Anti cd4 buv395

Single-cell suspensions of spleen, lymph node, and bone marrow cells were stained with the following antibody panel: PerCP5.5-anti-CD21/CD35, APC-anti-CD11c, AF700-anti-CD19, BV510-anti-CD45R/B220, BV605-anti-CD11b, BV650-anti-IgD, BV711-anti-CD44, BV785-anti-Ter119, PE/Cyanine5-anti-Ly-6G/Ly-6C (GR-1), PE/Cyanine7-anti-CD23 (BioLegend, San Diego, California, United States), BV750-anti-CD5, BUV395-anti-CD4, BUV496-anti-CD3e, BUV563-anti-CD43, BUV615-anti-CD8a, BUV661-anti-CD93, and BUV737-anti-IgM (Becton-Dickinson, Franklin Lakes, United States).
Data were acquired on a BD FACSymphony A5 flow cytometer (Becton-Dickinson) and analyzed using FlowJo software (TreeStar, Ashland, Oregon, United States).

The intracellular cytokine assay was performed as previously described [26 (link),30 (link)]. Briefly, CD8+ T cell lines were stimulated with cognate peptide pools or 10 μM individual peptides (Genscript, Hong Kong, China) and incubated for 5 h in the presence of GolgiPlug (BD Biosciences), GolgiStop (BD Biosciences) and anti-CD107a-AF488 (BD Biosciences/eBioscience, Melbourne, Australia). Following incubation, the cells were surface stained for 30 min with anti-CD8-PerCP-Cy5.5 (BD Biosciences/eBioscience), anti-CD4-BUV395 (BD Biosciences), anti-CD14-APCH7, CD19-APCH7 and Live/Dead Fixable Near-IR Dead Cell Stain (Life Technologies, Melbourne, Australia). The cells were then fixed and permeabilised for 20 min using BD Cytofix/Cytoperm solution (BD Biosciences) and intra-cellularly stained with anti-IFN-γ-BV421 and anti-TNF-PE-Cy7 (all BD Biosciences) for a further 30 min. The cells were acquired on a BD LSRFortessa with FACSDiva software (version 6.1.3, BD Biosciences). The analysis was performed using FlowJo software (version 10.7.1, BD Biosciences), where cytokine levels identified in the R0 control condition were subtracted from corresponding test conditions.

We applied BD FACSCalibur and BD FACSAria II instruments for FCM detection. BD FACSDiva software and FlowJo software were used for analysis. FCM detection was performed according to the schedule shown in Figure 2C. CD3, CD4, CD8, and CAR expression were evaluated on days 6, 10, and 14, and memory T subsets were evaluated on days 10 and 14. We used His-tagged recombinant CEACAM5 (CEA-His; Sino Biological Inc., 11077-H08H-50) and biotin-protein L (GenScript, M00097) for specific detection of CAR expression. The following antibodies were used: anti-CD3-PE-Cy7 (BioLegend, 300420), anti-CD4-BUV395 (BD, 564724), anti-CD8-BV510 (BioLegend, 344732), anti-CD25-BV421 (BioLegend, 302630), anti-CD45RA-BV421 (BioLegend, 304130), anti-CD45RO-PerCP-Cy5.5 (BioLegend, 304222), AF-647-conjugated IgG fraction of mouse monoclonal anti-biotin (Jackson, 200-602-211) and anti-CD197-PE (BioLegend, 353204).

For analysis of surface markers, T cells were collected at indicated time points after activation with CD3/CD28 Dynabeads (11131D, Thermo Fisher). For analysis of CD11b expression on T cells, activated T cells were collected at day 8, washed in Flow Cytometry Staining Buffer (00-4222-26, Thermo Fisher) and incubated with anti-CD11b biotin antibody (553309, BD Biosciences; 1:100) for 30 min at 4 °C. Cells where then washed and incubated with anti-CD3 BUV805 (612894, BD Biosciences; 1:100), anti-CD4 BUV395 (563550, BD Biosciences; 1:200) anti-CD8 APC/H7 (566855, BD Biosciences; 1:200) and streptavidin PE (12-4317-87, Thermo Fisher; 1:200) for 30 min at 4 °C in the dark. After washing, cells were resuspended in 200 µL staining buffer containing DAPI (1:10,000) (D1306, Invitrogen).
For STn surface staining, cells were incubated with anti-STn primary antibody (ab115957, Abcam; 1:100) for 30 min at 4 °C. Cells were then washed and incubated with an anti-mouse IgG-FITC secondary antibody (406001, Biolegend; 1:200) for 30 min at 4 °C in the dark. After a final wash step, cells were resuspended in 200 µL of 2 % BSA in PBS with DAPI (1:10,000). Data were collected on a FACSymphony flow cytometer and analyzed using FlowJo (BD Biosciences).