CD8⁺ regulatory T cells (Tregs) were the first suppressive cells reported in 1970, but they were put aside for years due to a lack of markers to properly define them. Our team demonstrated that CD8⁺ Tregs identified by low and/or negative expression of CD45RC, one of the isoforms of the CD45 molecule, show potent suppressive activity in vitro and in vivo, while cells expressing high levels of CD45RC do not. Herein, we addressed the heterogeneity within CD8⁺CD45RC^low/- Tregs and identified new markers.

We performed single cell RNA-sequencing on more than 10000 non-stimulated CD8⁺CD45RC^low/- Tregs of 3 healthy volunteers (HV) and a CD screen analysis of 360 membrane molecules on different cells subsets of 4 HV (CD8⁺CD45RC^high; non-stimulated, stimulated or expanded CD4⁺CD25^highCD127^low and CD8⁺CD45RC^low/- Tregs). Using these technologies, we were able to characterize the transcriptomic heterogeneity at a single cell level and the proteomic heterogeneity from non-stimulated CD8⁺CD45RC^low/- Tregs. We thus identified sub-populations of cells and one cluster of particular interest expressing critical markers and molecules involved in tolerance. Further work on two of these promising membrane markers, i.e., TNFR2 and CD29, using cell sorting and suppressive assays highlighted CD8⁺CD45RC^low/-TNFR2⁺CD29^low Tregs subset as the most suppressive subsets within CD8⁺CD45RC^low/- Tregs. Here, we reveal the importance of the combination of TNFR2 and CD29 as highly specific membrane markers for human CD8⁺ Tregs and highlighted their potential roles as targets for treatments in tolerance. Other new promising markers identified in our study showed an interesting role in CD8⁺ Tregs function; their precise roles are still under investigation. To date, to our knowledge, this is the largest characterization study of human CD8⁺ Tregs, this huge data resource will help in the current revival of CD8⁺ Tregs in research.