The most significant finding in these studies was

The most significant finding in these studies was the 10-fold enhanced expansion of transplantable HSCs in mice engrafted with IK6-transduced human CD34+ CB cells. This was indicated both by the higher numbers of CD34+CD38− Decitabine Supplier regenerated from the IK6-transduced cells after 6 months in primary recipients and by the functional ability of these cells to reconstitute hematopoiesis in secondary mice for another 7.5 months. To our knowledge, this effect matches or exceeds any strategy for experimentally expanding human HSCs thus far reported. It also stands in sharp contrast to the reported suppression of mouse HSC expansion by disruption of IKAROS activity (Nichogiannopoulou et al., 1999; Papathanasiou et al., 2009), thus underscoring the importance of using primary human hematopoietic cells to interrogate the role of IKAROS in their normal regulation. Also striking was the consistent IK6-induced in vivo enhancement of human B-lineage cell generation, in contrast to the impaired B cell outputs obtained by loss of IKAROS activity in mouse cells (Joshi et al., 2014; Schwickert et al., 2014) also confirmed here. These effects of IK6 on human B cell development were presaged by the premature activation of a B-lineage transcriptional program in the human HSC-enriched CD34+CD38− compartment, which did not interfere with their HSC activity or subsequent execution of normal B cell differentiation. We also saw different effects on mouse and human T cell outputs, which were enhanced by IK6 expression in mouse cells, consistent with previous reports, whereas human T cell outputs were unaffected. Historical limitations inherent to previous human modeling systems may explain why we did not observe the IK6-induced suppression of human B cell production previously reported in engrafted NOD/SCID mice (Tonnelle et al., 2001). One potential explanation for the difference in effects of IK6 on mouse and human lymphopoiesis could be species-specific differences in the downstream events activated, as exemplified by the discrepancies in B cell phenotypes of human inherited immunodeficiency syndromes when compared with their equivalent mouse models (Conley et al., 2000; Mestas and Hughes, 2004). Dominant-negative isoforms may also differentially suppress wild-type IKAROS activity in the different models thus far described, raising the interesting possibility that the antiproliferative and pro-B-lineage differentiation roles of IKAROS may not be tightly linked. In the GM lineages, the observed enhancing effect of IK6 expression on human cells could be traced to several specific stages of GM progenitors and was associated with a selectively increased sensitivity to two granulopoietic growth factors, IL-3 and GM-CSF. These cytokines share a common signaling receptor component and hence activate many common downstream intermediates (Gubina et al., 2001). Hyperactivation of the transcription factor CREB, and its downstream targets FOS and Cyclin B1, was also revealed, consistent with a role of IKAROS in normally inhibiting the proliferation of primitive human hematopoietic cells. In agreement with previous findings (Dijon et al., 2008), an opposite effect on erythropoietic cells was noted. In summary, we provide multiple lines of evidence for a stimulatory role of IK6 in primitive normal human hematopoietic cells, associated with an enhanced activation of certain signaling intermediates involved in GM-CSF and IL-3 responses. This finding, reinforced by the demonstration of IKAROS protein expression in all known stages of primitive human hematopoietic cells, strongly implicates IKAROS as a cell-intrinsic negative regulator of HSCs and their immediate progeny in humans. However, none of these sequelae appear sufficient to lead to the induction of leukemia in human cells, suggesting that mutations targeting IKZF1 require additional alterations in order to produce such an outcome.
Experimental Procedures
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