Selective engraftment of donor CD4⁺25^high FOXP3-positive T cells in IPEX syndrome after nonmyeloablative hematopoietic stem cell transplantation

To the editor:

Mutations of FoxP3 result in the disturbance of FoxP3 expression and lack of functional CD4⁺CD25^high regulatory T cells in humans, causing immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. The only curative approach for IPEX syndrome, which is fatal within the first year of life in many cases, is allogeneic hematopoietic stem cell transplantation (HSCT). We monitored lineage-specific chimerism in a FoxP3-deficient patient after nonmyeloablative HSCT and graft rejection over 6 years. The patient lost donor chimerism in T cells, B cells, NK cells, monocytes, and granulocytes 1 year after transplantation, but remained clinically healthy without immunosuppression. To elucidate the immunologic basis of his continuing remission, we performed detailed lymphocyte subset analyses and detected FoxP3-expressing CD4⁺CD25^high T cells, comprising 1% to 2% of the CD4⁺ T cells (∼ 10/μL), which were more than 90% donor-derived. The patient is disease-free and shows no signs of autoimmunity, suggesting that stable, selective engraftment of regulatory T cells is possible and associated with cure from IPEX syndrome.

HSCT may be a curative approach for IPEX syndrome.^1,–3  However, the required levels of donor chimerism and conditioning intensity are unknown. Myeloablative conditioning is associated with substantial transplantation-related mortality,^2,,–5  whereas nonmyeloablative conditioning carries an increased risk of rejection because of dysregulated effector T-cell function.⁶ 

Here we report a boy with IPEX syndrome who underwent peripheral blood HSCT at the age of 11 months from an unrelated 10/10-matched donor (2 × 10⁷/kg CD34⁺, 10⁹/kg CD3⁺ cells) after nonmyeloablative conditioning according to the local protocol (fludarabine 6 × 30 mg/m²; melphalan 140 mg/m²; alemtuzumab 5 × 0.2 mg/kg). The post-HSCT course was uncomplicated with complete engraftment on day +12. All IPEX-linked symptoms resolved until day +30. Acute graft-versus-host disease (skin, II°) was treated with steroids until day +56; cyclosporin A was tapered 1 year after transplantation. At this time, in vitro responses to vaccine antigens and phytohemagglutinin were normal. Six years posttransplantation the patient remains insulin-dependent due to irreversible islet cell damage but has no active autoimmunity. The clinically healthy boy shows no increased frequency or severity of infections (Table S1; available on the Blood website; see the Supplemental Materials link at the top of the online article). Chimerism was determined in peripheral blood cell subsets regularly (described elsewhere⁶ ). Approval was obtained from the St Anna Children's Hospital (Vienna, Austria) Institutional Review Board for these studies. Informed consent was obtained in accordance with the Declaration of Helsinki.

After 100% donor cell engraftment, there was a continuous decline throughout the first year to an overall and subset-specific donor chimerism of 5% to 10% (± 6%; Figure 1A) without any clinical symptoms of relapse. Five-, 5.5-, and 6-year posttransplantation chimerism was determined additionally in the CD4⁺CD25^high, CD4⁺CD25^med, and CD4⁺CD25^neg T-cell subsets (Figure 1B). In the CD4⁺CD25^high subset, comprising 1% to 2% of CD4⁺ cells (∼ 10 cells/μL), donor chimerism was 53% to 91% (± 6%) at separate occasions within 6 months, whereas all other CD4⁺ T-cell subsets were of recipient origin (Figure 1C). FoxP3 protein expression of fresh CD4⁺ patient cells was restricted to the CD25^high (predominantly donor-derived) CD127^low compartment (Figure S1), in line with the inability of recipient-type cells to express FoxP3 due to a translational start-site mutation. FoxP3mRNA was detectable in full blood and CD4⁺CD25^med and CD4⁺CD25^high cells (not shown; for methods, see Seidel et al⁷ ).

In this patient, FoxP3 expression and levels of CD4⁺CD25^highCD127^lowFoxP3-expressing cells are detectable at a low-normal range after HSCT. That the patient has no signs of autoimmunity 6 years after HSCT suggests that the donor-derived CD4⁺CD25^highCD127^lowFoxP3-expressing T cells have regulatory function in vivo, although we cannot exclude that the highly immunosuppressive conditioning regimen for HSCT per se caused persistent remission. Despite lacking in vitro analyses for regulatory T (T_reg) function, which were not feasible because of the low cell frequency in this patient, we speculate that selective long-term engraftment of active donor T_reg cells occurred and has been responsible for the continuing disease-free state of the patient.

Although these descriptive observations might lack mechanistic insight, they suggest that (1) severe IPEX syndrome may be cured with nonmyeloablative HSCT; (2) long-term remission is achievable with a minimal donor cell population restricted to approximately 10/μL CD4⁺CD25⁺CD127^lowFoxP3⁺ cells; and (3), because remission of immune dysregulation is observed after HSCT without FoxP3 reconstitution in other lymphocyte subsets, T_reg-independent FoxP3 function within T-effector cells might be less relevant than hypothesized in vivo.^4,8,–10