Lag3 and PD-1 pathways regulate NFAT-dependent TCR signalling programmes during early CD4+ T cell activation
Here we perform QuantSeq 3' mRNA sequencing of RNA extracted from flow sorted splenic CD4+ T cell from Tg4 Nr4a3-Tocky Tiger mice that been reactivated in the presence of Isotype, anti-PD-L1, anti-Lag3 or combination therapy. Transcriptional analysis reveals strong T cell receptor signalling, T helper cell bias and metabolic priming in response to combination therapy. Overall design: Tg4 Nr4a3-Tocky Il10-GFP mice were immunized through subcutaneous injection of 4 mg/kg [4Y] MBP peptide PBS into the flank. 24 hrs later antibodies were administrated via the intraperitoneal (i.p.) route with isotype control (pool or rat IgG1 and rat IgG2a), 0.5 mg of anti-PD-L1 antibody (clone MIH5 73), in vivo grade anti-Lag3 (clone C9B7W) or r combination therapy (anti-PD-L1 and anti-Lag3 pooled at 1:1 ratios).  30 minutes after immunotherapy administration a second dose of 0.4 mg/kg [4Y]-MBP peptide in PBS was administered subcutaneously to the contralateral flank. Mice were then euthanized 12 hours later and CD4+ Nr4a3-Timer Red+ Nr4a3-Timer Blue+ T cells isolated and RNA extracted for 3' mRNA sequencing. 3 biological replicate mice were obtained for the 4 experimental groups. Each sample was run on 4 lanes, resulting in 48 FASTQ files (4 per experimental replicate).