Anti cd28 antibody clone 37

GFP⁺ Treg cells were sorted from pooled LNs and spleens isolated from Foxp3^GFP mice and cultured in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (GE Healthcare Life Sciences), 2 mM l-glutamine, 1% penicillin-streptomycin (both from Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich) at 37 °C with 5% CO₂. For in vitro expansion, Treg cells were cultured in 96-well plates coated with 1 μg/ml anti-CD3 antibody (clone 17A2, prepared in-house) and 1 μg/ml anti-CD28 antibody (clone 37.51, eBioscience) in RPMI medium supplemented with 300 U/ml hIL-2 (Sigma) for 14 days. The cells were refed with fresh medium every second day. Total CD4⁺ T cells were isolated using the MACS CD4 Negative Isolation Kit II (Miltenyi Biotec) and labeled with the cell proliferation dye eFluor^TM 670 (eBioscience) according to the manufacturer’s instructions. To assess the suppressive capacity of Tregs in vitro, total CD4⁺ cells were stimulated with Dynabeads^TM Mouse T-Activator CD3/CD28 (Thermo Fisher) in the presence of WT or Cd25^Y129H Treg cells for 72 h.

The effects of Cd137l-Fc on T cells were investigated by sandwich ELISA measurement of IL-2 production and a cell proliferation assay. T cells were isolated from splenocytes which were removed from 6-week-old male C3H/HeJ mice (CLEA Japan, Tokyo, Japan). CD4⁺ and CD8⁺ cells were sorted using FACS Aria (BD Biosciences, San Jose, CA, USA). CD4⁺ and CD8⁺ cells were seeded at 5.0 × 10⁵ or 2.5 × 10⁵ cells/500 μl medium/well, respectively, in 24-well culture plates precoated with 5.0 μg/ml anti-CD3 antibody (clone 145-11 C, Biolegend) in the presence of 20 μg/ml Cd137l-Fc or 20 μg/ml mock-Fc. Anti-CD28 antibody (clone 37.51, eBioscience, San Diego, CA, USA, 1.5 μg/ml) was used as a positive control. After 48 h, IL-2 production was determined by ELISA (mouse IL2 DuoSet ELISA, R and D systems, Minneapolis, MN, USA) for the above culture supernatants. All assays were performed in triplicate.