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    STEMCELL Technologies Inc easyseptm human nk cell enrichment kit
    Cell-to-cell contact and soluble priming factors on MDM are modulated by HIV-1 infection as well as <t>NK</t> <t>cells</t> (A) Experimental setup of <t>MDM-NK</t> <t>cell</t> co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture at a ratio of 1:1, according to the seeding density of CD14 + monocytes. After 24 h, NK-cell ligand expression on MDM was assessed. (B–D) MDM infection frequencies (CD4 − p24+ cells) after HIV-1 infection with 89.6 and THRO.c with and without 24 h NK-cell co-culture are depicted. Differences in infection frequencies between MDM with and without NK cell co-culture for each strain were analyzed using the Wilcoxon signed-rank test (89.6: n = 8; THRO.c: n = 9). Representative histogram of HLA-E (C) and HLA-ABC (D) surface expression on unstimulated MDM (left). Median fluorescence intensity (MdFI) of HLA-E and HLA-ABC on unstimulated, HIV-1-infected and bystander MDM is compared between MDM cultured without (NK-) or with NK <t>cells</t> <t>(NK+)</t> (right). Differences in MdFI values between infected and bystander MDM and between NK- and NK + MDM in each strain were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (unstained (us): n = 19; unstim.: n = 21; 89.6 NK+: n = 14; 89.6 NK-: n = 9; THRO.c NK+: n = 11; THRO.c NK-: n = 10). (E–G) Scheme showing the cytokine analysis of supernatants from 24 h MDM-NK cell co-culture supernatants. Soluble IL-15 (F), IL-23 (G), and IL-18 (H, left) concentrations in MDM-NK cell co-culture supernatants from HIV-1 89.6, THRO.c and LPS/IFNγ conditions compared to unstimulated co-cultures. Differences in cytokine concentrations to unstimulated were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (89.6: n = 4; THRO.c: n = 9; LPS/IFNγ: n = 10). Only significant p -values are shown. (H, right) Relation between IL-18 concentrations in 89.6- and THRO.c-infected MDM-NK co-cultures and the corresponding infection frequency. Spearman correlation coefficient was used to analyze the correlation between infection frequency and cytokine concentration (89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels A and E (B, C right, D) and panel H (C left) . n.s., nonsignificant. See also .
    Easyseptm Human Nk Cell Enrichment Kit, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/easyseptm+human+nk+cell+enrichment+kit/easysep+human+t+cell+isolation+kit/pmc12268675-97-0-7
    Average 90 stars, based on 1 article reviews
    easyseptm human nk cell enrichment kit - by Bioz Stars, 2026-09
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    1) Product Images from "HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production"

    Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

    Journal: iScience

    doi: 10.1016/j.isci.2025.112879

    Cell-to-cell contact and soluble priming factors on MDM are modulated by HIV-1 infection as well as NK cells (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture at a ratio of 1:1, according to the seeding density of CD14 + monocytes. After 24 h, NK-cell ligand expression on MDM was assessed. (B–D) MDM infection frequencies (CD4 − p24+ cells) after HIV-1 infection with 89.6 and THRO.c with and without 24 h NK-cell co-culture are depicted. Differences in infection frequencies between MDM with and without NK cell co-culture for each strain were analyzed using the Wilcoxon signed-rank test (89.6: n = 8; THRO.c: n = 9). Representative histogram of HLA-E (C) and HLA-ABC (D) surface expression on unstimulated MDM (left). Median fluorescence intensity (MdFI) of HLA-E and HLA-ABC on unstimulated, HIV-1-infected and bystander MDM is compared between MDM cultured without (NK-) or with NK cells (NK+) (right). Differences in MdFI values between infected and bystander MDM and between NK- and NK + MDM in each strain were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (unstained (us): n = 19; unstim.: n = 21; 89.6 NK+: n = 14; 89.6 NK-: n = 9; THRO.c NK+: n = 11; THRO.c NK-: n = 10). (E–G) Scheme showing the cytokine analysis of supernatants from 24 h MDM-NK cell co-culture supernatants. Soluble IL-15 (F), IL-23 (G), and IL-18 (H, left) concentrations in MDM-NK cell co-culture supernatants from HIV-1 89.6, THRO.c and LPS/IFNγ conditions compared to unstimulated co-cultures. Differences in cytokine concentrations to unstimulated were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (89.6: n = 4; THRO.c: n = 9; LPS/IFNγ: n = 10). Only significant p -values are shown. (H, right) Relation between IL-18 concentrations in 89.6- and THRO.c-infected MDM-NK co-cultures and the corresponding infection frequency. Spearman correlation coefficient was used to analyze the correlation between infection frequency and cytokine concentration (89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels A and E (B, C right, D) and panel H (C left) . n.s., nonsignificant. See also .
    Figure Legend Snippet: Cell-to-cell contact and soluble priming factors on MDM are modulated by HIV-1 infection as well as NK cells (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture at a ratio of 1:1, according to the seeding density of CD14 + monocytes. After 24 h, NK-cell ligand expression on MDM was assessed. (B–D) MDM infection frequencies (CD4 − p24+ cells) after HIV-1 infection with 89.6 and THRO.c with and without 24 h NK-cell co-culture are depicted. Differences in infection frequencies between MDM with and without NK cell co-culture for each strain were analyzed using the Wilcoxon signed-rank test (89.6: n = 8; THRO.c: n = 9). Representative histogram of HLA-E (C) and HLA-ABC (D) surface expression on unstimulated MDM (left). Median fluorescence intensity (MdFI) of HLA-E and HLA-ABC on unstimulated, HIV-1-infected and bystander MDM is compared between MDM cultured without (NK-) or with NK cells (NK+) (right). Differences in MdFI values between infected and bystander MDM and between NK- and NK + MDM in each strain were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (unstained (us): n = 19; unstim.: n = 21; 89.6 NK+: n = 14; 89.6 NK-: n = 9; THRO.c NK+: n = 11; THRO.c NK-: n = 10). (E–G) Scheme showing the cytokine analysis of supernatants from 24 h MDM-NK cell co-culture supernatants. Soluble IL-15 (F), IL-23 (G), and IL-18 (H, left) concentrations in MDM-NK cell co-culture supernatants from HIV-1 89.6, THRO.c and LPS/IFNγ conditions compared to unstimulated co-cultures. Differences in cytokine concentrations to unstimulated were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (89.6: n = 4; THRO.c: n = 9; LPS/IFNγ: n = 10). Only significant p -values are shown. (H, right) Relation between IL-18 concentrations in 89.6- and THRO.c-infected MDM-NK co-cultures and the corresponding infection frequency. Spearman correlation coefficient was used to analyze the correlation between infection frequency and cytokine concentration (89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels A and E (B, C right, D) and panel H (C left) . n.s., nonsignificant. See also .

    Techniques Used: Infection, Co-Culture Assay, Expressing, Fluorescence, Cell Culture, Concentration Assay

    Priming by HIV-1-infected macrophages does not change NK-cell phenotype (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture. After 24 h, NK cell activation and receptor expression was assessed. (B) Flow cytometric detection of the two major NK cell subsets (CD56 br CD16 − and CD56 dim CD16 + ). (C) Concatenated contour plots showing the determination of activated NK cells as measured via CD69 upregulation (left) and frequency of CD69 + NK cells in bulk NK cells after 24 h co-culture with MDM in the different conditions (unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9) (right). Representative contour plots and expression (MdFI) of (D) intracellular perforin and (E) granzyme B, (F) 2B4, and (G) NKG2D in CD56 br and CD56 dim NK cells after co-culture with MDM in the different conditions ( Perforin : unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9; Granzyme B, 2B4 and NKG2D : unstim.: n = 10; LPS/IFNγ: n = 10; 89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. (C–G) Differences in percent positives or MdFI values of the different conditions compared to unstimulated in each NK cell subset were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) and only significant p values are depicted. Stainings were performed with antibody panels B and G (C, D, E) or panel B (F, G) . n.s., nonsignificant. ICS, intracellular staining. See also .
    Figure Legend Snippet: Priming by HIV-1-infected macrophages does not change NK-cell phenotype (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture. After 24 h, NK cell activation and receptor expression was assessed. (B) Flow cytometric detection of the two major NK cell subsets (CD56 br CD16 − and CD56 dim CD16 + ). (C) Concatenated contour plots showing the determination of activated NK cells as measured via CD69 upregulation (left) and frequency of CD69 + NK cells in bulk NK cells after 24 h co-culture with MDM in the different conditions (unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9) (right). Representative contour plots and expression (MdFI) of (D) intracellular perforin and (E) granzyme B, (F) 2B4, and (G) NKG2D in CD56 br and CD56 dim NK cells after co-culture with MDM in the different conditions ( Perforin : unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9; Granzyme B, 2B4 and NKG2D : unstim.: n = 10; LPS/IFNγ: n = 10; 89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. (C–G) Differences in percent positives or MdFI values of the different conditions compared to unstimulated in each NK cell subset were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) and only significant p values are depicted. Stainings were performed with antibody panels B and G (C, D, E) or panel B (F, G) . n.s., nonsignificant. ICS, intracellular staining. See also .

    Techniques Used: Infection, Co-Culture Assay, Activation Assay, Expressing, Staining

    In vitro HIV-1 infection of macrophages primes NK-cell effector function toward cytokine release (A) Experimental setup of assessment of NK cell function after MDM co-culture. After co-culture with HIV-1-infected (89.6 and THRO.c (TH)), LPS/IFNγ-stimulated, and unstimulated MDM for 24 h, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation, measured by CD107a surface expression, and cytokine production, measured by intracellular CCL4, TNF, and IFNγ expression, were assessed. (B) Concatenated flow cytometry contour plots displaying CD107 and CCL4 expression of NK cells after MDM co-culture with and without targets (left). Representative contour plots for TNF and IFNγ plotted against CCL4 and determination of cytokine-positive NK cells (right). (C) Frequencies of CD107a+, CCL4+, TNF+, and IFNγ+ bulk (CD56 br+dim ) NK cells in response to 721.221 targets after co-culture with MDM in the different conditions. Differences in the functional response to targets in the different conditions to the unstimulated MDM priming condition were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg), only significant FDR-adjusted p values are shown ( p < 0.05) (89.6: n = 14; THRO.c: n = 12; LPS/IFNγ: n = 13). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels C and F (B, C) . See also .
    Figure Legend Snippet: In vitro HIV-1 infection of macrophages primes NK-cell effector function toward cytokine release (A) Experimental setup of assessment of NK cell function after MDM co-culture. After co-culture with HIV-1-infected (89.6 and THRO.c (TH)), LPS/IFNγ-stimulated, and unstimulated MDM for 24 h, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation, measured by CD107a surface expression, and cytokine production, measured by intracellular CCL4, TNF, and IFNγ expression, were assessed. (B) Concatenated flow cytometry contour plots displaying CD107 and CCL4 expression of NK cells after MDM co-culture with and without targets (left). Representative contour plots for TNF and IFNγ plotted against CCL4 and determination of cytokine-positive NK cells (right). (C) Frequencies of CD107a+, CCL4+, TNF+, and IFNγ+ bulk (CD56 br+dim ) NK cells in response to 721.221 targets after co-culture with MDM in the different conditions. Differences in the functional response to targets in the different conditions to the unstimulated MDM priming condition were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg), only significant FDR-adjusted p values are shown ( p < 0.05) (89.6: n = 14; THRO.c: n = 12; LPS/IFNγ: n = 13). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels C and F (B, C) . See also .

    Techniques Used: In Vitro, Infection, Cell Function Assay, Co-Culture Assay, Cell Culture, Expressing, Flow Cytometry, Functional Assay

    KIR+ NKG2A+ NK cells from PLWH respond to macrophage priming, whereas the CD56 neg subset remains dysfunctional (A) Set-up of NK cell and MDM co-culture from peripheral blood of PLWH. After 24 h co-culture with LPS/IFNγ-stimulated (stimulation for 24 h prior to co-culture) and unstimulated MDM, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation (measured by CD107a), and cytokine production, measured by intracellular CCL4, TNF, and IFNγ staining were assessed. (B) Flow cytometry staining of phenotypic MDM markers after 9 days of differentiation. (C) Expression of pSTAT1 (MdFI) in MDM with and without LPS/IFNγ stimulation. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (D) Representative dot plot of the three different subsets of NK cells (CD56 br , CD56 dim and CD56 neg ) (left) and respective frequencies among bulk NK cells from PLWH (right) ( n = 7). (E) Frequencies of CD107a-, CCL4-, TNF-, and IFNγ-expressing bulk (CD56 br+dim+neg ) NK cells with or without 721.221 targets after MDM co-culture. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (F) Gating strategy for NKG2A, NKG2C, CD57, and KIRs (any KIR Boolean gate: KIR2DL1/S5 OR KIR2DL2/L3 OR KIR3DL1/S1) in CD56 br , CD56 dim , and CD56 neg NK cells from PLWH. (G) Heatmap showing median frequencies of CD107a-, CCL4-, TNF-, and IFNγ-positive NK cells from PLWH of indicated subsets after MDM co-culture in response to targets. Values were clustered unsupervised according to the CD107a response, corresponding frequencies of the detected subsets in total NK cells are depicted to the right ( n = 7). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panel E (B, C), panel F (D, E, G), or panels C and F (F) . See also .
    Figure Legend Snippet: KIR+ NKG2A+ NK cells from PLWH respond to macrophage priming, whereas the CD56 neg subset remains dysfunctional (A) Set-up of NK cell and MDM co-culture from peripheral blood of PLWH. After 24 h co-culture with LPS/IFNγ-stimulated (stimulation for 24 h prior to co-culture) and unstimulated MDM, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation (measured by CD107a), and cytokine production, measured by intracellular CCL4, TNF, and IFNγ staining were assessed. (B) Flow cytometry staining of phenotypic MDM markers after 9 days of differentiation. (C) Expression of pSTAT1 (MdFI) in MDM with and without LPS/IFNγ stimulation. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (D) Representative dot plot of the three different subsets of NK cells (CD56 br , CD56 dim and CD56 neg ) (left) and respective frequencies among bulk NK cells from PLWH (right) ( n = 7). (E) Frequencies of CD107a-, CCL4-, TNF-, and IFNγ-expressing bulk (CD56 br+dim+neg ) NK cells with or without 721.221 targets after MDM co-culture. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (F) Gating strategy for NKG2A, NKG2C, CD57, and KIRs (any KIR Boolean gate: KIR2DL1/S5 OR KIR2DL2/L3 OR KIR3DL1/S1) in CD56 br , CD56 dim , and CD56 neg NK cells from PLWH. (G) Heatmap showing median frequencies of CD107a-, CCL4-, TNF-, and IFNγ-positive NK cells from PLWH of indicated subsets after MDM co-culture in response to targets. Values were clustered unsupervised according to the CD107a response, corresponding frequencies of the detected subsets in total NK cells are depicted to the right ( n = 7). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panel E (B, C), panel F (D, E, G), or panels C and F (F) . See also .

    Techniques Used: Co-Culture Assay, Cell Culture, Staining, Flow Cytometry, Expressing

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    Cell Viability Assay:

    Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins RPMI 1640 Medium, GlutaMAXTM Supplement Gibco Cat# 61870127 DMEM, high glucose, GlutaMAXTM Supplement Gibco Cat# 10566024 HEPES Gibco Cat#15630106 MEM Non-Essential Amino Acids Solution (100X) Gibco Cat# 11140076 Sodium Pyruvate (100 mM) Gibco Cat# 11360070 heat-inactivated fetal bovine serum Hyclone Cat# SH30071.03HI Penicillin-Streptomycin (5,000 U/mL) Gibco Cat# 15070063 2-Mercaptoethanol Gibco Cat# 21985023 Ficoll-PaqueTM PLUS density gradient media Cytiva Cat# 17144003 PMA/ionomycin cocktail plus transporter inhibitors eBioscience Cat# 00-4975 PMA/ionomycin cocktail eBioscience Cat# 00-4970 GW280264X MedChemExpress Cat# HY-115670 Golgi-Plug (BD, 555029) BD Biosciences Cat# 555029 RIPA lysis buffer ThermoScientific Cat# 89900 protease and phosphatase inhibitor ThermoScientific Cat# 78842 4-12% Bis-Tris gel Invitrogen Cat# NP0336BOX PVDF membrane Invitrogen Cat# IB24002 PierceTM ECL Plus Western Blotting Substrate ThermoScientific Cat# 32132 Liberase TL Research Grade Sigma-Aldrich Cat# 5401020001 Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# D4263-1VL High Sensitivity RNA Screen Tape Agilent Cat# 5067-5579 High Sensitivity RNA Screen Ladder Agilent Cat# 5067-5581 High Sensitivity RNA Screen Buffer Agilent Cat# 5067-5580 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 human recombinant IL-2 STEMCELL Technology Cat # 78036.2 human NCR1(22-254)-His AcroBio Cat# NC1-H52H4 biotin- human NCR1(22-254)-His Amgen, Inc N/A mouse NCR1(22-255)-His AcroBio Cat# NC1-M52H7 mouse CD19(19-287)-His AcroBio Cat# CD9-M52H8 anti-huIgG Fc Jackson Immuno Research Cat#109-005-098 biotinylated anti-huIgG Fc Amgen Inc N/A Human HER2(23-653)-6xHis Amgen Inc N/A biotin human HER2(23-653)-6xHis Amgen Inc N/A human CLL1(67-265)-FLAG Amgen Inc N/A human FcγRIIIa (CD16a158v)-GST-His6 Amgen Inc N/A human FcγRIIIa (CD16a158F)-GST-His6 Amgen Inc N/A human FcγRIIIb (CD16b)-His Amgen Inc N/A biotin-human CD3e (1-27)-human Fc Amgen Inc N/A NCR1xHER2 Amgen Inc In this paper CD3xHER2 Amgen Inc In this paper huCLL1 TCE Amgen Inc In this paper NKCE-I Amgen Inc In this paper NKCE-II Amgen Inc In this paper NKCE-III Amgen Inc In this paper NKCE-IV Amgen Inc In this paper NKCE-V Amgen Inc In this paper muCD19 TCE Amgen Inc In this paper (Continued on next page) e2 Cell Reports Medicine 6, 102117, May 20, 2025 Article ll OPEN ACCESS .. REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS .. Human PBMC from healthy volunteers was obtained with informed consent through the Amgen Research Donor Program.

    Staining:

    Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins RPMI 1640 Medium, GlutaMAXTM Supplement Gibco Cat# 61870127 DMEM, high glucose, GlutaMAXTM Supplement Gibco Cat# 10566024 HEPES Gibco Cat#15630106 MEM Non-Essential Amino Acids Solution (100X) Gibco Cat# 11140076 Sodium Pyruvate (100 mM) Gibco Cat# 11360070 heat-inactivated fetal bovine serum Hyclone Cat# SH30071.03HI Penicillin-Streptomycin (5,000 U/mL) Gibco Cat# 15070063 2-Mercaptoethanol Gibco Cat# 21985023 Ficoll-PaqueTM PLUS density gradient media Cytiva Cat# 17144003 PMA/ionomycin cocktail plus transporter inhibitors eBioscience Cat# 00-4975 PMA/ionomycin cocktail eBioscience Cat# 00-4970 GW280264X MedChemExpress Cat# HY-115670 Golgi-Plug (BD, 555029) BD Biosciences Cat# 555029 RIPA lysis buffer ThermoScientific Cat# 89900 protease and phosphatase inhibitor ThermoScientific Cat# 78842 4-12% Bis-Tris gel Invitrogen Cat# NP0336BOX PVDF membrane Invitrogen Cat# IB24002 PierceTM ECL Plus Western Blotting Substrate ThermoScientific Cat# 32132 Liberase TL Research Grade Sigma-Aldrich Cat# 5401020001 Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# D4263-1VL High Sensitivity RNA Screen Tape Agilent Cat# 5067-5579 High Sensitivity RNA Screen Ladder Agilent Cat# 5067-5581 High Sensitivity RNA Screen Buffer Agilent Cat# 5067-5580 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 human recombinant IL-2 STEMCELL Technology Cat # 78036.2 human NCR1(22-254)-His AcroBio Cat# NC1-H52H4 biotin- human NCR1(22-254)-His Amgen, Inc N/A mouse NCR1(22-255)-His AcroBio Cat# NC1-M52H7 mouse CD19(19-287)-His AcroBio Cat# CD9-M52H8 anti-huIgG Fc Jackson Immuno Research Cat#109-005-098 biotinylated anti-huIgG Fc Amgen Inc N/A Human HER2(23-653)-6xHis Amgen Inc N/A biotin human HER2(23-653)-6xHis Amgen Inc N/A human CLL1(67-265)-FLAG Amgen Inc N/A human FcγRIIIa (CD16a158v)-GST-His6 Amgen Inc N/A human FcγRIIIa (CD16a158F)-GST-His6 Amgen Inc N/A human FcγRIIIb (CD16b)-His Amgen Inc N/A biotin-human CD3e (1-27)-human Fc Amgen Inc N/A NCR1xHER2 Amgen Inc In this paper CD3xHER2 Amgen Inc In this paper huCLL1 TCE Amgen Inc In this paper NKCE-I Amgen Inc In this paper NKCE-II Amgen Inc In this paper NKCE-III Amgen Inc In this paper NKCE-IV Amgen Inc In this paper NKCE-V Amgen Inc In this paper muCD19 TCE Amgen Inc In this paper (Continued on next page) e2 Cell Reports Medicine 6, 102117, May 20, 2025 Article ll OPEN ACCESS .. REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS .. Human PBMC from healthy volunteers was obtained with informed consent through the Amgen Research Donor Program.

    Fluorescence:

    Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins RPMI 1640 Medium, GlutaMAXTM Supplement Gibco Cat# 61870127 DMEM, high glucose, GlutaMAXTM Supplement Gibco Cat# 10566024 HEPES Gibco Cat#15630106 MEM Non-Essential Amino Acids Solution (100X) Gibco Cat# 11140076 Sodium Pyruvate (100 mM) Gibco Cat# 11360070 heat-inactivated fetal bovine serum Hyclone Cat# SH30071.03HI Penicillin-Streptomycin (5,000 U/mL) Gibco Cat# 15070063 2-Mercaptoethanol Gibco Cat# 21985023 Ficoll-PaqueTM PLUS density gradient media Cytiva Cat# 17144003 PMA/ionomycin cocktail plus transporter inhibitors eBioscience Cat# 00-4975 PMA/ionomycin cocktail eBioscience Cat# 00-4970 GW280264X MedChemExpress Cat# HY-115670 Golgi-Plug (BD, 555029) BD Biosciences Cat# 555029 RIPA lysis buffer ThermoScientific Cat# 89900 protease and phosphatase inhibitor ThermoScientific Cat# 78842 4-12% Bis-Tris gel Invitrogen Cat# NP0336BOX PVDF membrane Invitrogen Cat# IB24002 PierceTM ECL Plus Western Blotting Substrate ThermoScientific Cat# 32132 Liberase TL Research Grade Sigma-Aldrich Cat# 5401020001 Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# D4263-1VL High Sensitivity RNA Screen Tape Agilent Cat# 5067-5579 High Sensitivity RNA Screen Ladder Agilent Cat# 5067-5581 High Sensitivity RNA Screen Buffer Agilent Cat# 5067-5580 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 human recombinant IL-2 STEMCELL Technology Cat # 78036.2 human NCR1(22-254)-His AcroBio Cat# NC1-H52H4 biotin- human NCR1(22-254)-His Amgen, Inc N/A mouse NCR1(22-255)-His AcroBio Cat# NC1-M52H7 mouse CD19(19-287)-His AcroBio Cat# CD9-M52H8 anti-huIgG Fc Jackson Immuno Research Cat#109-005-098 biotinylated anti-huIgG Fc Amgen Inc N/A Human HER2(23-653)-6xHis Amgen Inc N/A biotin human HER2(23-653)-6xHis Amgen Inc N/A human CLL1(67-265)-FLAG Amgen Inc N/A human FcγRIIIa (CD16a158v)-GST-His6 Amgen Inc N/A human FcγRIIIa (CD16a158F)-GST-His6 Amgen Inc N/A human FcγRIIIb (CD16b)-His Amgen Inc N/A biotin-human CD3e (1-27)-human Fc Amgen Inc N/A NCR1xHER2 Amgen Inc In this paper CD3xHER2 Amgen Inc In this paper huCLL1 TCE Amgen Inc In this paper NKCE-I Amgen Inc In this paper NKCE-II Amgen Inc In this paper NKCE-III Amgen Inc In this paper NKCE-IV Amgen Inc In this paper NKCE-V Amgen Inc In this paper muCD19 TCE Amgen Inc In this paper (Continued on next page) e2 Cell Reports Medicine 6, 102117, May 20, 2025 Article ll OPEN ACCESS .. REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS .. Human PBMC from healthy volunteers was obtained with informed consent through the Amgen Research Donor Program.

    Quantitation Assay:

    Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins RPMI 1640 Medium, GlutaMAXTM Supplement Gibco Cat# 61870127 DMEM, high glucose, GlutaMAXTM Supplement Gibco Cat# 10566024 HEPES Gibco Cat#15630106 MEM Non-Essential Amino Acids Solution (100X) Gibco Cat# 11140076 Sodium Pyruvate (100 mM) Gibco Cat# 11360070 heat-inactivated fetal bovine serum Hyclone Cat# SH30071.03HI Penicillin-Streptomycin (5,000 U/mL) Gibco Cat# 15070063 2-Mercaptoethanol Gibco Cat# 21985023 Ficoll-PaqueTM PLUS density gradient media Cytiva Cat# 17144003 PMA/ionomycin cocktail plus transporter inhibitors eBioscience Cat# 00-4975 PMA/ionomycin cocktail eBioscience Cat# 00-4970 GW280264X MedChemExpress Cat# HY-115670 Golgi-Plug (BD, 555029) BD Biosciences Cat# 555029 RIPA lysis buffer ThermoScientific Cat# 89900 protease and phosphatase inhibitor ThermoScientific Cat# 78842 4-12% Bis-Tris gel Invitrogen Cat# NP0336BOX PVDF membrane Invitrogen Cat# IB24002 PierceTM ECL Plus Western Blotting Substrate ThermoScientific Cat# 32132 Liberase TL Research Grade Sigma-Aldrich Cat# 5401020001 Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# D4263-1VL High Sensitivity RNA Screen Tape Agilent Cat# 5067-5579 High Sensitivity RNA Screen Ladder Agilent Cat# 5067-5581 High Sensitivity RNA Screen Buffer Agilent Cat# 5067-5580 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 human recombinant IL-2 STEMCELL Technology Cat # 78036.2 human NCR1(22-254)-His AcroBio Cat# NC1-H52H4 biotin- human NCR1(22-254)-His Amgen, Inc N/A mouse NCR1(22-255)-His AcroBio Cat# NC1-M52H7 mouse CD19(19-287)-His AcroBio Cat# CD9-M52H8 anti-huIgG Fc Jackson Immuno Research Cat#109-005-098 biotinylated anti-huIgG Fc Amgen Inc N/A Human HER2(23-653)-6xHis Amgen Inc N/A biotin human HER2(23-653)-6xHis Amgen Inc N/A human CLL1(67-265)-FLAG Amgen Inc N/A human FcγRIIIa (CD16a158v)-GST-His6 Amgen Inc N/A human FcγRIIIa (CD16a158F)-GST-His6 Amgen Inc N/A human FcγRIIIb (CD16b)-His Amgen Inc N/A biotin-human CD3e (1-27)-human Fc Amgen Inc N/A NCR1xHER2 Amgen Inc In this paper CD3xHER2 Amgen Inc In this paper huCLL1 TCE Amgen Inc In this paper NKCE-I Amgen Inc In this paper NKCE-II Amgen Inc In this paper NKCE-III Amgen Inc In this paper NKCE-IV Amgen Inc In this paper NKCE-V Amgen Inc In this paper muCD19 TCE Amgen Inc In this paper (Continued on next page) e2 Cell Reports Medicine 6, 102117, May 20, 2025 Article ll OPEN ACCESS .. REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS .. Human PBMC from healthy volunteers was obtained with informed consent through the Amgen Research Donor Program.

    Sequencing:

    Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins RPMI 1640 Medium, GlutaMAXTM Supplement Gibco Cat# 61870127 DMEM, high glucose, GlutaMAXTM Supplement Gibco Cat# 10566024 HEPES Gibco Cat#15630106 MEM Non-Essential Amino Acids Solution (100X) Gibco Cat# 11140076 Sodium Pyruvate (100 mM) Gibco Cat# 11360070 heat-inactivated fetal bovine serum Hyclone Cat# SH30071.03HI Penicillin-Streptomycin (5,000 U/mL) Gibco Cat# 15070063 2-Mercaptoethanol Gibco Cat# 21985023 Ficoll-PaqueTM PLUS density gradient media Cytiva Cat# 17144003 PMA/ionomycin cocktail plus transporter inhibitors eBioscience Cat# 00-4975 PMA/ionomycin cocktail eBioscience Cat# 00-4970 GW280264X MedChemExpress Cat# HY-115670 Golgi-Plug (BD, 555029) BD Biosciences Cat# 555029 RIPA lysis buffer ThermoScientific Cat# 89900 protease and phosphatase inhibitor ThermoScientific Cat# 78842 4-12% Bis-Tris gel Invitrogen Cat# NP0336BOX PVDF membrane Invitrogen Cat# IB24002 PierceTM ECL Plus Western Blotting Substrate ThermoScientific Cat# 32132 Liberase TL Research Grade Sigma-Aldrich Cat# 5401020001 Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# D4263-1VL High Sensitivity RNA Screen Tape Agilent Cat# 5067-5579 High Sensitivity RNA Screen Ladder Agilent Cat# 5067-5581 High Sensitivity RNA Screen Buffer Agilent Cat# 5067-5580 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 human recombinant IL-2 STEMCELL Technology Cat # 78036.2 human NCR1(22-254)-His AcroBio Cat# NC1-H52H4 biotin- human NCR1(22-254)-His Amgen, Inc N/A mouse NCR1(22-255)-His AcroBio Cat# NC1-M52H7 mouse CD19(19-287)-His AcroBio Cat# CD9-M52H8 anti-huIgG Fc Jackson Immuno Research Cat#109-005-098 biotinylated anti-huIgG Fc Amgen Inc N/A Human HER2(23-653)-6xHis Amgen Inc N/A biotin human HER2(23-653)-6xHis Amgen Inc N/A human CLL1(67-265)-FLAG Amgen Inc N/A human FcγRIIIa (CD16a158v)-GST-His6 Amgen Inc N/A human FcγRIIIa (CD16a158F)-GST-His6 Amgen Inc N/A human FcγRIIIb (CD16b)-His Amgen Inc N/A biotin-human CD3e (1-27)-human Fc Amgen Inc N/A NCR1xHER2 Amgen Inc In this paper CD3xHER2 Amgen Inc In this paper huCLL1 TCE Amgen Inc In this paper NKCE-I Amgen Inc In this paper NKCE-II Amgen Inc In this paper NKCE-III Amgen Inc In this paper NKCE-IV Amgen Inc In this paper NKCE-V Amgen Inc In this paper muCD19 TCE Amgen Inc In this paper (Continued on next page) e2 Cell Reports Medicine 6, 102117, May 20, 2025 Article ll OPEN ACCESS .. REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS .. Human PBMC from healthy volunteers was obtained with informed consent through the Amgen Research Donor Program.

    DNA Library Preparation:

    Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins RPMI 1640 Medium, GlutaMAXTM Supplement Gibco Cat# 61870127 DMEM, high glucose, GlutaMAXTM Supplement Gibco Cat# 10566024 HEPES Gibco Cat#15630106 MEM Non-Essential Amino Acids Solution (100X) Gibco Cat# 11140076 Sodium Pyruvate (100 mM) Gibco Cat# 11360070 heat-inactivated fetal bovine serum Hyclone Cat# SH30071.03HI Penicillin-Streptomycin (5,000 U/mL) Gibco Cat# 15070063 2-Mercaptoethanol Gibco Cat# 21985023 Ficoll-PaqueTM PLUS density gradient media Cytiva Cat# 17144003 PMA/ionomycin cocktail plus transporter inhibitors eBioscience Cat# 00-4975 PMA/ionomycin cocktail eBioscience Cat# 00-4970 GW280264X MedChemExpress Cat# HY-115670 Golgi-Plug (BD, 555029) BD Biosciences Cat# 555029 RIPA lysis buffer ThermoScientific Cat# 89900 protease and phosphatase inhibitor ThermoScientific Cat# 78842 4-12% Bis-Tris gel Invitrogen Cat# NP0336BOX PVDF membrane Invitrogen Cat# IB24002 PierceTM ECL Plus Western Blotting Substrate ThermoScientific Cat# 32132 Liberase TL Research Grade Sigma-Aldrich Cat# 5401020001 Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# D4263-1VL High Sensitivity RNA Screen Tape Agilent Cat# 5067-5579 High Sensitivity RNA Screen Ladder Agilent Cat# 5067-5581 High Sensitivity RNA Screen Buffer Agilent Cat# 5067-5580 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 human recombinant IL-2 STEMCELL Technology Cat # 78036.2 human NCR1(22-254)-His AcroBio Cat# NC1-H52H4 biotin- human NCR1(22-254)-His Amgen, Inc N/A mouse NCR1(22-255)-His AcroBio Cat# NC1-M52H7 mouse CD19(19-287)-His AcroBio Cat# CD9-M52H8 anti-huIgG Fc Jackson Immuno Research Cat#109-005-098 biotinylated anti-huIgG Fc Amgen Inc N/A Human HER2(23-653)-6xHis Amgen Inc N/A biotin human HER2(23-653)-6xHis Amgen Inc N/A human CLL1(67-265)-FLAG Amgen Inc N/A human FcγRIIIa (CD16a158v)-GST-His6 Amgen Inc N/A human FcγRIIIa (CD16a158F)-GST-His6 Amgen Inc N/A human FcγRIIIb (CD16b)-His Amgen Inc N/A biotin-human CD3e (1-27)-human Fc Amgen Inc N/A NCR1xHER2 Amgen Inc In this paper CD3xHER2 Amgen Inc In this paper huCLL1 TCE Amgen Inc In this paper NKCE-I Amgen Inc In this paper NKCE-II Amgen Inc In this paper NKCE-III Amgen Inc In this paper NKCE-IV Amgen Inc In this paper NKCE-V Amgen Inc In this paper muCD19 TCE Amgen Inc In this paper (Continued on next page) e2 Cell Reports Medicine 6, 102117, May 20, 2025 Article ll OPEN ACCESS .. REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS .. Human PBMC from healthy volunteers was obtained with informed consent through the Amgen Research Donor Program.

    RNA sequencing:

    Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins RPMI 1640 Medium, GlutaMAXTM Supplement Gibco Cat# 61870127 DMEM, high glucose, GlutaMAXTM Supplement Gibco Cat# 10566024 HEPES Gibco Cat#15630106 MEM Non-Essential Amino Acids Solution (100X) Gibco Cat# 11140076 Sodium Pyruvate (100 mM) Gibco Cat# 11360070 heat-inactivated fetal bovine serum Hyclone Cat# SH30071.03HI Penicillin-Streptomycin (5,000 U/mL) Gibco Cat# 15070063 2-Mercaptoethanol Gibco Cat# 21985023 Ficoll-PaqueTM PLUS density gradient media Cytiva Cat# 17144003 PMA/ionomycin cocktail plus transporter inhibitors eBioscience Cat# 00-4975 PMA/ionomycin cocktail eBioscience Cat# 00-4970 GW280264X MedChemExpress Cat# HY-115670 Golgi-Plug (BD, 555029) BD Biosciences Cat# 555029 RIPA lysis buffer ThermoScientific Cat# 89900 protease and phosphatase inhibitor ThermoScientific Cat# 78842 4-12% Bis-Tris gel Invitrogen Cat# NP0336BOX PVDF membrane Invitrogen Cat# IB24002 PierceTM ECL Plus Western Blotting Substrate ThermoScientific Cat# 32132 Liberase TL Research Grade Sigma-Aldrich Cat# 5401020001 Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# D4263-1VL High Sensitivity RNA Screen Tape Agilent Cat# 5067-5579 High Sensitivity RNA Screen Ladder Agilent Cat# 5067-5581 High Sensitivity RNA Screen Buffer Agilent Cat# 5067-5580 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 human recombinant IL-2 STEMCELL Technology Cat # 78036.2 human NCR1(22-254)-His AcroBio Cat# NC1-H52H4 biotin- human NCR1(22-254)-His Amgen, Inc N/A mouse NCR1(22-255)-His AcroBio Cat# NC1-M52H7 mouse CD19(19-287)-His AcroBio Cat# CD9-M52H8 anti-huIgG Fc Jackson Immuno Research Cat#109-005-098 biotinylated anti-huIgG Fc Amgen Inc N/A Human HER2(23-653)-6xHis Amgen Inc N/A biotin human HER2(23-653)-6xHis Amgen Inc N/A human CLL1(67-265)-FLAG Amgen Inc N/A human FcγRIIIa (CD16a158v)-GST-His6 Amgen Inc N/A human FcγRIIIa (CD16a158F)-GST-His6 Amgen Inc N/A human FcγRIIIb (CD16b)-His Amgen Inc N/A biotin-human CD3e (1-27)-human Fc Amgen Inc N/A NCR1xHER2 Amgen Inc In this paper CD3xHER2 Amgen Inc In this paper huCLL1 TCE Amgen Inc In this paper NKCE-I Amgen Inc In this paper NKCE-II Amgen Inc In this paper NKCE-III Amgen Inc In this paper NKCE-IV Amgen Inc In this paper NKCE-V Amgen Inc In this paper muCD19 TCE Amgen Inc In this paper (Continued on next page) e2 Cell Reports Medicine 6, 102117, May 20, 2025 Article ll OPEN ACCESS .. REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS .. Human PBMC from healthy volunteers was obtained with informed consent through the Amgen Research Donor Program.



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    Cell-to-cell contact and soluble priming factors on MDM are modulated by HIV-1 infection as well as <t>NK</t> <t>cells</t> (A) Experimental setup of <t>MDM-NK</t> <t>cell</t> co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture at a ratio of 1:1, according to the seeding density of CD14 + monocytes. After 24 h, NK-cell ligand expression on MDM was assessed. (B–D) MDM infection frequencies (CD4 − p24+ cells) after HIV-1 infection with 89.6 and THRO.c with and without 24 h NK-cell co-culture are depicted. Differences in infection frequencies between MDM with and without NK cell co-culture for each strain were analyzed using the Wilcoxon signed-rank test (89.6: n = 8; THRO.c: n = 9). Representative histogram of HLA-E (C) and HLA-ABC (D) surface expression on unstimulated MDM (left). Median fluorescence intensity (MdFI) of HLA-E and HLA-ABC on unstimulated, HIV-1-infected and bystander MDM is compared between MDM cultured without (NK-) or with NK <t>cells</t> <t>(NK+)</t> (right). Differences in MdFI values between infected and bystander MDM and between NK- and NK + MDM in each strain were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (unstained (us): n = 19; unstim.: n = 21; 89.6 NK+: n = 14; 89.6 NK-: n = 9; THRO.c NK+: n = 11; THRO.c NK-: n = 10). (E–G) Scheme showing the cytokine analysis of supernatants from 24 h MDM-NK cell co-culture supernatants. Soluble IL-15 (F), IL-23 (G), and IL-18 (H, left) concentrations in MDM-NK cell co-culture supernatants from HIV-1 89.6, THRO.c and LPS/IFNγ conditions compared to unstimulated co-cultures. Differences in cytokine concentrations to unstimulated were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (89.6: n = 4; THRO.c: n = 9; LPS/IFNγ: n = 10). Only significant p -values are shown. (H, right) Relation between IL-18 concentrations in 89.6- and THRO.c-infected MDM-NK co-cultures and the corresponding infection frequency. Spearman correlation coefficient was used to analyze the correlation between infection frequency and cytokine concentration (89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels A and E (B, C right, D) and panel H (C left) . n.s., nonsignificant. See also .
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    Cell-to-cell contact and soluble priming factors on MDM are modulated by HIV-1 infection as well as NK cells (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture at a ratio of 1:1, according to the seeding density of CD14 + monocytes. After 24 h, NK-cell ligand expression on MDM was assessed. (B–D) MDM infection frequencies (CD4 − p24+ cells) after HIV-1 infection with 89.6 and THRO.c with and without 24 h NK-cell co-culture are depicted. Differences in infection frequencies between MDM with and without NK cell co-culture for each strain were analyzed using the Wilcoxon signed-rank test (89.6: n = 8; THRO.c: n = 9). Representative histogram of HLA-E (C) and HLA-ABC (D) surface expression on unstimulated MDM (left). Median fluorescence intensity (MdFI) of HLA-E and HLA-ABC on unstimulated, HIV-1-infected and bystander MDM is compared between MDM cultured without (NK-) or with NK cells (NK+) (right). Differences in MdFI values between infected and bystander MDM and between NK- and NK + MDM in each strain were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (unstained (us): n = 19; unstim.: n = 21; 89.6 NK+: n = 14; 89.6 NK-: n = 9; THRO.c NK+: n = 11; THRO.c NK-: n = 10). (E–G) Scheme showing the cytokine analysis of supernatants from 24 h MDM-NK cell co-culture supernatants. Soluble IL-15 (F), IL-23 (G), and IL-18 (H, left) concentrations in MDM-NK cell co-culture supernatants from HIV-1 89.6, THRO.c and LPS/IFNγ conditions compared to unstimulated co-cultures. Differences in cytokine concentrations to unstimulated were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (89.6: n = 4; THRO.c: n = 9; LPS/IFNγ: n = 10). Only significant p -values are shown. (H, right) Relation between IL-18 concentrations in 89.6- and THRO.c-infected MDM-NK co-cultures and the corresponding infection frequency. Spearman correlation coefficient was used to analyze the correlation between infection frequency and cytokine concentration (89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels A and E (B, C right, D) and panel H (C left) . n.s., nonsignificant. See also .

    Journal: iScience

    Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

    doi: 10.1016/j.isci.2025.112879

    Figure Lengend Snippet: Cell-to-cell contact and soluble priming factors on MDM are modulated by HIV-1 infection as well as NK cells (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture at a ratio of 1:1, according to the seeding density of CD14 + monocytes. After 24 h, NK-cell ligand expression on MDM was assessed. (B–D) MDM infection frequencies (CD4 − p24+ cells) after HIV-1 infection with 89.6 and THRO.c with and without 24 h NK-cell co-culture are depicted. Differences in infection frequencies between MDM with and without NK cell co-culture for each strain were analyzed using the Wilcoxon signed-rank test (89.6: n = 8; THRO.c: n = 9). Representative histogram of HLA-E (C) and HLA-ABC (D) surface expression on unstimulated MDM (left). Median fluorescence intensity (MdFI) of HLA-E and HLA-ABC on unstimulated, HIV-1-infected and bystander MDM is compared between MDM cultured without (NK-) or with NK cells (NK+) (right). Differences in MdFI values between infected and bystander MDM and between NK- and NK + MDM in each strain were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (unstained (us): n = 19; unstim.: n = 21; 89.6 NK+: n = 14; 89.6 NK-: n = 9; THRO.c NK+: n = 11; THRO.c NK-: n = 10). (E–G) Scheme showing the cytokine analysis of supernatants from 24 h MDM-NK cell co-culture supernatants. Soluble IL-15 (F), IL-23 (G), and IL-18 (H, left) concentrations in MDM-NK cell co-culture supernatants from HIV-1 89.6, THRO.c and LPS/IFNγ conditions compared to unstimulated co-cultures. Differences in cytokine concentrations to unstimulated were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) (89.6: n = 4; THRO.c: n = 9; LPS/IFNγ: n = 10). Only significant p -values are shown. (H, right) Relation between IL-18 concentrations in 89.6- and THRO.c-infected MDM-NK co-cultures and the corresponding infection frequency. Spearman correlation coefficient was used to analyze the correlation between infection frequency and cytokine concentration (89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels A and E (B, C right, D) and panel H (C left) . n.s., nonsignificant. See also .

    Article Snippet: EasySepTM Human NK Cell Enrichment Kit , StemCell Technologies , Cat#19055.

    Techniques: Infection, Co-Culture Assay, Expressing, Fluorescence, Cell Culture, Concentration Assay

    Priming by HIV-1-infected macrophages does not change NK-cell phenotype (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture. After 24 h, NK cell activation and receptor expression was assessed. (B) Flow cytometric detection of the two major NK cell subsets (CD56 br CD16 − and CD56 dim CD16 + ). (C) Concatenated contour plots showing the determination of activated NK cells as measured via CD69 upregulation (left) and frequency of CD69 + NK cells in bulk NK cells after 24 h co-culture with MDM in the different conditions (unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9) (right). Representative contour plots and expression (MdFI) of (D) intracellular perforin and (E) granzyme B, (F) 2B4, and (G) NKG2D in CD56 br and CD56 dim NK cells after co-culture with MDM in the different conditions ( Perforin : unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9; Granzyme B, 2B4 and NKG2D : unstim.: n = 10; LPS/IFNγ: n = 10; 89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. (C–G) Differences in percent positives or MdFI values of the different conditions compared to unstimulated in each NK cell subset were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) and only significant p values are depicted. Stainings were performed with antibody panels B and G (C, D, E) or panel B (F, G) . n.s., nonsignificant. ICS, intracellular staining. See also .

    Journal: iScience

    Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

    doi: 10.1016/j.isci.2025.112879

    Figure Lengend Snippet: Priming by HIV-1-infected macrophages does not change NK-cell phenotype (A) Experimental setup of MDM-NK cell co-culture. After 5 days of HIV-1 infection or 24 h of LPS/IFNγ-stimulation of MDM, autologous NK cells were added to the culture. After 24 h, NK cell activation and receptor expression was assessed. (B) Flow cytometric detection of the two major NK cell subsets (CD56 br CD16 − and CD56 dim CD16 + ). (C) Concatenated contour plots showing the determination of activated NK cells as measured via CD69 upregulation (left) and frequency of CD69 + NK cells in bulk NK cells after 24 h co-culture with MDM in the different conditions (unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9) (right). Representative contour plots and expression (MdFI) of (D) intracellular perforin and (E) granzyme B, (F) 2B4, and (G) NKG2D in CD56 br and CD56 dim NK cells after co-culture with MDM in the different conditions ( Perforin : unstim.: n = 14; LPS/IFNγ: n = 10; 89.6: n = 8; THRO.c: n = 9; Granzyme B, 2B4 and NKG2D : unstim.: n = 10; LPS/IFNγ: n = 10; 89.6: n = 4; THRO.c: n = 9). All data are shown as median ± IQR with each dot representing one biological replicate. (C–G) Differences in percent positives or MdFI values of the different conditions compared to unstimulated in each NK cell subset were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg) and only significant p values are depicted. Stainings were performed with antibody panels B and G (C, D, E) or panel B (F, G) . n.s., nonsignificant. ICS, intracellular staining. See also .

    Article Snippet: EasySepTM Human NK Cell Enrichment Kit , StemCell Technologies , Cat#19055.

    Techniques: Infection, Co-Culture Assay, Activation Assay, Expressing, Staining

    In vitro HIV-1 infection of macrophages primes NK-cell effector function toward cytokine release (A) Experimental setup of assessment of NK cell function after MDM co-culture. After co-culture with HIV-1-infected (89.6 and THRO.c (TH)), LPS/IFNγ-stimulated, and unstimulated MDM for 24 h, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation, measured by CD107a surface expression, and cytokine production, measured by intracellular CCL4, TNF, and IFNγ expression, were assessed. (B) Concatenated flow cytometry contour plots displaying CD107 and CCL4 expression of NK cells after MDM co-culture with and without targets (left). Representative contour plots for TNF and IFNγ plotted against CCL4 and determination of cytokine-positive NK cells (right). (C) Frequencies of CD107a+, CCL4+, TNF+, and IFNγ+ bulk (CD56 br+dim ) NK cells in response to 721.221 targets after co-culture with MDM in the different conditions. Differences in the functional response to targets in the different conditions to the unstimulated MDM priming condition were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg), only significant FDR-adjusted p values are shown ( p < 0.05) (89.6: n = 14; THRO.c: n = 12; LPS/IFNγ: n = 13). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels C and F (B, C) . See also .

    Journal: iScience

    Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

    doi: 10.1016/j.isci.2025.112879

    Figure Lengend Snippet: In vitro HIV-1 infection of macrophages primes NK-cell effector function toward cytokine release (A) Experimental setup of assessment of NK cell function after MDM co-culture. After co-culture with HIV-1-infected (89.6 and THRO.c (TH)), LPS/IFNγ-stimulated, and unstimulated MDM for 24 h, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation, measured by CD107a surface expression, and cytokine production, measured by intracellular CCL4, TNF, and IFNγ expression, were assessed. (B) Concatenated flow cytometry contour plots displaying CD107 and CCL4 expression of NK cells after MDM co-culture with and without targets (left). Representative contour plots for TNF and IFNγ plotted against CCL4 and determination of cytokine-positive NK cells (right). (C) Frequencies of CD107a+, CCL4+, TNF+, and IFNγ+ bulk (CD56 br+dim ) NK cells in response to 721.221 targets after co-culture with MDM in the different conditions. Differences in the functional response to targets in the different conditions to the unstimulated MDM priming condition were analyzed using the Wilcoxon signed-rank test with FDR correction (Original Benjamini and Hochberg), only significant FDR-adjusted p values are shown ( p < 0.05) (89.6: n = 14; THRO.c: n = 12; LPS/IFNγ: n = 13). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panels C and F (B, C) . See also .

    Article Snippet: EasySepTM Human NK Cell Enrichment Kit , StemCell Technologies , Cat#19055.

    Techniques: In Vitro, Infection, Cell Function Assay, Co-Culture Assay, Cell Culture, Expressing, Flow Cytometry, Functional Assay

    KIR+ NKG2A+ NK cells from PLWH respond to macrophage priming, whereas the CD56 neg subset remains dysfunctional (A) Set-up of NK cell and MDM co-culture from peripheral blood of PLWH. After 24 h co-culture with LPS/IFNγ-stimulated (stimulation for 24 h prior to co-culture) and unstimulated MDM, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation (measured by CD107a), and cytokine production, measured by intracellular CCL4, TNF, and IFNγ staining were assessed. (B) Flow cytometry staining of phenotypic MDM markers after 9 days of differentiation. (C) Expression of pSTAT1 (MdFI) in MDM with and without LPS/IFNγ stimulation. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (D) Representative dot plot of the three different subsets of NK cells (CD56 br , CD56 dim and CD56 neg ) (left) and respective frequencies among bulk NK cells from PLWH (right) ( n = 7). (E) Frequencies of CD107a-, CCL4-, TNF-, and IFNγ-expressing bulk (CD56 br+dim+neg ) NK cells with or without 721.221 targets after MDM co-culture. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (F) Gating strategy for NKG2A, NKG2C, CD57, and KIRs (any KIR Boolean gate: KIR2DL1/S5 OR KIR2DL2/L3 OR KIR3DL1/S1) in CD56 br , CD56 dim , and CD56 neg NK cells from PLWH. (G) Heatmap showing median frequencies of CD107a-, CCL4-, TNF-, and IFNγ-positive NK cells from PLWH of indicated subsets after MDM co-culture in response to targets. Values were clustered unsupervised according to the CD107a response, corresponding frequencies of the detected subsets in total NK cells are depicted to the right ( n = 7). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panel E (B, C), panel F (D, E, G), or panels C and F (F) . See also .

    Journal: iScience

    Article Title: HIV-1 infection of macrophages differentially primes NK-cell cytotoxicity and proinflammatory cytokine production

    doi: 10.1016/j.isci.2025.112879

    Figure Lengend Snippet: KIR+ NKG2A+ NK cells from PLWH respond to macrophage priming, whereas the CD56 neg subset remains dysfunctional (A) Set-up of NK cell and MDM co-culture from peripheral blood of PLWH. After 24 h co-culture with LPS/IFNγ-stimulated (stimulation for 24 h prior to co-culture) and unstimulated MDM, NK cells were co-cultured with 721.221 (targets) for 6 h in the presence of brefeldin A and anti-CD107a antibody. NK cell degranulation (measured by CD107a), and cytokine production, measured by intracellular CCL4, TNF, and IFNγ staining were assessed. (B) Flow cytometry staining of phenotypic MDM markers after 9 days of differentiation. (C) Expression of pSTAT1 (MdFI) in MDM with and without LPS/IFNγ stimulation. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (D) Representative dot plot of the three different subsets of NK cells (CD56 br , CD56 dim and CD56 neg ) (left) and respective frequencies among bulk NK cells from PLWH (right) ( n = 7). (E) Frequencies of CD107a-, CCL4-, TNF-, and IFNγ-expressing bulk (CD56 br+dim+neg ) NK cells with or without 721.221 targets after MDM co-culture. Differences were analyzed using the Wilcoxon signed-rank test ( n = 7). (F) Gating strategy for NKG2A, NKG2C, CD57, and KIRs (any KIR Boolean gate: KIR2DL1/S5 OR KIR2DL2/L3 OR KIR3DL1/S1) in CD56 br , CD56 dim , and CD56 neg NK cells from PLWH. (G) Heatmap showing median frequencies of CD107a-, CCL4-, TNF-, and IFNγ-positive NK cells from PLWH of indicated subsets after MDM co-culture in response to targets. Values were clustered unsupervised according to the CD107a response, corresponding frequencies of the detected subsets in total NK cells are depicted to the right ( n = 7). All data are shown as median ± IQR with each dot representing one biological replicate. Stainings were performed with antibody panel E (B, C), panel F (D, E, G), or panels C and F (F) . See also .

    Article Snippet: EasySepTM Human NK Cell Enrichment Kit , StemCell Technologies , Cat#19055.

    Techniques: Co-Culture Assay, Cell Culture, Staining, Flow Cytometry, Expressing