Recombination activating gene 1 (RAG1) is essential for variable diversity joining recombination during early T- and B-cell development. Null mutations cause a complete block in receptor rearrangement, resulting in T-B- severe combined immunodeficiency (SCID). Patients with RAG1-SCID require hematopoietic stem cell transplantation for survival. Our phase I/II clinical trial (NCT04797260) is currently evaluating lentiviral RAG1 gene addition in autologous hematopoietic stem and progenitor cells (HSPCs). However, studying early human T-cell development is challenging due to limited access to thymic tissue. The artificial thymic organoid (ATO) system offers a promising in vitro model to study human T-cell differentiation. Here, we show that ATO cultures efficiently support T-cell development from healthy donor HSPCs derived from umbilical cord blood or mobilized peripheral blood, yielding not only αβ but also γδ T cells with a polyclonal T-cell receptor (TCR) repertoire. In contrast, noncorrected RAG1-deficient HSPCs from 3 RAG1-SCID patients show a developmental arrest before or at the aberrant CD4+CD8dim double-positive stage, characterized by minimal or absent CD1a upregulation and CD7 downregulation, absence of TCRβ rearrangement, and only partial TCRγ and TCRδ rearrangement. Lentiviral RAG1 gene addition using the clinical vector rescues T-cell development in these patient-derived HSPCs and restores TCR repertoire diversity. These findings highlight the ATO system as a valuable model for dissecting human T-cell development and for the preclinical development and evaluation of gene therapy.

Reticular dysgenesis (RD) is a rare genetic disorder caused by mutations in the adenylate kinase 2 (AK2) gene. It is characterized by a T-B- severe combined immunodeficiency, agranulocytosis, and sensorineural deafness. We established and characterized a haematopoiesis-specific conditional Ak2-knockout mouse model to provide a model system to study the molecular pathophysiology of RD. As expected from the human phenotype of RD, haematopoiesis-specific AK2-deficient embryos had a small, atrophic thymus consisting mainly of epithelial cells. No recognizable T-cell component was observed, but B-cell lineage precursor cells were present in the foetal liver. The effects of AK2 deficiency on myelopoiesis were less severe in mice than in humans. The absolute numbers of monocytes, macrophages, granulocytes and megakaryocytes in foetal liver as well as colony-forming precursors were not reduced. In contrast to humans, haematopoiesis-specific Ak2-knockout mice exhibit embryonic lethality between E13 and E15 due to severe anaemia caused by an early block in definitive erythropoiesis. Murine erythroid progenitors mainly express AK2 and only low levels of functionally related kinases, which are unable to compensate for AK2 deficiency, in contrast to human erythroid progenitors.

Although absence of interleukin-7 (IL-7) signaling completely abrogates T and B lymphopoiesis in mice, patients with severe combined immunodeficiency caused by mutations in the IL-7 receptor α chain (IL-7Rα) still generate peripheral blood B cells. Consequently, human B lymphopoiesis has been thought to be independent of IL-7 signaling. Using flow cytometric analysis and single-cell RNA sequencing of bone marrow samples from healthy controls and patients who are IL-7Rα deficient, in combination with in vitro modeling of human B-cell differentiation, we demonstrate that IL-7R signaling plays a crucial role in human B lymphopoiesis. IL-7 drives proliferation and expansion of early B-cell progenitors but not of pre-BII large cells and has a limited role in the prevention of cell death. Furthermore, IL-7 guides cell fate decisions by enhancing the expression of BACH2, EBF1, and PAX5, which jointly orchestrate the specification and commitment of early B-cell progenitors. In line with this observation, early B-cell progenitors of patients with IL-7Rα deficiency still expressed myeloid-specific genes. Collectively, our results unveil a previously unknown role for IL-7 signaling in promoting the B-lymphoid fate and expanding early human B-cell progenitors while defining important differences between mice and humans. Our results have implications for hematopoietic stem cell transplantation strategies in patients with T- B+ severe combined immunodeficiency and provide insights into the role of IL-7R signaling in leukemogenesis.

Increased immunogloblulin-E (IgE) levels associated with eosinophilia represent a common finding observed in Omenn syndrome, a severe immunodeficiency caused by decreased V(D)J recombination, leading to restricted T- and B-cell receptor repertoire. V(D)J recombination is initiated by the lymphoid-restricted recombination-activating gene (RAG) recombinases. The lack of RAG proteins causes a block in lymphocyte differentiation, resulting in T-B- severe combined immunodeficiency. Conversely, hypomorphic mutations allow the generation of few T and B cells, leading to a spectrum of immunological phenotypes, in which immunodeficiency associates to inflammation, immune dysregulation, and autoimmunity. Elevated IgE levels are frequently observed in hypomorphic RAG patients. Here, we describe the role of RAG genes in lymphocyte differentiation and maintenance of immune tolerance.

Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. Artemis activation by DNA-PKcs resolves hairpin DNA ends formed during V(D)J recombination. Artemis deficiency disrupts development of adaptive immunity and leads to radiosensitive T- B- severe combined immunodeficiency (RS-SCID). An activated state of Artemis in complex with DNA-PK was solved by cryo-EM recently, which showed Artemis bound to the DNA. Here, we report that the pre-activated form (basal state) of the Artemis:DNA-PKcs complex is stable on an agarose-acrylamide gel system, and suitable for cryo-EM structural analysis. Structures show that the Artemis catalytic domain is dynamically positioned externally to DNA-PKcs prior to ABCDE autophosphorylation and show how both the catalytic and regulatory domains of Artemis interact with the N-HEAT and FAT domains of DNA-PKcs. We define a mutually exclusive binding site for Artemis and XRCC4 on DNA-PKcs and show that an XRCC4 peptide disrupts the Artemis:DNA-PKcs complex. All of the findings are useful in explaining how a hypomorphic L3062R missense mutation of DNA-PKcs could lead to insufficient Artemis activation, hence RS-SCID. Our results provide various target site candidates to design disruptors for Artemis:DNA-PKcs complex formation.