Computational protocol: Angiotensin II type-1 receptor (AT1R) regulates expansion, differentiation, and functional capacity of antigen-specific CD8+ T cells

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Protocol publication

[…] All fluorochrome-conjugated monoclonal anti-mouse antibodies were purchased from eBioscience or BD unless stated otherwise: CD45.1 (clone A20); PE-Cy7-conjugated CD8 (clone 53–6.7), CD25 (clone PC61.5), CD122 (clone 5H4) CD69 (H1.2F3), CD160 (clone CNX46–3), CD44 (clone IM7), CD62L (clone MEL-14), KLRG1 (clone 2F1), IL-7Rα (clone A7R34), PD-1 (clone J43), LAG-3 (clone C9B7W), CTLA-4 (clone UC10–4B9), IFN-γ (clone XMG1.2), IL-2 (clone JES6–5H4), TNF-α (clone MP6-XT22), and CD107a (clone 1D4B). PerCP/PE/FITC-conjugated IgG1 and IgG2 isotype controls were all purchased from BD Pharmingen. All results were collected with CellQuest software on a FACSCalibur (Becton Dickinson), and multi-cytokine experiments were performed on a LSRII flow cytometer (Becton Dickinson).For flow cytometry, isolated AT1R+/+ or AT1R−/− OT-I cells from recipient C57BL/6 mice were incubated with Fc block (anti-mouse CD16 and CD32 antibodies) to block non-specific binding sites for 30 min at 4 °C. Later, the cells were washed and incubated with the appropriate concentration of antibodies cited above. IgG isotypes were used as irrelevant antibodies to define positive populations as indicated in the gate strategy (). At least 105 cells per sample were acquired. Analysis of surface cell markers and intracellular cytokines was performed using FlowJo software (TreeStar). All data were collected and presented in a log scale of fluorescence intensity and presented as plots. The percentage of OT-I cells was determined in a gate of CD8+ CD45.1+ cells and each analysis was made in relation to the total OT-I cells (gated on CD8+ CD45.1+ cells). as showed in the gate strategy (). The MFI data were determined by calculating the median fluorescence intensity in the total OT-I cells (gated on CD8+ CD45.1+ cells) considering the fluorescence of the isotype control, using FlowJo software (TreeStar) (). [...] El4 cells (T-cell lymphoma cell line of C57BL/6 (H-2b) origin) were pulsed with SIINFEKL peptide (10 μg/ml), and control El4 cells (non-pulsed) were incubated at 37 °C for 1 h. Lymphocytes harvested from the spleen of γ-spz immunized mice at days 7, 12, 20, or 32 p.i. were co-cultured with El4 cells pulsed or not for 4 h at 37 °C in the presence of 1:400 brefeldin A (GolgiPlug; BD Bioscience), 1:600 monensin (GolgiStop; BD Bioscience), and anti-CD107a-FITC, a marker of cytotoxic activity. Cells were then permeabilized and fixed using a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions and stained for intracellular cytokines using anti-IFN-γ-PE-Cy7, anti-TNF-α-Pacific Blue, and anti-IL-2-APC (clone JES6–5H4) at pre-determined concentrations. After staining, cells were washed, diluted and analyzed on a LSR II flow cytometer (BD Bioscience). Functional characterization is based on the capacity of these cells to simultaneously produce or not different cytokines and also express CD107a on the cell surface, which indicates cytotoxic activity. Polyfunctional analysis was done by Boolean combination of gates with FlowJo software, and the data were exported to PESTLE and SPICE software for analysis. […]

Pipeline specifications

Software tools FlowJo, SPICE
Application Flow cytometry
Organisms Mus musculus, Rattus norvegicus, Plasmodium berghei
Diseases Ataxia Telangiectasia, Malaria
Chemicals Angiotensin II