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First published online 16 August 2006
doi: 10.1242/dev.02510

Development 133, 3619-3628 (2006)
Published by The Company of Biologists 2006
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Fetal spleen stroma drives macrophage commitment

Julien Y. Bertrand1,2,*, Guillaume E. Desanti1,*, Richard Lo-Man3, Claude Leclerc3, Ana Cumano1 and Rachel Golub1,{dagger}

1 Unité du Développement des Lymphocytes, INSERM U668, Institut Pasteur, 25, Rue du Dr Roux, 75724 Paris cedex 15, France.
2 University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0380, USA.
3 Unité de Biologie des Regulations Immunitaires, INSERM E352, Institut Pasteur, 25, Rue du Dr Roux, 75724 Paris cedex 15, France.


Figure 1
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  		Fig. 1. Colonization and maintenance of LTR-HSCs in the FS. (A) Sub-lethally irradiated Rag{gamma}c-/- mice (H-2k) were injected with one or two embryo equivalents (ee) of E13 FL, or with one, two, four, eight or 16 embryo equivalents of E13 FS, explanted from C57BL/6 embryos (H-2b). After 6 months, recipient BM was scored for the presence of H-2b+ donor-derived cells. Gr-1/Mac1 FACS plots are gated on live donor-derived H-2b+ cells to show the contribution of donor cells to the myeloid compartment. (B) E13 FL and FS explants (CD45.2) were cultured on filter for 4 days. FLOC and FSOC cell suspensions were injected into sub-lethally irradiated Rag{gamma}c-/- recipients (CD45.1). After 5-6 months, recipient BM and spleen were scored for the presence of donor-derived (CD45.2+) B and myeloid cells (n=6). (C) E13 FL and FS explants (CD45.2 H-2b) were irradiated, reconstituted by the same number of E10.5 AGM cells (total of six embryo equivalents of CD45.1 H-2b) and, after 4 days, FSOC and FLOC (3/injection) were dissociated and tested for LTR in Rag{gamma}c-/- (CD45.1 H-2k). The BM of recipients was scored for the presence of AGM-HSC-derived B (CD19) and myeloid (granulocytes, Gr-1) cells (n=6).

 

Figure 2
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  		Fig. 2. AGM cell differentiation potential in FL and FS environments. (A) Differentiation of AGM-HSCs in the FS environment. CD45.1+ FS explants were seeded with E10.5 CD45.2+ AGM cells. After 8 days on filters, FSOC were scored for the presence of donor-derived B (CD19) and myeloid (Mac1) cells (n=12). Plots are gated on live PI- cells. (B) Differentiation of E13 FL precursors in the FS environment. CD45.1+ FS explants were seeded with E13 FL Ter119-depleted cell suspension (CD45.2+; n=12). Analyses are identical to A. (C) Differentiation of AGM-HSCs in the FL environment. CD45.2+ FL explants were seeded with E10.5 CD45.1+ AGM cells (n=12). In this microenvironment, AGM-HSCs could differentiate into CD19+ B cells, a few of them reaching the mature IgM+ stage by the end of the coculture (7 days only). Plots show cells gated on live donor-derived (CD45.1+ PI-) cells.

 

Figure 3
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  		Fig. 3. FS colonization by B committed precursors at E13. (A) CD45+ Ter119- cells from E13.5 FS and FL were sorted and analyzed by RT-PCR for the expression of transcription factors involved in B cell commitment: E47, Ebf and Pax5. E15 FL cells depleted of erythrocytes were used as a positive control. (B) FS and FL from E13.5 Rag2GFP transgenic embryos were scored by FACS for B220 and CD43. We also obtained B220+ CD43+ B progenitors that express GFP.

 

Figure 4
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  		Fig. 4. Fetal spleen stroma drives HSCs to the sole macrophage lineage. (A) HSCs were sorted from E14 FL and cultured for 8-10 days onto three different FSS lines (n=5). (B) To understand the lineage relationship between these two cell subsets, sorted CD45lo F4/80- cells were cultured for 7 days on FSS cells. Only CD45+ F4/80+ macrophages were obtained. Both F4/80- and F4/80+ populations were sorted and scored for the expression of myeloid/macrophage-related genes by RT-PCR. (C) For the RT-PCR, we used as positive control, two populations: E15 FL cells and macrophages from the peritoneal cavity (PeC). (D) Analysis of cell surface proteins by cytometry showing that macrophages are highly auto-fluorescent. The expression of indicated markers (gray line) was compared to a negative control (dashed line) that corresponds to the same unstained population (respectively, CD45lo F4/80- and CD45+ F4/80+).

 

Figure 5
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  		Fig. 5. Ontogeny of macrophage populations in the spleen. (A) CD45+ FS cells were gated and then the presence of macrophages was scored at different time-points [E15, newborn (NB), 5 days after birth (P5) and adulthood], by the expression of F4/80 and Mac1 molecules. At E15, macrophages account for half of the FS cell suspensions. (B) E15.5 FS (explanted from Rag2GFP embryos) was analyzed by immunohistochemistry for the presence and localization of F4/80 macrophages (red, arrows). Sections were observed with a Zeiss Axioplan 2 imaging microscope and the section shown is representative of many independent FS sections. We could determine no organization between macrophages (red), lymphoid progenitors (green, arrowheads) and erythrocytes-anucleated (delineated zones) or nucleated (asterisks)-although these latter seem to be in very close contact with macrophage. Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. Bone marrow precursor proliferation is highly enhanced by FSS supernatant. Total bone marrow cells (106) were cultured for 7 days with supernatant from either fetal spleen stromal cell lines (FSS) or a bone marrow stromal cell line (S17), or with recombinant growth factors, alone or mixed, as indicated. (A) Cell differentiation was analyzed by FACS for macrophages (CD11c- Mac1+ F4/80+ or F4/80-) and DCs (CD11c+); the percentage of each population is indicated. (B) Proliferation of total bone marrow precursors was evaluated for each condition by counting the absolute number of live cells (Trypan blue exclusion). To evaluate whether FSS supernatant selectively acts on the proliferation and differentiation of particular precursors, the number of each cell type obtained was determined. The percentage of macrophages and DCs in the various conditions of culture (obtained by FACS analysis in A) was multiplied by the absolute number of live cells.

 

Figure 7
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  		Fig. 7. FSS supernatant is a strong inhibitor of T cell proliferation. (A) T cell proliferation assays were carried out using 104 OT-II CD4+ T cells stimulated with 104 of different putative APCs (BM derived macrophages and DCs, FSS derived macrophages and FSS) in the absence (white bars) or presence (black bars) of 1 µg/ml of OVA323-336 peptide (pOVA) for 4 days. (B) Supernatants from serially diluted putative APC (10 to 105 cells), from the experiments shown in A, were added to a new proliferation assay performed with 104 OT-II CD4+ T cells stimulated with 104 BM-derived DCs. T cell proliferation was measured by [3H]-thymidine incorporation for a period of 18 hours and is expressed as the mean of cpm of duplicates.

 




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