted islets tration of donor antigen and one cycle of [ 141.However, in our experience, the ability CsA slightly but significantly prolonged graft of allogeneic islets cultured for 5 days at survival to 14.0 f 5.8 days, compared to 37°C to induce lymphoproliferation, as Group 2 which received one cycle of CsA judged by [3H]thymidine incorporation, is treatment alone (P = 0.05). Administration modestly reduced compared with freshly isoof three cycles of CsA combined with a single lated islets [ 151. This study suggests that a dose of antigen further prolonged graft sur- brief 5-day culture at 37°C certainly does not vival to a greater extent (MST > 57.7 f 5 1.4 eliminate the immunogenicity of Lewis islets days) with 50% of recipients still normoglyin AC1 donors. The 5-day culture regimen cemic at 60 days after transplantation. was employed in these experiments to standardize islet preparation and storage and not Skin graft. At 60 days posttransplantation, two recipients administered cyclic CsA and to gain any special immunological advanone dose Ag were challenged with skin grafts. tage. Nine doses of CsA prolonged the surBoth donor Lewis and third-party Brown- vival of Lewis islets while three doses of 10 Norway (RTl”) skin transplants were re- mg/kg/day CsA did not. Combination therapy with a 3&y course jected by 10 days. In addition, both rats became diabetic by 11 days after skin grafts. of CsA and one dose of donor-soluble antigen has been shown to prolong the survival of kidney allografts [7]. Multiple, intermitDISCUSSION tent courses of CsA with antigen further proIn the rat, the survival of intraportal islet longed the renal allograft survival time in allografts transplanted across a major histowhich 50% of recipients showed long-term compatibility barrier was found to be unsurvival [lo]. Presentation of donor antigen changed with doses of CsA up to 20 mg/kg/ with low-dose cyclosporine seemsto provoke day given for 14 days continuously [5, 61. a special immune recognition which confers The present study demonstrated that a short privilege to that antigen. CsA inhibits helper course of CsA administration ( 10 mg/kg/day T, while sparing suppressor T cells [2, 161 on Days - 1, 0, and 1) did not change the and donor antigen stimulates suppressor T cells preferentially [8,9]. Thus, the combina40 60 60 DAYS 10 20 30 tion of CsA and donor antigen is synergistic loo I,ii;.in prolonging graft survival by inducing par‘i:i I i tial immune tolerance. i____,i ; i-y”......... In related experiments with islet trans7Ii..: i, i.........................................I....5 i.? plantation, Nelken and Morse [ 171 demonI fi strated that the injection of donor liver extract combined with antilymphocyte serum 1:I i 1 and pertussis culture filtrate extended allo1 2 3 graft survival to 37.7 f 18.9 days in rats. FIG. 1. Effect of CsA and 5 mg soluble antigen on The present study confirms the efficacy of Lewis islet allograf? survival in AC1 recipients. Group 1, the combination of a short course of CsA no treatment; Group 2, CsA - 1, 0, I days; Group 3, CsA with donor antigen in rat islet allotransplan-I,O,ldaysplusAg-lday,Group4,CsA-1,0,1,7, tation. The survival time of allografted islets 8, 9, 14, 15, 16 days; Group 5, CsA -i,O, 1, 7, 8, 9, 14, IS, 16days plus Ag -1 day. was slightly but significantly prolonged by a intervals of 7 days (Group 4) showed significantly prolonged survival (MST = 26.4 f 30.3) compared to the untreated Group 1 (P < 0.002) and to Group 2 which received only the single course of CsA (P < 0.009).
KAKIZAKI
ET AL.: DONOR-SPECIFIC ANTIGEN
3-day course of CsA combined with Ag. When multiple, intermittent courses of CsA were administered with one dose of Ag, the prolongation of survival time was dramatic (MST > 57.7 days). Three out of six recipients have not rejected allogeneic islet grafts by 60 days after transplantation. The rats with long-surviving islet grafts did not accept donor strain skin grafts, suggesting that a state of indefinite donor-specific unresponsiveness was not established by CsA-Ag treatment. Moreover, the rats became diabetic after receiving a skin allograft. This observation is consistent with other evidence for a tenuous graft tolerance in islet grafting. For example, injection of donor splenocytes or peritoneal exudate cells can induce rejection of established allografted islets [17, 181. In conclusion, three cycles of CsA prolongs the survival of allografted islets. Combination of one dose of 5 mg 3 M KC1 extracted donor-soluble antigen with a short course of CsA treatment prolongs graft survival more than CsA therapy alone. Fifty percent of recipients given three cycles of CsA with Ag showed long-term survival of islet allografts. The present study establishes the efficacy of the combination of donor antigen with a short course of CsA in rat islet allotransplantation. REFERENCES 1. Ballinger, W. F., and Lacy P. E. Transplantation of intact pancreatic islets in rats. Surgery 72: 175, 1972. 2. Trimble, E. R., Karakash, C., Malaisse-Lagque, E., Vassuhne, E., Orci, L., and Renold, A. C. Effects of intraportal islet transplantation, the transplanted tissue and the recipient pancreas. Diabetes 29: 34 1, 1980. 3. Kahan, B. D. Cyclosporine: The agent and its actions. Transplant. Proc. Suppl. I 17: 5, 1985. 4. Morris, P. J. Cyclosporin A. Transplantation 32: 349, 1981. 5. Gray, D. W. R., and Morris, P. J. Cyclosporine and pancreas transplantation. World J. Surg. 8: 230, 1984. 6. Garvey, J. F., McShane, P., Poole, M. D., Millard, P. R., and Morris, P. J. The effect of cyclosporin A on experimental pancreas allografts in the rat. Transplant. Proc. 12: 266, 1980.
AND CYCLOSPORINE
497
7. Yasumura, T., and Kahan, B. D. Prolongation of rat kidney allografts by pretransplant administration of donor antigen extract or whole blood transfusion combined with a short course of cyclosporine. Transplantation 36: 603, 1983. 8. Yoshimura, N., and Kahan, B. D. The immunosuppressive action of suppressor cells from antigen-cyclosporine-treated hosts on renal allograft survival. Transplantation 40: 384, 1985. 9. Yoshimura, N., and Kahan, B. D. Nature of the suppressor cells mediating prolonged graft survival alter administration of extracted histocompatibility antigen and cyclosporine. Transplantation 39: 162, 1985. 10 Yasumura, T., and Kahan, B. D., Prolongation of allograft survival by repeated cycles of donor antigen and cyclosporine in rat kidney transplantation. Transplantation 38: 418, 1984. 11 Lernmark, A., Nathans, A., and Steiner, D. F. Preparation and characterization of plasma membraneenriched fractions from pancreatic islets. J. Cell Biol. 71: 606, 1976. 12 Reisfeld, R. A., and Kahan, B. D. Biological and chemical characterization of human histocompatibility antigens. Fed. Proc. 29: 2034, 1970. 13. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72: 248, 1972. 14. Tze, W., and Tai, J. Prolongation of islet allograft and xenograft in nonimmunosuppressed rat recipients. Metabolism 32: 279, 1983. 15. Shizuru, J., Trager, D., and Merrell, R. C. Structure, function, and immune properties of reassociated islet cells. Diabetes 341 898, 1985. 16. Orasz, C. G., Roopenian, D. C., Widmer, M. B., and Bach, F. H., Analysis of cloned T cell function: II. Differential blockade of various cloned T cell func36: 706, tions by cyclosporine. Transplantation 1983. 17. Nelken, D., and Morse, S. I. Prolonged survival of allotransplanted islets of Langerhans cells in the rat. Transplantation
22: 74, 1976.
18. Faustman, D., Hauptfeld, V., Lacy, P., and Davie, J. Demonstration of active tolerance in maintenance of established islet of Langerhans allografis. Proc. Natl. Acad. Sci. USA 79: 4 153, 1982.
19. Lacy, P. E., Davie, J. M., and Finke, E. H. Induction of rejection of successful allografts of rat islets by donor peritoneal exudate cells. Transplantation 28: 415, 1979. 20. Hess, A. D., Danneberg, A., and Tutschke, P. J. Effect of cyclosporine on human lymphocyte responsesin vitro: Analysis of responding T-lymphocyte subpopulations in primary MLR with monoclonal antibodies. J. Immunol. 130: 717, 1983. 2 1. Lacy, P. E., and Kostianovsky, M. Method for isolating of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35, 1967.
OF SURGICAL
RESEARCH
Donor-Specific
42,494-491(1987)
Antigen and Cyclosporine
in Rat Islet Allografts
M.D., RALPH DIDLAKE, M.D., GIACOMO BASADONNA, M.D., BARRY D. KAHAN, PH.D., M.D., AND RONALD C. MERRELL, M.D.
KJZNJI KAKTZAKT, Department
of Surgery, The University
of Texas Medical School at Houston, 6431 Fannin, Houston, Texas 77030
Presented at the Annual Meeting of the Association for Academic Surgery, Washington, D.C., November 5-8, 1986 Combination therapy with one dose of 3 M KC1 extracted donor-soluble antigen (Ag) and a short course of cyclosporine (GA) has proven to prolong the survival of kidney allografts by enhancing specific T-suppressor populations. This regimen is tested in rat islet allografis in this study (Lewis to ACI). A 3-day perioperative course of 10 mg/kg/day CsA on Days - 1, 0, and 1 did not prolong graft survival (MST = 10.7 + 2.5 days vs 9.4 + 1.2 days in controls). When this course of CsA therapy was combined with a single dose of donor antigen on Day - 1, the survival time was prolonged slightly but significantly (MST = 14.0 + 5.8 days). Three cycles of a 3&y course of CsA therapy at 7-day intervals, a total of nine dosesof 10 mg/kg/day CsA, were effective in delaying rejection of islet allogratts (MST = 26.4 + 30.3). Moreover, combined therapy with donor antigen and three cycles of a 3day course of CsA prolonged the survival of islet allografts (MST = 57.7 f 5 1.4 days) with 50% of recipients still normoglycemic at 60 days after transplantation. These findings indicate that the combination therapy of donor antigen with a short course of CsA has a powerful effect to prevent the rejection of islet allografts, as shown in kidney allografts, in rats. 0 1987 Academic Press.Inc.
INTRODUCTION
It has been repeatedly demonstrated that isotransplantation of rat pancreatic islets can ameliorate pharmacologically induced diabetes mellitus [ 1, 21. However, results of allotransplantation of rat islets are currently very disappointing since the immunogenicity of isolated islets is apparently extreme. Even cyclosporine (CsA), which has proven to be a powerful immunosuppressive agent to prolong the survival of various organ allografts [3, 41, does not permit successful islet transplantation across sharp histocompatibility barriers [5, 61. A 3 M KC1 extracted donor soluble antigen (Ag) combined with a 3-day perioperative course of CsA prolongs the survival of rat renal allogratts (Buffalo, RTlb to Wistar Furth RTla), by enhancing specific suppressor T populations [7]. The effect of the combination of CsA with Ag is synergistic and donor specific [ 7-91. In this study, the combination of a short, 3-day perioperative course of CsA with Ag was tested in rat islet allografts (Lewis RT 1’ to ACI, RTl’). In addition, multiple short 0022-4804/87 %1.50 Copyright 0 1987 by Academic Press,Inc. All rights of reproduction in any form reserved.
cycles of CsA after the combined Ag-CsA regimen [ lo] have been shown to produce long-term survival of renal allografis (Buffalo, RT 1b to Wistar Furth RT 1”). Therefore, the benefit of repeated short cycles of CsA combined with one dose of Ag was also assessedin rat islet allografis. METHODS
Animals. Male AC1 rats (RT l*) were made diabetic by intravenous injection of streptozotocin (65 mg/kg). Nonfasting blood glucosewas determined on blood obtained from the tail vein using a YSI glucose analyzer. A rat was used as a recipient only if its blood glucose exceeded 350 mg/dl for more than 3 weeks. Islet isolation and transplantation. Islets were isolated from male Lewis rats (RT 1’) by the collagenase method [4] and Ficoll gradient separation [ 1I] with subsequent handpicking under a dissecting microscope. Isolated islets were maintained in tissue culture medium (RPM1 1640 with 10% calf serum) at 37°C for 5 days and then 1000 islets were transplanted into the liver via portal vein.
494
KAKIZAKI
ET AL.: DONOR-SPECIFIC ANTIGEN AND CYCLOSPORINE
Donor-soluble antigen. Donor-soluble antigen was prepared from Lewis spleen cells by the method of Reisfeld and Kahan [ 121. Spleens were minced with scissors and single-cell suspensions were obtained by pressing through fine mesh. Following removal of erythrocytes by osmotic shock, the cell pellet was resuspended in 3 M KC1 dissolved in phosphate-buffered saline (PBS) at 4°C for 16 hr. Cellular debris was removed by ultracentrifugation at 164,OOOg.The crude supernatant was concentrated by dialysis against 50% sucrose and then dialyzed against PBS. The protein concentration of extracts was estimated by the Bradford method [ 131.Rats were injected intravenously with a dose of 5 mg protein of antigen on the day prior to transplantation. Cyclosporine (GA). CsA was dissolved in olive oil at a concentration of 20 mg/ml. Rats were administered CsA 10 mg/kg/day by gavage. Experimental groups. The recipient rats were divided into the following groups. Group 1 (controls) received no treatment. Group 2 was treated with one cycle of CsA for 3 days by gavage on the day prior to (- I), on the day of (0) and on the day after (+ 1)
495
transplantation. Group 3 received 5 mg of soluble antigen intravenously on Day -1 plus CsA on Days - 1, 0, and 1. Group 4 had three cycles of intervals of 7 days (on Days -1,O,and1;7,8,and9;and14,15,and16). Group 5 received 5 mg of soluble antigen intravenously on Day - 1 plus three cycles of CsA on Days - 1, 0, and 1; 7, 8, and 9; and 14, 15, and 16. Criteria for rejection. Nonfasting blood glucose levels were determined daily and rejection was considered to have occurred when the blood glucose levels exceeded 200 mg/dl on two consecutive determinations. Gehan’s modification of the Wilcoxson rank sum test was used for data analysis. RESULTS
Eflect of cyclosporine therapy. The outcome of transplantation in each group is summarized in Table 1. Untreated AC1 hosts rejected Lewis grafts at 9.4 + 1.2 days. Administration of one cycle of CsA, on Days - 1, 0, and 1, did not affect graft survival. The mean survival time (MST) of 10.7 + 2.5 days (Group 2) was not significantly different from the untreated group (Group 1). On the other hand, hosts given three cycles of CsA at
TABLE I EFFECTSOF SOLUBLE ANTIGEN WITH CYCLICCsA THERAPYON ISLETALLOGRAFTSURVIVAL Group
Aga
CsA cyclesb
nc
1
None
None
7
Day of graft failure 6, 8, 9, 9, 9, 10, 10
MST + SDd
P value’
9.4 2 1.2
Graft function at 60 days 0
0.11 2
None
1
7
7,8,9, 11, 11, 11, 13
10.7 f 2.5
0 0.05
3
+
1
11
4
None
3
6
8, 8, 9, 10, 12, 14, 15, 15, 17, 21, 24 10, 13, 17, 18, 24, >60
14.0 ? 5.8
0
26.4 +- 30.3
16.7% 0.09
5
+
3
6
9, 15,26,>60 x 3
57.7 + 5 1.4
50%
Note. Group 1 vs 2, not significant; Group 1 vs 3, P = 0.018; Group 1 vs 4, P = 0.002; Group 1 vs 5, P = 0.007; Group 2 vs 3, P = 0.05; Group 2 vs 4, P = 0.009; Group 3 vs 5, P = 0.005; Group 3 vs 5, P = 0.002; Group 4 vs 5, not significant. ’ Rats received 5 mg donor antigen intravenously on the day prior to transplantation. b CsA was administered by gavage at 10 m&kg suspendedin olive oil either in one cycle on days - 1, 0, and + 1 or in three cycles on days -1, 0, and 1; 7, 8, and 9; 14, 15, and 16. ’ Number of animals in each group. d Mean survival time + standard deviation. eP value determined by Gehan’s modification of the Wilcoxson rank sum test.
496
JOURNAL OF SURGICAL RESEARCH: VOL. 42, NO. 5, MAY 1987
survival time of allograhed islets, but three cycles of 3-day CsA courses at intervals of 7 days (a total of nine doses of 10 mg/kg/day CsA) prolonged the graft survival to 26.4 days. In vitro culture of islets for 7- 14 days at Efect of combination of soluble antigen 37°C prior to transplantation does not prowith cyclic CsA therapy, Combined adminis- long the survival of allotransplanted islets tration of donor antigen and one cycle of [ 141.However, in our experience, the ability CsA slightly but significantly prolonged graft of allogeneic islets cultured for 5 days at survival to 14.0 f 5.8 days, compared to 37°C to induce lymphoproliferation, as Group 2 which received one cycle of CsA judged by [3H]thymidine incorporation, is treatment alone (P = 0.05). Administration modestly reduced compared with freshly isoof three cycles of CsA combined with a single lated islets [ 151. This study suggests that a dose of antigen further prolonged graft sur- brief 5-day culture at 37°C certainly does not vival to a greater extent (MST > 57.7 f 5 1.4 eliminate the immunogenicity of Lewis islets days) with 50% of recipients still normoglyin AC1 donors. The 5-day culture regimen cemic at 60 days after transplantation. was employed in these experiments to standardize islet preparation and storage and not Skin graft. At 60 days posttransplantation, two recipients administered cyclic CsA and to gain any special immunological advanone dose Ag were challenged with skin grafts. tage. Nine doses of CsA prolonged the surBoth donor Lewis and third-party Brown- vival of Lewis islets while three doses of 10 Norway (RTl”) skin transplants were re- mg/kg/day CsA did not. Combination therapy with a 3&y course jected by 10 days. In addition, both rats became diabetic by 11 days after skin grafts. of CsA and one dose of donor-soluble antigen has been shown to prolong the survival of kidney allografts [7]. Multiple, intermitDISCUSSION tent courses of CsA with antigen further proIn the rat, the survival of intraportal islet longed the renal allograft survival time in allografts transplanted across a major histowhich 50% of recipients showed long-term compatibility barrier was found to be unsurvival [lo]. Presentation of donor antigen changed with doses of CsA up to 20 mg/kg/ with low-dose cyclosporine seemsto provoke day given for 14 days continuously [5, 61. a special immune recognition which confers The present study demonstrated that a short privilege to that antigen. CsA inhibits helper course of CsA administration ( 10 mg/kg/day T, while sparing suppressor T cells [2, 161 on Days - 1, 0, and 1) did not change the and donor antigen stimulates suppressor T cells preferentially [8,9]. Thus, the combina40 60 60 DAYS 10 20 30 tion of CsA and donor antigen is synergistic loo I,ii;.in prolonging graft survival by inducing par‘i:i I i tial immune tolerance. i____,i ; i-y”......... In related experiments with islet trans7Ii..: i, i.........................................I....5 i.? plantation, Nelken and Morse [ 171 demonI fi strated that the injection of donor liver extract combined with antilymphocyte serum 1:I i 1 and pertussis culture filtrate extended allo1 2 3 graft survival to 37.7 f 18.9 days in rats. FIG. 1. Effect of CsA and 5 mg soluble antigen on The present study confirms the efficacy of Lewis islet allograf? survival in AC1 recipients. Group 1, the combination of a short course of CsA no treatment; Group 2, CsA - 1, 0, I days; Group 3, CsA with donor antigen in rat islet allotransplan-I,O,ldaysplusAg-lday,Group4,CsA-1,0,1,7, tation. The survival time of allografted islets 8, 9, 14, 15, 16 days; Group 5, CsA -i,O, 1, 7, 8, 9, 14, IS, 16days plus Ag -1 day. was slightly but significantly prolonged by a intervals of 7 days (Group 4) showed significantly prolonged survival (MST = 26.4 f 30.3) compared to the untreated Group 1 (P < 0.002) and to Group 2 which received only the single course of CsA (P < 0.009).
KAKIZAKI
ET AL.: DONOR-SPECIFIC ANTIGEN
3-day course of CsA combined with Ag. When multiple, intermittent courses of CsA were administered with one dose of Ag, the prolongation of survival time was dramatic (MST > 57.7 days). Three out of six recipients have not rejected allogeneic islet grafts by 60 days after transplantation. The rats with long-surviving islet grafts did not accept donor strain skin grafts, suggesting that a state of indefinite donor-specific unresponsiveness was not established by CsA-Ag treatment. Moreover, the rats became diabetic after receiving a skin allograft. This observation is consistent with other evidence for a tenuous graft tolerance in islet grafting. For example, injection of donor splenocytes or peritoneal exudate cells can induce rejection of established allografted islets [17, 181. In conclusion, three cycles of CsA prolongs the survival of allografted islets. Combination of one dose of 5 mg 3 M KC1 extracted donor-soluble antigen with a short course of CsA treatment prolongs graft survival more than CsA therapy alone. Fifty percent of recipients given three cycles of CsA with Ag showed long-term survival of islet allografts. The present study establishes the efficacy of the combination of donor antigen with a short course of CsA in rat islet allotransplantation. REFERENCES 1. Ballinger, W. F., and Lacy P. E. Transplantation of intact pancreatic islets in rats. Surgery 72: 175, 1972. 2. Trimble, E. R., Karakash, C., Malaisse-Lagque, E., Vassuhne, E., Orci, L., and Renold, A. C. Effects of intraportal islet transplantation, the transplanted tissue and the recipient pancreas. Diabetes 29: 34 1, 1980. 3. Kahan, B. D. Cyclosporine: The agent and its actions. Transplant. Proc. Suppl. I 17: 5, 1985. 4. Morris, P. J. Cyclosporin A. Transplantation 32: 349, 1981. 5. Gray, D. W. R., and Morris, P. J. Cyclosporine and pancreas transplantation. World J. Surg. 8: 230, 1984. 6. Garvey, J. F., McShane, P., Poole, M. D., Millard, P. R., and Morris, P. J. The effect of cyclosporin A on experimental pancreas allografts in the rat. Transplant. Proc. 12: 266, 1980.
AND CYCLOSPORINE
497
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