Cd8 magnetic beads

CD8⁺ T cells were isolated from whole blood using CD8⁺ magnetic beads (Miltenyi) and a QuadroMACS^TM separator (Miltenyi). In total, 5 × 10⁶ cells/mL were stained with the CellTrace^TM violet proliferation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Next, anti-CD3 and anti-CD28 T cell activation beads were added to the isolated T cells before placing them in a round-bottom 96-well plate. A total of 5 × 10⁵ T cells was pipetted into each well. After 24 h of activation, the same number of autologous monocytes was added to the T lymphocytes. Prior to addition, monocytes were treated with conditioned media of untreated PBMCs or ATG-treated PBMCs for 24 h and subsequently washed twice. In addition, we incubated PDL-1⁺ monocytes for 1 h with durvalumab (Imfinzi; MedImmune, AstraZeneca, Cambridge, UK), a monoclonal antibody directed against PDL-1, to assess its contribution to the inhibition of CD8⁺ T cell proliferation. Experiments were performed with four independent donors. After 4 days of co-culture, cells were collected and stained for CD8-PECy5 (BioLegend) and assessed for cell proliferation with flow cytometry. Conditioned media of co-cultured cells were collected and stored at −20 °C until further use.

Isolation of CD8⁺ T cells from HD samples was performed with CD8 magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer’s instructions. Freshly isolated CD8⁺ T cells, anti-CXCR3 (10 ng/mL, Abcam), or Stattic (10 nM; S7024; Selleck Chem, Houston, TX)-treated CD8⁺ T cells as well as peripheral blood mononuclear cells (PBMCs) from HDs were seeded on the upper chambers of a 5.0-μm pore Transwell. Supernatants of tumor tissue, anti-CXCL10 (100 ng/mL, Abcam), or recombinant human (rh) CXCL10 (Peprotech, Rocky Hill, NJ) were added to the lower chambers. CD8⁺ T cells in the upper chambers were pretreated with rhIL-17A (20 ng/mL), Stattic (10 nM), and/or the supernatants of enriched Th17 cells (5 × 10⁶) with or without anti-IL-17A (10 ng/mL) for 48 h in vitro. Migrated cells in the lower chambers were then counted and analyzed by flow cytometry after 12 h.

Peripheral blood mononuclear cells (PBMCs) from healthy human donors were isolated using Lymphoprep density gradient centrifugation (Stemcell, 07851). PBMCs were cultured in six-well plates at a density of 3 × 10⁶ per well with H1734 or H1975 cell lysate (0.5 mg/ml) and IL-2 (20 ng/ml) for 72 h to promote T cell activation. Stimulated PBMCs were then harvested and purified by Lymphoprep density gradient centrifugation, and co-cultured with the H1734 or H1975 cells at a 10:1 ratio for 16 h. The CD8 positive expressing T cells were purified by CD8 magnetic beads (Miltenyi, 130-096-730). The flow cytometry was then performed to analysis the expression of TNF-α, IFN-γ, IL-10, and IL-1β in CD8⁺ T cells. Co-culture media were assayed for TNF-α, IFN-γ, IL-2, IL-10, IL-1β, and TGF-β using a cytokine ELISA assay.

Spleens from OT-1 transgenic mice were collected and cells were separated and filtered through a 100μm filter into a falcon tube (50mL) containing 10mL of PBS. RBC were lysed with 5mL of ammonium chloride buffer (4.15 g NH₄Cl, 0.5 g KHCO₃, 0.0186 g EDTA, 500 mL milli-Q water, pH 7.4) for 3 min at 37°C. Cells were washed (300 x g, 5 min), in PBS (20mL) and counted before adding MACS buffer (1X DPBS, 0.5% BSA, 2 mM EDTA, Filter sterilized) and CD8 magnetic beads (Miltenyi Biotec Inc.) (90μL of buffer plus 10μL of beads per 10⁷ cells). Cells were incubated on ice for 30min and washed (300 x g, 5 min) with 1.5mL of MACS buffer per 10⁷ cells. The cells were resuspended in 500μL of MACS buffer per 10⁸ cells and T cells selected on an AutoMACS Pro Separator (Miltenyi Biotec) using the positive selection program according to the manufacturer's instructions. The positive fraction was collected and cells were washed twice in PBS (20mL) (300 x g, 5 min). The percentage of positive CD8+ OT-1 T cells after separation was >95% (data not shown).

Human CD8⁺ T cells were isolated from PBMCs using CD8 magnetic beads (Miltenyi Biotec). Human tumor-infiltrating lymphocytes (TILs) were isolated from TNBC patient biopsy samples (National Taiwan University Hospital, IRB No: 2014121193RINC). Isolated CD8⁺ T cells and TILs were activated with T cell TransAct (Miltenyi Biotec) for 3 days. Following activation, T cells were treated with Ab/IL-10 fusion proteins for 3 days and restimulated with anti-CD3 (Biolegend) for 4 h. Concentrations of IFN-γ and granzyme B in cell culture media were measured by ELISA (Biolegend) according to the manufacturer’s instructions.

All included patients were treated with conditioning chemotherapy including fludarabine (FLU, 30 mg/m² × 3 days) in combination with cyclophosphamide (CY, 300 mg/m² × 3 days) before the intravenous infusion of cryopreserved CD19 CAR-T cells at a dose of 1 × 10⁶ cells/kg body weight. The CAR in this study consists of an CD19-specific single-chain antibody fragment (scFv) derived from FMC63 fused to a modified IgG4-hinge spacer, a costimulatory molecule including CD28 alone or both CD28 and CD137 (4-1BB), and a CD3ζ signaling domain. Enrichment of CAR-T cells from patient’s PBMCs was done using CD4-magnetic beads (Miltenyi Biotec GmbH) and CD8-magnetic beads (Miltenyi Biotec GmbH).