Clonal Acquisition of Inhibitory Ly49 Receptors on Developing NK Cells Is Successively Restricted and Regulated by Stromal Class I MHC Claude Roth, James.

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Clonal Acquisition of Inhibitory Ly49 Receptors on Developing NK Cells Is Successively Restricted and Regulated by Stromal Class I MHC Claude Roth, James R Carlyle, Hisao Takizawa, David H Raulet Immunity Volume 13, Issue 1, Pages 143-153 (July 2000) DOI: 10.1016/S1074-7613(00)00015-7

Figure 1 Clonal Growth of Sorted Bone Marrow–Derived Cell Populations Employed for Analysis of NK Cell Development Percoll gradient purified bone marrow cells were further purified by cell sorting for the indicated markers and cultured at limiting dilution in the presence of stromal cells and cytokines. (A) Gates for three-color sorting of NK1.1+CD3−CD19−Ly49A/C/F/G2/I− cells that express DX5 are shown (left panels). Reanalysis of the sorted NK1.1+DX5+CD3−CD19−Ly49A/C/F/G2/I− population revealed a 91% pure population (right panels). (B) Gates for three-color sorting of c-kit+Sca-2+ cells that are also negative for lineage markers, including NK1.1, are shown (left panels). Reanalysis of the sorted c-kit+Sca-2+Lin− population revealed a 91%–93% pure population (right panels). (C) Clonal growth of sorted NK1.1+DX5+Ly49A/C/F/G2/I− and c-kit+Sca-2+Lin− precursor cells. Both populations were cultured at limiting dilution with either irradiated B6 bone marrow stromal cells (closed circles) or irradiated OP9 stromal cells (open circles) in the presence of c-kit ligand, IL-7, flt-3 ligand, and IL-15. The number of growing wells containing NK1.1+ cells was determined. The frequency of wells that did not contain growing NK1.1+ cells at day 10 is plotted against the number of input cells. Results are representative of three independent experiments each. Immunity 2000 13, 143-153DOI: (10.1016/S1074-7613(00)00015-7)

Figure 2 Ly49 Expression by Clones Derived In Vitro from NK1.1+DX5+Ly49A/C/F/G2/I− or c-kit+Sca-2+Lin− Precursor Cells (A) Representative clones derived from NK1.1+DX5+Ly49A/C/F/G2/I− precursor cells, derived at 1 input cell per well (clones F3, D2, and B1) or 0.5 cell per well (clones F5, B12, H3) are shown. Percentages are indicated in the quadrants. (B) Representative clones derived from c-kit+Sca-2+Lin− precursor cells, derived at 32 input cells/well (clones F6, H1, H5, H6), 16 cells/well (clones C3, G8), or 8 cells/well (clone A8) are shown. For H1 and H5 clones, double staining with anti-Ly49G2 and anti-Ly49C/I is shown on gated NK1.1+ cells. Some clones contained a CD19+ population. Percentages correspond to percent of NK1.1+ cells in the colonies. Immunity 2000 13, 143-153DOI: (10.1016/S1074-7613(00)00015-7)

Figure 3 Sorted Ly49+ Bone Marrow Cell Populations Employed for Analysis of NK Cell Development (A) CD3−CD19−NK1.1+ B6-derived bone marrow cells were sorted for the expression of Ly49A, but not Ly49F, G2, C/I. Post-sort analysis indicated a purity of 92%. (B) CD3−CD19−NK1.1+ B6-derived bone marrow cells were sorted for the expression of Ly49G2, but not Ly49A, F, C/I. Post-sort analysis indicated a purity of 95%–98%. Immunity 2000 13, 143-153DOI: (10.1016/S1074-7613(00)00015-7)

Figure 4 Influence of Stromal Class I MHC on the Cumulative Expression of Ly49G2 on Clonally Differentiating Ly49Atg c-kit+Sca-2+Lin− Precursors (A and B) The percentage of NK cells expressing Ly49G2 in each colony from Ly49Atg multipotent precursors that differentiated on class I MHC-different stromal cells are indicated. Bone marrow precursors (Ly49Atgc-kit+Sca-2+Lin−) were sorted and cloned onto irradiated bone marrow stromal cells derived from B6 (H-2b), B10.D2 (H-2d), and β2m−/− mice. Colonies were analyzed for Ly49G2 expression (on NK1.1+ cells) by flow cytometry. Each circle represents one colony; the mean percentages (thick bars) ± SEM are shown. Statistical significance values based on the two-tailed Student's t test are indicated. The dotted line represents the ≤5% threshold used for plotting (C) and (D). (C and D) The frequency of Ly49Atg clones that generated ≤5% Ly49G2+ NK cells per colony (see [A] and [B], dotted line) are indicated for clones derived on each type of stroma. Statistical significance values based on the two-tailed chi-square (χ2) test are indicated. (E and F) The levels of Ly49G2 surface expression are indicated for clones derived on each type of stroma. Colonies were analyzed for the mean fluorescence intensities (MFI ± SEM) of Ly49G2 expression (on NK1.1+Ly49G2+ cells). Statistical significance values based on the two-tailed Student's t test are indicated. Immunity 2000 13, 143-153DOI: (10.1016/S1074-7613(00)00015-7)