Quietly hiding: PD-L1 checkpoint expression in melanocyte stem cell quiescence and aging

Cellular quiescence is a reversible and tightly regulated stem cell function that is essential for healthy aging. However, the control elements of tissue-specific renewal, quiescence, and aging remain poorly understood. Using melanocyte stem cells (McSCs) to model and test the regulation of tissue-specific quiescence, we find that stem cell quiescence is neither a singular nor static process. McSCs display remarkable heterogeneity, with a fraction expressing the immune checkpoint protein PD-L1. Differential gene expression profiling identifies tissue-specific control of this immune privilege, specifically during melanocyte stem cell dormancy. In vitro quiescence assays confirm that inducing quiescence is sufficient to drive PD-L1 expression in melanoblasts and PD-L1 subsequently regulates aspects of melanoblast cell cycling. In vivo, a portion of McSCs appear to leverage this immune checkpoint as a key aspect of their dynamics during the dormant stage of the hair cycle. With age, this dynamic becomes unbalanced, tipping towards a higher proportion of PD-L1-expressing McSCs and a more deeply quiescent McSC pool. Collectively, these findings demonstrate that immune checkpoint expression is a physiological attribute of McSC quiescence and offer PD-L1-expressing quiescent stem cells as molecular targets for potential reactivation in regenerative and gerontological medicine. Overall design: Dermis from postnatal day 0.5, 8-10-week, and 24-month mouse body skin was dissociated and processed using flow cytometry to obtain enriched populations of melanoblasts, adult quiescent melanocyte stem cells (qMcSCs) and aged qMcSCs, respectively. Cells of the melanocyte lineage were identified by their surface expression of the receptor KIT and the absence of the immune cell marker CD45. Melanoblasts in the P0.5 dermis are located within developing hair follicles. Enrichment for qMcSCs at both the adult and aged timepoints was achieved by isolating KIT+CD45- cells from mouse skin whose hair was in the telogen stage of the hair cycle. McSC enrichment at telogen is dependent on the fact that telogen hairs contain only McSCs and do not contain differentiated melanocytes. The telogen stage of the hair cycle was confirmed by the pinkness of the skin. Female mice were used for all melanoblast and McSC enrichments as it is technically challenging to dissociate and obtain cells of high quality from male skin. Melanoblasts and McSCs from several animals were pooled to generate enough RNA to serve as one biological replicate. Amplified cDNA was created from 20 ng total RNA using the Ovation RNA-Seq System V2 (Tecan Genomics). The cDNA was fragmented using a Covaris E210 before proceeding to library construction with 1000ng cDNA using TruSeq RNA Sample Prep Kit, version 2 (Illumina) using 10 PCR cycles. Libraries were pooled in equimolar ratio and sequenced together on a HiSeq 2500 with version 3 flow cells and sequencing reagents. At least 60 million 126-base read pairs were generated for each library. Data were processed using RTA 1.18.64 and CASAVA 1.8.2. RNA-Seq reads that passed the Illumina platform quality check were used for downstream analyses.