# A Thymus-Independent Artificial Organoid System Supports Complete Thymopoiesis from Rhesus Macaque-Derived Hematopoietic Stem and Progenitor Cells

## TL;DR

Scientists created an artificial thymus system using rhesus macaque cells to study T cell development outside the body.

## Contribution

The first artificial thymic organoid system for non-human primates that supports complete T cell development ex vivo.

## Key findings

- RhATO organoids reproduced all stages of T cell development, including immature and mature subsets.
- TCR-selected T cells derived from RhATO showed functional properties and cytokine responses.
- The system mirrors human T cell ontogeny and supports recent thymic emigrant phenotypes.

## Abstract

Background: T cell regeneration in the thymus is intrinsically linked to the T cell-biased lineage differentiation of hematopoietic stem and progenitor cells (HSPCs). Although nonhuman primates (NHPs) serve as indispensable models for studying thymic output under physiological and pathological conditions, a non-animal technology facilitating efficient TCR-selected T cell development and evaluating T cell output from NHP-derived HSPCs has been lacking. To address this gap, we established a rhesus macaque-specific artificial thymic organoid (RhATO) modeling primary thymus-tissue-free thymopoiesis. Methods: The RhATO was developed by expressing Rhesus macaque (RM) Delta-like Notch ligand 1 in mouse bone marrow stromal cell line (MS5-RhDLL1). The bone marrow-derived HSPCs were aggregated with MS5-RhDLL1 and cultured forming 3D artificial thymic organoids. These organoids were maintained under defined cytokine conditions to support complete T cell developmental ontogeny. T cell developmental progression was assessed by flow cytometry, and TCR-selected subsets were analyzed for phenotypic and functional properties. Results: RhATOs recapitulated the complete spectrum of thymopoietic events, including emergence of thymus-seeding progenitors, CD4+CD3− immature single-positive and CD4+CD8+ double-positive early thymocytes, and mature CD4+ or CD8+ single-positive subsets. These subsets expressed CD38, consistent with the recent thymic emigrant phenotype, and closely mirrored canonical T cell ontogeny described in humans. RhATO-derived T cells were TCR-selected and demonstrated cytokine expression upon stimulation. Conclusions: This study provides the first demonstration of an NHP-specific artificial thymic technology that faithfully models thymopoiesis. RhATO represents a versatile ex vivo platform for studying T cell development, immunopathogenesis, and generating TCR selected T cells.