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The measurement of outcomes in kidney transplantation has been more accurately documented than almost any other surgical procedure result in recent decades. With significant improvements in short- and long-term outcomes related to optimized immunosuppression, outcomes have gradually shifted away from conventional clinical endpoints (ie, patient and graft survival) to surrogate and composite endpoints. This article reviews how outcomes measurements have evolved in the past 2 decades in the setting of increased data collection and summarizes recent advances in outcomes measurements pertaining to clinical, histopathological, and immune outcomes. Finally, we discuss the use of composite endpoints and Bayesian concepts, specifically focusing on the integrative box risk prediction score, in conjunction with machine learning to refine prognostication.

There is debate whether simultaneous lung-liver transplant (LLT) long-term outcomes warrant allocation of 2 organs to a single recipient. We hypothesized that LLT recipients would have improved posttransplant survival compared with matched single-organ lung recipients with an equivalent degree of liver dysfunction.The Organ Procurement and Transplant Network/United Network for Organ Sharing STAR file was queried for adult candidates for LLT and isolated lung transplantation from 2006 to 2016. Waitlist mortality and transplant odds were calculated for all candidates. Donor and recipient demographic characteristics were compiled and compared. The LLT recipients were matched 1:2 with a nearest neighbor method to single-organ lung recipients. Kaplan-Meier methods with log-rank test compared long-term survival between groups. Univariate regression was used to calculate the association of LLT and mortality within 6 months of transplant. A proportional hazards model was used to calculate risk-adjusted mortality after 6 months posttransplantation.Thirty-eight LLT patients were matched to 75 single-organ lung recipients. After matching, no differences in baseline demographics or liver function were observed between cohorts. Length of stay was significantly longer in LLT recipients compared to isolated lung recipients (45.89 days vs 22.44 days, P0.001). There was no significant difference in survival probability between LLT and isolated lung transplant (1 y, 89.5% vs 86.7%; 5 y, 67.0% vs 64.6%; P = 0.20).After matching for patient characteristics and level of liver dysfunction, survival in simultaneous LLT was comparable to isolated lung transplantation. Although this population is unique, the clinical picture prompting liver transplant is not clear. National guidelines to better elucidate patient selection are needed.

The purpose of this review is to discuss immunologic tolerance as it applies to solid organ transplantation and to identify barriers that hinder the achievement of this long-term goal. First, the definition of tolerance and an introduction of mechanisms by which tolerance exists or can be achieved will be discussed. Next, a review of contemporary attempts at achieving transplant tolerance will be described. Finally, a discussion of the humoral barriers to transplantation tolerance and potential ways to overcome these barriers will be presented., In this review regarding tolerance induction in organ transplantation, the authors highlight the current attempts of induction with their mechanisms, the contemporary barriers and the specific role of humoral immunity

Crosstalk between B and T cells in transplantation is increasingly recognized as being important in the alloimmune response. T cell activation of B cells occurs by a 3-stage pathway, culminating with costimulation signals. We review the distinct T cell subtypes required for B-cell activation and discuss the formation of the germinal center (GC) after transplantation, with particular reference to the repopulation of the GC after depletional induction, and the subsequent effect of immunosuppressive manipulation of T cell-B cell interactions. In addition, ectopic GCs are seen in transplantation, but their role is not fully understood. Therapeutic options to target T cell-B cell interactions are of considerable interest, both as immunosuppressive tools, and to aid in the further understanding of these important alloimmune mechanisms.

Background. Interleukin (IL)-15 is a chemotactic factor to T cells. It induces proliferation and promotes survival of activated T cells. IL-15 receptor blockade in mouse cardiac and islet allotransplant models has led to long-term engraftment and a regulatory T-cell environment. This study investigated the efficacy of IL-15 receptor blockade using Mut-IL-15/Fc in an outbred non-human primate model of renal allotransplantation. Methods. Male cynomolgus macaque donor-recipient pairs were selected based on ABO typing, major histocompatibility complex class I typing, and carboxy-fluorescein diacetate succinimidyl ester-based mixed lymphocyte responses. Once animals were assigned to one of six treatment groups, they underwent renal transplantation and bilateral native nephrectomy. Serum creatinine level was monitored twice weekly and as indicated, and protocol biopsies were performed. Rejection was defined as a increase in serum creatinine to 1.5 mg/dL or higher and was confirmed histologically. Complete blood counts and flow cytometric analyses were performed periodically posttransplant; pharmacokinetic parameters of Mut-IL-15/Fc were assessed. Results. Compared with control animals, Mut-IL-15/Fc-treated animals did not demonstrate increased graft survival despite adequate serum levels of Mut-IL-15/Fc. Flow cytometric analysis of white blood cell subgroups demonstrated a decrease in CD8+ T-cell and natural killer cell numbers, although this did not reach statistical significance. Interestingly, two animals receiving Mut-IL-15/Fc developed infectious complications, but no infection was seen in control animals. Renal pathology varied widely. Conclusions. Peritransplant IL-15 receptor blockade does not prolong allograft survival in non-human primate renal transplantation; however, it reduces the number of CD8+ T cells and natural killer cells in the peripheral blood.

Despite the success of immunosuppressive drug therapy to reduce the incidence of acute rejection in organ transplantation, chronic rejection is still an impediment to long-term graft survival and tolerance. There is a growing body of evidence that B-cell production of alloantibody is an important element in the genesis of chronic rejection. Effector function of B cells in transplantation is not specifically targeted by current T-cell-directed therapeutic approaches. We briefly discuss the origin, animal models, diagnostic methods, and currently available B cell reagents that might be used in combination with existing immunosuppressive regimens to address B-cell-mediated allograft injury.

Background. The best induction agent for simultaneous pancreas-kidney transplantation (SPKT) remains the subject of debate. Alemtuzumab is effective in preventing acute cellular rejection (ACR) in SPK recipients and has been used to prevent antibody-mediated rejection (AMR) in sensitized kidney transplant candidates. Methods. A retrospective cohort study was performed including 136 SPK recipients receiving maintenance immunosuppression with tacrolimus, mycophenolic acid prodrugs, and prednisone. Two groups were compared: those who received induction with alemtuzumab (n=97) and those induced with basiliximab (n=39). Results. Kidney ACR was more frequent in SPKT induced with basiliximab (2-year 12.8% vs. 3.1%, P=0.04), but the incidence of AMR was similar (2-year 18% with basiliximab vs. 13.8% with alemtuzumab, P=NS). Kidney rejection was associated with clinical pancreas rejection in 70% of cases, without differences between the groups. Postrejection kidney graft survival was similar in both groups (2-year basiliximab/alemtuzumab 94.7%/91.2%), but death-censored kidney graft survival was lower with alemtuzumab (100%/91.2%, P=0.056). In the basiliximab group, the predominant cause of kidney loss was death-with-function, whereas in the alemtuzumab group AMR accounted for all losses. Pancreas graft survival was similar in both groups, yet more pancreas losses due to acute rejection occurred in alemtuzumab-treated patients (4 vs. 1). Conclusions. Kidney AMR is more common than ACR in SPKT recipients treated with alemtuzumab, tacrolimus, mycophenolic acid, and steroids. ACR is better prevented by alemtuzumab than basiliximab, but no relevant difference is found in prevention of AMR. Despite the high incidence of AMR, survival rates are excellent in both groups.

Background. An increase in the incidence of autoimmune diseases has been described in patients receiving alemtuzumab. Methods. To determine whether induction with alemtuzumab increases recurrence of glomerular disease, we performed a retrospective study in 443 patients with biopsy-proven glomerular diseases undergoing kidney transplantation. Patients receiving alemtuzumab (n=161) were compared with those receiving interleukin (IL)-2-receptor antagonists (n=217) or antithymocyte globulin (n=64). Results. Biopsy-proven glomerular disease recurrence was similar in patients induced with alemtuzumab or IL-2 receptor antagonists. Patients receiving antithymocyte antibody had a lower recurrence rate than patients treated with other induction agents, with borderline significance (hazard ratio [HR] 0.13, 95% confidence interval [95% CI] 0.02−0.98, P=0.047). Patients with systemic lupus treated with alemtuzumab had a similar re-emergence of autoreactive antibodies to patients treated with other agents. Recurrent disease increased the risk of allograft failure (HR 2.36, 95% CI 1.28−4.32, P=0.0056). The development of acute rejection and the use of deceased (vs. living) donor kidneys were also significant factors influencing graft survival. A greater risk of mortality was detected in those patients with recurrent glomerular disease (HR 3.76, 95% CI 1.37−10.35, P=0.01), whereas increased age at transplantation (HR 1.05) and the use of deceased (vs. living) donor kidneys (HR 3.20) also increased mortality. No specific induction agent significantly affected graft loss or mortality when using adjusted or unadjusted hazard ratios. Conclusions. In this retrospective analysis, induction with alemtuzumab did not increase the rate of re-emergence of autoantibodies or biopsy-proven recurrence of glomerular disease. A slight reduction in the incidence of recurrence was observed in patients treated with thymoglobulin, yet this observation can only be validated in a prospective randomized trial.

BACKGROUND Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. METHODS Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. RESULTS The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. CONCLUSIONS Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.

Background A fundamental limitation of in vitro immunologic tests in the field of transplantation is that existing functional tests poorly correlate with in vivo immune responses such as rejection, tolerance, or absence of rejection due to immunosuppression. It would be helpful to have a measure of T lymphocyte responsiveness that reliably reflects these conditions. Methods C57BL/6J mice received skin transplants from BALB/c donors with: a) no treatment, b) treatment with CsA, or c) treatment with CTLA-4Ig, alpha-CD40L mAb, and alpha-CD25 mAb. Syngeneic skin transplants served as controls. Recipient splenocytes were co-cultured with irradiated donor splenocytes and culture supernatant was harvested once a day for 5 consecutive days. IFN-gamma levels were measured by ELISA. Results Splenocytes obtained from non-transplanted mice responded to specific alloantigen stimulation (primary response) at least 2 days later than the splenocytes from mice which had rejected skin grafts (effector/memory response). Splenocytes from mice treated with CsA after skin transplants had no response to third-party alloantigen, but showed an effector/memory pattern of IFN-gamma elaboration with donor cell stimulation (immunosuppression), although the IFN-gamma levels were not as high as those mice with unmodified graft rejection. Mice treated with combined CTLA4Ig, alpha-CD40L and alpha-CD25 accepted skin grafts without further immunosuppression. Splenocytes from these tolerant mice showed a primary response to the third-party and failed to secrete detectable IFN-gamma in the presence of donor cells (tolerance). Conclusion This assay clearly differentiated the functional status of the alloreactive T cells, including primary alloimmune response, effector/memory response, immunosuppressed T cell response, and donor specific tolerance.

BACKGROUND Kidney transplant patients given Campath-1H (Alemtuzumab) immunodepletion therapy and long-term rapamycin monotherapy have excellent graft survival and function at three years. As an initial step in understanding the characteristics of repopulated T lymphocytes in these patients, we performed several assays to assess alloreactivity. METHODS We measured T-cell responses using CFSE-labeled recipient lymphocytes in a direct one-way MLR, and also analyzed the kinetics of expression of IFN-gamma. We examined the T-cell responses of Campath-treated transplant patients on monotherapy versus those treated with anti-CD25 (Basiliximab) induction therapy and maintenance immunosuppression consisting of cyclosporine A, mycophenolate mofetil, and steroids. RESULTS On average, proliferative responses to donor antigen were equal between Campath and control groups. However, the Campath group displayed a greater response to third party compared to donor antigen (CD3 P = 0.04, CD4 P = 0.07, CD8 P < 0.01), whereas the control group did not display a greater response to third party (CD3 P = 0.69, CD4 P = 0.72, CD8 P = 0.60). Interestingly, more Campath patients (4 of 15) than control patients (0 of 8) displayed donor specific unresponsiveness as gauged by IFN-gamma expression and T-cell proliferation (P = 0.15). CONCLUSIONS These studies suggest that Campath-1H in conjunction with rapamycin monotherapy retains intact immune responses to third party alloantigen, yet may promote hyporesponsiveness to donor antigen.

Background. In this study, we determined whether Campath-1H induction followed by sirolimus monotherapy inhibited alloantibody production in renal transplantation. Second, we evaluated the correlation between human leukocyte antigen (HLA) antibody production and serum creatinine levels. Methods. Sera were taken 1 to 24 months after transplantation from 24 patients treated with Campath-lH and sirolimus and tested for serum creatinine and HLA-specific antibody by using flow cytometry and enzyme-linked immunosorbent assay. Results. Ten (42%) of the 24 patients treated with Campath-1H and sirolimus produced HLA antibodies. Six of these 10 developed both donor-specific antibodies (DSAs) and non-donor-specific antibodies (NDSAs), whereas only NDSAs were detected in the other four patients. In patients with biopsy-diagnosed humoral rejection (C4d+ serum levels of both DSA and NDSA significantly correlated with patient serum creatinine levels. Rejection treatment successfully reduced both DSAs and NDSAs and reversed humoral rejection. Conclusions. The numeric relationship between serum creatinine and DSA levels suggests a causal relationship between alloantibody and transplant rejection.

Background.This study assesses the safety and efficacy of the novel human anti-human CD154 monoclonal antibody ABI793 in rhesus monkeys.Methods.Outbred rhesus monkeys were used for renal transplantation from major histocompatibility complex-mismatched donors. Seven recipients were treated with ABI79

It is clear that both humans and nonhuman primates can do without immunosuppression for long periods of time before rejecting their allogeneic organ transplants. Immune cell depletion, particularly lymphocyte depletion, is an effective clinical strategy for achieving a tolerant state. Based on nonhuman primate and human studies, evidence suggests that metastable tolerance develops over time in a donor-specific manner and is mediated at least in part by donor antigen-specific regulatory T cells. Suppression of effector T cells by CD8+ and CD4+ T-regulatory cells is mediated by transforming growth factor (TGF)-beta or interleukin 10, and the presence of CD4+ TGF beta(latent) T-cell infiltrates may be a useful diagnostic marker for metastable tolerance. Loss of these cells has been shown to correlate with loss of tolerance. Future efforts to withdraw immunosuppressive drugs and establish a tolerant state will depend importantly on development of such diagnostic immunologic monitoring tools. Some of the reasons that tolerance remains such a challenging goal in clinical transplantation are discussed herein.

Background. Chronic allograft nephropathy (CAN) remains a major barrier to long-term allograft survival. The authors retrospectively compared the development of CAN in recipients of cadaveric (CAD), living-related donor (LRD), and living-unrelated donor (LURD) transplants at their center. Methods. The authors retrospectively examined the impact of various factors on the incidence of CAN using univariate and multivariate proportional hazards analysis in a single-center kidney transplant population. Results. Between 1 January 1990 and 31 May 2000, 2,140 kidney-alone transplants were performed at the authors' center. The overall 5-year incidence of biopsy-proven CAN was 12.2% (n=203). Risk factors for CAN included the number of transplants (P=0.0001), acute rejection (P=0.0001), panel reactive antibody (P=0.0001), discharge creatinine (P=0.0001), 1-year creatinine (P=0.0015), delayed graft function (P=0.007), total human leukocyte antigen (HLA)-B and -DR mismatches (P=0.0005), recipient age (P=0.003), black donor race (P=0.001), black recipient race (0.0457), donor age (P=0.0053), cold storage time (P=0.019), and cytomegalovirus infections (P=0.002). Interestingly, although the LRD HLA-identical recipients had a significantly lower incidence of CAN (P=0.0015), the incidence of CAN in CAD and HLA-nonidentical LRD recipients did not differ. Graft survival was significantly worse in CAD recipients compared with all other groups (P

Background Transplantation of the pancreas has become the treatment of choice for selected patients with type 1 diabetes mellitus. With the current shortage of cadaver donors and the increasing number of diabetic patients on the transplant waiting list, there is a critical need to optimally use all available pancreas grafts for transplantation. We have therefore explored the use of traditionally "less-than-ideal" pancreas donors, including pediatric (4-10 years), older (>or=45 years), obese (weight >or=200 lb), and non-heart-beating donors and donors with an elevated amylase (75% greater than normal values). Methods A total of 620 primary simultaneous pancreas-kidney transplantations were performed at our center. We analyzed the ratio of livers to pancreata transplanted at our center and compared this to the United Network for Organ Sharing database. Using univariate and multivariate analyses, we then assessed the impact of these less-than-ideal donors on patient survival, graft survival, and postsurgical complications after simultaneous pancreas-kidney transplantation. Results A substantial nationwide underutilization of pancreata from donor procurements is demonstrated in the United Network for Organ Sharing database. By using these less-than-ideal donors, the ratio of liver to pancreata procured can be reduced to 1.25:1. Graft survival was not significantly different in patients receiving transplants from obese, non-heart-beating, pediatric, or hyperamylasemic donors compared with grafts from ideal donors. However, grafts from donors 45 years of age or older had significantly lower 1- and 5-year graft survival rates (76% and 65% vs. 90% and 80%, P=0.006). Conclusions This study demonstrates that utilization of pancreas grafts from selected, less-than-ideal donors results in good overall outcomes and could potentially expand the organ donor pool.

Background. The discovery of new immunosuppressive agents has enhanced short-term graft survival. However, current immunosuppressants often induce toxicities that limit their clinical use. Thus, there is a need for new immunosuppressants for use in clinical transplantation. Piceatannol blocks Syk and ZAP-70, tyrosine kinases involved in immune cell activation. We examined whether piceatannol prolongs kidney allograft survival in the stringent ACI-to-Lewis rat model. Methods. Kidney recipients were divided into four groups. Group 1 (n=8) received piceatannol 30 mg/kg per day intravenously and cyclosporine A (CsA) 2 mg/kg per day intramuscularly from day -3 to day 7 after transplantation. At day 8, piceatannol was reduced to 10 mg/kg per day and the combined treatment continued until day 60. Group 2 (n=9) received 2 mg/kg per day CsA alone from day -3 to day 60. Group 3 (n=4) received piceatannol alone as in group 1. Group 4 (n=2) received only the vehicle dimethyl sulfoxide from day -3 to day 60. Graft rejection was defined as either a serum creatinine level more than 2 mg/dL or animal death. Results. Group 1 animals survived for at least 115 days (n=8, P

Background. The T-cell receptor (TCR)/CD3 complex is the target of therapeutic strategies aimed at prolonging allograft survival. The immunotoxin FN18-CRM9, composed of the anti-CD3 monoclonal antibody FN18 and the mutated diphtheria toxin CRM9, is useful for prolonging allograft survival in preclinical models of transplantation. To explore the influence of conjugation of the mutated diphtheria toxin on functional activation of the TCR/CD3 complex, we compared the effects of FN18-CRM9 and unconjugated FN18 on protein tyrosine phosphorylation and ligand/ receptor internalization in purified monkey peripheral blood T cells, Methods. Purified normal rhesus monkey T cells were incubated with unconjugated FN18 or conjugated FN18-CRM9 and examined for differences in antibody binding, tyrosine phosphorylation, and CD3 internalization. Results. Binding cross-inhibition studies demonstrated that both compounds were able to inhibit fluorescein isothiocyanate-FN18 binding to CD3 with similar efficacy and potency. However, FN18-CRM9 was more potent than FN18 in triggering the phosphorylation of several proteins on tyrosine residues and in inducing CD3 internalization. The tyrosine kinase inhibitor genistein blocked FN18-CRM9-induced protein tyrosine phosphorylation and CD3 internalization, suggesting that tyrosine phosphorylation is involved in the internalization of the immunotoxin. Interestingly, in FN18-CRM9- but not FN18-treated cells, there was a gradual decrease in cellular CD3 protein levels within 24 and 48 hr; such a decrease was not observed with the control protein Csk. Conclusions. Our findings suggest that the conjugation of the mutated diphtheria toxin CRM9 to FN18 modulates the monoclonal antibody-mediated crosslinking of the TCR/CD3 complex, leading to a stronger protein tyrosine phosphorylation and CD3 internalization. This may in turn contribute to the greater efficacy of the immunotoxin in prolonging allograft survival.

Background. Initiated in 1995, the Studies of Pediatric Liver Transplantation (SPLIT) registry database is a cooperative research network of pediatric transplantation centers in the United States and Canada. The primary objectives are to characterize and follow trends in transplant indications, transplantation techniques, and outcomes (e.g., patient/graft survival, rejection, growth parameters, and immunosuppressive therapy.) Methods. As of June 15, 2000, 29 centers registered 1144 patients, 640 of whom received their first liver-only transplant while registered in SPLIT. Patients are followed every 6 months for 2 years and yearly thereafter. Data are submitted to a central coordinating center. Results. One/two-year patient survival and graft loss estimates are 0.85/0.82 and 0.77/0.72, respectively. Risk factors for death include: in ICU at transplant (relative risk (RR) = 2.63, P

Background. Anti-CD3 immunotoxin (IT), a T-cell-depleting agent, prolongs survival of renal allografts in a rhesus monkey model without the need for long-term immunosuppression. In this study we sought to further prolong allograft survival by giving short-term conventional immunosuppression simultaneous with IT administration. Methods. MHC class II mismatched, juvenile rhesus monkeys were paired as donor and recipient for renal transplantation. Recipients received two to three daily doses of IT starting on the day of transplantation. Additional immunosuppression was given for no more than 60 days. Graft function was monitored by serum creatinine and renal biopsies. Flow cytometry was used to monitor T-cell recovery. Results. Graft survival time (GST) in animals receiving IT was prolonged compared with controls with 50% of IT-treated monkeys surviving >100 days. Animals treated with IT plus mycophenolate mofetil (MMF) and steroids had significantly enhanced GST (mean GST, 305 days) compared with those treated with IT alone (mean GST, 94 days). In contrast, addition of cyclosporine or 40-O-[2-Hydroxyethyl]rapamycin did not significantly increase graft survival time. A comparison among animals from all treatment groups with short ( 100 days) GST demonstrated that those with the shorter GST had a higher blood T-cell count 2 weeks after transplantation. Full recovery of CD4 + T cells required longer than 6 months. Conclusions. A combination with MMF and steroids given for 4 days after renal allograft transplantation significantly increases GST in IT-treated monkeys. We hypothesize that MMF and steroids suppress the initial T-cell activation mediated by IT.

BACKGROUND: Several conventional forms of immunosuppression have been shown to antagonize the efficacy of anti-CD154 monoclonal antibody- (mAb) based costimulatory molecule blockade immunotherapy. Our objective was to determine if allograft recipients treated with a conventional immunosuppressive regimen could be sequentially converted to anti-CD154 mAb monotherapy without compromising graft survival. METHODS: Outbred juvenile rhesus monkeys underwent renal allotransplantation from MHC-disparate donors. After a 60-day course of triple therapy immunosuppression with steroids, cyclosporine, and mycophenolate mofetil, monkeys were treated with: (1) cessation of all immunosuppression (control); (2) seven monthly doses of 20 mg/kg hu5C8 (maintenance), or; (3) 20 mg/kg hu5C8 on posttransplant days 60, 61, 64, 71, 79, and 88 followed by five monthly doses (induction+maintenance). Graft rejection was defined by elevation in serum creatinine>1.5 mg/dl combined with histologic evidence of rejection. RESULTS: Graft survival for the three groups were as follows: group 1 (control): 70, 75, >279 days; group 2 (maintenance): 83, 349, >293 days, and; group 3 (induction+maintenance): 355, >377, >314 days. Acute rejection developing in two of four monkeys after treatment with conventional immunosuppression was successfully reversed with intensive hu5C8 monotherapy. CONCLUSIONS: Renal allograft recipients can be successfully converted to CD154 blockade monotherapy after 60 days of conventional immunosuppression. An induction phase of anti-CD154 mAb appears to be necessary for optimal conversion. Therefore, although concurrent administration of conventional immunosuppressive agents including steroids and calcineurin inhibitors has been shown to inhibit the efficacy of CD154 blockade, sequential conversion from these agents to CD154 blockade appears to be effective.

Background. In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival.The role of endothelial cell alteration in chronic rejection was examined in our model. Methods. Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale. Results. Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection. Conclusion. Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

BACKGROUND: Advances in perioperative care and immunosuppression have enabled clinicians to broaden the indications for organ transplantation. Advanced age is no longer considered a contraindication to transplantation at most centers. Although short-term studies of elderly liver transplant recipients have demonstrated that the incidence of complications and overall patient survival are similar to those of younger adults, transplant center-specific, long-term data are not available. METHODS: From August of 1984 to September of 1997, 91 patients 60 years of age or older received primary liver transplants at the University of Wisconsin, Madison. This group of patients was compared with a group of younger adults (n=387) ranging in age from 18 to 59 years who received primary liver transplants during the same period. The most common indications for transplantation in both groups were Laennec's cirrhosis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, and cryptogenic cirrhosis. There was no difference in the preoperative severity of illness between the groups. Results. The length of hospitalization was the same for both groups, and there were no significant differences in the incidence of rejection, infection (surgical or opportunistic), repeat operation, readmission, or repeat transplantation between the groups. The only significant difference identified between the groups was long-term survival. Five-year patient survival was 52% in the older group and 75% in the younger group (P

BACKGROUND Hypoalbuminemia is associated with poorer outcomes in renal transplantation. Diabetes can compound hypoalbuminemia's detrimental effects. Kidney-pancreas transplantation alters the diabetic milieu; yet, some patients continue to be hypoalbuminemic. METHODS We retrospectively analyzed 232 patients who underwent simultaneous kidney-pancreas transplantation (SPK) between 1993 and 1997 to determine the incidence and clinical correlates of hypoalbuminemia in SPK recipients. Post-SPK hypoalbuminemia was defined as a serum albumin level < or =3.5 g/dl. Univariate analyses were performed to determine whether post-SPK hypoalbuminemia was associated with pre-SPK variables. The effect of albumin level and hypoalbuminemia on the risk of post-SPK events (cardiac events, cytomegalovirus [CMV] infection, rejection, readmission, kidney and pancreas graft failure, and death) was examined with a Cox proportional hazards model. RESULTS The study population consisted of 149 men and 83 women. Average follow-up was 2.0+/-1.3 years. Hypoalbuminemia (serum albumin level < or =3.5 g/dL) was most common early after SPK (3 months: 44% of evaluable patients were hypoalbuminemic; 12 months: 15.3%; 36 months: 8.3%). Acute rejection episodes and readmission were the most common adverse events after SPK transplantation. There were 24 episodes of renal allograft loss and only 5 cardiac events. Ten SPK recipients died during the study time period. SPK-related hypoalbuminemia was associated with an increased risk for CMV infection (risk ratio [RR] 2.5; P

Background. Although bladder drainage of the pancreas remains the most common site for drainage of exocrine secretions, enteric drainage is becoming more common in the United States. The most common cause of morbidity after pancreas transplantation is infection, particularly recurrent urinary tract infection. Methods. We examined the incidence of infectious complications for enteric-drained (ED) versus bladder-drained (BD) simultaneous pancreas-kidney transplants (PTx) to determine the incidence of posttransplant infection. The patient cohort included simultaneous pancreas-kidney PTx recipients from June 1995 through August 1997 using a similar induction protocol with antithymocyte globulin, mycophenolate mofetil, prednisone, and Neoral. During this time period, 48 BD PTx and 78 ED PTx were performed. Demographic data including age of transplant, gender, race, and duration of initial hospital stay were similar. However, mean follow-up for the BD PTx was 1.9 years vs. 0.9 years for ED PTx. Rejection, infection, and graft and patient survival rates were estimated by the method of Kaplan and Meier. Results. For the entire cohort, 1-year patient survival was 98%, kidney survival 94%, and pancreas survival 93%. There was no difference in survival between ED or BD PTx. At 6 months, kidney transplant rejection had occurred in 38% of BD PTx vs. 30% of ED PTx. Steroid resistant rejection was similar (BD 19%, ED 17%). Postoperative pancreatic leak occurred in 12% BD PTx and 5% ED PTx (P=0.06). There was no significant difference in time to first infection or first abdominal infection between groups. Opportunistic infections were much less likely to occur in ED recipients by 1 year (12% vs. 31%, P=0.002). Both cytomegalovirus infection rates (BD 21% vs. ED 8%, P=0.04) and fungal infection rates (BD 17% vs. ED 4%, P=0.04) were lower in ED PTx. The rate of first urinary tract infection was dramatically decreased with ED. At 1 year, only 20% of ED PTx developed a urinary tract infection vs. 63% of BD PTx (P=0.0001). Conclusion. Enteric drainage of the pancreas is more physiologic, has similar results to bladder drainage, but has less infectious complications, particularly urinary tract infections.

Background. Mycophenolate mofetil (MMF; Cell-Cept) is a potent and selective inhibitor of B and T lymphocyte proliferation that has proven effective in reducing the incidence of acute rejection in cadaveric kidney transplant recipients in several randomized, blinded clinical studies. Because the frequency and characteristics of rejection episodes may be different and more severe after combined pancreas-kidney transplantation, we hypothesized that MMF would have a significant impact on pancreas-kidney rejection and graft outcome. Therefore, we compared the efficacy of MMF versus azathioprine (AZA) in cyclosporine-treated simultaneous pancreas-kidney transplantations. Methods. A retrospective comparison of 358 consecutive primary SPK transplantations performed from 1990 to 1997 was conducted. Patients received either MMF (n=109, 3 g/day) or AZA (n=249, 2 mg/kg q.d.) in combination with cyclosporine-based immunosuppression. All patients received a quadruple-drug sequential induction protocol with either OKT3 or At-gam. Several outcome parameters, including patient and graft survival rates and frequency of rejection, were analyzed. Results. MMF-treated patients demonstrated a markedly reduced rate of biopsy-proven kidney rejection (31 vs. 75% AZA, P=0.0001), clinically significant pancreas rejection (7 vs. 24% AZA; P=0.003), and steroid-refractory rejection (15 vs. 52% AZA; P=0.01). As a result, kidney and pancreas allograft survival was significantly better in MMF patients compared with AZA patients (2-year survival rates: kidney, 95 vs. 86%; and pancreas, 95 vs. 83%). Although surgical infections after transplantation were more frequent in MMF patients, MMF patients were more likely to have undergone enteric drainage. Importantly, we did not observe an increased incidence of any of the bacterial, fungal, or viral infections that typically plague immunosuppressed transplant recipients. Conclusions. This retrospective study demonstrates that MMF is a highly effective immunosuppressant in SPK transplantation. It is not associated with an increased risk of opportunistic infections when a balanced immunosuppressive management approach is used. MMF strikingly reduces the frequency of acute cellular and steroid-resistant rejection. As a result of this combined experience, it is not unexpected then that we observe significantly improved graft survival rates in MMF-treated SPK patients compared with patients receiving a more traditional immunosuppressive regimen.

Background. Thymoglobulin, a rabbit anti-human thymocyte globulin, was compared with Atgam, a horse anti-human thymocyte globulin for the treatment of acute rejection after renal transplantation. Methods. A multicenter, double-blind, randomized trial with enrollment stratification based on standardized histology (Banff grading) was conducted. Subjects received 7-14 days of Thymoglobulin (1.5 mg/kg/ day) or Atgam (15 mg/kg/day). The primary end point was rejection reversal (return of serum creatinine level to or below the day 0 baseline value). Results. A total of 163 patients were enrolled at 25 transplant centers in the United States. No differences in demographics or transplant characteristics were noted. Intent-to-treat analysis demonstrated that Thymoglobulin had a higher rejection reversal rate than Atgam (88% versus 76%, P=0.027, primary end point). Day 30 graft survival rates (Thymoglobulin 94% and Atgam 90%, P=0.17), day 30 serum creatinine levels as a percentage of baseline (Thymoglobulin 72% and Atgam 80%; P=0.43), and improvement in posttreatment biopsy results (Thymoglobulin 65% and Atgam 50%; P=0.15) were not statistically different. T-cell depletion was maintained more effectively with Thymoglobulin than Atgam both at the end of therapy (P=0.001) and at day 30 (P=0.016). Recurrent rejection, at 90 days after therapy, occurred less frequently with Thymoglobulin (17%) versus Atgam (36%) (P=0.011).

Background. FN18-CRM9 is a CD3-specific immunotoxin that is capable of depleting CD3 + T cells. Pretreatment of rhesus monkeys with this agent before transplantation can induce donor-specific tolerance and split tolerance to renal allografts. Methods. Heterotopic renal transplants were performed on monkeys that received posttransplant FN18-CRM9. Histological and immunohistological staining, as well as analysis of the intragraft cytokine profile by reverse transcriptase polymerase chain reaction, was performed on percutaneous allograft biopsies. Results. Experimental monkeys had significant prolongation of allograft survival. Although an interstitial, mononuclear cell infiltrate was seen in all of the renal transplants, there was minimal evidence of acute cellular rejection. Histological evidence of alloantibody-mediated damage was detected 3 to 5 months after transplantation in the monkeys treated with FN18-CRM9. Immunohistology demonstrated the reappearance of CD3 + and CD4 + T cells, as well as CD20 + B cells, in the grafts. Cytokine analysis demonstrated expression of interferon-γ. An intact anti-donor IgG response was seen. Conclusion. Treatment of monkeys with FN18-CRM9 immediately after transplantation significantly prolongs renal allograft survival. Allograft biopsies demonstrate a lack of acute cellular rejection; however, alloantibody-mediated graft damage and rejection occur, with an intact anti-donor IgG response. The intragraft expression of the interferon-γ may reflect this ongoing humoral rejection. These data suggest that even a brief period of T-cell allosensitization may lead to humorally mediated allograft damage. Efforts to achieve tolerance with posttransplant FN18-CRM9 will require modification of the protocol to deplete T cells before allosensitization exposure or to supplement the posttransplant immunomodification strategy.

Background. Renal allografts were performed in rhesus monkeys using FN18-CRM9, a potent immunotoxin capable of depleting T cells to less than 1% of baseline levels in blood and lymph nodes, as a preparative agent. We have recently reported that animals pretreated with FN18-CRM9 1 week before transplantation without further immunosuppression had prolonged graft survival time compared with control animals, and frequently became tolerant. Methods. This report examines the alloimmune responses of recipient monkeys to the donor, including cytotoxic T lymphocyte precursor (CTLp) frequency, mixed lymphocyte response, and antidonor IgG response. Results. CTLp frequencies declined significantly (P 150 days), an increase in anti-third-party CTLp was detected 1 month after grafting with third-party skin. No change was seen in the antidonor CTLp frequency after donor skin grafting, indicating that a specific defect in the antidonor CTL response had developed. Conclusions. These data suggest that FN18-CRM9 treatment of rhesus monkeys allows the development of specific down-regulation of antidonor CTL activity in renal allograft recipients.

Background Transplant tolerance, rather than immunity, may be favored in the setting of a lower mature lymphoid mass in the recipient induced by anti-T cell agents. A novel immunosuppressive agent, FN18-CRM9, known to specifically kill T cells with great potency, was evaluated in a transplant model. Methods In order to ablate recipient T cells, the immunotoxin FN18-CRM9 was administered to rhesus monkey recipients of MHC-mismatched renal allografts. Donor lymphocytes were injected intrathymically into some animals. Results All monkeys with T-cell depletion by immunotoxin had prolonged allograft survival, and tolerance confirmed by skin grafting has been confirmed in five of six long-surviving recipients. Conclusions In this clinically relevant model, profound but transient T-cell depletion by a single agent substantially promotes tolerance.

Background. Exposure to hepatitis C virus (HCV) and subsequent infection after renal transplantation lead to significant clinical hepatitis in approximately 50% of graft recipients. Methods. One hundred thirty-two consecutive renal allotransplant patients, who underwent transplantation of kidneys from HCV-positive cadaveric donors, were studied to investigate the relationship between donor and recipient HLA type and the risk of developing clinical hepatitis. Specific attention was directed toward the DR3 and DR4 alleles, as these had previously been associated with worse prognoses in autoimmune and viral hepatitis. Results. Overall, 42% of patients receiving kidneys from donors seropositive for HCV developed clinical hepatitis. This was unrelated to preoperative recipient HCV serum reactivity (P=0.65). Patients receiving kidneys from seropositive donors with HCV RNA as detected by PCR were more likely to develop hepatitis than those receiving kidneys from PCR-negative donors (56% vs. 11%; P=0.005). The presence of the DR3 allele was associated with a significant risk of clinical hepatitis (P=0.025); 80% of DR3-positive recipients (n=34) progressed to hepatitis compared with 42% of DR3-negative patients. No other recipient HLA type was significantly related to prognosis. All patients receiving a donated kidney that expressed the B41 allele developed hepatitis, compared with 55% of recipients of non-B41 grafts (P=0.039). No association between the development of clinical hepatitis and HLA compatibility was found. Conclusions. These results suggest that both HLA type and viral presence as assayed by polymerase chain reaction, influence the risk of disease progression after transplantation of HCV-positive kidneys. Application of these associations may decrease the relative risk of a recipient contracting HCV hepatitis after cadaveric renal transplantation.

With the ultimate goal of modulating the host immune response in organ transplantation, gene therapy studies have demonstrated that direct plasmid DNA injection into transplanted myocardium can result in detectable levels of transgene expression. However, the restricted distribution and low level of transgene expression evident in these studies have limited its application. Recently, replication-defective adenovirus vectors have been shown to be an efficient gene-transfer vehicle in vivo whose infection does not require target-cell proliferation. In the present study, adenovirus vectors encoding reporter genes were delivered into transplanted hearts by either direct injection into the myocardium or perfusion via aorta of the donor hearts. The efficacy and stability of the transgene expression by perfusion and by direct injection were examined and compared. Using the adenovirus vector encoding the firefly luciferase gene, we found that a higher level of transgene expression was achieved by direct injection, but that more evenly distributed transgene expression was observed in hearts perfused with viral vector. These results were further confirmed by 5-bromo-4-chloro-3-indolyl-beta-d-galactoside histochemical staining of another adenoviral vector encoding beta-galactosidase. The transgene expression was not stable and decreased within 1 month with either delivery method. Nevertheless, these results indicate that adenovirus-mediated gene transfer can result in short-term expression of the gene throughout the heart and may be useful as a gene vector in organ transplantation.

We studied multiple determinants of graft survival at a single center and the effects of nonimmunologic graft loss on transplant survival. This retrospective study examined the results of 589 cadaver donor transplants performed between 1986 and 1992. Graft survival rates were calculated using Kaplan-Meier estimates for both overall graft survival (all causes of graft loss) and immunologic graft survival (function lost due to acute or chronic rejection and noncompliance). Cadaver graft survival was significantly poorer with an increasing degree of DR mismatch (P=0.02). An analysis of pretransplant variables showed graft loss risk was highest with greater DR mismatches, two B-antigen mismatch, higher donor serum creatinine, and younger recipient age. After transplantation, acute rejection was the most significant factor associated with long-term graft survival. Our data demonstrate a significant advantage for zero DR and one DR mismatch cadaver donor transplants, with excellent immunologic graft survival. This study suggests that a combination of immediate graft function, prevention of acute rejection by appropriate early immunosuppressive therapy, and acceptable DR match enhances cadaveric graft survival.

B7.75-84, a 10-amino-acid peptide derived from the HLA-B7 molecule, prolongs rat heterotopic cardiac allograft survival time (GST) when used with cyclosporine in the Lewis-to-ACI strain combination. We evaluated the ability of B7.75-84 to prolong GST in other strain combinations without cyclosporine and studied the effect of B7.75-84 on the immune response in the Wistar-Furth (WF)-to-ACI strain combination. GST was markedly prolonged in most low-responder (ACI) recipients but only slightly prolonged in the high-responder (Lewis) recipient. Cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) limiting dilution assays (LDA) were performed 10 days after cardiac allografts from WF donors were placed in ACI recipients treated with B7.75-84. HTL-LDA assays at 10 days posttransplant showed a slight decrease in HTL precursor frequency and a decrease in their IL-2 production in B7.75-84 treated recipients with prolonged GST in response to donor antigen as well as third-party (Lewis) antigen. CTL-LDA assays at day 10 showed no difference in CTL precursor frequency among treated recipients but did show a significant decrease in CTL killing activity against donor cells in recipients with prolonged GST. No significant difference in CTL killing activity was seen against third- party cells. Antibody analysis was performed at day 8 in treated recipients. Serum from B7.75-84-treated recipients with prolonged graft survival generally showed no detectable IgG antibody response against donor MHC class I antigen. All B7.75-84 treated recipients showed a strong IgM response against donor antigen regardless of allograft outcome. Our results suggest that the immunosuppressive effect of B7.75-84 in rats is greater using a low-responder RT1 haplotype. Furthermore, B7.75-84 induces a nonspecific decrease in HTL function while producing a donor-specific decrease in CTL function and a diminished antidonor MHC class I IgG response.

Factors influencing the incidence of recurrent glomerulonephritis following renal transplantation are poorly understood. Bilateral pretransplant native nephrectomy has been advocated to reduce the likelihood of recurrence after renal transplant. However, there is significant morbidity of native nephrectomy in the uremic population. Therefore, we sought to determine the effect of pretransplant native nephrectomy on the incidence of recurrent primary glomerulonephritis and the attendant risk of graft failure due to recurrent disease. Three hundred sixty-four consecutive cadaveric (n = 214), living-related (n = 137), and living-unrelated (n = 13) renal transplants were performed in 319 patients with a diagnosis of primary glomerulonephritis. Specific diagnoses included were focal segmental glomerulosclerosis (FSGS), rapidly progressive glomerulonephritis/idiopathic crescentic glomerulonephritis (RPGN/ICG), IgA nephropathy (IgA), mesangioproliferative glomerulonephritis, type I and II (MPG), anti-glomerular basement membrane nephritis (anti-GBM), and membranous glomerulonephritis (MGN). Rates of recurrence and graft loss were compared between patients treated with bilateral native nephrectomy (n = 61) and those who were not (n = 303). Bilateral nephrectomy did not prevent or delay the onset of recurrent glomerulonephritis in the renal allograft. In fact, there was a significantly increased five- and ten-year risk of recurrence in patients undergoing pretransplant nephrectomy vs. no nephrectomy (25.2% and 42% vs. 13.9% and 19.4%, P < 0.02, respectively). The increased rate of recurrence was evident in the CAD/LUD recipients, but not in recipients of LRD transplants. Of the specific diseases, FSGS and MGN recurred more commonly (20.2% and 20.3%, respectively). A detrimental effect of pretransplant nephrectomy on recurrence rates and incidence of graft loss due to recurrent disease independent of other variables could be demonstrated only for FSGS patients. Based on these findings, we no longer recommend native nephrectomy in the prospective renal transplant recipient at high risk for developing recurrent glomerulonephritis.

The current liver allocation system has been criticized, since available organs go to those who are the most critically ill. These recipients have the poorest overall survival. Identification of pretransplant risk factors for mortality would allow better allocation of donor livers. This study was a retrospective analysis of pretransplant clinical and laboratory parameters and subsequent postoperative liver transplant mortality to identify high-risk subgroups. Of 347 consecutive consecutive primary liver transplant recipients, 59 (17%) met United Network for Organ Sharing (UNOS) criteria for status 4. Pretransplant factors included liver function, coagulation, albumin and ammonia levels, renal function, the presence of ascites, and etiology of liver disease. Overall 1-year patient survival was significantly worse for the status 4 recipients (89.0fi vs. 67.7% ; P=0.01). In a univariate analysis of pretransplant risk factors for all recipients, elevated creatinine (P=0.008) and ammonia (P=0.009), and UNOS status 4 (P=0.01) significantly affected postoperative survival. In multivariate analysis of pretransplant risk factors for all recipients, elevated creatinine (P=0.003) was the only factor to significantly affect postoperative survival. In UNOS status 4 patients, univariate analysis of pretransplant risk factors and their influence on patient survival demonstrated that prolonged coagulation partial thromboplastin time (P=0.04) and a higher grade of encephalopathy (P=0.02) significantly affected postoperative survival. Advanced encephalopathy (P=0.009) and prolonged partial thromboplastin time (P=0.01) were the only significant risk factors by multivariate analysis in status 4 patients. In status 4 and non-status 4 patients, we identified risk factors that adversely affected patient survival, but their predictive power was insufficient to deny transplantation. Despite the higher mortality in status 4 recipients, their long-term survival is only slightly worse than that of non-status 4 patients. Until better predictors of survival are ascertained, our data do not support limiting the use of donor livers in UNOS status 4 recipients.

The current organ shortage has made utilization of organs from less-than-ideal donors more common. Although several transplant centers use kidneys from non-heart-beating donors (NHBDs), there has been reluctance to extend the use of these donors to extrarenal organs. Of the 130 donors referred to our organ procurement organization between January 1993 and May 1994, 16 (12.3%) were NHBDs. Organ retrieval from 10 of these resulted in extrarenal donation, 5 resulted in renal donation only, and 1 resulted in no retrieval as a result of prolonged warm ischemia (> 2 hr). A total of 39 organs were transplanted from these NHBDs. A rapid en bloc retrieval technique was used for extrarenal NHBDs. The mean warm ischemic time was 15.4 min; preservation times were similar for both NHBDs and heart-beating donors. After liver transplantation (n = 5), one episode of primary nonfunction that was technical in origin required retransplantation. Following simultaneous pancreas-kidney transplantation (n = 6), all patients were insulin independent and free of graft pancreatitis; one patient required hemodialysis (16.7%). After isolated renal transplantation (n = 21), 3 patients (14.3%) required hemodialysis. Three of 4 liver recipients are alive after a mean follow-up period of 12.7 months; all simultaneous pancreas-kidney and renal transplant recipients are alive after a mean follow-up period of 8.4 and 8.3 months, respectively. Three liver allografts, 5 pancreas and kidney allografts, and 19 renal allografts are functioning. The lung allograft was lost to rejection 81 days after transplantation; however, the recipient is alive 3 months after retransplantation. Our results demonstrate that in controlled situations, extrarenal organs can be utilized from NHBDs and can be expected to function similarly to organs retrieved from heart-beating donors. We increased the number of transplanted organs by 8.6% using NHBDs for both renal and extrarenal donation. Continued application of these techniques will likely further increase the number of organs retrieved for transplantation.

Background. The in vivo effects of immunosuppressants on T cells are classically determined using animal models of organ transplantation. These methods are technically difficult and time consuming. A simple in vivo method is needed for screening new immunosuppressants. Methods. Donor mouse spleen cells were labeled with a fluorescent dye, carboxy-fluorescein diacetate succinimidyl ester (CFSE), and then injected into the blood of recipient severe combined immunodeficiency mice. Three days after the injection, spleen cells of the recipient mice were isolated and the proliferating alloreactive T cells were analyzed by flow cytometry. Results. In control recipient mice, 50% of the T cells were proliferating, consisting of both CD4 + and CD8 + T cells. In cyclosporine- or FK506-treated mice, T-cell proliferation was suppressed in the CD4 subset but not in the CD8 subset. On the contrary, T-cell proliferation was significantly reduced in the CD8 subset but not in the CD4 subset in recipient mice treated with rapamycin. Conclusion. The present mouse model using carboxy-fluorescein diacetate succinimidyl ester labeling is simple and fast. It is useful for screening new immunosuppressants and for examining the effect on T-cell subsets.

Induction of tolerance to allogeneic MHC antigens has been a goal in the field of transplantation because it would reduce or eliminate the need for generalized immunosuppression. Although encouraging results have been obtained in experimental models by exposing recipient thymus to donor cells before transplantation, donor cells are not typically available at that time, and the donor antigens responsible for the effect are poorly defined. In the present study, thymic tolerance was demonstrated without using donor cells. Recipient thymus was injected before transplantation with autologous myoblasts and myotubes that were genetically modified to express allogeneic donor-type MHC class I antigen. Donor-specific unresponsiveness was induced to a completely MHC-disparate liver transplant and to a subsequent donor-type cardiac allograft, but not a third-party allograft. In vitro, recipient CTL demonstrated a 10-fold reduction in killing of donor cells, but not of third-party cells. Our results demonstrate: (1) that recipient muscle cells can be genetically engineered to induce donor-specific unresponsiveness when given intrathymically, and (2) transfected recipient cells expressing only donor MHC class I antigen can induce tolerance to a fully allogeneic donor.