Cd25 pe clone m a251

Matched NI, I, and TDLNs and primary tumors from patients with breast cancer were processed for RNA and/or TCR sequencing. T cells were first isolated using the Pan T Cell isolation kit (Miltenyi Biotec, 130-096-535). Non-specific binding was blocked using anti-CD32 (Stem Cell), and cells were stained with CD25−PE (clone M-A251; BD Biosciences), CD4-PE-CF594 (clone RPA-T4; BD Biosciences), CD8-Alexa700 (clone 3B5; Life Technologies), CD27-APC (clone L128; BD Biosciences) or CD45RA-PE-Cy7 (clone HI100; BD Biosciences) and then with 4′,6-diamidino-2-phenylindole (DAPI) LIVE/DEAD stain. CD4+ CD45RA-CD25− (Memory CD4+ Tconv), CD4+ CD45RA-CD25^high (Memory Treg), were sorted by flow cytometry in a BD FACS ARIA II cell sorter, with a purity of 98–99. Cells were collected and lysed with TCL buffer (Qiagen) with 1% of β-mercaptoethanol and stored at −80 °C until subsequent analysis. RNA was isolated using a Single Cell RNA purification kit (Norgen), including RNase-Free DNase Set (Qiagen) treatment. The RNA integrity number was evaluated with an Agilent RNA 6000 pico kit. All samples were assessed according to the manufacturer’s instructions.

Cells were washed with PBS and incubated with antibodies diluted in PBS/10% HS/0.01% NaN3 (sodium azide) for 30 min on ice. Surface antibodies were anti-CD4 PerCp (clone SK3), CD3 FITC (clone UCHT1), and CD25 PE (clone M-A251, all BD Biosciences). Intracellular staining with anti-Foxp3 APC (clone PCH101, eBiosciences) was performed using the eBioscience fixation/permeabilization buffer kit. A minimum of 10⁵ events in the lymphocyte gate was acquired using a FACScalibur flow cytometer for 4-color analysis and analyzed using WEASEL software (WEHI, Melbourne, VIC, Australia). Cells were gated first based on forward and side scatter to excluded dead cells and cell debris. T cells in the lymphocyte gate were identified based on CD3 expression, further sub-gated on CD4+ T cells (Figure 1A) and CD25+ cells then subdivided into Foxp3hi and Foxp3int cells (Figure 1B).

Nanfang Hospital of Guangzhou approved this study, and the experiment was performed in accordance with relevant guidelines and regulations. All donors gave written informed consent. Latently infected CD4⁺ T cell experiments were performed as described previously by Lewin’s group (27 (link)), with minor modifications. Briefly, PBMCs from a single healthy donor were isolated from buffy coats, and primary CD4⁺ T cells were isolated from the PBMCs by using an EasySep negative selection kit (StemCell Technologies, Inc., Vancouver, BC, Canada) according to the manufacturer’s instructions. After purification, the cells were maintained in complete RPMI 1640 medium supplemented with 29 nM CCL19 (R&D Systems) for 3 days. The cells were washed and infected with the HIV-1_NL4-3 virus (100 ng of p24 viral equivalents per million cells) by spinoculation for 2 h at 1,000 × g. After infection, the cells were washed three times and cultured in the presence of interleukin-2 (IL-2; 10 IU/ml; Roche) for 6 days. Next, resting CD4⁺ T cells were isolated with a positive CD4⁺ T-cell selection kit (StemCell Technologies, Inc., Vancouver, BC, Canada) using a cocktail of antibodies against CD25-PE (clone M-A251; BD Pharmingen), CD69-PE (BD Pharmingen, clone FN50), and HLA-DR-PE (clone G46-6; BD Pharmingen).