Journal: Communications Biology

Article Title: Tumor-derived NKG2D ligand sMIC reprograms NK cells to an inflammatory phenotype through CBM signalosome activation

doi: 10.1038/s42003-021-02440-3

Figure Lengend Snippet: Primary human NK cells enriched from PBMCs were stimulated with 100 ng/ml of recombinant sMICA in the absence or presence of sMIC-clearing monoclonal antibody B10G5 for 18 h. Total RNA was isolated for bulk RNA-sequencing analysis. a Heatmap representing the overall number of differentially expressed genes in human NK cells stimulated with sMICA, compared to the cells stimulated with sMICA in presence of B10G5. Primary mouse NK cells isolated from spleens of Rag −/− mice were stimulated with 100 ng/ml of recombinant sMICB in the absence or presence of B10G5 for 18 h. Total RNA was isolated for bulk RNA-sequencing analysis. b Heatmap representing the overall number of differentially expressed genes in mouse NK cells stimulated with sMICB, compared to the cells stimulated with sMICB in presence of B10G5. c Heatmap highlighting the key differentially expressed genes related to cytotoxicity and inflammatory response pathways in human NK cells upon stimulation with sMICA and sMICA+B10G5. d Heatmap highlighting the key differentially expressed genes related to cytotoxicity and inflammatory response pathways in mouse NK cells upon stimulation with sMICB and sMICB+B10G5. e Validation of representative genes associated with NK cell cytotoxicity and pro-inflammatory function (represented in ( b ) and ( d )) by qRT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (Student’s t test; two tailed).

Article Snippet: Mouse NK cells were isolated from Rag1 −/− mice, cultured in presence of IL-2 for 5 days (as described above) with a purity of more than 98% NK1.1 + cells, and stimulated with recombinant soluble MICB (Sino biologicals, 10759-H08H) in the absence or presence of B10G5 for 18 h. Cells were collected.

Techniques: Recombinant, Isolation, RNA Sequencing Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Tumor-derived NKG2D ligand sMIC reprograms NK cells to an inflammatory phenotype through CBM signalosome activation

doi: 10.1038/s42003-021-02440-3

Figure Lengend Snippet: Tumors from sMICB hi and sMICB lo TRAMP/MIC mice were harvested, and single-cell suspension was obtained using enzymatic digestion. CD45 + tumor-infiltrating lymphocytes were isolated by cell sorting. Single-cell RNA-sequencing analysis of tumor-infiltrating lymphocytes was conducted using the 10× genomics platform. a Representative images of (left panel) sMICB lo well-differentiated (WD) and (right panel) sMIC hi poorly differentiated (PD) tumors from TRAMP/MICB mice. b Serum levels of sMICB in WD tumors ( n = 6) and PD tumors ( n = 4) from TRAMP/MICB mice. c UMAP plots illustrating different tumor-infiltrating immune cell populations identified by clustering algorithms in well-differentiated sMICB lo and poorly differentiated sMICB hi tumors. d Heatmap representing key gene ontology (GO) pathways affected in NK cells in sMICB lo and sMICB hi tumors identified using gene set enrichment analysis (GSEA). e UMAP plots illustrating identified NK cell cytotoxic and pro-inflammatory clusters in the sMICB lo and sMICB hi tumors. f Heatmaps representing the gene expression of cytotoxic and pro-inflammatory markers at the single NK cell level in the identified NK cell cytotoxic sub-cluster (top panel) and pro-inflammatory sub-cluster (bottom panel) in the sMICB lo and sMICB hi tumors. g Box plots representation of average cytotoxic marker gene expression level (top panel) and average pro-inflammatory marker gene expression level (bottom panel) in NK cells and their comparison between sMICB lo and sMICB hi tumors. h Functional analysis of tumor-infiltrating NK cells from sMICB lo and sMICB hi tumors in response to ex vivo PMA/Ionomycin stimulation assessed by flow cytometry. Representative flow cytometry dot plots and histograms demonstrating higher NK1.1 + cell population, higher IFNγ, Granzyme B, and TNFα-producing NK cells from sMICB lo tumors compared to sMICB hi tumors. ** P < 0.01 (Student’s t test; two-tailed).

Article Snippet: Mouse NK cells were isolated from Rag1 −/− mice, cultured in presence of IL-2 for 5 days (as described above) with a purity of more than 98% NK1.1 + cells, and stimulated with recombinant soluble MICB (Sino biologicals, 10759-H08H) in the absence or presence of B10G5 for 18 h. Cells were collected.

Techniques: Isolation, FACS, RNA Sequencing Assay, Expressing, Marker, Functional Assay, Ex Vivo, Flow Cytometry, Two Tailed Test

Journal: Communications Biology

Article Title: Tumor-derived NKG2D ligand sMIC reprograms NK cells to an inflammatory phenotype through CBM signalosome activation

doi: 10.1038/s42003-021-02440-3

Figure Lengend Snippet: a , b NK cells isolated from Rag1−/− mice and cultured in presence of IL-2 for 5 days before co-culturing with tumor cell lines TC2, TC2-sMICB, and TC2-mMICB. The supernatant was collected from the 24 h co-culture for quantitative 31-plex mouse cytokine array assay (Eve Technologies). a Heatmap representing significantly higher levels of pro-inflammatory cytokines and chemokines produced by mouse NK cells when stimulated with sMICB compared to stimulation with mMICB. Heatmap data are represented as a percentage change in cytokine/chemokine levels produced by NK cells stimulated with sMICB and mMICB, respectively, versus NK cells stimulated with MIC negative control TC2 cells (baseline) and normalized to 0 to 1 scale. b Bar graphs showing differential levels of GM-CSF, CCL4, CCL3, and IL-10 produced by mouse NK cells when stimulated with sMICB vs. mMICB. Data points in the bar graph are mean values from two independent experiments. c , d Primary human NK cells were co-cultured tumor cell lines C1R, C1R-sMICA, and C1R-mMICA. The supernatant was collected from the 24 h co-culture for quantitative 42-plex human cytokine array analyses (Eve Technologies). c Heatmap representing significantly higher levels of pro-inflammatory cytokines and chemokines produced by human NK cells stimulated with sMICA compared to stimulation with mMICA. Heatmap data are represented as percentage changes in cytokine/chemokine levels produced by NK cells stimulated with sMICA and mMICA, respectively, versus NK cells stimulated with MIC negative control C1R cells (baseline) and normalized to 0 to 1 scale. d Bar graphs showing significantly higher levels of cytokines and chemokines IL-10, GM-CSF, CCL4, CCL3 in the culture supernatant of human NK cells stimulated with sMICA vs mMICA. Data shown are representative of three independent experiments. ** P < 0.01, *** P < 0.001 with two-tailed Student’s t test.

Article Snippet: Mouse NK cells were isolated from Rag1 −/− mice, cultured in presence of IL-2 for 5 days (as described above) with a purity of more than 98% NK1.1 + cells, and stimulated with recombinant soluble MICB (Sino biologicals, 10759-H08H) in the absence or presence of B10G5 for 18 h. Cells were collected.

Techniques: Isolation, Cell Culture, Co-Culture Assay, Produced, Negative Control, Two Tailed Test