Figure 1.

CD47 deficiency increases immature NK-lineage cells in spleen. (A) Time line showing C57BL/6 mice received i.p. injections of 750 μl PBS or CD47-morpholino (10 μmol/L in PBS) and were analyzed 2 weeks later. (B) Morphology of spleens at day 14 of treated and control mice. (C,D) Splenocytes were stained for lineage (Lin: B220, CD19, CD11b, CD11c, CD49b, CD105, MHC-II, and Ter119), CD3, CD4, and CD8 and acquired in a flow cytometer. DAPI was used to discriminate live/dead cells. Representative contour plots (values indicate percentage of parent population), frequency and counts of live Lin⁻CD3⁻CD4⁻CD8⁻ cells in PBS vs. CD47-morpholino treated mice are shown, n = 3. (E) Morphology of spleens was depicted from WT and Cd47^−/− littermate mice. (F–G) Splenocytes were enriched for lineage-negative cells and stained for CD3, CD4, and CD8. FACS plots and ImageStream analysis show staining for live Lin⁻-CD4⁺, -CD8⁺, -CD3⁺CD4⁻CD8⁻ (-CD3⁺DN) and -CD3⁻CD4⁻CD8⁻ (-CD3⁻DN) cells in WT and Cd47^−/− spleens. (H) RT-qPCR analysis of Lin⁻-CD4⁺, -CD8⁺, -CD3⁺DN and -CD3⁻DN populations sorted from spleens of Cd47^−/− mice was performed, β-Actin and Gapdh used as reference genes and relative normalized expressions are shown, n = 3. Representative contour plots (values indicate percentage of parent population) and counts of live FcR-blocked (I,J) CD45.2⁺CD3⁻CD4⁻CD8⁻NK1.1⁺NKp46⁺ cells and (K,L) CD45.2⁺Lin (CD11b, CD11c, CD19, B220, CD49b, CD105, MHC-II, and Ter119)⁻CD3⁻CD4⁻CD8⁻NK1.1⁺CD122⁺ cells in the spleens of WT and Cd47^−/− littermate mice are shown. Data are representative of two experiments involving 3–4 mice per experiment (Mean ± SEM).