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Blood, Vol. 113, Issue 4, 807-815, January 22, 2009
Reductive isolation from bone marrow and blood implicates common lymphoid progenitors as the major source of thymopoiesis
Blood Serwold et al.
113: 807
Supplemental materials for: Serwold et al
Files in this Data Supplement:
- Figure S1. Reanalysis of sorted BM populations (JPG, 201 KB)
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BM was separated as shown in Fig. 1, and reanalyzed to assess purity. The top row shows the staining profile of lineage depleted BM as in Fig. 1, and gating hierarchy is indicated by arrows. All remaining rows show reanalysis of sorted populations, gated on total live cells.
- Figure S2. The Flk2+CD27+ compartment in BM and blood does not contain B220hi or Thy1hi cells (JPG, 183 KB)
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BM and blood were stained to reveal the Flk2+CD27+ subset, as shown in the left panels. Arrows in top panels indicate gating. The last column is gated on total live cells, and is useful for comparison of B220 and Thy1 staining levels. In both BM and blood, IL-7R+ and IL-7R− fractions could be detected, as seen in the second column, indicating that phenotypic CLP are present in the blood. The top two rows show the c-Kit versus B220 profiles of the indicated populations, while the bottom two rows display the c-Kit versus Thy1 profiles of the same populations. These data show that there are no B220hi or Thy1hi cells within the BM or blood Flk2+CD27+ population.
- Figure S3. Quantification of the percentage of donor cells at the DN (CD4−CD8−) stages of development over time (JPG, 29.1 KB)
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Data are pooled from the three experiments presented in Fig. 2. Each symbol represents one mouse, and the bar represents the average. Note that the CLP-derived thymocytes present at day 7 are largely at the DN stage. By day 14, very few CLP-derived DN cells remain. In contrast, the majority of MPP-derived thymocytes present at day 14 are still at the DN stage of development. As determined by the student’s t test, the differences in DN chimerism were significant for days 7, and 14 (p= .004, and .002 respectively).
- Figure S4. c-Kit and Flk2 levels are reduced in the BM seven days post-irradiation (JPG, 71.8 KB)
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FACS plots are gated on lineage−. Changes in the c-Kit and Flk2 profiles seven days after irradiation reflect either reduced surface levels of these proteins following irradiation or ablation of the cells expressing these markers. Since these changes could also affect donor cells, it was not possible to accurately determine donor Flk2+CD27+ BM chimerism seven days after transplant into irradiated recipients.
- Figure S5. MPP yields thymic chimerism eighteen days after transfer into non-irradiated IL-7R−∕− recipients (JPG, 112 KB)
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CLP and MPP were sorted, and BM equivalents (2.5 × 104 CLP and 1.4 × 104 MPP) were injected into unirradiated IL-7R−∕− recipients. Thymic chimerism was assessed eighteen days later, and MPP recipients had high levels of chimerism at this timepoint. Representative plots are shown. The left column is gated on live CD45+ events, and the right two columns are gated on the Thy1.1+ events from the left column.
- Figure S6. Donor-derived splenic lineages over time (JPG, 257 KB)
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Spleens from the timecourse experiments presented in Fig. 2 were analyzed for donor-derived lineages. Data are representative of 3 mice per group per timepoint in each of three experiments. Arrows in the top panels indicate gating hierarchy. Note that MPP-derived myeloid readout was evident at all timepoints, while very little CLP-derived myeloid readout could be detected. NK cells, DCs, and B cells were derived from both MPP and CLP, but there is a notable lag in B-cell production from MPP at the day 7 timepoint.
- Figure S7. Lineages found in the spleens of mice transplanted with blood Lin− Flk-2+ CD27+ or non–Flk-2+CD27+ cells (JPG, 100 KB)
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Spleens of animals transplanted with the populations described in Fig. 7 were analyzed for donor lineages. Blood Flk2+CD27+ cells yielded mostly B lineage cells in the spleen, but also gave a detectable CD11c+ population and a large Mac-1+, myeloid population, supporting the hypothesis that both MPP and CLP are present in this population. The non-Flk2+CD27+ population did not give nearly as many B cells, but did give similar numbers of Mac-1+ myeloid cells.
- Figure S8. MPP and CLP both proliferate and generate T-lineage cells seven days after intrathymic transplantation (JPG, 96.1 KB)
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1,000 CLP or MPP were double-sorted and injected intrathymically into sublethally irradiated CD45 congenic recipients. Seven days later, thymuses were analyzed for donor chimerism and for lineages. Gating hierarchy is indicated in top row. Both MPP and CLP made thymocytes at the DN2–DN3 stages. In addition, both generated CD11c+ dendritic cells. MPP also demonstrated significant monocyte/macrophage potential.
- Figure S9. Within the CLP population, both c-Kit+Sca-1+ and c-KitloSca-1lo subsets give rise to thymic chimerism seven days post transplantation (JPG, 80.1 KB)
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CLP were sorted as in Fig. 1C, and were then subjected to a second round of sorting in which they were divided into cells that fell within the c-Kit+Sca-1+ gate used to define MPP and those that did not fall in that gate. Cells not within the c-Kit+Sca-1+ gate are equivalent to the original phenotypic definition of CLP. Left plots show post-sort analysis of these populations. Plots are gated on lineage−Flk2+CD27+ cells. BM equivalents (1.2 × 104 non–c-Kit+Sca-1+ CLP and 1.1 × 103 c-Kit+Sca-1+ CLP) were injected intravenously into sublethally irradiated congenic recipients. Right plots show representative thymic chimerism after seven days. Arrows indicate gating hierarchy.
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