FIGURE 2

Characterization of cellular and soluble ligand(s) for Mill1 and Mill2. (a) BM cells from B6 mice were stained with either ULBP3-Fc (control), Mill1-Fc or Mill2-Fc (as indicated). (b) B6 BM gated on live (FCS/SSC) B220⁺ cells and subfractioned into (1) early pre-B cells, (2) late pre-B cells and (3) immature B cells. Staining of fractions 1–3 from (b) with Mill2-Fc (filled histograms) is depicted in (c). (d) B6 BM subfractioned into four subsets based on CD71 and Ter119 as follows: (1) pro-erythroblasts, (2) basophilic erythroblasts, (3) chromatophilic erythroblasts and (4) ortho-chromatophilic erythroblasts and erythrocytes. Staining of fractions 1–4 from (d) with Mill2-Fc (filled histograms) is shown in (e). (f) B6 splenocytes were activated with anti-CD3 for 48 h, gated on CD4⁺ and CD8⁺ cells (as indicated) and assessed for labelling with Mill1-Fc and Mill2-Fc (as marked). In the upper panel of (g), B6 splenocytes were activated for 72 h with LPS, gated on CD19⁺ cells and assessed for labelling with Mill2-Fc. In the bottom four panels, BM cells were cultured in GM-CSF (MDC) or Flt-3L (PDC) for 7 or 9 days, respectively and stained with Mill2-Fc prior to or after activation for 24 h (as indicated). For MDC, cells were gated as CD11b⁺/CD11c⁺. For PDC, cells were gated as CD11c⁺/CD11b^-. (h) B6 BM was stained with Mill2-Fc in the presence of the indicated molecules. Staining with ULBP3-Fc (control) is shown as the black histogram. (i) B6 BM was stained with Mill1-Fc or Mill2-Fc at pH 7 or pH 6.8 (as marked). For (c), (e–g) and (i), staining with ULBP3-Fc (control) is shown as open histograms. BM, bone marrow; GM-CSF, granulocyte-macrophage colony-stimulating factor; MDC, myeloid dendritic cells; PDC, plasmacytoid dendritic cells.