Fitc anti mouse cd8

APC anti-mouse CXCR3 and APC anti-mouse CCR5 antibodies were purchased from BioLegend (San Diego, CA). APC anti-mouse CCR2 was from R&D Systems, Inc. (Minneapolis, MN). FITC anti-mouse CD8, PE anti-mouse CD4 and anti-mouse CD16/32 antibodies were from BD Pharmingen (San Diego, CA). APC anti-mouse CD62L antibody was from BioLegend (San Diego, CA).

Splenocytes (10⁷ cells/well) were cultivated in the presence of RBD (10 μg/mL) as a specific antigen or Con A (20 μg/mL) as a positive control. Unstimulated samples were used as a negative control. The stimulated cells were grown at 37 °C for 72 h and then incubated for an additional 5 h with Brefeldin A (BioLegend, San Diego, CA, USA). After incubation, the stimulated cells were harvested, treated with TruStain FcX (Anti-mouse CD16/32 antibody, BioLegend, San Diego, CA, USA) and subjected to surface staining with PE-Cy7 anti-mouse CD3, PE anti-mouse CD4 and FITC anti-mouse CD8 (BD Biosciences) antibodies. Subsequently, cells were processed with a fixation/permeabilization kit (BD Biosciences, San Diego, CA, USA), followed by staining with APC anti-mouse IFN-γ (BioLegend, San Diego, CA, USA). The percentages of positive cells were enumerated by flow cytometry. Simultaneously, aliquots of the culture supernatant were harvested at 12, 24, 48 and 72 h of treatment, and were then subjected to IFN-γ, IL-2 and IL-4 detection using ELISA (BioLegend, San Diego, CA, USA)

Peptide-specific cytotoxic T-cells were quantified by flow cytometry analysis. Single cell suspensions of excised spleens were obtained using 70 μm cell strainers (BD Falcon). Subsequently, 2*10⁶ splenocytes were plated per well in 48-wells culture plates (Greiner) and restimulated with 50 ng M1_58–66 peptide for 18 h at 37°C 5% CO₂. Next, Golgi-plug (1:1000, BD) was added to the cells to inhibit cellular protein and cytokine transport, and cells were incubated for another 4 h. Subsequently, cells were transferred to a 96-wells plate, washed twice with FACS buffer (PBS, 0.5% BSA), and stained with anti-mouse CD4-PE (BD Biosciences), anti-mouse CD8-FITC (BD Biosciences) and Live/dead-Aqua (Invitrogen). Next, cells were washed twice with FACS buffer, and fixed with fixation-permeabilization buffer (BD Biosciences). Subsequently cells were washed with permeabilization wash buffer (BD Biosciences), and intracellular staining was performed with IFN-γ-APC (Biolegend). Finally, cells were washed with FACS buffer and 1.5 to 2 million cells were measured on a FACS Canto II flow cytometer (BD Biosciences). Data was analyzed using FlowJo software version 9 (Tree Star) for Mac OSX.

Bone marrow and mediastinal lymph nodes were aseptically removed at the time of necropsy (n=4 of each group). Single-cell suspensions were obtained by flushing the bone marrow or gently pressing the lymph node against a 70-μm strainer into PBS. Cells were washed in fluorescence-activated cell sorting (FACS) buffer after lysing with ammonium chloride potassium (ACK) buffer. Cell surface staining was done using anti-mouse Gr-1-FITC, CD3-PerCP, CD4-PE, CD8-FITC, and CD19-APC (BD Biosciences, USA). For influenza-specific CD8+ cells, mediastinal lymph node cells were stimulated overnight with formalin-inactivated Influenza A PR/8/34 H1N1 (MOI=4). Brefeldin A (BFA, 1 μmol/L) was added after overnight stimulation and cells were incubated for an additional 5 h. After that, cells were surface stained with anti-mouse CD8-FITC, then permeabilized with Cytofix-Cytoperm solution (BD Biosciences, USA) and stained with intracellular anti-mouse IFN-γ-APC. Flow cytometry data were collected on the FACS Canto (BD Biosciences, USA) and analyzed using FlowJo version 7.6.1.

T cell populations were analyzed by flow cytometry. In short, single-cell suspensions of splenocytes were plated at a concentration of 2×10⁶ cells in a 48-well plate in RPMI medium (Life Technologies) with 10% Hyclone fetal calf serum (FCS, Thermo Scientific), and stimulated overnight with either medium, 50 ng peptide or 50 ng PR8 WIV. Cytokine transport was blocked by incubating with Golgi-plug (BD Biosciences) for the last 4 h. Cells were stained with anti-mouse CD8-FITC (BD Biosciences), anti-mouse CD4-PE (BD Biosciences), and Live-dead-Aqua (Invitrogen), fixated with fixation/permeabilization buffer (BD Biosciences), and washed with permeabilization wash buffer (BD Biosciences). Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ⁺ CD8⁺ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences). Acquired data were analyzed with FlowJo version 10 for Mac OSX (TreeStar Inc.). Gating strategy for the quantification of CD8⁺ IFN-γ⁺ T cells is shown in Figure S2 in Supplementary Material.