- Email: [email protected]
Development of Anti–Human Leukocyte Antigen Class 1 Antibodies Following Allogeneic Islet Cell Transplantation
Development of Anti–Human Leukocyte Antigen Class 1 Antibodies Following Allogeneic Islet Cell Transplantation P.I. Lobo, C. Spencer, W.D. Simmons, K.D. Hagspiel, J.F. Angle, S. Deng, J. Markmann, A. Naji, S.E. Kirk, T. Pruett, and K.L. Brayman ABSTRACT Currently there is minimal concern that islet allograft failure could result from the development of anti– human leukocyte antigen (HLA) antibodies reactive to the allograft. We report here a case of islet allograft failure where the recipient developed immunoglobulin G anti-HLA class I antibodies reactive to HLA antigens present in two of the three islet cell donors. The patient had no detectable anti-HLA antibodies prior to the transplant but these antibodies were detected approximately 4 months posttransplant. Of concern, these antibodies developed despite induction with anti-IL2R antibodies (Zenapex) prior to intraportal islet cell infusion, low-dose tacrolimus (12-hour troughs 3 to 5 ng/mL) and rapammune (target troughs 12 to 15 ng/mL). The patient was not presensitized with blood products or a previous allograft. Her husband, however, shared antigens present in one of the islet donors and the recipient could have been presensitized to her husband during her two pregnancies. This case clearly demonstrates that islet allografts can lead to development of anti-HLA antibodies, which can cause islet allograft failure, as is the case with solid organ transplants, and hence emphasizes the need to monitor for such antibodies pre- and posttransplant. Additionally it appears that currently recommended immunosuppression may not be sufficient to inhibit a humoral response to both alloantigens and autoantigens.
I
SLET CELL TRANSPLANTATION has become a clinical reality thanks to the investigative efforts of Ricordi et al to develop an automated method for retrieving sufficient number of islets from a single human donor pancreas and research efforts from other centers to improve the technology of islet preservation and to avoid immunosuppression-induced islet cell toxicity.1–3 Strategies that have contributed to improvement in islet cell transplantation (since 1990) have included the following: (1) using a donor pancreas with a cold ischemia time of ⬍8 hours; (2) isolating and culturing islet cells for 24 to 72 hours (prior to transplantation) to render them less immunogenic; (3) infusing islets into the intraportal circulation; (4) infusing a minimum of ⬎9000 islet equivalents (IEQ) per kilogram of recipient’s weight; and (5) optimizing immunosuppression to include antibody induction prior to islet infusion and avoiding steroids and minimizing calcineurin inhibitors, both of which are toxic to islet cells (reviewed in Stock and Bluestone4). With this approach, the Edmonton group has shown that 9 of 15 patients (ie, approximately 60%) are insulin inde-
pendent at more than 1 year while at the University of Minnesota, five of eight patients have remained insulin-free during the first year, and at the University of Pennsylvania six of the seven recipients have not required insulin during the first year.5–7 Known causes of islet allograft loss include problems related to donor organ procurement, inadequate preservation of islets during isolation, and development of autoantibodies to donor islet cells. Of the six patients with islet cell graft loss in the Edmonton Center, three were found to have autoantibodies to islet cells, while it was unclear why the other three lost the grafts, especially since none of these From the Departments of Internal Medicine and Surgery (P.I.L., C.S., W.D.S.), Radiology (K.D.H., J.F.A.), Internal Medicine (S.E.K.), Surgery (T.P., K.L.B.), University of Virginia Health System, Charlottesville, Virginia, USA; and Surgery (S.D., J.M., A.N.), University of Pennsylvania, Philadelphia, USA. Address reprint requests to Peter Lobo, Department of Internal Medicine, University of Virginia Health System, Charlottesville, VA 22908. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.065
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3438
Transplantation Proceedings, 37, 3438 –3440 (2005)
DEVELOPMENT OF ANTI-HLA ANTIBODIES
3439
patients had detectable anti– human leukocyte antigen (HLA) antibodies as evaluated by a cytotoxic assay to a panel of HLA typed cells (PRA).5 Similarly the single patient from the University of Pennsylvania with islet allograft failure did not develop anti-HLA antibodies using the PRA technique.7 Oberholzer et al identified immunoglobulin (Ig) M antibodies cytotoxic to B lymphocytes in all four patients with islet allograft failure but did not define the antibody specificity.8 However, Olack et al had three patients with a prior kidney allograft who developed IgG anti-HLA antibodies with loss of islet cell function, but it is unclear from their findings whether prior sensitization with the kidney allograft played a role in induction of anti-HLA antibody after the islet cell transplant.9 Stock and Bluestone, in a recent review, have stated the following: “There has been no sensitization of islet recipients to HLA antigens, despite the infusion of islets isolated from multiple donors. The lack of sensitization reflects effective immunosuppression from the alloimmune response.”4 Here, we present a case who only received an islet allograft and clearly demonstrate that the islet allograft induced IgG anti-HLA antibodies, contributing to islet cell failure.
CASE REPORT The patient is a 47-year-old Caucasian woman weighing 41 kg, with type I diabetes mellitus for 31 years who received two sets of intraportal islet infusions performed 3 months apart. The islets were obtained from the University of Pennsylvania, and the technique for recovery of donor pancreas, islet isolation procedure, and islet culture has previously been described in detail.7 Table 1 details the islet cell mass she received with each infusion as well as her insulin requirements posttransplant. Immunosuppression consisted of Zenapex (anti-IL2R antibody) induction given initially prior to islet infusion and followed with four more doses at 2-week intervals, and low-dose Prograf to maintain 12-hour trough levels of 3 to 5 ng/mL and Rapammune to maintain a target trough of 12 to 15 ng/mL. As noted in Table 1, her insulin requirements decreased by about 50% with the first islet infusion, while the second islet infusion failed to further reduce the insulin requirement. In investigating the cause for the lack of a beneficial effect on insulin requirement after the second infusion, we found that she had developed IgG anti-HLA antibodies despite continued immunosuppression. These antibodies were reactive to HLA class I antigens present in one of the two donors from the first islet infusion and the donor in the second islet infusion (Table 2). The serum for identifying anti-HLA antibodies was obtained 1 month after the second infusion. Serum Table 1. Correlating Islet Infusion Mass With Insulin Requirement at 5 Weeks Postinfusion Islet cell mass (IEQ/kg)
Pretransplantation 1st islet infusion (two donors) 2nd islet infusion (one donor)
Insulin requirement (u/d)
24–30 15,000
13–17
9778
15–20
Table 2. Development of Anti-HLA Antibodies Posttransplant Correlating With Sensitizing Event (Husband’s HLA)
Recipient 1st islet infusion Donor no. 1 Donor no. 2 2nd islet infusion Donor no. 3
HLA-A
HLA-B
HLA-DR
1, —
8, —
17, 52
3, 25 3, 26
18, 35 57*, 62*
1, 15 7, 15
2, 28*
44, 60
4, 13
Presensitization events: no blood transfusions, two pregnancies (husbandA3, -32; B18, -62, DR4, -9). *Donor HLA specificities reactive to anti-HLA antibody in recipient.
tested at 3 months, 1 month, and 1 day prior to the first infusion had no anti-HLA antibodies.
DISCUSSION
Two methods were used to detect and identify the specificity of IgG anti-HLA antibody: (1) A complement-dependent lymphocytotoxicity assay with a panel of HLA-typed lymphocytes from different individuals (PRA). The recipient’s serum was treated with dithiothreitol to denature IgM antibodies, which rarely have anti-HLA reactivity. (2) A flowcytometry assay that detects binding of IgG antibodies to purified HLA (class I or II) complexed to beads (Flow PRA, One-Lambda, Calif, USA). With these assays, the patient was found to have developed anti-HLA-B57 and -B62 reactive to these antigens present on donor no. 2 from the first islet infusion (see Table 2) and anti-HLA-A28 reactive to this antigen present on donor no. 3 from the second islet infusion. In Table 2, it is apparent that the only presensitizing event were two pregnancies, and the patient could have been presensitized to her husband’s HLA-B62, which cross-reacts with HLAB57. Anti-HLA-B62 and -B57 were clearly undetectable pretransplant but the islet cells from donor no. 2 could have provoked a humoral response as a result of this presensitization event (ie, despite immunosuppression that is currently felt to be “effective” in inhibiting a humoral response). However, the humoral response to HLA-A28 present in donor no. 3 cannot be ascribed to prior sensitization to the husband’s HLA-A3, -A32. Both the donors in the first islet infusion had an HLA-A3 and hence prior sensitization to the husband’s HLA-A3 and -A32 should have provoked a humoral response to HLA-A3 as well as to -A25. Hence these data would indicate that the anti-HLAA28 antibody developed de novo as a result of a direct sensitization by islets from donor no. 3. This case clearly demonstrates that islet allografts, like blood transfusions or solid organ transplants, can provoke an alloantibody response, which can lead to graft failure either acutely (as in this case) or on a chronic basis (as seen in solid-organ transplants).10,11 Strategies to prevent antiHLA antibody-mediated islet allograft loss could include (1) prescreening of potential recipients for presence of anti-HLA antibodies resulting from previous sensitizing events (eg, blood transfusions, pregnancies, and previous
3440
allograft transplantation); (2) avoidance of islet allograft donors known to have HLA antigens to which the recipient may have been sensitized (eg, the husband’s antigens as in this case or antigens from a previous allograft); (3) performing islet cell transplants in anti-HLA antibody-positive recipients only when there is no antibody reactivity to the islet donor lymphocytes (ie, a negative donor-specific crossmatch); and (4) monitoring of recipients posttransplant for development of anti-HLA antibodies and islet cell autoantibodies especially since there is therapy (eg, pooled intravenous IgG immunoglobin [IVIG], Rituximab, plasmapheresis) to inhibit formation of such antibodies. Finally, this case emphasizes the need to reevaluate current immunosuppressive protocols for islet cell transplantation. We could consider using induction agents to more effectively prevent allorecognition (eg, thymoglobulin, as the lack of steroids, low-dose Prograf, and Rapammune may not effectively prevent activation of T cells important in providing B-cell help). It is not uncommon for islet cell transplants to function for a period and then to fail over time with insulin requirements increasing but not returning to pretransplant levels. The development of anti-HLA antibodies to some but not all donor tissue may provide an explanation for some of these cases. REFERENCES 1. Ricordi C, Lacy PE, Fink EH, et al: Automated method for isolation of human pancreatic cells. Diabetes 31:413, 1988
LOBO, SPENCER, SIMMONS ET AL 2. Bretzel RG, Brandhorst D, Brandhorst H, et al: Improved survival of intraportal pancreatic islet cell allografts in patients with type-1 diabetes mellitus by refined peritransplant management. J Mol Med 77:140, 1999 3. Shapiro AM, Lakey JR, Ryan EA, et al: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230, 2000 4. Stock PG, Bluestone JA: Beta-cell replacement for type 1 diabetes. Annu Rev Med 55:133, 2004 5. Ryan EA, Lakey JRT, Paty BW, et al: Continued insulin reserve provides long-term glycaemic control. Diabetes 51:2148, 2002 6. Hering BJ, Kandaswamy R, Ansite JD, et al: Successful single donor islet transplantation in type 1 diabetes. Am J Transpl 3(suppl 5):A567, 2003 (Abstract) 7. Markmann JF, Deng S, Huang X, et al: Insulin independence following isolated islet transplantation and single islet infusions. Ann Surg 237:741, 2003 8. Oberholzer J, Triponez F, Mage R, et al: Human islet transplantation: lessons from 13 autologous and 13 allogeneic transplants. Transplantation 69:1115, 2000 9. Olack BJ, Swanson CJ, Flavin KS, et al: Significance of HLA in human islet transplantation. Sensitization to HLA antigens in islet recipients with failing transplants. Transplant Proc 29:2268, 1997 10. Lobo PI, Spencer CE, Stevenson WC, et al: Evidence demonstrating poor kidney survival when acute rejections are associated with IgG donor-specific lymphocytotoxins. Transplantation 59:357, 1995 11. Reinsmoen NL, Nelson K, Zeevi A: Anti-HLA antibody analysis and crossmatching in heart and lung transplantation. Transpl Immunol 13:63, 2004
I
SLET CELL TRANSPLANTATION has become a clinical reality thanks to the investigative efforts of Ricordi et al to develop an automated method for retrieving sufficient number of islets from a single human donor pancreas and research efforts from other centers to improve the technology of islet preservation and to avoid immunosuppression-induced islet cell toxicity.1–3 Strategies that have contributed to improvement in islet cell transplantation (since 1990) have included the following: (1) using a donor pancreas with a cold ischemia time of ⬍8 hours; (2) isolating and culturing islet cells for 24 to 72 hours (prior to transplantation) to render them less immunogenic; (3) infusing islets into the intraportal circulation; (4) infusing a minimum of ⬎9000 islet equivalents (IEQ) per kilogram of recipient’s weight; and (5) optimizing immunosuppression to include antibody induction prior to islet infusion and avoiding steroids and minimizing calcineurin inhibitors, both of which are toxic to islet cells (reviewed in Stock and Bluestone4). With this approach, the Edmonton group has shown that 9 of 15 patients (ie, approximately 60%) are insulin inde-
pendent at more than 1 year while at the University of Minnesota, five of eight patients have remained insulin-free during the first year, and at the University of Pennsylvania six of the seven recipients have not required insulin during the first year.5–7 Known causes of islet allograft loss include problems related to donor organ procurement, inadequate preservation of islets during isolation, and development of autoantibodies to donor islet cells. Of the six patients with islet cell graft loss in the Edmonton Center, three were found to have autoantibodies to islet cells, while it was unclear why the other three lost the grafts, especially since none of these From the Departments of Internal Medicine and Surgery (P.I.L., C.S., W.D.S.), Radiology (K.D.H., J.F.A.), Internal Medicine (S.E.K.), Surgery (T.P., K.L.B.), University of Virginia Health System, Charlottesville, Virginia, USA; and Surgery (S.D., J.M., A.N.), University of Pennsylvania, Philadelphia, USA. Address reprint requests to Peter Lobo, Department of Internal Medicine, University of Virginia Health System, Charlottesville, VA 22908. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.065
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3438
Transplantation Proceedings, 37, 3438 –3440 (2005)
DEVELOPMENT OF ANTI-HLA ANTIBODIES
3439
patients had detectable anti– human leukocyte antigen (HLA) antibodies as evaluated by a cytotoxic assay to a panel of HLA typed cells (PRA).5 Similarly the single patient from the University of Pennsylvania with islet allograft failure did not develop anti-HLA antibodies using the PRA technique.7 Oberholzer et al identified immunoglobulin (Ig) M antibodies cytotoxic to B lymphocytes in all four patients with islet allograft failure but did not define the antibody specificity.8 However, Olack et al had three patients with a prior kidney allograft who developed IgG anti-HLA antibodies with loss of islet cell function, but it is unclear from their findings whether prior sensitization with the kidney allograft played a role in induction of anti-HLA antibody after the islet cell transplant.9 Stock and Bluestone, in a recent review, have stated the following: “There has been no sensitization of islet recipients to HLA antigens, despite the infusion of islets isolated from multiple donors. The lack of sensitization reflects effective immunosuppression from the alloimmune response.”4 Here, we present a case who only received an islet allograft and clearly demonstrate that the islet allograft induced IgG anti-HLA antibodies, contributing to islet cell failure.
CASE REPORT The patient is a 47-year-old Caucasian woman weighing 41 kg, with type I diabetes mellitus for 31 years who received two sets of intraportal islet infusions performed 3 months apart. The islets were obtained from the University of Pennsylvania, and the technique for recovery of donor pancreas, islet isolation procedure, and islet culture has previously been described in detail.7 Table 1 details the islet cell mass she received with each infusion as well as her insulin requirements posttransplant. Immunosuppression consisted of Zenapex (anti-IL2R antibody) induction given initially prior to islet infusion and followed with four more doses at 2-week intervals, and low-dose Prograf to maintain 12-hour trough levels of 3 to 5 ng/mL and Rapammune to maintain a target trough of 12 to 15 ng/mL. As noted in Table 1, her insulin requirements decreased by about 50% with the first islet infusion, while the second islet infusion failed to further reduce the insulin requirement. In investigating the cause for the lack of a beneficial effect on insulin requirement after the second infusion, we found that she had developed IgG anti-HLA antibodies despite continued immunosuppression. These antibodies were reactive to HLA class I antigens present in one of the two donors from the first islet infusion and the donor in the second islet infusion (Table 2). The serum for identifying anti-HLA antibodies was obtained 1 month after the second infusion. Serum Table 1. Correlating Islet Infusion Mass With Insulin Requirement at 5 Weeks Postinfusion Islet cell mass (IEQ/kg)
Pretransplantation 1st islet infusion (two donors) 2nd islet infusion (one donor)
Insulin requirement (u/d)
24–30 15,000
13–17
9778
15–20
Table 2. Development of Anti-HLA Antibodies Posttransplant Correlating With Sensitizing Event (Husband’s HLA)
Recipient 1st islet infusion Donor no. 1 Donor no. 2 2nd islet infusion Donor no. 3
HLA-A
HLA-B
HLA-DR
1, —
8, —
17, 52
3, 25 3, 26
18, 35 57*, 62*
1, 15 7, 15
2, 28*
44, 60
4, 13
Presensitization events: no blood transfusions, two pregnancies (husbandA3, -32; B18, -62, DR4, -9). *Donor HLA specificities reactive to anti-HLA antibody in recipient.
tested at 3 months, 1 month, and 1 day prior to the first infusion had no anti-HLA antibodies.
DISCUSSION
Two methods were used to detect and identify the specificity of IgG anti-HLA antibody: (1) A complement-dependent lymphocytotoxicity assay with a panel of HLA-typed lymphocytes from different individuals (PRA). The recipient’s serum was treated with dithiothreitol to denature IgM antibodies, which rarely have anti-HLA reactivity. (2) A flowcytometry assay that detects binding of IgG antibodies to purified HLA (class I or II) complexed to beads (Flow PRA, One-Lambda, Calif, USA). With these assays, the patient was found to have developed anti-HLA-B57 and -B62 reactive to these antigens present on donor no. 2 from the first islet infusion (see Table 2) and anti-HLA-A28 reactive to this antigen present on donor no. 3 from the second islet infusion. In Table 2, it is apparent that the only presensitizing event were two pregnancies, and the patient could have been presensitized to her husband’s HLA-B62, which cross-reacts with HLAB57. Anti-HLA-B62 and -B57 were clearly undetectable pretransplant but the islet cells from donor no. 2 could have provoked a humoral response as a result of this presensitization event (ie, despite immunosuppression that is currently felt to be “effective” in inhibiting a humoral response). However, the humoral response to HLA-A28 present in donor no. 3 cannot be ascribed to prior sensitization to the husband’s HLA-A3, -A32. Both the donors in the first islet infusion had an HLA-A3 and hence prior sensitization to the husband’s HLA-A3 and -A32 should have provoked a humoral response to HLA-A3 as well as to -A25. Hence these data would indicate that the anti-HLAA28 antibody developed de novo as a result of a direct sensitization by islets from donor no. 3. This case clearly demonstrates that islet allografts, like blood transfusions or solid organ transplants, can provoke an alloantibody response, which can lead to graft failure either acutely (as in this case) or on a chronic basis (as seen in solid-organ transplants).10,11 Strategies to prevent antiHLA antibody-mediated islet allograft loss could include (1) prescreening of potential recipients for presence of anti-HLA antibodies resulting from previous sensitizing events (eg, blood transfusions, pregnancies, and previous
3440
allograft transplantation); (2) avoidance of islet allograft donors known to have HLA antigens to which the recipient may have been sensitized (eg, the husband’s antigens as in this case or antigens from a previous allograft); (3) performing islet cell transplants in anti-HLA antibody-positive recipients only when there is no antibody reactivity to the islet donor lymphocytes (ie, a negative donor-specific crossmatch); and (4) monitoring of recipients posttransplant for development of anti-HLA antibodies and islet cell autoantibodies especially since there is therapy (eg, pooled intravenous IgG immunoglobin [IVIG], Rituximab, plasmapheresis) to inhibit formation of such antibodies. Finally, this case emphasizes the need to reevaluate current immunosuppressive protocols for islet cell transplantation. We could consider using induction agents to more effectively prevent allorecognition (eg, thymoglobulin, as the lack of steroids, low-dose Prograf, and Rapammune may not effectively prevent activation of T cells important in providing B-cell help). It is not uncommon for islet cell transplants to function for a period and then to fail over time with insulin requirements increasing but not returning to pretransplant levels. The development of anti-HLA antibodies to some but not all donor tissue may provide an explanation for some of these cases. REFERENCES 1. Ricordi C, Lacy PE, Fink EH, et al: Automated method for isolation of human pancreatic cells. Diabetes 31:413, 1988
LOBO, SPENCER, SIMMONS ET AL 2. Bretzel RG, Brandhorst D, Brandhorst H, et al: Improved survival of intraportal pancreatic islet cell allografts in patients with type-1 diabetes mellitus by refined peritransplant management. J Mol Med 77:140, 1999 3. Shapiro AM, Lakey JR, Ryan EA, et al: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230, 2000 4. Stock PG, Bluestone JA: Beta-cell replacement for type 1 diabetes. Annu Rev Med 55:133, 2004 5. Ryan EA, Lakey JRT, Paty BW, et al: Continued insulin reserve provides long-term glycaemic control. Diabetes 51:2148, 2002 6. Hering BJ, Kandaswamy R, Ansite JD, et al: Successful single donor islet transplantation in type 1 diabetes. Am J Transpl 3(suppl 5):A567, 2003 (Abstract) 7. Markmann JF, Deng S, Huang X, et al: Insulin independence following isolated islet transplantation and single islet infusions. Ann Surg 237:741, 2003 8. Oberholzer J, Triponez F, Mage R, et al: Human islet transplantation: lessons from 13 autologous and 13 allogeneic transplants. Transplantation 69:1115, 2000 9. Olack BJ, Swanson CJ, Flavin KS, et al: Significance of HLA in human islet transplantation. Sensitization to HLA antigens in islet recipients with failing transplants. Transplant Proc 29:2268, 1997 10. Lobo PI, Spencer CE, Stevenson WC, et al: Evidence demonstrating poor kidney survival when acute rejections are associated with IgG donor-specific lymphocytotoxins. Transplantation 59:357, 1995 11. Reinsmoen NL, Nelson K, Zeevi A: Anti-HLA antibody analysis and crossmatching in heart and lung transplantation. Transpl Immunol 13:63, 2004