Fluorescence tracking Treg movement identifies anti-CCR8 and radiation as a therapeutic combination

Using 10X Genomics single-cell RNA sequencing, we investigated the migration of regulatory T cells (Tregs) following radiation therapy (RT). Employing the Kaede photoconvertible mouse model, we tracked Tregs migrating from the tumor-draining lymph node (TdLN) to the tumor within the post-RT tumor microenvironment. This study serves to identify how RT influences the migrating Treg phenotypes change within the tumor in order to better inform and improve thearaputic strategies for targeting Tregs in combination with RT. Overall design: MC38 tumors established in Kaede mice. On day 14 post tumor innoculation, TdLNs were photoconverted followed by immediate treatment with 12Gy of radiation. Mice were also injected intraperitoneally with FTY720 on day 15. Tumors were harvested on either 24 hours (day 15) or 72 hours (day 17) post-RT. Tumors were processed into single cell suspensions, magnetically enriched for CD8/CD4 TIL, and stained for with viability dye, CD90.2-Alexa Fluor 700, and Totalseq hashtag antibodies. Kaede green or red live CD90.2+ cells were sorted using a Symphony S6 cell sorter. Each experimental condition had 2 replicates each containing 4 tumors pooled from 4 mice. Samples were immediately processed and libraries were constructed using the Chromium GEM-X Single Cell 5' Reagent v3 protocol. Libraries were sequenced using the Illumina NovaSeq 6000 with the NovaSeq 6000 S1 Reagent Kit (v1.5). All cDNA pools and resulting libraries were measured using the Agilent Fragment Analyzer with the DNF-474 HS NGS Fragment Kit and Qubit High Sensitivity assays.