Finally we tested whether the B cell differentiation defect

Finally, we tested whether the B cell differentiation defect in the OsxCre;iDTR mutants could be rescued in vivo by intravenous administration of recombinant IL-7 and IGF-1. Both controls and mutants were subjected to daily injections of DT, DT + IL-7, or DT + IGF-1 for 12 days. At the end of the rescue regimen, bone marrow cells were evaluated by flow cytometry (Figures 7A–7H). IL-7 administration increased the C′ and C″ pro-B populations (Figures 7C and 7D) but did not rescue later B stages (Figures 7F–7H). In contrast, IGF-1 had no effect at the early pro-B stages (Figures 7A–7E) but rescued the pre-B and mature B levels (Figures 7F–7H). These results suggest that while IL-7 supports the differentiation of early B precursors, IGF-1 is required for downstream B cell maturation and that Osx cells regulate this process via production of IGF-1.
Discussion These and our recent publication (Yu et al., 2015a) demonstrate that mesenchymal cell populations in the niche have stage-specific functional interactions with the hematopoietic system. Specifically, while primitive mesenchymal cells regulate HSC, we have recently shown that Ocn+ cells modulate production of T cells through the regulation of thymus-seeding progenitors. Those data indicate that T lymphoid progenitor specification and production were diminished with Ocn+ deletion; DLL4 was the identified Ocn+ cell product responsible for the production of these T cell precursors. Here we show that specific steps of B cell differentiation are affected by the partial loss of Osx cells. The importance of osteolineage cells in regulating PD 0332991 within the bone marrow has been known from reports documenting the roles of IL-7 and CXCL12, among other factors (Ding and Morrison, 2013; Greenbaum et al., 2013; Visnjic et al., 2004; Wu et al., 2009; Zhu et al., 2007). Previous studies reported that IGF-1 promotes B cell expansion in vitro (Taguchi et al., 2006) and that activation of the IGF-1 receptor is needed for immunoglobulin production (Baudler et al., 2005). Local IGF-1 production may be most important for marrow B lymphopoiesis, as prior studies of either liver or pituitary-specific IGF-1 deficiency only observed altered B cell numbers in the spleen and not within the bone marrow (Montecino-Rodriguez et al., 1997; Welniak et al., 2004). Our data indicate that osteoprogenitors secrete IL-7 and IGF-1; both are necessary to support full B lineage differentiation in the bone marrow (Figure 7I). Deletion of Osx cells lead to minimal changes in IL-7 serum levels despite significant knockdown of Il7 transcripts. It is likely that other cell types compensate for the production of IL-7 in the niche. Rescue experiments showed that IGF-1 is required together with IL-7 to restore the full differentiation program of B cells. These data suggest that IL-7 controls early stages of B cell differentiation, in agreement with previous data (Tokoyoda et al., 2004), while IGF-1 is necessary downstream of IL-7 for the differentiation into pro-B and mature B cells. Taken together, our data suggest that marrow B lymphopoiesis requires localized IGF-1 production while extramedullary maturation is more affected by systemic IGF-1. Having defined through the triple transgenic model that Osx cells are distinctive molecularly from Ocn+ cells, we tested their selective function. The model we used was intentionally of short duration to avoid the progression of Osx cells to Ocn-expressing, ++ cells. In vivo, these cells are dynamic and are lost after ∼90 days (Park et al., 2012). Our data demonstrate that the Osx cells have no effect on CLP or the T competent progenitors, Ly6D−CLP (Figure 4C) in contrast to the Ocn+ cells. Nor do they produce DLL4 at levels comparable with those in Ocn+ cells. However, they do express abundant IL-7 and IGF-1 and through these molecules regulate the maturation of B cell through the pro-B cell stage. This maturation step is critical, as blunting B cell progression by deleting Osx support cells resulted in B lymphopenia and functional compromise of immunoglobulin production.