Cd8 clone rpa t8

Using lymphocyte separation medium (Corning) density gradient centrifugation, we isolated peripheral blood mononuclear cells (PBMCs). Staining of CX3C chemokine receptor 1 (CX3CR1) in T cells was performed as described before [23 (link), 24 (link)]. In brief, fresh or cryopreserved PBMCs were incubated with Fc block with human immunoglobulin G (IgG) (Sigma) at 12 mg/mL for 20 min. Anti-human CD3 (clone UCHT1), CD8 (clone RPA-T8), and CX3CR1 (clone 2A9-1) antibodies were obtained from BioLegend, and anti-CD4 (clone RPA-T4) antibody was from BD Biosciences for flow cytometry. LSRFortessa (BD) was used for sample acquisition, and FlowJo software v10.1.5 (FlowJo LLC) was used for sample analysis.

The day prior to the assay, 3×10⁴ JEG-3 cells were labeled with 1 µM of CFSE (CellTrace, Thermo Fisher) and seeded in flat-bottom 96-well microplates. On the day of the assay, CAR-T cells were added at various E:T ratios to either the plated CFSE-labeled JEG-3 cells or 3×10⁴ CFSE-labeled K562/K562-HLA-G1 cells. After 24 hours incubation, medium was collected and cells recovered, washed and labeled with antibodies against CD4 (clone RPA-T4, Biolegend), CD8 (clone RPA-T8, Biolegend), CD19 (HIB-19, Biolegend), CD25 (clone M-A251, BD Bioscience), CD69 (clone FN-50, BD Bioscience), PD-1 (EH12.2h7, Biolegend) and a viability dye (Invitrogen). For degranulation assays, co-cultures were set-up at an E:T ratio of 10:1. Anti-CD107a (clone eBioH4A3, Biolegend) was added at the start of the experiment, GolgiStop (BD Bioscience) was added after 1 hour. Five hours after the beginning of the assay, cells were collected and labeled with antibodies directed against CD4, CD8, CD19 and a viability dye. Acquisition was performed with a fluorescence-activated cell sorting (FACS) Attune (Thermo Fisher), and results were analyzed with FlowJo software.

Co-cultures were set up with CAR-T cells and K562-HLA-G1 cells at an E:T ratio of 10:1 with no IL-2. Twenty-four hours after stimulation, 50 µL of medium were taken, stored for cytokine secretion analysis and replaced by complete RPMI. Seventy-two hours after stimulation, half of the medium was changed with complete RPMI supplemented with 50 U/mL of IL-2. Cells were maintained at 10⁶/mL. Twelve days after stimulation, some cells were used for flow cytometry analysis, whereas the rest was used for a new round of stimulation. A total of three consecutive stimulations were performed. For flow cytometry analysis, cells were labeled with antibodies directed against CD4 (clone RPA-T4, Biolegend), CD8 (clone RPA-T8, Biolegend), CD19 (HIB-19, Biolegend), CD62L (clone DREG-56, BD Bioscience) and CD45RA (clone HI-100, Invitrogen). In these experiments, the phenotype of CAR-T cells and activated NT autologous controls was also established 24 hours prior to the assay. At the end of the experiment, all recovered medium was analyzed for IFNγ, TNFα and IL-2 secretion using a cytometric bead array (CBA) kit (BD Biosciences).

The CAR T cells were suspended with pre-warmed R10 medium containing brefeldin A (20 μL/mL, Sigma-Aldrich) and GolgiStop (1.4 μL/mL, BD). The target cells were added into the wells to stimulate the CAR T cells for 6 hr in the incubator. After stimulation, the cells were stained with the viability dye followed by surface staining with CD3 (Clone OKT3), CD4 (Clone OKT4), and CD8 (Clone RPA-T8) antibodies (BioLegend, San Diego, CA, USA). After washing with PBS, the cells were permeabilized with Cytofix/Ctyoperm buffer (BD Biosciences) for 20 min at room temperature and then washed with 1 × BD Perm/Wash buffer twice. Next, the cells were stained with the intracellular antibodies, including TNF-α (Clone MAb11) and IFN-γ (Clone B27; both BioLegend).