Anti cd28 antibodies clone 37.51

Ovalbumin (OVA) and human CD19-expressing MC38 colon adenocarcinoma (MC38^OVA and MC38^hCD19) cells were grown at 37 °C and 5% CO₂ in standard DMEM medium supplemented with 10% FBS (Sigma), 2 mM glutamine, 1 mM sodium pyruvate, 1% penicillin/streptomycin, 2 mM Hepes and 0.1 mM non-essential amino acids (NEAA, all Gibco). 5 x 10⁵ MC38^OVA cells were subcutaneously (s.c.) injected into the flanks of 8-12 weeks old C57BL/6 mice. Six days after tumor inoculation, mice were irradiated sublethally (6 Gy). The following day, mice were injected intravenously (i.v.) with 2 x 10⁶ OT-I CD8⁺ T cells that were isolated from the spleen and peripheral lymph nodes of OT-1 mice and stimulated with 0.5 μg/ml anti-CD3 (clone 145-2C1), 1 μg/ml anti-CD28 antibodies (clone 37.51, both Bio X Cell) and 10 ng/ml rhIL-2 (Peprotech) for 72 h with or without 1 mM 2-desoxyglucose (2-DG, Sigma). Tumor size was assessed every day using a caliper. For some experiments, tumor tissue was harvested and digested with 1 mg/ml collagenase type I and 100 μg/ml DNase I (both Roche) and T-cell infiltration analyzed by flow cytometry. Alternatively, Rag1^–/– mice were s.c. injected with 1 × 10⁶ MC38^hCD19. After 7 days, 2 x 10⁶ anti-hCD19 CAR T cells^{33 (link)} treated with and without 2 mM 2-DG for 2 days were adoptively transferred i.v.

Total and CD4⁺ T cells were isolated from the spleens and LNs using the Mouse pan T Cell Enrichment Kit or the Mouse CD4⁺ T Cell Enrichment Kit, respectively (both STEMCELL Technologies; cat. nos 19852 and 19751). CD4⁺ T cells were stimulated with 1 μg ml⁻¹ plate-bound anti-CD3 (clone 2C11) plus 1 μg ml⁻¹ anti-CD28 antibodies (clone 37.51; both Bio X Cell). For differentiation of naive CD4⁺ T cells into T_H1, T_H17 and T_H2 cells, T cells were polarized for 3 days with 10 ng ml⁻¹ IL-12 (PeproTech) and 2 μg ml⁻¹ anti-IL-4 (eBioscience) for T_H1; 20 ng ml⁻¹ IL-6 (PeproTech), 0.5 ng ml⁻¹ hTGFβ1 (PeproTech), 2 μg ml⁻¹ anti-IL-4 (clone 11B11) and 2 μg ml⁻¹ anti-IFNγ (clone XMG1.2; both eBioscience) for T_H17 and 100 ng ml⁻¹ IL-4 (PeproTech), 5 μg ml⁻¹ anti-IL-12 and 20 μg ml⁻¹ anti-IFNγ (both eBioscience) for T_H2 cells in IMDM or RPMI medium (both Cellgro). Both media contained 2 mM L-glutamine, 50 mM 2-ME, 100 U ml⁻¹ penicillin/streptomycin and 10% FCS. For cytokine expression, cells were (re-)stimulated with 1 μM ionomycin plus 20 nM phorbol myristate acetate (both Calbiochem) for 6 h in the presence of brefeldin A (BioLegend) and analysed by flow cytometry as described below.

A total of 5 × 10⁶ T cells were loaded with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen; cat. no. C34554) according to the manufacturer's instructions. Cells were blocked with 50% FBS, washed twice with RPMI and stimulated with 1 μg ml⁻¹ plate-bound anti-CD3 (clone 2C11) plus 1 μg ml⁻¹ anti-CD28 antibodies (clone 37.51; both Bio X Cell) with or without 50 U ml⁻¹ IL-2 (NIH) for 4 days. CFSE dilution was monitored daily using an LSRII flow cytometer and FACSDiva Software (BD Biosciences) and further analysed with FlowJo Software (Tree Star).