Cd45ro fitc

The following mAbs were used for staining and flow cytometric measurements: CD3-VioGreen (BW264/56), CD4-allophycocyanin-Vio770 (VIT4), CD8-PE-Vio770 (BW135/80), CD14-peridinin chlorophyll protein (PerCP) (TÜK4), CD20-PerCP (LT20), CD27-FITC (M-T271), CD28-FITC (15E8), CD45RA-VioBlue (T6D11), CD45RO-FITC (UCHL1), CD62L-FITC (145/15), CD107a-PE (1D4B), CD127-FITC (MB15-18C9), CD137-allophycocyanin, CD137-PE (4B4-1), CD154-VioBlue (5C8), CD178-PE (NOK-1), CD197 (CCR7)-allophycocyanin, CD197 (CCR7)-PE (150503), CD279-allophycocyanin (PD1.3.1.3), anti-IL-2-PE-Vio770 (N7.48A), anti-IL-4-PE (7A3-3), anti-IL-17-FITC (CZ8-23G1), anti-IFN-γ-FITC, anti-IFN-γ-PE-Vio770 (45-15), anti-TNF-α-PE, and anti-TNF-α-VioBlue (cA2) (all Miltenyi Biotec).
Soluble biotinylated pHLA-A*0201 molecules loaded with WT1₃₇ (VLDFAPPGA), WT1₁₂₆ (RMFPNAPYL), WT1₁₈₇ (SLGEQQYSV), WT1₂₃₅ (CMTWNQMNL), or cytomegalovirus (CMV) pp65₄₉₅ (NLVPMVATV) were produced as described previously.14 (link) Tetramerization was achieved by binding to streptavidin-PE or streptavidin-allophycocyanin (both BioLegend, San Diego, CA). In each case, 2·5 × 10⁶ cells were stained with 1 µg/ml tetramer for 45 min at 4°C.
Data were acquired using a MACSQuant flow cytometer and analysed with MACSQuantify software (both Miltenyi Biotec).

Lysed cells were acquired and subsequently stained for 30 min at 4 °C with the following fluorescein-conjugated monoclonal antibodies: CD3-PE (Bio- Legend, San Diego, CA, USA), CD4-APC (eBioscience, San Diego, CA, USA), CD8a-Percp/Cy5.5 (eBioscience), CD45RO-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CCR7-APC/Cy7 (BioLegend). The T cell subsets were defined as previous study reported [10 (link)]: Naive T cells being CCR7⁺ and CD45RO⁻; central memory T cells as CD45RO⁺ and CCR7⁺; effector memory T cells as CD45RO⁺ and CCR7⁻, and effector memory RA (EMRA) T cells as CD45RO⁻ and CCR7⁻ (Figure S1). A total of 200,000 events were acquired by the BD LSRFortessa™ flow cytometer (BD Bioscience, San Jose, CA, USA). The data analysis was carried out with Flowjo v10.1 Software (Tree Star, Ashland, OR). The absolute number of each T cell subset was calculated as follows: (percentage of each cell population among total lymphocytes) × (total lymphocytes count)/100.

On the day of blood drawing, blood samples mixed with heparin anticoagulant were lysed with red blood cell lysis solution and 0.1 mM EDTA and prepared for flow cytometry analysis with the following fluorescein-conjugated monoclonal antibodies: CD3-PE (Bio- Legend, San Diego, CA, USA), CD4-APC (eBioscience, San Diego, CA, USA), CD8a-Percp/Cy5.5 (eBioscience), CD45RO-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and CCR7-APC/Cy7 (BioLegend). The relative expression of CD45RO and CCR7 was used to identify naïve T cell (T_Naïve, CD45RO⁻ CCR7⁺), central memory T cell (T_CM, CD45RO⁺ CCR7⁺), effector memory T cell (T_EM, CD45RO⁺ CCR7⁻), and T effector memory CD45RA cell (T_EMRA, CD45RO⁻ CCR7⁻) subsets of CD4⁺ or CD8⁺ T cells. These markers were selected according to previous studies (7 (link), 17 (link)). The immunophenotyping methods and gating strategy have been elaborated in the supplementary materials (Figure S1).