- Email: [email protected]
31: Mixed hematopoietic chimerism prevents lung allograft rejection in miniature swine
The Journal of Heart and Lung Transplantation Volume 26, Number 2S
and their reasons. Subsequent biopsy results were recorded. Sustained significant rejection (SSR) was defined as ISHLT ⱖ 2A on two successive biopsies. Students T-test was used to compare rates of rejection between populations. A p-value of ⱕ 0.05 was defined as significant. Results: 189 patients were followed between 1999 and 2006. The mean age at transplant was 48 years, and 80% were male. 98% of patients had MMF as part of their anti-rejection regimen, and 71% of these patients had their medication dose reduced at some point. The most common reasons for dose reduction of MMF were leucopenia (46%), GI intolerance (33%), and infections (23%). The prevalence of sustained significant rejection (SSR) in the GI intolerant group versus those patients who had no MMF dose reductions was 66% vs 35% (p⫽.002). The SSR rate for patients with MMF dose reductions due to leukopenia or infection was not significantly different compared to those patients who never had a dose reduction (Leukopenia: 35% v 36%, p⫽1.0; Infection: 35% v 43%, p⫽0.5). The rate of SSR was significantly higher in patients with MMF dose reductions due to GI intolerance versus those with dose reductions for non-GI reasons (67% vs 35%, p⫽0.003). Conclusions: MMF dose reduction due to GI intolerance was associated with a significantly increased rate of sustained rejection compared to patients maintained on target doses or who had dose reduction for non-GI reasons. MMF dose reductions for GI intolerance require careful follow-up due to this increased risk of rejection. 29 TROUGH LEVEL ADAPTED MMF IN COMBINATION WITH EITHER TACROLIMUS OR CYCLOSPORINE – THE PAN-EU-HTX TRIAL B. Meiser,1 J. Van Cleemput,2 F. Perez Villa,3 A. Zuckermann,9 M. Crespo,4 M. Goenen,5 K. Karason,6 J.F. Delgado,7 R. Voss,8 B. Reichart,1 1Cardiac Surgery, University of Munich, Munich, Germany; 2Cardiology, University Hospital Gasthuisberg, Leuven, Belgium; 3Heart Transplantation Program, University of Barcelona, Barcelona, Spain; 4Cardiac Surgery, Juan Canalejo Hospital, La Coruna, Spain; 5Cardiovascular Surgery, University of Louvain, Brussels, Belgium; 6Cardiology, Sahlgrenska University Hospital, Goteborg, Sweden; 7Cardiology, Hospital Doce de Octubre, Madrid, Spain; 8Cardiology, Univ. of Giessen, Giessen, Germany; 9Cardio-Thoracic Surgery, Univ. of Vienna, Vienna, Austria Purpose: Nineteen centers from 6 European countries participated in the Pan-European-HTx Trial. This prospective, centrally randomized investigator driven trial (sponsored by Roche and Astellas) was initiated to compare tacrolimus/MMF versus (vs) cyclosprine/MMF. In contradistinction to previous trials, MMF dosage was adapted to MPA trough level measurements in all patients (pts). Methods and Materials: Altogether 218 pts were randomized, 199 entered the study. Pts received either tacrolimus/MMF/steroids (n⫽ 104) or cyclosporine/MMF/steroids (n⫽95). Mean recipient age (years) was 53.2 vs 53.7, the male/female ratio (%) was 84/16 vs 78/22. Results: In an interim analysis, 183 pts could be evaluated revealing the following data (tacrolimus (n⫽95) vs cyclosporine (n⫽88)): Survival: 80% vs 88%, rejection incidence per patient: 0.2 vs 0.47, % of pts. rejection free: 83 vs 72, serum creatinine (mg/dl): 1.46 vs 1.53, blood glucose (mg/dl): 115 vs 109, diastol. blood pressure (mmHg): 87 vs 88, systol. blood pressure (mmHg): 129 vs 134. Conclusions: The interim analysis indicates a better survival in the cyclosporine group, while tacrolimus treated pts have a pronounced lower rejection incidence. Remarkably is the high percentage of pts. completely free of rejection in both groups which might be due to the MPA level adapted MMF application. Until the annual meeting, a more
Abstracts
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complete statistical analysis, including causes of death, no. of treatment switches, etc will be available. Research grants for clinical studies, consultant and medical advisor; Roche, Astellas. 30 TACROLIMUS WITH SIROLIMUS OR MYCOPHENOLATE MOFETIL (MMF) COMPARED WITH CALCINEURININHIBITORFREE IMMUNOSUPPRESSION (SIROLIMUS/MMF) AFTER HEART TRANSPLANTATION: 1-YEAR RESULTS I. Kaczmarek,1 O. Delgado,1 S. Michel,1 S. Sadoni,1 M. Schmoeckel,1 B. Reichart,1 B. Meiser,1 1Cardiac Surgery, University Hospital Grosshadern, LMU Munich, Munich, Germany Purpose: Despite improvements in immunosuppressive therapy the most advantageous combination for cardiac transplant recipients has yet to be found. Therefore we performed a randomized trial to evaluate the efficacy and safety of three immunosuppressive protocols. Methods and Materials: Between April 2003 and October 2005, 78 de novo cardiac transplant recipients were randomized on a 2:2:1 basis to receive either tacrolimus (TAC) ⫹ sirolimus (SRL)(n⫽31), TAC ⫹ mycophenolate mofetil (MMF)(n⫽31) or SRL ⫹ MMF (n⫽16). Antilymphocyte induction therapy was given for 5 days in the SRL ⫹ MMF group. Steroids were withdrawn after 6 months. Results: Freedom from biopsy proven rejection or hemodynamic compromise rejection requiring treatment at 1 year was TAC/MMF 100%, TAC/SRL 93.5%, SRL/MMF 86.7% resulting in a significantly lower incidence of rejection in the TAC/MMF group when compared to SRL/MMF (p ⫽ 0.042). Survival at 1 year was TAC/MMF 90.1%, TAC/SRL 100%, SRL/MMF 93.3% resulting in a trend to superior survival with TAC/SRL when compared to the TAC/MMF group (p⫽0.076). Causes of death in the TAC group were primary graft failure, pneumonia and sudden cardiac death. Two cases of allograft vasculopathy were detected after one year, both in the TAC/MMF group. Mean levels of serum creatinine at 1 year were TAC/MMF 1.7⫾0.7 mg/dL , TAC/SRL 1.6⫾0.6 mg/dL and SRL/MMF 1.3⫾0.5 mg/dL, revealing a trend to superior renal function in the SRL/MMF group. Total cholesterol at 1 year was TAC/MMF 160⫾47 mg/dL, TAC/SRL 185⫾40 mg/dL and SRL/MMF 197⫾36 mg/dL (p⫽0.036). In the SRL/MMF group 6 patients discontinued SRL treatment due to side effects (delayed wound healing in 3, rejection in 2 and ulcus ventriculi in 1). Conclusions: Both tacrolimus groups proved to be efficacious for the prevention of acute rejection. The side effect profile for the SRL/MMF group is inferior to the TAC groups resulting in a high discontinuation rate while this calcineurininhibitor-free protocol results in less nephrotoxicity. 31 MIXED HEMATOPOIETIC CHIMERISM PREVENTS LUNG ALLOGRAFT REJECTION IN MINIATURE SWINE H. Sahara,1 M.J. Weiss,1 K.J. Wikiel,1 J.K. Sayre,1 A.C. Pujara,1 C.Y. Ng,1 P.L. Cho,1 B.M. Horner,1 S.L. Houser,1 J.C. Wain,1 D.H. Sachs,1 J.C. Madsen,1 C.A. Huang,1 J.S. Allan,1 1 Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA Purpose: The induction of mixed hematopoietic chimerism is a robust method of achieving tolerance in a variety of experimental and clinical settings. The aim of this study is to determine whether the establishment of stable mixed chimerism through hematopoietic cell transplantation (HCT) can prevent lung allograft rejection in a pre-clinical miniature swine model.
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Abstracts
Methods and Materials: SLAad swine (class Ia/d, class IIa/d) received a one-haplotype, fully mismatched SLAac (class Ia/c, class IIa/c) HCT using a non-myeloablative conditioning regimen, consisting of 1 Gy of total body irradiation, T-cell depletion, and a 45-day course of cyclosporine. Three engrafted animals that maintained multi-lineage chimerism for over 28 weeks then received left lung allografts from the HCT donor without any immunosuppression. As a control, two SLAad animals received SLAac lungs without immunosuppression. Donor cell chimerism and engraftment were assessed by flow cytometry and polymerase chain reaction. In vitro reactivity to donor cells was tested with assays of mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML). Results: Both control recipients acutely rejected their grafts on POD 6. In contrast, the chimeric recipients have shown longer graft survival (⬎312, ⬎33 days [experiment in progress], and ⬎49 days [graft loss due to obstructive pneumonitis without rejection]). All animals have maintained engraftment and multi-lineage chimerism after lung transplantation. MLR and primary CML assays have shown donor-specific hyporesponsiveness; and results from secondary CML co-culture assays are consistent with both deletional and regulatory mechanisms of tolerance. Conclusions: Mixed chimerism induced by HCT can induce longterm acceptance of lung allografts without ongoing immunosuppression in a pre-clinical large-animal model. In vitro data suggest that the observed tolerance may be due to a combination of deletion and regulation. Further observation is required to determine whether this approach can prevent chronic rejection. 32 HIGH SUPPRESSIVE POTENCY OF ISOLATED CD25ⴙ T CELLS IN PORCINE ALLOGENEIC LUNG TRANSPLANTATION IN VITRO S. Thissen,1 B. Kruse,1 G. Warnecke,1 M. Avsar,1 R. Reinhard,1 C. Peters,1 A.R. Simon,1 A. Haverich,1 M. Strueber,1 1Div. of Thoracic and CV Surgery, Hannover Medical School, Hannover, Germany Purpose: In vivo, an increased frequency of CD25⫹ T-cells is associated with markedly prolonged allograft survival in our porcine model. Here, we wished to study the in vitro suppressive potency of CD25⫹ T-cells. Methods and Materials: Left sided single lung transplantation from MHC-mismatched donors was performed in 8 adult minipigs. Animals received a 28 day course of immunosuppression with methylprednisolone and azathioprine, in combination with either cyclosporine⫹everolimus or tacrolimus. Some pigs underwent whole body irradiation and thymic irradiation before surgery. According to allograft survival, animals were divided into an acutely rejecting group (n⫽4; allograft survival ⬍90 post operative days, POD) and long term survivors (n⫽4; ⬎200 POD). CD25⫹ lymphocytes were positively selected from PBMC by magnetic beads separation. Purity monitored by FACS analysis was 85 %. In vitro suppression was determined by MLR. Recipient PBMC were incubated at a 1:1 ratio with irradiated donor, third party PBMCs or control mitogen. CD25⫹ lymphocytes were added in a 1:7, 1:10, 1:20 and 1:70 ratio. Results: Numbers of circulating CD25⫹CD4⫹ T cells were significantly higher in long term survivors than in rejectors. CD25⫹ lymphocytes in a 1:7 ratio suppressed MLR responses to donor antigen significantly both in long term survivors (LTS: stimulation index SI 3.23⫾3.67, CD25⫹ 1:7 SI 0.01⫾0.44; p⫽0.026) and rejectors (R: SI 4.98⫾1.81, CD25⫹ 1:7 SI 0.02⫾0.44; p⫽0.0003). The same effect was obvious for third party antigen as well as control mitogen in both groups. An attenuating suppressive function was detectable in both groups in a 1:10 and 1:20 ratio, but not in a 1:70 concentration.
The Journal of Heart and Lung Transplantation February 2007
Conclusions: CD25⫹ lymphocytes in vitro have a high suppressive, dose dependent potency. Suppression is comparable in long term surviving and rejecting animals. Prolonged allograft survival is more likely to depend on higher numbers of CD25⫹ T-cells in vivo than on a more efficient suppressive effect of these cells in long term surviving animals. 33 ACUTE REJECTION IS ASSOCIATED WITH HIGH FOXP3 MRNA EXPRESSION LEVELS IN THE GRAFT, BUT NOT IN THE PERIPHERAL BLOOD, OF HEART TRANSPLANT RECIPIENTS I.E. Dijke,1 K. Caliskan,2 S.S. Korevaar,1 A.H. Balk,2 P.E. Zondervan,3 A.P. Maat,2 W. Weimar,1 C.C. Baan,1 1Internal Medicine, Erasmus MC, Rotterdam, Netherlands; 2Thorax Center, Erasmus MC, Rotterdam, Netherlands; 3Pathology, Erasmus MC, Rotterdam, Netherlands Purpose: Regulatory FOXP3⫹ T cells are required for the control of immune responses of antigen-activated T cells. Yet, the role of FOXP3⫹ T cells and their dynamics in the regulation of anti-donor responses after clinical heart transplantation are still poorly understood. In this study, we examined the FOXP3 mRNA levels in the allograft and in peripheral blood of heart transplant patients to reveal the associated immunological processes of rejection or quiescence and whether the FOXP3 expression levels could provide clues for non-invasive detecting of acute rejection. Methods and Materials: Quantitative real-time PCR was used to analyze the mRNA expression levels of the regulatory T-cell marker FOXP3 as well as the levels of CD3⑀, CD25 and the effector cytokine IL-2, in endomyocardial biopsies (EMB) (n⫽85) and in PBMC samples (n⫽97) of cardiac allograft recipients. Results: The FOXP3 mRNA levels were higher in EMB taken during acute rejection (n⫽12, ISHLT 2R or more) than in EMB without evidence of rejection (p⫽0.01). No relationship was found between the FOXP3 mRNA levels and the time after transplantation. In addition, the levels of CD3⑀, CD25 and IL-2 were elevated in the rejecting EMB compared with the non-rejecting biopsies (p⫽0.01, p⫽0.02 and p⫽0.006, respectively). In PBMC, however, no association was detected between mRNA expression levels of FOXP3, CD3⑀, and CD25 and ISHLT rejection grades or time after transplantation. Interestingly, the mRNA levels of IL-2 were lower in the PBMC of rejecting patients than in the patients with ISHLT 1R (p⫽0.04), suggesting that IL-2-producing cells may have migrated from the blood to the inflamed allograft. Conclusions: We conclude that, in contrast to peripheral blood, FOXP3 mRNA levels were specifically elevated within the allograft during acute rejection. These findings suggest that rejection of the transplanted organ induces FOXP3⫹ T cells in the graft in an attempt to control anti-donor responses. 34 FOXP3ⴙ T CELLS CAN BE EXPANDED FROM REJECTING HUMAN CARDIAC ALLOGRAFTS I.E. Dijke,1 J.H. Velthuis,1 A.H. Balk,2 P. de Kuiper,1 M. Klepper,1 K. Caliskan,2 W. Weimar,1 C.C. Baan,1 1Internal Medicine, Erasmus MC, Rotterdam, Netherlands; 2Cardiology, Erasmus MC, Rotterdam, Netherlands Purpose: In organ-transplant recipients, we and others have shown that immune responses directed at the graft may consist of two components: a destructive and a protective one, the latter mediated by FoxP3⫹ regulatory T cells. We examined whether these FoxP3⫹ T cells can be grown from endomyocardial biopsies (EMB).
and their reasons. Subsequent biopsy results were recorded. Sustained significant rejection (SSR) was defined as ISHLT ⱖ 2A on two successive biopsies. Students T-test was used to compare rates of rejection between populations. A p-value of ⱕ 0.05 was defined as significant. Results: 189 patients were followed between 1999 and 2006. The mean age at transplant was 48 years, and 80% were male. 98% of patients had MMF as part of their anti-rejection regimen, and 71% of these patients had their medication dose reduced at some point. The most common reasons for dose reduction of MMF were leucopenia (46%), GI intolerance (33%), and infections (23%). The prevalence of sustained significant rejection (SSR) in the GI intolerant group versus those patients who had no MMF dose reductions was 66% vs 35% (p⫽.002). The SSR rate for patients with MMF dose reductions due to leukopenia or infection was not significantly different compared to those patients who never had a dose reduction (Leukopenia: 35% v 36%, p⫽1.0; Infection: 35% v 43%, p⫽0.5). The rate of SSR was significantly higher in patients with MMF dose reductions due to GI intolerance versus those with dose reductions for non-GI reasons (67% vs 35%, p⫽0.003). Conclusions: MMF dose reduction due to GI intolerance was associated with a significantly increased rate of sustained rejection compared to patients maintained on target doses or who had dose reduction for non-GI reasons. MMF dose reductions for GI intolerance require careful follow-up due to this increased risk of rejection. 29 TROUGH LEVEL ADAPTED MMF IN COMBINATION WITH EITHER TACROLIMUS OR CYCLOSPORINE – THE PAN-EU-HTX TRIAL B. Meiser,1 J. Van Cleemput,2 F. Perez Villa,3 A. Zuckermann,9 M. Crespo,4 M. Goenen,5 K. Karason,6 J.F. Delgado,7 R. Voss,8 B. Reichart,1 1Cardiac Surgery, University of Munich, Munich, Germany; 2Cardiology, University Hospital Gasthuisberg, Leuven, Belgium; 3Heart Transplantation Program, University of Barcelona, Barcelona, Spain; 4Cardiac Surgery, Juan Canalejo Hospital, La Coruna, Spain; 5Cardiovascular Surgery, University of Louvain, Brussels, Belgium; 6Cardiology, Sahlgrenska University Hospital, Goteborg, Sweden; 7Cardiology, Hospital Doce de Octubre, Madrid, Spain; 8Cardiology, Univ. of Giessen, Giessen, Germany; 9Cardio-Thoracic Surgery, Univ. of Vienna, Vienna, Austria Purpose: Nineteen centers from 6 European countries participated in the Pan-European-HTx Trial. This prospective, centrally randomized investigator driven trial (sponsored by Roche and Astellas) was initiated to compare tacrolimus/MMF versus (vs) cyclosprine/MMF. In contradistinction to previous trials, MMF dosage was adapted to MPA trough level measurements in all patients (pts). Methods and Materials: Altogether 218 pts were randomized, 199 entered the study. Pts received either tacrolimus/MMF/steroids (n⫽ 104) or cyclosporine/MMF/steroids (n⫽95). Mean recipient age (years) was 53.2 vs 53.7, the male/female ratio (%) was 84/16 vs 78/22. Results: In an interim analysis, 183 pts could be evaluated revealing the following data (tacrolimus (n⫽95) vs cyclosporine (n⫽88)): Survival: 80% vs 88%, rejection incidence per patient: 0.2 vs 0.47, % of pts. rejection free: 83 vs 72, serum creatinine (mg/dl): 1.46 vs 1.53, blood glucose (mg/dl): 115 vs 109, diastol. blood pressure (mmHg): 87 vs 88, systol. blood pressure (mmHg): 129 vs 134. Conclusions: The interim analysis indicates a better survival in the cyclosporine group, while tacrolimus treated pts have a pronounced lower rejection incidence. Remarkably is the high percentage of pts. completely free of rejection in both groups which might be due to the MPA level adapted MMF application. Until the annual meeting, a more
Abstracts
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complete statistical analysis, including causes of death, no. of treatment switches, etc will be available. Research grants for clinical studies, consultant and medical advisor; Roche, Astellas. 30 TACROLIMUS WITH SIROLIMUS OR MYCOPHENOLATE MOFETIL (MMF) COMPARED WITH CALCINEURININHIBITORFREE IMMUNOSUPPRESSION (SIROLIMUS/MMF) AFTER HEART TRANSPLANTATION: 1-YEAR RESULTS I. Kaczmarek,1 O. Delgado,1 S. Michel,1 S. Sadoni,1 M. Schmoeckel,1 B. Reichart,1 B. Meiser,1 1Cardiac Surgery, University Hospital Grosshadern, LMU Munich, Munich, Germany Purpose: Despite improvements in immunosuppressive therapy the most advantageous combination for cardiac transplant recipients has yet to be found. Therefore we performed a randomized trial to evaluate the efficacy and safety of three immunosuppressive protocols. Methods and Materials: Between April 2003 and October 2005, 78 de novo cardiac transplant recipients were randomized on a 2:2:1 basis to receive either tacrolimus (TAC) ⫹ sirolimus (SRL)(n⫽31), TAC ⫹ mycophenolate mofetil (MMF)(n⫽31) or SRL ⫹ MMF (n⫽16). Antilymphocyte induction therapy was given for 5 days in the SRL ⫹ MMF group. Steroids were withdrawn after 6 months. Results: Freedom from biopsy proven rejection or hemodynamic compromise rejection requiring treatment at 1 year was TAC/MMF 100%, TAC/SRL 93.5%, SRL/MMF 86.7% resulting in a significantly lower incidence of rejection in the TAC/MMF group when compared to SRL/MMF (p ⫽ 0.042). Survival at 1 year was TAC/MMF 90.1%, TAC/SRL 100%, SRL/MMF 93.3% resulting in a trend to superior survival with TAC/SRL when compared to the TAC/MMF group (p⫽0.076). Causes of death in the TAC group were primary graft failure, pneumonia and sudden cardiac death. Two cases of allograft vasculopathy were detected after one year, both in the TAC/MMF group. Mean levels of serum creatinine at 1 year were TAC/MMF 1.7⫾0.7 mg/dL , TAC/SRL 1.6⫾0.6 mg/dL and SRL/MMF 1.3⫾0.5 mg/dL, revealing a trend to superior renal function in the SRL/MMF group. Total cholesterol at 1 year was TAC/MMF 160⫾47 mg/dL, TAC/SRL 185⫾40 mg/dL and SRL/MMF 197⫾36 mg/dL (p⫽0.036). In the SRL/MMF group 6 patients discontinued SRL treatment due to side effects (delayed wound healing in 3, rejection in 2 and ulcus ventriculi in 1). Conclusions: Both tacrolimus groups proved to be efficacious for the prevention of acute rejection. The side effect profile for the SRL/MMF group is inferior to the TAC groups resulting in a high discontinuation rate while this calcineurininhibitor-free protocol results in less nephrotoxicity. 31 MIXED HEMATOPOIETIC CHIMERISM PREVENTS LUNG ALLOGRAFT REJECTION IN MINIATURE SWINE H. Sahara,1 M.J. Weiss,1 K.J. Wikiel,1 J.K. Sayre,1 A.C. Pujara,1 C.Y. Ng,1 P.L. Cho,1 B.M. Horner,1 S.L. Houser,1 J.C. Wain,1 D.H. Sachs,1 J.C. Madsen,1 C.A. Huang,1 J.S. Allan,1 1 Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA Purpose: The induction of mixed hematopoietic chimerism is a robust method of achieving tolerance in a variety of experimental and clinical settings. The aim of this study is to determine whether the establishment of stable mixed chimerism through hematopoietic cell transplantation (HCT) can prevent lung allograft rejection in a pre-clinical miniature swine model.
S72
Abstracts
Methods and Materials: SLAad swine (class Ia/d, class IIa/d) received a one-haplotype, fully mismatched SLAac (class Ia/c, class IIa/c) HCT using a non-myeloablative conditioning regimen, consisting of 1 Gy of total body irradiation, T-cell depletion, and a 45-day course of cyclosporine. Three engrafted animals that maintained multi-lineage chimerism for over 28 weeks then received left lung allografts from the HCT donor without any immunosuppression. As a control, two SLAad animals received SLAac lungs without immunosuppression. Donor cell chimerism and engraftment were assessed by flow cytometry and polymerase chain reaction. In vitro reactivity to donor cells was tested with assays of mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML). Results: Both control recipients acutely rejected their grafts on POD 6. In contrast, the chimeric recipients have shown longer graft survival (⬎312, ⬎33 days [experiment in progress], and ⬎49 days [graft loss due to obstructive pneumonitis without rejection]). All animals have maintained engraftment and multi-lineage chimerism after lung transplantation. MLR and primary CML assays have shown donor-specific hyporesponsiveness; and results from secondary CML co-culture assays are consistent with both deletional and regulatory mechanisms of tolerance. Conclusions: Mixed chimerism induced by HCT can induce longterm acceptance of lung allografts without ongoing immunosuppression in a pre-clinical large-animal model. In vitro data suggest that the observed tolerance may be due to a combination of deletion and regulation. Further observation is required to determine whether this approach can prevent chronic rejection. 32 HIGH SUPPRESSIVE POTENCY OF ISOLATED CD25ⴙ T CELLS IN PORCINE ALLOGENEIC LUNG TRANSPLANTATION IN VITRO S. Thissen,1 B. Kruse,1 G. Warnecke,1 M. Avsar,1 R. Reinhard,1 C. Peters,1 A.R. Simon,1 A. Haverich,1 M. Strueber,1 1Div. of Thoracic and CV Surgery, Hannover Medical School, Hannover, Germany Purpose: In vivo, an increased frequency of CD25⫹ T-cells is associated with markedly prolonged allograft survival in our porcine model. Here, we wished to study the in vitro suppressive potency of CD25⫹ T-cells. Methods and Materials: Left sided single lung transplantation from MHC-mismatched donors was performed in 8 adult minipigs. Animals received a 28 day course of immunosuppression with methylprednisolone and azathioprine, in combination with either cyclosporine⫹everolimus or tacrolimus. Some pigs underwent whole body irradiation and thymic irradiation before surgery. According to allograft survival, animals were divided into an acutely rejecting group (n⫽4; allograft survival ⬍90 post operative days, POD) and long term survivors (n⫽4; ⬎200 POD). CD25⫹ lymphocytes were positively selected from PBMC by magnetic beads separation. Purity monitored by FACS analysis was 85 %. In vitro suppression was determined by MLR. Recipient PBMC were incubated at a 1:1 ratio with irradiated donor, third party PBMCs or control mitogen. CD25⫹ lymphocytes were added in a 1:7, 1:10, 1:20 and 1:70 ratio. Results: Numbers of circulating CD25⫹CD4⫹ T cells were significantly higher in long term survivors than in rejectors. CD25⫹ lymphocytes in a 1:7 ratio suppressed MLR responses to donor antigen significantly both in long term survivors (LTS: stimulation index SI 3.23⫾3.67, CD25⫹ 1:7 SI 0.01⫾0.44; p⫽0.026) and rejectors (R: SI 4.98⫾1.81, CD25⫹ 1:7 SI 0.02⫾0.44; p⫽0.0003). The same effect was obvious for third party antigen as well as control mitogen in both groups. An attenuating suppressive function was detectable in both groups in a 1:10 and 1:20 ratio, but not in a 1:70 concentration.
The Journal of Heart and Lung Transplantation February 2007
Conclusions: CD25⫹ lymphocytes in vitro have a high suppressive, dose dependent potency. Suppression is comparable in long term surviving and rejecting animals. Prolonged allograft survival is more likely to depend on higher numbers of CD25⫹ T-cells in vivo than on a more efficient suppressive effect of these cells in long term surviving animals. 33 ACUTE REJECTION IS ASSOCIATED WITH HIGH FOXP3 MRNA EXPRESSION LEVELS IN THE GRAFT, BUT NOT IN THE PERIPHERAL BLOOD, OF HEART TRANSPLANT RECIPIENTS I.E. Dijke,1 K. Caliskan,2 S.S. Korevaar,1 A.H. Balk,2 P.E. Zondervan,3 A.P. Maat,2 W. Weimar,1 C.C. Baan,1 1Internal Medicine, Erasmus MC, Rotterdam, Netherlands; 2Thorax Center, Erasmus MC, Rotterdam, Netherlands; 3Pathology, Erasmus MC, Rotterdam, Netherlands Purpose: Regulatory FOXP3⫹ T cells are required for the control of immune responses of antigen-activated T cells. Yet, the role of FOXP3⫹ T cells and their dynamics in the regulation of anti-donor responses after clinical heart transplantation are still poorly understood. In this study, we examined the FOXP3 mRNA levels in the allograft and in peripheral blood of heart transplant patients to reveal the associated immunological processes of rejection or quiescence and whether the FOXP3 expression levels could provide clues for non-invasive detecting of acute rejection. Methods and Materials: Quantitative real-time PCR was used to analyze the mRNA expression levels of the regulatory T-cell marker FOXP3 as well as the levels of CD3⑀, CD25 and the effector cytokine IL-2, in endomyocardial biopsies (EMB) (n⫽85) and in PBMC samples (n⫽97) of cardiac allograft recipients. Results: The FOXP3 mRNA levels were higher in EMB taken during acute rejection (n⫽12, ISHLT 2R or more) than in EMB without evidence of rejection (p⫽0.01). No relationship was found between the FOXP3 mRNA levels and the time after transplantation. In addition, the levels of CD3⑀, CD25 and IL-2 were elevated in the rejecting EMB compared with the non-rejecting biopsies (p⫽0.01, p⫽0.02 and p⫽0.006, respectively). In PBMC, however, no association was detected between mRNA expression levels of FOXP3, CD3⑀, and CD25 and ISHLT rejection grades or time after transplantation. Interestingly, the mRNA levels of IL-2 were lower in the PBMC of rejecting patients than in the patients with ISHLT 1R (p⫽0.04), suggesting that IL-2-producing cells may have migrated from the blood to the inflamed allograft. Conclusions: We conclude that, in contrast to peripheral blood, FOXP3 mRNA levels were specifically elevated within the allograft during acute rejection. These findings suggest that rejection of the transplanted organ induces FOXP3⫹ T cells in the graft in an attempt to control anti-donor responses. 34 FOXP3ⴙ T CELLS CAN BE EXPANDED FROM REJECTING HUMAN CARDIAC ALLOGRAFTS I.E. Dijke,1 J.H. Velthuis,1 A.H. Balk,2 P. de Kuiper,1 M. Klepper,1 K. Caliskan,2 W. Weimar,1 C.C. Baan,1 1Internal Medicine, Erasmus MC, Rotterdam, Netherlands; 2Cardiology, Erasmus MC, Rotterdam, Netherlands Purpose: In organ-transplant recipients, we and others have shown that immune responses directed at the graft may consist of two components: a destructive and a protective one, the latter mediated by FoxP3⫹ regulatory T cells. We examined whether these FoxP3⫹ T cells can be grown from endomyocardial biopsies (EMB).