Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Measure of STAT6 mRNA level in HT-29 cells 24 hours post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (B) STAT6 protein level analysis at 2, 5 and 7 days post-transfection in HT-29 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Measure of STAT6 mRNA level in ZR-75-1 cells 3 days post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (D) STAT6 protein level analysis after 4 and 7 days of transfection in ZR-75-1 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. E) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells (top) and ZR-75-1 (bottom) cells. STAT6 siRNA sequences and non-targeting siRNA were used at 100 nM as the final concentration. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Concentration Assay

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A and B) Number of live HT-29 cells measured at 5 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (C) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent experiments (n) shown in A and B. (D and E) Number of live ZR-75-1 cells measured at 4 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (F) The graph illustrates how ZR-75-1 cells grew over time and represents the mean ± SEM of the multiple independent experiments (n) shown in D and E. The number of live cells was calculated as detailed in the material and methods using NucleoCounter NC-100. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the transfection with STAT6 siRNA sequences. STAT6 and non-targeting (NT) siRNA sequences were used at 100 nM as the final concentration. Non-transfected cells served as negative control and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Comparison, Concentration Assay, Negative Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Apoptosis analysis in HT-29 cells. (B) Apoptosis analysis in ZR-75-1 cells. In both cases, the graphs represent from left to right: early (Annexin V + /PI - ), late (Annexin V + /PI + ) and total (Annexin V + ) apoptosis. The graphs represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Representative flow cytometry plots in HT-29 cells. (D) Representative flow cytometry plots in ZR-75-1 cells. The X axes represent Annexin V and the Y axes represent PI fluorescence intensity. Quadrants were set according to cells independently stained with Annexin V or PI. Apoptosis was studied at 7 days post-transfection in both cell lines. Data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Flow Cytometry, Fluorescence, Staining, Transfection, Software, Sequencing, Concentration Assay, Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: STAT6 siRNA transfection was carried out at day 1 of cell culture with (A) STAT6.1 and (B) STAT6.4 at 100 nM. A second transfection was carried out in both cases with STAT6.1 and STAT6.4 at the same concentration 7 days after the first transfection. The graphs represent the number of live cells over time measured at day 7 and 14 days post-first transfection counted using NucleoCounter NC-100 as detailed in the material and methods section. The mean ± SEM of 3 independent experiments is represented in each graph. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the individual transfection with STAT6 siRNA sequences, and sequential transfection with NT (NT+NT) vs . sequential transfection with STAT6.1 and STAT6.4.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Cell Culture, Concentration Assay, Control, Comparison

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) STAT6 expression at protein level in HT-29 cells at day 2 and 7 post-transfection measured by flow cytometry. The graph represents the mean ± SEM of multiple independent experiments (n). Data was analysed using Flowjo Software. The percentage of STAT6 positive cells is represented on the Y axis. (B) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells. (C) Number of live HT-29 cells measured at day 7 post-transfection by NucleoCounter NC100. The graph represents the mean ± SEM of multiple independent experiments (n). (D) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent number of experiments shown in C. (E, F and G) The graphs show early (E) (Annexin V + /PI - ), late (F) (Annexin V + /PI + ) and total (G) (Annexin V + ) apoptosis, and represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. Apoptosis was studied at 7 days post-transfection. Data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Expressing, Transfection, Flow Cytometry, Software, Control, Sequencing, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation

doi: 10.1084/jem.20070157

Figure Lengend Snippet: Quantitative RT-PCR and IHC analyses demonstrate reduced expression of IL-1F6 as the skin phenotype resolves. (A) Expression of IL1F5 , -1F6 , -1F8 , and -1F9 in the skin decreases over the P1–21 time course. (B) Expression of IL1RL2 is elevated in transgenic skin relative to control skin at P1 and 3. Expression of IL1RL2 at P5–21, and expression of IL1RAP from P1–21 is not different in transgenic and nontransgenic skin. (C) Expression of IL23A , CSF2 , TNF, IL1A , CCL2 , KRT14 , CXCL2 , TGA2 , HBEGF , and AREG is given in both transgenic and nontransgenic skin from P1 to 21. Total skin RNA was isolated from K14/ IL1F6 transgenic and nontransgenic pups ( n = 3) and was analyzed by quantitative RT-PCR. Expression of specific genes is relative to β-actin. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars in A–C represent the mean ± the SD. (D) Protein expression of IL-1F6 in K14/ IL1F6 transgenic pup skin is elevated at P1 and P5 relative to nontransgenic control skin, but is decreased to the endogenous level by P10. (E) K14 protein was detected throughout the epidermis in transgenic pups at P1 and 5, and is observed only in the basal layer at P10. Skin samples were fixed in neutral buffered formalin, sectioned, analyzed by IHC using a monoclonal antibody against mouse IL-1F6 or a polyclonal antibody against K14. Bar, 100 μm.

Article Snippet: IHC was performed on selected skin sections using the following antibodies: 2.5 μg/ml K6 (Covance), 50 ng/ml K14 (Covance), 2 μg/ml CD54 (ICAM-1; Millipore), 10 μg/ml CD205 (DEC205; Serotec), 5 μg/ml CD3 (DakoCytomation), 20 μg/ml F4/80 (BM8; Serotec), and 15 μg/ml IL-1F6 (Amgen).

Techniques: Quantitative RT-PCR, Expressing, Transgenic Assay, Control, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation

doi: 10.1084/jem.20070157

Figure Lengend Snippet: IL-1F5 is an antagonist of IL-1F6 in vivo. (A) K14 /IL1F6 pups with reduced IL1F5 gene dosage exhibit an altered gross appearance 3 d after birth. The skin phenotype and runting are most severely affected in K14 /IL1F6 Tg + , IL1F5 −/− pups. From left to right: K14 /IL1F6 Tg − , IL1F5 −/− ; K14 /IL1F6 Tg + , IL1F5 +/− ; K14 /IL1F6 Tg + , IL1F5 −/− . (B) K14 /IL1F6 pups with reduced IL1F5 gene dosage have a thickened epidermis, increased dermal infiltrate, and parakeratosis compared with K14 /IL1F6 , IL1F5 +/+ pups. HE staining of skin is from 3-d-old pups. (C) Adult K14 /IL1F6 , IL1F5 +/− mice have skin abnormalities, whereas K14 /IL1F6 , IL1F5 +/+ mice do not. HE staining of skin is from 14-wk-old mice. (D, left) K14/ IL1F6 , IL1F5 +/− skin exhibits parakeratotic hyperkeratosis; note the nucleated keratinocytes in the corneal layer (arrowhead). (right) K14/ IL1F6 , IL1F5 +/− skin contains dilated superficial dermal blood vessels (arrowheads) and a mixed inflammatory infiltrate composed predominantly of neutrophils and macrophages, with fewer lymphocytes and rare eosinophils. Bars, 100 μm.

Article Snippet: IHC was performed on selected skin sections using the following antibodies: 2.5 μg/ml K6 (Covance), 50 ng/ml K14 (Covance), 2 μg/ml CD54 (ICAM-1; Millipore), 10 μg/ml CD205 (DEC205; Serotec), 5 μg/ml CD3 (DakoCytomation), 20 μg/ml F4/80 (BM8; Serotec), and 15 μg/ml IL-1F6 (Amgen).

Techniques: In Vivo, Staining

Journal: The Journal of Experimental Medicine

Article Title: Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation

doi: 10.1084/jem.20070157

Figure Lengend Snippet: Model for the role of IL-1F6 and -1F5 in the K14/ IL1F6 skin phenotype. (A) Proposed mechanism of action (MOA) for IL-1F6 and -1F5 in the regulation of a competent signaling complex containing RP2 and AcP, and downstream events. IL-1F6 activates a receptor complex, resulting in signal transduction, induction of cytokine and chemokine expression, and mobilization of neutrophils and macrophage into the dermis. This process is antagonized by IL-1F5. (B) Model for the critical balance between IL-1F6 and -1F5 expression in pup skin inflammation. In wild-type pups, a balance between IL-1F6 agonist activity and IL-1F5 antagonist activity results in normal skin. Increasing IL-1F6 expression by transgenic expression in an IL1F5 +/+ background tips the balance toward skin inflammation. Reducing IL1F5 gene dosage ( IL1F5 +/− ) in the K14 /IL1F6 mice exacerbates skin inflammation. Complete deletion of IL1F5 further shifts the balance in K14 /IL1F6 mice, resulting in an even more severe inflammation.

Article Snippet: IHC was performed on selected skin sections using the following antibodies: 2.5 μg/ml K6 (Covance), 50 ng/ml K14 (Covance), 2 μg/ml CD54 (ICAM-1; Millipore), 10 μg/ml CD205 (DEC205; Serotec), 5 μg/ml CD3 (DakoCytomation), 20 μg/ml F4/80 (BM8; Serotec), and 15 μg/ml IL-1F6 (Amgen).

Techniques: Transduction, Expressing, Activity Assay, Transgenic Assay

Journal: Blood Advances

Article Title: IL-15/IL-15Rα/CD80-expressing AML cell vaccines eradicate minimal residual disease in leukemic mice

doi: 10.1182/bloodadvances.2018019026

Figure Lengend Snippet: Appropriate cellular processing and expression of IL-15, IL-15Rα, and CD80 are observed after lentiviral transduction of 293T and 32Dp210 cell lines. (A) Levels of IL-15 secretion after lentiviral transduction of 293T cells. Murine IL-15 secretion was quantified by ELISAs of tissue culture supernatants 48 hours after transfection. On the left are designations of lentiviral vector constructs generated and used for transduction studies. Asterisks denote vectors that were used to generate the final 32Dp210 vaccines. Lanes 1 to 5 on the vertical axis depict IL-15 secretion detected as nanograms per milliliter as detected by ELISA for cells transduced with each of the designated constructs. Bars represent the mean cytokine concentration ± SEM. (B) Levels of IL-15 secretion after lentiviral transduction of 32Dp210 cells are comparable pre- and postirradiation. 32Dp210 cells transduced with lentiviral vectors containing mIL-15/IL-15Rα or CD80, or both IL-15/IL-15Rα and CD80, were expanded, and populations were selected that express similar levels of cell-surface IL-15Rα and/or CD80 by fluorescence-activated cell sorter. High-expressing populations were purified. Levels of IL-15 in culture supernatants (ng/mL) were measured before (red bars) and 48 hours after irradiation with 40 Gy (blue bars). 32Dp210, to the left of the uppermost 2 lanes, indicates IL-15 secretion in untransduced controls. As previously, designations to the left of the graph indicate the genes (IL-15, IL-15Rα, or CD80, or all 3 cassettes) contained in the lentiviral vector used to transduce the 32Dp210 cells analyzed. Levels of IL-15 detected in cell culture supernatants are indicated (ng/mL) on the horizontal axis. Bars represent the mean cytokine concentration ± SEM. (C) Transduced 32Dp210 cells exhibit high level cell-surface expression of IL-15Rα, CD80, and IL-15 after purification of transduced 32Dp210 populations. Lentivirally transduced, purified 32Dp210-derived cells were stained with anti-IL-15, anti-IL-15Rα, and anti-CD80 antibodies and subjected to flow cytometric analyses. 32Dp210 on the left indicates the untransduced parental cell line used as a control. The genes encoded by each lentiviral vector used to transduce the 32Dp210 cells are indicated to the left of the flow analysis, and the antibody used to stain cells is indicated above each flow plot. (D) Cell-surface expression of IL-15Rα and CD80 on 32Dp210 vaccines is stable 48 hours after irradiation with doses up to 50 Gy. Lentivirally transduced, purified 32Dp210 cells were stained with anti-IL-15Rα and anti-CD80 antibodies, and the effects of different doses of radiation on IL-15Rα and CD80 expression analyzed. The lentiviral vector used to transduce 32Dp210 cells is indicated above each plot, and the dose of irradiation indicated on the vertical axis. Antibodies used to analyze cell surface expression are indicated below the plots. Data showing cell surface IL-15Rα expression are shown in the left 2 graphs, and cell surface staining with anti-CD80 antibodies is presented in the right 2 graphs.

Article Snippet: Flow cytometric analyses Antibodies to H-2D K (15-5-5), H-2K k (36-7-5), H-2Kb (AF6-88.5), CD3 (2C11), CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61), CD335 (29A-1.4), CD11b (M1/70), Ly-6G and Ly-6C (Gr-1), CD80 (16-10A1), IL-15 (16-7154-85), IL-15Rα (DNT15Ra), CD279 (J43), CD44 (IM7), CD62L (MEL-14), FoxP3 (FM23), interferon γ (IFN-γ; 14-4-4s) and Fc Block (2.4G2), PD-L1 (BV711 [Rat anti-mouse CD274, clone MIH5], matched BV711-conjugated isotype control [mAb; rat IgG 2a]), and PD-1 (BV421 [Hamster anti-mouse CD279, clone J43]) were purchased from BD Biosciences (San Jose, CA).

Techniques: Expressing, Transduction, Transfection, Plasmid Preparation, Construct, Generated, Enzyme-linked Immunosorbent Assay, Concentration Assay, Fluorescence, Purification, Irradiation, Cell Culture, Derivative Assay, Staining

Journal: Blood Advances

Article Title: IL-15/IL-15Rα/CD80-expressing AML cell vaccines eradicate minimal residual disease in leukemic mice

doi: 10.1182/bloodadvances.2018019026

Figure Lengend Snippet: Serial vaccination with 32Dp210-derived whole cell vaccines in non-tumor-bearing mice stimulates robust antileukemic cytolytic activity. Naïve C3H mice were treated with 4 vaccinations with 2 × 106 irradiated 32Dp210-Luc, 32Dp210-mIL-15-IL-15Rα, 32Dp210-CD80, or 32Dp210-IL-15/IL-15Rα/CD80. In the first experimental group of mice, splenocytes were harvested and restimulated in vitro with irradiated (100 Gy) 32Dp210 parent cells for 5 days. Thereafter, cells were harvested and purified by Ficoll density gradient for ex vivo assays (A-F). In a parallel experimental group, vaccinated mice were challenged with IV inoculation of 32Dp210 leukemia and monitored for survival (G). (A) Cytolytic activity of splenocytes in ex vivo assays is greatest after stimulation by 32Dp210-IL-15/IL-15Rα/CD80 vaccine. Splenic effectors cells and 1 × 105 CellTrace DDAO-SE labeled 32Dp210 target cells were cocultured at different ratios (red bars, 1:1; blue bars, 5:1; green bars, 10:1) for 48 hours. X-axis: naïve: indicates splenocytes from uninjected C3H mice; 32Dp210: splenocytes from mice vaccinated with the parent 32Dp210 cell line; vaccine expressing IL-15/IL-15Rα, CD80, or IL-15/IL-15Rα/CD80 (below graph): depict assays with splenocytes from C3H mice treated with lentivirally transduced 32Dp210 vaccines expressing the indicated transgenes. The mean percentage of apoptotic cells, defined by detection of activated caspase-3 by antibody staining, is depicted on the y-axis (± SEM). (B) ELIspot assay of IFN-γ expression in splenocytes from vaccinated mice shows increased frequencies of cytotoxic cells after treatment with all transduced 32Dp210 vaccines. Splenocytes from unvaccinated mice (naïve), or from mice vaccinated with irradiated parent cells 32Dp210 cells, or with engineered 32Dp210 cells expressing each of the transgene cassettes, indicated below the bar graphs, were cocultured with irradiated 32Dp210 cells, and the frequency of IFN-γ positive cells was quantified. The mean number of spots per well per containing 3 × 105 cells in triplicate samples is depicted on the y-axis ± SEM. (C and D) Highest levels of intracellular IFN-γ expression are observed in CD3+CD8+ and CD3+CD4+ T cells from 32Dp210-IL-15/IL-15Rα/CD80-vaccinated mice after secondary stimulation, whereas stimulation with BCR-ABL-loaded splenocytes produces IFN-γ levels comparable to unstimulated controls. In panels C and D, the y-axis depicts the percent IFN-γ positive CD3+CD8+ T cells (C) and CD3+CD4+ T cells (D) in splenocytes from vaccinated non-tumor-bearing mice. X-axis: naïve = unvaccinated mice; 32Dp210 = vaccinated weekly, 4 times with unmodified irradiated 32Dp210 parent cells; -IL-15/IL-15Rα = vaccinated with 32Dp210-IL-15/IL-15Rα; -CD80 = vaccinated with 32Dp210-CD80; -IL-15/IL-15Rα/CD80 = vaccinated with 32Dp210-IL-15/IL-15Rα/CD80. Splenocytes were stimulated for 20 hours with either media alone, C3H splenocytes loaded with an irrelevant control peptide, or BCR-ABL peptide, or with unmodified 32Dp210 cells as indicated. (E) Comparative ELIspot assays of IFN-γ expression in splenocytes from vaccinated mice confirm increased frequencies of cytotoxic cells after treatment with all transduced 32Dp210 vaccines but no significant stimulation by BCR-ABL-loaded cells. ELIspot assays were performed as in panel A. C3H splenocytes were loaded with either BCR-ABL peptide or control peptide and cocultured with splenocytes from vaccinated animals as described in panel A, depicted on the x-axis. The mean number of spots per well per containing 3 × 105 cells in triplicate samples is depicted on the y-axis (± SEM). (F) Splenocytes from non-tumor-bearing mice treated with the 32Dp210-derived vaccines show high levels of lytic activity to 32Dp210 targets, but not to human BCR-ABL peptide loaded syngeneic C3H cells. Splenocytes from vaccinated non-tumor-bearing C3H mice, as described in panel A were stimulated ex vivo for 5 days with irradiated 32Dp210 cells and used as effectors. Unmodified 32Dp210 cells, BCR-ABL, or control peptide-loaded splenocytes from naïve C3H mice were used as targets. Ex vivo lytic activity was assayed by intracellular staining of active caspase-3 after coculture of effectors and targets at an effector-to-target ratio = 10:1 for 24 hours. X-axis depicts cells from different experimental vaccine groups as in panel A. The mean percentage of apoptotic cells, defined by detection of activated caspase-3 by antibody staining, is depicted on the y-axis (± SEM). (G) Treatment with transduced 32Dp210-derived vaccines confers greater survival after leukemia challenge than does administration of untransduced irradiated 32Dp210 cell vaccines. Mice were treated weekly 3 times with 32Dp210-derived vaccines as described previously. Thereafter, they were inoculated IV with 1 × 104 32Dp210-luc cells and longitudinally monitored by in vivo bioluminescence imaging for tumor progression and survival. Percent survival is depicted on the y-axis, and duration of survival on the x-axis. Animals surviving the initial leukemia challenge were rechallenged with a second IV inoculation of 32Dp210 leukemia (indicated by downward arrow), 150 days after the initial tumor challenge.

Article Snippet: Flow cytometric analyses Antibodies to H-2D K (15-5-5), H-2K k (36-7-5), H-2Kb (AF6-88.5), CD3 (2C11), CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61), CD335 (29A-1.4), CD11b (M1/70), Ly-6G and Ly-6C (Gr-1), CD80 (16-10A1), IL-15 (16-7154-85), IL-15Rα (DNT15Ra), CD279 (J43), CD44 (IM7), CD62L (MEL-14), FoxP3 (FM23), interferon γ (IFN-γ; 14-4-4s) and Fc Block (2.4G2), PD-L1 (BV711 [Rat anti-mouse CD274, clone MIH5], matched BV711-conjugated isotype control [mAb; rat IgG 2a]), and PD-1 (BV421 [Hamster anti-mouse CD279, clone J43]) were purchased from BD Biosciences (San Jose, CA).

Techniques: Derivative Assay, Activity Assay, Irradiation, In Vitro, Purification, Ex Vivo, Labeling, Expressing, Staining, Enzyme-linked Immunospot, In Vivo, Imaging

Journal: Blood Advances

Article Title: IL-15/IL-15Rα/CD80-expressing AML cell vaccines eradicate minimal residual disease in leukemic mice

doi: 10.1182/bloodadvances.2018019026

Figure Lengend Snippet: Administration of lentivirally engineered 32Dp210 vaccines in 32Dp210 leukemia-bearing mice confers greater progression-free and overall survival. (A) Overall survival of mice with 32Dp210 leukemia is greatest after serial vaccination with 32Dp210-IL-15/IL-15Rα/CD80: mice were inoculated with 1 × 104 32Dp210 leukemia cells, and vaccination was initiated 3 days later (depicted in the schema above the graph). Experimental groups included mice inoculated with tumor that received no further treatment (no vaccine, filled gray circles, n = 15) and mice vaccinated with irradiated parental 32Dp210 cells (open red circles, n = 15), 32Dp210-IL-15/IL-15Rα (filled blue squares, n = 15), 32Dp210-CD80 (open green squares, n = 15), or 32Dp210-IL-15/IL-15Rα/CD80 vaccines (filled purple triangles, n = 25). Percent survival is depicted on the y-axis, and duration of survival (days), beginning on day 0 with tumor inoculation, is shown on the x-axis. Data represent the results of 3 independent experiments. (B) In vivo, antibody-mediated depletion of CD8+ cells abrogates 32Dp210-IL-15/IL-15Rα/CD80 vaccine effects on overall survival in leukemic mice. After tumor inoculation on day 0, mice underwent 3 weekly vaccinations with 32Dp210-IL-15/IL-15Rα/CD80. Filled purple triangles depict vaccination without antibody depletion. Filled gray circles depict unvaccinated control mice inoculated with 32Dp210 leukemia. Antibody-mediated in vivo depletion of CD8+ cells (filled red diamonds, n = 10), CD4+ cells (open blue squares, n = 5), and asialo GM1+ cells (filled green squares, n = 10) are shown according to the schema at the top of the figure. Percent survival is depicted on the y-axis, and duration of survival (days) on the x-axis.

Article Snippet: Flow cytometric analyses Antibodies to H-2D K (15-5-5), H-2K k (36-7-5), H-2Kb (AF6-88.5), CD3 (2C11), CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61), CD335 (29A-1.4), CD11b (M1/70), Ly-6G and Ly-6C (Gr-1), CD80 (16-10A1), IL-15 (16-7154-85), IL-15Rα (DNT15Ra), CD279 (J43), CD44 (IM7), CD62L (MEL-14), FoxP3 (FM23), interferon γ (IFN-γ; 14-4-4s) and Fc Block (2.4G2), PD-L1 (BV711 [Rat anti-mouse CD274, clone MIH5], matched BV711-conjugated isotype control [mAb; rat IgG 2a]), and PD-1 (BV421 [Hamster anti-mouse CD279, clone J43]) were purchased from BD Biosciences (San Jose, CA).

Techniques: Irradiation, In Vivo

Journal: Blood Advances

Article Title: IL-15/IL-15Rα/CD80-expressing AML cell vaccines eradicate minimal residual disease in leukemic mice

doi: 10.1182/bloodadvances.2018019026

Figure Lengend Snippet: 32Dp210-IL-15/IL-15Rα/CD80 vaccines administered postremission induction have efficacy in eradicating MRD. (A) Leukemic burden of groups 32Dp210-luc-HSV-TK leukemic mice before and after 5 days of GCV treatment destined for postremission vaccination with 32Dp210-IL-15/IL-15Rα/CD80 or no further therapy. Mice were inoculated with 1 × 105 32Dp210 cells on day 0 as indicated by the schema at the top of the figure. On day 10, mice underwent in vivo bioluminescence imaging, and average photon counts per animal were plotted (upper set of bar graphs). N1 to N5 represent normal, non-tumor-bearing mice injected with luciferin on the same day, to establish background luminescence levels. Lanes 1 to 13 indicate mice inoculated with leukemia and subsequently treated with GCV according to the schema at the top of the figure. Blue bars in lanes 1 to 5 indicate analyses of mice that would receive 14 days GCV treatment and no further intervention. Green bars in lanes 6 to 13 depict analyses of mice treated with 14 days of GCV treatment that would undergo serial vaccination with irradiated 32Dp210-IL-15/IL-15Rα/CD80 cells beginning at day 17. Lower panel of bar graphs depicts semiquantitative IVIS studies after 5 days of GCV administration (day 15) in the same animals analyzed in the upper panel of bar graphs. (B) Survival of 32Dp210-luc-HSV-TK leukemia bearing mice after remission induction with GCV treatment is predicated on administration of irradiated 32Dp210-IL-15/IL-15Rα/CD80 vaccine. Purple filled circles: leukemia inoculation with no treatment (n = 5); open circles: 14 days GCV treatment beginning day 10 after leukemia injection with no additional treatment (n = 5); filled green squares: GCV-mediated remission induction followed by serial vaccination with irradiated 32Dp210-IL-15/IL-15Rα/CD80 cells (n = 10).

Article Snippet: Flow cytometric analyses Antibodies to H-2D K (15-5-5), H-2K k (36-7-5), H-2Kb (AF6-88.5), CD3 (2C11), CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61), CD335 (29A-1.4), CD11b (M1/70), Ly-6G and Ly-6C (Gr-1), CD80 (16-10A1), IL-15 (16-7154-85), IL-15Rα (DNT15Ra), CD279 (J43), CD44 (IM7), CD62L (MEL-14), FoxP3 (FM23), interferon γ (IFN-γ; 14-4-4s) and Fc Block (2.4G2), PD-L1 (BV711 [Rat anti-mouse CD274, clone MIH5], matched BV711-conjugated isotype control [mAb; rat IgG 2a]), and PD-1 (BV421 [Hamster anti-mouse CD279, clone J43]) were purchased from BD Biosciences (San Jose, CA).

Techniques: In Vivo, Imaging, Injection, Irradiation