Anti cd8 pe

The blood and tumor tissues were collected, and the corresponding single-cell suspensions were generated. The following reagents were used for the identification of CD8+ T cell, CD4+ T cell, and MAIT in the blood and tumor: anti-CD3-FITC (#561798), anti-CD4-PB450 (#562891), anti-CD8-PE (#553033), anti-CD3-APC-A750 (#557596), anti-TCR beta-APC-A700 (#560705), and MR1 (#361106) (26 (link), 27 (link)); all these antibodies were purchased from Becton Dickinson (San Jose, CA, USA). Briefly, the cells were blocked using the isotype control antibodies. After washing, the fluorescence-conjugated antibodies were added into the single-cell suspensions and incubated for 30 min at room temperature. The cells were washed and resuspended. The labeled cells were determined using the CytoFLEX (Beckman Coulter, Brea, CA, USA), and data were analyzed using FlowJo cell analysis software (FlowJo, LLC, Ashland, OR).

For CD8⁺NKG2D⁺ cells analysis, cells were harvested and stained with anti-CD3-FITC (E10472-1633, eBioscience, USA), anti-CD8-PE (561946, BD, USA), anti-NKG2D-APC (130-099-216, Miltenyi Biotec, Germany), anti-CD27-PE (560985, BD, USA), and anti-CD57-APC (560845, BD, USA) antibodies. To detect the infiltration of CD8 or NK cells in the tumor mass, tumors were removed and crushed into single cell suspension. Then cells were stained with mouse anti-CD8-PErCp (46-0083-80, eBioscience, USA) or anti-NK1.1-FITC (553164, BD, USA) antibodies. To confirm the depletion of CD8 or NK cells, cells were isolated from peripheral blood and stained with anti-CD8-PErCp (46-0083-80) or anti-NK1.1-PerCp antibodies (45-5941-82) (all from eBioscience, USA). The staining was performed on ice for 30 min. Samples were acquired on the FACS Calibur (BD, USA) and data was analyzed using FlowJo software (Version 8.5.3, Tree Star Inc., USA).

T cell subset populations and memory T lymphocytes in splenocytes were analyzed by flow cytometry. Splenocytes were seeded into six-well plates at a density of 5 × 10⁶ cells/mL and stimulated with or without purified RVFV Gn head protein (10 μg/mL) for 36 h at 37°C and 5% CO₂. Cells were then labeled with equal volumes of 1:250 dilutions of anti-CD3-PE-Cy5, anti-CD4-FITC, anti-CD8-PE, anti-CD44-APC, and anti-CD62-PerCP-Cy5 (BD Biosciences, United States) for 30 min at 4°C. After washing, labeled cells were analyzed in a FACSAria™ Cell Sorter (BD Biosciences, United States).

Whole blood (50 μl) was stained for 15 minutes at room temperature in Trucount tubes (BD Biosciences) with anti-CD8 PE (Clone SK1), anti-CD4 BV510 (clone L200), anti-CD45 PerCP-Cy5.5 (clone D058-1283), anti-CD3 PE-CF594 (clone Sp34-2), anti-CD20 Alexa700 (clone 2H7), anti-CD159a APC (Clone z199, Beckman Coulter) and anti-HLA-DR APC-Cy7 (clone L243). The samples were then treated for 15 minutes at room temperature with 450 μl BD FACS lysing solution to lyse erythrocytes and fix mononuclear cells. The cells were analyzed by flow cytometry, collecting at least 5000 fluorescent bead events. Absolute counts for NK cells (CD45⁺CD3^-CD20^-CD8⁺), B cells (CD45⁺CD20⁺), CD4⁺ T cells (CD45⁺CD3⁺CD4⁺) and CD8⁺ (CD45⁺CD3⁺CD8⁺) T cells were determined by dividing the number of gated cellular events by the number of bead events and multiplying by the bead concentration.

Spleen cells from 6-7 week-old naïve B6 mice were pooled after lysing red blood cells. Cells were then stained with anti-CD8-PE, anti-CD122-FITC, anti-PD-1-PerCP and anti-CD3-APC mAbs (BD Biosciences, Mountain View, CA), and CD8+CD122+PD-1+ Tregs or CD3+ T cells were sorted out using a FACSAria III (BD Biosciences). The purity of the sorted cells was typically > 95%.

Antibodies used in the current study were Anti-CD11b PE (eBioscience M1/70)/APC (BD,M1/70), anti-GR-1 FITC (BD RB6-8C5), anti-Ly6G FITC (RB5-8C5) APC (RB5-8C5), anti-Ly6C PreCP-Cr™5.5 (BD AL-21), anti-IL10 FITC (BD), anti-IL12 (P40/P70) (BD), anti-p-Stat3 PE (BD PY705), anti-NOS2 PE (eBioscience CXNFT), arginase FITC (R&D), anti-CD8 PE (BD), anti-IFNγ FITC (BD).
The numbers of IFN-γ-secreting CD8⁺ T cells were analyzed by flow cytometry after adding Golgi plug (1 μg/mL, BD Pharmingen) for 8 hours. Analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton-Dickinson Immunocytometry System, Mountain View, CA) and Flowjo 10 software.