Identification of a Developmentally-Restricted Hematopoietic Stem Cell That Gives Rise to Innate-like Lymphocytes

The generation of innate-like immune cells distinguishes fetal hematopoiesis from adult hematopoiesis, but the cellular mechanisms underlying differential cell production during development remain to be established. Specifically, whether differential lymphoid output arises as a consequence of discrete hematopoietic stem cell (HSC) populations present during development or whether the fetal/neonatal microenvironment is required for their production remains to be established. We recently established a Flk2/Flt3 lineage tracing mouse model wherein Flk2-driven expression of Cre recombinase results in the irreversible switching of a ubiquitous dual-color reporter from Tomato to GFP expression. Because the switch from Tom to GFP expression in this model involves an irreversible genetic excision of the Tomato gene, a GFP+ cell can never give rise to Tom+ progeny. Using this model, we have definitively demonstrated that all functional, adult HSC remain Tomato+ and therefore that all developmental precursors of adult HSC lack a history of Flk2 expression. In contrast, adoptive transfer experiments of Tom+ and GFP+ fetal liver Lin-cKit+Sca1+ (KLS) fractions demonstrated that both Tom+ and GFP+ fetal HSC support serial, long-term multilineage reconstitution (LTR) in irradiated adult recipients. We have therefore identified a novel, developmentally restricted HSC that supports long-term multilineage reconstitution upon transplantation into an adult recipient but does not normally persist into adulthood. Developmentally-restricted GFP+ HSC display greater lymphoid potential, and regenerated both innate-like B-1 lymphocytes and Vg3-expressing T lymphocytes to a greater extent than coexisting Tom+ FL and adult HSC. Interestingly, whereas developmental regulation of fetal-specific B-cell subsets appears to be regulated cell-instrinsically, as fetal HSC generated more innate-like B-cells than adult HSC even within an adult environment, T-cell development may be regulated both cell intrinsically and extrinsically, as both the cell-of-origin and the fetal microenvironment regulated the generation of innate-like T-cells. Our results provide direct evidence for a developmentally restricted HSC that gives rise to a layered immune system and describes a novel mechanism underlying the source of developmental hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, these findings are critical for understanding the development of autoimmune diseases, allergies, and tolerance induction upon organ transplantation. Furthermore, by uncoupling self-renewal capacity in situ with that observed upon transplantation, our data suggests that transplantation- and/or irradiation-induced cues may allow for the engraftment of developmental HSC populations that do not normally persist in situ. As LTR upon transplantation has served as the prevailing definition of adult HSC origin during development, our data challenge the current conceptual framework of adult HSC origin.