Journal: Cancers

Article Title: TGFβ Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion

doi: 10.3390/cancers10110423

Figure Lengend Snippet: The chronic stimulation by transforming growth factor-beta (TGFβ) during natural killer (NK) cell expansion and activation generates NK cells with increased cytokine secretion. NK cells were cultured for 14 days in 50 IU/mL IL-2 or 50 IU/mL IL-2 plus 10 ng/mL TGFβ (TGFβi) with a weekly stimulation with K562 mbIL-21 feeder cells. ( A ) NK cell proliferation was compared by determining the fold change of control and TGFβi total NK (CD3 − /CD56 + and CD3 − /CD16 + ) cells at Day 14 and the viability of total cells was determined using Tonbo Viability dye. ( B ) After 14 days of expansion, NK cells were rested overnight with 50 IU/mL IL-2 (− TGFβ, baseline) or 50 IU/mL IL-2 plus 10 ng/mL TGFβ (+ TGFβ, acute TGFβ treatment). NK cells were then co-cultured with tumor targets for 3 h in fresh media (under identical cytokine conditions as used in the overnight rest) and supernatants were collected to measure interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) cytokine secretion using Cytometric Bead Array analysis. Individual data points are depicted for MG63 (osteosarcoma) (IFNγ: n = 12, TNFα: n = 9), DAOY (medulloblastoma) ( n = 12), and CHLA-255 (neuroblastoma) ( n = 5). ( D ) The control and TGFβi NK cells were stimulated with 10 µg/mL of PHA at 2 e6 NK cells/mL for 4 h and cytokine secretion was measured by cytometric bead array (CBA) or a MACSPlex Cytokine 12 Kit. Individual data points depicted. Lines and bars represent Mean ± SD. ( E ) TGFβi and control NK cell anti-tumor cytokine secretion following overnight treatment in fresh media with 50 IU/mL IL-2 was assessed against DAOY at Day 7 and Day 14 of expansion, and after removal from expansion conditions at Day 21, 35 and 47 +/− 1 day as described for B , C . (Day 7 n = 5, Day 14 and 21 n = 6, Day 35 and 47, n = 2)). Median with min to max whiskers depicted. Control in black, TGFβi in red. Statistical differences were determined by paired t -test ( A , D , E ) and two-way repeated measures ANOVA with Holm–Sidak’s multiple comparisons test for all others. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. See also .

Article Snippet: Human NK cells were purified with a RosetteSep Human NK Cell Enrichment Cocktail (Stem Cell Technologies, 15065, Vancouver, BC, Canada) as described in Reference [ ].

Techniques: Activation Assay, Cell Culture, Control

Journal: Cancers

Article Title: TGFβ Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion

doi: 10.3390/cancers10110423

Figure Lengend Snippet: NK cell activation is required for TGFβ induced cytokine hypersecretion. NK cells were cultured with IL-2 alone or IL-2 plus 10 ng/mL TGFb and ( A ) K562mbIL-15 (IFNγ: n = 4, TNFα: n = 3) or ( B ) no feeder cell ( n = 6), or ( C ) parental K562 ( n = 4) for 7–14 days. Following culture, anti-tumor cytokine secretion or production by CBA or intracellular flow cytometry was assessed against an MG63 tumor target. ( D ) NK cells were stimulated overnight with 10 ng/mL IL-12, 50 ng/mL IL-15, and 50 ng/mL IL-18 plus or minus IL-2 and TGFβ. Following overnight stimulation, the NK cells were cultured with 1 ng/mL IL-15 plus or minus IL-2 and TGFβ. After 7–14 days of culture, anti-tumor IFNγ and TNFα production in response to MG63 was measured by intracellular flow cytometry ( n = 4). Percent anti-tumor IFNγ+ and TNFα+ NK cells normalized to no target depicted for (B) and (D). Individual data points depicted for all. Lines and bars represent Mean ± SD. Statistical differences were determined by paired t-test for 2B and two-way repeated measures ANOVA with Holm–Sidak’s multiple comparisons test for all other graphs. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. See also .

Article Snippet: Human NK cells were purified with a RosetteSep Human NK Cell Enrichment Cocktail (Stem Cell Technologies, 15065, Vancouver, BC, Canada) as described in Reference [ ].

Techniques: Activation Assay, Cell Culture, Flow Cytometry

Journal: Cancers

Article Title: TGFβ Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion

doi: 10.3390/cancers10110423

Figure Lengend Snippet: TGFβi generates NK cells with increased degranulation but transiently impairs cytotoxicity. ( A ) Control and TGFβi NK cells from K562mbIL-21 expansions were rested overnight in IL-2 or IL-2 + TGFβ and subsequently stimulated with tumor targets for 3 h (MG63, n = 6; DAOY, n = 4; HOS, n = 5) and assessed for degranulation by CD107a. %CD107a + NK cells are corrected for no target controls. ( B ) The control and TGFβi NK cell cytotoxicity was measured using a 4-h calcein-release cytotoxicity assay, following overnight treatment in IL-2 alone or IL-2 plus TGFβ. (MG63, n = 9; DAOY, n = 3; HOS, n = 5; CHLA-255, n = 3). ( C ) TGFβi NK cells were removed from TGFβ for 7 days (±1 day) and cytotoxicity against CHLA-255, MG63, and DAOY following overnight treatment with IL-2 or IL-2 plus TGFβ was measured using a calcein-release assay ( n = 3). Individual data points depicted for all. Lines and bars represent Mean ± SD. Statistical differences were determined by two-way repeated measures ANOVA with Holm-Sidak’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Human NK cells were purified with a RosetteSep Human NK Cell Enrichment Cocktail (Stem Cell Technologies, 15065, Vancouver, BC, Canada) as described in Reference [ ].

Techniques: Control, Cytotoxicity Assay, Release Assay

Journal: Cancers

Article Title: TGFβ Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion

doi: 10.3390/cancers10110423

Figure Lengend Snippet: TGFβi remodels K562mbIL-21 expanded NK cell receptor expression. ( A ) Cell surface protein expression on the control and TGFβi NK cells was measured using flow cytometry. Geometric median fluorescent intensity (gMFI) normalized to viability only stained NK cells are shown. Flow data from one representative donor is depicted. Control in black, TGFβi in red. ( B ) mRNA expression of NK cell receptors was assessed by RNA-seq ( n = 4). ( C ) Chromatin accessibility of TNSF10 and FCGR3A was determined at Day 14 using ATAC-seq. Control in black, TGFβi in red. Individual Data points depicted. Lines and bars represent Mean ± SD. Statistical differences were determined by paired t -test and DESeq2 for RNA-seq. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. See also .

Article Snippet: Human NK cells were purified with a RosetteSep Human NK Cell Enrichment Cocktail (Stem Cell Technologies, 15065, Vancouver, BC, Canada) as described in Reference [ ].

Techniques: Expressing, Control, Flow Cytometry, Staining, RNA Sequencing

Journal: Cancers

Article Title: TGFβ Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion

doi: 10.3390/cancers10110423

Figure Lengend Snippet: TGFβi decreases granzyme A and perforin expression. ( A ) Granzyme and perforin protein expression in TGFβi NK cells were measured by flow cytometry. Percent positive NK cells and relative expression (gMFI) were depicted. Granzyme A and perforin ( n = 10), granzyme B ( n = 5). A representative flow plot is depicted. ( B ) mRNA ( n = 4 for RNA) of granzymes and perforin was measured using RNA-seq. ( n = 4 for RNA). ( C ) Chromatin accessibility of GZMA and PRF1 loci as measured by ATAC-seq. Control in black, TGFβi in red. Data are Mean ± SD. Statistical differences were determined by paired t -test and RNA-seq by DESeq2. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. See also .

Article Snippet: Human NK cells were purified with a RosetteSep Human NK Cell Enrichment Cocktail (Stem Cell Technologies, 15065, Vancouver, BC, Canada) as described in Reference [ ].

Techniques: Expressing, Flow Cytometry, RNA Sequencing, Control

Journal: Cancers

Article Title: TGFβ Imprinting During Activation Promotes Natural Killer Cell Cytokine Hypersecretion

doi: 10.3390/cancers10110423

Figure Lengend Snippet: TGFβ imprinting modifies the NK cell IFNγ regulation. See also . ( A ) RNA expression of significantly changed IFNγ regulatory genes as assessed using RNA-seq (all n = 4). ( B ) Expression of IFNγ regulatory genes shown using Clustvis. ( C ) T-bet protein expression level (gMFI) was assessed using flow cytometry. Representative donor depicted ( n = 5). Chromatin accessibility of TBX21 (T-bet) was determined using ATAC-seq. Control in black, TGFβi in red. ( D ) SMAD3 and E4BP4 ( n = 4) protein expressions were measured by western blot. Chromatin accessibility of SMAD3 was measured by ATAC-seq. See also . Statistical differences were determined using DESeq2 for RNA-seq and paired t -test for flow cytometry. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Human NK cells were purified with a RosetteSep Human NK Cell Enrichment Cocktail (Stem Cell Technologies, 15065, Vancouver, BC, Canada) as described in Reference [ ].

Techniques: RNA Expression, RNA Sequencing, Expressing, Flow Cytometry, Control, Western Blot

Journal: Med (New York, N.y.)

Article Title: Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination

doi: 10.1016/j.medj.2021.03.014

Figure Lengend Snippet:

Article Snippet: Primary human NK cells were purified from buffy coats from healthy donors using the RosetteSep human NK cell enrichment cocktail (StemCell, #15065), then resuspended in R-10 media containing 10 μg/mL brefeldin A (Sigma, #B7651), GolgiStop (BD Biosciences, #554724), and fluorescent anti-CD107a.

Techniques: Recombinant, Purification, Plasmid Preparation, Software, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Flow Cytometry