Anti trinitrophenol clone 2a3

At 6 weeks of age, C57BL/6J mice were fed ND or HFD for 18 weeks to induce ATT impairment. At 24 weeks of age, mice were given injections of anti–mouse PD1 (catalog CD279, clone RMP1-14, Bio X Cell) or IgG2a isotype control, anti-trinitrophenol (clone 2A3, Bio X Cell) at 10 mg/kg every third day for 4 total injections. The day after the last injection, systemic metabolism was assessed by GTT. After refeeding and resting overnight, mice were sacrificed, and a fraction of fresh SVF was stained for flow cytometry analysis to assess basal T cell differences and ensure the PD1 antibody entered adipose tissue and blocked PD1. The following antibodies were used: AF488-CD4 (catalog 100423) and APCcy7-CD8 (catalog 100713) from BioLegend and PerCPcy5.5-CD3 (catalog 45-0031-82), PE-FoxP3 (catalog 12-4771-82), and Alex Fluor 647 anti–rat IgG2a (catalog 407511) from eBioscience. The remaining SVF was used for an ATT activation assay to assess whether PD1 blockade restored ATT inflammatory potential.

Three days prior to primary vaccination, mice were treated with 500 μg of In Vivo Plus anti-mouse CD8a antibody (53-6.7, BioXCell, Lebanon, NH, USA, Cat. #BP0004-1) injected IP (100 μL in PBS) followed by weekly injections of 250 μg. In Vivo Plus rat IgG2a isotype control, anti-trinitrophenol (clone 2A3, BioXCell Cat. #BP0089) was administered as a control. Two to three days post-treatment, single-cell suspensions from peripheral blood were isolated and stained as described above for CD8 depletion analysis.