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Combination of Donor-Specific Blood Transfusion With Anti-CD28 Antibody Synergizes to Prolong Graft Survival in Rat Liver Transplantation
Combination of Donor-Specific Blood Transfusion With Anti-CD28 Antibody Synergizes to Prolong Graft Survival in Rat Liver Transplantation H. Urakami, D.V. Ostanin, T. Hünig, and M.B. Grisham ABSTRACT Donor-specific blood transfusion (DST) has been shown to effectively induce tolerance to certain allografts. In addition, it is well known that blockade of costimulatory signals reduces the ability of T cells to respond to alloantigens, prolonging allograft survival in some transplant models. We assessed the effects of single or multiple DSTs in the absence or presence of anti-CD28 monoclonal antibodies (mAbs) on graft function and host survival in rat liver transplantation (LTx). Fully MHC-mismatched adult male Dark Agouti (DA) and Lewis (LEW) rats were used as donors and recipients, respectively. Heparinized DA blood was administered to naïve LEW rats 7 days before LTx [DST(⫺7d)], 14 and 7 days before LTx [DST(1⫻2)], twice a week for 2 weeks prior to LTx [DST(2⫻2)] and once a week for 4 weeks prior to LTx [DST(1⫻4)]. For some experiments, two different monoclonal antibodies (mAb) to rat CD28 (JJ316 and JJ319) were administered in combination with some DST treatments. We found that DST administration induced a time- and dose-dependent increase in host survival. Treatment of LEW rats with JJ316 or JJ319 mAb alone failed to prolong graft survival over untreated rats; however, the combination of DST(1⫻2) with JJ316 or JJ319 mAb induced indefinite survival at 100 days following surgery. We found that this protective effect was associated with increased numbers of splenic CD4⫹CD45RC⫺ but not CD4⫹CD25⫹foxp3⫹ T-cells in long-term survivors. Our data suggest that the combination of suboptimal DST with CD28 mAb induces donor-specific tolerance that correlates with enhanced numbers of regulatory T-cells.
T
HE DEVELOPMENT of potent immunosuppressive drugs has improved long-term outcome in clinical transplantation. However, graft loss related to chronic rejection, drug toxicity, or infections remains a substantial concern. Donor-specific blood transfusion (DST) is an efficient strategy to induce hyporesponsiveness without additional immunotherapy, both clinically and experimentally.1,2 Recent studies have demonstrated that a single DST failed to induce long-term graft acceptance; however, multiple DSTs successfully induced long-term graft acceptance in a mouse skin graft model.3 it has been suggested that DST generates antispecific regulatory T cells and inhibits graft rejection; however, the underlying mechanisms have not been defined. For effective T-cell activation, naïve T cells require two distinct signals: the engagement of a T-cell receptor against antigen-specific signals, and the costimulatory signals. The CD28 molecule has been identified as a major receptor for costimulatory ligands, such as
CD80 and CD86. JJ316 and JJ319 are rat CD28-specific monoclonal antibodies (mAbs) with identical costimulatory potency; however, the JJ316 mAb has been shown to promote selectively the expansion of regulatory T cells in vivo and in vitro.4,5 The objective of this study was to assess the effects of single or multiple DST treatments in the absence or presence of anti-CD28 mAbs on graft function and host survival in a model of rat liver transplantation (LTx).
From the Department of Molecular and Cellular Physiology (H.U., D.V.O., M.B.G.), Louisiana State University Health Sciences Center, Shreveport, Louisiana; and Institute for Virology and Immunobiology (T.H.), University of Würzburg, Germany. Address reprint requests to Hidejiro Urakami, MD, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130. E-mail: [email protected]
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.042
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3244
Transplantation Proceedings, 38, 3244 –3246 (2006)
DONOR-SPECIFIC BLOOD TRANSFUSION WITH ANTI-CD28
3245
MATERIALS AND METHODS Fully MHC-mismatched adult male Dark Agouti (DA, RT1a) and Lewis (LEW, RT11) rats were used as donor and recipients, respectively. One milliliter of heparinized DA blood was administered intravenously to naïve LEW rats 7 days before LTx [DST(⫺7d)], 14 and 7 days before LTx [DST(1⫻2)], twice a week for 2 weeks prior to LTx [DST(2⫻2)], and once a week for 4 weeks prior to LTx [DST(1⫻4)]. Brown Norway (BN, RT1n) rat blood was used as third-party blood donor to examine the donor specificity of DST. One milligram of two different mAbs to rat CD28 (JJ316 and JJ319) were administered 4 days prior to transplantation in combination with DST treatments in the DST(1⫻2) and DST(2⫻2) groups. Orthotopic rat liver transplantation was performed without hepatic artery reconstruction. Rats were monitored daily for signs of illness or distress for more than 100 days after surgery. Graft rejection was defined by death of the recipient. Long-term survivors were harvested 100 days after surgery, and flow cytometric analyses of the splenocytes were done regarding rat T-cell surface markers such as CD4, CD25, and CD45RC as well as intracellular marker such as foxp3 to examine which subset of T cells would contribute to prolong graft survival.
RESULTS
We found that DST administration induced a time- and dose-dependent increase in host survival ranging from 0% survival for rats that did not receive DST to 25%, 50%, 75%, and 88% survival for the DST (⫺7d), DST (1⫻2), DST (2⫻2), and DST (1⫻4) treated animals, respectively, at 100 days following LTx (Fig 1). LEW rats treated with third-party BN blood prior to LTx all died within 11 to 12 days postsurgery (Fig 1). On the other hand, treatment of LEW rats with JJ316 or JJ319 mAb alone (4 mg/kg) failed to prolong graft survival over untreated rats; however, the combination of DST (1⫻2) with JJ316 or JJ319 mAb induced indefinite survival (100%) at 100 days following surgery (Fig 2). In addition, the combination of JJ319 mAb with DST (⫺7d) treatment induced indefinite survival, whereas the combination of JJ316 mAb with DST (⫺7d) induced 50% survival (Fig 2). We found that this protective
Fig 1. DST administration induced a time- and dosedependent increase in host survival.
Fig 2. Treatment of LEW rats with JJ316 or JJ319 mAb alone (4 mg/kg) failed to prolong graft survival over untreated rats; however, the combination of DST (1⫻2) with JJ316 or JJ319 mAb induced indefinite survival (100%) at 100 days following surgery.
effect was associated with increased numbers of splenic CD4⫹CD45RC⫺ but not CD4⫹CD25⫹foxp3⫹ T-cells in the long-term survivors. DISCUSSION
The mechanisms of the blood transfusion effect were not clearly understood; however, DST remains a promising strategy to establish tolerance.6 – 8 In this study, we developed a rat liver transplant model in which tolerance is induced by DST alone without the need of immunosuppression. In addition, those effect was time and dose dependent of DST administration as shown in Fig 1. Moreover, we found that neither JJ316 nor JJ319 mAb alone prolonged graft survival over untreated rats; however, the combination of DST with JJ316 or JJ319 mAb successfully prolonged graft survival. Some groups say that CD28 mAb effectively extended the graft survival in a skin and kidney transplantation model; however, those effects were quite limited. We believe that these effects are related to the organ specificity such as liver passenger leukocytes and biliary system in liver. In addition, we speculate that this shows that CD28 mAb enhances the donor-specific tolerance in combination with DST compared with DST alone. Thus we did not investigate the effect of CD28 mAb in a third-party blood transfused group. In this series, we demonstrated that treatment of JJ319 mAb leads to long-term graft survival compared with JJ316 mAb under the cover of DST as shown in Fig 2. The long-term survivors showed a trend toward increased numbers of CD4⫹CD45RC⫺ T cell unlikely in the CD4⫹CD25⫹foxp3⫹ T-cell subset, which is generally recognized as regulatory T cell. This was not statistically significant. Taken together, our data suggest that the combination of suboptimal DST with CD28 mAb generates donor-specific tolerance that correlates with enhanced numbers of CD4⫹CD45RC⫺regulatory T cells.
3246
REFERENCES 1. Quigley RL, Wood KJ, Morris PJ: Transfusion induces blood donor-specific suppressor cells. J Immunol 142:463, 1989 2. Flye MW, Burton K, Mohanakumar T, et al: Donor-specific transfusions have long-term beneficial effects for human renal allografts. Transplantation 60:1395, 1995 3. Bushell A, Karim M, Kingsley CI, et al: Pretransplant blood transfusion without additional immunotherapy generates CD25⫹CD4⫹ regulatory T cells: a potential explanation for the blood-transfusion effect. Transplantation 76:449, 2003 4. Tacke M, Hanke G, Hanke T, et al: CD28-mediated induction of proliferation in resting T cells in vitro and in vivo
URAKAMI, OSTANIN, HU¨NIG ET AL without engagement of the T cell receptor: evidence for functionally distinct forms of CD28. Eur J Immunol 27:239, 1997 5. Lin CH, Hunig T: Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist. Eur J Immunol 33:626, 2003 6. Potter DE, Portale AA, Melzer JS, et al: Are blood transfusions beneficial in the cyclosporine era? Pediatr Nephrol 5:168, 1991 7. van Twuyver E, Mooijaart RJ, ten Berge IJ, et al: Pretransplantation blood transfusion revisited. N Engl J Med 325:1210, 1991 8. Auchincloss H Jr: In search of the elusive Holy Grail: the mechanisms and prospects for achieving clinical transplantation tolerance. Am J Transplant 1:6, 2001
T
HE DEVELOPMENT of potent immunosuppressive drugs has improved long-term outcome in clinical transplantation. However, graft loss related to chronic rejection, drug toxicity, or infections remains a substantial concern. Donor-specific blood transfusion (DST) is an efficient strategy to induce hyporesponsiveness without additional immunotherapy, both clinically and experimentally.1,2 Recent studies have demonstrated that a single DST failed to induce long-term graft acceptance; however, multiple DSTs successfully induced long-term graft acceptance in a mouse skin graft model.3 it has been suggested that DST generates antispecific regulatory T cells and inhibits graft rejection; however, the underlying mechanisms have not been defined. For effective T-cell activation, naïve T cells require two distinct signals: the engagement of a T-cell receptor against antigen-specific signals, and the costimulatory signals. The CD28 molecule has been identified as a major receptor for costimulatory ligands, such as
CD80 and CD86. JJ316 and JJ319 are rat CD28-specific monoclonal antibodies (mAbs) with identical costimulatory potency; however, the JJ316 mAb has been shown to promote selectively the expansion of regulatory T cells in vivo and in vitro.4,5 The objective of this study was to assess the effects of single or multiple DST treatments in the absence or presence of anti-CD28 mAbs on graft function and host survival in a model of rat liver transplantation (LTx).
From the Department of Molecular and Cellular Physiology (H.U., D.V.O., M.B.G.), Louisiana State University Health Sciences Center, Shreveport, Louisiana; and Institute for Virology and Immunobiology (T.H.), University of Würzburg, Germany. Address reprint requests to Hidejiro Urakami, MD, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130. E-mail: [email protected]
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.042
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3244
Transplantation Proceedings, 38, 3244 –3246 (2006)
DONOR-SPECIFIC BLOOD TRANSFUSION WITH ANTI-CD28
3245
MATERIALS AND METHODS Fully MHC-mismatched adult male Dark Agouti (DA, RT1a) and Lewis (LEW, RT11) rats were used as donor and recipients, respectively. One milliliter of heparinized DA blood was administered intravenously to naïve LEW rats 7 days before LTx [DST(⫺7d)], 14 and 7 days before LTx [DST(1⫻2)], twice a week for 2 weeks prior to LTx [DST(2⫻2)], and once a week for 4 weeks prior to LTx [DST(1⫻4)]. Brown Norway (BN, RT1n) rat blood was used as third-party blood donor to examine the donor specificity of DST. One milligram of two different mAbs to rat CD28 (JJ316 and JJ319) were administered 4 days prior to transplantation in combination with DST treatments in the DST(1⫻2) and DST(2⫻2) groups. Orthotopic rat liver transplantation was performed without hepatic artery reconstruction. Rats were monitored daily for signs of illness or distress for more than 100 days after surgery. Graft rejection was defined by death of the recipient. Long-term survivors were harvested 100 days after surgery, and flow cytometric analyses of the splenocytes were done regarding rat T-cell surface markers such as CD4, CD25, and CD45RC as well as intracellular marker such as foxp3 to examine which subset of T cells would contribute to prolong graft survival.
RESULTS
We found that DST administration induced a time- and dose-dependent increase in host survival ranging from 0% survival for rats that did not receive DST to 25%, 50%, 75%, and 88% survival for the DST (⫺7d), DST (1⫻2), DST (2⫻2), and DST (1⫻4) treated animals, respectively, at 100 days following LTx (Fig 1). LEW rats treated with third-party BN blood prior to LTx all died within 11 to 12 days postsurgery (Fig 1). On the other hand, treatment of LEW rats with JJ316 or JJ319 mAb alone (4 mg/kg) failed to prolong graft survival over untreated rats; however, the combination of DST (1⫻2) with JJ316 or JJ319 mAb induced indefinite survival (100%) at 100 days following surgery (Fig 2). In addition, the combination of JJ319 mAb with DST (⫺7d) treatment induced indefinite survival, whereas the combination of JJ316 mAb with DST (⫺7d) induced 50% survival (Fig 2). We found that this protective
Fig 1. DST administration induced a time- and dosedependent increase in host survival.
Fig 2. Treatment of LEW rats with JJ316 or JJ319 mAb alone (4 mg/kg) failed to prolong graft survival over untreated rats; however, the combination of DST (1⫻2) with JJ316 or JJ319 mAb induced indefinite survival (100%) at 100 days following surgery.
effect was associated with increased numbers of splenic CD4⫹CD45RC⫺ but not CD4⫹CD25⫹foxp3⫹ T-cells in the long-term survivors. DISCUSSION
The mechanisms of the blood transfusion effect were not clearly understood; however, DST remains a promising strategy to establish tolerance.6 – 8 In this study, we developed a rat liver transplant model in which tolerance is induced by DST alone without the need of immunosuppression. In addition, those effect was time and dose dependent of DST administration as shown in Fig 1. Moreover, we found that neither JJ316 nor JJ319 mAb alone prolonged graft survival over untreated rats; however, the combination of DST with JJ316 or JJ319 mAb successfully prolonged graft survival. Some groups say that CD28 mAb effectively extended the graft survival in a skin and kidney transplantation model; however, those effects were quite limited. We believe that these effects are related to the organ specificity such as liver passenger leukocytes and biliary system in liver. In addition, we speculate that this shows that CD28 mAb enhances the donor-specific tolerance in combination with DST compared with DST alone. Thus we did not investigate the effect of CD28 mAb in a third-party blood transfused group. In this series, we demonstrated that treatment of JJ319 mAb leads to long-term graft survival compared with JJ316 mAb under the cover of DST as shown in Fig 2. The long-term survivors showed a trend toward increased numbers of CD4⫹CD45RC⫺ T cell unlikely in the CD4⫹CD25⫹foxp3⫹ T-cell subset, which is generally recognized as regulatory T cell. This was not statistically significant. Taken together, our data suggest that the combination of suboptimal DST with CD28 mAb generates donor-specific tolerance that correlates with enhanced numbers of CD4⫹CD45RC⫺regulatory T cells.
3246
REFERENCES 1. Quigley RL, Wood KJ, Morris PJ: Transfusion induces blood donor-specific suppressor cells. J Immunol 142:463, 1989 2. Flye MW, Burton K, Mohanakumar T, et al: Donor-specific transfusions have long-term beneficial effects for human renal allografts. Transplantation 60:1395, 1995 3. Bushell A, Karim M, Kingsley CI, et al: Pretransplant blood transfusion without additional immunotherapy generates CD25⫹CD4⫹ regulatory T cells: a potential explanation for the blood-transfusion effect. Transplantation 76:449, 2003 4. Tacke M, Hanke G, Hanke T, et al: CD28-mediated induction of proliferation in resting T cells in vitro and in vivo
URAKAMI, OSTANIN, HU¨NIG ET AL without engagement of the T cell receptor: evidence for functionally distinct forms of CD28. Eur J Immunol 27:239, 1997 5. Lin CH, Hunig T: Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist. Eur J Immunol 33:626, 2003 6. Potter DE, Portale AA, Melzer JS, et al: Are blood transfusions beneficial in the cyclosporine era? Pediatr Nephrol 5:168, 1991 7. van Twuyver E, Mooijaart RJ, ten Berge IJ, et al: Pretransplantation blood transfusion revisited. N Engl J Med 325:1210, 1991 8. Auchincloss H Jr: In search of the elusive Holy Grail: the mechanisms and prospects for achieving clinical transplantation tolerance. Am J Transplant 1:6, 2001