Recombinant interleukin 2 ril 2

We used superparamagnetic polystyrene beads (Miltenyi Biotec GmbH, Germany) coated with a mouse anti-rat CD4 mAb (isotype: mouse IgG2a, κ; clone: OX-38) to harvest CD4⁺ T cells from spleen-derived lymphocytes, which were extracted using rat tissue lymphocyte isolation kit according to the protocol. The purity of CD4⁺ T cells was > 90%, as determined by a flow cytometry assay (Supplementary Figure 3). Then CD4⁺ T cells were stimulated to differentiate Th1 or Th2 with stimulating factors for 6 days, followed by published studies previously [12 (link), 47 (link)–49 (link)]. Briefly, for Th1 polarization, 10 ng/ml recombinant interleukin-2 (rIL-2), 5 ng/ml rIL-12 and 30 μg/ml anti-IL-4 were used; for Th2 polarization, 10 ng/ml rIL-2, 40 ng/ml rIL-4, 30 μg/ml anti-IL-12 and 30 μg/ml anti-IFN-γ were used (all cytokines and antibodies against cytokines were purchased from PeproTech Inc., NJ, USA). After the differentiation into Th1 and Th2 cells, we detected the levels of IFN-γ and IL-4, IL-5 and IL-13, which could be viewed as the signature cytokines of Th1 and Th2 respectively [50 (link)] to identify the purity of Th1 and Th2 cells (Supplementary Figure 4).

CD4⁺CD25⁺ Tregs were isolated from the peripheral blood of healthy volunteers using a MACS separation kit (MiltenyiBiotec, Bergisch Gladbach, Germany), as previously reported [54 (link)]. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Paque by density gradient centrifugation. CD4⁺ T cells were enriched from PBMCs by immune-magnetic depletion using a CD4⁺ T Cell Biotin-Antibody Cocktail and Anti-Biotin MicroBeads with an LD column (MiltenyiBiotec). Enriched CD4⁺ T cells were incubated with CD25 MicroBeads and then applied to an MS column to collect CD4⁺CD25⁺ Tregs. The isolated CD4⁺CD25⁺ Tregs were then expanded for 2 weeks in round-bottom 96-well plates with CD3/CD28 MACSiBead Particles (MiltenyiBiotec) at an initial bead-to-cell ratio of 4:1 and recombinant interleukin 2 (rIL-2) (Peprotech) with a concentration of 500 IU/mL. The expanded Tregs were fluorescently stained with FITC-conjugated antihuman CD4 (Biolegend, San Diego, CA, USA), BV510-conjugated antihuman CD25 (Biolegend), and Alexa Fluor647-conjugated antihuman FOXP3 (Biolegend) antibodies to confirm final purity. The number of Tregs in the cell co-culture systems was in a 2–3:1 proportion of 11Z or HESCs.