Cd45ra pecy7 clone hi 100

Freshly isolated PBMCs (1 × 10⁵) from Omicron COVID-19 cases (n = 19) were used for surface staining of NK cells, NKT-like cells, B cells, T cells, memory B, and memory T cells using cocktail of antihuman antibodies (CD19 PercpCy5.5 (clone HB-19), CD27 PECy7 (clone M-T271), CD3 APCH7 (clone SK7), CD4 BV480 (clone SK3), CD8 FITC (clone RPA-T8), CD45RA PECy7 (clone HI 100), CD62L APC (clone DREG-56), CCR7 PE (clone 2-LI-A) and NK Tritest (CD3 FITC: clone SK7, CD56 PE:clone NCAM 16.2, CD16 PE: clone B73.1, CD 45 RA: C8Mab-1)) all from BD Biosciences following a previously described protocol [18 (link)–23 (link)]. PE-Cy™7 Mouse IgG1 κ Isotype Control (BD Biosciences, San Jose, CA, USA) was used as the negative control.
Briefly, PBMCs were incubated with fluorochrome-tagged antihuman monoclonal antibodies for 30 min in the dark. After washing, the cells were fixed with 2% paraformaldehyde. The cells were acquired on FACS Aria II (BD Biosciences, San Jose, CA, USA). For each experiment, 50,000 events were acquired within the lymphocyte gate with appropriate isotype control. Data were analyzed using FACS Diva software (Becton Dickinson, San Jose, CA, USA), and results are expressed as the percentage of positive cells in the gated population. The gating strategy is depicted in Figure 1.

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.