Project Summary/Abstract: Continuous functional T and B lymphoid output is critical to overall health and life, as delayed and reduced lymphopoietic output has been associated with serious illness and even mortality. The lymphoid system is particularly sensitive to hematopoietic perturbation, both in the context of steady state and regeneration. Hematopoietic stem cell transplantation (HSCT) into irradiated recipients and stress from infection or other insults in native hematopoietic systems have been shown to negatively regulate lymphoid output. One of the challenges to developing clinical therapies to restore healthy immune function is that we don’t have enough information on lymphoid development in vivo. This is true for both the embryonic immune system which hPSC blood differentiation systems attempt to reproduce and the adult immune system. Understanding the mechanisms by which lymphoid output from HSPCs is controlled at clonal, transcriptional, and epigenetic levels is critical for revealing how lymphoid output is established and regulated in steady state, how it is suppressed with regeneration, and whether there is a specific signature associated with reduced lymphoid output that is conserved between native and non-native systems. It was recently shown that the HSC specific transcription factor Tcf15 is indispensable for long term self-renewal, following HSCT. Intriguingly, I have demonstrated that Tcf15 also negatively regulates both T and B lymphoid differentiation. This was true both in native hematopoiesis and regeneration. It is well established that HSCs display multilineage priming. This allows HSCs to readily regenerate the hematopoietic system, upon perturbation and to differentiate into lineage restricted progenitor cells to meet native hematopoietic demands. In addition, reduced lymphoid lineage priming has been shown to promote HSC expansion. For the mentored K99 phase of this proposal, I will transition from a focus on Tcf15 in native hematopoiesis, to a focus on Tcf15 in lymphopoiesis, a focus that will allow me to establish research independence from my mentor. With the evidence of multilineage HSC priming, the evidence of native antagonism between HSC self-renewal and lymphoid differentiation programs, and my preliminary data hypothesize that Tcf15 expression attenuates lymphoid lineage priming in HSCs and that this is conserved between steady state and regeneration. I propose to test this hypothesis in two aims: In Aim 1, I will apply scRNA- seq, scATAC-seq, and proteomic analyses to elucidate the transcriptional and epigenetic program by which Tcf15 regulates T and B lymphoid differentiation from HSPCs. In Aim 2, I will use Tcf15 reporter and CRISPR- based lineage tracing (CARLIN) mouse models generated in our lab to explore the effect of endogenous Tcf15 on T and B cell specification from HSCs. For the R00 component of this award, I will seek to continue my examination of regulation of lymphoid lineage priming, but down...