Yoshiro Kamachi

Identification of Severe Combined Immunodeficiency by T-Cell Receptor Excision Circles Quantification Using Neonatal Guthrie Cards

OBJECTIVE To assess the feasibility of T-cell receptor excision circles (TRECs) quantification for neonatal mass screening of severe combined immunodeficiency (SCID). STUDY DESIGN Real-time PCR based quantification of TRECs for 471 healthy control patients and 18 patients with SCID with various genetic abnormalities (IL2RG, JAK3, ADA, LIG4, RAG1) were performed, including patients with maternal T-cell engraftment (n = 4) and leaky T cells (n = 3). RESULTS TRECs were detectable in all normal neonatal Guthrie cards (n = 326) at the levels of 10(4) to 10(5) copies/microg DNA. In contrast, TRECs were extremely low in all neonatal Guthrie cards (n = 15) and peripheral blood (n = 14) from patients with SCID, including those with maternal T-cell engraftment or leaky T cells with hypomorphic RAG1 mutations or LIG4 deficiency. There were no false-positive or negative results in this study. CONCLUSION TRECs quantification can be used as a neonatal mass screening for patients with SCID.

Identification of FOXP3-negative regulatory T-like (CD4+CD25+CD127low) 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.

Omenn Syndrome—Review of Several Phenotypes of Omenn Syndrome and RAG1/RAG2 Mutations in Japan

Omenn syndrome (OS) is a form of severe combined immunodeficiency (SCID) characterized by erythrodermia, hepatosplenomegaly, lymphadenopathy, and alopecia. In patients with OS, B cells are mostly absent, T-cell counts are normal to elevated, and T cells are frequently activated and express a restricted T-cell receptor (TCR) repertoire. Thus far, inherited hypomorphic mutations of the recombination activating genes either 1 or 2 (RAG1/2) have been detected in most OS patients. We have recently experienced a rare case of OS showing the revertant mosaicism due to multiple second-site mutations leading to typical OS clinical features with RAG1 -deficient SCID. In this review, we will focus on the variation of several phenotypes of OS.

Necrotizing toxoplasmic encephalitis in a child with the X-linked hyper-IgM syndrome

Abstract We report on a 9-year-old boy with the hyper-IgM syndrome who presented with rapid impairment of consciousness. The brain CT scan showed multiple round lucencies, and the brain histology revealed necrotizing toxoplasmic encephalitis. This patient, whose CD40/CD40 ligand system was impaired, indicates the importance of this system for defence against toxoplasmic infection. Conclusion Although disseminated toxoplasmosis is a rare complication of the hyper-IgM syndrome, it must be included in the differential diagnosis of infections.

Clonal haematopoiesis in children with acquired aplastic anaemia

Summary. The methylation pattern of three X‐linked genes, phosphoglycerate kinase (PGK), hypoxanthine phosphoribosyl transferase (HPRT) and DXS255 detected by hypervariable M27β probe, was analysed to determine the proportion of aplastic anaemia (AA) with clonal haematopoiesis in Japanese children. Methylation analysis was performed on DNA from separated granulocytes and compared to that of bone marrow derived fibroblasts to exclude selective lyonization in all somatic cells. Of 20 female patients examined, the methylation pattern of at least one gene was informative in granulocyte DNA from 18 patients (90%). Of these, 8/20 patients (40%) were heterozygous for PGK, 8/18 (44%) were heterozygous for HPRT and 17/18 (94%) were heterozygous for DXS255. In 14/18 patients both alleles were equally methylated. Four patients exhibited a unilateral methylation pattern in their granulocytes. The same unilateral pattern was again demonstrated in fibroblasts from two of the four patients suggesting that in the latter one X chromosome was selectively inactivated in all of the somatic cells. The remaining two patients showed a unilateral methylation pattern that was restricted to their granulocytes, suggesting the existence of true clonal haematopoiesis. They responded well to antilymphocyte globulin (ALG) and presently have no evidence of a clonal disorder such as myelodysplastic syndrome (MDS) or paroxysmal nocturnal haemoglobinuria (PNH). Although these results indicate that some children with AA exhibit clonal haematopoiesis, analysis of a greater number of subjects will be required to establish the clinical value of clonal haematopoiesis in patients with AA.

Engraftment of NOD/SCID/γcnull 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/γcnull 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.

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

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.

Analysis of mutations and recombination activity in RAG-deficient patients.

Mutations in the recombination activating genes (RAG1 or RAG2) can lead to a variety of immunodeficiencies. Herein, we report 5 cases of RAG deficiency from 5 families: 3 of Omenn syndrome, 1 of severe combined immunodeficiency, and 1 of combined immunodeficiency with oligoclonal TCRγδ(+) T cells, autoimmunity and cytomegalovirus infection. The genetic defects were heterogeneous and included 6 novel RAG mutations. All missense mutations except for Met443Ile in RAG2 were located in active core regions of RAG1 or RAG2. V(D)J recombination activity of each mutant was variable, ranging from half of the wild type activity to none, however, a significant decrease in average recombination activity was demonstrated in each patient. The reduced recombination activity of Met443Ile in RAG2 may suggest a crucial role of the non-core region of RAG2 in V(D)J recombination. These findings suggest that functional evaluation together with molecular analysis contributes to our broader understanding of RAG deficiency.

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.

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA–peptide complexes

BACKGROUND Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.