PEDIATRICS Vol. 121 No. 4 April 2008, pp. e998-e1002 (doi:10.1542/peds.2007-1863)
EXPERIENCE & REASON |
Immune Reconstitution and Recovery of FOXP3 (Forkhead Box P3)-Expressing T Cells After Transplantation for IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked) Syndrome
a Institute of Child Health
b Great Ormond Street Hospital, London, United Kingdom
c Warwick Medical School, Coventry, United Kingdom
d Lucia Perroni, Human Genetics, Galliera Hospital, Genova, Italy
ABSTRACT
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome is a rare X-linked disorder that usually presents in early childhood with immune enteropathy, diabetes mellitus, and other autoimmune complications. The disease is caused by mutations in the forkhead box P3 gene, a transcription factor that is essential for the development and function of regulatory T cells. This population of cells plays an essential role in controlling immune responses and preventing autoimmunity. Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome is often initially treated with immunosuppressive drugs, but only allogeneic hematopoietic stem cell transplantation has offered the possibility of cure. We recently performed an unrelated donor transplant in a child with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome by using a reduced-intensity conditioning regimen. This transplant provided a rare opportunity to gain valuable insight into the regeneration of the immune system after transplantation. Clinical recovery was associated with the emergence of regulatory T cell populations, the majority of which expressed memory phenotype markers and raised important questions about the origin and longevity of the FOXP3+ regulatory T cell pool.
Key Words: IPEX syndrome • immune dysregulation • polyendocrinopathy • enteropathy • X-linked syndrome • regulatory T cells • Tregs • forkhead box P3 • FOXP3 • reduced-intensity conditioning
Abbreviations: IPEX, immune dysregulation, polyendocrinopathy, enteropathy, X-linked • FOXP3, forkhead box P3 • Tregs, regulatory T cells • HSCT, hematopoietic stem cell transplantation • PCR, polymerase chain reaction • GvHD, graft-versus-host disease • PHA, phytohemagglutinin • Ig, immunoglobulin • TREC, T-cell receptor excision circle
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare but often life-threatening disorder, presenting in the first few months of life with diarrhea and failure to thrive. Additional manifestations include dermatitis, insulin-dependent diabetes mellitus, thyroid disease, and hemolysis; however, the most serious and consistent finding is that of autoimmune enteropathy.1,2 The molecular basis of the condition maps to the forkhead box P3 (FOXP3) gene on the X chromosome. FOXP3 encodes a transcription factor essential for the generation and function of regulatory T cells (Tregs).3 These cells play a central role in controlling immune responses, maintaining tolerance, and preventing autoimmunity, and have been intensely studied in recent years.
Hematopoietic stem cell transplantation (HSCT) has been undertaken in a handful of children with mutation-proven IPEX syndrome, but the outcomes have yielded mixed results. Baud et al4 reported matched-sibling donor transplantation in a child with IPEX syndrome by using myeloablative conditioning (busulphan, 20 mg/kg; cyclophosphamide, 200 mg/kg; rabbit antithymocyte globulin, 50 mg/kg). The conditioning procedure itself induced the rapid reversal of insulin dependence, and, within a few weeks, there was resolution of enteropathy and notable clinical improvement. Stable mixed chimerism (up to 30% in T cells) was achieved but, unfortunately, 29 months after the procedure, the child unexpectedly developed a progressive and fatal hemophagocytic syndrome. A number of groups have now reported the beneficial effects of transplantation in reducing symptoms, but the procedures have been associated with an unusually high mortality rate.1 One regimen aimed at reducing transplant-related mortality is that of reduced-intensity conditioning, and we have previously reported the benefits of nonmyeloablative conditioning in children who have undergone transplantation for primary immunodeficiency.5 The use of such conditioning regimens in IPEX syndrome was recently reported6, and it has also been used in 1 child who underwent umbilical cord blood stem grafting for the condition.7 Here, we describe a child with a novel FOXP3 mutation who underwent successful allogeneic HSCT by using a modified version of our reduced-intensity conditioning regimen. We have tracked his immune reconstitution in detail, including the regeneration of FOXP3-expressing Tregs over a 30-month period. A variety of cell surface markers have been used to characterize and track Tregs and, recently, it has become possible to detect FOXP3 expression in T cells by flow cytometry using intracellular staining techniques. FOXP3+ Tregs can be identified within the CD4 T cell compartment as expressing high levels of CD25 (the IL-2 receptor 
-chain) and low levels of CD127 (The IL-7 receptor -chain). We have undertaken serial flow cytometry by using these parameters and quantitative polymerase chain reaction (PCR) for FOXP3 levels, and have tracked this patient's immune recovery for nearly 3 years.
CASE REPORT
A white male infant of nonconsanguinous parents was born prematurely at 29 weeks'gestation after placental abruption. At 4 to 5 months of age, he was checked for chronic diarrhea and failure to thrive, and was diagnosed as having multiple food allergies (with elevated serum immunoglobulin E [IgE] and positive skin-prick tests for dairy products and wheat). Subsequent investigations included duodenal biopsy, which showed crypt hyperplastic villous atrophy, with a minimal increase of intraepithelial lymphocytes. Clinical suspicions of IPEX syndrome were confirmed on mutation analysis of the FOXP3 gene. A novel mutation (T380I) in exon 10 of the forkhead domain was detected (and was not found on polymorphic screening of 200 X chromosomes).
At the time, immunologic studies revealed normal lymphocyte subset profiles, including the presence of CD4+CD25+ T cells, intact T cell responses to mitogens, and normal immunoglobulin levels. It is interesting to note that we were able to detect the presence of the mutated FOXP3 protein by using monoclonal antibody directed against the unaffected upstream repressor domain. Similar findings have been recently revealed in other IPEX syndrome patients.8 Despite the use of exclusion diets, nutritional supplements, and extended immunosuppression with prednisolone, azathioprine, and tacrolimus, symptoms progressed, which required extended hospital stays and a dependence on total nutrition provided by the parent.
At the age of 5 months, we undertook matched unrelated donor peripheral blood stem cell transplantation by using nonmyeloablative conditioning (Table 1) . A combination of fludarabine at 150 mg/m2, cyclophosphamide at 1200 mg/m2, alemtuzumab at 0.6 mg/kg, and anti-CD45 monoclonal antibodies (YTH24/54 at 1600 µg/kg, which was kindly provided by Dr Geoff Hale, Therapeutic Antibody Centre, Oxford, United Kingdom) was used because of his continuing severe enteropathy. The graft contained a CD34 stem cell dose of 2.7 x 107/kg and 8.0 x 108/kg of CD3+ T cells. Preexisting therapy with prednisolone (0.25 mg/kg per day) was continued, although tacrolimus was substituted for ciclosporin and mycophenolate mofetil as a prophylaxis against graft-versus-host disease (GvHD) in the period after transplantation. He received granulocyte colony-stimulating factor at 5 µg/kg per day (until neutrophil recovery was above 1 x 109/L) and antipathogen prophylaxis with aciclovir, itraconazole, cotrimoxazole, and penicillin. Blood and urine samples were screened by using DNA PCR to detect cytomegalovirus adenovirus and Epstein-Barr virus. Engraftment studies were performed by using a variable number of tandem repeat analyses as described previously.5
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Immune reconstitution was characterized by using flow cytometry analysis of peripheral blood mononuclear cells (using fluorescein isothiocyanate-phycoerythrin–labeled antibodies against CD3, CD4, CD16/CD56, and CD19) and measuring IgG, IgA, and IgM levels. In addition, the phytohemagglutinin >(PHA) stimulation index was measured for T cell mitogen responses (defined as the ratio of baseline-maximal stimulated levels of 3H-thymidine uptake in a 3-day culture of peripheral blood mononuclear cells stimulated with a range of concentrations of PHA). T-cell receptor excision circle (TREC) analysis was performed to quantify thymic output by using CD4+ and CD8+ cells isolated by bead selection. TREC levels were analyzed by real-time quantitative PCR assay as described previously.5 Tregs were quantified by using flow cytometry for CD4+CD25+FOXP3+ cells and quantitative PCR for FOXP3 expression, which was normalized against glyceraldehyde-3-phosphate dehydrogenase expression as described previously.9
The transplant procedure was well tolerated, and stable full-donor chimerism was achieved. Grade II/III GvHD of the skin and gut required additional immunosuppression, including methylprednisolone and a course of therapy with monoclonal antibodies against CD25 (dacluzimab at 25 mg per week) and tumor necrosis factor (infliximab at 200 mg per week). The kinetics of immune reconstitution were slow compared with the usual rate after reduced-intensity transplantation, which reflects the extended use of immunosuppression to control GvHD. By 18 months there was evidence of emerging cellular immune reconstitution (Fig 1) and the presence of cells with a regulatory CD4+CD25hi+FOXP3+ phenotype. These populations continued to increase during the subsequent 12 months and, as GvHD resolved, it became possible to reduce immunosuppression. At the same time, clinical and histologic resolution of small bowel pathology allowed for a return to enteral nutrition.
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Evidence of de novo T cell production was provided by high levels of TREC (at >70000 per 106 µL of CD4+ cells) and the presence of circulating naive T cells (CD4+CD45RA+CD27+). Interestingly, FOXP3 expression was predominant in memory phenotype cells, as highlighted in Fig 2A, where memory and naive populations are stained for expression of FOXP3. Coexpression of CD127 (IL-7
receptor, IL-7R) has recently been shown to be reduced in FOXP3-expressing cells,10 and provides a useful alternative comarker for Tregs. The bias of FOXP3 expression in memory cells was confirmed at the molecular level by quantitative PCR evaluation of FOXP3 messenger RNA in memory and naive cells sorted by flow cytometry (Fig 2B). Overall, immune reconstitution has been excellent, and successful revaccination with childhood immunizations was recently confirmed by the detection of protective anti-Haemophilus influenzae type b and tetanus responses.
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Before transplantation, the patient was hospitalized, dependent on parental nutrition, and had a poor quality of life. At 30 months after transplantation, the patient was well and at home, tolerated supplemented enteral nutrition, and engaged in normal daily activities.
DISCUSSION
IPEX syndrome provides a fascinating insight into how Tregs are essential for health from a young age. A diverse range of immunosuppressive therapeutic strategies have been attempted in children with IPEX syndrome but have been of limited benefit. Immune modulation by using extended courses of corticosteroids and T-cell suppressants (such as ciclosporin or tacrolimus) have been successful in inducing remission, but longer-term effectiveness has varied results. Other strategies have included strict exclusion diets, pancreatic enzyme therapy, the administration of fresh frozen plasma, intravenous immunoglobulin, infliximab, and retuximab (anti-CD20 monoclonal antibody). Procedures designed to reconstitute T cell immunity by using HLA-matched allogeneic HSCT have offered the most promising avenue of corrective therapy, but questions have remained about the longer-term efficacy of this approach.
We used 2 approaches to track the recovery of Tregs populations, based on flow cytometry for FOXP3 after intracellular staining and quantitative PCR for FOXP3 mRNA. It is of interest to note that by using both techniques, we found that FOXP3 expression was highest in memory phenotype CD4+ cells. This result raises interesting questions regarding the origins of Tregs after transplant for IPEX syndrome. After successful transplantation, the Tregs pool is assumed to be derived from 2 main sources: donor-derived mature T cells carried by the graft and de novo T cells emerging from the host thymus after stem cell engraftment and migration. The emergence of significant numbers of thymic emigrants can take several months, and these populations can be considered as naive T cells. The relative proportion of Tregs derived from naive T cells and memory T cells is not known. In patients who are undergoing transplantation for hematologic malignancy, it has been suggested that FOXP3-expressing Tregs are largely contained in populations of recent thymic emigrants (naive cells), and that recovery of these cells is associated with control of GvHD.11 Sophisticated studies in healthy adults have suggested that the memory pool plays an important role in replenishing the regulatory pool in normal healthy individual,12 but the contribution that these cells make to the regulatory pool after transplantation is not known. After transplantation, our patient has consistently been found to have full-donor chimerism with no evidence of autologous T cell recovery. Treatment for GvHD delayed his immune reconstitution, but he now has increasing numbers of naive T cells and good evidence of active thymopoiesis, with high TREC levels. Of interest is the fact that the majority of FOXP3-expressing cells lack CD45RA expression, suggesting they are long-lived memory T cells. Presumably mature donor-derived T cells that were adoptively transferred at the time of grafting, have expanded in vivo, and reconstituted the Treg pool sufficiently to achieve regulatory control, reverse gut autoimmunity, and control GvHD.
In the scurfy mouse model of IPEX syndrome, there has been controversy as to whether FoxP3 gene expression is restricted to thymocytes, or if expression is also required in host thymus stroma for effective thymopoiesis.13 The issue is highly relevant to children with IPEX syndrome, because continued aberrant expression of FOXP3 in host thymic stromal tissue could serve as a block to the generation of naive cells and, thus, the generation of new Treg populations from donor T-cell progenitors might be compromised. We have demonstrated highly effective thymopoiesis after transplantation in IPEX syndrome and the regeneration of a competent immune system with documented vaccine responses. However, our findings indicate that the Tregs compartment is predominantly of the memory phenotype and, to date, naive T cells have played a lesser role in replenishing the regulatory pool. Careful long-term follow-up will help determine the longevity and functional durability of memory Tregs in IPEX syndrome and other childhood conditions treated by stem cell transplantation.
FOOTNOTES
Accepted Aug 30, 2007.
Address correspondence to Hong Zhan, MD, PhD, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom. E-mail: w.qasim{at}ich.ucl.ac.uk
The authors have indicated they have no financial relationships relevant to this article to disclose.
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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics
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