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Blood, Vol. 112, Issue 9, 3753-3761, November 1, 2008
Lymphoid precursors are directed to produce dendritic cells as a result of TLR9 ligation during herpes infection
Blood Welner et al.
112: 3753
Supplemental materials for: Welner et al
Files in this Data Supplement:
- Figure S1. Lymphoid progenitors acquire myeloid differentiation potential as a result of TLR9 ligation while NK cell formation is unchanged (JPG, 28.7 KB)
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CLP were sorted to purity, stimulated with or without CpG for 48 h in culture, harvested and re-plated in Methocel with recombinant growth and differentiation factors (A). Colonies were counted on day 10. Similarly, pre-cultured CLP were placed in B or NK supportive serum-free cultures (B). Data are representative of three independent experiments.
- Figure S2. TLR9 ligation of myeloid progenitors does not stimulate DC formation (JPG, 33.2 KB)
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Sorted CMP were stimulated for 48 h with medium alone or CpG before re-culture in the presence of Flt3 ligand (left and middle panels) or recombinant FL, G-CSF, IL-3 and IL-6 (right panel). Cells were harvested after an additional 8 days and evaluated by flow cytometry for B220−CD19−CD11c+CD11b+ cDCs (left panel), B220+CD19−CD11c+CD11b+ pDCs + NK-like IKDC (middle panel) and CD19−CD11b+GR-1+ myeloid cells (right panel). Data are representative of three independent experiments.
- Figure S3. Responsiveness of common lymphoid progenitors (CLP) to IL-7 is suppressed after short term exposure to CpG (JPG, 44.3 KB)
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CLPs were isolated from normal mice and cultured for 24 h with medium or 0.6 µg/ml of CpG. Surface IL-7Rα was determined by flow cytometry (A). After washing and resting for 20 min, subgroups of the cells were stimulated with IL-7 for 20 min. All aliquots were then fixed and stained for phospho-STAT5 and examined by flow cytometry. One representative histogram is shown from three separate experiments (B). STAT5b-CA transgenic mice were used to supply a constituently active signal from IL-7Rα. Highly purified CLPs from these mice were treated with LAL water or CpG for 48 h and then cultured in the presence of IL-7, SCF and FL to support B lymphopoiesis. Flow cytometry was used to evaluate the differentiation of mature cells from these cultures (C). Data are representative of two independent experiments.
- Figure S4. Cells responding to a TLR9 agonist do not influence neighboring progenitors in culture (JPG, 32.2 KB)
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CLP were isolated to high purity from BM of normal CD45.1+ and TLR9−/− CD45.2+ mice before 48 h of culture with LAL water or CpG. They were washed, mixed as indicated and incubated in serum-free, stromal cell-free cultures with SCF, FL and IL-7 for 8 days before flow cytometry analyses. CD45.1+ and TLR9−/− CD45.2+ cells were cultured separately as controls (not shown), while results from 50:50 mixture cultures are given. The CD45 staining profiles allow discrimination of the cells made from CD45.2− normal and CD45.2+ TLR9−/− mice. Data are representative of three independent experiments.
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