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The results of immunologic investigations of IPEX1 are inconsistent with any of the known X-linked immunodeficiency diseases.13 Severe infections have been observed in patients with IPEX, but they may have been complications of immunosuppressive therapy.4 Massive infiltration of T lymphocytes into the skin and gastrointestinal tract and high serum levels of autoantibodies against blood, thyroid, and pancreatic cells suggest that IPEX is an autoimmune disorder. Specific antibodies against a 75-kd antigen in intestinal and renal epithelial cells have been identified in one patient.14 Mutations of the FOXP3 gene have recently been identified in patients with IPEX, confirming that IPEX is a distinct X-linked immune disorder.15,16,17 Scurfy mice, in which a disease similar to IPEX develops, have a mutation in an analogous gene.18
Supportive therapy with total parenteral nutrition, insulin, and blood transfusions is beneficial in patients with IPEX, and prolonged immunosuppressive therapy with steroids and cyclosporine has been attempted.19,20 Nevertheless, the prognosis is very poor, and most reported cases have been fatal. Transient clinical improvement occurred in three children with continued parenteral nutrition and prolonged immunosuppressive therapy.2,5,10,15 Given the immune pathogenesis of IPEX and the poor prognosis for patients with this condition, we performed transplantation of allogeneic bone marrow from an HLA-identical family member in a four-month-old boy with the disease.
Case Report
We studied a child with IPEX born to parents from Morocco (Figure 1). The mother had been pregnant seven times. She had had an unexplained spontaneous abortion during the fourth month of one pregnancy and had four healthy daughters who were 11, 16, 18, and 19 years of age at the time of this report. One son had died at 4.5 months of age from intractable diarrhea, thrombocytopenia, insulin-dependent diabetes mellitus, and ichthyosiform dermatitis. A biopsy of the duodenal mucous membrane from this child showed villous atrophy, and autopsy showed infiltration by lymphocytes and plasmocytes of the liver, small bowel, colon, and pancreas.
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The numbers of CD19+, CD3+, CD4+, and CD8+ lymphocytes in the blood were within normal ranges; the in vitro T-lymphocyte response to phytohemagglutinin and the serum IgG, IgA, and IgM concentrations were also within normal ranges at four months of age. In contrast, serum IgE concentrations were very high (up to 1750 IU per milliliter [4200 µg per liter]). Titers of serum antibodies against antigens used for vaccination (tetanus toxoid, poliovirus) were normal.
IPEX was suspected clinically, and the diagnosis was confirmed by the presence of a mutation in the FOXP3 gene.15 The mutation was a substitution of guanine for thymine at nucleotide position 1113 (exon 10), resulting in the substitution of cysteine for phenylalanine at amino acid position 371 (F371C). The patient's mother was a heterozygous carrier of the F371C mutation.
Immunosuppressive treatment was initiated (250 mg of methylprednisolone per square meter of body-surface area per day for a total of 1250 mg per square meter per week, followed by 2 mg of methylprednisolone per kilogram of body weight per day plus 25 mg of methylprednisolone per kilogram per week and 0.3 mg of tacrolimus per kilogram per day).
There was a transient clinical response, but then the child's condition worsened. At four months of age, he was therefore considered for allogeneic transplantation of bone marrow from his healthy, 18-year-old, HLA-identical sister, who did not carry the F371C mutation. The conditioning regimen consisted of intravenous rabbit anti–T-lymphocyte globulin (Pasteur-Merieux, Lyons, France; 10 mg per kilogram per day on days 14 to 10 before transplantation), oral busulfan (Glaxo Wellcome, Paris; 5 mg per kilogram per day on days 9 to 6 before transplantation), and intravenous cyclophosphamide (Asta Medica, Paris; 50 mg per kilogram per day on days 5 to 2 before transplantation).
A total of 435x107 bone marrow mononuclear cells per kilogram and 217x106 CD34+ cells per kilogram were infused. For prophylaxis against graft-versus-host disease, the patient received cyclosporine intravenously (until day 21) and then orally (until day 180), so that the serum concentration of the drug was maintained at 150 to 200 µg per liter. Intravenous acyclovir (Glaxo Wellcome), at a dose of 1600 mg per square meter per day, was given for prophylaxis against cytomegalovirus infection until day 60. The blood products used tested negative for cytomegalovirus and were irradiated.
Results
On day 11 after transplantation, the polymorphonuclear neutrophil count in the blood was more than 500 per cubic millimeter. Analysis of sex chromosomes by fluorescence in situ hybridization on day 19 showed that 95 percent of white cells were of the female XX genotype. There was no evidence of venous occlusion, graft-versus-host disease, or infection.
Diarrhea improved as early as three days before bone marrow transplantation, and stools were normal one month after transplantation. Two weeks later, enteral feeding was successfully reinstated with an elemental diet without cow's-milk proteins or gluten. Two months after bone marrow transplantation, solid foods were slowly introduced into the diet. Stools remained normal in number and appearance. Parenteral nutrition was discontinued one month later. Six months after bone marrow transplantation, the patient's diet was normal and was associated with a normal intestinal transit time and no apparent gastrointestinal problems. Duodenal, jejunal, and colonic biopsies showed normal tissues with recovered villous architecture and mucous membranes; only rare lymphocytes were detectable in the lamina propria (Figure 2).
The conditioning regimen was associated with a progressive improvement in blood glucose control, and insulin therapy was stopped seven days before bone marrow transplantation. Autoantibodies directed against pancreatic islet cells and GAD65 were no longer detected by day 180. Blood products (red cells and platelets) were not required after day 21. Moderate hepatitis occurred from day 42 to day 50. Six months after bone marrow transplantation, mild eczematous lesions were observed on the face and forearms, and the diagnosis was confirmed by a skin biopsy. These skin lesions improved rapidly when treated with a corticosteroid cream for three to four weeks. After a new series of vaccinations (against tetanus and poliomyelitis) were administered six months after bone marrow transplantation, the patient's T-lymphocyte responses in vitro to phytohemagglutinin and vaccine antigens were within the normal range.
Twenty-three months after bone marrow transplantation, the patient resumed normal feeding and was progressing very well, with normal hematologic and immunologic function, feeding, stools, glucose concentrations, and skin (Table 1). Stable mixed chimerism was found at 16 and 23 months after transplantation in red cells (CcK– and CCK+ red cells) by blood typing and in leukocytes (28 percent XX at 23 months), polymorphonuclear cells (15 percent XX), resting CD3+ T lymphocytes (30 percent XX), phytohemagglutinin–interleukin-2–driven CD3+ T-lymphocyte blasts (25 percent XX), and CD19+ B cells (3 percent XX) by fluorescence in situ hybridization analysis. The patient's weight, height, and head circumference 23 months after bone marrow transplantation were normal.
While the child was still in remission, fever, splenomegaly, and lymphadenopathy developed, along with hyponatremia, elevated concentrations of liver enzymes, and thrombocytopenia. Most peripheral-blood T lymphocytes were activated. Hemophagocytosis was found in the bone marrow. The results of an extensive search for infectious agents, including cytomegalovirus and Epstein–Barr virus, were negative. Despite treatment with broad-spectrum antibiotics and high-dose methylprednisolone (5 mg per kilogram per day), the child died. The death occurred 29 months after bone marrow transplantation, when the child was nearly 3 years of age. An autopsy was not performed.
Discussion
IPEX is usually fatal; there are only three known long-term survivors of the disease.2,5,10,15 Tissue infiltration by lymphocytes, the presence of serum autoantibodies, and the mutations in FOXP3 that is expressed in lymphocytes in patients with IPEX strongly suggest that the pathogenesis of the disease involves the immune system, prompting the use of immunosuppressive therapy for this condition.19,20 In one case, cyclosporine had partial, transient efficacy.10 Azathioprine failed to induce a remission in one child.21 Combination therapy with tacrolimus, betamethasone, and verapamil was transiently beneficial in one patient, but the patient died.22 Our results show that bone marrow transplantation should be considered as a potentially curative option in patients with IPEX.
In our patient, bone marrow transplantation was followed by a complete remission. Interestingly, the conditioning regimen itself controlled most of the clinical and biologic features of the disease, including the diarrhea, hyperglycemia, and dermatitis. Engraftment probably facilitated a sustained remission, and the patient remained in clinical and biologic remission for more than two years after bone marrow transplantation. Given that there is a germ-line mutation in IPEX, it is unlikely that autologous bone marrow transplantation would produce a sustained remission. In our patient, irreversible insulin-dependent diabetes had not yet occurred; this is important, because a lack of pancreatic islets has been found at autopsy in several patients with IPEX. Interestingly, partial donor chimerism was sufficient to maintain a complete remission, suggesting that a minority of healthy donor leukocytes is sufficient to control the autoimmune process. Bone marrow transplantation in other children with IPEX would be required to assess the stability and efficacy of transplantation.
FOXP3 is expressed primarily in CD4+ T lymphocytes. Studies in scurfy mice, which have a similar mutation, have shown that CD4+ T-helper lymphocytes are important in the development of the lesions.23,24 These observations suggest that mouse scurfy and human IPEX are disorders of T-helper–lymphocyte regulation. Our success with bone marrow transplantation is consistent with these findings and demonstrates that hematopoietic cells have an essential role in the pathogenesis of IPEX.
The occurrence of diabetes mellitus and eczematous ichthyosis distinguishes IPEX from other inherited autoimmune conditions such as complement deficiencies,25 autoimmune lymphoproliferative syndrome,26 and autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy.27,28 Transplantation of haploidentical bone marrow has been successfully attempted in two children with the autoimmune lymphoproliferative syndrome caused by complete Fas deficiency.29,30 Together with our results, these data suggest that allogeneic bone marrow transplantation may be a therapeutic option in other severe inherited autoimmune conditions.
We have no clear explanation for the development of a rapidly progressive hemophagocytic syndrome, which proved fatal, in our patient. There was no evidence of an infectious agent, and whether the hemophagocytic syndrome was a complication of the bone marrow transplantation, was related to the fact that the child had mixed hematopoietic chimerism, or both, is unclear. Hemophagocytosis is not a known feature of IPEX. Bone marrow transplantation, in our opinion, remains a potentially curative option for patients with IPEX, even though the possibility of long-term complications, such as hemophagocytosis, should be carefully considered.
We are indebted to Professors Ichiro Kobayashi and Ernest Seidman for helpful discussions. This article is dedicated to the memory of the child with IPEX and to his family.
Source Information
From the Service d'Immunologie et d'Hématologie Pédiatriques (O.B., S.D.-G., A.F., J.-L.C.), Service de Gastro-enterologie Pédiatrique (O.G.), Service d'Anatomie Pathologique (D.C., N.B.), Laboratoire INSERM Unité 429 (F.L.D., M.C.-C., A.F.), Laboratoire de Cytogénétique Hématologique (I.R.), Laboratoire INSERM E9925 (N.C.-B.), and Laboratoire Université René Descartes–INSERM Unité 550 (J.-L.C.), Hôpital Necker–Enfants Malades, Paris; and the Service de Pédiatrie 2, Hôpital Arnaud de Villeneuve, Montpellier, France (D.R.).
Other authors were Christine Rivet, M.D., Service de Gastro-enterologie Pédiatrique, and Christine Bodemer, M.D., Service de Dermatologie Pédiatrique, Hôpital Necker–Enfants Malades, Paris; and Robert Wildin, M.D., Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland.
Address reprint requests to Dr. Casanova at the Unité d'Immunologie et d'Hématologie Pédiatriques, Hôpital Necker–Enfants Malades, 149 rue de Sèvres, 75015 Paris, France, or at casanova{at}necker.fr.
References
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