The present study was conducted to evaluate the efficacy of autologous SCT in treatment-refractory autoimmune disease. After a median follow up of 14 months (range10-21 months) four patients with polychondritis or SLE are in clinical remission. Following the immunoablative regimen, including cyclophosphamide, ATG and steroids, the disease-specific titres of autoantibodies became negative in patients 2-4, who had SLE. No symptoms of recurrent autoimmune reactivity have been detected in these patients. Obviously, the rigorous regimen of immunoablation, consisting of high-dose cyclophosphamide in combination with ATG, was successful in achieving rather complete aplasia. This combination increased the efficacy of immunoablation in comparison with that achieved by other studies [12,13], by grossly reducing the number of autoreactive immune cells. Plasma cells can be found resting in bone marrow for more than 90 days , and apparently were eliminated in the patients with SLE in the present study under treatment with ATG, presumably by the recognition of specific surface antigens by immunoglobulins of the ATG.
Side effects during immunoablation may be severe, and flares of the autoimmune disease are particularly detrimental to the patient's condition. Comparable with a previously reported observation of the induction of flares by G-CSF in a patient with rheumatoid arthritis , in the present trial flares of SLE were diagnosed in patient 2 who was in aplasia after the immunoablative regimen (Table 1). Flares may be attributed to `cytokine-primed' clinical situations in which G-CSF is released from macrophages and residual lymphocytes during bone marrow aplasia. Flares can be controlled by high-dose steroids. After gradual reduction in the dose of steroids and without further treatment of patients 1-4, recurrence of the flares was not observed during the follow up of more than 12 months. The flares that occurred in patient 2, in aplasia and during septicaemia, may represent a `burn-out' of autoreactivity in terms of an exhaustion of mediators participating in autoimmunity. In order to avoid the induction of flares, G-CSF was not applied after termination of the immunoablative regimen and during the phase of haematological reconstitution.
In a previous study in five patients with haematological malignancies or solid tumours and concomitant refractory autoimmune disease, who were treated by autologous SCT , the disease persisted or relapsed within 3 months. In that investigation the transplantations were performed without in vivo/ex vivo depletion of T cells, emphasizing the importance of effective immunoablation. Successful immunoablation in vivo, combined with less intensive purging of the transplant (2-3 log depletion of CD3+ cells) was reported to halt disease progression in patients with multiple sclerosis, rheumatoid arthritis or SLE . The further reduction in CD3+ cells, as applied in the present study, but not in the previous investigation , did not lead to a higher incidence of severe infections. Thus, it is tempting to speculate that in future studies even lower amounts than 1×104 CD3+ cells/kg body weight, as applied in the present study, may be tolerated.
In the present trial, preparations with greatly reduced numbers of CD3+cells were used for autologous SCT. By application of the recently developed CliniMACS™ device , a highly efficient technology was introduced for the selection of haematopoietic stem cells. High-gradient magnetic cell sorting was able to purify CD34+ cells effectively from G-CSF-mobilized peripheral blood, resulting in 4.5-5 log depletions of CD3+ T cells, thus minimizing the risk of retransplantation of autoreactive T cells. In fact, after autologous SCT no relapse of autoreactivity was observed in the patients with polychondritis or those with SLE, even though the absolute counts of CD4+, CD8+ and all other leucocytes had reached pretreatment levels. On the other hand, the large-scale depletion of T cells from the transplants did not provoke life-threatening infections before and during immunological reconstitution. The relevance of the in vivo/ex vivo depletion procedure used in our investigation may be confirmed by controlled studies.
Stem-cell support is essential in shortening the duration of aplasia and in the reconstitution of haemopoiesis after immunoablation by a regimen of cyclophosphamide and ATG. Any immunoablative treatment without subsequent autologous SCT is associated with the risk of severe infections during neutropenia and thrombopenic bleeding. This appears to be in contrast to the results of a recent approach with high-dose cyclophosphamide followed by G-CSF but without autologous SCT , in which two patients with SLE had follow-up periods of 12 or 14 months with complete or partial remission. The results appear to be due to the G-CSF-induced priming after high-dose cyclophosphamide. In patients with aplastic anaemia treated with allogeneic bone marrow transplantation the advantage of a combined immunosuppression with cyclophosphamide and ATG was emphasized by a significant reduction in graft rejections . The present data on patients with polychondritis or SLE are in accord with the recent results achieved by high-stringency immunoablation followed by autologous SCT in a panel of 10 patients suffering from multiple sclerosis, rheumatoid arthritis or SLE . With follow-up periods of 6 and 12 months in that study the two patients with SLE were in remission at the time of publication.
In the present investigation, a rapid decrease in levels of pathological autoantibodies to normal values was observed in the SLE patients responding to autologous SCT. The early phase in the reconstitution of the immune system was marked by rapid recovery of granulocytes and platelets in all patients. During the second phase of reconstitution CD4+ cells, exclusively of the antigen-experienced memory/effector type, were observed 2-5 months after autologous SCT (CD45RO+, CD45RA-; Table 2). Similar kinetics of reconstitution were described in patients after allogeneic bone marrow transplantation for haematological malignancies . In the patients we studied, the activated T-helper cells may reflect the clonal expansion of persisting cells after the preparative regimen, which might have been stimulated by minor infections during reconstitution .
During the second phase of reconstitution, patient 2 in the present study suffered an episode of varizella-zoster infection. After in vitro incubation of mononuclear cells of that patient with varizella-zoster virus antigen, the secretion of IFN-γ by a subpopulation of T cells was observed (data not shown). This may exclude a persisting general deficiency of the immune system due to the aggressive immunoablation. Deficiency of the immune system was considered as a basic reason for self-tolerance in autoimmune disease after immunosuppression followed by autologous SCT .
The two patients with SSc who were evaluable for follow up showed no clinical and serological responses at 6 or 13 months after autologous SCT. We presume that insufficient immunoablation may have a role in the treatment failures. This is supported by the persistance of Scl-70 autoantibody, and by the early recovery of lymphocytes (patient 5) and natural killer cell reconstitution (patient 6). In SSc no correlation between the activity of the disease and the presence of autoantibodies has been shown, although serum Scl-70 is associated with poor prognosis with regard to pulmonary or cardiac involvement . Patients 5 and 6 were still positive for Scl-70 after autologous SCT, suggesting a need for intensification of treatment. However, the reason for insufficient immunoablation in the SSc patients is not clear, and may be related to the underlying pathophysiology that is different from that in SLE. SSc appears to be less responsive, at least at the advanced stage of tissue destruction, due to fibrotic processes that are not present in SLE.
The present results are in contrast to those of a previous report of a significant decline in ANAs in a SSc patient within 6 months after autologous SCT . The stable disease in patient 5 in the present study was accompanied by a pregnancy during the follow-up period. Pregnancy in patients with autoimmune disease has been postulated to be a reason for stability in SSc and multiple sclerosis [21,22]. Patient 7 of the present trial, who had a brief history of SSc, died 2 days after autologous SCT from cardiac failure due to massive pulmonary fibrosis. After 2 g/m2 cyclophosphamide was administered for stem-cell mobilization, she developed clinical and serological progress until autologous SCT. In hindsight, the advanced stage of pulmonary fibrosis was not foreseeable, and is to be considered the cause for the treatment-associated mortality.
In conclusion, the present study demonstrates that the induction of immune tolerance for disease-related antigens is feasible and can be achieved with immunoablation and subsequent autologous SCT in the case of refractory polychondritis and SLE. Effective ex vivo depletion of CD34-cells can be achieved with state-of-the-art technologies, and appears to be essential for sustained tolerance after immunological reconstitution. These results show unambiguously that a `reset' of the immune system was brought about, which was able to deal successfully with pathogens. The treatment consisted of one admission into hospital for stem-cell mobilization, and another one for immunoablation and autologous SCT, with median durations of hospitalization of 20 and 34 days, respectively. The high costs of the complex and intensive therapy performed in the present study may be acceptable when compared with those of disease-related long-term hospitalization and invalidity.