American Society of Hematology

Figure 6

$Figure 6. CBFβ is required at an early stage of NK-cell development in vivo. (A) Representative scatter plots of Lin− 15.5 dpc FL cells grown on OP9 cells in the presence of Flt3L, IL-7, and IL-15. Gr-1−CD19− cells were analyzed for CD122 and NK1.1 expression. (B) Percentage and absolute number of CD122+ cells (± SD) harvested from OP9 cultures established from 3 Cbfb+/+ and 4 Cbfbrss/− fetuses (nexperiments = 3). (C) NK cells in 15.5 dpc fetal thymi. (D) Total number of thymic NK1.1+ and NK1.1+CD122+ cells averaged from 3 Cbfb+/+ and 3 Cbfbrss/− fetuses (nexperiments = 3). (E-G) Lethally irradiated CD45.1 × CD45.2 F1 recipients were reconstituted with wild-type CD45.1+ BM and CD45.2+ FL cells from Cbfb+/+, Cbfbrss/rss, or Cbfbrss/− fetuses (expressing 100%, 30%, and 15% of normal CBFβ levels, respectively). (E) Bone marrow of recipient mice analyzed 10 months after reconstitution. The data are from gated FL-derived (CD45.2+) progenitors. A representative example is shown in each group (Cbfb+/+, n = 4; Cbfbrss/rss, n = 4; Cbfbrss/−, n = 5). Bone marrow from a 10-week-old C57BL/6 (B6) mouse is shown as control. CD19−CD3−TCRβ−NK1.1+DX5+ cells are mature NK cells, whereas immature NK lineage cells (iNK) have a NK1.1+Dx5− phenotype. (F) The spleen of recipient mice analyzed in a similar way, illustrating the absence of immature and mature NK cells among cells derived from Cbfbrss/− progenitors. (G) Primitive NK lineage committed progenitors (NKP) (CD122/IL2Rβ+NKG2D+NK1.1−Dx5−LinT−) (LinT = CD3CD4CD8αTCRβTCRγ) are preserved in the spleen of recipient mice. NKP express the IL-2/IL-15Rβ chain and have an NK1.1−Dx5−CD19−CD3−CD4−CD8a−TCRβ−TCRγ− phenotype. A fraction of these cells expresses NKG2D.3 (H) Expression Runx1, Runx3, and Cbfb by qRT-PCR in LSK (Lin = CD4, CD8, TCRβ, TCRγ, DX5, CD19, Mac-1, Ter119), iNK (CD4−CD8−TCRβ−TCRγ−CD19−CD49b−NK1.1+CD122+) and mature NK (CD4−CD8−TCRβ−TCRγ−CD19−CD49b+NK1.1+CD122+) cells sorted from BM of 8- to 12-week-old wild-type mice. The purity of post-sort populations was more than 98%. Expression of individual genes was normalized to Hprt. * Significant difference (P≤ .01) from LSK cell values.$

CBFβ is required at an early stage of NK-cell development in vivo. (A) Representative scatter plots of Lin⁻ 15.5 dpc FL cells grown on OP9 cells in the presence of Flt3L, IL-7, and IL-15. Gr-1⁻CD19⁻ cells were analyzed for CD122 and NK1.1 expression. (B) Percentage and absolute number of CD122⁺ cells (± SD) harvested from OP9 cultures established from 3 Cbfb^+/+ and 4 Cbfb^rss/− fetuses (n_experiments = 3). (C) NK cells in 15.5 dpc fetal thymi. (D) Total number of thymic NK1.1⁺ and NK1.1⁺CD122⁺ cells averaged from 3 Cbfb^+/+ and 3 Cbfb^rss/− fetuses (n_experiments = 3). (E-G) Lethally irradiated CD45.1 × CD45.2 F1 recipients were reconstituted with wild-type CD45.1⁺ BM and CD45.2⁺ FL cells from Cbfb^+/+, Cbfb^rss/rss, or Cbfb^rss/− fetuses (expressing 100%, 30%, and 15% of normal CBFβ levels, respectively). (E) Bone marrow of recipient mice analyzed 10 months after reconstitution. The data are from gated FL-derived (CD45.2⁺) progenitors. A representative example is shown in each group (Cbfb^+/+, n = 4; Cbfb^rss/rss, n = 4; Cbfb^rss/−, n = 5). Bone marrow from a 10-week-old C57BL/6 (B6) mouse is shown as control. CD19⁻CD3⁻TCRβ⁻NK1.1⁺DX5⁺ cells are mature NK cells, whereas immature NK lineage cells (iNK) have a NK1.1⁺Dx5⁻ phenotype. (F) The spleen of recipient mice analyzed in a similar way, illustrating the absence of immature and mature NK cells among cells derived from Cbfb^rss/− progenitors. (G) Primitive NK lineage committed progenitors (NKP) (CD122/IL2Rβ⁺NKG2D⁺NK1.1⁻Dx5⁻LinT⁻) (LinT = CD3CD4CD8αTCRβTCRγ) are preserved in the spleen of recipient mice. NKP express the IL-2/IL-15Rβ chain and have an NK1.1⁻Dx5⁻CD19⁻CD3⁻CD4⁻CD8a⁻TCRβ⁻TCRγ⁻ phenotype. A fraction of these cells expresses NKG2D.³ (H) Expression Runx1, Runx3, and Cbfb by qRT-PCR in LSK (Lin = CD4, CD8, TCRβ, TCRγ, DX5, CD19, Mac-1, Ter119), iNK (CD4⁻CD8⁻TCRβ⁻TCRγ⁻CD19⁻CD49b⁻NK1.1⁺CD122⁺) and mature NK (CD4⁻CD8⁻TCRβ⁻TCRγ⁻CD19⁻CD49b⁺NK1.1⁺CD122⁺) cells sorted from BM of 8- to 12-week-old wild-type mice. The purity of post-sort populations was more than 98%. Expression of individual genes was normalized to Hprt. * Significant difference (P≤ .01) from LSK cell values.

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