DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

Abstract

T cells are key mediators of the adaptive immune response and show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to serve as a consistent and renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we developed an efficient serum- and feeder-free protocol for differentiating human PSCs into hematopoietic progenitors and T cells. This defined method allowed us to study the impact of individual recombinant proteins on blood emergence and lineage potential. We demonstrate that the presence of DLL4 and VCAM1 during the endothelial-to-hematopoietic transition (EHT) enhances downstream progenitor T cell output by >80-fold. Using single cell transcriptomics, we showed that these two proteins synergise to drive strong notch signalling in nascent hematopoietic stem and progenitor cells and that VCAM1 additionally drives a pro-inflammatory transcriptional program. Finally, we applied this differentiation method to study the impact of cytokine concentration dynamics on T cell maturation. We established optimised media formulations that enabled efficient and chemically defined differentiation of CD8αβ+, CD4-, CD3+, TCRαβ+ T cells from PSCs.

T-Cell Progenitors As A New Immunotherapy to Bypass Hurdles of Allogeneic Hematopoietic Stem Cell Transplantation

“…In an effort to develop clinically compatible methods, feeder cell-free immobilized DLL1 or DLL4 microbead-based techniques have been developed to generate T-cell progenitors and T cells from iPSCs ( 75 , 90 ). Progenitor T-cell differentiation has also been shown to be improved by the induction of endothelial-to-hematopoietic transition on DLL4 and VCAM1 ( 99 ).…”

E-cadherin in developing murine T cells controls spindle alignment and progression through β-selection

“…Vascular cell adhesion molecule 1 (VCAM1) has the capacity to provide such an interaction, being presented by cortical stromal cells and binding the α 4 integrin on early T cell progenitors (19). The VCAM1-α 4 integrin interaction is critical for cells just before the β-selection stage [termed double negative 2 (DN2)] to migrate to the outer cortex where they commence β-selection (19) and synergizes with Notch ligands for in vitro differentiation to the DN3 stage (20,21). However, expression of the α4 integrin drops sharply from the DN2 stage (21), and polarization to the Notch ligand, delta-like 4 (DL4) is inhibited, rather than enhanced, by VCAM1 in DN3a undergoing asymmetric cell division (ACD) (6).…”

E-cadherin in developing murine T cells controls spindle alignment and differentiation during β-selection

“…VCAM1 has the capacity to provide such an interaction, being presented by cortical stromal cells and binding the α4 integrin on early T cell progenitors (Prockop, Palencia et al 2002). Indeed, the VCAM1-α4 integrin interaction is critical for cells just prior to the β-selection stage (termed DN2) to migrate to the outer cortex where they commence β-selection (Prockop, Palencia et al 2002), and synergizes with Notch ligands for in vitro differentiation to the DN3 stage (Shukla, Langley et al 2017, Michaels, Edgar et al 2021). However, expression of the α4 integrin drops sharply from the DN2 stage (Michaels, Edgar et al 2021), and polarisation to DL4 is inhibited, rather than enhanced, by VCAM1 in DN3a undergoing asymmetric cell division (Charnley, Ludford-Menting et al 2020).…”