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Donor specific spleen cells mediate allograft survival by a different mechanism than bone marrow
The Journal of Heart and Lung Transplantation Volume 18, Number 1
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DONOR SPECIFIC SPLEEN CELLS MEDIATE ALLOGRAFT SURVIVAL BY A DIFFERENT MECHANISM THAN BONE MARROW. Daniel RGoldstein, Theresa Chang, Scott D Sweeney, James K Kirklin, Judith M Thomas, and James F George. Univ of Alabama, Birmingham, USA. IntroductionC3H (H-2’) murine skin allograft survival can be augmented in B6AFl (H-2: Km, I-Ati, I-E-&, Dwd) recipient mice by peri operative, intra-peritoneal administration of anti-lymphocyte serum (AL&-days 11 and +2 r&&e to transnlantation) followed bv I.V. administration at dav +7 of 2.5 x 10’donor C3H bone’marrow &Is. We havk previously shown that t& mechanism of transplantation tolerance mediated by the donor bone marrow is Fas-ligand dependent. Since donor specific spleen cells (SPC) have also been shown to prolong allograft survival in this model, we investigated whether donor spleen cells mediate extended graft survival by similar mechanisms. Methods and Results. B6AFl mice were treated according to the standard protocol outlined above, except that on day +7, mice were randomly assigned to the following groups; i) no infusion (ALS controls) N =lO. ii) C3H donor specific infusion 5.0 x 10 SPC /mouse (wild type control) N= 8. iii) C3H;gld (mutant FAS ligand expressed on cell surface) donor specific infusion 5.0 x 10 SPC GLD /mouse N=9. Results show that, compared to ALS control (median survival 27 days), the SPC GLD, unlike bone marrow, significantly enhanced allograft survival (37 days, p =O.M. log-rank). Wild type control group also promoted allagraft survival (44 days, p=O.COOS). There was no statistical difference between wild type and GLD g&pa (p=O.l). In a second set of experiments, we determined wh&r donor SPC could induce awotosis of donor mecific T cells in vitro. Female T cell receotor transgenic mick &e used in wbi;h the majority of their T cells arc react& to the male H-Y antigen. ALS was given in the same standard protocol as stated above, relative to a skin transplant from an H-Y’ male donor. Six days after transplantation, H-Y’ female recipient lymph node cells were harvested. The following day, donor (i.e. H-Y’ male strain) spleen cells were added to the culture wells in five different dosages, H-Y‘ female spleen cells were used as a negative control. Apoptosis was measured by annexin V staining, and distinguished from dead cells using ‘I-ADD. Enumeration of apoptosis was by flow cytometry gated specifically on the CD8+ transgenic cells, which have previously been shown to be graft reactive in vitro. In comparison to the female SPC (negative control, mean apoptosis 6.186, S-.34), the mean level of apoptosis induced by male SPC was twofold higher at 11.83% (SE=1.76, p=O.O5). Conclusion. These results show that, in contrast to donor bone marrow, the prolongation of allograft survival mediated by donor SPC is not Fas-ligand dependent. The in vitro data suggest that donor spleen cells induce apoptosis via an alternative pathway.
FUNCTIONAL RESPONSES OF ANTI-CD25 BLOCKED T-CELLS DURING CARDIAC REJECTION C.C. Baan. T. van Gelder. A.H.M.M. Balk, A.P.W.M. Maat, and W. Weimar. Department of Internal Medicine and Thorax Center; University Hospital Rotterdam-Dijkzigt. The Netherlands Recently, we have shown that in vivo blockade of the IL-2/IL-2R signaling pathway by anti-CD25 (IL-2R a-chain) mAb treatment did not completely prevent rejection. In these cases the macrophage derived T-cell growth factor IL-15, which interacts with the IL-15R a-chain and the p and ychain of the IL-2R, was abundantly present in the graft. However, whether IL15 responsive T-cells indeed play an active role in these rejections is not known. The biological functions of IL-2 and IL-15 are exerted through their specific high affinity receptor. To test whether IL-2 and IL-15 can induce T-cell proliferation during anti-CD25 mAb treatment, we measured by limiting dilution analysis, the frequency of IL-2 and IL-15 responsive T-cells in peripheral blood from anti-CD25 treated patients during cardiac rejection (n=lO) and from healthy controls (n=7) in the absence and presence of the mouse an&CD25 mAb BT563. In the absence of anti-CD25 mAb, T-cells from patients were able to respond to fixed IL-2 (10 nglml) or IL-15 (10 nglml) concentrations but their frequencies were significantly lower than those measured in peripheral blood of healthy controls: median of IL-2 responding T-cells 86/10’ (range 31-210/106) vs 136/106 (range 129-484/10’, p=O.O02). and median of IL-15 responding T-cells T-cells 32110’ (range I-207/106) vs 128/106 (range 79-192/10’, p=O.O6). A completely different picture emerged when frequencies of IL-2 and IL-15 responding T-cells were determined in the presence of anti-CD25 mAb. The response to IL-2 was specifically inhibited by anti-CD25 mAb, while the proliferation of T-cells mediated by IL-15 was not affected. Frequencies of IL-2 responding T-cells from cardiac allograft recipients decreased with 85% (median inhibition, range 46-lOO%), whereas the frequencies of IL-15 responding T-cells were not influenced (median inhibition 4%, range O-50%, pcO.001). These in vitro studies show that in cardiac transplant patients the proliferative capacity of T-cells to respdnd to IL2 to IL-15 is impaired. The residual responsiveness of T-cells to IL-2 was abrogated by anti-CD25 mAb, while the responsiveness to IL-15 proved to be resistant to IL-2R blockade. We conclude that T-cell proliferation during cardiac rejection under anti-CD25 therapy is a IL-15 driven phenomenon.
LATE REJECTION GREATER THAN 1 YEAR AFTER PEDIATRIC HEART TRANSPLANTATION: AN OMINOUS FINDING. SA Webber, DC Naftel. J Parker, N Mulla, I B&four. L Cipriani, JK Kirklin, WR Morrow and the Pediatric Heart Transplant Study. Depts of Pediatrics and Surgery, Pittsburgh, Arkansas and Cardinal Glennon Children’s Hospitals, Loma Linda Medical Center and University of Alabama, USA.
Abstracts
69
Purpose: We sought to evaluate the incidence of late rejection episodes (z-1 year) after pediatric heart transplantation and to define risk factors for its occurrence as well as outcome. Methods: We analvzed data on 685 pediatric recipients (<18years) transplanted between lj1/93 and 12/31/97 al 18 centers in the Pediatric Heart Transplant Study (PHTS). Probability of freedom from late rejection was determined and tisk factors for late rejection and for death after late rejection were sought using univariate and multivariate analyses. Results: 431 patients were followed for > 1 year (median follow-up 32.9 mths) with 106 (24.6%) experiencing 1 or more late rejection episodes (total 178 episodes, 27 with severe hemodynamic compromise). Probability of freedom from first late rejection was 73% al 2 years and 65% al 3 years after transplantation. Risk factors (multivariate analysis) for first late rejection were >I episode of rejection in first year (P=O.OOS). recipient black mce (P=O.O002) and older age al time of transplant (P=O.O003). Only 4 of 325 (1.2%) children surviving beyond 1 year without late rejection died compared to 26 of 106 (24.5%) with late rejection (PcO.0001). Nine of these 26 died within one month of the first late rejection episode with 17 subsequent deaths secondary to acute rejection 5, sudden unexplained death 3, documented coronary disease 3 and other causes 6. Severe hemodynamic compromise with late rejection was identified as a risk factor for death among children with one or more episodes of late rejection (P=O.Ol). Inferences: Approximately one quarter of pediatric recipients in the PHTS surviving beyond 1 year have experienced late rejection episodes. Late rejection is associated with poor survival. esoeciallv when associated with hemodvnamic comoromise. Absence of iate reiection episodes is associated with ;ery low risk ‘of death during medium term follow-up after pediatric heart transplantation. Determination of risk factors for late rejection helps identify a cohort of children who may benefit from enhanced rejection surveillance and treatment.
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OUTCOME OF LISTING FOR CARDIAC TRANSPLANTATION FOR FAILED FONTAN: A MULTI-INSTITUTIONAL STUDY D Bernstein. DC Naftel. DT Hsu, LJ Addonizio, ED Blume. PL Gamberg, JK Kirklin. WR Morrow and the Pediatric Heart Transplant Study Group (PHTS). Stanford, Harvard, and Columbia Universities, Univ of Arkansas, Univ of Alabama, Birmingham, AL. The Fontan procedure is a successful palhation for children with single ventricle physiology. However, many patients eventually require heart transplantation (TX). The purpose of this study was to examine outcomes and risk factors for death while waiting and survival after undergoing TX. We analyzed data from 1002 patients listed for TX at 18 PHTS centers from Jan 1.1993 through Dee 31, 1997. Data was analyzed using actuarial. parametric, competing outcomes, and multi-variable risk factor analysis. 47 Fontan pts. lyr of age with previous sternotomy for congenital heart disease (CHD) or 387 children without congenital heart disease (NoCHD). Patients who were younger, Status 1 at listing, had shorter interval since FONT, or were on a ventilator at listing were more likely to die while waitmg. Using competing outcomes analysis at 6 mos post listing the probability of death while waiting was 10% for Status 2 compared to 34% for Status 1. 71% of patients listed after recent failed FONT (<3mos) died before transplant. 33 FONT pts. underwent transplantation. Actuarial survival post TX was 80% at 1 yr and 68% at 3 yr with no difference in suwival compared to CHD and NoCHD. Of the 8 deaths in FONT pts. infection predominated as the primaty cause of death(5pts). 16pts. were transplanted for protein losing enteropathy (PLE). In this group the mean serum albumin increased from 2.9gmldl pre transplant to 4.2gmldl post transplant (p=O.O02). In conclusion, heart TX is an effective therapy for pts. with failed FONT including those with PLE. The high risk of death while waiting in Status I FONT and those with recent FONT emphasizes the importance of early referral for TX before the onset of marked clinical deterioration Primary TX should be considered instead of FONT in selected previously palliated pts. with single ventricle physiology.
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DEATH AFTER HEARTTRANSPLANTATION IN CHILDREN: WHO DIES WHEN AND WHY EA Frazier, DC Naftel, CE Canter, MM Boucek. JK Kirklin, WR Monow, and the Pediatric Heart Transplant Study (PHTS). Univ of Alabama, Univ of Colorado, Washington Univ, and Univ of Arkansas, Liile Rock, AR, USA. Death following head bansplantation (TX) in children results from a vsriety of causes, each having a unique incidence over time. To belter understand the time related riik of death from any specific cause after TX, a competing outcomes snslysii wss used to estimate the time-related cause-specific mortality after pediatrk TX. Patkml Po~ulatlcn:
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DONOR SPECIFIC SPLEEN CELLS MEDIATE ALLOGRAFT SURVIVAL BY A DIFFERENT MECHANISM THAN BONE MARROW. Daniel RGoldstein, Theresa Chang, Scott D Sweeney, James K Kirklin, Judith M Thomas, and James F George. Univ of Alabama, Birmingham, USA. IntroductionC3H (H-2’) murine skin allograft survival can be augmented in B6AFl (H-2: Km, I-Ati, I-E-&, Dwd) recipient mice by peri operative, intra-peritoneal administration of anti-lymphocyte serum (AL&-days 11 and +2 r&&e to transnlantation) followed bv I.V. administration at dav +7 of 2.5 x 10’donor C3H bone’marrow &Is. We havk previously shown that t& mechanism of transplantation tolerance mediated by the donor bone marrow is Fas-ligand dependent. Since donor specific spleen cells (SPC) have also been shown to prolong allograft survival in this model, we investigated whether donor spleen cells mediate extended graft survival by similar mechanisms. Methods and Results. B6AFl mice were treated according to the standard protocol outlined above, except that on day +7, mice were randomly assigned to the following groups; i) no infusion (ALS controls) N =lO. ii) C3H donor specific infusion 5.0 x 10 SPC /mouse (wild type control) N= 8. iii) C3H;gld (mutant FAS ligand expressed on cell surface) donor specific infusion 5.0 x 10 SPC GLD /mouse N=9. Results show that, compared to ALS control (median survival 27 days), the SPC GLD, unlike bone marrow, significantly enhanced allograft survival (37 days, p =O.M. log-rank). Wild type control group also promoted allagraft survival (44 days, p=O.COOS). There was no statistical difference between wild type and GLD g&pa (p=O.l). In a second set of experiments, we determined wh&r donor SPC could induce awotosis of donor mecific T cells in vitro. Female T cell receotor transgenic mick &e used in wbi;h the majority of their T cells arc react& to the male H-Y antigen. ALS was given in the same standard protocol as stated above, relative to a skin transplant from an H-Y’ male donor. Six days after transplantation, H-Y’ female recipient lymph node cells were harvested. The following day, donor (i.e. H-Y’ male strain) spleen cells were added to the culture wells in five different dosages, H-Y‘ female spleen cells were used as a negative control. Apoptosis was measured by annexin V staining, and distinguished from dead cells using ‘I-ADD. Enumeration of apoptosis was by flow cytometry gated specifically on the CD8+ transgenic cells, which have previously been shown to be graft reactive in vitro. In comparison to the female SPC (negative control, mean apoptosis 6.186, S-.34), the mean level of apoptosis induced by male SPC was twofold higher at 11.83% (SE=1.76, p=O.O5). Conclusion. These results show that, in contrast to donor bone marrow, the prolongation of allograft survival mediated by donor SPC is not Fas-ligand dependent. The in vitro data suggest that donor spleen cells induce apoptosis via an alternative pathway.
FUNCTIONAL RESPONSES OF ANTI-CD25 BLOCKED T-CELLS DURING CARDIAC REJECTION C.C. Baan. T. van Gelder. A.H.M.M. Balk, A.P.W.M. Maat, and W. Weimar. Department of Internal Medicine and Thorax Center; University Hospital Rotterdam-Dijkzigt. The Netherlands Recently, we have shown that in vivo blockade of the IL-2/IL-2R signaling pathway by anti-CD25 (IL-2R a-chain) mAb treatment did not completely prevent rejection. In these cases the macrophage derived T-cell growth factor IL-15, which interacts with the IL-15R a-chain and the p and ychain of the IL-2R, was abundantly present in the graft. However, whether IL15 responsive T-cells indeed play an active role in these rejections is not known. The biological functions of IL-2 and IL-15 are exerted through their specific high affinity receptor. To test whether IL-2 and IL-15 can induce T-cell proliferation during anti-CD25 mAb treatment, we measured by limiting dilution analysis, the frequency of IL-2 and IL-15 responsive T-cells in peripheral blood from anti-CD25 treated patients during cardiac rejection (n=lO) and from healthy controls (n=7) in the absence and presence of the mouse an&CD25 mAb BT563. In the absence of anti-CD25 mAb, T-cells from patients were able to respond to fixed IL-2 (10 nglml) or IL-15 (10 nglml) concentrations but their frequencies were significantly lower than those measured in peripheral blood of healthy controls: median of IL-2 responding T-cells 86/10’ (range 31-210/106) vs 136/106 (range 129-484/10’, p=O.O02). and median of IL-15 responding T-cells T-cells 32110’ (range I-207/106) vs 128/106 (range 79-192/10’, p=O.O6). A completely different picture emerged when frequencies of IL-2 and IL-15 responding T-cells were determined in the presence of anti-CD25 mAb. The response to IL-2 was specifically inhibited by anti-CD25 mAb, while the proliferation of T-cells mediated by IL-15 was not affected. Frequencies of IL-2 responding T-cells from cardiac allograft recipients decreased with 85% (median inhibition, range 46-lOO%), whereas the frequencies of IL-15 responding T-cells were not influenced (median inhibition 4%, range O-50%, pcO.001). These in vitro studies show that in cardiac transplant patients the proliferative capacity of T-cells to respdnd to IL2 to IL-15 is impaired. The residual responsiveness of T-cells to IL-2 was abrogated by anti-CD25 mAb, while the responsiveness to IL-15 proved to be resistant to IL-2R blockade. We conclude that T-cell proliferation during cardiac rejection under anti-CD25 therapy is a IL-15 driven phenomenon.
LATE REJECTION GREATER THAN 1 YEAR AFTER PEDIATRIC HEART TRANSPLANTATION: AN OMINOUS FINDING. SA Webber, DC Naftel. J Parker, N Mulla, I B&four. L Cipriani, JK Kirklin, WR Morrow and the Pediatric Heart Transplant Study. Depts of Pediatrics and Surgery, Pittsburgh, Arkansas and Cardinal Glennon Children’s Hospitals, Loma Linda Medical Center and University of Alabama, USA.
Abstracts
69
Purpose: We sought to evaluate the incidence of late rejection episodes (z-1 year) after pediatric heart transplantation and to define risk factors for its occurrence as well as outcome. Methods: We analvzed data on 685 pediatric recipients (<18years) transplanted between lj1/93 and 12/31/97 al 18 centers in the Pediatric Heart Transplant Study (PHTS). Probability of freedom from late rejection was determined and tisk factors for late rejection and for death after late rejection were sought using univariate and multivariate analyses. Results: 431 patients were followed for > 1 year (median follow-up 32.9 mths) with 106 (24.6%) experiencing 1 or more late rejection episodes (total 178 episodes, 27 with severe hemodynamic compromise). Probability of freedom from first late rejection was 73% al 2 years and 65% al 3 years after transplantation. Risk factors (multivariate analysis) for first late rejection were >I episode of rejection in first year (P=O.OOS). recipient black mce (P=O.O002) and older age al time of transplant (P=O.O003). Only 4 of 325 (1.2%) children surviving beyond 1 year without late rejection died compared to 26 of 106 (24.5%) with late rejection (PcO.0001). Nine of these 26 died within one month of the first late rejection episode with 17 subsequent deaths secondary to acute rejection 5, sudden unexplained death 3, documented coronary disease 3 and other causes 6. Severe hemodynamic compromise with late rejection was identified as a risk factor for death among children with one or more episodes of late rejection (P=O.Ol). Inferences: Approximately one quarter of pediatric recipients in the PHTS surviving beyond 1 year have experienced late rejection episodes. Late rejection is associated with poor survival. esoeciallv when associated with hemodvnamic comoromise. Absence of iate reiection episodes is associated with ;ery low risk ‘of death during medium term follow-up after pediatric heart transplantation. Determination of risk factors for late rejection helps identify a cohort of children who may benefit from enhanced rejection surveillance and treatment.
143
OUTCOME OF LISTING FOR CARDIAC TRANSPLANTATION FOR FAILED FONTAN: A MULTI-INSTITUTIONAL STUDY D Bernstein. DC Naftel. DT Hsu, LJ Addonizio, ED Blume. PL Gamberg, JK Kirklin. WR Morrow and the Pediatric Heart Transplant Study Group (PHTS). Stanford, Harvard, and Columbia Universities, Univ of Arkansas, Univ of Alabama, Birmingham, AL. The Fontan procedure is a successful palhation for children with single ventricle physiology. However, many patients eventually require heart transplantation (TX). The purpose of this study was to examine outcomes and risk factors for death while waiting and survival after undergoing TX. We analyzed data from 1002 patients listed for TX at 18 PHTS centers from Jan 1.1993 through Dee 31, 1997. Data was analyzed using actuarial. parametric, competing outcomes, and multi-variable risk factor analysis. 47 Fontan pts.
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DEATH AFTER HEARTTRANSPLANTATION IN CHILDREN: WHO DIES WHEN AND WHY EA Frazier, DC Naftel, CE Canter, MM Boucek. JK Kirklin, WR Monow, and the Pediatric Heart Transplant Study (PHTS). Univ of Alabama, Univ of Colorado, Washington Univ, and Univ of Arkansas, Liile Rock, AR, USA. Death following head bansplantation (TX) in children results from a vsriety of causes, each having a unique incidence over time. To belter understand the time related riik of death from any specific cause after TX, a competing outcomes snslysii wss used to estimate the time-related cause-specific mortality after pediatrk TX. Patkml Po~ulatlcn: