Cell-Autonomous Defects in Dendritic Cell Populations of Ikaros Mutant Mice Point to a Developmental Relationship with the Lymphoid Lineage  Li Wu, Aliki Nichogiannopoulou, Ken Shortman, Katia Georgopoulos  Immunity  Volume 7, Issue 4, Pages 483-492 (October 1997) DOI: 10.1016/S1074-7613(00)80370-2

Figure 1 Lack of DCs in the Thymus and Spleen of Ikaros DN−/− Mutant Mice Pooled (four to eight) thymuses or spleens from the wild-type control mice and pooled spleens from Ikaros DN−/− mutant mice were subjected to dendritic cell extraction and enrichment procedures as described in Experimental Procedures. The enriched dendritic cell preparations were stained in three fluorescent colors with FITC–anti–MHC class II and PE–anti-CD8α and biotin anti-CD11c, then Red-670–avidin as the second stage. Pooled (four to eight) thymuses from Ikaros DN−/− mice were digested with collagenase and treated with EDTA, then stained with the same fluorescently labeled antibodies as above. The stained cells were analyzed on the FACScan. The fluorescence intensities are expressed on a four-decade logarithmic scale in all FACS profiles in this paper. Boxed DC populations are shown as percentages of the events collected. The MHC ClassII+ DCs are also CD11c+ (data not shown). Immunity 1997 7, 483-492DOI: (10.1016/S1074-7613(00)80370-2)

Figure 2 Hemopoietic Lineage Development in the Bone Marrow Chimeric Mice The bone marrow chimeric mice were made by cotransferring intravenously 5 × 104 host-type Ly 5.1 bone marrow cells together with 5 × 106 Ikaros DN−/− bone marrow or wild-type bone marrow cells (Ly 5.2) into irradiated Ly 5.1 recipients (detailed in Experimental Procedures). Four weeks after transfer, the thymuses and spleens from these chimeric mice were pooled, and single-cell suspensions were stained with fluorescently labeled antibody against donor type Ly 5.2 and with antibodies against different hemopoietic lineage markers. Thymocytes were stained with FITC–anti–Ly 5.2 and PE–anti-CD4. Spleen cells were stained with FITC–anti–Ly 5.2 and PE–anti-CD4 for T cells, or FITC–anti–Ly 5.2 and PE–anti-B220 for B cells, or FITC–anti–Ly 5.2 and biotin–anti-Mac-1 and followed with Red-670–avidin as the second stage for myeloid cells. The percentages of Ly 5.2 donor-derived T cell, B cell, and macrophage are indicated in the upper right quadrants. Immunity 1997 7, 483-492DOI: (10.1016/S1074-7613(00)80370-2)

Figure 3 Dendritic Cell Development in the Ikaros DN−/− Bone Marrow Chimeras The bone marrow chimeric mice described in Figure 2 were also analyzed for DC development 4 weeks after transfer. Pooled thymuses, spleens, and LNs from the recipient mice were subjected to DC extraction and enrichment procedures. The DC-enriched preparations were stained in two fluorescent colors with FITC-conjugated anti-donor type Ly 5.2 together with biotinylated anti-MHC class II, followed by Red-670–avidin as the second stage. The percentages of Ly 5.2 donor-derived DCs are indicated in the upper right quadrants. Immunity 1997 7, 483-492DOI: (10.1016/S1074-7613(00)80370-2)

Figure 4 The Presence of Low Levels of CD8α+DEC-205+ DCs in the Thymus and Spleen, and the Absence of CD8α−DEC-205− DCs in the Spleen of Ikaros Null C−/− Mice Pooled (five to seven) thymuses or spleens from Ikaros C−/− and wild-type siblings were subjected to DC extraction and enrichment procedures. The DC-enriched cell preparations were stained in three fluorescent colors with FITC–anti–MHC class II, PE–anti-CD8α, and biotin–anti-CD11c or biotin–anti-DEC-205, followed with Red-670–avidin as the second stage. The phenotypes of DCs were analyzed by gating for MHC class II+ cells. The expression of DC markers CD11c, CD8α, and DEC-205 on the gated MHC class II+ cells is represented by the solid lines in the histograms. The broken lines represent the background staining with isotype-matched control rat immunoglobulin. The absolute cell numbers of each DC population per thymus or per spleen are summarized in Table 5. Immunity 1997 7, 483-492DOI: (10.1016/S1074-7613(00)80370-2)

5 Immunity 1997 7, 483-492DOI: (10.1016/S1074-7613(00)80370-2)