Cd8 apc fire

Tumor cells isolated from mice were thawed and stained with LIVE/DEAD Fixable Violet Dead Staining Kit (ThermoFisher). Subsequently, the cells were divided and stained with cocktails of fluorochrome-conjugated monoclonal antibodies: CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD45 BV605, CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C PE, Ly6G APC-Cy7, MHC II FITC, CD80 PE-Cy7 (all from Biolegend) for myeloid cell identification and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, CD25 PE, CD44 PE-Cy7, CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using FACSFortessa flow cytometer with Diva software (Becton Dickinson).

Eight to ten-week old female C57BL/6 mice were subcutaneously inoculated in the right flank with MC38/0 cells (1.1 × 10⁶/0.2 ml/mouse). On the 14th, 15th and 17th day of the experiment, mice were injected i.t. with LVs encoding shRNA against IL-10 (shIL10–3, 2x10⁶TU/50 μl/mouse) or reference LVs encoding scrambled shRNA against human GAPDH (shN). Two days after the third injection, the mice were sacrificed and their tumor nodules were dissected and homogenized. Efficacy of transduction in tumors was measured by flow cytometry as the fluorescence intensity of EGFP among cells isolated from tumors. Concentration of IL-10 was estimated by ELISA in supernatants collected from 24 h culture of 5 mg tumor tissue/ml. Myeloid and lymphocyte populations in tumors were analyzed using LSR Fortessa with Diva software (Becton Dickinson) after staining with fluorochrome-conjugated antibodies: CD45 V500, CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C BV510, Ly6G BV605, MHC II APC-Cy7, for myeloid cell identification (all from Biolegend) and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, (all from BioLegend) for lymphocyte identification.

Spleen cells obtained from control or treated tumor-bearing mice were restimulated during co-culture with mitomycin C-treated MC38 cells (50 mg MitC/3 × 10⁶ cells; 30 min., 37 °C) in presence of recombinant human IL-2 (200 U/ml). After 5 days, the cells and supernatants were collected. Cytotoxic activity of restimulated splc toward DiO-labeled MC38 target cells, as well as the ability of effector cells to secrete lytic granules were measured as previously described [26 (link)]. Cytotoxic cells were identified using anti-CD107 APC, anti-CD8 PE-Cy7 and anti-CD49b PE monoclonal antibodies (BioLegend). In order to determine the polarization of systemic immune response followed by applied treatment, Tbet and FoxP3 expression, and IFN-γ production by T cells was measured. Spleen cells, obtained from treated and control mice, were stimulated with ConA (0.5 μg/ml; Sigma) and IL-2 (200 U/ml) for 48 h. Then cells were harvested and after staining with fluorochrome-conjugated antibodies: CD4 FITC and CD8 APC-Fire (BioLegend) were fixed and permeabilized to intracellular staining of Tbet, FoxP3 and IFN-γ with following antibodies: anti-Tbet PE-Cy7, anti-FoxP3 APC, anti-IFN-γ PE (eBioscience). Flow cytometry analyses were performed using FACSFortessa with FACSDiva software (Becton Dickinson).