Anti ccr7 pe cy7

Purified CD8⁺ T cells were stained with the LIVE/DEAD Fixable Aqua Dead Cell Stain kit and the following antibodies: anti–CD3 Alexa Fluor 700, anti–CD8 APC Cy7, anti–CCR7 PE Cy7, anti–CD45RA BV421, and anti–CD27 PE (all from BD Biosciences). Viable central memory, transitional memory, effector memory, and terminal effector CD8⁺ T cells were bulk sorted using the BD FACSAria III (BD Biosciences) and left to rest for at least 6 hours in complete medium before treatment with the GSK3 inhibitor or vehicle control. Then, cells were washed and stimulated with plate-bound anti-CD3/anti-CD28 antibodies (1 μg/mL) for 48 hours. After culturing, cells were counted, and a total of 2500 cells per condition were placed in 96-well plates containing VILO Reaction Mix, SUPERase-In, and NP40 (all from Thermo Fisher Scientific). Plates were snap-frozen and stored at –80°C. Analysis of gene expression was performed as previously described (8 (link)), using Delta Gene primers, 96.96 Dynamic Array chips, and a Biomark instrument for microfluidics-based quantitative PCR (Fluidigm). Linear derivative mode baseline correction was applied. Data were normalized to GAPDH as the housekeeping gene, and the ΔCt method was applied. Gene expression values are plotted as log 2^–ΔCt.

Dendritic cells were harvested, and 1 × 10⁶ cells were washed with cold 1× DPBS and then incubated with anti-CD40-APC (BD Biosciences; San Jose, CA, USA), anti-CD80-PE (eBioscience; San Diego, CA, USA), anti-CD83-PE-Cy5 (BD Biosciences; San Jose, CA, USA), anti-CD86-PE (eBioscience; San Diego, CA, USA), anti-HLA-DR-PE (eBioscience; San Diego, CA, USA), and anti-CCR7-PE-Cy7 (BD Biosciences; San Jose, CA, USA) labeled antibodies (BD Biosciences; San Jose, CA, USA) for 30 min on ice. After washes, cells were fixed with 1% paraformaldehyde (Fisher Scientific; Waltham, MA, USA) and data were collected on an LSRII flow cytometer (BD Biosciences; San Jose, CA, USA). Expression of cell surface markers was analyzed on gated GFP positive cells using FlowJo 6.3.4 software (FlowJo; Ashland, OR, USA).

T cell responses to TBEV were assessed using multi-color flow cytometry, and the monoclonal antibodies (mAbs) used were; anti-CD107a FITC, anti-CD4 Pacific Blue, anti-CD8 PerCP, anti-HLA-DR PerCP, anti-Ki67 FITC, anti-Ki67 Alexa Fluor 700, anti-Bcl2 PE, anti-CCR7 PE-Cy7, anti-MIP-1β PE, anti-CD14 BD horizon V500, anti-CD19 BD horizon V500, anti-perforin FITC and anti-granzyme B APC, anti-granzyme B PE-CF594 all from BD Biosciences (San Jose, CA). Anti-CD45RA APC-Cy7, anti TNF pacific blue, anti-IFN-γ Brilliant Violet 570, anti-CD27 Brilliant Violet 785, anti-CD27 Brilliant Violet 421, anti-Helios Pacific Blue, anti-T-bet Alexa Fluor 488, anti-T-bet PE-Cy7, anti-CCR7 Brilliant Violet 785, anti-CD279 Brilliant Violet 711, anti-CD27 biotin and anti-CD127 Brilliant Violet 570 were all from Biolegend (San Diego, CA). Anti-CD38 Alexa Fluor 700, anti-CD38 eFluor 650, anti-CD127 Alexa Fluor 780, anti-PD-1 (CD279) PE, anti-Eomes eFluor 660 and IgM eFluor 650 were all from eBioscience (San Diego, CA). Anti-CD4 Qdot 605, anti-CD8 Qdot705, anti-CD8 Qdot 605, Streptavidin-Qdot 585, anti-CD57 pure and Aqua Live/Dead were all from Invitrogen (Carlsbad, CA). Anti-CD3 ECD, anti-CD3 PE-Cy5, HLA-A2 CMV pp65 tetramer in PE and anti-CD56 ECD were from Beckman Coulter (Brea, CA). HLA-A2 ILLDNITTL tetramer in PE was kindly provided by the NIH Tetramer core facility.

For surface staining, T cells were washed in FACS buffer containing PBS and 5% foetal bovine serum followed by incubation for 20 minutes at 4°C with fluorophore-conjugated antibodies against the several surface markers selected. The following antibodies were used: anti-CD4 APC-Cy7, anti-CD8 Pacific Blue, anti-TMEM123 APC and PE, anti-LFA1 APC, anti-ICAM1 APC, anti-CD44 FITC, anti-CD62L PE-Cy-5, anti-CCR7 PE-Cy-7, anti-PD1 BV711, anti-CD39 BUV563, anti-TIM-3 BV650, anti-CD69 PE-Cy-7 (all from BD Biosciences). Cytokine production was assessed by intracellular staining. The cells were fixed and permeabilized with Cytofix/Cytoperm (BD Bioscience) and then stained with anti-IFN-γ PE/Cy-5, anti-TNFα APC, anti-IL-2 FITC and anti-IL22 PE-Cy-7 (from BD Biosciences). Samples were acquired using a FACS Canto-II flow cytometer (BD Biosciences) and data were analysed using FlowJo software version 10 (LLC).

PE-conjugated HLA-A2 tetramers refolded around the CMV pp65_495-503 epitope NLVPMVATV (NLV) were generated as described previously^{48 (link),49 (link)} and used at a final concentration of 10 μg/mL. Directly conjugated mAbs were obtained from commercial suppliers: (i) anti-CD3-APC-H7, anti-CD8-BV786, anti-CD14-BV510, anti-CD19-BV510, anti-CD45-RA-PerCP-Cy5.5, anti-CD57-APC, anti-CD127-BV421, anti-CD160-PerCP-Cy5.5, anti-CCR7-PE-Cy7, anti-CTLA-4-PE-Cy7, anti-BTLA-APC, anti-2B4-FITC, anti-2B4-PE, and anti-Tim-3-AF700 (BD Biosciences); (ii) anti-CD27-BV605 and anti-PD-1-PE/Dazzle 594 (BioLegend); and (iii) a panel of anti-TCR-Vβ-FITC and anti-TCR-Vβ-PE mAbs (Beckman Coulter). The acute effects of dasatinib administration were monitored using an IOTest Beta Mark TCR Repertoire Kit (BD Biosciences). Non-viable cells were excluded from the analysis using LIVE/DEAD Fixable Aqua (Life Technologies). Data were acquired using a Fortessa X-20 flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.0.7 (Tree Star). Viable CD3⁺ CD8⁺ TCR-Vβ⁺ cells were sorted using an Influx^TM Cell Sorter (BD Biosciences).

The following monoclonal antibodies were used to characterize MoDC: anti-CD86 FITC, anti-CD1a PE, anti-HLA-DR PERCP, anti-CD83 APC, anti-CD14 APC-H7, anti-CD80 FITC, anti-CD40 PE, anti-HLA-I APC, anti-CCR7 PE-Cy7, anti-CCR5 APC-H7 from BD Biosciences, and anti-BT3A.1 PE from BioLegend. To evaluate γδ T lymphocytes phenotype we used anti-Vδ2 FITC, anti-CD3 PE, anti-CD69 PERCP, anti-CD45RA PE-Cy7, anti-CD27 APC, anti-CD16 PACIFIC BLU, anti-CD25 APC-H7 (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide, and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 4% paraformaldehyde, and analyzed using a FACS Canto II (Becton Dickinson). Since the presence of 2 purified populations, the gating strategy was performed as follow: dead cells were excluded by scatter characteristics; MoDC were identified by morphological parameters (FSC vs SSC); gated cells were then analyzed for the expression of the molecules described above. T lymphocytes were first gated by using morphological parameters; in this gate Vγ9Vδ2 T cells were identified as Vδ2+CD3+. Analysis was carried out by using Facs Diva software (Becton Dickinson). The histogram overlays were performed by FlowJo software (TreStar, Olten, Switxìzerland).