Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors.

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells.

Selective expansion of donor-derived regulatory T cells after allogeneic bone marrow transplantation in a patient with IPEX syndrome.

IPEX syndrome is a rare and fatal disorder caused by absence of regulatory T cells (Tregs) due to congenital mutations in the Forkhead box protein 3 gene. Here, we report a patient with IPEX syndrome treated with RIC followed by allogeneic BMT from an HLA-matched sibling donor. We could achieve engraftment and regimen-related toxicity was well tolerated. Although the patient was in mixed chimera and the ratio of donor cells in whole peripheral blood remained relatively low, selective and sustained expansion of Tregs determined as CD4+CD25+Foxp3+ cells was observed. Improvement in clinical symptoms was correlated with expansion of donor-derived Tregs and disappearance of anti-villin autoantibody, which was involved in the pathogenesis of gastrointestinal symptoms in IPEX syndrome. This clinical observation suggests that donor-derived Tregs have selective growth advantage in patients with IPEX syndrome even in mixed chimera after allogeneic BMT and contribute to the control of clinical symptoms caused by the defect of Tregs.

B-cell function after unrelated umbilical cord blood transplantation using a minimal-intensity conditioning regimen in patients with X-SCID.

Patients with X-linked severe combined immunodeficiency (X-SCID) suffer from severe and persistent infections, and usually die early in life unless treated by hematopoietic stem cell transplantation. If a patient has an HLA-identical sibling donor, preparative conditioning is not necessary for T-cell engraftment and B-cell function. However, in the absence of such a donor, long-term reconstitution of full B-cell function is often problematic, leading in many cases to a lifetime requirement for immunoglobulin replacement therapy. Preparative myeloablative conditioning has been shown to improve long-term B-cell function, but may aggravate pre-existing infection and transplant-related toxicity. It is thus important to determine the minimum intensity of conditioning that assures immunoglobulin production. In the present study, we performed reduced-intensity conditioning (RIC), consisting of fludarabine 125 mg/m(2) and melphalan 80 mg/m(2), prior to unrelated umbilical cord blood transplantation (UCBT) for five patients with X-SCID, none of them had an HLA-identical donor. Four patients survived more than 4 years without sequelae, and none required long-term immunoglobulin replacement therapy. One patient succumbed to sepsis in conjunction with severe GVHD. Our result demonstrates that the RIC regimen described above in combination with UCBT is an effective and less toxic conditioning to correct B-cell function in patients with X-SCID.

Wiskott-Aldrich syndrome presenting with a clinical picture mimicking juvenile myelomonocytic leukaemia.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency caused by defects of the WAS protein (WASP) gene. Patients with WAS typically demonstrate micro-thrombocytopenia. PROCEDURES: The report describes seven male infants with WAS that initially presented with leukocytosis, monocytosis, and myeloid and erythroid precursors in the peripheral blood (PB) and dysplasia in the bone marrow (BM), which was initially indistinguishable from juvenile myelomonocytic leukaemia (JMML). RESULTS: The median age of affected patients was 1 month (range, 1-4 months). Splenomegaly was absent in four of these patients, which was unusual for JMML. A mutation analysis of genes in the RAS-signalling pathway did not support a diagnosis of JMML. Non-haematological features, such as eczema (n = 7) and bloody stools (n = 6), ultimately led to the diagnosis of WAS at a median age of 4 months (range, 3-8 months), which was confirmed by absent (n = 6) or reduced (n = 1) WASP expression in lymphocytes by flow cytometry (FCM) and a WASP gene mutation. Interestingly, mean platelet volume (MPV) was normal in three of five patients and six of seven patients demonstrated occasional giant platelets, which was not compatible with WAS. CONCLUSIONS: These data suggest that WAS should be considered in male infants presenting with JMML-like features if no molecular markers of JMML can be detected.

Identification of FOXP3-negative regulatory T-like (CD4(+)CD25(+)CD127(low)) cells in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune disorder caused by mutations in the FOXP3 gene, which plays a key role in the generation of CD4(+)CD25(+)regulatory T (Treg) cells. We selected CD127 as the surface marker of Treg cells to illustrate the development and function of Treg cells in IPEX syndrome. CD4(+)CD25(+)FOXP3(+) T cells, the putative Treg cells, were almost completely absent in all patients. Importantly, a substantial number of CD4(+)CD25(+)CD127(low) T cells were observed in 3 IPEX patients with hypomorphic mutations in the FOXP3 gene. We demonstrated that CD4(+)CD25(+)CD127(low) T cells isolated from these 3 patients exhibited an appreciable suppressive activity on effector T cell proliferation, although less than that displayed by Treg cells from healthy controls. These results suggest that genetically altered FOXP3 can drive the generation of functionally immature Treg cells, but that intact FOXP3 is necessary for the complete function of Treg cells.

Allergic bronchopulmonary aspergillosis in a 2-year-old asthmatic boy with immune dysregulation, polyendocrinopathy, enteropathy, X-linked.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is caused by impaired function of CD4(+)CD25(+) regulatory T cells that play an important role in controlling exaggerated Th2 responses. The pathogenesis of allergic bronchopulmonary aspergillosis (ABPA) appears to be closely associated with the Th2 response. We report the first case of ABPA in a 2-year-old asthmatic boy with IPEX, this being an unusually young age for the development of ABPA.

Wiskott-Aldrich syndrome is an important differential diagnosis in male infants with juvenile myelomonocytic leukemialike features.

A newborn presented with thrombocytopenia at birth and subsequently developed leukocytosis, monocytosis, and mild hepatomegaly. The bone marrow was normocellular with dysplasia and spontaneous granulocyte-monocyte colony formation was demonstrated. These findings fulfilled the diagnostic criteria of juvenile myelomonocytic leukemia. Then he developed atopic dermatitislike eczema, which led to the consideration of Wiskott-Aldrich syndrome (WAS). Lack of intracellular WASP expression and WASP gene mutation confirmed the diagnosis of WAS. After stem cell transplantation, he is alive in good condition with normal WASP expression. WAS should be considered as a differential diagnosis in male infants with juvenile myelomonocytic leukemialike features.

PTPN11, RAS and FLT3 mutations in childhood acute lymphoblastic leukemia.

PTPN11, the gene which encodes protein tyrosine phosphatase SHP-2, plays an important role in regulating intracellular signaling. Germline mutations in PTPN11 were first observed in Noonan syndrome, while somatic mutations were identified in hematological myeloid malignancies. Recently, PTPN11 mutations have been reported in children with acute lymphoblastic leukemia (ALL). In the present study, we investigated the prevalence of mutations in PTPN11, RAS and FLT3 in samples from 95 Japanese children with ALL. We observed exon 3 and 8 missense mutations of PTPN11 in 6 children with B precursor ALL. One patient with Down syndrome and ALL had PTPN11 mutation. We also identified RAS mutations in ten patients and FLT3 internal tandem duplication (FLT3/ITD) in one patient. None of the patients had simultaneous mutations in PTPN11 and RAS, while one patient had both PTPN11 and FLT3 mutations. These data suggest that PTPN11 mutation may play an important role for leukemogenesis in a proportion of children with ALL, particularly B precursor ALL.

Engraftment of NOD/SCID/gammac(null) mice with multilineage neoplastic cells from patients with juvenile myelomonocytic leukaemia.

Several lines of evidence indicate the clonal nature of juvenile myelomonocytic leukaemia (JMML), involving myeloid, erythroid, megakaryocyte and B-lymphoid lineages. However, it is unclear whether the T-lymphocyte lineage is involved. We demonstrated that cells from six patients with JMML repopulated in non-obese diabetic/severe combined immunodeficient/gammac(null) mice and differentiated into granulocytes, monocytes, erythrocytes, B lymphocytes, T lymphocytes and natural killer cells. The percentage of human CD45 antigen-positive cells ranged from 41% to 73% in the murine bone marrow 12 weeks after transplantation. To examine the involvement of lymphocyte subpopulations, we purified human CD3(+), CD19(+) and CD56(+) cells from murine bone marrow cells transplanted from a patient with monosomy 7. Fluorescence in situ hybridization (FISH) showed the clonal marker in 96-100% of purified CD3(+), CD19(+) and CD56(+) subpopulations. These findings support the concept that JMML originates in transplantable multilineage haematopoietic stem cells. This novel murine xenotransplant model should be useful for investigating the nature of stem cells and testing new therapies for patients with JMML.