Single-cell RNA sequencing on peripheral blood PBMCs from 4T1 mouse model treated with STING agonist deliveredy by PEG biopsy marker does not reveal peripheral T cell clonal expansion.

To explore whether immune escape could be secondary to a failure of STING agonist to induce T-cell clonality and expansion, we performed single-cell RNA sequencing (scRNAseq) and T-cell receptor sequencing (TCR-seq) on peripheral PBMCs from animals treated with PEG markers with PBS or 100 µg ADU-S100, using the low tumor burden model of 4T1. Cell types were clustered based on gene signature analysis with 9 resultant clusters. We then analyzed the frequency of unique clonotypes that were expanded following PBS versus ADU-S100 treatment. In both groups, most of the cells that had a unique clonotype did not expand, showing a frequency of 1. There were no specific clonotypes that had a frequency over 5, indicating that ADU-S100 treatment of 4T1 mice does not result in peripheral T cell clonal expansion. Overall design: Balb/c mice were inoculated with 10K 4T1 cells followed by PEG with PBS or 100 µg ADU-S100 implantation on Day 4. On Day 30, another PEG marker with PBS or 100 µg ADU-S100 was implanted. We then collected peripheral blood from one mouse in each group on Day 50 and performed scRNAseq/TCRseq.