Mhc class 2

Splenic DCs were purified from CD11c^creIFNγR2^fl/fl or WT mice by using CD11c beads (Miltenyi Biotech) with >90% purity. As readouts of maturation, purified DCs were stained for MHC class II and CD86 (all eBioscience) 24hr after LPS treatment (50ng/ml). In T cell proliferation assay, CFSE labeled OT II T cells (1x10⁵) were cultured with aforementioned DCs (4x10⁴) pulsed with OVA protein in 96 well plate for 60hr. For in vitro polarization assay, FACS sorted CD4⁺CD25^-CD62L^hi naïve T cells (2.5x10⁵) were incubated with purified DCs (5x10⁵) for 4 days in the presence of IL-12(10U/ml) or IL-4 (20ng/ml) with anti-IFNγ (10μg/ml) and anti-IL-12 (10μg/ml) under Th1 or Th2 conditions, respectively. Cytokine production was assessed by flow cytometry with intracellular IFNγ and IL-4 staining.

Cells were incubated with fixable viability dye (eFluor 455UV; eBioscience, UK) followed by CD16/CD32 antibody (Fc-block; clone 2.4G2, BD Bioscience, UK), then stained with directly conjugated antibodies (10 μg/ml and analysed using an LSR II device (BD Bioscience, UK). The following antibodies were purchased from BD Bioscience, UK unless stated otherwise: MHC class II #553623, CD4 #552051, CD11b #550993, CD11c #550261, CD135 #562537, CD40 #562846, CD86 #560581, CD25 #553866, CCR7 #563596, PDL-1 #564716, Zbtb46 #565832, SIRP-1α #740159, c-kit #560185, CD115 #25–1152-82 (eBioscience) and CX3CR1 #149023 (Biolegend). Transcription factor buffer (BD Bioscience, USA, catalog #562574) was used in accordance with the manufacturer’s instructions to test for transcription factors. FlowJo software (TreeStar, Inc., Ashland, OR, USA) was used for data analysis.

Bone marrow MSCs were isolated as previously described [14 (link)]. Briefly, bone marrow cells were flushed out from the bone cavity of femurs and tibias of mice with Dulbecco’s modified Eagle's medium low glucose (L-DMEM, Hyclone, USA) containing 10 % fetal bovine serum (FBS; Gibco, USA). A single-cell suspension of all nucleated cells was obtained by passing all bone marrow cells through a 70-μm cell strainer (BD Bioscience, USA). All the single cells were seeded at 1 × 10⁶ into 100-mm culture dishes (Corning, USA) and initially incubated for 48 hours at 37 °C and 5 % CO₂. To eliminate the nonadherent cells, the cultures were washed with phosphate-buffered saline (PBS; Gibco) twice on the second day. The attached cells were cultured with L-DMEM supplemented with 15 % FBS, 2 mM L-glutamine (Invitrogen, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Cell surface marker analysis confirmed that MSCs were positive for CD44 and Sca-1, and negative for CD11b, CD31, CD34, CD45, major histocompatibility complex (MHC) class I and MHC class II (eBioscience, USA).

Cells were incubated for 30 min with Phycoerythrin (PE)-labeled monoclonal antibodies recognizing either CD40, CD83, CD86, MHC Class II, or CD1d (eBioscience), washed with staining buffer, and analyzed by flow cytometry on a FACSCalibur (BD Biosciences, Franklin Lakes, NJ).

Spleens were extracted and manually digested. CD11c+ cells were isolated using Miltenyi Biotec Inc. (San Diego, CA) MACS magnetic cell separation with positive selection for CD11c (CD11c, Biolegend, cat. no. 117304). Subsequently, the CD11c- splenic fraction was used to negatively select for naïve CD4+ T cells using the Miltenyi MACS system with the following antibodies: CD11c (Biolegend, cat. no. 117304); CD45R (eBioscience, cat. no. 13-0452-86); CD11b (eBioscience, cat. no. 13-0112-86); CD25 (eBioscience, cat. no. 36-0251-85); CD49b (eBioscience, cat. no. 13-5971-85); CD69 (eBioscience, cat. no. 13-0691-85); CD8a (eBioscience, cat. no. 13-0081-86); Ly-6G (eBioscience, cat. no. 13-5931-86); MHC class II (eBioscience, cat. no. 13-5321-85); TER-119 (eBioscience, cat. no. 13-5921-85). CD11c+ and CD4+ cells were cultured at a ratio of 1:2 in 96-well round-bottom plates for 24 hr, 108 hr (for proliferation assay), or 3.5 hr (for ERK phosphorylation staining). Peptides were added at indicated concentrations with the CD11c+ and CD4+ cells in DMEM supplemented with 10% Fetal Bovine Serum. For sequencing experiments, CD4+ cells were re-isolated from the culture using the Miltenyi MACS system and the same set of antibodies as above less CD25 and CD69. For phospho-ERK staining, whole splenic cells were used, rather than purified CD11c+ and CD4+ cells.