Cd8a percp

Antibodies were CD45-PE-Cy7, CD3-AF647, CD4-PE, CD25-BV510, Foxp3-AF488, CD8a-PerCP from Biolegend and CD11b-AF700, CD68-BV510 from AbD serotec and CCR7-AF647, CD206-FITC from Bioss and CD163-PE from LSBio. Single cell suspensions were first stained with 50 μl antibody mixture for the surface markers CD45, CD11b, CD163, CD206, CCR7 (macrophage panel), or CD45, CD3, CD4, CD25, CD8 (T cell panel), for 30 minutes in room temperature. Cells were then washed once and incubated with Cytofix/Cytoperm for intracellular staining according to the manufacturer’s instructions (BD Biosciences) at 4 °C for 20 min. Fixed and permeabilized samples were stained with intracellular markers CD68 (for macrophage panel) or Foxp3 (for T cell panel) diluted in Perm/Wash buffer for 30 minutes. Cells were then washed 3 times with Perm/Wash buffer before being fixed in 1% paraformaldehyde. Data were acquired using Gallios Flow Cytometer (Beckman Coulter) and fluorescence minus one (FMO) samples was used to set the gates. Evaluation was performed using FlowJo v10 software.

All flow cytometry and analysis was done on an Aria FACS Diva flow cytometer equipped with 3 lasers, and all analysis was done with the FACS Diva software. The following antibodies were used in specific phenotypic panels: T-Cell Panel: CD4-APC-Cy7 (Biolegend, San Diego, CA, USA), CD8a-V450 (BD Horizon San Jose, CA, USA), CD44-FITC (ThermoFisher Scientific, Waltham, MA, USA), CD62L-eFluor660 (eBiosciences, San Diego, CA, USA), KLRG1-Biotin (BD Pharmingen, San Jose, CA, USA) with Streptavadin-Alexa Fluor 430 (ThermoFisher Scientific) as the secondary, and CD127-PE (R&D Systems, Minneapolis, MN, USA). NK Cell Panel: CD56-Alexa Fluor 700 (Novus Biologicals, Littleton, CO, USA), CD314-PE (ThermoFisher Scientific), CD3-FITC, CD161-APC, CD8a-PerCP all from Biolegend. DC Panel: CD11c-FITC (Bio-Rad, Hercules, CA, USA), CD86-PE (Biolegend), and MHC Class II-APC (Miltenyi Biotec, Teterow GER). Treg Panel: FoxP3-PE, CD4-APC and CD25-FITC all from Biolegend. All antibodies were used at the company’s suggested concentration or titrated for optimal use.

ART (purity: 99.83%, Selleck Chemicals, California, USA) was dissolved in DMSO at 100 mM for storage and use. Antibodies used for immunohistochemical staining included anti-CD3 (ab16669,1:800), anti-CD8 (ab217344,1:400), anti-CD68 (ab31630, 1:1600), anti-Foxp3 (ab22510, 1:400; all from Abcam, Cambridge, England), anti-CD4 (Cell Signaling Technology, Shanghai, China; D7D2Z, 1:800), and anti-rat C4d (Hycult Biotech, Uden, Netherlands; HP8034, 1:400). Anti-rat antibodies for flow cytometry were from BioLegend, including CD45-Pacific Blue, CD3-FITC, CD4-APC/Cy7, CD8a-PerCP, and CD11b- PE/Cy7. B220-PE was purchased from Thermo Fisher.

Leukocytes from lymph nodes, blood, spleen and peritoneal cavity wash were collected and isolated as described [56 (link)], and analysed by flow cytometry. Anti-mouse CD16/CD32 antibody (1:50, BD Biosciences, clone #2.4G2) was used to block non-specific binding. The following fluorochrome-conjugated antibodies were used: CD3e-AF700 (1:200, BD Biosciences, clone #500A2), CD4-BUV395 (1:100, BD Biosciences, clone #RM4-5), CD8a-PerCP (1:100, BioLegend, clone #53-6.7), CD25-PE-CF594 (1:100, BD Biosciences, #PC-61), FoxP3-APC (1:50, eBioscience, clone #FJK-16S), CD11b-APC (1:200, BioLegend, clone #MI/71), CD11c-BV421 (1:50, BD Bioscience, clone #HL3), GR1-PerCP Cy5.5 (1:200, BD Biosciences, clone #RB6-8C5), F4/80-BV711 (1:100, BioLegend, clone #BM8) and MHCII-APC/Cy7 (1:500, BioLegend, clone #M5/114.15.2). Single-stained beads were used to set compensation controls, and fluorescence-minus-one (FMO) controls were used to define population gates. Data were acquired using a BD LSRFortessa X-20 (BD Biosciences), and were analysed using FlowJo software v10.5.0 (BD Biosciences).