Anti cd28 ab

Splenocytes from WT and CD38-/- mice were left unstimulated or stimulated with 1 µg/mL of anti-CD3ε Ab (BD Biosciences, San Diego, CA, USA), 1 µg/mL of anti-CD28 Ab (BD Biosciences, San Diego, CA, USA), and 200U rhIL-2 (Sigma Aldrich) or Phorbol Myristate Acetate (50 ng/mL) plus Ionomycin (500 ng/mL) for 72 h. According to the manufacturer’s protocol, supernatant concentrations of IFN-γ and IL-10 were evaluated by Mouse ELISA MAXTM DELUXE Set (Biolegend, San Diego, CA, USA).

Splenocytes from naïve Balb/c mice were labelled with 1 μM CFSE and placed in triplicates into a U-bottom 96-well plates (1 × 10⁵) with or without the presence of 1 μg ml⁻¹ anti-CD3 Ab (#553057, BD Biosciences) and 5 μg ml⁻¹ anti-CD28 Ab (#557393, BD Biosciences). Labelled Splenocytes were co-cultured with purified MDSCs for 72 h and CFSE dilution of CD4⁺ T-cell fractions was analysed by flow cytometry.

The procedure of cell staining and flow cytometry was previously described (5 (link)). For intracellular staining of IL-21, gastric CD4⁺T cells were restimulated with 1 μg/ml anti-CD3 Ab (Clone UCHT1, BD), 1 μg/ml anti-CD28 Ab (Clone CD28.2, BD), and 3 μg/ml brefeldinA (eBioscience, CA, USA) for 12 h. The intracellular fixation/permeabilization buffer set (eBioscience, CA, USA) was used in IL-21 staining. Flow cytometric detection was performed with FACS Canto II (BD, NJ, USA). Data was analyzed by the FlowJo software (Tree Star). The anti-human antibodies used in this study were purchased from Biolegend, BD Biosciences, or Miltenyi Biotec, namely: CD3 (Clone UCHT1, BioLegend), CD4 (Clone OKT4, BioLegend), GITR (Clone 108-17, BioLegend), GITRL (Clone REA841, Miltenyi), IL-21 (Clone 3A3-N2.1, BD), BCL6 (Clone 7D1, BioLegend), STAT3 Phospho (Ser727) (Clone A16089B, BioLegend), BTLA (Clone MIH26, BioLegend), CXCR5 (Clone J252D4, BioLegend), and PD-1 (Clone EH12.2H7, BioLegend).