Glogiplug

Spleen homogenates obtained from vaccinated mice were filtrated through a 40 µm cell strainer (BD Biosciences) in RPMI-1640 medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Biowest, Nuaille, France). All erythrocytes were lysed in RBS lysis buffer (Sigma-Aldrich, Merck KGaA) for 5 min and washed with RPMI-1640 medium. Cell suspensions, diluted to 2 × 10⁶ cells/mL, were seeded onto 48-well cell culture plates (SPL) and stimulated with 20 µg/mL of GBS NSP 14-358 lysate in the presence of 0.5 µg/mL GolgiStop (eBioscience, San Diego, CA, USA) and 0.5 µg/mL GlogiPlug (eBioscience) for 12 h at 37 °C. The cells were washed with cold PBS and stained for T cell-surface markers using a LIVE/DEAD staining kit (Aqua vitality dye, InvivoGen, San Diego, CA, USA), anti-CD4-BV421 (1; 200), and anti-CD8a (1; 200) antibodies (BD Bioscience) and then washed in cold PBS. The cell suspensions were subsequently permeabilized using a Cytofix/Cytoperm kit (BD Bioscience) for 30 min at 4 °C. Finally, the cells were stained intracellularly with anti-IFN-γ (PE, BD Biosciences), anti-IL-5 (APC, BD Biosciences), and anti-IL17A (PE-Cy7, BD Biosciences) antibodies. All stained cells were analyzed using a BD FACSverse flow cytometer (BD Biosciences).

PBMCs were cultured in RPMI 1640 containing 10% fetal bovine serum and then stimulated with anti-CD3 (1 ng/mL) (T&L Biological Technology, Beijing, China) and anti-CD28 (1 ng/mL) (BioLegend) monoclonal antibodies for 6 h or an HIV pol, gag, and env peptide pool (1 μg/mL each) (JPT, Berlin, Germany) for 6h for different experiments. The cells were also treated with or without AZD4635 (0.2 µM) (Selleck Chemicals, Houston, USA) and/or an anti-PD-L1 antibody (10 µg/mL) (eBioscience). For analyses of cytokine production in stimulated cells, GlogiPlug (eBioscience) was added 6h before harvest. The cells were collected and the intracellular expression levels of IL-2 and IFN-γ were measured by flow cytometry, as described above.