Strategies for Investigating Intrathymic Human T-Cell Development Using Humanized Mouse Models

T-cells play a critical role in mediating antigen-specific and long-term adaptive immunity against viral and bacterial pathogens. The thymus provides a highly specialized microenvironment for the generation of T-cells. T-cell immunodeficiency, or T-lymphopenia, can result from perturbations to the thymic microenvironment due to aging or irradiation required for cancer treatment, or from infections that directly target T-cells. Previous work by our group supports the co-transplant of in vitro-generated T-cell progenitors (proT-cells) for accelerating long-term hematopoietic stem cell (HSC)-derived T-cell reconstitution during periods of T-lymphopenia after HSC transplantation. However, an understanding of the mechanisms underlying proT mediated effects within the thymus, as well as systems for the generation of clinically scalable numbers of proT-cells, remain to be fully elucidated. I revealed a potential mechanism by which receptor activator of NF-ÎşB (RANKL)-expressing proT-cells reorganize the stromal cell compartment within the thymus of NOD/SCID/Îłcnull (NSG), creating chemokine-rich niches that likely favor the recruitment of HSC-derived progenitors. Given that proT-cells have important implications for T-cell regeneration during periods of lymphopenia, I established a scalable differentiation system, which enabled >250-fold expansion of hematopoietic stem/progenitor cells (HSPCs), for generation of increased quantities of proT-cells from a single HSPC donor. Both CD34+CD7+ and CD34-CD7+ proT-cells were generated using this strategy and while both subsets had thymus-engrafting capabilities, CD34+CD7+ cells exhibited higher thymic reconstitution. Building on the ability to manipulate HSPCs ex vivo, I also gene-modified HSPCs to express a secreted HIV entry inhibitor, soluble CD4 (sCD4). When tested in vivo, NSG hosts supported multi-lineage differentiation of human gene-modified HSPCs. Upon infection with HIV, I observed that humanized mice capable of secreting sCD4 demonstrated a reduction in viral load over time compared to control humanized mice. Taken together, this work demonstrates how large quantities of in vitro-generated proT-cells can be generated from ex vivo manipulated HSPCs for intrathymic repair and immunotherapeutic application.Ph.D