Donor apoptotic cell–based therapy for effective inhibition of donor‐specific memory T and B cells to promote long‐term allograft survival in allosensitized recipients

Allosensitization constitutes a major barrier in transplantation. Preexisting donor‐reactive memory T and B cells and preformed donor‐specific antibodies (DSAs) have all been implicated in accelerated allograft rejection in sensitized recipients. Here, we employ a sensitized murine model of islet transplantation to test strategies that promote long‐term immunosuppression‐free allograft survival. We demonstrate that donor‐specific memory T and B cells can be effectively inhibited by peritransplant infusions of donor apoptotic cells in combination with anti‐CD40L and rapamycin, and this treatment leads to significant prolongation of islet allograft survival in allosensitized recipients. We further demonstrate that late graft rejection in recipients treated with this regimen is associated with a breakthrough of B cells and their aggressive graft infiltration. Consequently, additional posttransplant B cell depletion effectively prevents late rejection and promotes permanent acceptance of islet allografts. In contrast, persistent low levels of DSAs do not seem to impair graft outcome in these recipients. We propose that B cells contribute to late rejection as antigen‐presenting cells for intragraft memory T cell expansion but not to alloantibody production and that a therapeutic strategy combining donor apoptotic cells, anti‐CD40L, and rapamycin effectively inhibits proinflammatory B cells and promotes long‐term islet allograft survival in such recipients.     

B cells: a rational target in alloantibody-mediated solid organ transplantation rejection

Loss of allografts over time remains a barrier to achieving successful outcomes in solid organ transplantation. Although the role of donor-specific alloantibody-mediated mechanisms in hyperacute rejection is well known, research and management of early and late post-transplant rejection have traditionally focused on T cell-mediated mechanisms. However, available agents that affect T-cell pathways have minimal impact on long-term graft survival, suggesting that other effector mechanisms are involved. A growing body of evidence now supports a role for alloantibody-mediated mechanisms in early and late graft rejection, which can significantly impact on long-term graft survival. The important role of B cells in generating and perpetuating alloantibody production provides a rationale for B-cell depletion therapy as an approach to prevent or reduce alloantibody formation before transplantation and to treat or prevent early and late alloantibody-mediated rejection. The use of monoclonal antibodies that directly target B cells, in combination with standard alloantibody-depleting therapies and/or immunosuppression, has been investigated in several small non-controlled studies. Promising results suggest that this strategy warrants further investigation in larger controlled studies.     

Patient and renal allograft survival in the late posttransplant period.

Finding the proper balance between too much and not enough immunosuppression is just as important in the late posttransplant period as it is during the first year after transplantation. In general, too much immunosuppression leads to an increase in patient mortality, whereas inadequate immunosuppression can lead to an inordinately high rate of allograft failure (Fig 5). In the late posttransplant period, patient and allograft survival are both critically dependent on the degree of immunosuppression and on the long-term side effects of the agents used to achieve this immunosuppression. Adequate immunosuppression is important in treating and preventing the acute allograft rejection episodes that are common during the first year after transplantation (Fig 6). Some data suggest that the severity of early acute rejection episodes may influence the development of chronic rejection, the most common cause of graft failure in the late posttransplant period. Otherwise, the role of immunosuppression in treating and preventing chronic rejection is unclear. The discontinuation of immunosuppression by noncompliant patients is a major cause of late graft failure. Whether the nephrotoxicity of CsA will also result in graft failure in the very late posttransplant period is still unknown. The agents used to achieve immunosuppression, along with decreased graft function and proteinuria, contribute to hypercholesterolemia, hypertension, and hyperglycemia. These and other risk factors have a negative impact on both graft and patient survival. Thus, immunosuppression is directly, or indirectly linked to most of the common causes of death and graft failure after renal transplantation. Although potent new immunosuppression protocols have increased the rate of short-term patient and allograft survival after renal transplantation, future advances in long-term survival after renal transplantation will depend on improvements that are effective in the late posttransplant period. Currently, the best approach to preventing complications in the late posttransplant period is to maintain a vigilant, comprehensive program of on-going medical care. The minimal amount of immunosuppression required to prevent allograft rejection should be used, while adhering to the principle that it is better to lose the graft than to lose the patient.     

Memory T Cells in Transplantation: Generation, Function, and Potential Role in Rejection

The adaptive immune system is endowed with long‐lived memory to recall previous antigen encounters and respond more effectively to them. Memory immune responses are mediated by antigen‐specific memory T lymphocytes that exhibit enhanced function compared with naïve T cells that have never encountered antigen. While the generation of memory T cells specific for pathogens is beneficial in providing protective immunity, memory T cells specific for alloantigens can be deleterious to the recipient of a transplanted organ. In graft rejection, memory T cells mediate accelerated, ‘second‐set’ rejection and their presence has been associated with increased propensity for early rejection. Recent findings have demonstrated that alloreactive memory T cells can be generated via exposure to alloantigens, as well as stimuli that are cross‐reactive with alloantigens, and are therefore likely present in ‘naïve’ individuals. This review focuses on the characteristics of memory T cells which make them of special interest to the transplant community, including differential activation requirements, broad homing properties, and resistance to tolerance induction. The multiple ways in which memory T cells can contribute to early and late graft rejection are discussed, as well as potential targets for combating alloreactive memory to be considered in the future design of tolerance induction strategies.     

Studies on the mechanism of chronic graft rejection in mice

Acute and chronic types of rejection after transplantation are distinguished. The exact mechanism, however, by which the different types of rejections have been brought about are not clarified. In this study, experimental data are presented on some characteristics of the chronic type of graft rejection. Allografting was performed between isogeneic mouse strains differing at their strong histocompatibility antigens. Recipients were treated with antithymocyte serum (ATS). Those recipients which showed a chronic type of graft rejection were selected and regrafted with a second-set or a third party allograft. It was found that, in most cases, the chronic rejection of first-set allografts did not results in a sensitization (= accelerated rejection), but rather in a partial specific tolerance (= prolonged graft survival) to a second-set allograft. Furthermore, transfer of spleen cells from mice of chronic rejection into normal, adult, ATS-treated, allografted mice did not result in any significant change of the mean graft survival as compared to mice not receiving spleen cell transfer. On the other hand, transfer of spleen cells from mice with acutely rejected grafts resulted in a shortened graft survival. It is supposed that the partial specific tolerance observed after chronic graft rejection may be maintained rather by a specific suppressor mechanism than clonal deletion.     

Indirect allorecognition: not simple but effective

Donor dendritic cells are potent but short-lived stimulators of early transplant rejection, and recipient antigen presenting cells presenting donor major histocompatibility complex peptides sustain immunoreactivity and contribute to chronic rejection. We briefly consider how CD4 T cells that recognize allopeptide can provide help for effector and regulatory responses and highlight the implications for promoting graft survival.     

Lack of Improvement in Renal Allograft Survival Despite a Marked Decrease in Acute Rejection Rates Over the Most Recent Era

Acute rejection is known to have a strong impact on graft survival. Many studies suggest that very low acute rejection rates can be achieved with current immunosuppressive protocols. We wanted to investigate how acute rejection rates have evolved on a national level in the U.S. and how this has impacted graft survival in the most recent era of kidney transplantation. For this purpose, we analyzed data provided by the Scientific Registry of Transplant Recipients regarding all adult first renal transplants between 1995 and 2000. We noted a significant decrease in overall acute rejection rates during the first 6 months, during the first year, and also in late rejections during the second year after transplantation. Despite this decrease in the rate of acute rejection, there was no significant improvement in overall graft survival; furthermore, we noted a statistically significant trend towards worse death-censored graft survival. There was also a trend for a greater proportion of rejection episodes to fail to recover to previous baseline function after treatment. Our data suggest that decreasing acute rejection rates between 1995 and 2000 have not led to an increase in long-term graft survival. Part of this discordance might be related to a higher proportion of acute rejections which have not resolved with full functional recovery in more recent years. However, the etiology of this concerning trend for worse death censored graft survival in recent years will warrant further investigation.     

Intravenous and portal venous administration of modified donor antigen prolongs rat parathyroid allograft survival

The effect of pretransplant (day -6) systemic intravenous or portal venous immunization with modified donor antigen combined with cyclophosphamide treatment (75 mg/kg on day -4) on rat parathyroid allograft survival was evaluated. Systemic intravenous preimmunization of Buffalo recipients with 10(8) untreated Lewis donor spleen cells plus cyclophosphamide resulted in 100% accelerated rejection of Lewis parathyroid allografts (mean survival time, 7.3 +/- 0.9 days vs 10.8 +/- 1.1 days for controls). Portal venous administration of untreated cells plus cyclophosphamide reduced accelerated rejection to 40% but could not prolong graft survival (10.8 +/- 2.7 days). Intravenous or portal venous preimmunization with heat-inactivated cells (45 degrees C for 60 minutes) plus cyclophosphamide also did not prolong graft survival, with accelerated rejection occurring in 20% and 40% of recipients, respectively. In contrast, preimmunization by either route with ultraviolet B-irradiated cells (UVB; 12,000 joule/m2) plus cyclophosphamide significantly prolonged graft survival (intravenous = 22.2 +/- 6.0 days and portal venous = 21.4 +/- 7.2 days; p less than 0.005), with no accelerated rejection. Preimmunization with UVB cells combined with cyclophosphamide was synergistic, because neither treatment alone prolonged allograft survival (UVB cell preimmunization only = 10.8 +/- 1.3 days; cyclophosphamide only = 12.6 +/- 2.6 days). The effect of UVB preimmunization was donor specific because third-party Wistar-Furth UVB cells had no effect on Lewis graft survival (12.5 +/- 2.9 days). We conclude that pretreatment with UVB-modified donor antigen plus cyclophosphamide induces allospecific immune hyporesponsiveness and prolongs parathyroid allograft survival.     

Long-term survival and late graft loss in pediatric liver transplant recipients—a 15-year single-center experience

Increasing numbers of children undergo successful liver transplantation. Limited data exist on long-term survival and late graft loss. Survival and graft loss were studied in 376 primary liver graft recipients who survived more than 3 months after transplantation (80.5% of all primary graft recipients). Patient records were reviewed retrospectively for causes of graft loss. Risk factors were identified by analyzing graft, recipient, and posttransplant variables using multivariate Cox regression. One-, 5-, and 10-year actuarial graft survival rates in the study population were 94.6%, 87.3%, and 86.3%, respectively. Corresponding patient survival rates were 95.7%, 91.4%, and 90.4%. Forty-seven (12.5%) grafts were lost subsequently, 15 by patient death with preserved graft function. Survival rate after late retransplantation was 63.3%. Causes of late graft loss were infection (21.2%), posttransplant lymphoproliferative disease (PTLD, 21.2%), chronic rejection (17%), biliary complications (14.8%), and recurrence of malignant disease (8.5%). Independent risk factors for late graft loss and patient death included liver malignancy as primary disease, steroid resistant rejection, and PTLD. Graft loss rate was significantly increased for reduced-size grafts. Patients undergoing transplantation after 1991 and recipients of full-size grafts were more likely to survive. In conclusion, the long-term outcome for pediatric primary liver graft recipients surviving the early postoperative period is excellent except for patients with liver malignancy. There is no increased risk of late graft loss with the use of split or living related donor grafts. Technical complications are only a minor factor in late graft loss, but complications related to immunosuppression and infection remain a major hazard and must be addressed. (Liver Transpl 2002;8:615-622.)     

Early versus late acute rejection episodes in renal transplantation

BACKGROUND: Acute rejection is a major complication after renal transplantation and the most important risk factor for chronic rejection. We investigated whether the timing of the last treated acute rejection episode (ARE) influences long-term outcome and compared the risk profiles of early versus late ARE. METHODS: A cohort of 654 patients who underwent cadaveric renal transplants (1983-1997) that functioned for more than 6 months was studied. In 384 of 654 transplant recipients, one or more treated AREs were documented; the last ARE occurred in 297 of 384 transplant recipients within 3 months and in 87 of 384 after 3 months. Applying multivariate logistic regression analysis, we compared the predictor variables of the two groups with transplants without AREs. RESULTS: Ten-year graft survival rates censored for causes of graft loss other than chronic rejection were 94%, 86%, and 45% for patients without ARE, with early ARE, and with late ARE, respectively. Delayed graft function, odds ratio (OR) 2.37 (1.55-3.62), and major histocompatibility complex (MHC) class II incompatibility, OR 2.28 (1.62-3.20) per human leukocyte antigen (HLA)-DR mismatch, were independent risk factors for early ARE. In contrast, recipient age, OR 0.75 (0.61-0.93) per 10-year increase, donor age, OR 1.28 (1.07-1.53) per 10-year increase, female donor gender, OR 1.74 (1.03-2.94), and MHC class I incompatibility, OR 1.35 (1.07-1.72) per mismatch of cross reactive groups, were associated with late ARE. CONCLUSIONS: Late ARE has a detrimental impact on long-term graft survival and is associated with MHC class I incompatibility, whereas early ARE is correlated with HLA-DR mismatches and has a better prognosis. These data are consistent with the role of direct and indirect allorecognition in the pathophysiology of early and late ARE, respectively.     

Surgical experience with retroperitoneal heterotopic heart transplantation in the large white domestic swine.

Accelerated coronary atherosclerosis following heart transplantation is the main limiting factor for long-term survival, aside from graft failure and complications due to immunosuppression. Graft coronary vasculopathy is due to chronic rejection of the vascular wall leading to intimal hyperplasia in coronary arteries. Numerous heterotopic heart transplantation models have been used in different species to study the immunology and pathophysiology following graft implantation. This study reports our experience with the retroperitoneal heterotopic heart transplantation in Large White domestic swine using immunological typing. This approach mimics the kinetics of slow low-grade rejection in clinical human heart transplantation. One hundred and fifty-four retroperitoneal (n = 154) heterotopic heart transplantations were performed using Large White swine sampled for the major histocompatibility class (MHC) class I antigen and blood type using the microlymphotoxicity technique. Acute rejection studies were performed by intentional mismatch of the swine lymphocyte alloantigen (SLA) and chronic rejection studies were done in allografts implanted in donor-recipients matched for blood type and class I antigen to assess the effects of rejection per se, hypercholesterolemia, intracoronary L-NAME infusion, and endothelial denudation on the development of graft coronary vasculopathy. Assessment of in vitro coronary arterial vascular reactivity in standard organ chamber experiments comprised the core of vascular biology studies in this large animal model. Eighty (52%) transplanted recipients survived until the elective date of sacrifice at 60 days, 14 (9.1%) died during the surgery, 21 (13.6%) died <24 h after the transplant, and 8 (5.2%) died of late deaths. The retroperitoneal heterotopic heart transplantation model with blood typing and determination of the SLA class I antigen is a useful model for the study of immunological and vascular events due to graft rejection after heart transplantation.     

A personal perspective: 100-year history of the humoral theory of transplantation

The humoral theory states that antibodies cause the rejection of allografts. From 1917 to 1929, extensive efforts were made to produce antibodies against tumors. It was finally realized that the antibodies were produced against the transplant antigens present on transplantable tumors, not against the tumor-specific antigens. To get around this problem, inbred mouse strains were developed, leading to identification of the transplant antigens determined by the H-2 locus of mice. The antibodies were hemagglutinating and cytotoxic antibodies. The analogous human leukocyte antigen system was established by analysis of lymphocytotoxic alloantibodies that were made by pregnant women, directed against mismatched antigens of the fetus. The human leukocyte antigen antibodies were then found to cause hyperacute rejection, acute rejection, and chronic rejection of kidneys. Antibodies appeared in almost all patients after rejection of kidneys. With Luminex single antigen bead technology, donor-specific antibodies could be identified before rise in serum creatinine and graft failure. Antibodies were shown to be predictive of subsequent graft failure in kidney, heart, and lung transplants: patients without antibodies had superior 4-year graft survival compared with those who did have antibodies. New evidence that antibodies are also associated with chronic failure has appeared for liver and islet transplants. Four studies have now shown that removal or reduction of antibodies result in higher graft survival. If removal of antibodies prevents chronic graft failure, final validation of the humoral theory can be achieved.     

The impact of late acute rejection after cadaveric kidney transplantation

BACKGROUND: Acute graft rejection (AR) following renal transplantation results in reduced graft survival. However, there is uncertainty regarding the definition, aetiology and long-term graft and patient outcome of AR occurring late in the post-transplant period. AIM: To determine if rejection episodes can be classified by time from transplantation by their impact on graft survival into early acute rejection (EAR) and late acute rejection (LAR). MATERIALS AND METHODS: 687 consecutive adult renal transplant recipients who received their first cadaveric renal transplant at a single centre. All received cyclosporine (CyA)-based immunosuppression, from 1984 to 1996, with a median follow-up of 6.9 yr. Details were abstracted from clinical records, with emphasis on age, sex, co-morbid conditions, HLA matching, rejection episodes, patient and graft survival. ANALYSIS: Patients were classified by the presence and time to AR from the date of transplantation. Using those patients who had no AR (NAR) as a baseline, we determined the relative risk of graft failure by time to rejection. The characteristics of patients who had no rejection, EAR and LAR were compared. RESULTS: Compared with NAR, the risk of graft failure was higher for those patients who suffered a rejection episode. A much higher risk of graft failure was seen when the first rejection episode occurred after 90 d. Thus, a period of 90 d was taken to separate EAR and LAR (relative risk of 3.06 and 5.27 compared with NAR as baseline, p<0.001). Seventy-eight patients (11.4%) had LAR, 271 (39.4%) had EAR and 338 (49.2%) had NAR. The mean age for each of these groups differed (LAR 39.6 yr, EAR 40.8 yr compared with NAR 44 yr, p<0.003). The 5-yr graft survival for those who had LAR was 45% and 10-yr survival was 28%. HLA mismatches were more frequent in those with EAR vs. NAR (zero mismatches in HLA-A: 36 vs. 24%, HLA-B: 35 vs. 23% and HLA-DR: 63 vs. 41%, p<0.003). There was no difference in mismatching frequency between NAR and LAR. CONCLUSIONS: AR had a deleterious impact on graft survival, particularly if occurring after 90 d. AR episodes should therefore be divided into early and late phases. In view of the very poor graft survival associated with LAR, it is important to gain further insight into the main aetiological factors. Those such as suboptimal CyA blood levels and non-compliance with medication should be further investigated with the aim of developing more effective immunosuppressive regimens in order to reduce the incidence of LAR.     

Characterization of intra-graft B cells during renal allograft rejection

Intra-graft CD20(+) B-cell clusters are found during acute rejection of renal allografts and correlate with graft recovery following rejection injury. Here using archived kidney tissue we conducted immunohistochemical studies to measure specific subsets of pathogenic B cells during graft rejection. Cluster-forming CD20(+) B cells in the rejected graft are likely derived from the recipient and are composed of mature B cells. These cells are activated (CD79a(+)), and present MHC Class II antigen (HLADR(+)) to CD4(+) T cells. Some of these clusters contained memory B cells (CD27(+)) and they did not correlate with intra-graft C4d deposition or with detection of donor-specific antibody. Further, several non-cluster forming CD20(-) B-lineage CD38(+) plasmablasts and plasma cells were found to infiltrate the rejected grafts and these cells strongly correlated with circulating donor-specific antibody, and to a lesser extent with intra-graft C4d. Both CD20(+) B cells and CD38(+) cells correlated with poor response of the rejection to steroids. Reduced graft survival was associated with the presence of CD20 cells in the graft. In conclusion, a specific subset of early lineage B cells appears to be an antigen-presenting cell and which when present in the rejected graft may support a steroid-resistant T-cell-mediated cellular rejection. Late lineage interstitial plasmablasts and plasma cells may also support humoral rejection. These studies suggest that detailed analysis of interstitial cellular infiltrates may allow better use of B-cell lineage specific treatments to improve graft outcomes.     

The role of the regional lymph node in the response to secondarily vascularized grafts

Studies using neonatal hearts grafted into the foot pads of adult rats have shown significant differences in the tempo of rejection in various RT1-incompatible combinations of donor and recipient rats. The model allows simultaneous study of events in the graft and in the regional node draining the graft. Removal of the regional node in the inductive stages of the immune response resulted in highly significant prolongation of graft survival. This effect was not due to lymphatic interruption per se or to clonal deletion. The effect was independent of the presence of the primary graft. Second grafts implanted in animals from which both the original graft and its regional node had been removed showed prolonged survival. Once survival of the original graft, from which the node was removed, was established, survival of second grafts bearing the same antigens was also prolonged, although third-party grafts were rejected in first set time. The data suggest that the microenvironment and anatomical connections of the lymph node that first receives antigen, or the cells that have contacted antigen in the graft, or both, play a vital role in an orderly sequence of cellular interaction and migration that culminates in graft rejection. Interruption of this sequence by node removal appears to divert the alloimmune response toward specific enhancement of the graft.     

Kidney Transplant Rejection in Australia and New Zealand: Relationships Between Rejection and Graft Outcome

Although acute rejection rates have fallen over time, how this relates to graft outcomes is not known. Using data from the ANZDATA Registry, we examined associations of rejection within six months of transplantation with graft and patient outcomes among kidney-only transplants performed between April 1997 and December 2004 in Australia and New Zealand. Associations of biopsy histology with outcomes of the rejection episode were also examined. Outcomes were examined among 4325 grafts with 1961 rejection episodes in total. Crude rejection rates have fallen by one-third over that time, but rates of graft survival are constant. The occurrence of acute rejection was associated with an increased risk of graft loss after 6 months (HR, adjusted for donor and recipient characteristics, 1.69 [1.36-2.11], p<0.001). Late rejection (first rejection >or=90 days) was associated with higher risk of graft loss (adjusted HR 2.46 [1.70-3.56], p<0.001). Vascular rejection was also associated with a higher risk of graft loss 2.07 [95% CI 1.60-2.68], p<0.001. The occurrence of acute rejection is associated with an ongoing increased risk of graft loss, particularly if that episode occurred late or included vascular rejection. The reduced rates of rejection have not been associated with improved graft survival.     

CMV infection, class II antigen expression, and human kidney allograft rejection

After successful transplantation, the major histocompatibility complex (MHC) antigens of kidney parenchymal cells are lost and no longer detectable in the graft, presumably due to administration of glucocorticosteroids. During rejection, the MHC antigens reappear in the graft parenchymal cells. The upregulation is possibly due to gamma-interferon released in situ by the allograft activated T (blast) cells. In this communication we demonstrate that cytomegalovirus (CMV) infection is invariably associated with an upregulation of the antigen display in the graft. In 12 of 14 (86%) cases with proved CMV disease, the display of class II antigens was associated with a cytological and/or clinical episode of rejection. In 223 transplant recipients without proved CMV disease transplanted during the same period, the frequency of late rejections was 17% (P less than 0.001). The results suggest that the display of class II antigens on the graft, mediated presumably by gamma-interferon as a consequence of CMV infection, is the reason for graft rejection in context of CMV disease.     

Potential T regulatory cell therapy in transplantation: how far have we come and how far can we go?

Graft survival has been lately improved by the introduction of efficient immunosuppressive drugs. However, late graft loss caused by chronic rejection and the side effects of long‐term immunosuppression remain major obstacles for successful transplantation. Operational tolerance, which is defined by the lack of acute and chronic rejection and indefinite graft survival with normal graft function in the absence of continuous immunosuppression, represents an attractive alternative. Nevertheless, tolerance after allogeneic transplantation is commonly considered the ‘mission impossible’ for both immunologists and clinicians. One of the mechanisms involved in tolerance is the suppression of graft‐specific alloreactive T cells, which largely mediate graft rejection, by regulatory T cells (Tregs) or by soluble factors produced by Treg cells. With this review, I will make an effort to collect and describe the significant studies performed in transplanted patients, and not in animal models or in in vitro systems, with the attempt to: (i) understand how tolerance is achieved, (ii) define whether and how Treg cells influence transplant tolerance, (iii) describe the first clinical trials with Treg cells in humans (i.e. how far have we come) and (iv) predict the future of Treg cell‐based therapy in humans (i.e. how far can we go).     

Living donor kidney transplantation: the effects of donor age and gender on short- and long-term outcomes

BACKGROUND: The influence of donor age and sex on acute rejection episodes and short- and long-term graft survival in living donor (LD) kidney transplantation has not been well characterized. METHODS: This prospective cohort study includes 739 first time LD transplantations with median follow-up time of 55.1 months. Death censored graft survival according to donor age and sex was compared with Kaplan-Meier plots. Cox regression was performed to estimate the association between different risk factors and graft survival and acute rejection episodes. RESULTS: Graft survival was not affected by donor age above 50 years as long as these recipients did not experience an early acute rejection episode. Acute rejection episodes increased in recipients of grafts from donors > or =65 years (P=0.009). Donor age > or =65, recipient age less than 50 years, human leukocyte antigen (HLA)-DR matching, and female donor gender were risk factors for early acute rejection episodes. In multivariate analysis donor age > or =65 years was a risk factor for graft loss in all time periods after transplantation. During the first 5 years after transplantation a steroid resistant rejection episode was an additional risk factor. More than 5 years after transplantation male donor gender was the only additional risk factor for graft loss. CONCLUSION: These results support the continued use of older male and female living donors who meet carefully constructed medical criteria and who are highly motivated to donate. Furthermore, donor age seems to be a more important predictor of graft loss than donor sex.