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Quartal 1999 Prope
(almost) Tolerance and the WOFIE Hypothesis Tolerance to organ allografts in primates including man has been elusive, although in rodents and pigs tolerance can be achieved to organ allografts with relatively short courses of immunosuppressive treatment. Liver allografts in pigs and rats may induce tolerance without any immunosuppression. This is the only example of the production of tolerance in immunologically mature animals across an MHC barrier without immunosuppressant treatment (1). After allografting the organ undergoes a moderate to severe rejection crisis which resolves spontaneously. Then the animals may be fully tolerant as judged by indefinite survival of a skin graft from the donor. In clinical bone marrow transplantation complete ablation of the bone marrow followed by repopulation with donor stem cells results in macrochimaeric tolerance. Patients with long surviving liver allografts may be able to stop immunosuppression without rejection of their organs. The longest survivor has had no immunosuppression for the 14 years and is 25 years posttransplant (Starzl, personal communication). Cautious weaning from immunosuppression of long-term liver allograft recipients would suggest that some 30% can come off all immunosuppression without penalty after five years of good function of the allograft. Stopping immunosuppression in recipients of other organ allografts usually results in their rejection. In man the liver also protects other organs from the same donor source from rejection (2). We have studied the liver phenomenon for many years and a crucial series of experiments performed by Voravit Sriwaranawongsa showed that both hepatocyte parenchymal cells and bone marrow derived cells in the liver were requirements for the production of full tolerance in liver allografted rats. Neither alone would produce tolerance as tested by donor skin grafting. We also found that soluble donor class 1 MHC antigen is produced constantly in large quantities by an allografted liver and the liver is a source of much of the detectable circulating Class 1 MHC in normal people (3). Recently soluble Class 1 molecules have been shown to induce apoptosis in alloreactive cytotoxic T lymphocytes (4). In all varieties of graft acceptance that do not require full dose maintenance immunosuppression, immunological engagement of donor and recipient and an early unstable period have been observed. On the basis of the hypothesis that elimination of aggressive T-cell function should tip the balance in favour of tolerant state, experiments have been performed using donor bone marrow derived cells in renal allografted, MHC mismatched pigs. An interrupted course of seven 25 mg/lcg intravenous doses of Cyclosporin was given and a period of 2 to 3 days without immunosuppression. In kidney transplants between mismatched pigs, prolonged survival was observed in more than half the animals without any evidence of chronic rejection. This protocol with hypothetical "window of opportunity for immunological engagement" (WOFIE) could be suitable for clinical application. Excessive immunosuppression might prevent this engagement and the emergence of a tolerant state. Knechtle and his colleagues (5) have produced tolerance in a very difficult rhesus monkey model using a powerful Anti-CD3 monoclonal antibody linked to a modified diphtheria immunotoxin. Three doses of the immunotoxin were given intravenously, on three successive days, starting seven days prior to renal transplantation. The total dose of 2 mg per kg given over the three days produced little toxicity and tolerance was produced in most animals receiving mismatched kidneys. Donor skin grafts were usually accepted long-term. The immunotoxin produced profound lymphocyte depletion which was slow to recover and it was postulated that depletion of T-lymphocytes throughout the body was necessary in addition to purging the blood. In clinical organ grafting there is a spectrum of immunological engagement from tolerance to hyperacute rejection. Most patients with organ grafts require continuous dosage with immunosuppressive drugs. The objective would be to shift the curve to the left so that the majority of patients are "operationally tolerant" or "almost tolerant" and require only minimal immunosuppression. Requirement for tolerance: 1. Best possible HLA match 2. Minimum organ ischaemia 3. Temporary destruction or inactivation of potential aggressive T-cells. Current clinical protocols of immunosuppression probably do not achieve this aim. Circulatory lymphocytes are less than 3% of the lymphocyte pool. Excessive and prolonged high dose immunosuppression may: 1. Prevent the immunological host/ graft engagement necessary for tolerance. 2. Cause lethal infection or lymphoproliferative disease. These and collateral observations support an hypothesis that, in the absence of an aggressive T-cell response, an engagement of certain donor and recipient bone marrow derived cells can be beneficial, leading to tolerance. A window of opportunity for immunological engagement (WOFIE) may be needed for this to take place. The powerful immunosuppression produced by the immunotox used by Knechtle and colleagues had its most beneficial effect when given seven days before a kidney transplant. If the immunotox was given after transplantation there was prolongation of graft survival but rejection occurred with an antibody component. This they attributed to the lack of effect of B cells of their immunotox. A humanised antibody produced in Cambridge with the unique target of CD52 is a powerful depletor of T and B lymphocytes and also monocytes but not bone marrow stem cells. In view of its effect on B cells and also the impossibility in cadaveric transplants of giving the antibody 7 days before grafting, a protocol was established which we have investigated in 30 recipients of renal allografts, giving the antibody after allografting. 13 of these have been followed up now for between 14 and 18 months (6). The remainder have a shorter follow-up. No immunosuppression is given until the patient returns to the ward after the transplant operation. Then with a preceding dose of intravenous hydrocortisone to control any cytokine release syndrome, the patients were given 20 mgs of Campath IH intravenously. The following day a second and last dose was given. A period of 48 hours was then left without any immunosuppression and then daily cyclosporin Neoral was given to achieve a trough blood level of around 100 nanograms. No other immunosuppression was used unless there was evidence of rejection, in which case the patient was treated initially with three daily doses of lgm of prednisolone. If this did not rapidly reverse the rejection the patients were managed with dual therapy of Neoral and steroids. Infection prophylaxis was no different from standard management in our clinic. To date, there have been no serious infections and no malignancy and no patient has lost the kidney. There have been four disturbances of renal function attributed to rejection, two with a characteristic cellular infiltrate and two with a vascular type of rejection. All acute rejection responses were controlled with steroids but three of the four patients have been maintained with cyclosporin and steroids. These early results are encouraging. We have called the protocol "Prope Tolerance", or Almost Tolerance. Low-dose immunosuppression is the safeguard for acute rejection being precipitated by a viral infection or an allergy, and also provides the clinician with a safety net in that Neoral is a standard drug and the dose can be increased if necessary. The advantages to this protocol are complete avoidance of steroids in the majority of patients and considerable reduction in the cost of maintenance immunosuppression. The protocol is consistent with the concepts discussed at the beginning of this article but does not in any way constitute a proof of the WOFIE hypothesis. Nevertheless, one could argue that the Campath temporarily wipes out all circulating lymphocytes for approximately a month leaving the "slate clean" and the slow recovery, particularly of CD4 cells in the presence of the established graft can lead to some form of tolerance "mechanism(s)". We do not yet know the degree of lymphocyte depletion in lymphocyte depots scattered throughout the body, nor do we have any data on the nature of cellular response and cytokine production of lymphocytes when they return in the circulation and the behaviour of dendritic cells of the donor. Nor for that matter whether there is any change in the presentation of donor antigens. Details of these questions are now being studied including the kinetics of the immune recovery in these patients and a randomised trial comparing the above protocol with standard treatment is being planned. References [1] Calne RY et al. (1969) Nature Medicine 223: 472 [2] Rasmussen A et al. (1995) Transplantation 59: 919 [3] Zavazava N (1996) Nature Med 2: 1005 [4] Calne RY et al. (1994) Transplantation 57: 1433 [5] Armstrong N et al. (1998) Transplantation 66: 5-13 [6] Calne RY et al. (1998) Lancet 351: 1701-2 Prof. Sir R. Calne, M.D.
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