Ethylene diamine tetraacetic acid (edta)

Transduced T cells were sorted using the FACSAria II (BD Biosciences). Before sorting, cells were labeled with the Live/Dead near-IR fixable dye and resuspended in sterile PBS, supplemented with 2% BSA and 2 mM EDTA (Sigma, Gillingham, UK). The gating strategy was based on forward scatter (FSC) and side scatter (SSC), exclusion of debris and aggregates, and Live/Dead dye exclusion. ZsGreen⁺ cells were then sorted in sterile PBS, supplemented with 2% BSA and 2 mM EDTA, and cultured in X-Vivo 15 (Lonza), supplemented with 5% human serum (Seralabs) and 500 U/mL of Proleukin (Novartis) at a cell density of 1 × 10⁶ cells/mL for 16–24 h before being cryopreserved in CryoStor CS10 (Sigma) until use. Before processing the cells in the C1 Single-Cell Auto Prep system, the cells were thawed and incubated for 12–16 h at 37°C and 5% CO₂ in complete medium.

Tumors and tumor-draining LN were dissected from mice, mechanically disrupted and digested with 250 µg/mL collagenase D (Roche, Basel, Switzerland) and 300 µg/mL DNase I (Roche) in Hank’s Salt Solution (w/o Mg2⁺, Ca2⁺, PAN-Biotech) supplemented with 2% FCS. Tumors were digested for 45 min at 37°C and tumor-draining LN were digested for 25 min at 37°C. Digestion was stopped with 5 mM EDTA (Lonza, Basel, Switzerland) and tissue pieces were pressed through a 100 µm cell strainer (Corning, New York, USA) with a 2 mL syringe plunger (BD Biosciences, Franklin Lakes, New Jersey, USA) to obtain single-cell suspensions for flow cytometry analysis. Tumor-draining LN single-cell suspensions for the assessment of T-cell responses were generated by passing the tissue through a 100 µm cell strainer without prior digestion.

For adoptive cell transfer of OVA-specific OT-I and OT-II cells, CD8⁺ T cells were isolated from CD45.1⁺ OT-I T-cell receptor (TCR) transgenic mice79 (link) and CD4⁺ T cells from CD45.1⁺ OT-II TCR transgenic mice.80 (link) LN and spleen were harvested and passed through a 100 µm cell strainer (Corning) to obtain single cell suspensions. Red blood cells were lysed using ammonium chloride lysis buffer (1.68 mM NH₄Cl (Roth, Karlsruhe, Germany), 1 mM KHCO₃ (Roth), and 0.1 mM EDTA (Lonza)) for 3 min at RT. CD8⁺ T cells were isolated by using anti-mouse-CD8-α (Ly-2) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4⁺ T cells by using anti-mouse-CD4 (L3T4) MicroBeads (Miltenyi Biotec) according to the manufacturers’ instructions. For in vivo T-cell proliferation assays, isolated CD8⁺ T cells and CD4⁺ T cells were labeled with 0.4 µM CellTrace Violet (Thermo Fisher Scientific) in PBS for 3 min at RT and injected intravenously (1×10⁶ cells/mouse) into CD45.2⁺ D4M-OVA bearing recipient mice. Tumor-draining LN of recipient mice were harvested and CD8⁺ T cell and CD4⁺ T-cell proliferation and activation was analyzed by flow cytometry, 3 days and 5 days after the adoptive cell transfer, respectively.

Flow cytometry was performed on MSCs (s.c.ASC, o.ASC, and BMSC) at passage 3. The cells were trypsinized, subsequently centrifuged at 1,400 rpm for 5 min, and washed with PBS containing 0.5% bovine serum album (Sigma-Aldrich) and 0.5 M EDTA (Lonza). The number of cells was determined by hemocytometer. A total of 2 × 10⁶ cells were incubated with different fluorochrome-conjugated anti human monoclonal antibodies for 30 min. The following CD surface markers were tested: CD45/APC-Cy7, CD90/APC, CD105/FITC, CD73/PE, CD235a/PE-Cy5, CD34/AF700 (BD Biosciences, San Jose, CA, USA), CD31/PE-Cy7 (BioLegend), CD33/PC5, CD14/PC5 (Beckman Coulter, Brea, CA, USA), and CD146/VioBlue (Miltenyi Biotec). For each antigen 10,000 events were collected on an LSRII flow cytometer (Becton Dickinson). Analysis was conducted using Flow Jo software (Tree Star).

Collected intestinal tissue (ileum) was incubated for 30 minutes at 37°C in PRMI-1640 (Sigma) complete medium containing collagenase V from Clostridium histolyticum (Sigma, St. Louis, MO) at the concentration of 0.1 mg/mL followed by vigorously vortexing for 1 min, single-cell suspensions were obtained by filtration through 40-μm cell strainers (BD Biosciences, San Jose, CA). Cells were washed with MACS buffer consisting of phosphate-buffered saline, 0.5% bovine serum albumin (Hyclone Laboratories), and 2 mM EDTA (Lonza), and finally re-suspended in MACS buffer performing surface and intracellular staining using specific antibodies for further flow cytometric analysis.