Multiomics atlas-assisted discovery of transcription factors enables specific cell state programming.

The same types of cells can assume diverse states with varying functionalities. Effective cell therapy can be achieved by specifically driving a desirable cell state, which requires the elucidation of key transcription factors (TFs). Here, we integrated epigenomic and transcriptomic data at the systems level to identify TFs that define different CD8 + T cell states in an unbiased manner. These TF profiles can be used for cell state programming that aims to maximize the therapeutic potential of T cells. For example, T cells can be programmed to avoid a terminal exhaustion state (Tex Term ), a dysfunctional T cell state that is often found in tumors or chronic infections. However, Tex Term exhibits high similarity with the beneficial tissue-resident memory T states (T RM ) in terms of their locations and transcription profiles. Our bioinformatic analysis predicted Zscan20 , a novel TF, to be uniquely active in Tex Term . Consistently, Zscan20 knock-out thwarted the differentiation of Tex Term in vivo , but not that of T RM . Furthermore, perturbation of Zscan20 programs T cells into an effector-like state that confers superior tumor and virus control and synergizes with immune checkpoint therapy. We also identified Jdp2 and Nfil3 as powerful Tex Term drivers. In short, our multiomics-based approach discovered novel TFs that enhance anti-tumor immunity, and enable highly effective cell state programming. One sentence summary: Multiomics atlas enables the systematic identification of cell-state specifying transcription factors for therapeutic cell state programming.

In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells.

We report a serum-free, 3D murine artificial thymic organoid (M-ATO) system that mimics normal murine thymopoiesis with the production of all T cell stages, from early thymic progenitors to functional single-positive (CD8SP and CD4SP) TCRαß and TCRγδ cells. RNA sequencing aligns M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATOs initiated with Rag1-/- marrow produce the same differentiation block as seen in the endogenous thymus, and Notch signaling patterns in M-ATOs mirror primary thymopoiesis. M-ATOs initiated with defined hematopoietic stem cells (HSCs) and lymphoid progenitors from marrow and thymus generate each of the downstream differentiation stages, allowing the kinetics of T cell differentiation to be tracked. Remarkably, single HSCs deposited into each M-ATO generate the complete trajectory of T cell differentiation, producing diverse TCR repertoires across clones that largely match endogenous thymus. M-ATOs represent a highly reproducible and efficient experimental platform for the interrogation of clonal thymopoiesis from HSCs.

Pleiotropic Roles of VEGF in the Microenvironment of the Developing Thymus.

Neonatal life marks the apogee of murine thymic growth. Over the first few days after birth, growth slows and the murine thymus switches from fetal to adult morphology and function; little is known about the cues driving this dramatic transition. In this study, we show for the first time (to our knowledge) the critical role of vascular endothelial growth factor (VEGF) on thymic morphogenesis beyond its well-known role in angiogenesis. During a brief window a few days after birth, VEGF inhibition induced rapid and profound remodeling of the endothelial, mesenchymal and epithelial thymic stromal compartments, mimicking changes seen during early adult maturation. Rapid transcriptional changes were seen in each compartment after VEGF inhibition, including genes involved in migration, chemotaxis, and cell adhesion as well as induction of a proinflammatory and proadipogenic signature in endothelium, pericytes, and mesenchyme. Thymocyte numbers fell subsequent to the stromal changes. Expression patterns and functional blockade of the receptors VEGFR2 and NRP1 demonstrated that VEGF mediates its pleiotropic effects through distinct receptors on each microenvironmental compartment of the developing mouse thymus.

Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells.

The ability to generate T cells from pluripotent stem cells (PSCs) has the potential to transform autologous T cell immunotherapy by facilitating universal, off-the-shelf cell products. However, differentiation of human PSCs into mature, conventional T cells has been challenging with existing methods. We report that a continuous 3D organoid system induced an orderly sequence of commitment and differentiation from PSC-derived embryonic mesoderm through hematopoietic specification and efficient terminal differentiation to naive CD3+CD8αß+ and CD3+CD4+ conventional T cells with a diverse T cell receptor (TCR) repertoire. Introduction of an MHC class I-restricted TCR in PSCs produced naive, antigen-specific CD8αß+ T cells that lacked endogenous TCR expression and showed anti-tumor efficacy in vitro and in vivo. Functional assays and RNA sequencing aligned PSC-derived T cells with primary naive CD8+ T cells. The PSC-artificial thymic organoid (ATO) system presented here is an efficient platform for generating functional, mature T cells from human PSCs.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αß+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vß expression, consistent with allelic exclusion of Vß-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.