A Rapid and Scalable Subcutaneously Administered Murine Thymus Micro-organoid for Generating Functional T cells

Abstract

Thymic function can decline due to age-related involution, congenital disorders, acute infections or chemo/radiation therapy. Decline in thymic function leads to decreased T cell production and weakened immunity. To address these thymic insufficiencies, we sought to develop a transplantable and scalable micro-organoid system using fibroblasts and thymic cells. We have developed a reliable and rapid method to generate thymic micro-organoids using selectively screened fibroblasts and murine thymic cells. The thymic micro-organoids are cryo-preservable, injectable, and give rise to T cells both in vitro and in vivo. Thymic organoids expressed key genes required to sustain T cell development and maturation: ccl25, dll-1, dll-4, foxn-1, il-7, scf. When injected into T cell-deficient Prkdcscid mice, the organoids gave rise to functional {beta}, {gamma}{delta}, natural killer T (NKT) cells, and FoxP3+ regulatory T cells. Organoid-derived T cells expressed a diverse T cell receptor (TCR) repertoire in vivo and respond to stimulation with anti-CD3/28, Concanavalin-A, or Phytohemagglutinin. Thymic organoids derived from pmel-1 thymocytes gave rise to V{beta}13+ T cells that delayed the growth of B16 melanoma and enhanced activation of T and NK cells. This approach presents a valuable tool for mechanistic studies and addressing current therapeutic gaps in diseases associated with thymic decline and insufficiencies.