Anti cd8 fitc

To study the phenotypes of CD8⁺ Treg cells, lymphocytes were incubated with the following antibodies: anti-CD8-FITC, anti-CD25-PE-Cy7, anti-CD127-AlexaFluor 647 and anti-CTLA4-PE (BD Biosciences). To analyze FOXP3 level, cells were fixed, permeabilized and labeled with anti-FOXP3-AlexaFluor-647 antibodies (BD Biosciences). The cells were analyzed by FACS Verse flow cytometer using FACS Suite software (BD Biosciences). To check phosphorylated-SMAD3, GATA3, RUNX3 level control CD8⁺/tumor-CD8⁺ Treg cells were incubated with pSMAD3, GATA3, RUNX3 primary antibody (Santa Cruz Biotechnology) followed by anti-mouse and anti-rabbit AlexaFluor 488 antibody and then level observed by flow cytometry (FACS Verse). For the determination of intracellular cytokine, cells were first stimulated with PMA (10 ng/ml) and ionomycin (1 µM) and then treated with 10 µg/ml Brefeldin A (Sigma-Aldrich, St. Louis, MO). Intracellular TGFβ, IL10, IL2, and IFNγ were analyzed with the PE-tagged antibody (BD Biosciences) in FACS Verse.

The staining antibodies were anti-CD4-APC, anti-TIM3-PerCP-Cy5.5, anti-IFN-γ-PerCP, anti-IL-2-PE, and anti-TNF-α-FITC antibodies (Biolegend, USA), and anti-CD8-FITC (BD Biosciences, CA, USA).

A blood sample of 200 μl was used for CD4⁺ memory T-cell phenotyping with the following antibodies: anti-CD8-FITC (1/10, clone RPA-T8), anti-PD1-FITC (1/5, clone MIH4), anti-CD122-PE (1/10, clone Mik-B3), anti-CD62L-V450 (1/10, clone DREG-56), anti-CD4-V500 (1/20, clone RPA-T4), anti-CD95-APC (1/10, clone DX2), anti-CD45RA-PE-Cy7 (1/20, clone HI100), anti-CD45RO-PerCPCy5.5 (1/10, clone UCHL1), anti-CCR7-PE-CF594 (1/10, clone 150503), anti-CXCR3-Alexa 700 (1/10, clone 1C6/CXCR3), anti-CD27-APC-H7 (1/10, clone M-T271) (all from BD Biosciences), and anti-CD3-eFluor 650NC (1/10, clone OKT3, eBioscience). After staining, the blood sample was fixed (fix/lyse solution, BD Biosciences) and cells were acquired on a BD LSR Fortessa cytometer (BD Biosciences). Data were analysed with Flow Jo software.

To evaluate the ability of the selected epitopes to trigger the immune cells by CD8+ T cell dependent manner, PBMCs (1 × 10⁶/ml) from five HLAA2 positive VL treated subjects were incubated for overnight in the presence or absence of peptide at 37 °C and 5% CO₂. Additionally, six HLA A2 negative VL treated subjects were also considered for this study. Stimulation with SLA was run in parallel to all experiments as described previously^{50 (link)}. After overnight incubation followed by 6 h incubation with brefeldin-A (1 mg/ml), cells were harvested, washed with PBS, and stained with anti-CD3-PerCP (BD Biosciences) and anti-CD8-FITC(BD Biosciences) conjugated antibodies for 30 min at 4 °C. The cells were then washed with stain buffer, fixed and permeabilized Cytofix-Cytoperm buffer (BD Biosciences) for 20 minutes at 4 °C. The intracellular level of IFN-γ was stained with anti-IFN-γ PE-conjugated antibody (BD Biosciences) for 30 minutes and washed with perm wash buffer (BD Biosciences). A logical gate set using was used to measure the co-expression of intracellular CD8+ ve IFN-γ. At least 30 000 cells were acquired for each analysis, and the results are shown in percentage gated value (% gated).

Stimulated or non-stimulated endothelial cells were stained extracellularly with anti-CD31-fluorescin isothiocyanate (FITC) and intracellularly (either before or after stimulation) with anti-SLP-76 allophycocyanin (APC) antibodies (BD Biosciences, Allschwil, Switzerland). As control, stimulated (anti-CD3) or non-stimulated enriched human CD8+ T cells (MACS cell separation, Miltenyi Bergisch Gladbach, Germany) were stained extracellularly with anti-CD8-FITC and intracellularly with anti-SLP-76 APC antibodies (BD Biosciences, Allschwil, Switzerland). Data were acquired on a LSR II Fortessa flow cytometer equipped with FacsDIVA software (BD Biosciences, Allschwil, Switzerland).