Percp labeled anti cd3

To examine PD-1, CTLA-4 and GRAIL expression, splenocytes or CD4 T cells from control and infected animals or in vitro infected CD4 T cells were washed with saline solution 2% FBS and incubated with anti-mouse CD32/CD16 antibody for 20 minutes at 4°C to block Fc receptors. Then, cells were incubated with APC labeled anti-CD4, PercP labeled anti-CD3 (BD Pharmingen), and with PE-labeled anti-PD-1 or anti-CTLA-4 (BD Pharmingen) for 20 min at 4°C.
For the assessment of intracellular GRAIL expression, cells were first stained with FITC-CD3 and APC-CD4 antibodies, and then fixed and permeabilized with Citofix/Citoperm (BD Biosciences,) for 30 min followed by reacting with rabbit anti-GRAIL primary Ab (Abcam) for 45 min. After washing, cells were stained for 20 min with PE–anti-rabbit IgG (Biolegend). Finally, cells were washed twice with saline solution of 2% FBS, and stored at 4°C in the dark until analysis using a FACS flow cytometer (FACS Canto II, BD Biosciences). The results were processed using Flow Jo software (version 7.6.2).

Cytokine-stimulated NK cells were tested in a degranulation assay against the NK cell-susceptible target cell line K562. The analysis of NK cell degranulation was performed by measuring the expression of CD107a/b after activation with target cells at ratio 1:1 in the presence of BD GolgiStop (BD Biosciences) and a mixture of FITC-labeled anti-CD107a and anti-CD107b mAbs. After 4 h, cells were stained with PE-labeled anti-CD56 and PercP-labeled anti-CD3 from BD Biosciences and analyzed by flow cytometry by measuring the frequency of CD107a/b expression on CD3⁻CD56⁺ NK cells. Spontaneous basal NK cell degranulation was always below 10%. Background expression of CD107a/b (CD107a⁺ NK cells in medium only) was subtracted from expression with target cells.

Cells were washed and suspended in 100 μL of PBS containing 0.1% BSA and 0.05% sodium azide. For surface staining, cells were incubated with the respective mAbs at 4°C in the dark for 30 min. For the detection of intracellular cytokines, cells were fixed with 4% paraformaldehyde and permeabilized in PBS buffer containing 0.1% saponin (Sigma-Aldrich, USA), 0.1%BSA and 0.05% sodium azide for at least 2 h or overnight at 4°C and stained with conjugated mAbs for intracellular cytokines. Flow cytometry data were acquired with FACS Canto II (BD Bioscience, USA) and analyzed with FlowJo software (Tree Star, USA). The following mAbs were used for cell surface or intracellular staining: FITC labeled anti-CD3, anti-CD45RA, anti-Perforin, APC-labeled anti-CD69, anti-TNF-a, anti-PD-1, PE-labeled anti-CD103, anti- IFN-γ, anti-GranzymeB, Percp labeled anti-CD3, PE-cy7 labeled anti-CD4, Apc-cy7 labeled anti-CD8 and isotype matched control antibodies were all purchased from BD Bioscience PharMingen (San Jose, CA, USA).