In type III bare lymphocyte syndrome (BLS) patients, defects in the RFX protein complex result in a lack of MHC class II and reduced MHC class I cell surface expression. Using type III BLS cell lines, we demonstrate that the RFX subunits RFX5 and RFXAP are crucial for constitutive and CIITA-induced MHC class I and beta2m transactivation. Similar to MHC class II, the promoters of MHC class I and beta2m contain an S-X-Y region of which the X1 box is crucial for constitutive and CIITA-induced MHC class I and beta2m transactivation. Thus, the RFX complex is part of a regulatory pathway linking the transactivation of MHC class I and II and their accessory genes.
Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized RAG1 to ensure optimal expression. We used Rag1 À/À mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with RAG1 expression. Mice with low RAG1 expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34 + cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.
The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ−/− xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.H ematopoietic stem cell transplantation (HSCT) has become common clinical practice in the treatment of leukemia, lymphoma, and certain inherited immune and metabolic disorders. After transplantation there is an immediate need for de novo production of granulocytes, erythrocytes, and platelets, referred to as "hematopoietic reconstitution," later followed by recovery of lymphocyte numbers, termed "immune reconstitution." Although often successful, HSCT is associated with a number of complications arising from poor immune reconstitution, which presents one of the most important causes of morbidity after HSCT (1, 2). Poor myeloid reconstitution is directly linked to low numbers of HSCs in the transplant, but the reasons for poor immune and, in particular, T-lymphocyte reconstitution are much less clear.A study on the application of antithymocyte globulin in cord blood transplantation showed that antithymocyte globulin administration in pretransplantation conditioning results in decreased T-cell reconstitution and survival (3), indicating the need for a better understanding of de novo T-cell development after HSCT. As T cells develop in the thymus, in contrast to all other blood lineages that develop in the bone marrow (BM), HSCderived progenitors need to seed the thymus. Earlier work in mice has indicated that relatively few progenitors seed the thymus (4, 5), yet their numbers and the subsequent fate of the progeny derived from an HSC clone has remained elusive. The nature of the thymusseeding cell has been subject of much debate, certainly in humans. CD34+ cells (6, 7), CD34 (8), and others have been proposed as thymus-seeding cells in the human situation. In contrast to mice, the earliest hu...
HLA-DR molecules were isolated from eight different HLA-DR3 homozygous B-cell lines by immunoprecipitation with monoclonal antibodies, and they were subsequently analyzed by two-dimensional gel electrophoresis. We found that HLA-DR3 homozygous B-cell lines of consanguineous origin express two types of HLA-DR molecules. One type of HLA-DR molecule was present in all the cell lines tested, whereas the second DR molecule appears to be polymorphic. DNA isolated from the different HLA-DR3 homozygous cell lines was studied by Southern blot analysis to determine whether any DR beta restriction fragment length polymorphism could be observed. Polymorphisms detected at both the product and genomic level have been compared to each other, and their relations to the serological (HLA-DR) and cellular (HLA-D and LB-Q1) typing data will be discussed.
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