Anti tigit percp cy5

PBMCs responding to both NAE and AE in an IFNγ ELISPOT assay were pulsed with the peptides at 10μM in the presence of anti-CD28 and anti-CD49d. Monensin and brefeldin A were added 1 hour after peptide stimulation. The cells were incubated for an additional 11h. Following incubation, cells were surface stained for 30min at 4°C with dead cell dye (Invitrogen), anti-CD3-Alexa 780 (eBioscience), anti-CD4-Qdot655 (Invitrogen), and anti-CD8-V500 (BD Pharmingen) in the following panels: (1) anti-TIGIT-Percp/CY5.5 (Biolegend), anti-CD160-Alexa488 (eBioscience), anti-PD1-Alexa700 (Biolegend), anti-TIM3-BV421 (Biolegend) and anti-LAG3-PECy7 (Biolegend) and (2) anti-CD28-FITC (BD Pharmingen), anti-CD27-PECy7, anti-CD38-v450 (eBioscience), anti-CD57- Percp/CY5.5 (Biolegend), and anti-CD69-Alexa700 (Biolegend). The cells were then permeabilized and stained with anti-IFNγ-PE at 4°C for 30 min. At least 10⁶ total events were acquired on an LSR II flow cytometer (BD Immunocytometry Systems), and analyzed using FlowJo (version 9.6.4; TreeStar Inc.). The criteria of positivity is the same as defined above for tetramer staining [73 (link)].

The following monoclonal antibodies were used for flow cytometry analysis and cell sorting; anti-CD8a-Alexa Fluor 488 (300916, BioLegend, 1:200), anti-CD8a-FITC (FITC-65135, Proteintech, 1:50), anti-CD3-PE (317307, BioLegend, 1:200), anti-CD4-APC (300552, BioLegend, 1:200), anti-CD45RA-FITC (304148, BioLegend, 1:200), anti-CD45RO-PerCP/Cy5.5 (304222, BioLegend, 1:200), anti-PD-1-APC/Cy7 (329921, BioLegend, 1:50), anti-CD57-PerCP/Cy5.5 (359621, BioLegend, 1:200), anti-IFNγ-APC (502511, Biolegend, 1:200), anti-TNFα-BV421 (502931, BioLegend, 1:100), anti-CCR7-PE (353203, BioLegend, 1:50), anti-TIGIT-PerCP/Cy5.5 (372717, BioLegend, 1:50), anti-pan HLA (M0736, Dako, 1:200), control mouse IgG2a (401501, BioLegend, 1:1111), and anti-mouse IgG-FITC (406001, BioLegend, 1:200). LIVE/DEAD Fixable-Near IR Dead Cell Stain Kit (L10119, Thermo Fisher Scientific, 1:400), LIVE/DEAD Fixable-Aqua Dead Cell Stain Kit (L34957, Thermo Fisher Scientific, 1:400), and Human BD Fc Block (564220, BD Pharmingen, 1:50) was also used. MHC tetramer was prepared using QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-PE (TB-7302-K1, MBL Life Science) and QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-BV421 (TB-7302-K4, MBL Life Science) with appropriate peptides. CMV pp65_341-349 peptide (TS-0020-P, MBL) was purchased and all the other peptides were synthesized with a purity of >95% (Scrum Inc., or Toyobo).