Accelerating Immune Reconstitution in HSCT Patients through iPSC-Derived Thymic Epithelial Cells

T cells are central to host defense and tolerance, yet the critical role of the thymus in facilitating T cell development and TCR repertoire selection is often overlooked. Thymic function declines in the second decade of life and is susceptible to a wide range of pathogenic insults. Compromise of thymic function is a frequent complication after HSCT as a result of radiation, anti-thymocyte globulin, CMV infection and acute GVHD. Thymic injury can lead to delayed immune reconstitution, opportunistic infections, autoimmunity and chronic GVHD. Reconstituting thymic function, e.g. through induced pluripotent stem cell (iPSC)-derived thymic epithelial cells (TECs), holds great therapeutic promise. However, the regenerative potential of the thymus remains clinically untapped as the signals that drive thymic ontogeny beyond the anterior foregut are incompletely understood.

In this study, we elucidated pathways instructing commitment and specialization of the human thymic epithelial stroma through orthogonal single cell transcriptomic analysis strategies. First, we inferred inductive cues that contribute to the initiation of thymic organogenesis by comparing gene regulatory networks expressed in human fetal epithelial cells of the thymus and other anterior foregut-derived organs. Then we defined lineage trajectories within the thymic epithelium by evaluating human thymus samples sequentially across embryonic, fetal, and early postnatal stages.

Our data revealed that responses to types I and II interferon distinguish epithelial cells of the thymus from other anterior foregut-derived organs. We further found that the downstream effects of interferon extend differentially within the cortical and medullary lineages, reflected in characteristic NFκB and IRF signatures (Figure 1A). We have translated the core set of gene regulatory networks identified in our transcriptomic study into a morphogen-based approach to advance the differentiation of iPSCs into TECs. TECs derived from this protocol demonstrate high transcriptional fidelity with primary human fetal intertypic TECs that can give rise to cortical and medullary lineages (Fig. 1B). To test the functional capacity of iPSC-derived TECs to give rise to human T cells, and specifically to execute positive and negative selection, we transplanted iPSC-derived TECs into humanized athymic NSG-nude mice (NSG-Foxn1 ^Null) (Figure 1C). 12 weeks after transplantation, flowcytometric analysis of dissociated TEC grafts showed robust human T cell development recapitulating the physiologic progression from double-negative to double-positive to TCRab-rearranged single CD4 and CD8 positive T cells observed in primary human thymus. (Figure 1D)

These results provide proof of concept that transplantable iPSC-derived TECs can promote T cell development in vivo and have potential as a regenerative thymic replacement therapy for patients suffering from complications of defective T cell immune reconstitution after HSCT.