Igg2b

Fluorescent marker conjugated antibodies CD105, CD90, CD73, CD34, and CD31 (R&D Systems, Minneapolis, MN, USA) were used to confirm the phenotypic characteristics and to verify the lack of contaminating cells, according to the markers proposed by the International Society for Cellular Therapy [4 (link)]. Non-specific background signal was measured using an isotype control cocktail consisting of mouse anti-human IgG2A, IgG2B and IgG1 (R&D Systems).
Cells were harvested and suspended in flow cytometry buffer (PBS containing 0.5% (w:v) BSA) at a concentration of 4 × 10⁶ cells/ml. Every 2x10⁵ cells were incubated with 10μl of fluorescent marker conjugated antibody at 4°C for 30 min in the dark. The cells were then washed and finally resuspended in 400μl of flow cytometry buffer for analysis. To determine the viability, 1μl Propidium iodide (PI; Sigma-Aldrich, Dorset, UK) was added to each tube just before the sample was analysed using BD FACS Canto-F60 cell sorter (BD Biosciences, Wokingham, UK). Data was analysed by Flowjo software (FlowJo LLC, Ashland, OR, USA).

In order to study the role of MHC molecules in Th2 cell and treated-MM cell interactions, cells were incubated with mouse MoAb against HLA-DR (IgG2b; 1 μg/mL; Millipore, Billerica, MA, USA), mouse MoAb against MHC--I (IgG2b; 5 μg/mL; R&D Systems, Minneapolis, MN, USA), or control IgG2b (5 μg/mL) In order to assess the myeloma cell-T cell contact dependence of tumor cell clonogenicity, Transwell polyester membrane inserts (CLS3460, Corning, Inc., Corning, NY, USA) separating treated-RPMI8266 cells from MM-Th2 cells were compared with control inserts separating MM-Th2 cells from complete RPMI-1640 medium only.

Freshly purified thymocytes, such as CD4+, CD4+CD25+, or CD4+CD25− T cells, were seeded into 24-well plates in an RPMI 1640 Glutamax I medium supplemented with 10% fetal calf serum at 5 × 10⁵ cells per well, alone or with adherent cells. As different adherent cell types differ in their proliferation properties, cells were seeded in 24-well plates at 5 × 10⁴ cells per well for MITCs and 1 × 10⁵ cells per well for mTECs and CACO2 cells to reach subconfluence after coculture.
In some experiments (Figures 2, 3, 4, 5, 6), T cells were separated from adherent cells using cell culture insert TWs (1 μm pore size, Becton Dickinson, Le-Pont-de-Claix, France), to prevent cell contact but to allow diffusion of soluble mediators.
In some experiments (Figures 6 and 7), blocking antibodies were used at the following concentrations: anti-IL-10 at 5 μg/ml, anti-IL-2 between 0 and 5 μg/ml, anti-TSLP at 0.1 μg/ml, anti-TGF-β at 5 μg/ml, and anti-ICOSL between 0.5 and 1 μg/ml. All antibodies were from R&D Systems, Lille, France. Control isotypes IgG1 and IgG2B (R&D Systems) were used at the same concentrations as their corresponding antibody.