Automacs instrument

CD4⁺ and CD8⁺ T cells were isolated from the spleen and cervical lymph nodes of TCR-HA or CL4 mice, and purified by negative selection, using the CD4⁺ and CD8⁺ T cell isolation kit and an AutoMACS instrument (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. Purified T cells were labeled with 2.5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and i.v. injected into the tail veins (1.5–1.8 × 10⁶ HA-specific CD4⁺ T cells, 5–7 × 10⁶ HA-specific CD8⁺ T cells) of recipient mice. Mice were sacrificed 3 days after T cell transfer and T cell proliferation in the bronchial lymph nodes (BLN), and spleen was assessed by flow cytometry.

Peripheral blood mononuclear cells were prepared from buffy coats obtained from healthy donors by centrifugation through Ficoll-Paque (GE healthcare, Uppsala, Sweden). CD4+ T cells were isolated by magnetic bead depletion of CD8, CD14+, CD15+, CD16+, CD19+, CD36+, CD56+, CD123+, T cell receptor-gamma/delta, and glycophorin A–positive cells (CD4+ T Cell Isolation Kit) on an AutoMACS instrument (Miltenyi Biotec). CD4+ T cells (5×10⁵ cells/well) were cultured with X-VIVO medium (Lonza, Walkersville, MD) containing 2% FBS and 5 µg/ml anti-CD28 antibody (clone CD28.2, BioLegend, San Diego, CA) in anti-CD3-precoated 24-well culture plates (clone OKT3, BioLegend). During in vitro proliferation of CD4+ T cells, human amnion-, chorion-, or bone marrow-derived (Lonza) MSCs were co-cultured at 5×10⁴ cells/well. After 5 days of co-culturing, T cells were separated from the monolayer MSCs and counted with an automated cell counter (Countess, Invitrogen).

Murine fibrocyte-like cells were isolated according to previously published methods69 (link). Murine lungs removed from male SCID mice were minced with scissors and incubated with DMEM including 1 mg ml⁻¹ BSA (Sigma, St Louis, MO), 1 mg ml⁻¹ collagenase IV (Roche, Basel, Switzerland) and 100 μg ml⁻¹ DNase 1 (Sigma, St Louis, MO) for 1 h at 37 °C. The single cell suspensions from whole lungs were incubated in DMEM supplemented with 20% FBS in 100 mm fibronectin-coated dishes. When cells reached 70% confluence, they were passaged using trypsin digestion. After 7 days, the trypsinized cells were incubated with anti-CD45 Abs coupled to magnetic beads (Miltenyi Biotech, Auburn, CA) for 15 min on ice. The labelled cells were separated into CD45⁺ (fibrocyte-like cells) and CD45⁻ (fibroblasts) populations using an Auto MACS instrument (Miltenyi Biotech, Auburn, CA) according to the manufacturer's instructions.

PBMCs were isolated from 50 mL of peripheral blood obtained from healthy donors, by Ficoll-Paque Plus (Thermo Fisher Scientific) density gradient centrifugation followed by erythrocyte lysis using Buffer EL (Qiagen). After CD4 enrichment using magnetic cell separation with the autoMACS instrument (Miltenyi Biotec) using CD4 MicroBeads (Miltenyi Biotec), cells were left overnight at 4°C in basal cell culture medium [RPMI 1640 Medium with GlutaMAX Supplement-10 (Thermo Fisher Scientific), 10% FCS (Corning), 50 nM 2-mercaptoethanol (Thermo Fisher Scientific), 1 mM pyruvate (Biochrom), and 25 mM HEPES (Gibco)]. The following day, CD4⁺ memory T cells (CD4⁺ CD25⁺ CD127⁺ CD45RO⁺) and Tregs (CD4⁺ CD25⁺ CD127^–) were FACS-sorted with a BD FACSAria II SORP (Becton Dickinson). Sorted CD4⁺ memory T cells and Tregs were initially cultured in 96-well round-bottom plates (Greiner Bio-One) with the aforementioned cell culture media supplemented with 500 IU/mL rhIL-2 (R&D Systems) and 10 nM Rapamycin (STEMCELL Technologies) (day 0). The following day, cells were stimulated with anti-CD3/CD28 MACSiBead particles (Treg Activation/Expansion Kit, Miltenyi Biotec) according to manufacturer’s guidelines. Cells were stimulated at the same time points as the 1st and 2nd generation products, and collected for downstream DNA methylation analysis.

Six bones were collected from naïve donor mice (two tibias, two femurs, and two hip bones). Bones were then carefully crushed in HBSS media with 1% penicillin/streptomycin and HEPES. Filtered BM was RBC lysed using 5 min RBC lysis buffer (Biolegend, San Diego, CA, https://www.biolegend.com). Lysis buffer was washed out and cells were stained with anti-CD117 magnetic beads (eBiosciences, San Diego, CA, https://www.thermofischer.com) using the manufacturer’s protocol. CD117+ cells were positively selected using the AutoMACS instrument (Miltenyi). CD117+ cells were washed and stained for Lineage markers (Table 1) and Sca-1. After staining, HSPCs were purified by cell sorting.

Gut tissues were prepared as previously described (15 (link)). Briefly, cleaned gut tissues were deepithelialized in 5 mM ethylenediaminetetraacetic acid (EDTA), 15 mM Hepes, and 5% fetal bovine serum (FBS) in Hank’s Balanced Salt Solution (HBSS) before digestion with 0.5 mg/mL Collagenase Type IV (Sigma Aldrich) and 0.25 mg/mL DNase 1 in HBSS with 2% FBS. Lymphocytes enriched by passing the cell suspension sequentially through 100- and 40-μm strainers. For enzyme-linked immune absorbent spot (EliSPOT) experiments, CD4⁺ T cells were isolated from mLN using magnetic isolation (CD4 microbeads, Miltentyi Biotech), and dendritic cells were isolated from the spleens of naive SPF C57BL/6 mice (Jackson Laboratories) by magnetic isolation (CD11c microbeads, Miltenyi Biotech) using an AutoMACS instrument (Miltenyi Biotech) following the manufacturer’s instructions.