Journal: PLoS ONE

Article Title: Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug

doi: 10.1371/journal.pone.0066219

Figure Lengend Snippet: Whole splenocytes were activated by anti-CD3 antibody for 3 days under Th1 or Th17 polarization conditions in the presence or absence of Lodamin (vehicle, 1 nM and 10 nM), followed by stimulation with PMA and ionomycin in the presence of Brefeldin A; Cell were analyzed by FACS following intracellular cytokine staining of IL-17 and IFN-γ, all plots are gated on CD4+ cells. Data are representative of three independent results.

Article Snippet: CD4 + enriched cells were prepared using anti-CD4 Microbeads (#130-1-049-201; Miltenyi Biotec, Gladbach, Germany) and MS columns (#130-042-201; Miltenyi) with a MiniMACS™ separator (#130-090-312; Miltenyi Biotec).

Techniques: Staining

Journal: PLoS ONE

Article Title: Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug

doi: 10.1371/journal.pone.0066219

Figure Lengend Snippet: (A) Purified CD4+CD25- T cells from spleen were activated by anti-CD3 antibody for 3 days under Th1 or Th17 polarization conditions in the presence or absence of Lodamin (1 nM), followed by stimulation with PMA and ionomycin in the presence of Brefeldin A; all plots are gated on CD4+ cells. Polarization conditions: Th0; anti-IFN-g Ab (10ug/ml) and anti-IL-4 Ab (10ug/ml); Th1; IL-12 (10 ng/ml) and anti-IL-4 Ab (10 µg/ml); Th17; IL-6 (20 ng/ml), TGFβ (2 ng/ml), anti-IFNγ Ab (10 µg/ml) and anti-IL-4 Ab (10 µg/ml) (B) Lodamin inhibits hallmark cytokine of Th1/Th17 under each polarization condition. IFN-γ and IL-17 protein level as measured by ELISA from activated cells supernatants (as describe in panel A). (mean ± SEM, n = 3 , *** p <0.001).

Article Snippet: CD4 + enriched cells were prepared using anti-CD4 Microbeads (#130-1-049-201; Miltenyi Biotec, Gladbach, Germany) and MS columns (#130-042-201; Miltenyi) with a MiniMACS™ separator (#130-090-312; Miltenyi Biotec).

Techniques: Purification, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug

doi: 10.1371/journal.pone.0066219

Figure Lengend Snippet: (A) histological score of EAU evaluated on day 21 post immunization with IRBP 1–20 - in response for oral Lodamin treatment (30 mg/kg q.o.d) (○) or vehicle (•) ( n = 20 , mean ± SEM, * p <0.05 Mann-Whitney U test). (B) represented histopathological images of eyes enucleated from vehicle (control) or Lodamin-treated mice on day 21 post IRBP 1–20 immunization. The control mice exhibited uveitis with focal loss of the photoreceptor layer in the choroid as well as massive infiltrates of monocytes in the retina, vitreous, and choroid with retinal folding and focal granulomatous lesions (arrows). In contrast, in Lodamin treated group only a few inflammatory cells are observed. Scale bars = 100 µm. (C) FACS analysis of IFN-γ and IL-17 production by T cells from draining lymph node. CD4 + T cells from Lodamin-treated mice produced significantly less IRBP-specific IL-17 as well as IFN-γ than cells from untreated mice on day 21 post after IRBP 1–20 immunization (mean ± SEM, n = 3 , * p <0.05).

Article Snippet: CD4 + enriched cells were prepared using anti-CD4 Microbeads (#130-1-049-201; Miltenyi Biotec, Gladbach, Germany) and MS columns (#130-042-201; Miltenyi) with a MiniMACS™ separator (#130-090-312; Miltenyi Biotec).

Techniques: MANN-WHITNEY, Produced

Journal: PLoS ONE

Article Title: Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug

doi: 10.1371/journal.pone.0066219

Figure Lengend Snippet: On day 21 post IRBP 1–20 immunization, lymph nodes from control vehicle-administered mice and Lodamin treated mice were collected. (A) images of lymph nodes removed at day 21. (B) volume of inguinal lymph nodes calculated with two parameters; (width) 2 ×(length)×0.52. (C) diagrams (left) and statistics (right, mean ± SEM, n = 5, *** p<0.001 )) of FACS analysis of cell surface CD4 + CD62L + expression of single cell suspension from lymph nodes. (D) MetAP2 expression and p21 expression as evaluated by qPCR from purified lymph nodes CD4 + T cells, ( n = 5, * p<0.05) values represent means ± SEM.

Article Snippet: CD4 + enriched cells were prepared using anti-CD4 Microbeads (#130-1-049-201; Miltenyi Biotec, Gladbach, Germany) and MS columns (#130-042-201; Miltenyi) with a MiniMACS™ separator (#130-090-312; Miltenyi Biotec).

Techniques: Expressing, Suspension, Purification

Journal: PLoS ONE

Article Title: Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug

doi: 10.1371/journal.pone.0066219

Figure Lengend Snippet: (A) On day 21 after IRBP immunization, retinas were harvested and total RNA was extracted from naive mice (unimmunized control), EAU control group and Lodamin-administered group. The expression of IL-6, TNF, IFN-γ, and IL-17A were analyzed by realtime qPCR (mean ± SEM, n = 5–8, *** p <0.001). (B) Summary of Lodamin’s effects on T cell activity and differentiation in vitro and in EAU model (circled, red): Lodamin is taken up by T cells and (1) suppress the activation and differentiation of CD4 + T cells to Th1 and Th17. (2) reduces production of IL-17 and IFNγ in splenocytes and activated T cells (3) reduces IFNγ and IL-17 in Lymph T cells from EAU mice (4) increases the expression of homing receptor CD62 lymph node CD4+ T cells (5) do not effect Treg proliferation in lymph nodes (6) reduces the expression of retinal cytokines: IL-6. TNFα, IFNγ, IL-17A.

Article Snippet: CD4 + enriched cells were prepared using anti-CD4 Microbeads (#130-1-049-201; Miltenyi Biotec, Gladbach, Germany) and MS columns (#130-042-201; Miltenyi) with a MiniMACS™ separator (#130-090-312; Miltenyi Biotec).

Techniques: Expressing, Activity Assay, In Vitro, Activation 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