Tim 3 bv421

The generated short-term T cell lines were stimulated with mixed TAAs for 4 hours and cells without stimulation were used as negative controls. Then, cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) and surface markers, including CD3-AF700 (Bio Legend), CD4-FITC (BD Biosciences), CD8-APC-H7 (BD Biosciences), PD1-BV650 (BD Biosciences), and Tim3-BV421 (Bio Legend), fixed with 1 × CellFix solution (BD Biosciences) and acquired immediately on a BD LSR Fortessa. Fluorescence minus one (FMO) controls were applied accordingly in order to properly position gates.
In the validation cohort, 60 samples from 26 HCC patients were thawed and rested overnight. These cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) and surface markers, including CD3-BV786 (Bio Legend), CD4-BV711 (Bio Legend), CD8-Percp-cy5.5 (BD Biosciences), and CD39-PE-CF594 (Bio Legend), fixed with 1 × CellFix solution (BD Biosciences), and acquired immediately on a BD LSR Fortessa. Flow data were analyzed by FlowJo V.10.0.

We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4⁺, CD8⁺, and CD19⁺. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 10⁶ events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo^® software.

To analyze the content of immunomodulatory proteins on the surface of SEVs, a spectral flow cytometer with an enhanced small particle detection option (Cytek® Northern Lights™) at the FlowCore Mannheim was used. SEVs were diluted (1:20 in 100 µL PBS) and stained with 1 µL fluorescently-labeled antibodies for tetraspanins (CD9-, CD63-, CD81-FITC; BioLegend) and immunomodulatory proteins (PD-L1-PE, CTLA-4-APC, FasL-PE, TGF-β-APC, Tim3-BV421, LAG-3-BV421; BioLegend) for 30 min at 4 °C. Afterwards EVs were diluted 1:4 in PBS and 50 µL of sample was acquired. As negative controls, PBS only and PBS with antibodies were used. To prove that the signal was related to SEVs, we also disrupted the lipid bilayer of the vesicles using 0.5% sodium dodecyl sulfate (SDS). Next, EVs were gated according to ApogeeMix beads (#1527) to exclude particles or aggregates that are larger than 500 nm polystyrene beads (Suppl. Figure 2). Additionally, isotype and SDS controls were examined to exclude unspecific binding of the antibodies. The count of positive events for immunomodulatory proteins in the EV marker-positive population was calculated using FlowJo and normalized to the volume of 50 µL.

Cells were incubated with 1% BSA in PBS for 10 min, centrifuged and stained for 15 min with the following antibodies: CD3-FITC (cat no 300406), CD4-APC/Cy7 (cat no 300518), CD8-PE/Cy7 (cat no 301012), CD19-PerCP (cat no 302228), CCR7-BV421 (cat no 353208), CD45RA-APC/Cy7 (cat no 304128), CD27-PE (cat no 302808), CD28-PE/Cy7 (cat no 302926), Tim-3-BV421 (cat no 345007), PD-1-PE (cat no 329906) all purchased from Biolegend, α-CAR-DyLight649 AffiniPure F(ab')₂ Fragment Goat Anti-Human IgG (H+L) purchased from Jackson ImmunoResearch Europe LTD (cat no 109-496-088), or negative isotype control antibodies (Biolegend). Cells were washed with PBS. Analyzed on BD Canto II (BD Biosciences, San Jose, CA) and evaluated with FlowJo (Treestar, Ashland, OR, USA). The transduction efficiency of 3G CAR was lower than that of 2G CAR. Therefore, a correction factor was calculated based on the CAR expression of the 2G and 3G T cells from the same donor at the same time point. This correction factor was then used to normalize the values of the functional assays.