Journal: Cancer Immunology, Immunotherapy : CII

Article Title: miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy

doi: 10.1007/s00262-020-02507-w

Figure Lengend Snippet: Increased mRNAs and proteins from PD-L1, galectin-9 and HVEM genes in spleen DCs from breast cancer-bearing mice. a Detection of miRNAs encoding PD-L1, galectin-9 and HVEM by RT-qPCR. DCs were isolated from spleens of wild type and breast tumor-bearing mice using CD11c magnetically-labeled beads (Miltenyi Biotec, USA). CD11c cDNA was used to perform RT-qPCR and normalized to β-action expression. b Detection of inhibitory receptors ligand on CD11c+ DCs by flow cytometry. Spleen cells were collected from naive mice and breast tumor-bearing mice. Flow cytometry was performed with FITC-labeled CD11c, PerCP-eFluor 710-labeled PD-L1, PE-CY7-labeled galectin-9, APC-labeled HVEM antibody staining. Data are representative of three mice from at least three independent experiments. Two-tailed unpaired Student’s t tests were performed to determine statistical significance. Error bars represent standard deviation (*p < 0.05; n.s., p > 0.05)

Article Snippet: Isotype controls included rat anti-IgG2a κ-FITC and rat anti-IgG2b-PerCP-eFluor 710, mouse rat anti-IgG2a κ-PE-CY7, mouse anti-IgG1 κ-APC, anti-IgG2a κ-PE, and rat anti-IgG2a κ-PE-CY5 (eBioscience, San Diego, CA).

Techniques: Quantitative RT-PCR, Isolation, Labeling, Expressing, Flow Cytometry, Staining, Two Tailed Test, Standard Deviation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy

doi: 10.1007/s00262-020-02507-w

Figure Lengend Snippet: miR-5119 mimic-transfected DC vaccines suppress T cell exhaustion in vivo. a miR-5119 mimic-engineered DC vaccines increased exhausted CD8+ T cell proliferation in vivo. Spleen cells were isolated from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA engineered DC vaccine-treated breast tumor-bearing mice. Isolated cells were labeled with CFSE and seeded in 96 well plate at 1 × 106 cells/well. Wild type BALB/c bone marrow DCs were pulsed with 4T1 freeze–thaw antigen (50 ng/ml) and co-cultured with spleen cells from three groups (DC: T cells = 1:10) at 37 °C in a humidified atmosphere and 5% CO2 for 3 days. T-Cell proliferation was detected by flow cytometry. b miR-5119 mimic-engineered DC vaccines reduced apoptosis in exhausted CD8+ T cells in vivo. Spleen cells were isolated from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA DC vaccine-treated breast tumor-bearing mice. CD8+ T cells were purified from the collected splenocytes using magnetically-labeled beads. Apoptosis was detected by flow cytometry using FITC-Annexin V/PI detection. c miR-5119 mimic-engineered DC vaccines inhibited immune checkpoint molecule levels in exhausted CD8+ T cells in vivo. Spleen cells were collected from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA DC vaccine-treated breast tumor-bearing mice. Flow cytometry was performed after FITC-labeled CD8, PerCP-eFluor 710-labeled PD-1, PE-CY7-labeled TIM-3, APC-labeled BTLA antibody staining. Data are representative of three mice per group. One-way ANOVA analysis was performed to determine statistical significance. Error bars represent standard deviation (*p < 0.05; n.s., p > 0.05)

Article Snippet: Isotype controls included rat anti-IgG2a κ-FITC and rat anti-IgG2b-PerCP-eFluor 710, mouse rat anti-IgG2a κ-PE-CY7, mouse anti-IgG1 κ-APC, anti-IgG2a κ-PE, and rat anti-IgG2a κ-PE-CY5 (eBioscience, San Diego, CA).

Techniques: Transfection, Vaccines, In Vivo, Isolation, Labeling, Cell Culture, Flow Cytometry, Purification, Staining, Standard Deviation

Journal: ImmunoHorizons

Article Title: Depot medroxyprogesterone acetate administration alters immune markers for HIV preference and increases susceptibility of peripheral CD4 + T cells to HIV infection

doi: 10.4049/immunohorizons.1700047

Figure Lengend Snippet: PBMCs from subjects before and after Depo-Provera injection were isolated and the percentage and MFI of integrin α4β7 (A), HIV co-receptor CCR5 (B), and activation marker CD38 (C) were analyzed within the CD3+CD4+ T cell population by flow cytometry. Wilcoxon matched-pairs signed rank test was used to compare values from before Depo-Provera (visit 1), one month after Depo-Provera (visit 2), and 3 months after Depo-Provera (visit 3). Each dot represents one donor. Bars represent median and interquartile range. p≤0.05 was considered significant, p>0.05 was not significant (ns).

Article Snippet: V500-conjugated mouse IgG1κ anti-human CD45 (clone HI30), Alexa Fluor® 700-conjugated mouse IgG2a anti-human CD197 (CCR7) (clone 150503), APC-Cy™7-conjugated mouse IgG2aκ anti-human CD195 (CCR5) (clone 2D7/CCR5), APC-conjugated mouse IgG2bκ anti-human CD45RA (clone HI100), Alexa Fluor® 700-conjugated mouse IgG2aκ isotype control (clone G155-178), APC-Cy™7-conjugated mouse IgG2aκ isotype control (clone G155-178), and anti-mouse Ig BD™ CompBead set were from BD Biosciences.

Techniques: Injection, Isolation, Activation Assay, Marker, Flow Cytometry

Journal: ImmunoHorizons

Article Title: Depot medroxyprogesterone acetate administration alters immune markers for HIV preference and increases susceptibility of peripheral CD4 + T cells to HIV infection

doi: 10.4049/immunohorizons.1700047

Figure Lengend Snippet: The percentage and MFI of cell surface markers integrin α4β7, CCR5, and CD38 on CD3+CD4+ T cells from freshly isolated PBMCS was analyzed within patients who identified as either black (A) or Hispanic (B) by flow cytometry. Each dot represents one donor. Bars represent median and interquartile range. Wilcoxon matched-pairs signed rank test was used for analysis. p≤0.05 was considered significant, p>0.05 was not significant (ns).

Article Snippet: V500-conjugated mouse IgG1κ anti-human CD45 (clone HI30), Alexa Fluor® 700-conjugated mouse IgG2a anti-human CD197 (CCR7) (clone 150503), APC-Cy™7-conjugated mouse IgG2aκ anti-human CD195 (CCR5) (clone 2D7/CCR5), APC-conjugated mouse IgG2bκ anti-human CD45RA (clone HI100), Alexa Fluor® 700-conjugated mouse IgG2aκ isotype control (clone G155-178), APC-Cy™7-conjugated mouse IgG2aκ isotype control (clone G155-178), and anti-mouse Ig BD™ CompBead set were from BD Biosciences.

Techniques: Isolation, Flow Cytometry

Journal: ImmunoHorizons

Article Title: Depot medroxyprogesterone acetate administration alters immune markers for HIV preference and increases susceptibility of peripheral CD4 + T cells to HIV infection

doi: 10.4049/immunohorizons.1700047

Figure Lengend Snippet: PBMCs exposed to HIV-1BaL (MOI 0.01) were cultured for 10 days. In HIV infected PBMCs, cell surface marker expression of integrin α4β7, CCR5, and CD38 cell surface marker expression (A–C) and T cell subsets (D) were measured on HIV p24−CD4+ T cells and HIV p24+CD4+ T cells. Uninfected cells were included as a comparison. Each dot represents one donor. Bars represent median and interquartile range. The Mann-Whitney test was used to compare cell surface marker expression between uninfected, HIV p24− or HIV p24+CD4+ T cells. p≤0.05 was considered significant, p>0.05 was not significant (ns).

Article Snippet: V500-conjugated mouse IgG1κ anti-human CD45 (clone HI30), Alexa Fluor® 700-conjugated mouse IgG2a anti-human CD197 (CCR7) (clone 150503), APC-Cy™7-conjugated mouse IgG2aκ anti-human CD195 (CCR5) (clone 2D7/CCR5), APC-conjugated mouse IgG2bκ anti-human CD45RA (clone HI100), Alexa Fluor® 700-conjugated mouse IgG2aκ isotype control (clone G155-178), APC-Cy™7-conjugated mouse IgG2aκ isotype control (clone G155-178), and anti-mouse Ig BD™ CompBead set were from BD Biosciences.

Techniques: Cell Culture, Infection, Marker, Expressing, MANN-WHITNEY

Journal: ImmunoHorizons

Article Title: Depot medroxyprogesterone acetate administration alters immune markers for HIV preference and increases susceptibility of peripheral CD4 + T cells to HIV infection

doi: 10.4049/immunohorizons.1700047

Figure Lengend Snippet: Changes in integrin α4β7, CCR5, and CD38 cell surface marker expression (A–C) and CD4+ T cell subsets (D) between study visits were analyzed on HIV p24+CD4+ T cells by FACS analysis. Each dot represents one donor. Bars represent median and interquartile range. Wilcoxon matched-pairs signed rank test was used to compare differences between study visits. p≤0.05 was considered significant, p>0.05 was not significant (ns).

Article Snippet: V500-conjugated mouse IgG1κ anti-human CD45 (clone HI30), Alexa Fluor® 700-conjugated mouse IgG2a anti-human CD197 (CCR7) (clone 150503), APC-Cy™7-conjugated mouse IgG2aκ anti-human CD195 (CCR5) (clone 2D7/CCR5), APC-conjugated mouse IgG2bκ anti-human CD45RA (clone HI100), Alexa Fluor® 700-conjugated mouse IgG2aκ isotype control (clone G155-178), APC-Cy™7-conjugated mouse IgG2aκ isotype control (clone G155-178), and anti-mouse Ig BD™ CompBead set were from BD Biosciences.

Techniques: Marker, Expressing

Journal: Molecules

Article Title: Blockade of NF-κB Translocation and of RANKL/RANK Interaction Decreases the Frequency of Th2 and Th17 Cells Capable of IL-4 and IL-17 Production, Respectively, in a Mouse Model of Allergic Asthma

doi: 10.3390/molecules26113117

Figure Lengend Snippet: Effect of inhibitors of IKK (BMS-345541 (BMS)), NF-κB translocation (dehydroxymethylepoxyquinomicin (DHMEQ)) and RANK/RANK-L interaction (osteoprotegerin (OPG)), and glucocorticosteroid (methylprednisolone (MP)) administration on the number of IL-4- and IL-17-producing CD8 + T cells. The relative count ( A , D ) is expressed as a percentage of IL-4- or IL-17-producing cells within CD8 + T cells. The absolute count ( B , E ) represents the number of IL-4- or IL-17-producing CD8 + T cells per lung sample collected from individual mice. * p < 0.05, ** p < 0.01, *** p < 0.001. Examples of dot plot cytograms showing the distribution of IL-4- or IL-17-producing and non-producing cells within CD8 + T cells ( C and F , respectively).

Article Snippet: Subsequently, the cells were stained for surface antigens with fluorochrome-conjugated monoclonal antibodies (mAbs): PerCP-Cy 5.5 rat anti-mouse CD4 (clone RM4-5, IgG2a, κ), APC-Cy7 rat anti-mouse CD8a (clone 53-6.7, IgG2a, κ) and PE-Cy7 rat anti-mouse CD25 (clone PC61, IgG1, λ; all from BD Biosciences).

Techniques: Translocation Assay

Journal: Molecules

Article Title: Blockade of NF-κB Translocation and of RANKL/RANK Interaction Decreases the Frequency of Th2 and Th17 Cells Capable of IL-4 and IL-17 Production, Respectively, in a Mouse Model of Allergic Asthma

doi: 10.3390/molecules26113117

Figure Lengend Snippet: Gating strategy for flow cytometric data analysis and calculation of the absolute cell counts of lymphocyte subsets. Lymphocytes were identified based on forward and side scatter (FSC/SSC) properties, and then gated for expression of CD4 or CD8 surface receptors. CD4 + T cells were analyzed for expression/co-expression of CD25 and Foxp3. On this basis, Treg (Foxp3 + CD25 + CD4 + ) and non-Treg (the remaining CD4 + T cells, i.e., non-Foxp3 + CD25 + CD4 + T cells) cells were distinguished. Subsequently, IL-4-, IL-10-, IL-17- and TGF-β-producing cells were identified within particular cell subsets. Absolute cell counts of lymphocyte subsets (i.e., number of cells from particular subpopulations per mediastinal lymph node or lung sample) were calculated using the dual platform method, as shown above.

Article Snippet: Subsequently, the cells were stained for surface antigens with fluorochrome-conjugated monoclonal antibodies (mAbs): PerCP-Cy 5.5 rat anti-mouse CD4 (clone RM4-5, IgG2a, κ), APC-Cy7 rat anti-mouse CD8a (clone 53-6.7, IgG2a, κ) and PE-Cy7 rat anti-mouse CD25 (clone PC61, IgG1, λ; all from BD Biosciences).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Dengue immune sera enhance Zika virus infection in human peripheral blood monocytes through Fc gamma receptors

doi: 10.1371/journal.pone.0200478

Figure Lengend Snippet: PBMCs from healthy donors (n = 3) were treated with monoclonal antibodies (1 μg/ml, 3 μg/ml or 10 μg/ml) against each FcγR types or combined (1 μg/ml, 3 μg/ml or 10 μg/ml in total), or with IgG control antibody prior to performing the boosted ZIKV ADE assay with DENV-1 immune serum (D1-E01, 1/2,500 dilution). The percentage of anti-E positive Vero cells in the control group, where no anti-FcγR antibodies were applied at the ADE condition was defined as 100%. The mean and standard deviation of results from three independent experiments without blocking antibody treatment (A) or with blocking antibody treatment (B) were presented. Statistical significances were determined by comparison of the relative proportion of infected cells with blocking antibody treatment to that of the control IgG antibody treatment under the same antibody concentration using unpaired two-tailed Student’s t test and designated as “*”for p<0.05, “**” for p<0.01, and “***” for p<0.001.

Article Snippet: Isotype control murine immunoglobulin IgG1 κ (FITC; APC; clone P3.6.2.8.1), mouse IgG2a κ (PE; PE-Cy7; Alexa Fluor 488; clone eBM2a), mouse IgG2b κ (PE; clone eBMG2b) were purchased from eBioscience.

Techniques: Standard Deviation, Blocking Assay, Infection, Concentration Assay, Two Tailed Test