A strategy to achieve donor-specific hyporesponsiveness in cadaver renal allograft recipients by donor haematopoietic stem cell transplantation into the thymus and periphery
Hargovind L. Trivedi1,
Aruna V. Vanikar2,
Javed M. Vakil1,
Veena R. Shah3,
Pranjal R. Modi4 and
Varsha B. Trivedi2
1 Department of Nephrology and Clinical Transplantation, 2 Department of Pathology, Laboratory Medicine, Transfusion Services and Immuno Hematology, 3 Department of Anaesthesiology and Critical Care and 4 Department of Urology and Transplantation Surgery, Institute of Transplantation Sciences (ITS), Institute of Kidney Diseases and Research Centre (IKDRC), Ahmedabad, Gujarat, India
Correspondence and offprint requests to: Dr H. L. Trivedi, FRCP (C), Director, Professor, Institute of Transplantation Sciences (ITS) and Institute of Kidney Diseases and Research Centre (IKDRC), Civil Hospital Campus, Asarwa, Ahmedabad 380 016, Gujarat, India. Email: ikdrcad1{at}sancharnet.in
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Abstract
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Introduction. We designed a prospective, randomized clinical trial to evaluate the immune response to thymic and peripheral infusions of donor haematopoietic stem cells (HSCs) to create tolerance in recipients of cadaver renal allografts.
Method. We divided 24 patients into two equal groups. For group A, 350 ml of unfractionated bone marrow (BM) was aspirated from the anterior iliac crests of donor cadavers. A 2 ml aliquot of concentrated marrow was infused into the thymus of the subject and 100 ml into the BM before surgery; the remaining 250 ml was infused peripherally post-transplantation. The mean nucleated cell count inoculated into the thymus was 3.3 x 104 cells/cm3 and into the periphery 8.6 x 107 cells/kg body weight. Group B (controls) underwent renal transplantation directly. Recipients were lymphocytotoxicity cross-match negative in both groups. Group A received low dose prednisolone and cyclosporin; controls also received azathioprine.
Results. Over a mean follow-up of 703 days for both groups, group A had significantly better graft function with minimum acute rejection episodes or cytomegalovirus (CMV) infections, a mean serum creatinine (SCr) of 1.23 mg/dl and no graft or patient loss. Group B, with a mean SCr of 2.19 mg/dl had three patients with single acute rejection episodes, two of whom died following uncontrolled rejection-associated infections. The third patient maintained an SCr of 2.5 mg%. Actuarial graft survival was 87.5% in controls at the end of 2 years compared with group A with 100% graft survival at the end of 2 years.
Conclusion. This novel approach of introducing unfractionated HSCs into the thymus and periphery to create tolerance is safe and efficacious and gives significantly better graft function, minimum acute rejection and no CMV disease with monotherapy.
Keywords: donor-derived haematopoietic stem cells; cadaver renal transplantation; tolerance
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Introduction
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Cadaver organ transplantation is a recent addition to the transplantation scene in India, where live, related transplantation (LRD) is the commonly practised therapy for patients with end-stage renal failure. The Human Organ Transplantation Act was passed by the Indian parliament in 1994 and adopted by the legislative assemblies of different states thereafter (but not yet adopted by all states of India). However, the standard long-term maintenance immunosuppression with cyclosporin A (CsA; 812 mg/kg body weight/day), prednisolone (0.40.8 mg/kg body weight/day) and mycofenolate mofetil (2 g/day) or azathioprine (12 mg/kg body weight/day) is prohibitively expensive. Apart from these problems, the side effects of immunosuppression, infection- and malignancyrelated morbidity and mortality place transplantation out of reach for an average Indian patient. Hence strategies leading to better and stable allograft function with minimum or no immunosuppression would represent a significant advance in transplantation practice.
The utopian dream of immunological allotolerance has been achieved with reasonable success in experimental models. We are now trying to take this concept from the laboratory bench to the bedside. As the immunological mechanisms involved in allograft rejection are quite complex, to achieve tolerance, a combination of different protocols that address different pathways of rejection are required. We tailor-made a clinical protocol to address simultaneously the central and peripheral mechanisms of tolerance.
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Patients and methods
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Study design
Between June 2001 and September 2003, we performed a prospective, randomized, parallel, open-labelled, clinical study in accordance with the revised Declaration of Helsinki, to evaluate the effects of donor-derived haematopoietic stem cell (HSC) infusion into the thymus and peripheral circulation of our cadaveric renal allograft recipients (treated group A). Controls (group B) were transplanted without HSC infusion. From every donor cadaver, one ABO-compatible and lymphocytotoxicity cross-match-negative kidney was allocated to group A and one to group B. All patients were approached with the intent to treat, and their informed consent was obtained. Our institutional Ethics Committee approved the study protocol and the consent forms of the clinical trial.
Selection of patients
From the cadaveric transplantation list, 24 consecutive patients with end-stage renal disease (ESRD) were divided into two groups of 12 patients each. Group A received HSC infusions into the thymus, bone marrow and peripheral circulation, and controls (group B) underwent renal transplantation without any HSC infusion. Both groups were fairly balanced with respect to their clinical profiles (Table 1). They were followed-up at the same out-patient clinic at weekly intervals for the first 3 months, fortnightly for the next 3 months and monthly thereafter. At every visit, their renal and liver function status was monitored, blood counts were performed and CsA levels were measured. They were monitored at monthly intervals for human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) and cytomegalovirus (CMV) infection status (IgG and IgM antibodies) using the enzyme-linked immunosorbent assay (ELISA) technique.
Protocol design
Our aim was to inoculate concentrated bone marrow (BM) aspirate into the thymus and HSCs in the BM and periphery to mobilize and integrate the central and peripheral arms of clinical tolerance.
Stem cell collection, inoculation and infusion techniques
BM aspiration was performed from the anterior superior iliac crest of the donor cadavers. First, 6 ml of the aspirate was concentrated and 2 ml was kept for thymic infusion. Subsequently, out of 350 ml of the aspirated unfractionated BM, 100 ml was inoculated into the sternal BM of the recipient. The remaining 250 ml of the aspirate was infused intravenously (i.v.) into the peripheral circulation of the recipient at the end of the transplantation surgery. Cell counts (including CD34+) of the marrow aspirate for thymic inoculation and for the periphery and BM were performed separately. The average cell count of the thymic inoculum was 3.3 x 104 cells/cm3 (CD34+: 1.1%) and of the remaining aspirate was 1.9 x 104 cells/cm (CD34+: 0.7%).
Thymic inoculation
Under general anaesthesia, a 4 cm long incision was made into the right second intercostal space after the transplantation surgery. After incising all muscles, the mediastinal fascia was opened and the thymus identified in the retrosternal space. Then, the 2 ml of concentrated marrow was inoculated into the thymus with a 20 gauge needle, after which haemostasis was confirmed and the wound closed.
Recipient immunosuppression
Both groups were given prednisolone and CsA; controls also received azathioprine. CsA doses were adjusted with the intention of maintaining trough blood levels at
100 ng/ml, and the CsA levels were measured using the EMIT 2000 CsA assay (Syva Co., Dade Behring, USA, intended trough levels: 50176 ng/ml). Both groups were administered prednisolone, 0.5 mg/kg body weight/day for the first month post-transplantation followed by 0.2 mg/kg body weight/day subsequently. Azathioprine, 2.5 mg/kg body weight/day, was added in controls.
Rejection and treatment
Every recipient was biopsied if clinically suspected of acute rejection. Rejection was diagnosed according to the modified Banff criteria [1], and was treated with i.v. methylprednisolone, 250 mg/day for three consecutive days; tacrolimus was used for rescue.
Statistical analysis was performed using the Student paired t-test.
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Results
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One patient had procedure-related pneumothorax and required underwater intercostal drainage for 24 h following thymic inoculation.
Over a mean follow-up of 703 days, one patient (8.3%) in group A and three patients (25%) in group B had one acute rejection episode each. Two of them (one from each group) had borderline acute tubulo-interstitial rejection along with acute tubular necrosis (ATN); the other two patients from group B had moderate acute tubulo-interstitial rejections, type 1B with ATN. Their biopsies were performed at an average interval of 10 days post-transplantation. All patients in group A achieved early graft function and required no dialysis support. In group B, three patients (25%) were supported with dialysis three times a week until the functions of their renal allografts recovered. Serum creatinine (SCr) values were used as indicators of allograft function. For comparison, the SCr values of both groups were taken at 3, 6, 9, 12, 18 and 24 months post-transplantation (Figure 1). It was observed that the mean SCr was significantly better in group A than in group B. In group B, two patients (16.7%) died due to progressive uncontrolled rejection-associated infections. A KaplanMeier analysis was performed to evaluate the actuarial graft survival of eight patients of each group who had completed the 24 months of the study and qualified for this test. It was observed that 100% of patients in group A (with stable allograft function) and 87.5% in group B had surviving grafts at the end of 2 years. Graft vs host disease (GvHD) was not seen in group A. Tacrolimus administration was required in two patients in group B, and was found to be safe. None of the patients in group A had CMV or other infections.

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Fig. 1. Comparison of graft function of the treated group (group A) vs controls (group B) in terms of SCr values over a span of 2 years post-transplant.
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Discussion
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We developed a peri-operative, non-myeloablative, mega-dose, unfractionated HSC infusion protocol to create tolerance in cadaver renal allograft recipients who were without GvHD or hepatic dysfunction and almost acute rejection free, and who had stable graft function and very low incidence of CMV disease, with minimum immunosuppression [2,3]. Remuzzi et al. had found that the intrathymic inoculation of donor antigen and purging of donor-specific alloreactive T cells from the peripheral T-cell repertoire was necessary to create classical central tolerance [4]. We decided to validate their experience clinically in our cadaver renal allograft recipients by incorporating in our transplantation protocol the intrathymic insertion of donor HSCs. Owen's observation of naturally occurring mixed chimerism in paternal bovine twins inspired Billingham et al. to create the first experimental graft tolerance model in neonatal mice [5,6]. This seminal work of cell transplantation in a defenceless host became the basis for the BM transplantation of the future [7]. The experimental work of Posselt et al. proved that donor-specific hyporesponsiveness could be achieved by intrathymic inoculation of donor alloantigens [811]. Zinkernagel has envisaged the use of donor haemato-lymphopoietic cells as the key therapeutic manoeuvre to achieve stable, drug-free antigen-dependent T-cell exhaustion and chimerism [12]. It will therefore be logical to infer that chimerism is strongly predictive of successful tolerance induction.
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Conclusion
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Our innovative approach of thymic innoculation and peripheral infusion of unfractionated donor-derived HSCs to achieve donor-specific hyporesponsiveness in cadaver organ transplantation has proven itself safe and efficacious. It provides a stable and significantly better graft function with monotherapy immunosuppression, and is associated with minimum rejections and no CMV infections, as compared with controls.
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Acknowledgments
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We are indebted to Ms Priyadarshini Chaudhary, Ms Nalini Patel, Ms Jyotsna Patel and Mr Yazdi Wadia for updating patient charts, statistical analysis and the preparation of the manuscript.
Conflict of interest statement. None declared.
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References
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Received for publication: 24.10.03
Accepted in revised form: 6. 2.04