Article Figures & Data
Figures
- Figure 1.
Loss of CDK8 is dispensable for NK-cell development and maturation. A, Sorted Cdk8fl/fl and Cdk8fl/flNcr1Cre splenic NK cells (left) or poly(I:C)-treated Cdk8fl/fl and Cdk8fl/flMx1Cre splenocytes (right) were isolated and the protein level of CDK8 was analyzed by Western blot. B, Frequency of Lin−(CD3−CD19−Ly-6G−Ter119−) CD122+ NK cells in bone marrow and (C) percentages of CD3−NKp46+ NK cells in the spleen were assessed by flow cytometry. D, Bone marrow Lin−CD122+ cells were further divided into NK precursors (NKPs: NKp46−NK1.1−), immature NK cells (iNKs: NKp46−NK1.1+), and mature NK cells (mNKs: NKp46+NK1.1+). E, Splenic CD3−NKp46+ cells were analyzed for the expression of the maturation markers CD27 and CD11b. Shown are representative plots. The summarized data is presented in Supplementary Fig. S1. F, The abundance of KLRG1+, NKG2D+, and DNAM-1+ cells among CD3−NKp46+ NK cells was assessed in the spleen of Cdk8fl/fl, Cdk8fl/flNcr1Cre, and poly(I:C)-treated Cdk8fl/fl and Cdk8fl/flMx1Cre mice. Shown are representative histograms. The summarized data are presented in Supplementary Table S1. B–E, Bar graphs and values on the plots represent mean ± SEM of 2 independent experiments; n = 6–7.
- Figure 2.
CDK8-deficient NK cells show an increased cytotoxic capacity. A and B, IL2-expanded Cdk8fl/fl and Cdk8fl/flNcr1Cre NK cells were stimulated with IL12 or IL15 for 2 hours, and the expression of (A) CDK8, pSTAT1–S727, and total STAT1 and (B) CDK19 was analyzed by Western blot. C, MACS-purified Cdk8fl/fl and Cdk8fl/flNcr1Cre NK cells were plated in IL2 and the cell numbers were determined daily by manual cell counting using a Neubauer chamber. Symbols and error bars represent mean cell number ± SEM of technical triplicates from 1 out of 2 independent experiments with similar outcome. D, Cdk8fl/fl and Cdk8fl/flNcr1Cre splenocytes were stimulated with anti-NK1.1, IL2 + IL12, or IL2 + IL12 + IL15 for 4 hours and the percentage of IFNγ+ CD3−NKp46+ NK cells was analyzed by flow cytometry. Bar graph represents mean percentage of IFNγ+ NK cells ± SEM from 2 to 3 independent experiments; n = 4–6 per group. E, IL2 expanded Cdk8fl/fl and Cdk8fl/flNcr1Cre NK cells were stimulated with IL12 or IL15 for 2 hours and the expression of perforin and GZMB was analyzed by Western blot. F, Cdk8fl/fl and Cdk8fl/flNcr1Cre splenocytes were stimulated with IL2 + IL12 or IL2 + IL12 + IL15 for 4 hours and the expression of perforin and GZMB in CD3−NKp46+ cells was analyzed by intracellular staining. Representative histograms were overlaid. Bar graph represents mean MFI of perforin in NK cells or percentage of GZMB+ NK cells ± SEM from 2 independent experiments; n = 4 per group. G, IL2-expanded Cdk8fl/fl and Cdk8fl/flNcr1Cre NK cells were incubated for 3 to 4 hours with CFSE-stained YAC-1, B16F10, or v-abl+ target cells in effector-to-target ratios of 10:1, 5:1, and 1:1. The specific lysis of target cells was assessed by flow cytometry and a representative graph out of 2 independent experiments is shown. Symbols and error bars represent mean ± SEM of technical triplicates. *, P < 0.05; **, P < 0.01.
- Figure 3.
Mice lacking CDK8 in NK cells show reduced B16F10 lung metastasis. A,Cdk8fl/fland Cdk8fl/flMx1Cre mice were treated with poly(I:C) as described in the Materials and Methods section. On day 16, 5 × 104 B16F10 melanoma cells were injected i.v. into the mice. After 23 days, the number of pulmonary tumor nodules was assessed. B, 5 × 104 B16F10 melanoma cells were injected i.v. into Cdk8fl/fl and Cdk8fl/flNcr1Cre mice. After 23 days, the number of tumor nodules in the lung was assessed. A, B, Shown are representative lung pictures (left) and bar graphs representing mean ± SEM from 3 independent experiments; n = 11–12. *, P < 0.05.
- Figure 4.
Loss of CDK8 in NK cells results in improved antitumor response against v-abl+ leukemic cells. Cdk8fl/fl and Cdk8fl/flNcr1Cre mice were injected s.c. with 106 v-abl+ cells and after 10 days the tumor weight was assessed and tumor infiltrating NK cells were analyzed by flow cytometry. Shown are (A) representative tumor pictures and (B) bar graph representing mean tumor weight relative to body weight ± SEM from 2 independent experiments; n = 12–15. C, Representative plots of tumor infiltrating CD3−NKp46+ cells pregated on CD19− cells from Cdk8fl/fl (left panel) and Cdk8fl/flNcr1Cre mice (right panel) are shown and (D) their percentage among CD19− cells is presented as bar graph showing mean ± SEM from 2 independent experiments; n = 11. E and F, Tumor single-cell suspensions were left untreated or stimulated with IL2 + IL12 + IL15 for 4 hours and the percentage of IFNγ+CD3−NKp46+ NK cells was analyzed by flow cytometry. E, Representative histograms of unstimulated and stimulated tumor samples were overlaid. F, The bar graph represents mean percentage of IFNγ+ NK cells ± SEM from 1 experiment; n = 4 per group; *, P < 0.05; **, P < 0.01.
- Figure 5.
Loss of CDK8 in NK cells provides a survival benefit in a chronic leukemia model. Newborn Cdk8fl/fl (n = 10) and Cdk8fl/flNcr1Cre (n = 8) mice were injected s.c. with a replication-incompetent ecotropic retrovirus encoding for v-abl (A-MuLV). A, The Kaplan–Meier plot summarizes 2 independent experiments. B, The spleen/body weight ratio and the white blood cell count (WBC) at the survival endpoint are represented by bar graphs showing mean ± SEM of 2 independent experiments. C, Representative plots of bone marrow–infiltrating B cells (CD19+B220+) are shown, and the values represent mean ± SEM of one representative out of 2 independent experiments (n = 4–5). D, H&E stains of bone marrow from leukemia-bearing Cdk8fl/fl and Cdk8fl/flNcr1Cre mice were performed at the disease endpoint. E, The frequency of CD3−NKp46+ NK and CD3+ T cells amongst splenic, bone marrow or blood lymphocytes of leukemia-bearing mice at the disease endpoint were determined by flow cytometry. Bar graph represents mean ± SEM of one representative out of 2 independent experiments (n = 4–5). *, P < 0.05.
Additional Files
Volume 6, Issue 4
