Anti cd8 percp

PBMCs were cultured in 96 well plate in 5% CO2 incubator at 37°C for 6 hours in RPMI 1640 medium with or without 0.02 µm HTLV-1 tax peptide (Sigma-Aldrich, Japan) and brefeldin A (BD San Diego, CA) at a final concentration of 10 µg/ml. CD48-blocking antibodies were added at a final well concentration of 5 µg/ml [16] (link). Harvested cells were washed twice with phosphate buffered saline (PBS) and stained with anti-CD8 PerCP (BD Biosciences, San Jose, CA USA) or CD8-PC5 (Cytostat-Coulter, Fullerton USA), PE-conjugated HLA-A*0201 and HLA-A*2402 for 15 minutes at room temperature. Cells were washed and then permeabilized using FACS permeabilizing solution 2 (BD San Diego, CA) for 10 minutes at room temperature. Cells were again washed, suspended in saponin-containing PBS for 10 minutes at room temperature and then incubated with FITC-labeled perforin and isotype control IgG2b κ-FITC (BD) for 20 minutes at room temperature. Finally, cells were washed and analyzed with FACS Calibur flow cytometer and FlowJo Treestar software based on forward and side scatter in the lymphocyte gate as described [31] (link).

Anti-CD25-APC-Cy5, anti-CD127-Alexa-Fluor700, anti-CD4-FITC or Pacific blue, anti- CD45RA-FITC or PE-Cy5, anti-CCR7-PE, or PE-Cy7, anti-CD8-PerCP, anti-CD3-AmCyan, anti-IFN-gamma-FITC or APC-Cy7, anti-CD69-APC, anti-PD-1-PE and anti-PD-L1-PECy7, anti-FoxP3-PE were all products of BD Biosciences. Cells were analysed by FACSCanto II, and LSR II (BD Immunocytometry systems). At least 20 000 CD4 or CD8-gated events, and at least 100000 CD4 or CD8-gated events were collected for cell surface and intracellular studies, respectively.

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.

Osteoblasts were acquired and washed with PBS three times. All samples were divided into control and test tubes. Antibodies against mouse IgG1-FITC, IgG1-PE and IgG1-PerCP (BD Biosciences, US) were stained as a negative control. Antibodies against CD45-PerCP, CD138-FITC, and CD34-PE (BD Biosciences, US) were stained to identify the purity of osteoblasts. Peripheral blood samples were collected in EDTA- anticoagulant tubes. The number of immune subsets were measured by FCM using anti-CD4-FITC, anti-CD8-FE and anti-CD3-PerCP (to identify the subtypes of T lymphocytes); anti-CD4-PE, anti-CD25-FITC and anti-CD127-APC (regulatory T cells); anti-Lin-FITC, anti-HLA-DR-PerCP, anti-CD11c-PE and anti-CD123-APC (subtypes of dendritic cells, DC); anti-CD3-APC, anti-CD8-PerCP, anti-IFN-γ-FITC and anti-IL-4-PE (T helper cells, Th) (BD Biosciences, US). After incubation in the dark at 4°C for 30 min, the cells were incubated with 2 ml erythrocyte lytic solution (BD Biosciences) at room temperature for 10 min. The cells were then washed two times with PBS. At least 10,000-30,000 cells were acquired and analyzed on FACSCalibur flow cytometer (BD Biosciences).

The ex vivo Ag presentation assays were performed using a protocol adapted from Langlet et al. OVA-specific CD8⁺ T and CD4⁺ T cells were isolated from OTI and OTII transgenic mice, respectively, and labeled with 10 µM CFSE. 10⁵ OT-I T cells or 10⁵ OT-II T cells were subsequently co-cultured for 48 hours with respectively 10⁴ sorted Ly6C^hi monocytes, MHCII^hi DCs or MHCII^int DCs obtained from the DLN of mice previously (24 h) immunized with 20 µl of an OVA (100 µg/ml)/CpG (100 µg/ml) mixture. OT-I and OT-II proliferation was determined by the loss of CFSE fluorescence by flow cytometry. Sorts were performed using a FACSAria sorter (BD Biosciences).
In vivo antigen presentation assays were performed through adoptive transfer of 2 × 10⁵ CFSE labeled OT-I or OT-II cells to respectively WT C57BL/6 J mice, CCR2^−/− mice, CCR7^−/− mice and Flt3L^−/− mice 48 hours prior to OVA/CpG immunization. Four days post immunization, DLN were dissected, stained with anti-CD3-V450, anti-CD4-PerCP, anti-CD8-PerCP, anti-Vα2 TCR-PE, anti-CD19-APC-Cy7 (all BD Biosciences), PE-labeled SIINFEKL specific dextramers (Immudex) and measured on a LSRII flow cytometer (BD Biosciences).