Hiding in Plain Sight: Thymic CD8+FOXP3+Tregs sequester CD25 and are enriched in human tissues

For decades, regulatory T cell (Treg) research has focussed on CD4+FOXP3+ Tregs, while characterisation of CD8+FOXP3+ Tregs has been limited due to their scarcity in blood. Here, by analysing 95 tissue samples from 26 deceased transplant organ donors we show that, despite representing less than 5% of circulating Tregs, CD8+ Tregs are enriched in human tissue, particularly in non-lymphoid tissues and bone marrow. We further show that they are fully demethylated at the FOXP3 TSDR, indicating lineage stability, and demonstrate their presence in human thymic tissue and cord blood. Transcriptomic profiling revealed strong similarities to CD4+ Tregs, however at the protein level, they reside in tissue as surface CD25lo/-CD8+CD69+CD103+TLR9+HELIOS+FOXP3+ cells, expressing CD25 intracellularly. Surface CD25 was rapidly regained ex-vivo, allowing us to sort and expand them, and to subsequently demonstrate their therapeutic potential in a humanised mouse model of graft-vs-host disease. Additionally we report increased circulating CD8+Tregs in individuals with SLE and patients early following traumatic brain injury (TBI), underscoring their functional importance. We conclude that these under-studied cells likely play an essential but previously unappreciated role in maintaining peripheral tolerance. One Sentence SummaryFOXP3+CD8+ Tregs, expressing tissue residency markers and intracellular CD25, are enriched in human non-lymphoid tissues.