Complete rpmi medium

Non-parenchymal cells were isolated from mouse livers at 40 h post-infection. Cells were obtained by adapting a method previously described (21 (link)). Briefly, liver lobes were removed and perfused with liver perfusion medium (Gibco; Invitrogen) with 750 mg/l of Collagenase H (Roche) at 37°C. The resulting suspension was filtered through a 100 µm cell strainer (BD Falcon). The dissociated cells were suspended in liver perfusion medium and centrifuged for 10 min at 1,500 rpm, resuspended in RPMI complete medium (Gibco; Invitrogen), mixed in Percoll (GE Healthcare) solution to give a final concentration of 30% Percoll, and then centrifuged at 2,000 rpm for 10 min. The cell pellet was resuspended in RPMI and carefully laid on 30% Percoll solution and centrifuged at 2,000 rpm for 10 min. The cell pellet collected was washed and resuspended in ACK (NH₄Cl 0.15 M, KHCO₃ 10 mM, Na₂EDTA⋅2H₂O 0.1 mM and pH 7.2) for 3 min to lyse remaining erythrocytes. Cells were washed and centrifuged at 800 rpm for 20 s to discard the remaining hepatocytes; the supernatant was recovered, and NPCs were collected at 1,500 rpm for 5 min.

Spleen cells were obtained from a pool of 10 naïve mice. Spleens were harvested, perfused with RPMI complete medium (Gibco), and pressed gently through a sterile fine wire mesh. After red blood cells lysis (eBioscience), total splenocytes were suspended in 2 mM EDTA-PBS solution and separated using Lympholyte-M density gradient (Cedarlane Labs, Burlington, ON, Canada). Low-density cells at the interphase were isolated and then further FACS-purified for CD11c⁺ (APC-conjugated) with the FACSAria Fusion flow sorter (BD Biosciences, San Jose, CA, USA) using “low-recovery high-purity” sorting settings. A purity of >98% was obtained. Cells were allowed to rest for 1 h at 37°C with 5% CO₂. Afterward, cells were stimulated with S. suis strains P1/7 and ΔcpsF (initial MOI:1) or LPS at 1 µg/ml for 2, 4, and 18 h.

Heparinized venous blood was diluted in phosphate-buffered saline (PBS; pH 7.2 to 7.4); peripheral blood mononuclear cells (PBMCs) and plasma were separated by density gradient centrifugation using Ficoll-Isopaque (Pharmacia, Piscataway, NJ). Isolated plasma specimens were frozen at −20°C prior to use in immunological assays. PBMCs were suspended in RPMI complete medium (Gibco, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT) at a concentration of 1 × 10⁷ cells/ml and used immediately for assays.

DCs were generated as described previously from naïve mice [16 (link)]. Cell purity was 86–90% CD11c^high and F4/80^−/dim cells by FACS analysis as reported previously [16 (link)]. For purification of untouched CD4⁺ T cells, spleens (from either naïve or infected mice) were harvested, perfused with RPMI complete medium (Gibco), and pressed gently through a sterile fine wire mesh. After red blood cells lysis (eBioscience), total splenocytes were suspended in 2 mM EDTA-PBS solution and separated using Lympholyte-M density gradient (Cedarlane Lab.). Low-density cells at the interphase were purified by magnetic-activated cell sorting (MACS) negative selection (Miltenyi Biotec). The enriched CD4⁺ T cells had >96% purity by FACS analysis using CD3 and CD4 antibodies (data not shown). For all experiments, cells were incubated at 37°C, 5% CO₂.

Five different human cancer cell lines, namely, MCF-7 (breast carcinoma with estrogen receptor (ER+)), MDA-MB-231 (breast carcinoma without estrogen receptor (ER-)), HT-29 (colon adenocarcinoma), HCT-16 (colon carcinoma), and Reh (acute lymphoma leukemia), were employed in this study. MCF-7, HT-29, and HCT-116 cells were kind gifts by Dr. Amin Malik Shah Bin Abdul Majid (Universiti Sains Malaysia), while MDA-MB-213 and Reh cells were purchased from American Type Culture Collection (ATCC, USA). MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) complete medium (Gibco, USA) while MDA-MB-231 cells were cultured in Leibovitz's L-15 complete medium (Gibco, USA). HT-29, HCT-116, and Reh cells were cultured using RPMI complete medium (Gibco, USA). All complete media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, USA) and 100U/mL Penicillin-Streptomycin (Gibco, USA). All the cells were incubated at 37°C with a humidified atmosphere containing 5% of CO₂, except for MDA-MB-231 cells which were cultured without CO₂. All the cells were subcultured every 2-3 days to reach 90% confluence.

We collected venous blood from each participant in Vacutainer blood collection tubes (Becton Dickinson), containing sodium heparin anticoagulant. Samples were obtained at day 0 (before vaccination), day 7 (7 days after the first dose of vaccine), and day 21 (1 week after the second dose of vaccine). Following sample collection, blood was diluted in PBS (pH 7.4) and processed for separation of plasma and peripheral blood mononuclear cells (PBMCs). PBMCs were prepared by differential centrifugation in Leucosep tubes (Greiner Bio-One Ltd) filled with Ficoll-Isopaque (Pharmacia, Piscataway, NJ), and after two subsequent PBS washes, resuspended at a concentration of 5 x 10⁶ cells/mL in RPMI complete medium (Gibco, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT) and 1% Penicillin-Streptamycin (Sigma-Aldrich). These cells were used immediately as described below for measuring ASC responses. Plasma was shipped in a liquid nitrogen dry vapor unit and stored at -80°C prior to analysis in Boston.

Sodium-heparinized blood was diluted two-fold with phosphate-buffered saline (PBS, pH 7.2–7.4) and peripheral blood mononuclear cells (PBMCs), and plasma samples were isolated after centrifugation on Ficoll-Isopaque (Pharmacia, Piscataway, NJ) [27] (link). Plasma was stored at −20°C for further immunological assays. PBMCs were washed and resuspended at a concentration of 10⁷ cells/ml in RPMI complete medium (Gibco, Carlsbad, CA) with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT) [27] (link). The cells were then immediately used for the enzyme-linked immunosorbent spot (ELISPOT) assay to determine ASC responses, or cells were cultured for memory B cell responses as described below.