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Adenovirus-mediated gene transfer of CTLA4Ig to liver allografts results in prolonged survival and local T-Cell anergy
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Adenovirus-Mediated Gene Transfer of CTLA4Ig to Liver Allografts Results in Prolonged Survival and Local T-Cell Anergy K.M. Olthoff, X. Da Chen, A. Gelman, L. Turka, and A. Shaked
T
CELL-mediated alloimmune responses are an integral part of the rejection process. Activation of the T cell requires major histocompatibility complex presentation by the antigen-presenting cells to the T-cell receptor. Recent evidence has confirmed that an additional costimulatory signal, such as B7/CD28:CTLA4, is necessary for T-cell stimulation, and inhibition of this pathway during T cell activation produces a state of T-cell anergy.’ CTLA4Ig, a recombinant fusion protein containing the extracellular domain of human CTLA4 fused to the human IgGl region, effectively binds the B7 molecule, interrupting the costimulatory pathway.233 The use of CTLA4Ig in transplantation is currently being explored in several solid organ and animal models. Thus far, several studies have achieved prolonged survival and donor-specific transplantation tolerance in mouse and rat cardiac, renal, and islet cell allograft models with the use of CTLA41g.4-6 Viral-mediated gene transfer is an alternative approach to immunoregulation with local expression of functional genes within the graft. ‘,’ To compare local and systemic administration of CTLA4Ig on the survival and rejection pattern of liver allografts, we treated recipients with either systemic administration of CTLA4Ig or transplanted grafts engineered to locally express CTLA4Ig. METHODS Orthotopic rat liver transplant was performed in the highresponder ACI-to-Lewis combination. A replication-deficient adenoviral vector containing an expression cassette encoding murine CILA4Ig replacing the viral EI region was constructed. Ex vivo gene transfer into liver grafts was performed during cold preservation via perfusion of the portal vein with cold lactated Ringer’s solution containing AdmCTLA4Ig at a vector-to-hepatocyte ratio of 1OO:l. Systemic treatment with CTLA4Ig was achieved by treating the animals with a single intraperitoneal injection of 1 mg of recombinant CTLA4Ig on the second postoperative day (POD). Orthotopic liver transplant was performed in the following combinations: (1) Lew to Lew syngeneic controls; (2) AC1 to Lew allogeneic controls with grafts perfused only with lactated Ringer’s solution; (3) AC1 to Lew allogeneic grafts infused with AdmCTLA4Ig; (4) AC1 to Lew treated with systemic injection of CTLA4Ig on POD2; (5) AC1 to Lew allogeneic controls treated with systemic injection of 1 mg hlg on POD2. Six animals per group were transplanted and kept alive for survival studies. Additional animals in each group were sacrificed on postoperative days 2,4, 8, 14, 20, and 40 for histology and cytokine analysis. 0041-1315/97/$17.00 PII SO041 -1345(96)00355-7
Table 1. Experimental Groups and Survival Outcomes Group 1. Syngeneic
n
2. No treatment
2 6
3. AdmCTLA4lg, MOI 1OO:l 4. CTLA4lg 1 mg IP POD2 5. Human lg 1 mg IP POD2
6 6 6
Survival(d) >lOO, >lOO, ZlOO lO,lO, lO,ll, 11, 12 43, 280, >80, 280, 85, >lOO >40, 49, 63, 87, r100, >lOO 10, 11, ll,ll, 11, 12
Intragraft cytokine and adenoviral CTLA4Ig analysis was performed by semiquantitative reverse transcriptase polymerase chain reaction (rt-PCR). Cellular RNA isolates were prepared from quick-frozen tissue by homogenization and then purified over silica gel columns. Reverse transcription and DNA amplification were carried out with “‘P-labeled primers and then electrophoresed in agarose gels. RESULTS
Untreated controls and Ig-treated animals (control groups 2 and 5) acutely rejected their grafts and died by day 12. Successful transduction and local production of CTLA4Ig resulted in indefinite survival, similar to the syngeneic controls. Treatment with systemic injection of recombinant CTLA4Ig (group 4) also resulted in prolonged survival (Table 1). To analyze the pathologic effects on the allograft, control and transduced animals were sacrificed on POD 2, 4, 8, 14, 20, and 40 and evaluated histologically. Histologic evidence of progressive acute rejection resulting in graft failure was present in untreated allografts. This was manifested by a severe mononuclear infiltrate and apoptotic cell death by POD 4, with massive tissue destruction and complete obliteration of the portal triad by POD 8. The adenoviraltreated animals also demonstrated a significant mononuclear infiltrate by POD 8, associated with endothelitis and mild apoptosis. However, his infiltrate was delayed, less intense, and associated with less parenchymal damage than From the Departments of Surgery and Medicine (L.T.), University of Pennsylvania, Philadelphia, Pennsylvania. Supported in pat-l by the Thomas B. McCabe and Jeannette E. Laws McCabe Fund (KO) and NIH Grant DK 46311 (AS). Address reprint requests to Kim M. Olthoff, MD, Department of Surgery, University of Pennsylvania, 4 Silverstein, 3400 Spruce Street, Philadelphia, PA 19104.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1030
Transplantation
Proceedings,
29, 1030-l 031 (1997)
CTLA41G IN LIVER ALLOGRA!=TS
in the untreated controls. The inflammatory process then progressed to moderate-to-severe triaditis with mild parenchymal damage. By POD 20, this response had decreased, but the portal lymphocytes were still present, and the infiltrate persisted well beyond 40 days. Of interest was a marked bile ductular proliferation in the livers that survived long term. Similarly, the animals treated with the fusion protein CTLA4Ig demonstrated a periportal infiltrate and bile duct proliferation that also remained far beyond the transplant period. Intragraft cytokine levels were measured by rt-PCR. There was an initial surge in IFN-y and TNF-a levels within the graft that persisted in the untreated allogeneic controls until death but decreased to background levels by POD 20. CONCLUSIONS
These studies demonstrate that gene transfer with local expression of sequences encoding T-cell inhibitory proteins prolongs graft survival, as does systemic therapy with a single dose of CTLA4Ig. Local immunomodulation appears
to be as effective as systemic treatment. The persistent mononuclear infiltrate without parenchymal damage seen in the CTLA4Ig-treated grafts is consistent with a pattern of immune inhibition by T-cell anergy. An additional aspect of the mechanism of action may be interference with the cytokine cascade, as evidenced by the lower levels of IFN-y and TNF-IX in the treated animals.
REFERENCES
1. Schwartz RH: Science 248:1349, 1990 2. Wu Y, Guo Y, Liu Y: J Exp Med 178:1789, 1993 3. Tan P, Anasetti C, Hansen JA, et al: J Exp Med 177:165,1993 4. Boiling SF, Lin H, Wei RQ, et al: J Surg Res 57:60, 1994 5. Sayegh MH, Akalin E, Hancock Ww, et al: J Exp Med 181:1869, 1995 6. Lenschow DJ, Zeng Y, Thistlethwaite JR, et al: Science 257:789, 1992 7. Quin L, Chavin KD, Ding Y, et al: Ann Surg 220:508, 1994 8. Shaked A, Csete ME, Drazan KE, et al: Transplantation 57:1508, 1994
Adenovirus-Mediated Gene Transfer of CTLA4Ig to Liver Allografts Results in Prolonged Survival and Local T-Cell Anergy K.M. Olthoff, X. Da Chen, A. Gelman, L. Turka, and A. Shaked
T
CELL-mediated alloimmune responses are an integral part of the rejection process. Activation of the T cell requires major histocompatibility complex presentation by the antigen-presenting cells to the T-cell receptor. Recent evidence has confirmed that an additional costimulatory signal, such as B7/CD28:CTLA4, is necessary for T-cell stimulation, and inhibition of this pathway during T cell activation produces a state of T-cell anergy.’ CTLA4Ig, a recombinant fusion protein containing the extracellular domain of human CTLA4 fused to the human IgGl region, effectively binds the B7 molecule, interrupting the costimulatory pathway.233 The use of CTLA4Ig in transplantation is currently being explored in several solid organ and animal models. Thus far, several studies have achieved prolonged survival and donor-specific transplantation tolerance in mouse and rat cardiac, renal, and islet cell allograft models with the use of CTLA41g.4-6 Viral-mediated gene transfer is an alternative approach to immunoregulation with local expression of functional genes within the graft. ‘,’ To compare local and systemic administration of CTLA4Ig on the survival and rejection pattern of liver allografts, we treated recipients with either systemic administration of CTLA4Ig or transplanted grafts engineered to locally express CTLA4Ig. METHODS Orthotopic rat liver transplant was performed in the highresponder ACI-to-Lewis combination. A replication-deficient adenoviral vector containing an expression cassette encoding murine CILA4Ig replacing the viral EI region was constructed. Ex vivo gene transfer into liver grafts was performed during cold preservation via perfusion of the portal vein with cold lactated Ringer’s solution containing AdmCTLA4Ig at a vector-to-hepatocyte ratio of 1OO:l. Systemic treatment with CTLA4Ig was achieved by treating the animals with a single intraperitoneal injection of 1 mg of recombinant CTLA4Ig on the second postoperative day (POD). Orthotopic liver transplant was performed in the following combinations: (1) Lew to Lew syngeneic controls; (2) AC1 to Lew allogeneic controls with grafts perfused only with lactated Ringer’s solution; (3) AC1 to Lew allogeneic grafts infused with AdmCTLA4Ig; (4) AC1 to Lew treated with systemic injection of CTLA4Ig on POD2; (5) AC1 to Lew allogeneic controls treated with systemic injection of 1 mg hlg on POD2. Six animals per group were transplanted and kept alive for survival studies. Additional animals in each group were sacrificed on postoperative days 2,4, 8, 14, 20, and 40 for histology and cytokine analysis. 0041-1315/97/$17.00 PII SO041 -1345(96)00355-7
Table 1. Experimental Groups and Survival Outcomes Group 1. Syngeneic
n
2. No treatment
2 6
3. AdmCTLA4lg, MOI 1OO:l 4. CTLA4lg 1 mg IP POD2 5. Human lg 1 mg IP POD2
6 6 6
Survival(d) >lOO, >lOO, ZlOO lO,lO, lO,ll, 11, 12 43, 280, >80, 280, 85, >lOO >40, 49, 63, 87, r100, >lOO 10, 11, ll,ll, 11, 12
Intragraft cytokine and adenoviral CTLA4Ig analysis was performed by semiquantitative reverse transcriptase polymerase chain reaction (rt-PCR). Cellular RNA isolates were prepared from quick-frozen tissue by homogenization and then purified over silica gel columns. Reverse transcription and DNA amplification were carried out with “‘P-labeled primers and then electrophoresed in agarose gels. RESULTS
Untreated controls and Ig-treated animals (control groups 2 and 5) acutely rejected their grafts and died by day 12. Successful transduction and local production of CTLA4Ig resulted in indefinite survival, similar to the syngeneic controls. Treatment with systemic injection of recombinant CTLA4Ig (group 4) also resulted in prolonged survival (Table 1). To analyze the pathologic effects on the allograft, control and transduced animals were sacrificed on POD 2, 4, 8, 14, 20, and 40 and evaluated histologically. Histologic evidence of progressive acute rejection resulting in graft failure was present in untreated allografts. This was manifested by a severe mononuclear infiltrate and apoptotic cell death by POD 4, with massive tissue destruction and complete obliteration of the portal triad by POD 8. The adenoviraltreated animals also demonstrated a significant mononuclear infiltrate by POD 8, associated with endothelitis and mild apoptosis. However, his infiltrate was delayed, less intense, and associated with less parenchymal damage than From the Departments of Surgery and Medicine (L.T.), University of Pennsylvania, Philadelphia, Pennsylvania. Supported in pat-l by the Thomas B. McCabe and Jeannette E. Laws McCabe Fund (KO) and NIH Grant DK 46311 (AS). Address reprint requests to Kim M. Olthoff, MD, Department of Surgery, University of Pennsylvania, 4 Silverstein, 3400 Spruce Street, Philadelphia, PA 19104.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1030
Transplantation
Proceedings,
29, 1030-l 031 (1997)
CTLA41G IN LIVER ALLOGRA!=TS
in the untreated controls. The inflammatory process then progressed to moderate-to-severe triaditis with mild parenchymal damage. By POD 20, this response had decreased, but the portal lymphocytes were still present, and the infiltrate persisted well beyond 40 days. Of interest was a marked bile ductular proliferation in the livers that survived long term. Similarly, the animals treated with the fusion protein CTLA4Ig demonstrated a periportal infiltrate and bile duct proliferation that also remained far beyond the transplant period. Intragraft cytokine levels were measured by rt-PCR. There was an initial surge in IFN-y and TNF-a levels within the graft that persisted in the untreated allogeneic controls until death but decreased to background levels by POD 20. CONCLUSIONS
These studies demonstrate that gene transfer with local expression of sequences encoding T-cell inhibitory proteins prolongs graft survival, as does systemic therapy with a single dose of CTLA4Ig. Local immunomodulation appears
to be as effective as systemic treatment. The persistent mononuclear infiltrate without parenchymal damage seen in the CTLA4Ig-treated grafts is consistent with a pattern of immune inhibition by T-cell anergy. An additional aspect of the mechanism of action may be interference with the cytokine cascade, as evidenced by the lower levels of IFN-y and TNF-IX in the treated animals.
REFERENCES
1. Schwartz RH: Science 248:1349, 1990 2. Wu Y, Guo Y, Liu Y: J Exp Med 178:1789, 1993 3. Tan P, Anasetti C, Hansen JA, et al: J Exp Med 177:165,1993 4. Boiling SF, Lin H, Wei RQ, et al: J Surg Res 57:60, 1994 5. Sayegh MH, Akalin E, Hancock Ww, et al: J Exp Med 181:1869, 1995 6. Lenschow DJ, Zeng Y, Thistlethwaite JR, et al: Science 257:789, 1992 7. Quin L, Chavin KD, Ding Y, et al: Ann Surg 220:508, 1994 8. Shaked A, Csete ME, Drazan KE, et al: Transplantation 57:1508, 1994