ABSTRACT
BACKGROUND: Expression of the pre-B-cell receptor (pre-BCR) by pre-BII cells constitutes a crucial checkpoint in B-cell differentiation. Mutations that affect the pre-B-cell receptor result in early B-cell differentiation blockades that lead to primary B-cell immunodeficiencies. BLNK adaptor protein has a key role in the pre-B-cell receptor signaling cascade, as illustrated by the abnormal B-cell development in the 4 patients with BLNK gene defects reported to date. However, the BLNK protein's precise function in human B-cell differentiation has not been completely specified. METHODS: B-cell development, including IgVH and Vk chain repertoires analysis, was studied in the bone marrow of a new case of BLNK deficiency in vitro and in vivo. RESULTS: Here, we report on a patient with agammaglobulinemia, with a total absence of circulating B cells. We detected a homozygous mutation in BLNK, which leads to the complete abrogation of BLNK protein expression. In the bone marrow, we identified a severe differentiation blockade at the pre-BI- to pre-BII-cell transition. IgVH gene rearrangements and selection of the IgH repertoire were normal, whereas the patient's pre-BI cells showed very restricted usage of the IgVκ repertoire. Complementation of bone marrow progenitors from the patient with the BLNK gene and transplantation into NOD/SCID/γcko mice allowed the complete restoration of B-cell differentiation and a normal usage of the IgVκ genes.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Agammaglobulinemia/immunology , B-Lymphocytes/immunology , Adaptor Proteins, Signal Transducing/genetics , Agammaglobulinemia/genetics , Agammaglobulinemia/pathology , Animals , B-Lymphocytes/pathology , B-Lymphocytes/transplantation , Bone Marrow/immunology , Bone Marrow/pathology , Cell Differentiation , Gene Expression , Genetic Complementation Test , Humans , Immunoglobulin kappa-Chains/genetics , Immunoglobulin kappa-Chains/immunology , Mice , Mice, Inbred NOD , Mice, SCID , Mutation , Protein Precursors/genetics , Protein Precursors/immunology , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , Single-Domain Antibodies/genetics , Single-Domain Antibodies/immunology , Transplantation, HeterologousABSTRACT
An important proof of principle has been achieved with the development of an in vitro T-cell differentiation assay based on the coculture of hematopoietic progenitors with the OP9-Delta1 stromal cell line. The original murine T cell differentiation assay has since been adapted for human T-cell differentiation, however with lower efficiency. The choice of both medium and cytokines is crucial in this assay, therefore our work has been focused on these two factors. The use of freshly reconstituted medium, the optimization of interleukine-7 (IL-7) concentration, and the addition of stem cell factor (SCF) have allowed to improve the proliferation of progenitors and T-cell precursors as well as the yield of double positive CD4+CD8+ T cells, and mature γδ and αß T cells. These optimizations make the OP9-Delta1 system sensitive enough to perform both quantitative and qualitative assays with various type of progenitors, including those transduced by a retroviral vector. The improved OP9-Delta1 assay therefore constitutes an extremely useful test for basic research purposes and for translational medicine.
Subject(s)
Cell Differentiation/drug effects , Culture Media, Conditioned/pharmacology , Cytokines/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , Adjuvants, Immunologic/pharmacology , Cells, Cultured , Coculture Techniques , Cytokines/immunology , Hematopoietic Stem Cells/immunology , Humans , Interleukin-7/pharmacology , Stem Cell Factor/pharmacology , Stromal Cells/immunologyABSTRACT
The delayed reconstitution of the T-lymphoid compartment represents a major clinical challenge after HLA-mismatched hematopoietic stem cell transplantation. The generation of new T lymphocytes deriving from transplanted hematopoietic stem cells requires several months, a period associated with an increased risk of opportunistic infections and relapses. Recently, the early steps of human lymphopoiesis and the nature of the thymus-seeding progenitors were described. Moreover several scientific groups succeeded to generate T-cell precursors from murine and human hematopoietic stem cells in vitro by transitory exposition to Notch-ligands. Here we summarize and discuss these results and their possible usage in the development of new cell therapies to shorten the immunodeficient period following hematopoietic stem cell transplantation.
