Current immunosuppressive regimens used after organ transplantation have major side effects.1, 2 Therefore, the definition of therapeutic strategies able to promote transplantation tolerance, defined as long-term survival of an allograft in the absence of ongoing immunosuppression (IS), would be a significant advancement. Extensive experimental work and some clinical observations have indicated that tolerance to allograft could be promoted by infusion of donor-derived hematopoietic cells after host's myeloconditioning in the peritransplant period.3 For clinical application, living donor transplantation offers a crucial advantage as compared to deceased donor transplantation, as early identification of the donor gives the opportunity to harvest donor tolerogenic cells before transplantation and prepare them for posttransplant infusion. Here, we report on 3 patients prospectively included in a protocol designed to favor graft acceptance using posttransplant conditioning with high doses of antithymocyte globulin followed by donor-derived stem cells (SC) infusion in living donor liver transplantation (LDLT).
Long-term results of organ transplantation are still limited by serious side effects of immunosuppressive drugs. A major issue, therefore, is to elaborate novel therapeutic protocols allowing withdrawal or minimization of immunosuppressive therapy after transplantation. We report on 3 patients prospectively enrolled in an original protocol designed to promote graft acceptance in living donor liver transplantation, using posttransplant conditioning with high doses of antithymocyte globulin followed by injection of donor-derived stem cells. In 2 patients, early immunosuppression withdrawal was possible, without subsequent graft deterioration. In these 2 cases, in vitro studies showed indices of immunological tolerance as assessed by specific hyporesponsiveness to donor alloantigens in mixed lymphocytes culture. In the third patient, acute rejection rapidly occurred after discontinuation of immunosuppression, and minimal immunosuppression has to be maintained during long-term follow-up. In this case, a clearly distinct immunoreactive profile was observed as compared to tolerant patients, as no specific modulation of the antidonor response was observed in vitro. Of note, no macrochimerism could be detected in any of the 3 patients during the follow-up. In conclusion, these clinical observations demonstrated that, despite the absence of macrochimerism, donor stem cells infusion combined with recipient conditioning may allow early immunosuppression withdrawal or minimization after liver transplantation. Liver Transpt 12:1523–1528, 2006. © 2006 AASLD.
PATIENTS AND METHODS
Due to the experimental nature of this protocol, it was initially proposed to patients with advanced liver cancer, ineligible for liver transplantation according to the admitted criteria4 and without any other potentially curative options. All donors were living-related (son of patient 1, and wives of patients 2 and 3) and free of medical illness. Both donors and recipients were fully informed of the potential risks, particularly of cancer recurrence after transplantation. Inclusion criteria were (1) advanced liver cancer ineligible for liver transplantation, (2) absence of extrahepatic spreading, (3) absence of other contraindication to liver transplantation, and (4) informed consent from both donor and recipient. The study protocol was approved by the hospital ethical committee.
The protocol relies on the use of donor SC administered after LDLT as tolerogenic/suppressive cells5 (Fig. 1). For preoperative harvesting of SC, the donor received granulocyte colony stimulating factor (Neupogen, Amgen Thousand Oaks, CA) for 4 days for mobilization. Donor peripheral blood cells were then collected by cytapheresis, and CD34+ selection was performed using the CliniMACS CD34 medical device (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Purified donor CD34+ SC were then frozen. LDLT was performed on day 0.
Posttransplant IS and conditioning included steroids, rapamycin and antithymocyte globulin (Thymoglobulin, Sangstat-Genzyme, Boston, MA) (Fig. 1). Rapamycin was chosen instead of calcineurin inhibitors as several data have suggested it does not interfere with active tolerogenic mechanisms.6 On day 7, total amount of donor CD34+ SC was infused through a central line. From day 7, maintenance IS consisted of steroids and rapamycin (Fig. 1). IS withdrawal was collectively decided by the physicians involved in the protocol as soon as the recipient's clinical condition became stable and liver tests returned to normal. After IS withdrawal, patients were followed by serial clinical examination and blood testing. In case of suspicion of acute rejection (AR), liver biopsy was performed. AR was treated with 4 bolus of 500 mg methylprednisolone, followed by weaning in approximately 10 days. According to the clinical response, either second IS withdrawal or tacrolimus administration was chosen.
To monitor antidonor reactivity, mixed lymphocyte reactions (MLR) were performed between unseparated recipient whole blood cells as responders and autologous, donor, or third-party antigen presenting cells (APC) as stimulators, as previously described.7 As readouts of T-cell reactivity, proliferation and IL-2 production were measured in MLR. Interleukin (IL)-2 messenger RNA production was measured by real-time polymerase chain reaction (Roche Applied Science, Vilvoorde, Belgium). APC stimulators were prepared using CD3+ depletion on whole blood by positive selection using a RosetteSep human CD3+ cell depletion kit (StemCell Technologies, Vancouver, BC, Canada). Results of antidonor reactivity in MLR (recipient whole blood cells vs. donor APC) were compared with background response (recipient whole blood cells vs. autologous APC) and nonspecific third-party response (recipient whole blood cells vs. third-party APC).
Analysis of Chimerism
DNA from recipient whole blood was isolated by the salting-out method. Intermediate/low-resolution DNA typing was done at the human leukocyte antigen (HLA)-A, HLA-B, and DRB1 loci by polymerase chain reaction–sequence-specific oligonucleotide probing using commercially available reagents (Dynal Reli SSO, Dynal Biotech, Bromborough, UK; INNO-LiPA Innogenetics N.V., Ghent, Belgium).To resolve ambiguous typing, a sequence-specific primer–polymerase chain reaction assay for the DRB1 alleles assignment was performed using commercially available reagents (Dynal AllSet+-SSP, Dynal Biotech, Bromborough, UK). To detect macrochimerism the recipient's peripheral blood mononuclear cell (PBMC) were tested for the presence of the donor-specific HLA-DRB1 alleles. In case of donor/recipient class II identity, chimerism was investigated at the HLA-A or HLA-B loci level.
Patient 1 was a 62-year-old man with Child-Turcotte-Pugh C hepatitis C–related cirrhosis and multifocal hepatocellular carcinoma without extrahepatic dissemination. After selection, 7.7 × 106 CD34+ donor SC per kg of recipient weight were obtained, including 15 × 104/kg residual CD3+ T cells. LDLT was performed on day 0, using donor's right lobe. Pathology on hepatectomy specimen confirmed multifocal hepatocellular carcinoma with microvascular invasion. Protocol IS was administered without side effects and donor CD34+ SC were infused on day 7, without subsequent sign of graft vs. host disease (GVHD). On day 18, after normalization of the liver tests, IS was discontinued. On day 108, liver test results deteriorated and liver biopsy showed grade II AR. Four boluses of 500 mg methylprednisolone were given, followed by progressive weaning for 6 days. No further AR was observed, and the patient remained IS free throughout the entire follow-up period (Table 1). Unfortunately, on day 326, tumor recurrence was demonstrated in liver graft and the patient died on day 561 posttransplant due to metastatic disease.
|Patient 1||Patient 2||Patient 3|
|Indication for LDLT||Multifocal HCC||Multifocal HCC||Hilar cholangiocarcinoma|
|Conditioning||ATG-rapamycin post-LDLT||ATG-rapamycin post-LDLT||ATG-rapamycin post-LDLT|
|Donor CD34+SC infused on day 7||7.7 × 106 CD34/kg||10 × 106 CD34/kg||5.3 × 106 CD34/kg|
|Immunosuppression withdrawal||Day 18||Day 23||Day 213|
|Acute rejection||Yes (day 108)||Yes (day 80)||Yes (day 241)|
|Total period off IS||533/561 days||319/356 days||28/498 days|
|Follow-up||561 days||356 days||498 days|
|Status at last follow-up||Died from tumor recurrence, immunosuppression free||Died from tumor recurrence, immunosuppression free||Alive, without tumor recurrence, under TAC|
Patient 2 was a 51-year-old man with Child-Turcotte-Pugh C hepatitis C–related cirrhosis and multifocal hepatocellular carcinoma. After selection, 10 × 106 donor CD34+ SC per kg of recipient weight were obtained without detectable residual CD3+ T cells. LDLT was performed using donor right lobe, and postoperative course was uneventful. Pathology of hepatectomy specimen confirmed multifocal hepatocellular carcinoma with macrovascular invasion. Protocol IS was administered without side effects and donor CD34+ SC were infused on day 7, without subsequent GVHD. On day 23, after normalization of liver tests, IS was stopped. On day 80, liver test results deteriorated and 4 boluses of 500 mg methylprednisolone were given, followed by progressive weaning on 10 days, with favourable clinical response. No further AR was observed and the patient remained IS free during the entire follow-up period (Table 1). Unfortunately, on day 140, tumor recurrence was diagnosed (right lung metastasis), and the patient died on day 356 posttransplant.
Patient 3 was a 49-year-old man with hilar cholangiocarcinoma without extrahepatic dissemination. After selection, 5.3 × 106 donor CD34+ SC per kg of recipient weight were obtained, including 7.3 × 103 per kg of recipient weight residual CD3+ T cells. LDLT was performed on day 0, using donor's right lobe. Pathology of hepatectomy specimen confirmed cholangiocarcinoma without lymphatic or perineural invasion. Protocol IS was administered without side effects and donor CD34 + SC were infused on day 7, without subsequent GVHD. At day 15, IS consisted of rapamycin monotherapy. Postoperative course was characterized by early biliary leak, requiring revision laparotomy. Considering the risk of impaired wound healing induced by rapamycin, IS was switched to tacrolimus on day 30. Due to persistent biliary problems, IS withdrawal was delayed until day 213. At day 241, liver function tests worsened and liver biopsy demonstrated grade II AR. Accordingly, tacrolimus was reintroduced and maintained. To date, at 498 days after LDLT, the patient remains under minimal IS (2 mg tacrolimus/day and target through level of 5 ng/mL) (Table 1) without any evidence of tumor recurrence.
In patients 1 and 2, who accepted their grafts for a prolonged period without IS, MLR demonstrated a sustained posttransplant hyporesponse against donor APC (Fig. 2). Indeed, after an initial period of unspecific hyporeactivity, as indicated by low responses to either donor or third-party APCs, IL-2 production and proliferative responses against third-party APCs in patient 1 and anti-third-party proliferative response in patient 2 progressively recovered, whereas antidonor donor responses remained very low, suggesting donor-specific modulation (Fig. 2). In contrast, in patient 3, who experienced rapid graft rejection after IS withdrawal, no such selective inhibition of antidonor reactivity was observed. In this case, under maintenance IS, IL-2 production and proliferative response in MLR against donor and third-party APCs remained similar at different time points in the posttransplant period (Fig. 2).
Analysis of Chimerism
In all patients, no circulating cells from donor origin were detected at any time points following donor CD34+SC infusion (limit of sensitivity: 1% of circulating cells from donor origin).
Continuous efforts have been made these last years to develop novel therapeutic protocols aiming at minimizing or discontinuing IS after solid organ transplantation. Animal studies have notably shown that robust transplantation tolerance could be induced using host conditioning and donor hematopoietic cell infusion.3 From a clinical perspective, this approach has been validated in rare cases of patients becoming tolerant to a living donor liver or kidney graft several years after receiving a bone marrow transplant from the same donor.8, 9 In a step further, it has been reported that tolerance to kidney allograft could be intentionally induced by myeloablative conditioning and donor bone marrow cell infusion in patients with renal failure and hematological malignancies requiring bone marrow transplantation.10 Until now, however, the toxicity of preparative conditioning, using either irradiation or myeloablative chemotherapy, and the risk of GVHD after donor hematopoietic cell infusion have hampered the development of such protocols outside these rare indications. The need for preparative recipient conditioning to allow tolerogenic engraftment of donor cells has been clearly demonstrated in several clinical studies. Indeed, in the absence of preparative therapy, infusion of donor bone marrow cell has been shown to have no significant effect in LT on either the need for maintenance IS or the rate of rejection, despite an increased rate of donor chimerism.11, 12 It has been proposed also that posttransplant IS using antilymphoid antibodies like antithymocyte globulin13 or Alemtuzumab14 without donor cell infusion may have a tolerogenic effect, allowing a significant proportion of patients to discontinue IS during long-term follow-up. The hypothesis was that such depletive IS regimen may allow progressive engraftment of graft-derived donor cells, leading to donor specific tolerance.13, 14 In these trials, however, IS minimization or discontinuation was scheduled several months after LT, and the majority of patients still required minimal IS.13, 14 It should also be noted that spontaneous tolerance may occur in approximately 20% of liver recipients, independent of the posttransplant immunosuppressive regimen.15 However, this state of tolerance is reached only after prolonged exposition to immunosuppressive drugs, and in the absence of reliable markers to identify these occasionally tolerant patients, attempts of IS discontinuation may trigger severe rejections and graft loss.16 Upon this background, we developed the present protocol combining nonmyeloablative conditioning and donor SC infusion and hypothesized that, in such conditions, IS could be withdrawn in the early posttransplant period. As the toxicity of immunosuppressive agents is known to be maximal in the postoperative period, it could be anticipated that such an approach may offer a significant advantage. For this purpose, we used donor SC as tolerogenic cells, as these cells are unable to mediate GVHD and carry a high capacity of engraftment thus requiring mild preparative conditioning.5, 17 To our knowledge, the cases reported here represent the first patients treated with mild conditioning and donor SC infusion after LDLT, in the absence of hematological malignancies. On the one hand, these cases demonstrated the safety of this approach, as neither conditioning-related side effect nor GVHD after donor SC infusion occurred. In addition, no patients developed opportunistic infections, suggesting that the conditioning used did not induce prolonged severe global immunodeficiency. On the other hand, in 2 patients, early IS withdrawal was possible, at days 18 and 23 after LDLT. Both patients experienced 1 episode of AR, rapidly responding to standard steroid therapy. For a prolonged period, both patients remained IS free thereafter with functional graft and no signs of acute or chronic rejection.
Consistently, MLR experiments displayed indices of immunological tolerance, as indicated by sustained posttransplant inhibition of IL-2 messenger RNA production and proliferative response against donor antigens, while anti-third-party response progressively recovered (Fig. 2). In the absence of ongoing IS, these tests strongly indicate the absence of sensitization to donor antigens after LDLT. These MLR profiles in acceptor patients are in direct contrast with those reported in LT recipient patients under standard IS therapy, as the majority of patients developed vigorous posttransplant IL-2 response to donor alloantigens in these conditions.18 The reason for failure of IS withdrawal in patient 3 is unknown. It is possible that the prolonged period of IS after LDLT, justified by technical problems, particularly the introduction of calcineurin inhibitor, may have played a negative role by inhibiting active tolerogenic processes.19 Interestingly, in this case, the immunoreactive profile in MLR contrasted with the responses observed in the 2 other patients, as no selective inhibition of antidonor reactivity was demonstrated (Fig. 2). Even if these tests have to be prospectively validated in order to assess their potential predictive value, our observations suggest that MLR may help to identify patients in whom IS could be safely discontinued from those requiring maintenance IS. Further refinements of these immunological tests will be pivotal for establishing the safety of such protocols.20 Besides, no macrochimerism has been detected at any time point after donor SC infusion in either acceptor patients or the patient who required maintenance IS. While it has been shown, in comparable protocols, that clinical tolerance could be achieved despite progressive loss of chimerism after peritransplant donor cell infusion,10 this observation strongly contrasts with the generally accepted statement that at least transient macrochimerism is essential for establishment of long-term donor-specific nonreactivity (i.e., tolerance).3, 8–10 The reason for absence of macrochimerism in these cases is unknown. It should be first noted that, as compared with previous studies using similar approach,10 the conditioning applied in the present protocol avoided cyclophosphamide and thymic irradiation and used lower antithymocyte globulin doses and could therefore be insufficient to allow early engraftment of donor cells. Absence of detectable macrochimerism could be also due to an insufficient number of injected donor cells or, eventually, to trapping in a recipient's hematopoietic system. Whatever the reason for the absence of detectable circulating donor cells, this observation certainly raises the question of the level of chimerism needed for induction of donor-specific hyporesponsiveness in such protocols. While it could be speculated that, even if rejected in the posttransplant period, donor SC may play a tolerogenic/suppressive role at the engagement phase of the immune response, and the necessity of their infusion remains undetermined. This question could be answered only by a 2-arm study, using a control group receiving the same conditioning/immunosuppressive regimen but without donor SC infusion.
Finally, despite short exposition to IS, tumor relapses were observed in patients 1 and 2, who initially carried poor cancer prognosis. One can assume that these cancer recurrences are related to undetected micrometastases and/or circulating tumor cells at the time of transplantation. Of note, timing of recurrences in these 2 cases is very similar to what can be expected in such diseases and does not suggest a negative effect of the conditioning treatment.
In conclusion, these first clinical observations demonstrated that, even in the absence of macrochimerism, early IS withdrawal or minimization is possible after LDLT using recipient nonmyeloablative conditioning and donor SC infusion. The absence of toxicity related to conditioning and donor SC infusions led us to propose this strategy as an alternative to classical IS for LDLT candidates within the admitted criteria. In contrast, tumor recurrences observed despite a short period of IS require us to no longer include patients with extended cancer indications.
The authors thank Cécile Galle for her critical reading of the manuscript and Dr. Lucienne Chatenoud (Hôpital Necker, Paris, France) for her help as coordinator of clinical trials in the RISET consortium.