Hamster anti mouse cd28 clone 37.51

Spleen and lymph nodes of JEDI mice were dissected, and single cell suspensions of leukocytes were obtained by mechanical disruption and filtering through a 70-mm cell strainer. Red blood cells were lysed using ACK buffer (Lonza), and CD8⁺ T cells were negatively selected using MagniSort Mouse CD8⁺ T Cell Enrichment Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. For in vitro experiments described in the text using JEDI, cells were activated for 2 days with 5 μg/mL plate-bound purified hamster anti-mouse CD3e (clone 145–2C11, BD Biosciences), 1 μg/mL purified hamster anti-mouse CD28 (clone 37.51, BD Biosciences), and 20 ng/mL recombinant mouse IL-2 (Gemini Bio-Products) in RPMI with 10% FBS, 100 U/ml penicillin/streptomycin/ampicillin, and 50 μM 2-mercaptoethanol. For in vitro experiments described in the text using GFP-specific T cells, cells were first FACS purified using H-2K^d-HYLSTQSAL tetramer reagent produced by the NIH Tetramer Core Facility prior to activation as described above. For all in vivo experiments, freshly isolated tetramer-sorted GFP-specific T cells were used without activation.

96 well U-shape plates (Fisher Scientific) were pre-coated with 5 μg/ml hamster antimouse CD3e (clone 145–2C1) and 2 μg/ml hamster anti-mouse CD28 (clone 37.51) from BD Biosciences. T cell receptor transgenic (TCR Tg) ABM CD4⁺ T cells were purified from the spleen of ABM mice using CD4⁺ cell negative isolation kit (Miltenyi). ABM CD4⁺ T cells were labeled with 1 μM violet proliferation dye (BD Biosciences). 4×10⁴ B6 BMDMs and 4×10⁴ ABM CD4⁺ T cells were plated in the precoated plates with the addition of either 30 μg of indicated NP preparations or 1.2×10⁵ indicated SP preparations in RPMI-1640 supplemented with 10% FCS for 3 days. The rationale for using 30μg NP preparations or 1.2×10⁵ SP preparations in this assay was that according to the CBQCA quantification in Figure 1D, these doses would provide approximately the same amount of peptide antigen to the co-cultured BMDMs. Cells were then harvested for determination of ABM CD4⁺ T cell proliferation by dilution of the violet proliferation dye.