The role of BCL11B in T lineage fate during human thymopoiesis and pluripotent stem cell differentiation

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ABSTRACT/SUMMARY The functional limitations and logistical challenges of using patient-derived (autologous) products for adoptive T cell therapy has prompted the exploration of a universal source of “off-the-shelf” T cells generated from self- renewing PSCs which can be readily genetically engineered to enhance function and expanded without limit. However current PSC differentiation systems are characterized by low T cell output and concurrent production of innate lymphoid cells (ILCs). Our preliminary studies suggest that the earliest stages of T cell specification and commitment seen during PSC differentiation do not fully recapitulate either normal human thymopoiesis or in vitro models that use definitive hematopoietic stem and progenitor cells (HSPC) to initiate T cell development. The goal of this proposal is to understand the cellular and molecular differences between normal and PSC-derived T cell development, with a focus on the role of the transcription factor BCL11B. T cells are generated in the thymus after notch signaling from the microenvironment triggers a series of transcriptional events that initiate the T-lineage program in HSPCs; these events first produce early thymic progenitors (ETPs) (T lineage specification) and then extinguish alternative (non-T) lineage programs in multipotent ETPs (T lineage commitment). BCL11B is a critical regulator of both of these processes. Our published and preliminary data show that, in contrast to the mouse model, BCL11B is essential for T cell specification during human thymopoiesis and initiates the expression of several T-cell genes. Moreover, when BCL11B is overexpressed in cord blood HSPCs, the T cell program is launched more rapidly and efficiently, even in the absence of notch signaling. Surprisingly little is known about how the T cell lineage is generated from PSCs. Through scRNA-Seq analysis we have identified candidate ETPs and their immediate progeny as they emerge from PSC-derived hematopoiesis. We hypothesize that the rare PSC-derived ETPs in which the T cell program is launched are functionally and transcriptionally different from ETPs in the thymus, and that these intrinsic differences are detrimental for the generation of conventional T cells from PSCs. Further, we propose that chromatin remodeling induced by BCL11B mediates both T lineage specification and the fate decisions between the conventional T cell and innate lymphoid pathways. Specifically we will: 1. Define the earliest T lineage progenitors generated during PSC differentiation; 2. Determine the epigenetic underpinnings of T-cell specification in PSC-ATOs and in primary thymopoiesis; and 3. Define how BCL11B affects conventional T and innate lineage fate choices. These studies will yield new mechanistic insights about T-cell differentiation that are critical for the development of PSC-derived T-cell immunotherapies.