Journal: PLOS One

Article Title: Combining lenalidomide with IL-2 family of cytokines enhances activating receptor and perforin/granzyme expression in NK cells

doi: 10.1371/journal.pone.0344471

Figure Lengend Snippet: a) Flow cytometric phenotyping of primary healthy human PBMC, gated on NK cell subpopulations as shown from total live cells (total NK cells are defined as CD3-CD56+and percentage is shown on the side of gates, NK cell subsets: CD56dim and CD56bright from total NK and percentages are shown inside each gate) after treatment with 10μM lenalidomide (LEN), or DMSO vehicle for 7 days. Data representative of n = 6 healthy donor individual experiments (biological replicates). Pseudo-coloring denotes the density of cells on each marker. b ) Isolated primary human healthy NK cells and subsets: (CD56 + CD3 - total NK cells, CD56 dim and CD56 bright ) after treatment with lenalidomide, IL-2, IL-15 or their combination for 7 days. Data representative of n = 6 healthy donor individual experiments (biological replicates). Pseudo-coloring denotes the density of cells on each marker. c ) Viability of isolated primary NK cells treated with lenalidomide, IL-2, IL-15 or their combination for 72 hours by flow cytometric analysis using a live/dead dye. Proliferation of isolated primary NK subsets (d) CD56 bright and (e) CD56 dim by flow cytometric analysis of CellTrace Violet from cells gated as in “a” and “b” (n = 6 healthy donor biological replicates). f ) Representative figure of proliferation with CellTrace Violet of NK subsets as in d and e from primary isolated NK cells from healthy donor PBMC (representative of n = 6 biological replicates). Grey line shows the recorded data, and the blue lines represent the divisions as calculated by FlowJo. In all figures, error bars represent the SEM of biological replicates and were analyzed by ordinary one-way ANOVA with multiple comparison analysis. P values are shown as asterisk: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 as summarized in Graphpad Prism.

Article Snippet: Lenalidomide (cat# S1029, Selleckchem, Houston, Texas, United States) was dissolved first in dimethyl sulfoxide (DMSO, cat# D2650, Sigma-Aldrich, St. Louis, Missouri, United States) at a 10mM concentration and aliquots frozen at -80 o C until ready to use.

Techniques: Marker, Isolation, Comparison

Journal: PLOS One

Article Title: Combining lenalidomide with IL-2 family of cytokines enhances activating receptor and perforin/granzyme expression in NK cells

doi: 10.1371/journal.pone.0344471

Figure Lengend Snippet: Proliferation of YT cells measured by overnight tritium (3H) incorporation (1.0 μCi per well) after culture for 1,3,5 or 7 days in the presence of 10 or 20μM lenalidomide (LEN) in the (a) absence or (b) presence of human recombinant (hr) IL-2 (100U/mL) or (c) combined results of YT proliferation at 7 days for coculture of lenalidomide with IL-2 and/or IL-15. Results expressed as mean counts per minute (cpm) of triplicate wells for each experiment, and a total of n = 3 biological replicates. Cell cycle analysis of isolated primary NK cells (n = 6 healthy donor biological replicates), treated as indicated, measured via flow cytometric analysis of propidium iodide (PI) staining and expressed as percent of cells in (d) S, (e) G1, or (f) G2 phase. g) Cell cycle analysis of YT cells cultured with 20μM LEN (for up to 14 days) measured via flow cytometric analysis of propidium iodide (PI) staining and expressed as percent of cells in S, G1 and G2 phase. Error bars represent the SEM and were analyzed by 2way ANOVA. P values are shown as asterisk: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 as summarized in Graphpad Prism.

Article Snippet: Lenalidomide (cat# S1029, Selleckchem, Houston, Texas, United States) was dissolved first in dimethyl sulfoxide (DMSO, cat# D2650, Sigma-Aldrich, St. Louis, Missouri, United States) at a 10mM concentration and aliquots frozen at -80 o C until ready to use.

Techniques: Recombinant, Cell Cycle Assay, Isolation, Staining, Cell Culture

Journal: PLOS One

Article Title: Combining lenalidomide with IL-2 family of cytokines enhances activating receptor and perforin/granzyme expression in NK cells

doi: 10.1371/journal.pone.0344471

Figure Lengend Snippet: (a) Flow cytometric phenotyping of NK cell receptor expression in primary human healthy PBMC, gated on NK cell subpopulations (total NK cells CD3 - CD56 + , NK cell subsets CD56 dim and CD56 bright from total NK) after treatment with 10μM lenalidomide (LEN), or DMSO. Shown are: CD16, NKp46, KIR2DL3, KIR2DL1. Data representative of n = 5 independent experiments. (b) Western blot analysis of phosphorylated AKT (pAKT) in either primary isolated NK cells or YT cells treated as shown. Full raw gels are shown in supplemental figure 2. (c) Quantification of western blots showing the ratio of phosphor-AKT (phosphorylated threonine 308) to total AKT (n = 3) and representative figure for p85 in YT cells treated with 20μM of LEN, or a DMSO control, for 1, 3, 5, or 7 days. (d) Corroboration of phosphor AKT reduction by LEN treatment measured with phospho-flow. To adjust data across samples to minimize technical variation (batch effects) for biological comparisons the Y-axis overlay normalization in FlowJo was used in this analysis as shown. Error bars represent the SEM and were analyzed by ANOVA with multiple comparison analysis. P values are shown as asterisk: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 as summarized in Graphpad Prism.

Article Snippet: Lenalidomide (cat# S1029, Selleckchem, Houston, Texas, United States) was dissolved first in dimethyl sulfoxide (DMSO, cat# D2650, Sigma-Aldrich, St. Louis, Missouri, United States) at a 10mM concentration and aliquots frozen at -80 o C until ready to use.

Techniques: Expressing, Western Blot, Isolation, Control, Comparison

Journal: PLOS One

Article Title: Combining lenalidomide with IL-2 family of cytokines enhances activating receptor and perforin/granzyme expression in NK cells

doi: 10.1371/journal.pone.0344471

Figure Lengend Snippet: (a) Schematic representation of pathways targeted with inhibitors to understand how NKp46 is regulated. Created in BioRender. Eksioglu, E. (2025) https://BioRender.com/rg1nk09 . (b) NKp46 expression by flow cytometry in YT cells after treatment with either 20μM or 5μM lenalidomide (figure representative of n = 3 biological replicates). Legend inside gate shows the percentage of events inside the gate shown from total live cells measured. (c) NKp46 expression by flow cytometry in YT cells after treatment with the PI3K inhibitors Wortmannin (doses tested 0.2nM and 20nM) and LY294002 (doses tested 20nM and 2μM). Figure representative of n = 3 biological replicates. Legend inside gate shows the percentage of events inside the gate shown from total live cells measured. (d) NKp46 expression by flow cytometry in YT cells after inhibition of other AKT targeting pathways: U0126 (MEK inhibitor, 0.1μM), AG490 (JAK2 inhibitor, 25μM) and API-2 (AKT inhibitor, 10μM). Figure representative of n = 3 biological replicates. (e) Mechanistic validation of PI3K signaling in YT cells infected with viral vectors expressing a CD56 (control), DNp110, or CAp110; followed by treatment with lenalidomide for 4 days. Graph shows the percent of NKp46 positive cells after transfection with either CD56 (control), CAp110 or DNp110 in the presence or absence of lenalidomide measured by flow cytometry. Error bars represent the SEM of n = 4 biological replicates and were analyzed by ordinary ANOVA with multiple comparison analysis. P values are shown as asterisk: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 as summarized in Graphpad Prism.

Article Snippet: Lenalidomide (cat# S1029, Selleckchem, Houston, Texas, United States) was dissolved first in dimethyl sulfoxide (DMSO, cat# D2650, Sigma-Aldrich, St. Louis, Missouri, United States) at a 10mM concentration and aliquots frozen at -80 o C until ready to use.

Techniques: Expressing, Flow Cytometry, Inhibition, Biomarker Discovery, Infection, Control, Transfection, Comparison

Journal: PLOS One

Article Title: Combining lenalidomide with IL-2 family of cytokines enhances activating receptor and perforin/granzyme expression in NK cells

doi: 10.1371/journal.pone.0344471

Figure Lengend Snippet: (a) . Concentration of cytokines secreted in the supernatants of isolated NK cells treated with 20μM lenalidomide (LEN) or vehicle measured at 1, 3 or 6 days of culture using a cytometric bead array. (b) Primary NK cells cultured in 10μM lenalidomide or DMSO for 7 days followed by pulsing with K562 tumor targets at a 1:1 ratio for 5 hours prior to staining for CD107 granule mobilization or IFN-γ activation by flow. Lysis of K562 targets cells as in “b” by either (c) PBMC (effector to target ratio 10:1), ( d ) isolated NK cells, or ( Supplemental Fig. 4b ) YT cells as effectors cultured in the presence or absence of 20μM lenalidomide, or DMSO, with or without hrIL-2 (100U/mL) 7 days after pulsing (effector to target ratio 10:1). (e) Lysis of 721.221 targets cells as in “d” in the presence of 20μM lenalidomide, or DMSO for 7 days after pulsing (effector to target ratio 5:1). (f) NK cells cultured as in “a” to measure the mRNA expression of Perforin (PRF1) and Granzyme B (GZMB) by q-PCR. Values were calculated by the ΔΔCt method with control (DMSO) as the experimental control and the housekeeping gene ACTB as the internal control. (g) Immunohistochemistry of perforin and granzyme B of isolated primary NK cells treated with lenalidomide, IL-2, IL-15 or their combination as shown. Quantification of mean fluorescence intensity (MFI) of ( h ) granzyme and ( i ) perforin immunostaining (n = 4) from representative figure shown in . In all experiments in this figure, error bars represent the SEM of the mean and p-value was calculated by paired student t test (a and e) or ordinary one-way ANOVA with multiple comparisons (b, c and d) .

Article Snippet: Lenalidomide (cat# S1029, Selleckchem, Houston, Texas, United States) was dissolved first in dimethyl sulfoxide (DMSO, cat# D2650, Sigma-Aldrich, St. Louis, Missouri, United States) at a 10mM concentration and aliquots frozen at -80 o C until ready to use.

Techniques: Concentration Assay, Isolation, Cell Culture, Staining, Activation Assay, Lysis, Expressing, Control, Immunohistochemistry, Fluorescence, Immunostaining

Journal: PLOS One

Article Title: Combining lenalidomide with IL-2 family of cytokines enhances activating receptor and perforin/granzyme expression in NK cells

doi: 10.1371/journal.pone.0344471

Figure Lengend Snippet: (a) Western blot analysis of activation of STAT5 and total STAT5 analysis of healthy PBMCs treated with 10μM lenalidomide (LEN) or DMSO, for 7 days stand alone or in combination with 100U/mL of IL-2. (b) Western blot analysis of activation of STAT5 and total STAT5 analysis of flow sorted healthy NK cells treated with 10μM l enalidomide, or DMSO, for 7 days in the presence of 100U/mL IL-2 or 10ng/mL IL-15. B-actin served as the loading control. (c) Representative flow cytometric analysis of pSTAT5 + isolated NK cells treated with l enalidomide alone or in combination with IL-2 or IL-15 (representative figure). (d) Analysis of IL-2Rα (CD25), IL-2Rβ or IL-2γ receptor chains by flow cytometric analysis of NK cells (CD3 - CD56 + ) or its subsets (CD56 bright , CD56 dim ) in healthy PBMCs treated with 10μM lenalidomide, or DMSO, for 7 days in combination with 100U/mL of IL-2 or 10ng/mL of IL-15 as shown (representative figure of n = 3). To adjust data across samples to minimize technical variation (batch effects) for biological comparisons the Y-axis overlay normalization in FlowJo was used in this analysis as shown.

Article Snippet: Lenalidomide (cat# S1029, Selleckchem, Houston, Texas, United States) was dissolved first in dimethyl sulfoxide (DMSO, cat# D2650, Sigma-Aldrich, St. Louis, Missouri, United States) at a 10mM concentration and aliquots frozen at -80 o C until ready to use.

Techniques: Western Blot, Activation Assay, Control, Isolation

Journal: PLOS One

Article Title: Combining lenalidomide with IL-2 family of cytokines enhances activating receptor and perforin/granzyme expression in NK cells

doi: 10.1371/journal.pone.0344471

Figure Lengend Snippet: Schematic representation of the effect of lenalidomide in human NK cells. Our work demonstrates that lenalidomide effects can be enhanced by the addition of IL-2 family of cytokines in two ways: In the first one (left side), lenalidomide leads to the activation of STAT5 through phosphorylation (thin green arrow) which can be enhanced by IL-2 or IL-15 (thick green arrow). This is due to changes in the expression of each of the receptor’s subunits α, β or γ. This is important to differentiate because they both cytokines use mainly IL-2Rβ/γ, while IL-2Rα only enhances the sensitivity of cells to IL-2. STAT5 activation leads to the transcription of cytokine triggered genes, including those involved in growth and survival, and more importantly increased expression of perforin and granzyme which are critical for cytotoxic function. The second lenalidomide effect changed by addition of IL-2 family of cytokines (right side) is expression of activating receptors such as NKp46. Lenalidomide inhibits the activation of the P110 subunit of PI3K which leads to inhibition of NKp46 (red arrow) which can be overcome by the addition of IL-2 family of cytokines (green arrow). This reactivation leads to AKT phosphorylation and activation of AKT-transcribed genes, some of which have been already linked to NK cytotoxic activity. Created in BioRender. Eksioglu, E. (2025) https://BioRender.com/x925jkx .

Article Snippet: Lenalidomide (cat# S1029, Selleckchem, Houston, Texas, United States) was dissolved first in dimethyl sulfoxide (DMSO, cat# D2650, Sigma-Aldrich, St. Louis, Missouri, United States) at a 10mM concentration and aliquots frozen at -80 o C until ready to use.

Techniques: Activation Assay, Phospho-proteomics, Expressing, Inhibition, Activity Assay