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326: Allogeneic CD4+CD25+ T Cells Regulate Rejection of Bronchus Transplants in Porcinized Mice
Abstracts antigen, K-alpha-1 tubulin (KAT) and to an extracellular matrix protein, Collagen-V (ColV) in LTx recipients diagnosed with BOS. These data (along with recent literature evidence) led to the hypothesis that the binding of donor specific anti-HLA Abs to the airway epithelial cell antigens (AEC) induces inflammation and remodeling which can expose otherwise cryptic self antigens triggering an autoimmune response. We extended these findings to clinical LTx by serially analyzing lung allograft recipients during the post-LTx period for the development of autoimmune response. Methods and Materials: Post-LTx sera was obtained in accordance the Washington University guidelines. Western blot analysis, and T-cell stimulation studies were performed. Results: In our current study out of a total of 106 patients who developed BOS post- LTx, 34 patients were positive for ColV (32%). Further, of the 12 patients who developed BOS post-LTx, and were positive for anticollagen Abs all 12 were positive for ColV ␣1 Abs and two were positive for ColV ␣2. However, understandably those patients who were BOS⫹ but were negative for anti-ColV Ab were also negative for V␣1 and V␣2. Further, a serial analysis for ColV Abs in 3 patients (Bos⫹, anti-Col V⫹) has revealed that the patients were positive for anti-ColV ␣1 and ␣2 initially (⬎24 months prior to onset of clinical BOS). However, only anti-ColV␣1 Abs persisted (9 ⫾ 7 months) until the onset of BOS. We are currently in the process of identifying immunodominant epitopes of ColV␣1. Conclusions: Our data provide a novel mechanistic role for alloimmune responses to mismatched donor specific anti-HLA antigens in the induction of a post-transplant immune response to self antigens, which can lead to the pathogenesis of chronic rejection following human LTx. 324 De Novo Formation of Lymphoid Tissue in the Lung Evolves Post Transplant Obliterative Airway Disease D. Wagnetz,1 M. Sato,1 J. Yeung,1 S. Hirayama,1 T. Waddell,1 M. Liu,2 S. Keshavjee.1 1Toronto General Hospital, Toronto, ON, Canada; 2 Latner Thoracic Research Laboratories, Toronto, ON, Canada. Purpose: We have recently described lymphoid neogenesis associated with obliterative bronchiolitis in transplanted human lungs, however, the significance is poorly understood.. We herein demonstrate the impact of ectopic lymphoid tissue in the lung using an intrapulmonary tracheal transplantation model. Methods and Materials: The trachea of Balb/c or C57Bl/6 mice were implanted into the left lung of either C57Bl/6 mice or Lymphotoxin-␣ ⫺/⫺ mice (LT-␣ ⫺/⫺), lacking secondary lymphoid organs. The recipient mice were sacrificed at day 14, 28 and 56 after transplantation (n⫽8/group). Crossections of the left lung were stained for B⫺/ T-cells, PnaD and Ki-67. The percentage of obliteration of the tracheal graft and the size of lymphoid areas around the graft were quantified. BrdU labeling and flow cytometry were used to look for T-cell and B-cell proliferation in lungs and bone marrow. Results: The intrapulmonary allogenic trachea demonstrated a typical obliterative process reflected by infiltration with myofibroblasts, loss of epithelial cells and almost complete obliteration by day 28 (82.07 ⫾ 2.63%), whereas the isogenic trachea remained preserved at any timepoint. Lymphoid structures with defined T-and B-cell zones and PnaD⫹ HEVs were detected around allografts, but not isografts. If an allogenic trachea is implanted to the lung of a LT-␣ ⫺/⫺ mice the graft becomes obliterated by day 28 (93.27 ⫾ 2.28%), despite the lack of secondary lymphoid organs. Strikingly, once implanted to the subcutaneous tissue the allogenic trachea was open with preserved epithelium at day 28. Increased T-and B-cell proliferation was detected in the left lung with the implanted allogenic trachea but not in the right lung or the bone marrow of mice lacking secondary lymphoid organs. Conclusions: Using the mouse intrapulmonary trachea transplantation model, we demonstrated for the first time that the lung itself becomes a lymphoid organ, capable in initiating and driving the alloimmune response leading to obliterative airway disease even in the absence of secondary lymphoid tissue.
S109 325 cKit and MHC Class-I Expression in the Tolerizing Inoculum Dictates Donor-specific Tolerance vs Third-Party Cardiac Allograft Acceptance in a Neonatal Tolerance Model M. Jeyakanthan, K. Tao, L.J. West. University of Alberta, Edmonton, AB, Canada. Purpose: We showed that injection of neonatal mice with allogeneic cKit⫹/Lin⫺ fetal liver cells (FLC) induces acceptance of both donorspecific and unrelated cardiac allografts as adults. This study investigated the role of cKit receptor and MHC expression in the tolerizing cells. Methods and Materials: BALB/c (BALB, H-2d) FLC or bone marrow cells (BMC) from adult BALB were sorted by magnetic cell separation. Neonatal C3H/He (C3H, H-2k) mice were injected iv with either cKit blocking antibody (ACK2) treated c-Kit⫹/Lin⫺ or c-Kit⫺/Lin⫹ BALB FLC, followed at 6-8 weeks by transplantation of C57BL/6 (B6, H-2b) cardiac grafts. Similar groups were injected with cKit⫹/Lin⫺ BMC followed by B6 or BALB cardiac grafts. Untreated C3H controls received B6 cardiac grafts. MHC class I&II expression in FLC and BMC was studied by FACS. Results: While long-term graft survival (MST⬎100days) was achieved in the cKit⫹/Lin⫺ group, MST (⫽35.5 days) was significantly reduced when cKit⫹/Lin⫺ FLC were pre-treated with ACK2 antibody. cKit⫹/Lin⫺ BMC-treated group achieved long-term survival of BALB grafts (MST⬎100 days) but acutely rejected B6 grafts (MST⫽14 days). BMC expressed MHC class-Ihigh whilst only 7-16% of FLC expressed MHC class-Ilow. MHC class-II was very low or undetectable in both FLC and BMC. Conclusions: This study shows that c-Kit⫹/Lin⫺ cells from FLC, when injected into fully MHC-mismatched neonatal mice, induce non-donor specific cardiac allograft acceptance. These data suggest that the cKit receptor on FLC may be responsible for the alterations in neonatal immunity that result in graft acceptance as adults. Data also suggest that MHC class-I expression may dictate donor-specific tolerance induction versus third-party cardiac allograft acceptance. Further refinement of this strategy may offer a useful tool in transplantation when donor-specific tolerance induction is not possible due to the short time-frame available, as in cardiac transplantation. 326 Allogeneic CD4ⴙCD25ⴙ T Cells Regulate Rejection of Bronchus Transplants in Porcinized Mice A.-K. Knoefel, N. Madrahimov, G. Warnecke, M. Avsar, K. Dreckmann, M. Strueber, A. Haverich. Hannover Medical School, Hannover, Germany. Purpose: In our porcine allogeneic lung transplantation model, we have previously shown that long term graft acceptance correlated with the frequency of CD4⫹CD25⫹ regulatory T cells. It is not known whether this type of T cell regulation is the cause for, or an epiphenomenon of, long term allograft survival. Therefore, we investigated the role of T cell regulation in an adoptive transfer system using porcine bronchus and allogeneic PBMC transfer into immune deficient mice. Methods and Materials: Porcine bronchi were transplanted under the skin of NODrag⫺/⫺gammac⫺/⫺ mice. 5x106 porcine PBMC were injected per animal in 4 groups (n⫽4-10). Group A received no PBMC, for group B cells and vessels were collected from two MHC mismatched pigs, group C recipients received allogeneic PBMC depleted of CD4⫹CD25⫹ T cells; group D recipients received allogeneic PBMC enriched of CD4⫹CD25⫹ T cells. Alloinjury of the heterotopic bronchus grafts were assessed in histology samples on postoperative day 28. Results: In the control group A cell infiltration and luminal obstruction were absent and structural damage to the cartilage and the epithelium was low (n⫽4). In the positive control group B cell infiltration was pronounced and histological changes were severe (n⫽8). In group C these changes were even more severe (n⫽10). In group D, cell infiltration was reduced and histological damage to the allografts was less severe (n⫽4). Conclusions: Thus, heterotopic transplantation of a porcine bronchus graft and reconstitution with allogeneic porcine PBMC in NODrag⫺/⫺gam-
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The Journal of Heart and Lung Transplantation, Vol 29, No 2S, February 2010
mac⫺/⫺ mice represents a valuable tool for transplant research. In this model, we could show in vivo that rejection of bronchus transplants, that is histologically reminiscent of bronchiolitis obliterans, is controlled by CD4⫹CD25⫹ T cell regulation. 327
dichotomization by the top quartile of indexed LV mass and concentricity (Figure).[figure1] Conclusions: A history of rejection and diabetes are associated with increased LV mass. Increased LV mass, particularly with a concentric phenotype, is an independent risk factor for all-cause mortality following cardiac transplant.
Gender Does Matter – Gender Specific Outcome Analysis of 1000 Heart Transplants at a Single Centre I. Kaczmarek, S. Eifert, S. Kofler, B. Meiser, B. Reichart. Grosshadern University Hospital, Munich, Germany. Purpose: Gender differences between donor and recipient might have an impact on outcome after heart transplantation (HTx). Literature reviews and registries reveal controversial results. We reviewed the data of 1000 heart transplants at our center focusing on the influence of gender differences on short- and long-term outcome after HTx. Methods and Materials: We performed a retrospective data analysis of all HTx at our center from 1981 to 2008. Kaplan-Meier survival, log-rank-test, Chi-square-test, ANOVA and a Cox-proportional-Hazard model were the basis for statistical analysis. In contrast to other studies the data for gender differences were calculated with respect to actuarial and conditional survival (without 30-day-mortality). Results: One-year-survival was significantly inferior in male recipients of female donor hearts (m/f 73.7%) compared to females receiving male donor organs (f/m 90.9%; p⬍0.05). After 10 years survival differences remained significant between these two groups (53.1% m/f vs. 69.6% f/m; p⫽0.04). In the conditional survival these differences disappeared indicating that gender differences significantly influence short-term outcome. Furthermore the combination female recipient/female donor resulted in a superior 10-year-survival compared to male donor/male recipients (f/f 84.7% vs. m/m 66.3%; p⫽0.04). Conclusions: The combination male recipient/female donor carries a higher risk for early mortality whereas female recipients reveal favorable short-term results with male donor hearts. In the long-term follow-up female donor hearts reveal superior results, especially in the combination female recipient/female donor. 328
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Increased LV Mass and Concentricity as Assessed by Cardiac MRI Are Associated with Increased Mortality in Cardiac Transplant Recipients P.C. Patel,1 S.C. Reimold,1 F.G. Araj,1 P.A. Kaiser,1 R.M. Peshock,1 C.W. Yancy,2 W.S. Ring,3 S. Gupta,1 J.D. Mishkin,1 P.P.A. Mammen,1 D.W. Markham,1 M.H. Drazner.1 1University of Texas Southwestern Medical Center, Dallas, TX; 2Baylor University Medical Center, Dallas, TX; 3University of Texas Southwestern Medical Center, Dallas, TX.
Primary Graft Dysfunction Does Not Lead to Increased Cardiac Allograft Vasculopathy in Surviving Patients S. Wong,1 S. Wong,1 J. Moriguchi,2 M. Kawano,2 J. Kobashigawa,2 R. Shemin,1 M. Kwon.1 1David Geffen School of Medicine at UCLA, Los Angeles, CA; 2David Geffen School of Medicine at UCLA/Cedars Sinai Heart Institute, Los Angeles, CA.
Purpose: Although risk factors for left ventricular (LV) hypertrophy in the native heart are well known, as is its association with increased risk of adverse outcomes, such information is poorly defined in cardiac transplant recipients. Methods and Materials: We identified 140 transplant recipients who had a cardiac MRI assessing LV mass and concentricity between 5/2003 and 5/2006. Patients were followed until 12/31/08. Rejection was defined as ⱖ ISHLT grade 3A/2R cellular rejection; or hemodynamically significant acellular rejection. Clinical characteristics associated with the top quartile of indexed LV mass were determined by multivariable logistic models. The associations of LV mass and concentricity with all-cause mortality were assessed by multivariable Cox regression. Results: MRIs were obtained at a median of 6.0 years after transplant. There were 30 deaths with a median follow-up of 4.6 years after MRI acquisition. The top quartiles of indexed LV mass and concentricity were ⱖ 40.3 g/m2.7 and ⱖ 1.5 g/ml, respectively. History of rejection (OR 3.5, 95% CI 1.4-8.6, p⬍0.01) and diabetes during MRI acquisition (OR 3.0, 95% CI 1.3-7.1, p⫽0.01) were associated with increased LV mass. Continuous LV mass and concentricity were independently associated with increased mortality (HR 1.1 per g/m2.7; HR 4.4 per g/ml, p⬍0.01) after adjustment for potential confounders. These associations persisted after
Purpose: Early injury is associated with the development of cardiac allograft vasculopathy (CAV) in cardiac transplantation. We examined whether adult heart transplant recipients incurring primary graft dysfunction (PGD) were more susceptible to the development of CAV than their non-PGD counterparts. Methods and Materials: 857 patients undergoing heart transplantation between January 1994 and December 2008 at our institution were reviewed. PGD was defined as the need for extracorporeal membrane oxygenation, open-chest, or intra-aortic balloon pump placement within 72 hours of transplantation. CAV was defined as ⱖ 50% coronary artery stenosis in any vessel. Allograft survival was defined by patient death or need for re-transplantation. Results: The PGD group (n⫽42) compared to the non-PGD group (n⫽815) had similar mean recipient age (55 vs. 55 years, p⫽0.91) and mean ischemic time (219 vs. 208 minutes, p⫽0.15). Donor age was significantly higher in the PGD group (37 vs. 32 years, p⫽0.011). 5-year allograft survival for the PGD and non-PGD groups were 45% vs. 79% (p⬍0.001) (figure 1).[figure1]Within a 30-day post operative period there were more deaths in the PGD group compared to the non-PGD group (28.6% vs. 2.3%) and more re-transplants (4.8% vs. 0%). Of the surviving patients, only 2 (7.1%) of the PGD patients developed CAV vs. 157 (19.7%) in the non-PGD group.
S109 325 cKit and MHC Class-I Expression in the Tolerizing Inoculum Dictates Donor-specific Tolerance vs Third-Party Cardiac Allograft Acceptance in a Neonatal Tolerance Model M. Jeyakanthan, K. Tao, L.J. West. University of Alberta, Edmonton, AB, Canada. Purpose: We showed that injection of neonatal mice with allogeneic cKit⫹/Lin⫺ fetal liver cells (FLC) induces acceptance of both donorspecific and unrelated cardiac allografts as adults. This study investigated the role of cKit receptor and MHC expression in the tolerizing cells. Methods and Materials: BALB/c (BALB, H-2d) FLC or bone marrow cells (BMC) from adult BALB were sorted by magnetic cell separation. Neonatal C3H/He (C3H, H-2k) mice were injected iv with either cKit blocking antibody (ACK2) treated c-Kit⫹/Lin⫺ or c-Kit⫺/Lin⫹ BALB FLC, followed at 6-8 weeks by transplantation of C57BL/6 (B6, H-2b) cardiac grafts. Similar groups were injected with cKit⫹/Lin⫺ BMC followed by B6 or BALB cardiac grafts. Untreated C3H controls received B6 cardiac grafts. MHC class I&II expression in FLC and BMC was studied by FACS. Results: While long-term graft survival (MST⬎100days) was achieved in the cKit⫹/Lin⫺ group, MST (⫽35.5 days) was significantly reduced when cKit⫹/Lin⫺ FLC were pre-treated with ACK2 antibody. cKit⫹/Lin⫺ BMC-treated group achieved long-term survival of BALB grafts (MST⬎100 days) but acutely rejected B6 grafts (MST⫽14 days). BMC expressed MHC class-Ihigh whilst only 7-16% of FLC expressed MHC class-Ilow. MHC class-II was very low or undetectable in both FLC and BMC. Conclusions: This study shows that c-Kit⫹/Lin⫺ cells from FLC, when injected into fully MHC-mismatched neonatal mice, induce non-donor specific cardiac allograft acceptance. These data suggest that the cKit receptor on FLC may be responsible for the alterations in neonatal immunity that result in graft acceptance as adults. Data also suggest that MHC class-I expression may dictate donor-specific tolerance induction versus third-party cardiac allograft acceptance. Further refinement of this strategy may offer a useful tool in transplantation when donor-specific tolerance induction is not possible due to the short time-frame available, as in cardiac transplantation. 326 Allogeneic CD4ⴙCD25ⴙ T Cells Regulate Rejection of Bronchus Transplants in Porcinized Mice A.-K. Knoefel, N. Madrahimov, G. Warnecke, M. Avsar, K. Dreckmann, M. Strueber, A. Haverich. Hannover Medical School, Hannover, Germany. Purpose: In our porcine allogeneic lung transplantation model, we have previously shown that long term graft acceptance correlated with the frequency of CD4⫹CD25⫹ regulatory T cells. It is not known whether this type of T cell regulation is the cause for, or an epiphenomenon of, long term allograft survival. Therefore, we investigated the role of T cell regulation in an adoptive transfer system using porcine bronchus and allogeneic PBMC transfer into immune deficient mice. Methods and Materials: Porcine bronchi were transplanted under the skin of NODrag⫺/⫺gammac⫺/⫺ mice. 5x106 porcine PBMC were injected per animal in 4 groups (n⫽4-10). Group A received no PBMC, for group B cells and vessels were collected from two MHC mismatched pigs, group C recipients received allogeneic PBMC depleted of CD4⫹CD25⫹ T cells; group D recipients received allogeneic PBMC enriched of CD4⫹CD25⫹ T cells. Alloinjury of the heterotopic bronchus grafts were assessed in histology samples on postoperative day 28. Results: In the control group A cell infiltration and luminal obstruction were absent and structural damage to the cartilage and the epithelium was low (n⫽4). In the positive control group B cell infiltration was pronounced and histological changes were severe (n⫽8). In group C these changes were even more severe (n⫽10). In group D, cell infiltration was reduced and histological damage to the allografts was less severe (n⫽4). Conclusions: Thus, heterotopic transplantation of a porcine bronchus graft and reconstitution with allogeneic porcine PBMC in NODrag⫺/⫺gam-
S110
The Journal of Heart and Lung Transplantation, Vol 29, No 2S, February 2010
mac⫺/⫺ mice represents a valuable tool for transplant research. In this model, we could show in vivo that rejection of bronchus transplants, that is histologically reminiscent of bronchiolitis obliterans, is controlled by CD4⫹CD25⫹ T cell regulation. 327
dichotomization by the top quartile of indexed LV mass and concentricity (Figure).[figure1] Conclusions: A history of rejection and diabetes are associated with increased LV mass. Increased LV mass, particularly with a concentric phenotype, is an independent risk factor for all-cause mortality following cardiac transplant.
Gender Does Matter – Gender Specific Outcome Analysis of 1000 Heart Transplants at a Single Centre I. Kaczmarek, S. Eifert, S. Kofler, B. Meiser, B. Reichart. Grosshadern University Hospital, Munich, Germany. Purpose: Gender differences between donor and recipient might have an impact on outcome after heart transplantation (HTx). Literature reviews and registries reveal controversial results. We reviewed the data of 1000 heart transplants at our center focusing on the influence of gender differences on short- and long-term outcome after HTx. Methods and Materials: We performed a retrospective data analysis of all HTx at our center from 1981 to 2008. Kaplan-Meier survival, log-rank-test, Chi-square-test, ANOVA and a Cox-proportional-Hazard model were the basis for statistical analysis. In contrast to other studies the data for gender differences were calculated with respect to actuarial and conditional survival (without 30-day-mortality). Results: One-year-survival was significantly inferior in male recipients of female donor hearts (m/f 73.7%) compared to females receiving male donor organs (f/m 90.9%; p⬍0.05). After 10 years survival differences remained significant between these two groups (53.1% m/f vs. 69.6% f/m; p⫽0.04). In the conditional survival these differences disappeared indicating that gender differences significantly influence short-term outcome. Furthermore the combination female recipient/female donor resulted in a superior 10-year-survival compared to male donor/male recipients (f/f 84.7% vs. m/m 66.3%; p⫽0.04). Conclusions: The combination male recipient/female donor carries a higher risk for early mortality whereas female recipients reveal favorable short-term results with male donor hearts. In the long-term follow-up female donor hearts reveal superior results, especially in the combination female recipient/female donor. 328
329
Increased LV Mass and Concentricity as Assessed by Cardiac MRI Are Associated with Increased Mortality in Cardiac Transplant Recipients P.C. Patel,1 S.C. Reimold,1 F.G. Araj,1 P.A. Kaiser,1 R.M. Peshock,1 C.W. Yancy,2 W.S. Ring,3 S. Gupta,1 J.D. Mishkin,1 P.P.A. Mammen,1 D.W. Markham,1 M.H. Drazner.1 1University of Texas Southwestern Medical Center, Dallas, TX; 2Baylor University Medical Center, Dallas, TX; 3University of Texas Southwestern Medical Center, Dallas, TX.
Primary Graft Dysfunction Does Not Lead to Increased Cardiac Allograft Vasculopathy in Surviving Patients S. Wong,1 S. Wong,1 J. Moriguchi,2 M. Kawano,2 J. Kobashigawa,2 R. Shemin,1 M. Kwon.1 1David Geffen School of Medicine at UCLA, Los Angeles, CA; 2David Geffen School of Medicine at UCLA/Cedars Sinai Heart Institute, Los Angeles, CA.
Purpose: Although risk factors for left ventricular (LV) hypertrophy in the native heart are well known, as is its association with increased risk of adverse outcomes, such information is poorly defined in cardiac transplant recipients. Methods and Materials: We identified 140 transplant recipients who had a cardiac MRI assessing LV mass and concentricity between 5/2003 and 5/2006. Patients were followed until 12/31/08. Rejection was defined as ⱖ ISHLT grade 3A/2R cellular rejection; or hemodynamically significant acellular rejection. Clinical characteristics associated with the top quartile of indexed LV mass were determined by multivariable logistic models. The associations of LV mass and concentricity with all-cause mortality were assessed by multivariable Cox regression. Results: MRIs were obtained at a median of 6.0 years after transplant. There were 30 deaths with a median follow-up of 4.6 years after MRI acquisition. The top quartiles of indexed LV mass and concentricity were ⱖ 40.3 g/m2.7 and ⱖ 1.5 g/ml, respectively. History of rejection (OR 3.5, 95% CI 1.4-8.6, p⬍0.01) and diabetes during MRI acquisition (OR 3.0, 95% CI 1.3-7.1, p⫽0.01) were associated with increased LV mass. Continuous LV mass and concentricity were independently associated with increased mortality (HR 1.1 per g/m2.7; HR 4.4 per g/ml, p⬍0.01) after adjustment for potential confounders. These associations persisted after
Purpose: Early injury is associated with the development of cardiac allograft vasculopathy (CAV) in cardiac transplantation. We examined whether adult heart transplant recipients incurring primary graft dysfunction (PGD) were more susceptible to the development of CAV than their non-PGD counterparts. Methods and Materials: 857 patients undergoing heart transplantation between January 1994 and December 2008 at our institution were reviewed. PGD was defined as the need for extracorporeal membrane oxygenation, open-chest, or intra-aortic balloon pump placement within 72 hours of transplantation. CAV was defined as ⱖ 50% coronary artery stenosis in any vessel. Allograft survival was defined by patient death or need for re-transplantation. Results: The PGD group (n⫽42) compared to the non-PGD group (n⫽815) had similar mean recipient age (55 vs. 55 years, p⫽0.91) and mean ischemic time (219 vs. 208 minutes, p⫽0.15). Donor age was significantly higher in the PGD group (37 vs. 32 years, p⫽0.011). 5-year allograft survival for the PGD and non-PGD groups were 45% vs. 79% (p⬍0.001) (figure 1).[figure1]Within a 30-day post operative period there were more deaths in the PGD group compared to the non-PGD group (28.6% vs. 2.3%) and more re-transplants (4.8% vs. 0%). Of the surviving patients, only 2 (7.1%) of the PGD patients developed CAV vs. 157 (19.7%) in the non-PGD group.