L3t4 microbeads

Induced Tregs (iTregs) were generated as described previously.⁷ (link) Briefly, CD4⁺ cells were isolated from B6 spleen and lymph nodes by magnetic bead sorting (L3T4 Microbeads, Miltenyi Biotec), and cultivated in plates coated with 10 µg/ml anti-CD3 (145-2C11), 1 µg/ml anti-CD28 (37.51) (BD Pharmingen) in the presence of 100 U/ml interleukin-2 (IL-2; Sigma) and 5 ng/ml recombinant human transforming growth factor-beta (rhTGF-β; R&D Systems) for 5 days. At the end of culture, the Treg-enriched cell population was used for therapeutic administration without additional sorting steps. FoxP3 expression was usually >80%. Then 3 × 10⁶ cells were injected intravenously per mouse.

All mice were sacrificed 12 h after CLP modeling and spleens were surgically removed. Lymphocytes from the spleens were isolated according to the instructions of the mouse splenic lymphocyte isolation kit (P8860; Solarbio). Next, 90 μL PBS buffer containing 5% serum was added to the obtained spleen cell suspension for resuspension, and 10 μL magnetic beads (L3T4 MicroBeads; Miltenyi Biotec) coupled with CD4⁺ T cell antibody were then added, incubated at 4 °C for 20 min, and washed. After resuspending again, CD4⁺ T cells were isolated by negative selection using separation columns and centrifuged in RPMI-1640 medium, and their viability and number was quantified by trypan blue exclusion with a TC20 automated cell counter (Bio Rad).

CD4⁺ T cells were obtained using L3T4 microbeads (130-117-043, Miltenyi Biotec). A total of 3 × 10⁵ cells (1:1, 1.5 × 10⁵ each from C57BL/6 Cd45.1⁺Ccr9^+/+ and Cd45.2⁺Ccr9^−/− mice) were intraperitoneally injected into Rag2^−/− mice. IELs in mice were analyzed 6 weeks after the transfer.

CD4+ cells were isolated from the lungs of 7–12-week-old wild-type, Ifng^-/-, Nos2^-/- or Csf2^-/- mice 21–28 d after ~500 CFU aerosol infection with M. tuberculosis Erdman using mouse CD4 (L3T4) MicroBeads from Miltenyi Biotec (130-117-043) according to the manufacturer’s protocol, resuspended in RPMI with 10% FBS, 2 mM L-glutamine, 1 mM Sodium pyruvate, 1 mM NEAA, 20 mM HEPES, 55 μM 2-mercaptoethanol and 35 μg/mL kanamycin (T cell media) supplemented with 10% supernatant from 3T3–M-CSF cells and added to infected BMDMs in co-culture or in transwells. Unless indicated, CD4 T cells were added at a 5:1 T cell:BMDM ratio. In these experiments, T cell media supplemented with 10% supernatant from 3T3–M-CSF cells was used for untreated and IFN-γ-treated controls.

To obtain Treg cells, CD4+ T cells were isolated from the spleens of control (untreated mice) or CIA mice in a DBA1J background, using microbeads conjugated to monoclonal anti-mouse CD4 antibodies (L3T4 MicroBeads; Miltenyi Biotech, Bergisch Gladbach, Germany). CD4+ T cells (5 × 10⁵) were cultured with plate-bound anti-CD3 (1 μg/ml; BD Pharmingen), soluble anti-CD28 (1 μg/ml; BioLegend), anti-IFN-γ (5g/ml; R&D Systems, Minneapolis, MN), anti-IL-4 (5 μg/ml; R&D Systems), human recombinant transforming growth factor beta (TGF-β) (5 ng/ml) (PeproTech, London, UK), and retinoic acid (0.1 μM) (Sigma-Aldrich, St. Louis, MO, USA) for two days24 (link).
To obtain Tr1 cells, isolated CD4+ T cells (5 × 10⁵) from single-cell suspensions of splenocytes in control or CIA mice in a DBA1J background were cultured with plate-bound anti-CD3 (1 μg/ml), soluble anti-CD28 (1 μg/ml), vitamin D3 (Vit D3) 10⁻⁷ M (Sigma-Aldrich), and dexamethasone (Dex) 5 × 10⁻⁸ M (Sigma-Aldrich) for two days36 (link).
MSCs (5 × 10⁵) were co-cultured with irradiated (2000 rad) T (5 × 10⁵), Treg (5 × 10⁵), or Tr1 (5 × 10⁵) cells (1:1 ratio) in 10 ml of culture medium for 24 h. MSCs were purified from co-cultures by sorting using negative selection of CD4+ T cells by flow cytometry.

Tregs were generated as described previously [17 (link)]. Shortly, cells were isolated from spleen and lymph nodes of naïve B6 mice. For iTreg generation, CD4⁺ cells were isolated (L3T4 microbeads, Miltenyi Biotec) and cultured for 6 days (144 h) in precoated 24-well plates (100 μg/ml anti-CD3 (145-2C11), 10 μg/ml anti-CD28 (37.51); BD Pharmingen) in the presence of 100 U/ml IL-2 (Sigma) and 5 ng/ml rhTGFβ (R&D Systems) [24 (link)]. Human CTLA4Ig (abatacept, purchased from Bristol-Myers-Squibb) was added at different concentrations (low dose, LD 40 μg/ml; high dose, HD 200 μg/ml) for the length of culture or for the last 24 h of culture (HD 200 μg/ml). Due to intentionally introduced mutations to achieve higher avidity for human B7 molecules, belatacept lost effective binding capacity for murine B7; therefore, only abatacept is used in the current study [25 (link)]. Living cells were counted at indicated time points using CASY System (Innovatis). Purity of MACS-sorted populations was >90%. At the end of culture, the Treg-enriched cell populations were used for subsequent cell culture assays without additional sorting steps [17 (link)].