In G2 (rejector group), deaths were because of cardiovascular causes (n=5) (myocardial infarct [n=3], cerebrovascular accident [n=1], and pulmonary embolism [n=1]), recurrent hepatitis C virus (n=4), pneumonia-sepsis (n=4), dialysis-sepsis (n=1), bile leak-sepsis (n=1), colon necrosis-sepsis (n=1), and lung, colon, and esophageal cancer (n=1 each). One patient in the rejector group was retransplanted successfully twice during this time period because of chronic rejection (Fig. 1b). Until the end of the follow-up period, patient survival in the tolerant and the rejector group was 63.66% vs. 74.25% and graft survival was 63.66% vs. 72.96%, respectively. The average posttransplant survival of the 6 patients who died in the tolerant group versus the 19 patients who died in the rejector group was 13.84±0.62 vs. 10.42±0.75 years (P=.03), respectively, and the average postwithdrawal study survival of the same patients was 7.41±1.02 vs. 5.84±0.29 years, respectively.
After the end of the withdrawal study, in G2, 19 patients presented 19 clinically suspected and 7 biopsy-proven mild (n=4), moderate (n=2), or severe (n=1) rejection episodes. In G1, a tolerant patient presented a moderate rejection 5.3 years after immunosuppression withdrawal and was restarted on maintenance immunosuppression (Table 3). All episodes were treated successfully. Freedom from rejection in the tolerant and rejector groups was 95% and 72.98%, respectively (P<.05; Fig. 1c).
There was a trend for serum creatinine to be lower in tolerant transplant patients (P=not significant). In the rejector group, since the end of the withdrawal study, five patients received a kidney transplant and four more are on dialysis (Table 3). In the tolerant group, until the end of the follow-up period, one patient was on dialysis. Freedom from renal replacement in the tolerant and rejector groups was 83.33% vs. 44.58%, respectively (P=not significant; Fig. 1d). Two additional patients, one in the rejector group and one in the tolerant group, presented with acute deterioration of their renal function during hospitalization for sepsis and received short courses of dialysis before they died. In addition, 13 patients had sirolimus (n=7) or mycophenolate mofetil (n=7) added to their immunosuppressive regimen, and 5 more were converted to sirolimus/mycophenolate mofetil immunosuppression for nephrotoxicity. The posttransplant time until the renal replacement treatment of the patient who had dialysis in the tolerant group versus the 9 patients who had kidney transplant or dialysis in the rejector group was 13.9 vs. 9.67±1.2 years, respectively, and the average postwithdrawal study time until the renal replacement treatment was 7.95 vs. 4.17±1.1 years, respectively.
In the rejector group, 12 patients presented neoplasms since the end of the withdrawal study including squamous cell skin carcinoma (n=5), melanoma, uterine, colon, esophageal, pharynx and lung cancer (n=1 each), and prostate cancer (n=3) (Table 3). In the tolerant group, four patients presented with squamous cell skin cancer (n=1), throat (n=1), kidney (n=1), and lung cancer (n=1).
In the rejector group, immunosuppression was gradually decreased but not discontinued. At the end of the follow-up period, patients were on an average of 2.5 mg of tacrolimus daily dose with 13 of them receiving 1 mg or less/day.
In our previous report (3), we focused on the effect of donor bone marrow infusion during gradual immunosuppression withdrawal in a selected group of patients. A the time we published that report, poststudy follow-up was 1 to 3 years for the patients who attained operational tolerance and for those who had rejection episodes. In this study with an average poststudy follow-up of almost 8 years (7.3 years for the tolerant group and 7.8 years for the rejector group), we evaluated the outcomes regarding safety, benefits, if any, derived for the tolerant patients from the discontinuation of immunosuppression, and the extend to which operational tolerance can be maintained.
In our study population, gender is a variable that seems to have an association with operational tolerance. Our operationally tolerant group included 91% (21/23) male patients versus 59.25% (48/81) in the rejector group. We do not know whether this is because liver transplant recipients are most commonly male (61% of liver transplants) (1) or there are other gender-related factors such as sex-linked genes that could provide greater proportions of regulatory processes that allow for easier removal of immunosuppression.
Donor-recipient HLA matching seems to have also a correlation with operational tolerance and immunosuppression withdrawal in our study group. In six patients in the tolerant group (6/23, 26%), there was a better HLA match between donor and recipient (two or more common AB antigens) versus two patients in the rejector group (2/81, 2.5%), and this seemed to also improve the chances of removing immunosuppressive drugs from these patients. In theory, this is likely due to lower antidonor responses when there is greater HLA matching, and thus, the need for immunosuppression is less in these patients.
Both in the tolerant and the rejector group, approximately 50% of the patients had received donor bone marrow infusion at the time of the transplant procedure. This indicates that in our group of patients, bone marrow infusion had no obvious beneficial effect regarding immunosuppression withdrawal. We believe this is because despite the bone marrow infusion, robust levels of chimerism were never achieved in the peripheral blood. As we have previously published (3), peripheral blood chimeric levels were less than 1% in the patients who had received donor bone marrow infusions and 10 times less in the patients who did not receive any bone marrow at all. The origin of the scarce chimeric cells in the latter case was from the donor liver.
At this point, the critical variables that allow certain persons to be successfully withdrawn are not precisely known. There is an overall array of host factors that affect the ability to attain operational tolerance, likely interrelated in a complicated biologic algorithm that yet has to be clarified. Both groups, tolerants and rejectors, presented with morbidity and mortality complications during the follow-up period. Although there was no difference in patient survival (63.66% vs. 74.25% in the tolerant and rejector group respectively), our data suggest that mortalities in the rejector group occurred earlier than in the tolerant group. The average posttransplant survival of the 6 patients who died in the tolerant group versus the 19 patients who died in the rejector group was 13.84±0.62 vs. 10.42±0.75 years (P=0.03), respectively, and the average postwithdrawal study survival of the same patients was 7.41±1.02 vs. 5.84±0.29 years, respectively. The increased number of deaths because of cardiovascular reasons can also been explained from the advanced age of this group of patients at the time of death.
Renal function outcomes were not significantly different between the two groups; however, our data suggest that in our patient sample, intervention (dialysis or kidney transplant) for end-stage renal disease occurred earlier in the rejector group than in the tolerant group. The posttransplant time until the renal replacement treatment of the patient who had dialysis in the tolerant group versus the nine patients who had kidney transplant or dialysis in the rejector group was 13.9 vs. 9.67±1.2 years, respectively, and the average postwithdrawal study time until the renal replacement treatment was 7.95 vs. 4.17±1.1 years, respectively. Patients in the tolerant group were also exposed to immunosuppression treatment and its subsequent nephrotoxicity for a period of approximately 5 years from the time of the transplant until withdrawal was achieved. Many of the patients had also concomitant diseases such as hypertension or diabetes that aggravated even more their kidney function. Immunosuppression withdrawal may delay the progression of kidney disease but could not reverse it. This observation raises also the question of immunosuppression minimization and withdrawal as early as possible after liver transplantation to avoid long-term exposure and organ damage from calcineurin inhibitor toxicity.
There was no difference in the occurrence of neoplasms between the two groups. As with the other immunosuppression complications, the absence of significant differences might be due to the small sample size of our study and also because all patients were exposed to immunosuppression for a significant amount of time, more than a decade in the rejector group, approximately 5 years in the tolerant group. Neoplasms because of the immunosuppressed state might have been formed during that period of time and were clinically diagnosed later on.
Patients in the tolerant group presented with significantly less rejection episodes. More important, operational tolerance is not a permanent state; one patient presented with a moderate rejection after being immunosuppression free for more than 5 years. We do not know what was the triggering event that caused the rejection episode. The medical history and clinical examination of the patient at that time did not reveal any precipitating causes, no other coexisting viral diseases, or other diseases that could be responsible for an increased immune reactivity. This event brings up the safety issue of immunosuppression withdrawal. Vigilance and continuous follow-up are required to avoid catastrophes. Operationally tolerant patients have to continue their follow-up regularly, and the transplant team has to be always vigilant for a rejection episode. In our study, we implemented a rigorous follow-up of these patients to ensure their safety. One operationally tolerant patient who presented rejection was timely detected and treated successfully.
Not all patients were successful during their first attempt. Immunosuppression minimization and rejection or successful withdrawal is a two-way phenomenon, as we learned from the two patients who rejected during the initial immunosuppression withdrawal and were successful at their second attempt and from the patient who was tolerant for a period of 5 years before presenting a rejection episode.
It would be most beneficial for the safety of the patients to develop a screening assay to suggest which patient would be a good candidate for immunosuppression withdrawal and which one would most probably reject. Studies of operationally tolerant patients (8–10) have indicated that these patients share a tolerance genetic fingerprint. As in our series, we had patients who rejected initially before developing operational tolerance and vice versa, serial sampling and longitudinal study of this fingerprint overtime may elucidate mechanisms related to the tolerance state.
In our long-term follow-up of operationally tolerant liver transplant patients, we did not observe any statistical significance in the outcomes, compared with a control group who failed to wean off immunosuppression. The safety of the patients was not compromised being in the tolerant state because of the rigorous follow-up for early detection and timely treatment of rejection episodes, and their outcomes were at least as good as the ones of the control group. The lack of a difference in the outcomes might be due to the small study sample and to the fact that tolerance was induced late posttransplantation. The tolerant patients were exposed to the deleterious effects of immunosuppression for a long period of time before withdrawal.
Both patients and transplant teams should always remember that tolerance, when achieved, is not a permanent state, and continuous vigilance and follow-up are required to detect rejection episodes. The mechanisms of operational tolerance are not yet elucidated. Multicenter longitudinal immunosuppression withdrawal trials might help us to better understand this phenomenon.
2. Ojo AO, Held PJ, Port FK, et al. Chronic renal failure after transplantation of a nonrenal organ. N Engl J Med
2003; 349: 931.
3. Tryphonopoulos P, Tzakis AG, Weppler D, et al. The role of donor bone marrow infusions in withdrawal of immunosuppression in adult liver allotransplantation. Am J Transplant
2005; 5: 608.
4. Li Y, Zhao X, Cheng D, et al. The presence of Foxp3 expressing T cells within grafts of tolerant human liver transplant recipients. Transplantation
2008; 86: 1837.
5. Yoshitomi M, Koshiba T, Haga H, et al. Requirement of protocol biopsy before and after complete cessation of immunosuppression after liver transplantation
2009; 87: 606.
6. Koshiba T, Li Y, Takemura M, et al. Clinical, immunological, and pathological aspects of operational tolerance
after pediatric living- donor liver transplantation
. Transpl Immunol
2007; 17: 94.
7. Li Y, Koshiba T, Yoshizawa A, et al. Analyses of peripheral blood mononuclear cells in operational tolerance
after pediatric living donor liver transplantation
. Am J Transplant
2004; 4: 2118.
8. Martínez-Llordella M, Puig-Pey I, Orlando G, et al. Multiparameter immune profiling of operational tolerance
in liver transplantation
. Am J Transplant
2007; 7: 309.
9. Martínez-Llordella M, Lozano JJ, Puig-Pey I, et al. Using transcriptional profiling to develop a diagnostic test of operational tolerance
in liver transplant recipients. J Clin Invest
2008; 118: 2845.
10. Brouard S, Mansfield E, Braud C, et al. Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance
. Proc Natl Acad Sci USA
2007; 104: 15448.
11. Braud C, Baeten D, Giral M, et al. Immunosuppressive drug-free operational immune tolerance
in human kidney transplant recipients: Part I. Blood gene expression statistical analysis. J Cell Biochem
2008; 103: 1681.
12. Sivozhelezov V, Braud C, Giacomelli L, et al. Immunosuppressive drug-free operational immune tolerance
in human kidney transplants recipients. Part II. Non-statistical gene microarray analysis. J Cell Biochem
2008; 103: 1693.
Keywords:© 2010 Lippincott Williams & Wilkins, Inc.
Liver transplantation; Immunosuppression withdrawal; Tolerance