Anti human cd3

CD4 positive T cells were obtained from peripheral mononuclear using paramagnetic beads (StemCell Technology, Canada) according to manufacturer's protocol. Cells purity was > 90% confirmed by low Cytometry (LSR II, Becton Dickinson). To determine cell division, CD4⁺ T cells were labeled using Carboxyfluorescein succinimidylester (CFSE, Biolegend, USA) according to manufacturer's instruction. After labeled, T cells were cultured alone in the presence of anti-human CD3 (2.5 μg/ml BD Biosciences) and anti-human CD28 (2.5 μg/ml, BD Biosciences) for 2 days. Before co-culture, SGC-7901 was treated as described in upper functional assay section. Then CD4 positive T cells were collected and incubated with treated SGC-7901 (5:1) for another two days in the presence of anti-human CD3 (2.5 μg/ml, BD Biosciences) and anti-human CD28 (2.5 μg/ml, BD Biosciences). Cell division of CD4⁺ T cells was analyzed by Flow Cytometry (LSR II, Becton Dickinson).

Before co-culture, SGC-7901 was transfected with miR-152 mimic, inhibitor or mimic and inhibitor for 48 hours according to manufacturer's protocol then IFN-γ (20 ng/ml) was added for another 24 hours. After that, cells was treated with Mitomycin C (10 μg/ml, Sigma) for 1.5 hours. Meanwhile, T cells were cultured alone in the presence of anti-human CD3 (2.5 μg/ml, BD Biosciences) and anti-human CD28 (2.5 μg/ml, BD Biosciences) for 2 days then T cells (5*10⁵) were co-cultured with above SGC-7901 (5:1) in the presence of anti-human CD3 (2.5 μg/ml, BD Biosciences) and anti-human CD28 (2.5 μg/ml, BD Biosciences) for 2 days. Then T cells were collected and stained with flurochrome-conjugated-specific antibodies extracellularly. Then T cells were fixed and permeabilized by using Perm/Fix solution (eBioscience, USA) according to the manufacturer's instruction. After that, the intracellular cytokines were stained by flurochrome-conjugated-specified anti-human IL-2 and IFN-γ antibody (eBioscience, USA) according to the manufacturer's instruction.

Human peripheral blood mononuclear cells were isolated from healthy volunteers and stored at −80°C in cryovials at a 10⁷-cell/ml concentration in FBS containing 10% DMSO. Before plating, cells were washed in PBS twice, recounted, and plated at a 10⁶-cell/ml concentration in RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine. Cells were stimulated for 3 days as described previously (12 (link)) with anti-human CD3 (BD no.555336, 0.3 µg/ml), anti-human CD28 (BD no.555725, 2 µg/ml) and recombinant human TGF-β1 (R&D no. 240B002, 2.5 ng/ml).
Bacteria isolated from human chloroform-resistant cultures were resuspended in PBS supplemented with protease inhibitor (Roche no. 4693159001) and phosphatase inhibitor (Roche no. 4906845001), heat-inactivated at 65°C for 1 h and sonicated for 10 min as described previously (14 (link)). Protein concentration in the resulting suspension was measured using the Pierce BCA protein assay kit (Thermo Scientific no. 23227). Bacterial extracts were added to PBMCs at 1 µg/ml 1 h after plating as described previously (13 (link)). PBS with the same protease inhibitor and phosphatase inhibitor was added as the no-bacterium control. Each human in vitro experiment contained at least 6 independent donor bacterial samples and was repeated at least twice.