Cd4 pe cf594 clone rpa t4

Staining of unfractionated fresh blood was performed according to the lyse and stain approach using monoclonal antibodies: CD45 FITC (clone 2D1), CD4 PE/CF594 (clone RPA-T4, BD Biosciences, San Jose, CA, USA), CD3 PE-Cy7 (clone UCHT1), CD8 BV510 (clone SK1), CD16 PE (clone B73.1), HLADR BV786 (clone L243, BioLegend, San Diego, CA, USA) and the results read in the Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Living cells (stained with LIVE/DEAD Fixable Dead Cell Stain Kit with BV421 fluorochrome, Life Technologies Carlsbad, CA, USA) were evaluated. The gating was done using both CD45 and side scatter signals. NovoExpress Software (Agilent Santa Clara, CA, USA) was used for subpopulation analysis.

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.

CD4+, CD25+, and CD4+ CD25− T lymphocytes were first isolated using the CD4+ CD25+ regulatory T-cell isolation kit (Miltenyi Biotec, 130-091-301) and then stained with CD25−PE (clone M-A251; BD Biosciences) and CD4-PE-CF594 (clone RPA-T4; BD Biosciences). CD4+ CD25− (Tconv) and CD4+ CD25high (Treg) cells were sorted by flow cytometry using a BD FACS ARIA II cell sorter. Tconvs were stained with CFSE (5 µm) (Life Technologies) and co-cultured with autologous Tregs at varying concentrations in a 96-well round bottom plate (2.5 × 10⁴ Tconvs per well) in the presence of Dynalbeads CD3/CD28 T-cell expander (Life Technologies) at a ratio of 10 cells/bead. Cells were incubated at 37 °C for 4 days in Roswell Park Memorial Institute 1640 Medium (Life Technologies) supplemented with 10% AB-human-serum and 1% penicillin–streptomycin, and were then analyzed using flow cytometry (BD LSR-Fortessa). Proliferation index was calculated with FlowJo Proliferation Tool (TreeStar Inc.).