Effectiveness and risks of total body irradiation for conditioning in the treatment of autoimmune disease with autologous bone marrow transplantation

Series Editor: P. Woo

D. W. van Bekkum

IntroGene bv, PO Box 2048, 2301 CA Leiden, The Netherlands

Abstract

The results of experiments with the induced autoimmune diseases adjuvant arthritis and allergic encephalomyelitis in rats, which led to the discovery of the curative effect of autologous bone marrow transplantation following high-dose myeloablative treatment, are reviewed. The rationale is eradication of the autoreactive lymphocytes and memory cells, and the prevention of relapse due to transfer of lymphocytes with the autograft. Comparison of various conditioning regimens in the animal models indicates that a combination conditioning with low-dose total body irradiation (TBI) and high-dose cyclophosphamide is optimal. These findings were the basis for the conditioning currently employed in the treatment of polyarticular juvenile chronic arthritis (JCA) by the teams in Utrecht and Leiden, which consists of cyclophosphamide 50 mg/kg for 4 days, 4 Gy TBI and anti-thymocyte globulin (ATG). The use of TBI for the treatment of non-malignant disease is regarded as undesirable by many physicians in view of the risks, in particular, of growth inhibition in children and the induction of tumours. Experimental and clinical data show that a dose of 4 Gy does not cause significant inhibition of skeletal growth in infants. The risk of excess cancer due to TBI has been well established in quantitative terms and is compared with the expected risk of high-dose cyclophosphamide and the risk associated with the highly immunosuppressive regimens currently used for the treatment of JCA.

The current clinical exploration of autologous stem cell transplantation for the treatment of patients suffering from severe forms of various autoimmune diseases has its scientific foundations in our research with laboratory animals. In 1990, we observed that fully developed arthritis in rats can be cured by treatment with a myelolymphoablative regimen followed by rescue with autologous bone marrow cells [1]. Subsequently, we demonstrated that experimental allergic encephalomyelitis (EAE) also responds favourably to autologous bone marrow transplantation [2]. These findings were totally unexpected, in contrast to the curative effect obtained in these experimental diseases with allogeneic bone marrow transplants [35]. The latter observation was in line with incidental regression of an autoimmune disease after allogeneic bone marrow transplantation for the treatment of coincidental leukaemia or aplastic anaemia [6, 7].

The mechanism which underlies the occurrence of complete remissions of autoimmune disease seems obvious in the case of treatment with allogeneic bone marrow. The purpose of the myeloablative conditioning regimen is, as in leukaemia, to eradicate the unwanted cells. In the case of autoimmune disease, the `culprits' are autoreactive lymphocytes. Many autoimmune diseases are T-cell dominated, in others, such as systemic lupus erythematosus (SLE), both T and B cells are involved, and in some like myasthenia gravis (MG) the symptoms are caused exclusively by autoantibodies, although these are T-cell dependent. Therefore, we presently assume a pivotal role of T cells in most if not all autoimmune diseases. As a consequence, the conditioning has to be targeted at the T lymphocytes and to achieve lasting remissions or cures to the memory T lymphocytes.

The first and most important step in this treatment is, therefore, to design a conditioning regimen that maximally kills off T lymphocytes of the wrong kind in all parts of the body. For example, a lethal dose of total body irradiation (TBI) is effective in both the arthritis model and the EAE model, but partial body irradiation has only short-term effects, if any [3, 8].

Obviously, a lymphoablative regimen is also myeloablative so that rescue with a stem cell transplant is required. When allogeneic bone marrow is grafted, the lymphatic system is repopulated by donor type cells, which are derived from a healthy person and thus not likely to become autoreactive.

Equally beneficial long-term effects can only be expected with autologous bone marrow rescue when the number of autoreactive T lymphocytes in the graft is less than that required for a relapse. In other words, the use of autologous bone marrow or peripheral blood stem cells probably requires T-cell depletion. If there are no autoreactive T cells regrafted, and assuming that the recipient's autoreactive T cells have been eradicated, regeneration of the T-cell population has to derive from primitive precursors via the thymus. In the thymus, autoreactive cells are selected out, which presumedly prevents relapses. This process of proliferation and tolerization to self, also called `recapitulation of ontogenesis', is the most likely explanation for the success of autologous grafts.

With the clinical autoimmune diseases, the cause of relapse is not known. Is it due to an activation of residual clones of autoreactive lymphocytes by non-specific stimuli or to a re-exposure of the patient to the specific antigen? In the two animal models employed, adjuvant arthritis (AA) and EAE, we have recorded the incidence of both spontanous and induced relapses. The latter were provoked by reimmunization of the cured animals with the same dose of antigen as used for the initial induction of the disease. As will be discussed in the following sections, the two models differ greatly in relapse incidence. This may be due to the fact that in the buffalo (BUF) rat strain, AA is a chronic progressive disease [3], while EAE is a chronic remitting and relapsing condition [9].

Selection of animal models

Currently available data for autologous grafts are provided by experiments in rats with AA, EAE and experimental autoimmune myasthenia gravis (EAMG). There are ongoing disputes about whether the experimentally induced autoimmune diseases or the spontaneous ones are good models for the corresponding human diseases [6]. The defenders of the spontaneous models have not so far attempted to treat animals with fully developed disease with autologous bone marrow transplantation. They anticipate that such treatment would be futile, because they postulate that these diseases are caused by an inherited defect of the haemopoietic stem cells. This hypothesis is based upon the finding that in haemopoietic chimeras between the affected and normal strains, the development of the disease is dictated by the origin of the bone marrow graft [10].

However, abrogation of spontaneous autoimmune disease in mice has been reported following treatment with a sublethal dose of TBI and this suggests that more complete lympho-myeloablation and autologous stem cell grafts will do even better [11]. As the regeneration of the immune system after sublethal TBI derives from surviving precursors of the treated animal itself, the mechanism of regeneration is, in fact, similar to that following the transplantation of autologous stem cells.

Our experiments are based on the concept that the induced experimental autoimmune diseases are more realistic models of human disease, because both have genetic determinants of predisposition and require exposure to external factors for initiation of the disease [12]. Our choice of AA and EAE was made because these experimental diseases have many features in common with the various forms of inflammatory arthritis in man, and with multiple sclerosis. By selecting the BUF rat from among a number of susceptible strains and by manipulating the dose of antigens for induction, we could reproducibly induce chronic progressive polyarticular arthritis or relapsing encephalomyelitis.

The objective of our research was to investigate whether fully developed autoimmune disease could be favourably influenced by radical lympho-myeloablation and rescue with bone marrow transplantation. In our first experiments with AA, we used allogeneic bone marrow from a non-susceptible rat strain, expecting this to have a beneficial effect, which it had [3]. In fact, it induced complete and long-lasting remission in all animals. As a control, we rescued a group with syngeneic marrow from healthy BUF rats, expecting them to relapse soon. Surprisingly, this was not the case, and this led us to investigate autologous bone marrow, which proved to be similarly effective [1]. In the EAE model, we have compared the effects of allogeneic, syngeneic and autologous marrow grafts in great detail [2, 4, 5, 8]. The present discussion is largely restricted to the results obtained with autologous bone marrow grafts. Although the focus of this meeting is on rheumatoid diseases, we take the data from our work with EAE into consideration as well, as it is likely that there are common basic principles involved in the treatment of different autoimmune diseases.

Principles of treatment

It is generally accepted that both rheumatoid arthritis (RA) and MS are T-cell-dominated autoimmune diseases, and the two models AA and EAE share this feature. In both models, only the inflammatory stages can be successfully treated; scar tissue does not respond and the same is to be expected in patients. During the acute inflammatory stages, the lesions are brought about by activated T lymphocytes. We venture to compare this stage of autoimmune disease with newly diagnosed or relapsed leukaemia. In order to cure leukaemia, the tumour cell load of 1012 cells has to be reduced to a very small number, the exact value of which is unknown, but assumed to be of the order of <106 cells. An autologous bone marrow transplant may only be considered for leukaemic patients who are in complete remission, otherwise enough leukaemic cells will be returned with the graft to cause a rapid relapse. In the case of florid autoimmune disease, large numbers of activated T cells are to be expected in the bone marrow and even more so in the peripheral blood, so that, in the case of an autologous graft, T-cell depletion is mandatory.

The key question to be answered is thus: What proportion of (autoreactive) T cells of the patient have to be killed or permanently inactivated to achieve complete remission and to avoid subsequent relapse. The answer would also provide a reliable indication of the number of autologous T cells that can be safely reinfused.

The information from clinical experience is limited. A dose of TBI of 1.5 Gy delivered in 10 fractions over 5 weeks did not influence the course of progressive RA [13], pulsed therapy with medium high doses of cyclophosphamide (CP) was also inadequate [14]. Substantial responses were recently reported following treatment of patients with severe active resistant RA with 200 mg/kg CP, although complete remission was observed in only one of four patients (J. A. Snowden, J. C. Biggs et al., personal communication). Rescue was with unmanipulated peripheral blood stem cells.The follow-up of these cases is presently too short for proper evaluation (3–6 months).

A proportion of patients with severe aplastic anaemia—also considered to be an autoimmune disease—can be cured with a prolonged course of anti-lymphocytic globulin (ALG), but the non-responders require an allogeneic bone marrow transplant after conditioning with a total dose of 200 mg/kg CP divided over 4 days. It cannot be safely concluded that this regimen causes sufficient reduction of autoimmune T cells because the allogeneic graft may add to the effect by way of the graft-vs-host reaction. However, Brodsky et al. [15] reported complete and lasting remissions in seven out of 10 patients with severe aplasic anaemia following treatment with 45 mg/kg CP per day for 4 days without bone marrow transplantation. According to a previous report, the response rate to lower doses is much less. From these clinical data, it seems reasonable to conclude that patients with severe aplastic anaemia that is refractive to immunosuppressive treatments require 200 mg/kg of CP to achieve a high cure rate. The next question is what proportion of the T lymphocytes is killed by this dose. Unfortunately, direct determinations of the sensitivity of T cells to in vivo treatment with CP have not been published. The only source of information is from the animal models AA and EAE referred to earlier, in which we compared different conditioning agents and different doses. The experiments with EAMG by Pestronk et al. [16] may not apply so well because MG is an autoantibody-dominated disease and, furthermore, because only one dose was employed. However, as will be discussed later, this study provides interesting leads concerning the choice of conditioning agents.

Conditioning: experience gained from experiments with AA and EAE

The two models differ with regard to remission induction, and with regard to the incidence of both spontaneous and inducible relapses. In rats with AA, a chronic progressive disease, complete remissions can only be obtained with high-dose conditioning, lower doses induce partial remissions only [1]. EAE, a chronic relapsing disease, responds to lower doses with complete remissions, but much higher doses are required to minimize the eventual rate of relapse. Whereas spontanous relapses hardly ever occur in AA following a complete remission, the incidence is ~30% in EAE following treatment with either syngeneic or autologous bone marrow, and only 5% after transplantation of allogeneic bone marrow from a resistant donor. The explanation for the latter is that spontaneous relapses are mainly due to surviving activated T cells and their number is further reduced by the graft-vs-host reaction from the allograft. The inducible relapses are brought about by reimmunization at the time when spontaneous relapses no longer occur. The induced relapse rate is also a rare event in AA rats that have remitted completely, while in EAE its frequency is 85% in untreated controls, 72% after rescue with autologous marrow, 44% after syngeneic marrow and 12% after allogeneic marrow. Roughly half of these relapses are of the rapid type, suggesting that they are caused by the activation of sensitized or memory T cells.

The best curative results are obtained in AA with a TBI of 9 Gy and in EAE with 10 Gy TBI. This dose amounts to 3–4 log cell kill of lymphocytes (D0 =~1 Gy). If a similar reduction is assumed to be necessary in patients with severe autoimmune disease, and the total lymphatic cell mass of man is taken as 1012 , the residual number of lymphocytes after conditioning with 10 Gy TBI is 108 , of which ~30% are T cells. It follows that no more than 3x107 T cells should be reintroduced with the graft. Unmanipulated peripheral blood stem cells may contain as many as 2x1010 T lymphocytes.

CP was compared with TBI in rats with AA. At the highest tolerated dose of 160 mg/kg (divided over 2 days), CP was clearly less effective than 9 Gy TBI; the best guess at present is that this dose of CP is equivalent to 6 Gy TBI in T-cell killing. The combination of CP 120 mg/kg with 4 Gy TBI was as effective as 9 Gy TBI alone (D. W. van Bekkum, unpublished observations).

However, the difference between the curative effects of CP and TBI may be due to different specificities. Pestronk [16] provided evidence in rats with EAMG that CP is less effective than TBI in eliminating memory cells. Admittedly, these were predominantly B memory cells in his model, but there is some evidence from in vivo experiments in animals that a similar difference holds for T memory cells (Table 1Go). This notion is supported by the experience in allogeneic bone marrow transplantation where rejection of the graft is attributed to T-lymphocyte activity by the host. In non-sensitized patients with severe aplastic anaemia, a low rate of graft failures is seen after conditioning with high-dose CP alone, but in patients who are sensitized as a result of numerous blood transfusions, most allografts are rejected. A combination of high-dose TBI and CP (100 or 120 mg/kg) is needed in these patients to restore the take rate.


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TABLE 1.  Sensitivity of B and T lymphocytes to ionizing radiation and cyclophosphamide
 
Accordingly, a combination of high-dose CP with moderate-dose TBI (to inactivate memory cells) is recommended for conditioning in the treatment of severe autoimmune disease.

The use of a moderate dose of TBI with cyclophosphamide for conditioning of children with juvenile chronic arthritis

Few topics are more emotive than the use of ionizing radiation for the treatment of non-malignant diseases. The memories of Hiroshima and of the excess of leukaemia in patients with ankylosing spondylitis after therapeutic irradiation continue to haunt the medical profession.

The most notable long-term sequelae of the two principal agents, TBI and CP, are compared in Table 2Go with the current treatment of juvenile chronic arthritis (JCA).


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TABLE 2.  Expected adverse side-effects in children
 
The question of a safe dose of TBI for growing children arose >30 yr ago, when bone marrow transplantation was introduced for the treatment of leukaemia. Infant rhesus monkeys were irradiated with different doses of between 4 and 9 Gy, and subjected to skeletal measurements using a follow-up of up to 10 yr [20]. Doses above 7.5 Gy caused an inhibition of the growth of the long bones of both males and females, resulting in up to 20% shortening as compared to the controls. The inhibition was more pronounced when TBI was delivered before the age of 40 months, i.e. before the prepubertal growth spurt. Doses of 4–5 Gy did not influence skeletal growth significantly in the males and only marginally in the females.

The monkeys were also examined for the development of cataract. Among the high-dose TBI group, 17% of the monkeys examined at 3 yr after irradiation had developed cataracts; the incidence was 100% among those examined after 10–15 yr [21]. None of four animals irradiated with a dose of 4 Gy showed lens opacities when examined between 5 and 8 yr later. In contrast, 75% of 13 monkeys had developed cataract during the same period after exposure to doses from 7.5 to 9 Gy. These results strongly suggest that, for children, 4 Gy of TBI does not carry a significant risk of growth inhibition and cataract. This information has to be weighed against the well-established growth arrest caused by prolonged treatment with high doses of corticosteroids.

The risk of permanent sterilization after exposure of the gonads at an early age to a dose of 4 Gy or to CP cannot be assessed with much confidence because of the limited number of scattered clinical data.

As regards the long-term risk of malignancies, few iatrogenic effects have been better documented and analysed than the carcinogenic effects of radiation. The estimates of risk for the development of tumours after irradiation are based on life-long studies of the survivors of Hiroshima and Nagasaki, on numerous populations of patients who received radiotherapy or high doses of diagnostic radiation and on the epidemiology of workers exposed to significant amounts of various radiations in their environment. The risk of developing a malignancy is generally taken to be 2.6% per Gy for TBI and the risk of a fatal malignancy 1.3% [22]. These estimates are accepted world wide and constitute the basis of national laws regulating the permissible exposure of the populations to ionizing radiation. A dose of 4 Gy TBI is expected to entail a lifetime excess risk of developing a malignant tumour of 10% for a population of all ages. To account for the increased susceptibility of children, this value should be multiplied by two, yielding an excess lifetime risk of 20% (Table 2Go). Such a risk is definitely not negligible, but it has to be viewed in the light of the risks involved in continued immunosuppressive treatment to which JCA patients are currently exposed. In a recent survey of kidney transplant patients receiving immunosuppression with cyclosporin A, Dantal et al. [23] reported an incidence of 25% malignant tumours within 6 yr after transplantation.

The alternative to TBI in the conditioning regimen is to use even higher doses of CP or to combine CP with other drugs. Clinical as well as experimental data leave no doubt that drugs which act by damaging DNA are carcinogenic. However, the available data are by no means adequate to provide even remotely reliable risk estimates. Clinical studies are complicated by the use of combination chemotherapy, sequential treatments with different agents and the combination of chemotherapy with radiation therapy. In attempting to predict the risk of excess malignancies for patients with severe autoimmune disease, e.g. rheumatic diseases, additional uncertainties arise from the suggested increased cancer risk innate to the disease.

Cyclophosphamide, busulphan and melphalan are alkylating agents that cause damage to DNA similar to radiation, and there is no uncertainty that these agents are carcinogenic. In a large study with rats, Zurcher et al. [24] recorded the occurrence of malignancies following conditioning with different regimens consisting of CP 100 mg/kg, TBI 8.5 Gy and TBI plus CP, respectively, and rescue with autologous marrow grafts. The survival time of the rats recieving CP was shorter than that of those treated with TBI alone, which precluded a direct comparison. However, the CP groups exhibited an exeptionally high incidence of malignant nerve sheath tumours and correction for the reduced lifespan suggests that CP carries at least the same risk for secondary tumours as TBI.

Conclusions

The present introduction of lympho-myeloablation and rescue with autologous stem cells, for the treatment of patients with severe progressive autoimmune disease, is justified because of the relatively low transplantation-related mortality of <5% as observed in leukaemic and aplastic patients. This risk seems to outweigh the mortality risk associated with conventional treatments [25] and the deterioration of the quality of life that goes with refractory disease. Of course, it will be important to minimize the long-term risk of developing a malignancy by selecting, if possible, a less risky but equally effective conditioning regimen. At the present stage of the art, however, priority should be given to establishing that these diseases can be cured by employing a conditioning regimen that has been shown to offer the best chances of a permanent cure in the animal models. In the meantime, further research with animals into the efficacy of non-carcinogenic lymphoablative agents should be encouraged.

References

  1.  Knaan-Shanzer S, Houben P, Kinwel-Bohre EPM, van Bekkum DW. Remission induction of adjuvant arthritis in rats by total body irradiation and autologous bone marrow transplantation. Bone Marrrow Transplant 1991;8:333–8.[ISI][Medline]
  2.  Van Gelder M, van Bekkum DW. Effective treatment of relapsing experimental autoimmune encephalomyelitis with pseudoautologous bone marrow transplantation. Bone Marrow Transplant 1996;18:1029–34.[ISI][Medline]
  3.  Van Bekkum DW, Bohre EPM, Houben PFJ, Knaan-Shanzer S. Regression of adjuvant-induced arthritis in rats following bone marrow transplantation. Proc Natl Acad Sci USA 1989;86:10090–4.[Abstract]
  4.  Van Gelder M, van Bekkum DW. Treatment of relapsing experimental autoimmune encephalomyelitis in rats with allogeneic bone marrow transplantation from a resistant strain. Bone Marrow Transplant 1995;16:343–51.[ISI][Medline]
  5.  Van Gelder M, Mulder AH, van Bekkum DW. Treatment of relapsing experimental autoimmune encephalomyelitis with largely MHC-matched allogeneic bone marrow transplantation. Transplantation 1996;62:810–8.[ISI][Medline]
  6.  Van Bekkum DW. Review: BMT in experimental autoimmune diseases. Bone Marrow Transplant 1993;11: 183–7.[ISI][Medline]
  7.  Marmont AM, van Bekkum DW. Stem cell transplantation for severe autoimmune diseases: new proposals but still unanswered questions. Bone Marrow Transplant 1995;16:497–8.[ISI][Medline]
  8.  Van Gelder M, Kinwel-Bohré EPM, van Bekkum DW. Treatment of experimental allergic encephalomyelitis in rats with total body irradiation and syngeneic BMT. Bone Marrow Transplant 1993;11:233–41.[ISI][Medline]
  9.  Van Gelder M. Bone marrow transplantation for treatment of experimental autoimmune encephalomyelitis in rats. Prospects for therapy of severe multiple sclerosis. Thesis, Leiden University, 1995.
  10. Ikehara S, Yasumizu R, Inaba M et al. Long-term observations of autoimmune-prone mice treated for autoimmune disease by allogeneic bone marrow tranplantation. Immunology 1989;86:3306–10.
  11. Loor F, Jachez B, Montecino-Rodriguez E et al. Radiation therapy of spontaneous autoimmunity: a review of mouse models. Int J Radiat Biol 1988;53:119–36.[ISI]
  12. Van Gelder M, Kinwel-Bohré EPM, Mulder AH, Van Bekkum DW. Both bone marrow and non-bone marrow-associated factors determine susceptibility to experimental autoimmune encephalomyelitis of BUF and WAG rats. Cell Immunol 1996;168:39–48.[ISI][Medline]
  13. Crook PR, Lucraft HH, Evans RG, Griffiths ID. Lack of effect of total body irradiation in rheumatoid arthritis. Br J Rheumatol 1986;25:384–7.[ISI][Medline]
  14. Euler HH, Schroeder JO, Harten P, Zeuner RA, Gutschmidt HJ. Treatment-free remission in severe systemic lupus erythematosus following synchronization of plasmapheresis with subsequent pulse cyclophosphamide. Arthritis Rheum 1994;37:1784–94.[ISI][Medline]
  15. Brodsky RA, Sensenbrenner LL, Jones RJ. Complete remission in severe aplastic anemia after high-dose cyclophosphamide without bone marrow transplantation. Blood 1996;87:491–4.[Abstract/Free Full Text]
  16. Pestronk A, Drachman DB, Teoh R, Adams RN. Combined short-term immunotherapy for experimental autoimmune myasthenia gravis. Ann Neurol 1983;14: 235–41.[ISI][Medline]
  17. Orme IM. Active and memory immunity to Lysteria monocytogenes infection in mice is mediated by phenotypically distinct T cell populations. Immunology 1989; 68:93–5.[ISI][Medline]
  18. Rouse BT, Hartley D, Doherty PC. Consequences of exposure to ionizing radiation for effector T cell function in vivo. Vir Immunol 1989;2:69–78.[Medline]
  19. Uzawa A, Suzuki G, Nakata Y et al. Radiosensitivity of CD45RO+ memory and CD45RO– naive T cells in culture. Radiat Res 1994;137:25–33.[ISI][Medline]
  20. Solleveld P, van Bekkum DW. The effect of whole body irradiation on skeletal growth in Rhesus monkeys. Radiology 1979;130:789–91.[Abstract]
  21. Solleveld P, Peperkamp E, van Bekkum DW. Incidence of cataracts in Rhesus monkeys treated with whole body irradiation. Radiology 1979;133:227–9.[Abstract]
  22. Recommendations of the International Commission on Radiation Protection, ICRP-26. Annals of the ICRP. Vol 1, No. 3. Oxford: Pergamon Press, 1977.
  23. Dantal J, Hourmant M, Cantarovich D et al. Effect of long-term immunosuppression in kidney-graft recipients on cancer incidence: randomized comparison of two cyclosporin regimens. Lancet 1998;351:623–8.[ISI][Medline]
  24. Zurcher C, Varekamp AE, Solleveld HA et al. Late effects of cyclophosphamide and total body irradiation as a conditioning regimen for bone marrow transplantation in rats (a preliminary report). Int J Radiat Biol 1987;51: 1059–68.[ISI]
  25. Pincus T. The case for early intervention in rheumatoid arthritis. J Autoimmun 1992;5:209–26.[ISI][Medline]
  26. Radis CD, Kahl L, Baker GL et al. Effects of cyclophosphamide on the development of malignancy and on long term survival of patients with rheumatoid arthritis. A 20 year follow-up study. Arthritis Rheum 1995;38:1120–7.[ISI][Medline]
Accepted 15 March 1999