Macsibead particles

Jurkat E6.1 cells, 2.5 × 10⁵ cells/well were seeded out in six-well plates and transfected with ANC or syn-hsa-miR-34a-5p miScript miRNA Mimics (QIAGEN N.V.) as described for protein extraction. Twenty-four hours after transfection, cells were collected for activation. The transfected cells were transferred to a 48-well plate and seeded out to a count of 3.5 × 10⁵ cells/well in a volume of 350 µl fresh medium, supplemented with MACSiBead™ Particles (Bead:Cell ratio 1:2) from human T Cell Activation/Expansion Kit (Miltenyi Biotec GmbH) and 5 ng/ml of PMA (Sigma Aldrich) was added. Incubation time was additional 24 h before supernatants were collected and total cell count was estimated. IL-2 quantification was performed following the protocol of Human IL-2 DuoSet® ELISA of R&D Systems, Inc. (Minneapolis, Minnesota, USA).

Transduced CD4+ T cells were irradiated (3000 rad) using a X-Ray irradiator Cabinet and added at a 1∶6 transduced:responder ratio to untransduced CD8+ T cells (2×10⁶ cells/ml) from the same donor (kept frozen while CD4+ T cells were blasted and transduced). Cells were stimulated with loaded antibiotin MACSiBead Particles (human T-cell activation/expansion kit; Macs Miltenyi Biotec) at a 1∶1 bead-to-cell ratio and 5 µM EdU pulses were added at times 0 h, 24 h, 48 h and 72 h (a non EdU treated control was included as a negative staining control). At 96 h, cells were collected, stained with anti-CD8, and proliferation analyzed using Click-iT™ EdU Flow Cytometry Assay Kits (Life Technologies™) [65] (link), following manufacturer recommendation.

CRLM and liver suspensions were resuspended with 5 mL of PBS and overlayed on a Ficoll gradient (GE Healthcare, Uppsala, Sweden). After 20 min of centrifugation at 870× g without brake and with the lowest acceleration, the interphase enriched in leukocytes was collected, washed and labeled with 1μM Carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, USA) in 1 × 10⁶ cells/mL concentration for 7 min at +37 °C. Labeling was quenched with 1mL of FCS, followed by a PBS wash. CFSE-labeled cells were resuspended to 1 × 10⁶ cells/mL with complete media and cultured for 4 days with and without 2 μg of pooled peptides based on antigens from Cytomegalovirus, Epstein–Barr virus, Flu (influenza) virus, plus Tetanus toxoid (PepMix CEFT pool) (JPT Peptide Technologies, Berlin, Germany), or 2 μL of MACSiBead particles loaded with anti- CD2, CD3 and CD28 antibodies (Miltenyi Biotec, Auburn, CA, USA). Proliferating T cells, indicated by their dilution of CFSE dye, were assessed by flow cytometry.

Isolated MDSC-like cells and autologous CD8⁺ T cells were cocultured at the indicated ratios for 5 days in a U-bottom plate in complete RPMI. T cells were stimulated by the addition of 100 IU/mL IL-2 (proleukin) and anti-CD3/CD28 stimulation using loaded MACSiBead particles (Miltenyi Biotec) at a ratio of 1:1 beads to cells. Unstimulated T cells and stimulated T cells without MDSC addition were used as controls. After five days, the supernatants were frozen at −80 °C, and the cells were stained for flow cytometry.
For Siglec-9 blocking, a Siglec-9 blocking antibody (Clone 191240, R&D Systems) was added at a final concentration of 10 µg/mL. For sialidase treatment, MDSCs were pretreated and washed before being added to the wells as described below.

MC38 cells (5 × 10⁵) were injected subcutaneously into the right flank of mice and left to establish for one week with no further intervention. Then, splenic CD8⁺ T cells were isolated from tumor-bearing mice via magnetic cell sorting with negative selection (CD8a⁺ T cell isolation kit, Miltenyi Biotec). CD8⁺ T cells were activated using MACSiBead particles loaded with monoclonal anti-CD3e and CD28 antibodies (Miltenyi Biotec) in RPMI-1640 medium supplemented with human recombinant IL-2 (1000 U/mL), 2-mercaptoethanol (50 μM), and sodium pyruvate (1 mM) and cultured for 2 days. MC38 cells were preincubated with 100 nM rPAI-1 overnight and stained with PKH67 (Sigma Aldrich). Subsequently, 1 × 10⁴ MC38 cells (target) were co-cultured with the activated CD8⁺ T cells (effector) at E:T ratios of 6.13:1 to 50:1 for 4 h. The PKH67⁺ MC38 cells were stained with PE/Cy7-conjugated anti-Annexin V antibody (BioLegend) and analyzed via flow cytometry. The cytotoxicity proportion was calculated as follows:
where ET represents the percentage of PKH67⁺ Annexin V⁺ cells during the effector and target cell co-culture and T0 represents the percentage of PKH⁺ Annexin V⁺ cells in the target cell alone culture.

Treg cells with a purity of more than 98% CD4⁺CD25^highCD62L⁺ from wild-type as well as TNFR2^−/− mice were cultured in the presence of anti-CD3ε and anti-CD28 antibodies (MACSiBead particles, Miltenyi Biotec) and recombinant human IL-2 (Proleukin S, Novartis Pharma, Basel, Switzerland) with or without TNCscTNF80 according to the instructions of the manufacturer of the Treg cell expansion kit (mouse, Miltenyi Biotec) for 7 days.