Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1

Abstract

The molecular and cellular signals that guide T-cell development from hematopoietic stem and progenitor cells (HSPCs) remain poorly understood. The thymic microenvironment integrates multiple niche molecules to potentiate T-cell development in vivo. Recapitulating these signals in vitro in a stromal cell-free system has been challenging and limits T-cell generation technologies. Here, we describe a fully defined engineered in vitro niche capable of guiding T-lineage development from HSPCs. Synergistic interactions between Notch ligand Delta-like 4 and vascular cell adhesion molecule 1 (VCAM-1) were leveraged to enhance Notch signaling and progenitor T-cell differentiation rates. The engineered thymus-like niche enables in vitro production of mouse Sca-1cKit and human CD34 HSPC-derived CD7 progenitor T-cells capable of in vivo thymus colonization and maturation into cytokine-producing CD3 T-cells. This engineered thymic-like niche provides a platform for in vitro analysis of human T-cell development as well as clinical-scale cell production for future development of immunotherapeutic applications.

A new role for Notch in the control of polarity and asymmetric cell division of developing T cells

“…VCAM-1 contributes to stromal-DN3 interactions and its presence is associated with improved in vitro generation of T cells (Abe et al, 2010;Shukla et al, 2017). We therefore assessed the impact of Fc-VCAM-1 on DL4-mediated polarisation during division by functionalising the surfaces with both ligands.…”

“…Notch signalling is required for progression through β-selection and subsequent fate choices (Ciofani et al, 2004;Ciofani et al, 2005). We exploited two model systems to explore the role of Notch1 during division of developing T cells; the OP9-DL1 coculture, and surfaces functionalised with Fc-DL1 (Janas et al, 2010;Mohtashami et al, 2010;Schmitt et al, 2002;Shukla et al, 2017;Van de Walle et al, 2013). We show that the interaction of Notch1 with its ligands, DL1 and DL4, drives the polarisation of Notch1 itself, and consequently the polarisation of cell fate determinants, α-Adaptin and Numb.…”

Posttransplant chimeric antigen receptor therapy

“…[68][69][70][71][72][73] Stromal-free cell culture systems are emerging. 74 Thus, Notch-based culture systems may offer an opportunity to generate naïve T cells for cell-based immunotherapies. Several reports have demonstrated the efficient generation and therapeutic potential of T-cell precursors generated by Notchbased culture of hematopoietic progenitor cells.…”

Concise Review: Human Pluripotent Stem Cells to Produce Cell-Based Cancer Immunotherapy

“…More recently, Zúñiga‐Pflücker and coworkers developed a more defined coculture system based on the critical function of the Notch pathway in T cell development. Studies using hematopoietic progenitor cells isolated from different sources demonstrated that overexpressing Notch ligand Delta‐like‐1 (OP9‐DL1) or Delta‐like‐4 (OP9‐DL4) expressed by mouse bone marrow‐derived OP9 stromal cells provides a suitable microenvironment to initiate T lymphopoiesis . These studies have used cells from human fetal liver , human umbilical cord blood (UCB) , and mouse ESC‐derived hematopoietic cells .…”

“…Studies using hematopoietic progenitor cells isolated from different sources demonstrated that overexpressing Notch ligand Delta‐like‐1 (OP9‐DL1) or Delta‐like‐4 (OP9‐DL4) expressed by mouse bone marrow‐derived OP9 stromal cells provides a suitable microenvironment to initiate T lymphopoiesis . These studies have used cells from human fetal liver , human umbilical cord blood (UCB) , and mouse ESC‐derived hematopoietic cells . The in vitro‐derived CD8 + SP T cells exhibit standard pathway activation upon stimulation and are capable of killing specific target cells if engineered with CAR and TCR .…”