Abstract Text: The molecular mechanisms underlying regulatory T cell (Treg)-mediated modulation of early cancer development are underexplored. The Notch ligand Jagged1 (Jag1), highly expressed in skin Tregs, is critical for facilitating hair follicle (HF) regeneration by mediating HF stem cell activation. Here, we aim to elucidate the role of Jag1pos Tregs in mediating tumour infiltrating (TI)-immune cell and progenitor cell activity during early melanoma development. We mined public single cell RNA-sequencing (scRNA-seq) dataset ofTI-immune cells in D5, D8 and D11 B16F10 melanoma to asess Jag1pos Treg abundance during tumour development. B16F10 cells were subcutaneously implanted into the right flank of Foxp3eGFP-Cre/ERT2Jag1fl/fl (homozygous flox) mice at day 14 after tamoxifen (0.1 mg/g of body weight/ day) i.p. induction for 5 consecutive days (days 1–5). Flow cytometric immunophenotyping was performed to compare the activation status of Tregs and other TI-immune effector cells between Jag1pos Treg-sufficient and -deficient melanoma tumours in vivo. scRNA-seq analysis revealed highest levels of Jag1pos Tregs in early tumours, in line withour flow cytometric data. Intriguingly, these intratumoral Jag1pos Tregs also showed higher Ki67 expression than their Jag1neg counterparts. Conditional deletion of Jag1 on Tregs attenuated tumour growth by 3.5-fold in tamoxifen-treated homozygous flox mice relative to Foxp3eGFP-Cre/ERT2Jag1fl/wt (heterozygous flox) controls at day 15 post tumour-implantation. Analysis of tumour cell digests revealed a decrease of Ki67 expression in total Tregs in homozygous flox mice compared to heterozygous flox controls. Our results suggest that Jag1 may be necessary for the initial expansion of Tregs in early TME driving tumour progression.
