C-Kit Signaling Blocks B Cell Differentiation of Common Lymphoid Progenitor Cells by Negatively Regulating Stat5-Dependent Activation of Ebf Expressionc-Kit Signaling Blocks B Cell Differentiation of Common Lymphoid Progenitor Cells by Negatively Regulating Stat5-Dependent Activation of Ebf1 Expression

Abstract 1209 B-lymphocyte commitment from common lymphoid progenitor cells (CLP) is orchestrated by a number of transcription factors and external cues such as IL-7, stem cell factor (SCF) and Flt3 ligand. IL-7 signaling plays a critical role in promoting B-cell differentiation by inducing the expression of transcription factor Ebf1 via the Jak-Stat5 signaling pathway (Kikuchi et al., 2007; Tsapogas et al., 2011). Previously, it has been shown that Flt3 ligand acts synergistically with IL-7 during the earliest stages of B-cell commitment (CLP and pre-pro-B fraction)(Ahsberg et al., 2010), but is downregulated by the pre-B stage to ensure normal development of B lymphocytes (Holmes et al., 2006). The role of SCF signaling in this early cell fate decision, however, remains unclear. Here, we demonstrate that SCF dominantly inhibits IL-7-induced B-cell differentiation from CLP by negatively regulating the Jak-Stat5 signaling pathway. Co-culture of bone marrow Lin-Kit+Sca-1+(LSK) cells with OP9 feeder cells in the presence of IL-7 (10 ng/ml) and SCF (10 ng/ml) resulted in 1.5–2-fold reduction in the frequency and absolute numbers of B220+CD19+ cells compared to cultures stimulated with IL-7 alone. To test whether c-Kit signaling inhibits B cell commitment in vivo , BM progenitor cells overexpressing a constitutively active form of KIT (MSCV-Kit-V814D-IRES-VEX) were transplanted into lethally irradiated mice. VEX+ cells in reconstituted mice had a very limited potential for B-lymphoid lineage differentiation, as judged by severe reduction in the frequency of B220+ cells in BM at 7 weeks post-transplant [B220+CD19-IgM-: 1.5%±0.3 (control) vs 0.6%±0.2 (V814D), p<0.05, n=6); B220+CD19+: 19.4%± 2.7 vs 2.1%±0.6, p<0.001; CD19+IgM+: 6.7%±1.3 vs 0.9%±0.3, p<0.01)]. Analysis of CLP cells isolated from IL-7−/− mice revealed that co-culture with IL-7 and SCF inhibited IL-7-induced upregulation of Ebf1 as well as other Stat5 target genes ( Socs3 , Cish , Osm ). Furthermore, short-term treatment of BM cells isolated from IL-7−/− mice with a combination of SCF and IL-7 resulted in a significant reduction of phospho-Stat5 in CLP cells compared to cells treated with IL-7 alone. Class III receptor tyrosine kinases such as Flt3 and c-Kit are able to activate multiple signaling pathways via a juxtamembrane SH2-docking site. Mutation of the SH2 domain in the context of the Kit-V814D mutation (Kit-V814D-Y567/569F) did not rescue B-cell development when this mutant protein was expressed in primary murine bone marrow cells in vivo . In contrast, expression of a mutant form of Kit-V814D that inhibited binding of the SH2B adapter protein 2 (APS) (Kit-V814D-I570A/L937A) rescued B-lymphopoiesis, indicating that a signaling pathway coupled to APS dominantly suppresses B cell commitment when CLP are co-stimulated with SCF and IL-7. Based on the observations that APS is a known adapter for the E2-ubiquitin ligase, c-Cbl, which has been shown to negatively regulate Stat5 in other systems, we speculate that c-Kit signaling inhibits IL-7-induced Stat5 activation via an APS-Cbl pathway during early B-cell development. Further characterization of this inhibitory developmental pathway will be discussed. Disclosures: No relevant conflicts of interest to declare.