Macs pan t cell isolation kit

Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats of healthy donors using Lymphoprep (Fresenius Kabi Norge, AS, Berg i Østfold, Norway). T cells were negatively selected from PBMCs using a MACS Pan T Cell Isolation Kit (Miltenyi Biotec, Bergish Gladbach, Germany) and activated using microbeads coated with anti-human CD3, anti-human CD2, and anti-human CD28 antibodies (Miltenyi Biotec) at a 1:1 bead:cell ratio for 24 h in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 40 IU/ml interleukin-2 (IL-2), 10 mM HEPES, 2 mM glutamine, and 1% penicillin/streptomycin. Every 1*10⁶ T cells were transduced with 5–10 ml CAR lentiviral supernatants in the presence of 8 μg/ml polybrene (Sigma) for 5 h with 1 ml 10% FBS containing RPMI1640, and a continuous two rounds of transduction were conducted. After transduction, T cells were cultured in fresh media containing IL-2 (300 IU/ml). Subsequently, fresh media were added every 2–3 days to maintain cell density within the range of 0.5–1 × 10⁶/ml. Healthy PBMC donors who provided primary specimens gave informed consent for the use of their samples for research purposes, and all procedures were approved by the Research Ethics Board of Guangzhou Institutes of Biomedicine and Health (GIBH).

A part of the procedure was similar to that of the bacterial growth inhibition assay. After pneumococcal uptake and incubation with or without TJ-41, macrophages (MH-S, 1 × 10⁵ cells/well) were washed and fixed with 2% paraformaldehyde (PFA) in PBS for 20 min, and then washed three times. T cells from BALB/c mouse spleen were isolated using the MACS Pan T Cell Isolation Kit (Miltenyi Biotec). The cells (1 × 10⁶ cells/well) were then added to fixed macrophages and cultured for up to 3 days under Th17-polarizing condition using mouse IL-6 (20 ng/mL; BioLegend), mouse TGF-β (1 ng/mL; BioLegend), anti-mouse IFN-γ Ab (1 μg/mL; BioLegend), anti-mouse IL-4 Ab (1 μg/mL; BioLegend), anti-mouse CD3 Ab (10 μg/mL; BioLegend), and anti-mouse CD28 Ab (5 μg/mL). IL-17A production from T cells, reflecting the degree of antigen presentation by macrophages, was measured using mouse ELISA kits (R&D Systems, MN, USA) according to the manufacturer's protocols.

Deidentified leukocyte-enriched buffy coats were purchased from the Gulf Coast Regional Blood Center (Houston, Texas, USA). The mononuclear cell fraction was enriched over a Ficoll gradient (MilliporeSigma) following standard procedures. In brief, 25 mL of cells were gently layered on top of 15 ml Ficoll. The gradient was centrifuged at 400g for 30 minutes with no break. The buffy coat was removed and centrifuged at 300g for 6 minutes with a break and the pellet resuspended in 10 mL of PBS. T cells were purified by negative selection using the MACS Pan T cell Isolation Kit (Miltenyi Biotec 130-096-535) per the manufacturer’s instructions.
CyTOF, static cell adhesion assays, LIBS epitope analysis, cell spreading assays, cell migration assays, purified α₄β₁-binding assays, proliferation and IL-2 production, killing assays, molecular dynamics simulations, static cell adhesion assays with CXCL12 and pertussis toxin, and TCGA analysis are described in Supplemental Methods.

For T cell cytotoxicity assays, T cells were isolated from splenocytes using MACS pan-T cell isolation kit (Miltenyi Biotec, Cambridge, USA) according to manufacturer’s instructions. MACS-enriched T cells and mSGM salivary gland cells were cocultured at various effector-target ratios in complete medium containing RPMI (Gibco) supplemented with 10% FBS and 1x penicillin/streptomycin (Gibco).