Figure 4.
CML suppression of NK cell cytotoxicity is partially restored by targeting cytokine signaling. (A) The effect of CML soluble factors on target-specific NK cell degranulation. NK cells were sorted from healthy C57BL/6 CD45.2 mice and treated with peripheral blood serum samples from control or CML mice for 24 hours. Yac-1-specific degranulation was measured as described in Figure 2. (B) Cish transcript evaluation by RT-PCR in healthy wild-type NK cells exposed to serum from control or CML mice for 4 hours. Median with a range. (C) Target-specific degranulation in CishKD (shaded bars) or scrambled-treated NK cells preexposed to control or leukemic serum. Mean ± SD. (D) Soluble molecules differentially present in peripheral blood (PB serum) and bone marrow fluid of control and CML mice. Evaluated by enzyme-linked immunosorbent assay microarray from RayBiotech. All presented analytes besides those highlighted in gray in PB serum have significantly different concentrations between control and CML fluids. Highlighted in black (black arrow) are the cytokines previously described in patients with CML or mouse models. Cytokines highlighted in red (red arrow) signal through STAT5 and therefore have a potency to tonically induce Cish expression in NK cells. (E) NK cell “in vivo response to the TNFα” scores in control vs CML-exposed NK cells. Genes upregulated in NK cells upon in vivo treatment with TNFα are upregulated in CML-exposed NK cells,57 suggesting the sensitivity of NK cells to TNFα in leukemia in CML. Median. (F) Genes negatively regulated in NK cells upon in vivo treatment with TNFα are downregulated in CML-exposed NK cells.57 Median. (G) TNFα concentrations in blood serum samples of control and CML mice. Median with a range. (H) TNFR2+ cells in the blood and splenic NK cells of control and CML mice. Mean ± SD. (I) Target-specific degranulation in etanercept-treated (shaded) and untreated NK cells preexposed to control or leukemic serum. Mean ± SD. (J) Cytokines inducing Cish expression in NK cells in vivo.57 Z-scores are normalized to the baseline Cish expression in phosphate-buffered saline injection controls. Statistics and images were obtained at https://www.immune-dictionary.org/app/home. Only the cytokines and cytokine combinations upregulated in CML mice are displayed. (K) Cish expression in healthy wild-type NK cells, untreated or treated with etanercept (shaded), exposed to serum from control or CML mice for 6 hours. Median with a range. For all experiments, unpaired t test or 2-way ANOVA with Fisher's least significant difference test; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001; n = 3 to 14 mice per group. ns, nonsignificant.

CML suppression of NK cell cytotoxicity is partially restored by targeting cytokine signaling. (A) The effect of CML soluble factors on target-specific NK cell degranulation. NK cells were sorted from healthy C57BL/6 CD45.2 mice and treated with peripheral blood serum samples from control or CML mice for 24 hours. Yac-1-specific degranulation was measured as described in Figure 2. (B) Cish transcript evaluation by RT-PCR in healthy wild-type NK cells exposed to serum from control or CML mice for 4 hours. Median with a range. (C) Target-specific degranulation in CishKD (shaded bars) or scrambled-treated NK cells preexposed to control or leukemic serum. Mean ± SD. (D) Soluble molecules differentially present in peripheral blood (PB serum) and bone marrow fluid of control and CML mice. Evaluated by enzyme-linked immunosorbent assay microarray from RayBiotech. All presented analytes besides those highlighted in gray in PB serum have significantly different concentrations between control and CML fluids. Highlighted in black (black arrow) are the cytokines previously described in patients with CML or mouse models. Cytokines highlighted in red (red arrow) signal through STAT5 and therefore have a potency to tonically induce Cish expression in NK cells. (E) NK cell “in vivo response to the TNFα” scores in control vs CML-exposed NK cells. Genes upregulated in NK cells upon in vivo treatment with TNFα are upregulated in CML-exposed NK cells,57 suggesting the sensitivity of NK cells to TNFα in leukemia in CML. Median. (F) Genes negatively regulated in NK cells upon in vivo treatment with TNFα are downregulated in CML-exposed NK cells.57 Median. (G) TNFα concentrations in blood serum samples of control and CML mice. Median with a range. (H) TNFR2+ cells in the blood and splenic NK cells of control and CML mice. Mean ± SD. (I) Target-specific degranulation in etanercept-treated (shaded) and untreated NK cells preexposed to control or leukemic serum. Mean ± SD. (J) Cytokines inducing Cish expression in NK cells in vivo.57 Z-scores are normalized to the baseline Cish expression in phosphate-buffered saline injection controls. Statistics and images were obtained at https://www.immune-dictionary.org/app/home. Only the cytokines and cytokine combinations upregulated in CML mice are displayed. (K) Cish expression in healthy wild-type NK cells, untreated or treated with etanercept (shaded), exposed to serum from control or CML mice for 6 hours. Median with a range. For all experiments, unpaired t test or 2-way ANOVA with Fisher's least significant difference test; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001; n = 3 to 14 mice per group. ns, nonsignificant.

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