Ifn γ pe cy7 clone xmg1.2

Single-cell suspensions of splenocytes, obtained as in 2.14, were incubated with D1DCs that were loaded overnight with 5 µM synthetic peptides of the MC38 neoepitopes Adpgk (peptide sequence: HLELASMTNMELMSSIVHQ) and Rpl18 (peptide sequence: KAGGKILTFDRLALESPK) [59 (link),60 (link)], in presence of 2 µg/mL Brefeldin A for 8 h at 37 °C. The samples were then stained with antibody mixes for flow cytometry. Again, all cytometric analyses were performed on samples provided in FACS buffer on a Cytek Aurora 5-laser flow cytometer. The antibody panel consisted of granzyme B-V450 (clone NGZB; Thermo Fisher Scientific, Waltham, MA, USA), CD3-BV510 (clone 145-2C11; BD Biosciences, San Jose, CA, USA), TNFα-FITC (clone MP6-XT22; Thermo Fisher Scientific, Waltham, MA, USA), IL-2-PE (clone JES6-5H4; Thermo Fisher Scientific, Waltham, MA, USA), IFN-γ-PE-Cy7 (clone XMG1.2; BD Biosciences, San Jose, CA, USA), CD8α-APC-R700 (clone 53-6.7; BD Biosciences, San Jose, CA, USA), and 7AAD viability staining (Invitrogen, Waltham, MA, USA).

Lung and lymph nodes were examined for intracellular cytokine production. Single cells were obtained as described above. Samples were then stimulated with 10 μg/ml H37Rv cell lysate (BEI NR-14822), 5 μg/ml ESAT6 peptide array (BEI NR-34824), and 5 μg/ml CFP10 peptide array (BEI NR-34825) in RPMI media for 3 h in a 37 °C, 5% CO₂ humidified incubator. Stimulated cells were then washed, and surface stained with anti-mouse CD3-FITC (clone 17A2, BD Pharmingen), anti-mouse CD4-V450 (clone RM4-5, BD Horizon), and anti-mouse CD8-PE (clone RM4-5, BD Pharmingen). Subsequently, cells were fixed and permeabilized using BD Cytofix/Cytoperm solution kit (BD 554714) as per the manufacturer’s instructions. Permeabilized cells were stained for intracellular cytokines using anti-mouse IL-17A-AF647 (clone TC11-18H10, BD Pharmingen) and IFNγ-PECy7 (clone XMG1.2, BD Pharmingen). All antibodies were used at a 1:50 dilution. Samples were acquired using the LSRFortessa X-20 and analyzed using FlowJo software (v10.6.1).