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.

T cell development in the mouse thymus has been studied extensively, but less is known regarding T cell development in the human thymus. We used a combination of single-cell techniques and functional assays to perform deep immune profiling of human T cell development, focusing on the initial stages of prelineage commitment. We identified three thymus-seeding progenitor populations that also have counterparts in the bone marrow. In addition, we found that the human thymus physiologically supports the development of monocytes, dendritic cells, and NK cells, as well as limited development of B cells. These results are an important step toward monitoring and guiding regenerative therapies in patients after hematopoietic stem cell transplantation.

The mechanisms that regulate proliferation, fate decisions and differentiation of hematopoietic stem cells (HSC) and thymic stem cells are highly complex. Several signaling pathways including Wnt signaling have important roles during these processes. Both canonical and non-canonical Wnt signaling are important in normal and malignant hematopoiesis and lymphoid development, yet their precise roles are controversial. In a side-by-side comparison, we investigated the roles of the canonical and non-canonical Wnt pathway in hematopoiesis and thymopoiesis. As complete loss-of-function models for non-canonical Wnt signaling are not yet available and highly complex for canonical Wnt signaling, we decided to use a gain-of-function approach. To this end, Wnt3a and Wn5a, two well-known prototypical canonical and non-canonical Wnt ligands were produced in hematopoiesis supporting stromal assays. High levels of Wnt3a signaling blocked T-cell development at early stages, whereas intermediate levels accelerated T-cell development. In contrast, Wnt5a signaling prompted apoptosis in developing thymocytes, without affecting differentiation at a particular stage. To explore the role of Wnt3a and Wnt5a in vivo, we transduced HSCs isolated from fetal liver, transduced with Wnt3a and Wnt5a vectors, and performed reconstitution assays in irradiated C57Bl/6 mice. Wnt3a overexpression led to increased lymphopoiesis, whereas Wnt5a augments myelopoiesis in the bone marrow (BM) and spleen. Thus, the canonical and non-canonical Wnt signaling have discrete roles in hematopoiesis and thymopoiesis, and understanding their right dose of action is crucial for prospective translational applications.

Adequate responsiveness of CD8+ T cell populations is of utmost importance for the efficacy of many vaccines and immunotherapeutic strategies against intracellular pathogens and cancer. In this study, we show in mouse models that the relative number of IL-2–producing cells within Ag-specific CD8+ T cell populations predicts the population expansion capacity upon challenge. We further demonstrate that IL-2 producers constitute the best responding subset. Notably, we show that elevated production of IL-2 by CD8+ T cells results in concomitant improved population expansion capacity and immunity. The amount of IL-2 produced on a per-cell basis essentially connects directly to the superior CD8+ T cell population expansion. Together, our findings identified that autocrine IL-2 production operates in a dose-dependent fashion to facilitate the expansion potential of Ag-specific CD8+ T cell populations, which may instigate ways to augment therapies depending on fit CD8+ T cells.

Phillips have a patent pending for HLA-A*32:01 testing for vancomycin drug reaction and eosinophilia and systemic symptoms. S. Mallal and E. J. Phillips receive royalties from UpToDate and have equity in IIID Pty Ltd, which holds a patent for HLA-B*57:01 testing for abacavir hypersensitivity. E. J. Phillips receives consulting fees from Biocryst. The rest of the authors declare that they have no relevant conflicts of interest.