Artificial thymic organoids represent a reliable tool to study T-cell differentiation in patients with severe T-cell lymphopenia

Abstract

The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαβ+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.

Current and Future Therapeutic Approaches for Thymic Stromal Cell Defects

“…While there are differences in T-cell development between humans and mice ( 43 – 46 ), the murine model continues to offer fundamental insight into this process and some of these developmental stages have been reproduced during the in vitro differentiation of human hematopoietic stem cells (HSCs) in co-culture systems with Notch ligand-expressing murine stromal cells ( 47 – 49 ). Additionally, novel molecular tools, such as high-throughput RNA sequencing, efficient high-precision genome editing and the possibility to use HSCs and induced pluripotent stem cells (iPSCs) from certain PID patients, are further facilitating the study of human T-cell development in new ex vivo ( 50 – 52 ) and in vivo models ( 53 , 54 ).…”

“…We have taken advantage of a monolayer co-culture system of patients’ bone marrow-derived CD34 + cells together with OP9/DL1 stromal cells and growth factors ( 49 ). Recently, two groups reported the use of artificial thymic organoids aggregating DLL4-expressing stromal cell lines (OP9/DLL4 and MS5-hDLL4) to facilitate the differentiation of CD34 + cells, isolated either from the patients’ bone marrow aspirate ( 52 ) or from their peripheral blood ( 131 ). Using these three-dimensional tools, they have shown that CD34 + cells of two cDGS patients, one with a TBX1 mutation and the other with a deletion at chromosome 22q11.2, had the potential to differentiate into CD3 + mature T-cells, in line with their disorder not affecting the HSC.…”

Dynamics of thymus function and T cell receptor repertoire breadth in health and disease

“…TEC-like progenitors appropriate for this purpose have also been generated by direct conversion of embryonic fibroblasts by induced expression of the TEC transcription factor FOXN1 [ 253 ], as well as inducing TECs from embryonic ctem cells or iPS cells from both mouse and human [ 92 – 94 , 254 ]. Although the efficacy of ATOs have not yet been extensively evaluated in the setting of regeneration outside of mice, ATOs have recently been used to great effect to distinguish hematopoietic from intrinsic thymic defects in pediatric immunodeficiencies; thereby identifying patients with DGS that would be candidates for thymus transplant [ 190 ].…”

Inborn errors of thymic stromal cell development and function

“…T cell lymphopaenia may mistakenly be ascribed to an undefined haematopoietic defect and a thymic aetiology only suspected after absent immune reconstitution post-HSCT. Novel tools are emerging to facilitate the distinction of primary haematopoietic defects from inborn errors of thymic stromal cells in patients presenting with genetically undefined severe T cell lymphopaenia [ 126 , 129 , 146 , 147 ]. These include the use of artificial thymic organoids that express DLL4 and can therefore support the differentiation of CD34 + haematopoietic stem cells (HSCs) into mature T cells [ 146 , 147 ].…”

“…Novel tools are emerging to facilitate the distinction of primary haematopoietic defects from inborn errors of thymic stromal cells in patients presenting with genetically undefined severe T cell lymphopaenia [ 126 , 129 , 146 , 147 ]. These include the use of artificial thymic organoids that express DLL4 and can therefore support the differentiation of CD34 + haematopoietic stem cells (HSCs) into mature T cells [ 146 , 147 ]. In principle, HSCs from patients with thymic stromal cell defects should be able to generate mature T cells, whereas the T cell differentiation capacity of HSCs derived from patients with haematopoietic defects is generally expected to be impaired, although there may be exceptions as has been seen for adenosine deaminase deficiency [ 146 , 147 ].…”