In Vivo Generation of Thymus-Independent T Cells in a Tissue-Engineered T Cell Development Supporting Microenvironment

Tissue engineering approaches based on implantation of biomimetic three-dimensional (3D) tissue constructs have been used for more than a decade for in vivo regeneration of various tissues including skin, bone, and cartilage. Engineering of functional lymphoid tissues is challenging due to the specific requirements for lymphoid differentiation and has barely been accomplished to date. The aim of this study was to develop a method for engineering of a T cell development supporting 3D microenvironment and to assess if implantation of such a tissue construct is feasible and results in thymus-independent T cell development in vivo.

We fabricated 3D scaffolds using the FDA-approved biodegradable polyester polycaprolactone (PCL) as well as PCL/collagen composites. The scaffold architecture was designed to provide mechanical and geometrical properties necessary to support cell growth, proliferation, differentiation, and motility. We utilized a layer-by-layer assembly approach to fabricate structures that consisted of alternating thin nanofiber layers and thick microfiber layers (A, B). Nanofiber layers were fabricated by electrospinning from a PCL/collagen solution. These layers were functionalized by vascular endothelial growth factor incorporation to promote vascularization and seeded with OP9-DL1 stromal cells to provide Notch signaling. Microfiber layers were highly porous structures fabricated from PCL by salt leaching, and were encapsulated with a biomimetic dextran-based hydrogel containing in vitro generated T lineage committed lymphoid precursor cells as well as growth factors, cytokines and chemokines required for T cell development (A, B). The layer-by-layer assembled structure with alternating layers of nanofibers and microfibers provided optimal support for stromal cell growth while facilitating angiogenesis and T lineage cell expansion and migration enabled by the highly porous microfiber layers.

Subcutaneous implantation of layer-by-layer tissue constructs into athymic nude mice resulted in rapid vascularization. Implanted T lineage cells could be detected by in vivo bioluminescence imaging at the implantation site (C) for more than 1 month, and progeny of these cells efficiently reconstituted secondary lymphoid organs of both allogeneic and syngeneic recipients. Flow cytometric analysis of donor cells in lymph nodes, spleen, and the implant 4 weeks after implantation identified both CD4 and CD8 single positive T cells as well as CD4 and CD8 double positive T cells and NKT cells. The majority of T cells had an effector memory phenotype (but central memory and na•ve cells were also present).

We conclude that thymus-independent in vivo T cell generation in a tissue-engineered artificial microenvironment is feasible, and further development of this technology is expected to produce an exciting innovative strategy for the treatment of T cell deficiency.