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High dose immunosuppressive therapy with subsequent autologous stem cell transplantation (ASCT) has proved effective for treatment resistant autoimmune diseases.1 However, in a number of patients, relapses of autoimmune disease after initial improvement or even remission occur. Until recently, it was impossible to differentiate recent thymic emigrants from residual or peripherally expanded T cells. Douek and coworkers2 and later our group3 described a method for detection of T cell receptor excision circles, stable DNA episomes which are formed during T cell receptor rearrangement in the thymus which are not replicated during mitosis, but are diluted during cell divisions.4 This study aimed at investigating whether these flares are associated with de novo T cell development.
Peripheral blood mononuclear cells were collected from six patients, mean age 32.5 years, range 24–42 (four with systemic sclerosis (SSc), one with Wegener’s granulomatosis (WG), one with mutilating psoriatic arthritis (PsA)), after high dose immunosuppression with cyclophosphamide 50 mg/kg body weight plus anti-thymocyte globulin 20 mg/kg on days 1–4, with subsequent retransfusion of 4.2×106 (median) CD34+ selected (by CliniMacs device) cells/kg body weight. Blood collection was performed at week 2 and then, at bimonthly intervals, genomic DNA was extracted using the QIAmp DNA Blood Mini Kit. DNA was stored at −80°C until analysis. T cell receptor rearrangement excision circles (TRECs) were measured by real time polymerase chain reaction using the light cycler (Roche Diagnostics Corporation, Idaho, USA). The study was approved by the local ethics committee and patients’ informed consent obtained.
All patients improved considerably (all patients with SSc) or went into remission (PsA, WG) after ASCT. In all patients, previous immunosuppressive therapy was discontinued, except in the patient with WG, who still received ciclosporin A for her kidney transplant. Two patients relapsed after 7, and 19 months, respectively (one SSc, one PsA). CD3+/CD4+ T cells were recovered after 6–14 months (median 12), and CD3+/CD8+ T cells a maximum of 3 months after ASCT in all patients. Autoantibodies disappeared in four of five patients after a median of 6 months and their recurrence or disappearance later did not correlate with relapse or remission. TRECs could not be detected in the patients shortly after ASCT (week 2), but recurred 9–26 months (median 16 months) after ASCT. In the two patients who experienced relapses, TRECs became detectable at the time when the first symptoms of relapse were noticed (SSc, patient No 3: relapse after 7 months, TRECs after 9 months; PsA, patient no 4: relapse after 19, TRECs after 21 months—the blood collection did not always coincide exactly with the diagnosis of relapse, which explains the 2 months’ difference between diagnosis and first detection of TRECs) (fig 1⇓, table 1⇓).
Normally, immune reconstitution and TREC recurrence is considerably quicker in ASCT than in allogeneic SCT.5 TREC recurrence in the allogeneic setting is known to be delayed by clinical events such as graft versus host disease,6 whereas in ASCT CD34+ selection of the graft was associated with increased thymic output (“rebound phenomenon”).2 In the present study, T cell ontogenesis was delayed even in comparison with allogeneic SCT for haematological diseases (TREC recurrence: 6 months6). Premature immunosenescence may be one of the reasons for this delay in T cell ontogenesis after ASCT in autoimmune diseases.7,8
De novo T cell ontogenesis in the thymus may be a critical event inducing relapses of the autoimmune disease, which may not be caused by peripheral expansion of memory T cells which survived the conditioning treatment, but by newly developed T cells of thymic origin. Vice versa, as in four patients TRECs recurred without relapse of the autoimmune disease, one might also argue that the occurrence of TRECs is not causally associated with relapse. Further studies of the immune reconstitution, correlating the recurrence of TRECs with T cell subsets, T cell receptor diversity, and autoantibody production in patients with autoimmune diseases after ASCT will improve our understanding of the pathogenesis of these diseases and help in the development of new therapeutic concepts.