Anti cd8 percp cy5

Cells harvested from overnight stimulation cultures were incubated with live/dead aqua V510 for 15 min on ice. The cells were then surface stained for 30 min on ice using anti-CD3-FITC (BioLegend, clone UCHT1, 1:200, Cat# 300406), anti-CD4-APC-H7 (BD Pharmingen™, clone RPA-T4, 1:200, Cat# 560158), and anti-CD8-PerCPCy5.5 (BD Biosciences, clone RPA-T8, 1:200, Cat# 560662) antibodies. After fixation and permeabilization with Cytofix and Perm (BD Biosciences, Cat# 554714) on ice for 15 min, intracellular staining was performed on ice for 30 min using anti-TFNα-PE-Cy7 (BD, clone MAb11, 1:200, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, 1:200, Cat# 554702) antibodies. After the final wash step, the cells were resuspended in 200 µL of FACS buffer. Samples were acquired using an FACSAria III instrument (BD Biosciences, San Diego, CA, USA) and analysed using the FlowJo software (Treestar, San Carlos, CA, USA).

PBMCs were adjusted to 1 x10⁶ cells/mL in 24-well plate and stimulated with the antigens mentioned above for 22–24 hours at 37°C in 5% CO₂. Four to six hours before the end of this incubation, 200 μL aliquots of the supernatant were collected for cytokine measurement, and 200 μL of fresh RPMIc medium with Brefeldin A (10 μg/mL) were added to block cytokine secretion. At the end of the incubation, cells were collected, washed twice with PBS and stained with anti-CD3-Alexa488, anti-CD4-PE and anti-CD8-PercP-Cy5.5 conjugated antibodies (all from BD, San Jose CA, USA). After fixation and permeabilization with Cytofix-Cytoperm (BD, San Jose CA, USA), cells were further stained with anti-INFγ-PE-Cy7 and anti-IL-10-APC (BD, San Jose CA, USA) conjugated antibodies for 20 minutes at 4°C, then fixed with 4% paraformaldehyde. Isotype-matched antibodies and basal fluorescence were assayed as controls. 100,000 events were acquired in a FACSVerse cytometer and then analyzed in FlowJo vX.0.7 (S1 Fig). The ratio of antigen-specific T cells producing cytokines was obtained by dividing the percentage of cells expressing cytokines in response to antigens (experimental conditions) by the percentage of cells expressing cytokines in response to RPMI (unstimulated cells).

After 5-day incubation, cells were stained with anti-CD8 PerCP-Cy5.5, CD4 PE, CD25 APC and anti-BrdU FITC (BD, Franklin Lakes, NJ, USA); while regulatory T cells (Treg) were identified with anti-CD4 PE, FoxP3 PerCP-Cy5.5, CD25 APC (BD, Franklin Lakes, NJ, USA). Samples were analyzed by flow cytometry. 10,000 events were registered within the lymphocyte region identified according to their size (Forward-scatter, FCS) and complexity (Side-scatter, SSC), considering their activation status [46 (link)].
Data were analyzed with FlowJo software (v.10; Tree Star, Inc., Ashland, OR, USA).

Anti-CD3 FITC, anti-CD3 APC, anti-CD4 PerCP-cy5.5, anti-CD8-PerCP-cy5.5, anti-CD8 FITC, anti-CD45RO FITC, anti-CD45RO PE, anti-IFN-γ FITC, anti-IFN-γ APC, anti-IL-4 PE and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-22 PE was purchased from R&D Systems (Abingdon, UK). Anti-IL-17 APC was purchased from eBioscience (San Diego, CA, USA). Phorbol myristate acetate (PMA), ionomycin, saponin and Brefeldin A (BFA) were purchased from Sigma-Aldrich (Fluka, Sigma, USA).

SPCs (Lipoid S100) were obtained from Lipoid GmbH (Ludwigshafen, Germany) with a purity of 97.6%. It was used as received and stored at −20 °C. GDO was a kind gift from Croda, UK, which contained minimally 95% diglycerides according to the producer. All tool compounds were used as obtained. IR820 was purchased from Sigma. aPD-L1(catalog number BE0101) used in vivo purchased from Bio X Cell. Anti-CD3-PerCP-Cy5.5 (catalog number 551163), anti-CD4-FITC (catalog number 553046), anti-CD8-PE (catalog number 553032), anti-CD4-FITC (catalog number 553046), anti-CD25-APC (catalog number 557192), and anti-Foxp3-PE (catalog number 563101), anti-CD11b-FITC (catalog number 557396), anti-CD11c-FITC (catalog number 557400), anti-CD80-PE (catalog number 560016), anti-CD86-APC (catalog number 553692), anti-LY-6G/LY/6C-PE (catalog number 553128), anti-CD3-FITC (catalog number 553065), anti-CD8-PErCP-Cy5.5 (catalog number 553030), anti-CD62L-APC (catalog number 562910), and anti-CD44-PE (catalog number 559250) were purchased from BD Biosciences.

CD4, CD8, and CD56 positive cells in PBMCs isolated from healthy human donors were detected by flow cytometry on a fluorescence-activated cell sorter FACScan (Becton Dickinson, Walpole, MA, USA). Alexa Fluor 700 anti-CD4 (eBioscience, San Diego, CA, USA), anti-CD8-Per-CP-Cy5.5, and anti-CD56-Per-CP-Cy5.5 (BD Pharmingen, San Diego, CA, USA) antibodies were used to detect expression levels of CD4, CD8, and CD56, respectively. Approximately 1 × 10^⁶ pelleted PBMC cells were blocked in PBS buffer with 1% BSA for 20 min at room temperature. The cells were then stained with antibodies at 4 °C for 30 min, washed twice in PBS buffer with 1% BSA and resuspended in 0.5 ml staining buffer for FACScan analysis.