Cd28 cd28.2

Functional analysis was also performed on T4 retention samples described in the previous paragraph. Thawed CAR T-cells were resuspended at 1 million/mL in RPMI 1640 medium (ThermoFisher) supplemented with 10 mM glucose, 0.1 mM non-essential amino acids, 10 mM HEPES buffer, 1 mM sodium-pyruvate and 10% fetal calf serum (FCS; all Sigma-Aldrich). Brefeldin A (1 µg/mL; BioLegend) was added to block export of effector proteins. Cells were either rested or activated with plate bound CD3 (OKT3 1 µg/mL) and CD28 (CD28.2, 0.5 µg/mL) antibodies (all BioLegend) in a 96 well plate in an incubator overnight at 37°C, 5% CO_2. After washing in 1×PBS, cells were surface stained as before with: biotinylated anti-hEGF antibody (R&D systems, code BAF236), Streptavidin-BV510, CD4-APC-Cy7, CD8-Alexa Fluor 700, and live/dead dye. After fixation with Cytofix (BD Biosciences) for 20 min at 4°C, cells were stained with Granzyme-B-AF488 (GRZB; BioLegend), tumor necrosis factor (TNF)-α-APC and IFN-γ-BV421 (all BD Biosciences) in permeabilization buffer (1% FCS, 0.1% saponin in 1×PBS) for 30 min.

Cells were washed with fluorescent-activated cell sorting (FACS) buffer (2% FBS in PBS) twice, and were then stained with antibodies at 4 °C for 30 min. Data were acquired using a FACS Calibur or LSRfortessa (BD Biosciences) and analyzed with a FlowJo software (Tree Star Inc., Ashland, OR). Median fluorescence intensity (MFI) data were shown after the subtraction of corresponding isotype control values. Antibodies used in flow cytometry as follows: CD11c (B-ly6), CD80 (L307.4), CD83 (HB15e), CD86 (IT2.2), and HLA-DR (TU36) were purchased from BD Biosciences, CCR7 (3D12) and Rat IgG2a were purchased from eBioscience, and CD3 (HIT3a) and CD28 (CD28.2) were purchased from BioLegend.

Peripheral blood mononuclear cells were incubated with antibodies against CD3 (UCHT1) and CD28 (CD28.2) (all from Biolegend) to stimulate T cells, CD16 (3G8) (Biolegend) to stimulate NK cells, or F(ab′)2 anti-human IgM + IgG (eBioscience) to stimulate B cells in the presence of DMSO or inhibitors for 30 min on ice. Cells were washed and incubated with secondary Goat anti-mouse antibodies for 15 min, followed by stimulation for 2 min at 37°C. Cells were then fixed by BD Phosflow Fixation buffer and stained for extracellular markers with CD19-AF700 (HIB19), CD56-BV510 (NCAM16.2) and CD3-PerCP (UCHT1) (all from Biolegend). Cells were then permeabilized by BD Perm 2, stained with p-SLP-76 Y128 AF647 (J141-668.36.58) or p-SLP-65 Y84 AF647 (J117-1278) (both from BD) and analyzed by flow cytometry on a BD LSRFortessa.

Blood samples were collected at the indicated time points, PBMCs isolated through the research cell bank at FHCRC by standard Ficoll-Hypaque gradient, and viably cryopreserved. Cells were analyzed by flow cytometry after permeabilization, fixation and staining with fluorochrome-conjugated monoclonal antibodies to CD4 (SK3; Becton Dickinson), CD8 (3B5; Invitrogen), CD19 (H1B19; Becton Dickinson), CD16 (3G8; Becton Dickinson), CCR7 (G043H7; Biolegend), CD45RO (UCHL1; Becton Dickinson), CD28 (CD28.2; Biolegend), KLRG1 (SA231A2; Biolegend), CD27 (L128; Biolegend), CXCR3 (G025H7; Biolegend), CD127 (A019D5; Biolegend), PD1 (ED12.2H7; Biolegend), Lag3 (2DS223H; eBioscience), 4-1BB (4B4-1; Biolegend), BCL2 (100; Biolegend), CTLA-4 (L3D10) and the above described tetramers. Cells were analyzed on a Fortessa cytometer (Becton Dickinson) and data analysis performed with FlowJo. Staining, acquisition and analyses were performed on all samples in a batch on same day and negative controls included.