Journal: PLoS Pathogens

Article Title: Systemic Expression of Kaposi Sarcoma Herpesvirus (KSHV) Vflip in Endothelial Cells Leads to a Profound Proinflammatory Phenotype and Myeloid Lineage Remodeling In Vivo

doi: 10.1371/journal.ppat.1004581

Figure Lengend Snippet: Fourteen serum cytokines were analyzed in ROSA26.vFLIP;Cdh5(PAC).creER T2 mice by using a quantitative flow cytometry-based assay. Analysis was done in 2–3 month-old mice, about one month after i.p. injection of tamoxifen. Data are representative of at least three experiments with similar results (error bars, SEM); at least three TG and control animals were analyzed in each experiment. P -values derived from two-tailed unpaired Student’s t-test on the means (bars) of WT versus TG mice are shown. * P <0.05, ** P <0.01 and *** P <0.005

Article Snippet: After incubation of the capture beads with analytes and detection reagent, the PE mean fluorescence intensity (MFI) of the complex was measured and readings within the assay linear range were used to calculate the serum cytokines concentrations against cytokines standard curve for each analyte (Becton Dickinson).

Techniques: Flow Cytometry, Injection, Derivative Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Systemic Expression of Kaposi Sarcoma Herpesvirus (KSHV) Vflip in Endothelial Cells Leads to a Profound Proinflammatory Phenotype and Myeloid Lineage Remodeling In Vivo

doi: 10.1371/journal.ppat.1004581

Figure Lengend Snippet: Expression of vFLIP in either B-cells or endothelial cells activates several cytokines, both in vivo and in vitro , that lead to aberrant myeloid differentiation with the emergence of myeloid subsets well known to have a role in angiogenesis, tumor immune evasion and tumor progression. (Drawing of myeloid differentiation was modified from Gabrilovich et al)

Article Snippet: After incubation of the capture beads with analytes and detection reagent, the PE mean fluorescence intensity (MFI) of the complex was measured and readings within the assay linear range were used to calculate the serum cytokines concentrations against cytokines standard curve for each analyte (Becton Dickinson).

Techniques: Expressing, In Vivo, In Vitro, Modification

Journal: Scientific Reports

Article Title: Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning

doi: 10.1038/s41598-021-93842-0

Figure Lengend Snippet: Reduction of tumor burden, unlike cytokine serum accumulation, is CD3 affinity-independent in xenogenic tumor models. ( a ) NSG mice were injected with 5 × 10 6 human PBMCs before 1 × 10 6 ascites cells from the OVCAR-3/luc cell line were administered IP (day 0). Mice were dosed IV with MUC16xCD3 bispecific antibodies or isotype control antibodies at 1 mg/kg, and imaged multiple times throughout the study to track tumor burden; ( b ) serum samples from the OVCAR-3/luc experiment were collected at 4 h post-dose to examine cytokine concentrations by MSD; ( c ) NSG mice were subcutaneously implanted with a mixture of human peripheral blood cells (1 × 10E 6 cells) and MOLP-8 tumor cells (5 × 10E 6 cells), and animals were continuously dosed IP twice weekly with BCMAxCD3 bispecific antibodies or controls through day 21, at various doses (4 mg/kg, 0.4 mg/kg, 0.04 mg/kg); ( d ) four hours post-implantation, serum from the MOLP-8 experiment was harvested for cytokine analysis by MSD. statistical analysis by ordinary one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.002, and *** P < 0.0003. Avg average; BCMA B-cell maturation antigen; d day; IP intraperitoneally; IV intravenously ; luc luciferase; m moderate; MSD Meso Scale Diagnostics; MUC16 Mucin-16 for ovarian cancer; NSG NOD scid gamma; PBMC peripheral blood mononuclear cell; PBS phosphate-buffered saline; vw very weak; w weak.

Article Snippet: Serum samples were collected at 4 h post-dose to examine cytokine concentrations by Meso Scale Diagnostics (MSD; Rockville, MD).

Techniques: Injection, Control, Luciferase, Saline

Journal: Nature biomedical engineering

Article Title: Traceless aptamer-mediated isolation of CD8 + T cells for CAR-T cell therapy

doi: 10.1038/s41551-019-0411-6

Figure Lengend Snippet: a , 2 nd generation CD19 CAR T cell construct with EGFRt reporter used to retrovirally transduce isolated T cells. b , Flow cytometry analysis of EGFRt expression in antibody- and aptamer-isolated T cells 9-days post initial bead stimulation (S1D9) and 13-days post rapid expansion protocol (REP, S1R1D13) with irradiated CD19 + TM-LCL cells. Timeline of cell expansion is shown in . Chart indicates EGFRt reporter MFI at S1R1D13, with symbols as in - . Flow histograms are representative of 1 independent experiment. Data are mean ± s.d., n = 3, P > 0.05 (two-sided paired t-test). c, Growth of non-transduced mock T cells post bead stimulation. Symbols as in - , n = 3, P > 0.05 (paired two-way ANOVA with Bonferroni correction). Curves represent a least-squares fit to the exponential growth equation. d , Flow cytometry analysis of Ki-67 expression in mock and CD19 CAR T cells on S1D14 immediately before REP. Symbols as in - . Data are mean ± s.d., n = 3, P > 0.05 (paired two-way ANOVA with Sidak correction). e , Flow cytometry analysis PD1/TIM3/LAG3 expression in mock and CD19 CAR T cells on S1D14 immediately before REP. Individual donor values can be found in . Pie charts show the mean phenotype of the cells, n = 3 biologically independent samples, P > 0.05, * P < 0.05, and ** P < 0.01 (paired two-way ANOVA with Bonferroni correction). f, Flow cytometry analysis of CD62L/CD45RA expression in mock and CD19 CAR T cells on S1D14 immediately before REP and on S1R1D14 immediately before functional assays. Individual donor values can be found in . Pie charts show the mean phenotype of the cells, n = 3 biologically independent samples, P > 0.05 and * P < 0.05 (paired two-way ANOVA with Bonferroni correction). g , h , In vitro anti-tumor cytotoxicity and cytokine release of mock and CD19 CAR T cells. For h , symbols as in - . Data are mean ± s.d., n = 3 biologically independent samples, P > 0.05 ( g, paired two-way ANOVA with Bonferroni correction; h , paired two-way ANOVA with Sidak correction).

Article Snippet: Briefly, 1.5 × 10 6 CD19 CAR T cells were co-incubated with 10.5 × 10 6 irradiated CD19 + TM-LCL feeder cells in 25 mL complete RPMI supplemented with the aforementioned cytokine concentrations in T-25 flasks (Corning).

Techniques: Construct, Isolation, Flow Cytometry, Expressing, Irradiation, Functional Assay, In Vitro