Journal: Frontiers in Immunology

Article Title: Regulating Immunogenicity and Tolerogenicity of Bone Marrow-Derived Dendritic Cells through Modulation of Cell Surface Glycosylation by Dexamethasone Treatment

doi: 10.3389/fimmu.2017.01427

Figure Lengend Snippet: Neuraminidase treatment alters immunogenic properties of immature DCs (iDCs) and tolerogenic DCs (tDCs). To test the immunomodulatory properties of iDCs, niDCs, tDCs, and ntDCs, they were placed into MLRs for 5 days. (A) Schematic representation of experimental design. DA iDCs, niDCs, tDCs, and ntDCs were placed in cocultures for 5 days with allogeneic LEW lymphocytes isolated from the spleen and lymph nodes. (B) Representative gating strategy. Cells were selected according to size and granularity (i) followed by live/dead discrimination based on Sytox AADvanced TM negative cells (live) (ii). After single cell selection (iii) cells were selected by CD3 (PE) positivity (iv). Further selected by CD4 (APC) and CD8 (PE-CY7) and proliferation was measured by successive generations of CellTrace TM Violet positive cells. (C) The ability of iDCs, niDCs, tDCs, and ntDCs to stimulate allogeneic LEW T-cells was analyzed using unstimulated splenocytes/lymphocytes as a negative control ( n = 3). (i) Representative histograms and bar charts displaying CD4 + and CD8 + T cell proliferation following a 5-day coculture with iDCs and niDCs. (ii) Representative histograms and bar charts displaying CD4 + and CD8 + T cell proliferation following a 5-day coculture with tDCs and ntDCs. Error bars: mean ± SEM * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 one-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: T cells were washed with phosphate-buffered saline and stained in prewarmed (37°C) CellTrace™ Violet (CTV) phosphate-buffered saline staining solution (Invitrogen, Carlsbad, CA, USA) as per manufacturer’s instructions.

Techniques: Isolation, Selection, Negative Control

Journal: Immunotherapy Advances

Article Title: SMAC mimetics induce human macrophages to phagocytose live cancer cells

doi: 10.1093/immadv/ltaf026

Figure Lengend Snippet: SMAC-mimetics induce phagocytosis in human macrophages. (A) Representative flow cytometry plots from a single donor showing the CTV⁺CD45⁺ phagocytosis population within the total CD45⁺ macrophage population. (B) Macrophages from n =8 healthy donors were treated with the 500 nM of the indicated SMAC mimetics for 24 hours prior to co-culture with CellTrace Violet labeled MDA-MB-231 breast cancer cells. Phagocytosis was assessed by flow cytometry after 18 hours of co-culture. Results from each donor were normalized to the vehicle control from that donor. Error bars are SD. Statistical significance was determined using a pairwise one-way ANOVA with Sidak’s multiple comparison. (C) Immunoblot analysis of macrophage lysates treated with SMAC-mimetics (500 nM) for 24 hours. Blots were probed for cIAP1, XIAP, and GAPDH. (D) The frequency of macrophages recovered after 18 hours co-culture with tumor cells under each of the indicated treatment conditions. (E) Macrophages from n = 5 healthy donors were treated with the indicated concentrations of LCL161 for 24 hours prior to co-culture with CellTrace Violet labeled MDA-MB-231 breast cancer cells. Phagocytosis was assessed by flow cytometry after 18 hours of co-culture. Results from each donor were normalized to the vehicle control from that donor. Error bars are SD. Statistical significance was determined using an ordinary one-way ANOVA. (F) Immunoblot of protein lysates from macrophages treated for 24 hours with the indicated concentrations of LCL161.

Article Snippet: The next day, tumor cells were collected and labelled with CellTrace Violet (CTV) (Fisher Scientific, Cat# C34557 ) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Co-Culture Assay, Labeling, Control, Comparison, Western Blot

Journal: Immunotherapy Advances

Article Title: SMAC mimetics induce human macrophages to phagocytose live cancer cells

doi: 10.1093/immadv/ltaf026

Figure Lengend Snippet: Autocrine TNFa sustains the phagocytosis phenotype. (A) Healthy donor macrophages were treated with 500 nM LCL161 and 50 ng/mL IFNg or vehicle control. TNFa was measured by ELISA of 24 hour culture supernatants and reported as absorbance values. Error bars are SD. (B) Healthy donor macrophages from n = 3 independent donors were treated with DMSO, LCL161, or LCL161+IFNg for 18 hours of co-culture with CellTrace Violet labeled PANC-1 pancreatic cancer cells with or without TNFa blocking antibodies (1 mg/mL). Phagocytosis was assessed by flow cytometry after 24 hours of co-culture. Error bars are SD. Statistical significance was determined using a pairwise one-way ANOVA with Sidak’s multiple comparisons assuming sphericity. (C) Graphical overview of the follow-up experiment in which the antibody was added both during pre-treatment with DMSO, LCL161, or LCL161+IFNg for 24 hours, and during co-culture with CellTrace Violet labeled PANC-1 cells for 18 hours. Phagocytosis was again assessed by flow cytometry. (D) Phagocytic index of macrophages from n = 6 independent healthy donors. For each donor, data were normalized to vehicle + isotype. The Wilcoxon matched-pairs signed rank test was used to determine statistical significance. (E) Bone marrow-derived macrophages from a WT- and TNFa KO mouse were pre-treated with DMSO, LCL161, or LCL161+IFNg for 24 hours. Macrophages were co-cultured with 6694c2 mouse pancreatic cancer cells for an additional 18 hours. Flow cytometry was used to measure phagocytosis. Error bars are SD. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: The next day, tumor cells were collected and labelled with CellTrace Violet (CTV) (Fisher Scientific, Cat# C34557 ) according to the manufacturer’s instructions.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Labeling, Blocking Assay, Flow Cytometry, Derivative Assay, Cell Culture