Ctla4 apc

PBMC adhered to the insert were removed using ice-cold PBS-Wash (PBS containing 0.5% bovine serum albumin and 1% sodium azide) and centrifuged at 400 x g for 5 minutes. The supernatant was discarded, and the pellet containing the cells was distributed into cytometry tubes and incubated with monoclonal antibodies anti-CD4/PerCP (BD™), CD8/FITC (BD™), CD28/PE (BD™) and CTLA-4/APC (BD™) for lymphocytes, and anti-CD14/FITC (BD™), CD80/PE (BD™), HLA-DR/PerCP (BD™) for CD14+ cells, for 30 min at room temperature protected from light. After incubation, the cells were washed by centrifugation (400 x g for 5 min at room temperature). Then, the cells were fixed with BD Cytofix™ (BD Biosciences^®) for 15 min at 4°C. Furthermore, after washing (400 x g for 5 min at room temperature), the cells were resuspended with 300 μL of PBS-Wash/tube and stored at 4°C until the time of acquisition on the FACScalibur flow cytometer (Beckton Dickson). The lymphocyte population was selected FSC versus SSC plots, with 20,000 events acquired within the R1 lymphocyte window, and CD14+ cells were selected via FCS versus FL1, with 1,000 events acquired within the R2 window. After selecting the window of interest (R1) and (R2), CD4+ and CD8+ T lymphocytes and CD14+ cells and molecules were analyzed by obtaining two-dimensional plots of the spot fluorescence distribution using FlowJo^® software version 10.8.1.

Different immune cell subpopulations were immunophenotyped with flow cytometry from fresh PB samples with 6 panels of different cell-surface markers, including the following immune checkpoint receptors and cytotoxicity and migration markers: CD3-PerCP-Cy5.5 (BD, catalog 332771), CD4-PE-Cy7 (BD, catalog 560649), CD45-APC-H7 (BD, catalog 560178), CD8-BV510 (BD, 563919), CD56-BV421 (BD, 562751), CXCR1-FITC (BioLegend, catalog 341606), CD16-PE (BD, 561313), TCR γδ-APC (BD, catalog 555718), PD1-FITC (BD, catalog 557860), LAG3-PE (BD, catalog 125209), ICOS-PE-Cy7 (eBioscience, catalog 25-9948-42), CTLA-4–APC (BD, catalog 560938), HLA-DR-BB515 (BD, catalog 560938), CD27-PE (BD, catalog 555441), CD25-PE-Cy7 (BD, catalog 561405), CD11b-APC (BD, catalog 550019), NKG2C-AF488 (R&D Systems, catalog FAB138G), CD161-PE (BD, catalog 556081), NKG2D-PE-Cy7 (BD, catalog 562365), NKG2A-APC (R&D Systems, catalog FAB1059A), DNAM-BB515 (BD, catalog 565152), CD57-PE (BD, catalog 560844), NKp46-PE-Cy7 (BD, catalog 562101), NKp30-AF647 (BD, 558408), CXCR3-AF488 (BD, catalog 561730), CCR7-PE (R&D Systems, catalog FAB197P), CD45RO-PE-Cy7 (BD, catalog 560608), and CXCR4-APC (BD, catalog 560936). CD45⁺ lymphocytes were acquired with the BD FACS Verse, and the data were analyzed with FlowJo, version 10.4. The results are shown in Supplemental Table 1.

The following reagents were used: PD-1^FITC, CTLA-4^APC, CD4^{FITC/APC-H7/V500}, CD14^PerCP, CD19^PerCP, CD3^PerCP, Via Probe, Monensin, functional grade anti-CTLA-4 (BD Biosciences); CD244^FITC, PD-1^APC, CD127^APC-Cy7, IL-2^FITC, IFN-γ^PE, TNF-α^PE-Cy7, anti-PD-L1/2 and anti-IgG1 isotype control (eBioscience); TIM-3^APC (R&D Systems); CCR7^FITC, CD57^PacificBlue, CD45RA^PacificBlue, functional grade anti-TIM-3 (Biolegend). Micro-Beads^anti-PE (Miltenyi). KLRG1^{AlexaFluor488} (clone: 13F12F2) was generated as described [14] (link).