Transcriptome analysis of virus-specific H2-Db-GP33-41+CD8+ T cells from WT or PSGL-1 KO mouse spleens at day 9 post LCMV Cl 13 infection

Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1) that is upregulated on responding T cells. PSGL-1-deficient mice unexpectedly cleared the virus due to dramatic increases in the intrinsic survival of multifunctional effector T cells that had downregulated PD-1 and other inhibitory receptors. Notably, this response resulted in CD4+ T cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8+ T cells inhibited TCR and IL-2 signaling, and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of malignant melanoma where T cell dysfunction occurs, PSGL-1-deficiency led to PD-1 downregulation, improved T cell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology, and also functions as a checkpoint that regulates T cell responses in the tumor microenvironment. WT or PSGL-1 KO mice were infected with 2 x 10^6 PFU LCMV Clone 13. Spleens from 10 WT or 10 PSGL-1 KO animals were pooled and processed. CD8+ T cells were negatively enriched from WT or PSGL-1 KO spleens (EasySep Stemcell). Purified T cells were stained with propidium iodide (PI) for 10 minutes on ice, cells were washed. CD8+ T cells were stained with H2-Db-GP33-41 tetramers (NIH) and FACS sorted (BD FACS Aria). Sorted tetramer+ cells were PI negative and purity was >98%. Experiment was repeated twice to generate 2 WT (WT 1; WT 2) and 2 PSGL-1 KO (KO 1; KO 2) samples that represented 10 pooled spleens per sample.