Siinfekl

The APC assay was performed as previously described (Roser-Page et al., 2014 (link)). Splenic CD11c+
dendritic cells (DCs) sorted by immunomagnetic beads (Miltenyi Biotech) were
used as antigen presenting cells. CD8+ T cells expressing a monoclonal
ovalbumin (OVA)-specific transgenic TCR were purified from the spleens of
OT-I mice by negative selection. CD11c+ DCs were pulsed for 4 hours with 1
μM antigen (OVA peptide) (SIINFEKL, InvivoGen). After two washes with
medium, cells were used for APC assay. For induced Treg cells cocultures,
OVA presenting dendritic cells at 150,000/well were incubated with splenic
OT-I CD8+ T cells (1 million/well) with or without induced Treg cells (1
million/well or increasing dose of Treg cells from 0.5 to 4 million/well)
for 24 h. CD8+ T cells were separated by EasySep Mouse CD8a positive
Selection Kit II (StemCell Technologies, Auburn, CA) and dissolved in TRIzol
reagent for RNA isolation and real time RT-PCR of Wnt10bmRNA. For ChIP assay, the time of coculture of OVA presenting dendritic
cells, CD8+ T cells and induced Treg cells was 4 h. During CTLA-4Ig
treatment, OVA presenting dendritic cells at 150,000/ well were incubated
with splenic OT-I CD8+ T cells (1 million/well) with or without CTLA-4Ig
(100 μg/mL) for 4h or 24 h.

We evaluated the immunological responsiveness of T-cells from different organs by quantifying the intracellular IFN-γ production in the CD8⁺ and CD4⁺ T-cells isolated from the immunized mice using a previously reported method.^{[24 (link)]} Briefly, we prepared single cell suspensions from the lung, spleen and LN harvested 7 days after the boost immunization, using the procedure described above. Cells were cultured in Iscove modified Dulbecco medium media (Thermo Fisher Scientific) supplemented with 10% HI-FBS, 2 mM L-glutamine and 1% penicillin/ streptomycin at 37 °C. Cells from spleen and LN were plated on a 6-well plate at a 7 × 10⁶ cells/well and cultured in presence of monensin (Sigma) and 1 μg mL^−1 of SIINFEKL (InvivoGen) or 100 μg mL^−1 OVA (InvivoGen) for 6 hours to re-stimulate OVA-specific CD8⁺ or CD4⁺ T-cells, respectively. In parallel, cells from the lung were plated in the same manner and re-stimulated with 1 μg mL^−1 ionomycin and 50 ng mL^−1 PMA (InvivoGen) for 4 hours. Cells from all three organs were additionally treated with 5 μg mL^−1 brefeldin A (Thermo Fisher Scientific) for 2 and 3 hours to inhibit secretion of newly produced IFN-γ from CD8⁺ and CD4⁺ T-cells, respectively, prior to the cell collection. Cells were then stained for flow cytometric analysis.