Rapid Generation of hPSC‐Derived High Endothelial Venule Organoids with In Vivo Ectopic Lymphoid Tissue Capabilities

Abstract Bioengineering strategies for the fabrication of implantable lymphoid structures mimicking lymph nodes (LNs) and tertiary lymphoid structures (TLS) could amplify the adaptive immune response for therapeutic applications such as cancer immunotherapy. No method to date has resulted in the consistent formation of high endothelial venules (HEVs), which is the specialized vasculature responsible for naïve T cell recruitment and education in both LNs and TLS. Here orthogonal induced differentiation of human pluripotent stem cells carrying a regulatable ETV2 allele is used to rapidly and efficiently induce endothelial differentiation. Assembly of embryoid bodies combining primitive inducible endothelial cells and primary human LN fibroblastic reticular cells results in the formation of HEV‐like structures that can aggregate into 3D organoids (HEVOs). Upon transplantation into immunodeficient mice, HEVOs successfully engraft and form lymphatic structures that recruit both antigen‐presenting cells and adoptively‐transferred lymphocytes, therefore displaying basic TLS capabilities. The results further show that functionally, HEVOs can organize an immune response and promote anti‐tumor activity by adoptively‐transferred T lymphocytes. Collectively, the experimental approaches represent an innovative and scalable proof‐of‐concept strategy for the fabrication of bioengineered TLS that can be deployed in vivo to enhance adaptive immune responses.