7 aad

Cells (100,000/well in 12-well plate) were treated with DMSO or tigecycline, or Z-VAD-fmk (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone, Selleckchem, US). After 3 days treatment, apoptotic cells were labelled by using Annexin V-FITC and 7-AAD (BD Pharmingen, US) staining according to manufacture’s instructions. The percentage of apoptotic cells (Annexin V+/7-AAD- and Annexin V+/7-AAD+) was determined by using flow cytometry analysis (Beckman Coulter FC500).

Analysis of apoptosis was performed as described previously^{37 (link)}. Apoptotic cells were measured by Annexin V staining using an APC Annexin V apoptosis detection kit with 7-AAD according to the manufacturer's (BioLegend, San Diego, CA, USA) instructions. Annexin V positive but 7-AAD negative (early apoptotic cells) and Annexin V positive and 7-AAD positive (late-stage apoptosis) cells were determined using a Gallios flow cytometer and analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA).

The specificity of CA9 DATE for GBM cells, ccRCC cells and T cells were tested using flow cytometry analysis. CA9^hi GBMs, CA9^- GBMs, ccRCCs and T cells (isolated from human PBMCs for GBM study and Jurkat cells for ccRCC study) were resuspended in PBS plus 2 mM EDTA and were stained with CA9 DATEs followed by the secondary antibody, goat anti -human APC-Fab IgG (1:2000, Jackson ImmunoResearch, Cat#109-136-170) staining. GBM and ccRCC cells were incubated for 15 minutes at room temperature and for 20 minutes on ice, respectively followed by 15 minutes incubation at room temperature for the secondary antibody staining. Dead cells were excluded using the viability dye 7AAD (1:10; Beckman Coulter, Cat#A07704) and samples were run on MoFlo XDP Cell Sorter (Beckman Coulter) to assess the level of CA9 DATE binding to each of the above-mentioned lines.

GBM Tumorspheres were dissociated using 0.2 Wünsch unit/mL Liberase Blendzyme 3 (Millipore Sigma, Cat#5401119001) plus 10 μL DNase (Worthington Biochemical, Cat#LK003170) and adherent cultures were dissociated using dissociation enzyme TrypLE (ThermoFisher, Cat#12605028). The single cells were resuspended in PBS+2 mM EDTA (Invitrogen, Cat# AM9260G). Cells were then stained with APC conjugated mouse monoclonal human Carbonic Anhydrase 9 antibody (1:10) (R&D, Cat#FAB2188A) or a matched isotype control and CA9 DATEs followed by goat anti human APC-Fab IgG (1:2000, Jackson ImmunoResearch, Cat#109-136-170) and incubated for 15 minutes at room temperature. T cells were stained with CA9 DATEs (15 minutes RT) followed by goat anti human APC-Fab IgG (1:2000, Jackson ImmunoResearch, Cat#109-136-170), anti-CD25 (Miltenyi Biotech, Cat#130-113-283) and anti-CD69 (BD Bioscienecs, Cat#555533). Samples were run on a MoFlo XDP Cell Sorter (Beckman Coulter). Dead cells were excluded using the viability dye 7AAD (1:10; Beckman Coulter, Cat#A07704). Compensation was performed using mouse IgG CompBeads (BD Biosciences, Cat#552843). Samples were run on a MoFlo XDP Cell Sorter (Beckman Coulter) to assess the level of CA9 surface expression.

A six-colour flow cytometry panel was designed to specifically analyse the proliferation of T cells. Therefore, cells were pipetted through a 40 µm cell strainer (Cat# 43-10040-70, pluriSelect, Leipzig, Germany) and stained with CD45 FITC (Cat# A07782, Beckman Coulter, Krefeld, Germany), 7-AAD (Cat# A07704, Beckman Coulter, Krefeld, Germany), CD4 PC7 (Cat# 737660, Beckman Coulter, Krefeld, Germany), CD8 PC7 (Cat# 737661, Beckman Coulter, Krefeld, Germany) and CD5 APC (Cat# 345783, BD Biosciences, Heidelberg, Germany). For PBMCs freshly isolated on day 0, cells were incubated with 1 mL of 1× NH₄Cl-based erythrocyte lysing solution (contained in Cat# IM3630d, Beckman Coulter) for 10 min at room temperature. Analysis was performed using the FACSLyric Flow Cytometer and the FACSuite software 1.4 (BD Biosciences, Heidelberg, Germany).
In order to determine the extent of T cell proliferation, a gating strategy was established for CD45⁺/7AAD⁻/CD5⁺/CD4⁺ or CD8⁺ singlet cells. Those cells are further referred to as T cells. Then, the proportion of T cells that had divided at least once (fraction diluted, Dil as described by Roederer et al. [31 (link)]) was determined and the number of daughter generations arising from the initial population was counted. Mean values for each triplicate were calculated to determine the inhibition by MSCs using the following formula:

PBMCs were collected from patients A2 and D3 vaccinated with hTERT and AFP-derived peptides, respectively. The collected PBMCs were cultured in the presence of 10 μg/mL hTERT₄₆₁ or AFP₃₅₇ peptide, 10 ng/mL recombinant interleukin (rIL)−7, and 100 pg/mL rIL-12 (PeproTech, Inc., Rocky Hill, NJ, USA) in a 96-well U-bottom plate. On days 3, 10, and 17, half of the culture medium was replaced with 350 U/mL rIL-2 (PeproTech, Inc.)-containing medium. On days 7 and 14, half of the culture was replaced with medium containing 350 U/mL rIL-2, 20 μg/mL peptide, and mitomycin-C-treated autologous PBMCs. After 3 weeks of in vitro culture, the generated CTLs were stained with phycoerythrin-conjugated peptide MHC tetramers (MBL Co., Ltd., Aichi, Japan) after Fc receptor blocking (Clear Back; MBL Co., Ltd.), followed by a fluorescein isothiocyanate-conjugated anti-CD8α antibody (MBL Co., Ltd.) and 7-AAD (Beckman Coulter, Inc., Indianapolis, IN, USA) staining. Ag-specific CD8⁺ T cells were detected and collected as single cells using a FACSAria II (BD Bioscience, San Diego, CA, USA). cDNAs of TCR α and β chains were amplified by the 5ʹ rapid amplification of cDNA end method and reverse transcription-polymerase chain reaction from single cells. cDNAs were analyzed with repertoires after sequence analyses and confirmation of Ag specificities using an hTEC10 system as described previously¹⁴ .