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- Patients, Materials and Methods
Induction of allograft-specific tolerance and the detection of a “tolerance” state in recipients under immunosuppression with long-term stable graft function are major challenges in transplantation. Clinical “operational tolerance,” defined as stable and acceptable graft function without immunosuppression for years, is a rare event. There is no report on the clinical history of such patients. In this article, we report on the medical history of 10 kidney recipients who display an immunosuppressive drug-free “operational tolerance” for 9.4 ± 5.2 years. Clinical factors that may favor such a tolerant state are underlined. Firstly, most of the patients interrupted immunosuppression over a long time period (until 4 years), which mimics the procedure of intentional immunosuppression weaning following liver transplantation. Secondly, donor age was younger (median 25 years) than the one of the general transplanted population, suggesting that graft quality is one of the conditions favoring “operational tolerance.” Moreover, the “operationally tolerant” recipients may be ‘low responders’ to blood transfusions (PRA 6 ± 5.4%, six blood transfusions). We also show that “operational tolerance” occurs in the presence of anti-donor class II antibodies, as assessed in two patients. Finally, two patients degraded their renal function 9 to 13 years after treatment withdrawal, however only one presented histological lesions of chronic rejection.
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- Patients, Materials and Methods
The advent of renal transplantation has notably reduced morbidity and mortality in patients with end-stage renal disease. New immunosuppressive molecules have dramatically reduced the risk of acute rejection (1) and prolonged graft survival (2). However, immunosuppressive treatments have numerous side effects, including infectious complications (3), malignancies (4,5) and metabolic disorders (6). Calcineurin inhibitor-related-nephrotoxicity also greatly contributes to the development of chronic allograft nephropathy in long-term functioning grafts (7). Moreover, immunosuppression poorly influences chronic rejection, the main cause of graft loss in renal transplantation (8,9). Understanding the mechanisms of immune tolerance in humans is therefore crucial if induction of graft-specific tolerance is to become achievable.
Tolerance has rarely been established in large animals models and is rare in humans. “Operational tolerance” is a clinical situation defined as a stable graft function without clinical features of chronic rejection and in the absence of any immunosuppressive drugs, usually for more than 1 year. This is observed more frequently in liver transplant patients than in other organ recipients (10,11). Albeit rare, “spontaneous” operational tolerance has also been reported in kidney recipients (12–21), although unidentified non-compliance to treatment may lead to underestimation of the phenomenon.
Such patients offer a unique opportunity to study the clinical and biological characteristics potentially specific to and associated with tolerance. Despite an active research on their biological characteristics, no detailed clinical history of such patients has been reported to date. In this article, we report on the clinical description of a cohort of 10 kidney recipients displaying clinical “operational tolerance” to their graft years after interruption of their immunosuppressive treatment.
Patients, Materials and Methods
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- Patients, Materials and Methods
Clinical “operational tolerance” was defined as a stable kidney graft function, without clinical markers of chronic rejection, and without any immunosuppressive drugs for more than 1 year. A creatininemia and proteinuria below 150 μmol/l and 1g/24h, respectively were defined as acceptable thresholds. Graft biopsies were not performed on these normally functioning kidneys for ethical reasons. However, biopsies were performed in patients who, over time, failed to meet the criteria of “operational tolerance” mentioned above. Anti-HLA class I and II antibodies were screened by microlymphocytotoxicity on a selected panel (PRA) and, more recently, by flow cytometry (Luminex technology). Specificity was determined with a cytotoxicity assay. The characteristics of the 10 patients meeting these clinical criteria are detailed in the case reports and are summarized in Tables 1 and 2. Creatininemia and proteinuria evolution are depicted for each patient in Figure 1.
Table 1. Main characteristics of “operationally tolerant” patients
|Case (Year of transplantation)||Initial renal disease||Recipient age||Donor age||Pre-graft blood transfusions||Pre-graft PRA||HLA incompatibility number||HLA DR incompatibility number||Cold ischemia (hours)||DGF||Induction therapy||Acute rejection||Cancer||Calcineurin inhibitor exposure (years)||IS withdrawal (time to transplantation)||Reason for IS withdrawal||Tolerance duration (y)||anti-HLA antibody (since IS withdrawal)|
|1 (1973)||Membrano-proliferative glomerulopathy||42||25||ND||No||1*||ND||14||Yes (d15)||Cyclophosphamide||Yes||None|| ||12||Non-compliance||20||None|
|2 (1981)||Obstructive uropathy||15||7||6||7%||3*||ND||37||No||No||Yes||None|| ||8||Non-compliance||16||None|
|3 (1987)||Undetermined glomerulopathy||41||16||4||No||4||1||36||Yes (d8)||ATG||No||None||13||13||Non-compliance||5||None|
|4 (1987)||Obstructive uropathy||19||ND||ND||ND||4||2||ND||ND||ATG||Yes||None||10||10||Non-compliance||8||ND|
|5 (1991)||Obstructive uropathy||13||21||2||12%||3||2||29||No||ATG||Yes||PTLD||8||8||PTLD||6||None|
|6 (1993)||Interstitial nephropathy||64||39||24||9%||3||1||40||Yes (d19)||No||Yes||Basocellular carcinoma||1||1||Initial renal deagradation||11||anti-donor anti-class II (DR11)**|
|7 (1994)||Renal hypoplasia||27||30||5||15%||4||1||24||No||ATG||No||None||10||10||Non-compliance||1||anti-donor anti-class II (DR04)**|
|8 (1998)||Interstitial nephropathy (drug toxicity)||27||28||2||No||0||0||3||No||No||No||None||1.5||2||Non-compliance||5||None|
|9 (1980)||Hypocomplementemic glomerulopathy||43||17||2||7%||3||1||18||Yes (d3)||No||No||None|| ||9||Non-compliance||13||None|
|10 (1987)||Undetermined glomerulopathy||52||30||4||4%||3||0||38||Yes (d2)||anti-IL2R||No||PTLD||7||7||PTLD||9||suspected for class II|
Table 2. Current biological parameters of ‘operationally tolerant’ patients
|Case||Creatininemia (μmol/L)||Proteinuria (g/d)||WBC (/mm3)||Lymphocytes (/mm3)||Hb (g/dl)||γglobulin (g/L)||Treatment|
|1||113||0.22*||4100||980||12.3||11.5||calcic inhibitor, β-blocker|
|4||150||0.11||6850||2898||14.4||ND||β blocker and central anti-hypertensor|
|Mean ± SD||119 ± 20||0.20 ± 0.18||6595 ± 1195||2123 ± 924||14 ± 1.8||11 ± 1|| |
Figure 1. Creatininemia and proteinuria evolution since transplantation. Each graph indicates post-transplantation evolution of creatininemia (μmol/L) and proteinuria (g/d) for each patient. The horizontal arrow corresponds to the period of progressive immunosuppressive medication withdrawal. The grey part indicates the period off-immunosuppression. Creatininemia and proteinuria remain stable over time, except for cases 9 and 10 whose renal function degraded 14 and 9 years, respectively after immunosuppression cessation. In these two last cases the scale of the graph was adjusted to the level of creatinemia and proteinuria.
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Case 1: A.S., born in 1931, developed a membrano-proliferative glomerulopathy. He received a first living-related HLA-identical donor kidney, which was immediately lost because of an arterial thrombosis. This patient was not HLA-immunized (previous blood transfusions not documented). In 1973, he received a second renal transplant, from a 25-year-old deceased donor with one HLA class I incompatibility (donor class II undetermined). His immunosuppressive treatment consisted of an initial cyclophosphamide perfusion relayed with corticosteroids (CS) and azathioprine (AZA). Graft function was delayed until day 15. During this period an acute rejection was suspected but not biopsy-proven, and treated with Cyclophosphamide and CS boluses. A Hepatitis C Virus (HCV) infection was clinically quiescent and he spontaneously recovered from a Hepatitis B Virus (HBV). Five years after transplantation, a systematic graft biopsy showed a discrete interstitial mononuclear infiltrate, and C3 and IgM deposits on arteriolar walls, interpreted as a border-line rejection, but he was not treated at this time. Twelve years after transplantation, the patient progressively stopped taking his immunosuppressive medication (incompliance) over an estimated period of 4 years. Twenty-six years later, his renal function is stable (creatininemia 113 μmol/L) and he is only treated for arterial hypertension.
Case 2: F.J., born in 1966, had an obstructive uropathy. He was 15 years old when hemodialysis was started. Despite six blood transfusions, a weak anti-HLA immunization was detected only once (PRA 7%). A few months later, in 1981, he received a deceased, 3 HLA class I incompatible renal transplant from a 7-year-old donor (donor class II undetermined). His immunosuppresive treatment was composed of CS and AZA. Seven days after transplantation he was treated with CS boluses for a clinically suspected acute rejection (not biopsy-proven). He was found to be positive for HCV and this disease is still clinically quiescent. Because of non-compliance, he stopped his immunosuppressive treatment 16 years ago, over an estimated time period of 2 years. Since this withdrawal, his renal function has remained stable (creatininemia 139 μmol/L).
Case 3: M.R., born in 1945, presented an end-stage renal disease related to an undetermined glomerulopathy. He was transfused four times before grafting; no HLA immunization was detected. In 1987, he received a renal transplant from a 16-year-old deceased donor, with 4 HLA incompatibilities. He received ATG and his maintenance immunosuppressive regimen consisted of Ciclosporine (CsA), CS and AZA. The graft began functioning after 8 days. CS were stopped at 3 months. After 13 years, and following an estimated period of 2 years of inconsistent drug intake, the patient finally stopped taking his immunosuppressive medication (incompliance). Five years following total interruption, his creatininemia is at 89 μmol/L and he is without proteinuria. He is only treated for hypertension.
Case 4: Y.M.L, born in 1968, had an obstructive nephropathy. In 1987, he received a deceased 4 HLA incompatible renal transplant. His therapy consisted of an induction with ATG and a maintenance regimen of CsA, AZA and CS. An acute rejection was suspected on day 6 post-surgery and treated with CS boluses; no graft biopsy was performed. Nine years after transplantation, he started to space out his immunosuppressive drug intake and stopped it definitively 1 year later. Eight years after immunosuppression withdrawal, his renal function remains stable (creatininemia 150 μmol/L) and he is only treated for hypertension.
Case 5: S.P., born in 1978, had an obstructive uropathy. He presented a weak HLA immunization secondary to two blood transfusions. In 1991, 1 year after dialysis, he received a kidney transplant from a 21-year-old deceased donor with 3 HLA incompatibilities. He was given ATG and his maintenance treatment was an association of CsA, AZA and CS. He displayed no delayed graft function (DGF). He had six graft biopsies during the first 5 years post-transplantation, all because of moderate episodes of renal dysfunction. All, but one, showed only mild interstitial fibrosis. One biopsy showed moderate focal lymphoid infiltration, with mild tubulitis, suggesting borderline changes, which was treated with steroid boluses. He presented a primo-infection with Epstein-Barr Virus (EBV) the year following transplantation. CS were progressively withdrawn 7 years after transplantation. In 1999, he developed a multifocal post-transplantation lymphoproliferative disorder (PTLD) relative to EBV, and CsA and AZA were abruptly interrupted. PTLD treatment consisted of chemotherapy and radiotherapy. Currently, 6 years after PTLD treatment, he is being treated with valacyclovir and considered to be cured of his disease. His renal function remains stable (creatininemia 123 μmol/L).
Case 6: H.L., born in 1929, whose initial disease was an interstitial nephropathy, received a deceased kidney transplant in 1993 from a 39-year-old donor with 3 HLA incompatibilities. Despite having previously transfused 24 times and being pregnant twice, she presented low levels of PRA (9%) before transplantation. Her immunosuppressive treatment consisted of CsA, CS and mycophenolate mofetil (MMF), without induction. She displayed a DGF, which was interlinked with an acute rejection episode (Banff grade Ib) treated with CS boluses. CS were stopped 5 months after transplantation, and MMF 1 month later (cytopenia). A second acute rejection episode (Banff Ia) occurred 7 months later and 3 CS boluses were administered. During the first year of transplantation, she presented numerous bacteriological infections and a basocellular carcinoma. Because of poor graft function, hemodialysis was started 12 months after the transplantation. CS and CsA were subsequently stopped. At this time, anti-donor class II antibodies were detected. Surprisingly, after months of dialysis, her renal function progressively recovered and she no longer required dialysis. Renal-DMSA scintigraphy performed in 2002 showed no residual native kidney function, but a functional kidney graft. Eleven years after immunosuppression withdrawal, her creatininemia is 96 μmol/L and her proteinuria is below 0.5g/d.
Case 7: F.C.E, born in 1967, presented a renal failure related to renal hypoplasia. A weak pre-graft immunization was noted secondary to five blood transfusions. In 1994, she received a deceased kidney from a 30-year-old donor with 4 HLA incompatibilities. She received an induction therapy with ATG. Her maintenance regimen consisted of CsA, CS and AZA. She displayed no DGF. She progressively stopped her immunosuppressive treatment due to incompliance over a 4-year period. She displayed a donor-class II immunization during CsA monotherapy. She has now been without treatment for 1 year and her renal function remains stable (creatininemia 128 μmol/L).
Case 8: I.K., born in 1966, suffered from a testicular cancer treated with chemotherapy and bone marrow auto-transplantation. He then developed renal failure attributed to drug toxicity and resulted in hemodialysis. He was transfused twice and did not develop PRA. In 1998, he received a renal transplant from his HLA-identical 28-year-old brother. He was treated with CsA, MMF and CS without induction therapy. CS were stopped 1 month later. Less than 1 year after transplantation, he spaced out his immunosuppressive drug administration due to incompliance and definitively interrupted his immunosuppressive medication 2 years after transplantation. He is now being treated only for arterial hypertension and, 5 years after immunosuppression withdrawal, continues to display a normal renal function (creatininemia 115 μmol/L).
Case 9: P.P., born in 1937, suffered from a hypocomplementemic glomerulopathy leading to renal failure. He received two blood transfusions and had low PRA levels. He had a chronic VHB assessed with the presence of AgHBs. In 1980, he received a kidney transplant from a 3 HLA incompatible 17-year-old deceased donor. He was treated with AZA and CS. He progressively stopped his immunosuppressive medication between 9 and 11 years after transplantation due to incompliance. After 13 years of “operational tolerance,” his renal function started to degrade. A subsequent graft biopsy revealed grade I chronic allograft nephropathy with mild nephroangiosclerosis without significant lymphoid infiltration or specific changes suggestive of chronic rejection (Figure 2). C4d staining was negative. No anti-HLA antibodies have been detected.
Figure 2. Graft biopsy performed following degradation of renal function in cases 9 and 10. Panels 2A and 2B correspond to the graft biopsy for patient 9 (Masson's trichrome coloration). It only revealed mild focal interstitial fibrosis and tubular atrophy (Figure 2A). Minimal arterial fibrous intimal thickness was detected and one glomeruli presented with a retracted floculus. The other ones showed no glomerulitis or double contours (Figure 2B). Lesions suggested nephroangiosclerosis rather than chronic allograft rejection. No acute component was present in this biopsy. Panels 2C and 2D correspond to graft biopsy of patient 10 (Masson's trichrome coloration for Figure 2C and PAS coloration for Figure 2D). It showed focal fibro-edema associated with mild mononuclear infiltration. Glomeruli showed double contours in 26 to 50% of peripheral capillary loops, which, together with the presence of parietal IgM and C1q deposits (not shown), were in favor of allograft glomerulopathy. There was no acute rejection lesion but moderate arteriolar hyalinosis (not shown) was detected.
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Case 10: M.C, born in 1935, presented an undetermined glomerulopathy. He was transfused four times and developed low PRA levels. In 1987, he received a kidney transplant from a 3 HLA incompatible 30-year-old deceased donor. He received an anti-IL2 receptor antibody together with CsA, AZA and CS. CS were stopped 2 months later. Seven years later he developed a PTLD that was treated with chemotherapy and his immunosuppression was abruptly withdrawn. His renal function remained stable for 7 years without immunosuppression, with an apparently cured PTLD, until a significant proteinuria appeared. His renal function progressively degraded, requiring dialysis in 2004. At this time, the presence of anti-HLA class II antibodies was suspected, but could not be confirmed on additional analysis. A graft biopsy, performed in 2002, showed grade Ib chronic allograft nephropathy with allograft glomerulopathy, but without C4d staining (Figure 2).
Donor and recipient ages, DGF and cold ischemia: The dates of transplantation for these patients are spread over a long period of time (1973 to 1998). Their median age at the time of transplantation was 34 years (range 13 to 64). Their donors (all but one deceased) were unusually young with a median age of 25 years (range 7 to 39). We compared the age of the donor for each recipient studied with the confident interval of the mean donor age in each corresponding year of graft. This test suggests a trend for younger donor in tolerant patients (i.e. outside of the confidence interval). However we realize that this approach is only informative as statistical significance was not reached. It was a first kidney transplantation for 9 out of 10 patients. Cold ischemia was on average 26.4 ± 12.8 hours. Four patients had a delayed graft function, defined as the requirement of at least one dialysis during the first week post-transplantation.
Blood transfusions, pre-graft immunization and HLA incompatibilities: Eight patients were multi-transfused before transplantation (6 ± 7.3 blood transfusions). Possible blood transfusions before transplantation could not be documented for the two other patients. Among the eight transfused patients, six presented a low anti-HLA immunization, with mean PRA levels of 9 ± 3.9% (range 4 to 15%). Taking into account the nine patients whose PRA had been tested before transplantation, the mean PRA was 6 ± 5.4%. Most of these blood transfusions were performed before 1990 and without leukocyte depletion. Mean HLA incompatibilities were 3 ± 1.5. The quality of matching varied between 1973 and 1998, and donor MHC class II was not determined for two patients transplanted in 1973 and in 1981 (Table 3).
Table 3. Immunological characteristics of “operationally tolerant” patients
|Blood transfusions||8 patients/8 (undetermined for 2)|
|Pre-graft immunization||6 patients/9 (undetermined for 1)|
|Mean PRA before transplantation||6 ± 5.4*|
|HLA incompatibilities||3 ± 1.5|
|Induction therapy||6 patients/10|
|Calcineurin inhibitor||7 patients/10|
|Acute rejection episode**||5 patients/10|
|Post-graft immunization||3 patients/9 (anti-donor class II for 2)|
|IS withdrawal (years from transplantation)||7.8 ± 4.3|
|Withdrawal through non-compliance||7 patients/10|
|Tolerance duration (years)||9.4 ± 5.7|
Induction and maintenance treatment: Six patients received an induction therapy: four with ATG, one with an anti-IL2 receptor antibody and one with cyclophosphamide. Three patients underwent transplantation before the ciclosporine era, and received a maintenance regimen of AZA and CS. All of the others received ciclosporine for a mean period of 7.2 ± 4.9 years before interruption.
Rejection and post-transplant immunization: Five patients were clinically diagnosed with acute rejection during the follow-up before immunosuppression withdrawal, but this was biopsy-proven for only two patients (one borderline). Histological findings of transplant glomerulopathy were present for only one of the two patients whose renal function finally degraded years after immunosuppression withdrawal. Lack of C4d staining suggested no humoral participation. Neither sample showed staining for anti-C4d, suggesting no significant involvement of antibodies, particularly in the patient with peripheral anti-class II antibodies. Interestingly, two other patients developed anti-donor class II antibodies: once for patient 6 (and this was not checked thereafter), and for four consecutive years for patient 7. No concurrent impairment of graft function was observed.
Infectious complications before and after treatment withdrawal (Table 4): Before immunosuppression withdrawal, three patients presented a symptomatic herpetic manifestation, one of them (patient 5) also suffered from an EBV primo-infection 1 year after transplantation and a zoster infection the following year. Four patients suffered from recurrent bacteriological infections, essentially urinary, pulmonary and ORL infections. Three patients presented tumoral disorders after transplantation: a skin basocellular carcinoma in one case (patient 6) and PTLD in two cases (patients 5 and 10).
Table 4. Infections and neoplasia during immunosuppressive treatment and after withdrawal
| ||During immunosuppression||After IS withdrawal||HBV status|
|Viral infections||Bacteriological infections||Neoplasia||Viral infections||Bacteriological infections||Neoplasia||EBV reactivation after IS withdrawal|
|1||Quiescent HCV||Abces||None||Quiescent HCV||Recurrent abces, infectious diarrhea, pneumopathy, sinusitis||None||ND||HBV infection spontaneously recovered|
|2||Quiescent HCV|| ||None||Quiescent HCV|| ||None||ND||Protective anti-HbS Ab (last control: 192 UI/ml)|
|3||Herpetic manifestation||Pneumopathy||None|| ||Pneumopathy||None||negative||Protective anti-HbS Ab (last control: 106 UI/ml)|
|4|| ||None|| ||Abces||None||ND||ND|
|5||EBV primoinfection, herpetic manifestation, zoster,||Urinary and pulmonary infections||EBV-induced PTLD|| ||Recurrent ORL infections,||None||positive (290/105PBMC)||Protective anti-HbS Ab (last control: 215 UI/ml)|
|6||Herpetic manifestation||Pulmonary and urinary infections, infectious colitis||Basocellular carcinoma|| ||None||ND||ND|
|7||Recurrent verruca||Recurrent sinusitis||None|| ||None||negative||Protective anti-HbS Ab (last control: 95 UI/ml)|
|8|| ||ORL infections||None|| ||None||negative||HBV infection spontaneously recovered|
|9|| ||None|| ||Arthritis||None||positive (low level)||HBV (AgHbS positive)|
|10|| ||PTLD|| ||None||ND||Protective anti-HbS Ab lost 10 years after IS withdrawal|
After treatment withdrawal, two patients (patients 1 and 5) continued to present recurrent bacteriological infections. EBV reactivation was checked for five patients several years after treatment withdrawal and was negative for three of them. One patient had a weak EBV reactivation and the other (patient 5 who had an EBV-induced PTLD) is still presenting significant EBV replication and is being treated with valacyclovir.
Concerning VHB, two patients presented a VHB infection from which they spontaneously recovered. One patient had a chronic VHB infection at the time of transplantation, but without significant cytolysis after transplantation. Six other patients retained protective anti-HBs titres after treatment withdrawal, but one of them (patient 10) lost his protective antibodies (10 years after immunosuppression withdrawal).
Overall, infections before and after the immunosuppressive drug-free phase were uncommon.
Causes and modalities of immunosuppression disruption, stability of operational tolerance: Immunosuppression was stopped at a mean of 7.8 ± 4.3 years post-transplantation. The reason for drug withdrawal was PTLD in two patients and incompliance in seven. The incompliant patients did not interrupt their drugs abruptly, but over a long time interval. Although no precise time period can be reported, the mean period of progressive interruption has been estimated to 1.8 ± 1.5 years. For the last patient (patient 6), immunosuppressive medication was withdrawn because of drug toxicity and a very poor clinical condition. The mean period of stable “operational tolerance” was 9.4 ± 5.7 years (range 1 to 20). Two out of 10 patients (patients 9 and 10) developed a renal failure after 9 to 13 years of stable “operational tolerance.” A biopsy on one patient (patient 10) showed transplant glomerulopathy (Figure 2). The eight other patients still display a stable renal function, with a creatininemia at 119 ± 20μmol/L (range 89 to 150) and a proteinuria at 0.20 ± 0.18 g/d (range 0.07 to 0.93) (cf: Table 2).
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In this article, we report on a cohort of 10 kidney recipients with clinical immunosuppression drug-free “operational tolerance.” For the first time, we present a detailed account of their medical history and their long-term clinical evolution. The median period of “operational tolerance” observed at the time of this study is 9.4 ± 5.7 years, with 8 out of 10 patients still “tolerant.” Anecdotal cases of long periods of “operational tolerance” previously reported in the literature (see Table 5 for review) (14–20). All but one recipient in our cohort received a kidney from a deceased mismatched donor (3 ± 1.5 HLA incompatibilities), contrasting with the previously described “operationally tolerant” kidney recipients, all but two of whom received grafts from living-related donors (14–20).
Table 5. Reported cases of “operationally tolerant” patients
|Patients||Donor||Numbers of HLA incompatibilities||Immunosuppressive treatment||Reason for IS withdrawal||Tolerance duration||Histology||Mechanisms||References|
|1||Living related||?||ND||Non-compliance||?|| ||(13)|
|6||Living related||0||ND||Non-compliance||3 to 8 years|| ||(14)|
|7||Living related||?||AZA (+CS for acute rejection)|| ||3 to 38 years||“normal”|| ||(15)|
|1||Living related||3 (haplo-identical)||DST and CsA+AZA+CS||Non- compliance||5 years||focal lymphocyte infiltrate and interstitial fibrosis, arteriolar intimal hyperplasia||Microchimerism, regulation, anergy||(16)|
|1||Living related||3 (haplo-identical)||ND||PTLD||> 3 years|| ||anti-donor hyporesponsivness (anergy? Clonal deletion?)||(17)|
|1||Deceased||6||Total lymphoid irradiation and ATG+ CS||Protocol||12 years|| ||no microchimerism, anti-donor reactivity restored in vitro||(19)|
|2||Living related (1) and deceased (1)||0 for one patient, 2 for the other patient||Induction therapy (OKT3) for one patient, no induction for the other||Non- compliance||5 and 27 years||Focal infiltrate confined to interstitium||regulation||(20)|
|1||ND||1 HLA class I mismatch (class II ND)||AZA+CS||Non- compliance||9 years|| ||(18)|
“Operational tolerance” in allotransplantation remains a clinically defined concept, with a stable graft function at a good or acceptable level for several months or years in the absence of immunosuppressive treatment. Clinical “operational tolerance” should be distinguished from “almost tolerance,” which corresponds to a stable graft function in minimally immunosuppressed recipients (low dose monotherapy) (22, 23). Graft biopsies were not proposed to these patients since they displayed normal renal function, without significant proteinuria. Despite this restriction, their acceptance of a mismatched deceased kidney, without any immunosuppressive drugs for years fulfils the most optimistic definition of tolerance and offers a unique opportunity to understand its mechanisms and to find a clinical and biological signature associated with tolerance in humans.
Before immunosuppression withdrawal, the clinical histories of these “operationally tolerant” patients do not seem to differ greatly from kidney recipients in general. However, although the relatively low number of such patients precludes relevant statistical analysis, the detailed clinical review of the patients described here revealed some interesting findings. Firstly, no evidence for a strongly biased proportion of original disease was observed, for example, glomerulopathy was the initial disease in 4 of the 10 patients, two undetermined) compared to 47% of the last 522 patients of our databank. Secondly, the main cause of immunosuppressive treatment cessation was non-compliance (7 out of 10 patients) and the patients usually discontinued their drugs progressively. They usually withdrew one drug and spaced out the administration of the remaining immunosuppressive drug, with several phases of ‘stop and start’ intake. The exact period of time during which treatment was spaced out is difficult to determine but in most cases probably lasted several years (1 to 4 years). This progressive “non-compliance procedure,” which mimics in some respects what is sometimes applied to liver recipients considered as being at low risk of rejection (10,11,24) may select these patients and favor a transition to a state of “operational tolerance.” The occurrence of such “operational tolerance” might be under-estimated because incompliance is more frequent than actually confessed (25,26), varying from 18% (27) to 52% (28). These patients are at a higher risk of late acute rejection and of increased serum-creatinine (29). However, incompliance also reveals states of clinical tolerance, as described in this article.
The third notable point concerns donor status. All the donors in our study were younger than the mean donor age of the corresponding year, and were also younger than the recipients themselves. Renal graft survival has been shown to be significantly reduced when the donors are older than 50 to 55 years, attributed in part to a reduced functional reserve (30–32). Kidneys from aging donors are usually more susceptible to ischemia-reperfusion lesions and to delayed graft function, and so may display higher antigenicity (32–34). One can hypothesize that good “quality” of the graft, with few age-related lesions (glomerulosclerosis, interstitial fibrosis), may provide better condition for the establishment of tolerance.
Finally, these “operationally tolerant” patients did not seem to be non-specifically immunosuppressed because they did not present significant opportunistic, frequent or severe viral infections or neoplasia following immunosuppression withdrawal. Preliminary data on the response of some of these “operationally tolerant” patients to influenza vaccination suggest that they present humoral and cellular responses not significantly different from those of healthy volunteers (35). Furthermore, their protective anti-HBs titres remain stable, also indirectly reflecting immunocompetence. Six patients out of nine had presented anti-HLA immunization after blood transfusions. Most of the transfusions were performed with blood non-depleted in leukocytes. This proportion of sensitized patients is comparable of what was usually observed in the literature, with range of immunized patients from 4.4% (36) to 90% (37) after five or more blood transfusions. However, the levels of PRA of “operationally tolerant” patients before the transplantation were low (6 ± 5.4%), nearly the half of the mean PRA observed by Fehrman et al. (mean PRA 11% after five blood transfusions) (38). Moreover, although three of these patients were treated for suspected or biopsy-proven (n = 2, with one borderline) acute rejection, all episodes were easily reversed by steroid pulses. Altogether, this could suggest that the “operationally tolerant” recipients described here, despite not being clinically immunoincompetent, may be low alloreactive responders. A larger cohort is needed to confirm this trend.
Although 8 of the 10 patients continue to display stable renal function, the renal function of two patients degraded during the study period despite no indication of poor graft quality (donor age below 30 years, no prolonged delayed graft function). These two patients did not present acute rejection. Graft biopsies performed when renal function started to degrade showed no signs of acute or chronic rejection for one patient (patient 9) but rather lesions suggestive of nephroangiosclerosis. In contrast, the biopsy from the other patient (patient 10) who presented one positive PRA test showed allograft glomerulopathy (Figure 2). Nevertheless, there was no active or humoral component, as suggested by an absence of C4d staining. Three previous cases of graft biopsies performed during the phase of “operational tolerance” have been reported in the literature. One of them presented a moderate and focal infiltrate with an arteriolar intimal hyperplasia, suggesting the beginnings of chronic allograft nephropathy lesions (16), another one only displayed a focal interstitial infiltrate (20) and the last one was described as normal (15). In our study, a biopsy was proposed only for patients with creatinine or proteinuria evolving outside of the defined values. Recommendation to not biopsy such patients presenting a stable function years following withdrawal of immunosuppression may appear questionable since the operational tolerant state is not necessarily permanent (Figure 1). Minimal lesions could be detected by histology, justifying immunosuppressive drug reintroduction. However, such a strategy would require serial biopsies (every year?) and such multiple procedures would certainly carry substantial risks (44). In addition, the reintroduction of immunosuppression based on the presence of minimal lesions is also questionable, particularly in view of the decade(s) of stable function enjoyed by these patients; alteration of active regulation could be also imagined.
Interestingly, two patients had anti-donor class II antibodies without showing degradation of renal function. We and others have shown that the late appearance of anti-donor antibodies is statistically associated with the secondary apparition of chronic rejection (39,40). However, the two patients who had anti-donor class II antibodies, still exhibit a stable renal function more than 4 and 10 years on.
The main mechanisms associated with experimental tolerance in allotransplantation are clonal deletion, sometimes associated with mixed chimerism, anergy and regulation (41). Regulation, alone or associated with anergy, has been suggested to play a role in previous reported cases of “operational tolerance” (16,19,42). VanBuskirk et al. have demonstrated an immune regulation implicating regulatory cell activity in some “operationally tolerant” kidney recipients using a “trans-vivo” delayed-type hypersensitivity assay (20). Clonal deletion does not seem to be crucial in “spontaneous tolerance,” as observed by Strober (19), as also suggested by the presence of anti-donor reactivity (anti-donor antibodies) in two patients of our cohort. We also showed that the T cells of “operationally tolerant” patients are characterized by low levels of cytokine transcripts, contrasting with profiles observed in recipients with chronic rejection (21). Blood cell phenotype studies suggest that the profile of “operationally tolerant” kidney recipients does not differ from those of healthy volunteers (43) whereas recipients with chronic rejection present significantly less CD4 + CD25hi cells (associated with regulatory cells) (Louis S. et al., submitted). In liver transplantation, “operationally tolerant” patients present a higher number of CD4 + CD25hi cells than healthy volunteers (45). Finally, although these patients presented a heterogeneous clinical history, they offer the opportunity to search for a common tolerance signature that would help to identify the state of “operational tolerance” in other recipients under classical immunosuppression (46). However, these observations are clearly not substantial enough for an immunosuppression weaning procedure to be undertaken in these patients. For this to be feasible safer, parameters of tolerance prediction will need to be available. What recommendations could be made for the follow-up of such “operationally tolerant” patients? One could try to monitor the renal function of these incompliant patients more closely (every month) and screen for panel reactive antibodies, which have been associated with an increased risk of developing chronic rejection (39). One could also recommend these patients to consult in the case of intercurrent infection episodes (47). Graft biopsies could be performed in the case of a proteinuria exceeding 1g/d, a creatininemia increase of more than 10% or the apparition of an anti-donor immunization.