Common lymphoid progenitors (CLPs) clonally produce both B- and T-cell lineages but have little myeloid potential in vivo. B- and T-cell development (ALP). Results and Discussion CLPs and the earliest B-cell progenitors reside at the transition from Kit-expressing progenitors to Cd19-expressing B-cell precursors and thus represent the ideal intermediates to test MiDReG using the established seed genes. To identify markers that could potentially separate B-lineage-committed from uncommitted progenitors within the CLP population we used the following seed conditions for MiDReG (Fig. Clemizole hydrochloride 1). For the first seed we used the logical combination “Kit high AND Mpl high” to represent the progenitors as both are expressed on early hematopoietic cells but not mature B cells. For the second seed we used “Cd19 high AND Cd3? low ” as CD3? is expressed only on T cells and therefore this combination would eliminate arrays of heterogeneous populations (e.g. whole-tissue arrays). We focused exclusively on genes encoding cell surface proteins as designated by the Gene Ontology (GO) database with commercially available antibodies suitable for flow cytometry. From this analysis MiDReG identified 26 genes encoding cell surface proteins that were predicted to be Clemizole hydrochloride differentially expressed during B-cell development: 19 up-regulated (Fig. 1B) and seven down-regulated (Fig. 1C). Figure 1. Prediction of surface markers up-regulated or down-regulated during B-cell development. (A) Prediction of genes encoding cell surface molecules up-regulated in B-cell development. The MiDReG algorithm uses Boolean implications from mouse data sets only. … We focused on four genes-Cd34 Cd27 Il1r1 Clemizole hydrochloride and Ly6d as antibodies to these proteins were readily available-and examined their surface expression during the progression through the MPP CLP pre-pro-B and Fraction B (Fr. B) stages. Because of the known presence of non-B-lineage cells within the pre-pro-B-cell population (Li et al. 1996; Nikolic et al. 2002) we first recharacterized this population as described in Supplemental Figure S1 to isolate only the B-cell progenitors within this population. Despite MiDReG’s prediction of down-regulation CD34 protein levels were not markedly different between the MPP CLP pre-pro-B and Fr. B stages (data not shown). The other surface proteins however revealed interesting expression patterns. CD27 is a known marker for early hematopoietic stem and progenitor cells but is not expressed in mature na?ve B cells. As we summarize in Supplemental Figure S2 CD27 is expressed on MPP CLP and pre-pro-B cells but separates Fr. B cells into CD27+ and CD27? subsets. Further characterization reveals that these subsets which we call Fr. B1 and Fr. B2 are functionally distinct and represent early and late stages of Fr. B respectively. Clemizole hydrochloride Interleukin-1 receptor 1 (Il1r1) is accurately predicted by MiDReG to be down-regulated during B-cell development (Supplemental Fig. S3). Furthermore we find that CLP pre-pro-B and Fr. B cells all respond differently to IL-1 in vitro. While CLPs develop exclusively into dendritic cells (DCs) in IL-1 cultures pre-pro-B cells die by apoptosis and Fr. B cells are unaffected (Supplemental Fig. S3). The fourth marker Ly6d was predicted by MiDReG to be up-regulated during B-cell development (Fig. 1B). Ly6d also known as ThB (Eckhardt and Herzenberg 1980) is expressed in all mature B cells and plasmacytoid DCs as well as developing but not mature thymocytes (Supplemental Fig. S4; Reese et al. 2001). We examined Ly6d expression in MPP CLP and pre-pro-B cells and found that MPPs were uniformly Ly6d? as were all hematopoietic stem cells while Ly6d was highly expressed on PROM1 pre-pro-B cells (Fig. 1D). Interestingly Ly6d expression divided the CLP population into two approximately equal subpopulations (Fig. 1D). Ly6d? CLPs expressed higher levels of Kit than Ly6d+ CLPs similar to the level of ckit expression observed for MPPs consistent with a progenitor/progeny relationship between these three populations (Fig. 1E; Supplemental Fig. S5). Ly6d? CLPs also expressed lower levels of IL7Rα than Ly6d+ CLPs (Supplemental Fig. S5). We also observed that Ly6d? CLPs developmentally precede Ly6d+ CLPs in vitro (Supplemental Fig. S6). As we show below only the Ly6d? subset of CLPs possesses full lymphoid potential. To distinguish this population from the original CLPs we designate this population ALPs. As the Ly6d+ subset is almost totally B-cell-committed we hereafter refer to this population as BLPs. We next examined the in vivo lymphoid potential of.