The earliest step in B lineage differentiation from common lymphoid progenitors is critically dependent upon interleukin 7 - PubMed (original) (raw)

The earliest step in B lineage differentiation from common lymphoid progenitors is critically dependent upon interleukin 7

Juli P Miller et al. J Exp Med. 2002.

Abstract

Little is known about the signals that promote early B lineage differentiation from common lymphoid progenitors (CLPs). Using a stromal-free culture system, we show that interleukin (IL)-7 is sufficient to promote the in vitro differentiation of CLPs into B220(+) CD19(+) B lineage progenitors. Consistent with current models of early B cell development, surface expression of B220 was initiated before CD19 and was accompanied by the loss of T lineage potential. To address whether IL-7 receptor (R) activity is essential for early B lineage development in vivo, we examined the frequencies of CLPs and downstream pre-pro- and pro-B cells in adult mice lacking either the alpha chain or the common gamma chain (gamma(c)) of the IL-7R. The data indicate that although gamma(c)(-/-) mice have normal frequencies of CLPs, both gamma(c)(-/-) and IL-7R(alpha)(-/-) mice lack detectable numbers of all downstream early B lineage precursors, including pre-pro-B cells. These findings challenge previous notions regarding the point in B cell development affected by the loss of IL-7R signaling and suggest that IL-7 plays a key and requisite role during the earliest phases of B cell development.

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Figures

Figure 1.

Figure 1.

Lin− IL-7Rα**+**AA4+ Sca-1low BM cells (CLPs) yield B lineage precursors when cultured in IL-7 without stromal cells. (A) 1,000 sorted CLPs were cultured in stromal-free conditions with 10 ng/ml IL-7 for the indicated times before staining with antibodies to CD45R/B220, CD19, and AA4. (B) Cell recoveries for triplicate cultures for the experiment shown in A. (C) 1,000 sorted CLPs were cultured in stromal-free conditions with or without the indicated cytokine for 5 d, and then analyzed as described in A. (D) 1,000 sorted CLPs were cultured in triplicate in the presence or absence of the indicated cytokines(s), each at 10 ng/ml, and then analyzed 5 d later as described in B. All cells remained AA4+ (not depicted). Data are representative of eight separate experiments. Proliferative indices for B and D were calculated by dividing the number of viable cells recovered by the input cell number.

Figure 2.

Figure 2.

Lin− IL-7Rα+ AA4+ Sca-1low BM cells (CLPs) are a relatively homogeneous population of B/T progenitors that lose T lineage potential when cultured in IL-7. (A) Representative flow cytometric data from a lobe cocultured with three CLPs and containing detectable donor-derived cells. Freshly sorted CLPs from B6.Ly5SJL adults were cocultured with irradiated fetal thymi in hanging drop culture at a dose of 100, 10, or 3 cells/lobe for 24 h before placement in the MLP FTOC assay as described in Materials and Methods. Individual lobes were assessed 16 d later for the presence of donor-derived (B6.Ly5SJL+) B and T lineage cells as determined by staining with antibodies to CD19 (1D3) and CD3 (2C11), respectively. (B) Each data point is derived from a total of 20 thymi. (B) (C) Intrathymic transfers were performed using 3,000 C57BL/6 (Ly5B6) CLPs either freshly isolated or cultured in IL-7 4 d before transfer into B6.Ly5SJL recipients. After 18 d, thymocytes from each recipient were stained for the expression of CD4, CD8, and the B6 allele of CD45/Ly5. (D) Percentage of donor-derived (Ly5B6+) cells present in each B6.Ly5SJL host thymi. Each dot represents one recipient. Data are representative of two separate experiments.

Figure 3.

Figure 3.

Lack of AA4+ B220+ CD43+ pro-B cells in adults lacking components of the IL-7R. BM cells from 8-wk-old C57BL/6, μMT−/−, IL-7Rα+, and γc −/− mice were stained with FL-CD43 (S7), PE-CD19 (1D3), APC-Cy7-B220, APC-AA4, and BI–anti-CD24/HSA (30F1) revealed with SA-PE-TR. 200,000 events per tube were subsequently analyzed on a MoFlo® flow cytometer as described in Materials and Methods. Data are representative of four separate experiments. Numbers indicate the fraction of events among the parent population falling within the indicated gate and were consistent with three or more mice per group.

Figure 4.

Figure 4.

Frequencies of very early B lineage precursors, but not CLPs, are diminished in adult mice lacking components of the IL-7R. (A) BM cells from 8-wk-old C57BL/6, μMT−/−, IL-7Rα+, and γc −/− mice were stained with FL lineage markers (see Materials and Methods), PE-anti–IL-7Rα, APC-AA4, and BI-anti–Sca-1. N.A., not applicable. (B) BM cells from the aforementioned adults were stained with FL-Ly6C (AL-21), PE-B220, APC-AA4, and BI-CD24/HSA (30F1). Biotinylated antibodies were revealed with SA-PerCP-Cy5.5 and 200,000 events per tube were collected on a FACSCalibur® flow cytometer as described in Materials and Methods. Numbers indicate the fraction of events among the parent population falling within the indicated gate and were consistent for three or more mice per group. Data are representative of four separate experiments.

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