Cd3 pe cy5

Expression of EGFR and other proteins on the tumor cell surface was detected by flow cytometry using the anti-EGFR antibody (BD, San Jose, CA, USA). In brief, the CAR T cells were collected from culture and assayed with monoclonal antibodies against human CD25-PC5 and CD69-PC5 (Beckman Coulter Inc., Indianapolis, IN, USA), CD3-PE-CY5, CD4-PE, CD8-FITC, CD45RO-PE-CY5, CD62L-PE, TIM3-PE, and LAG3-Alexa Fluor 647 (Biolegend, San Diego, CA, USA) and CCR7-FITC, CD107α-PE-CY5, and PD-1-PE (BD, San Jose, CA, USA) according to a previous study^{40 (link)} or the manufacturers’ instructions.
EGFR-modified CAR expression was detected by using an indirect method with biotinylated EGFR protein and streptavidin-coupled PE antibody (BD). Fluorescence was assessed using a Beckman Coulter Gallios™ flow cytometer, and the data were analyzed with the FlowJo vX.0.7 and Kaluza v1.5 software (Beckman Coulter Inc.).

For functional assays on cytokine production by T cells, thawed isolated PBMCs were stimulated for 16 h at 37 °C in a 5% CO2 atmosphere with anti-CD3/CD28 (1 μg/mL) in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, and 55 μM β-mercaptoethanol). For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein-transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson) and previously titrated concentration of CD107a-PE. After stimulation, cells were stained with LIVE-DEAD Aqua (ThermoFisher Scientific) and surface mAbs recognizing CD4 AF700, and CD8 APC-Cy7 (Biolegend, San Diego, CA, USA). Cells were washed with stain buffer and fixed and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Cells were next stained with previously titrated mAbs recognizing CD3 PE-Cy5, IL-17 BV421, TNF BV605, IFN-γ FITC, IL-4 APC, or granzyme-B BV421 (all mAbs from Biolegend). Then, a minimum of 100,000 cells per sample were acquired on a Attune NxT acoustic cytometer (ThermoFisher)^{53 (link)}. FCS data were acquired in list mode by Attune nxT Software v4.2. mAbs used are listed in Sup Data 6.

Peripheral blood samples were collected from matched patients with MIBC before RC and processed for the analysis of leukocytes. Briefly, 100 µL of blood were washed with 2 mL of PBS. The pellet was stained with a panel of antibodies to analyze leukocyte subpopulations: CD45 Vioblue (Miltenyi Biotec), CD3-PeCy5, CD8-PCy7, and PD-L1-APC and PD-1-PE (BioLegend). We used CD3 and CD8 antibodies to discriminate between CD4+ (CD3 + CD8−), CD8+ (CD3 + CD8+), CD3− CD8+, and CD3− CD8−. The expression of PD-L1 and PD-1 was determined in all cell population. The expression of PD-L1 and PD-1 was determined based on the limit of detection delimitated by FMO. Samples were acquired and analyzed with the MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec).