Anti cd4 fitc clone okt4

PBMCs thawed and cultured overnight in IMDM (Lonza, Basel, Switzerland), supplemented with 10% human serum (HS, Sigma-Aldrich) were labelled with anti-CD8-Alexa Fluor700 (Clone: RPA-T8, BD Biosciences, New Jersey, USA), anti-CD3-PE-Cy7 (Clone: UCHT1, Biolegend, San Diego, USA), anti-CD4-FITC(Clone OKT4, Biolegend) and 7AAD (Beckman Coulter, California, USA) and sorted on FACS Aria™ II (BD Biosciences) for 7AAD-CD3+CD8+ live T cells. Post-sorting analysis of purified subsets revealed greater than 98% purity. Mean CD8+ T cell count/μl was calculated as: (%CD8+ T cells gated from lymphocytes during sorting x total lymphocyte count/μl)/100 at the time of blood collection (± 2 days). Sorted CD8+ were pelleted by centrifugation, re-suspended in Trizol and immediately stored at -80°C. Total RNA was extracted using the miRNeasy Micro Kit (Qiagen, Hilden, Germany) and stored at -80°C after RNA quantification using a NanoDrop ND-1000 Spectrophotometer (Thermo scientific, Waltham, MA, USA). The phenotypic analysis was performed on FlowJo version 7.6.5 (Treestar, Ashland, USA).

Leukemia-reactive IFN-γ producing CD3⁺ T cells were evaluated after the coculture with DCs pulsed with leukemic lysate and matured with the cytokine cocktail containing PGE₂. After 4 h of incubation, brefeldin A was added (2 μg/mL, Sigma-Aldrich) and incubated overnight at 37°C. At the end of the incubation, cell-surface staining was performed as described above (anti-CD4 FITC: clone OKT4, anti-CD8 APC: clone HIT8a, Biolegend). Then, cells were fixed (30 min at 4°C in 2% paraformaldehyde (Sigma-Aldrich)) and the anti-IFN-γ antibody (clone B27; Biolegend) was added in 0.1% saponin and incubated for 30 min at 4°C.
Both assays were performed in the presence or absence of 1-MT-L (1 mM). At the end of the culture time, cultures were analyzed using BD FACSCanto II equipment (BD Biosciences).

Cells were harvested and washed twice with PBS containing 1% BSA (flow cytometry buffer). The following antibody cocktail was used to phenotype moDCs: anti-CCR7-APC (clone G043H7, BioLegend), anti-CD40-BV605 (clone 5C3, BD), anti-CD86-FITC (clone FUN-1, BD Pharmingen), anti-HLA-DR-PE-CY7 (clone G46-6, BD Pharmingen), and anti-HLA-A2-BV421 (clone BB7.2, BioLegend). Cells were also stained with 7-AAD (BioLegend) to discriminate live from dead cells. T cell phenotyping was performed using the following antibodies: anti-CD8-BV421 (clone RPA-T8, BioLegend), anti-CD45RA-PE (clone HI100, BD), anti-CD45RO-APC (clone UCHL1, BD) and MHC dextramer Mel-A-PE or -APC (HLA-A∗0201/ELAGIGILTV, WB2162, Immudex) for CD8⁺ T cells and anti-CD4-FITC (clone OKT4, BioLegend), anti-CD69-PerCP-CY5.5 (clone FN50, BioLegend) and anti-OX40-APC (clone ACT35, BioLegend) for CD4⁺ T cells. T cells were also stained with 7-AAD (BioLegend) to discriminate live from dead cells. Cells were acquired on the LSR Fortessa flow cytometer and analyzed with FlowJo software, version 10.0.