Single cell transcriptomic analysis for a better understanding of human CD8+ regulatory T cells
Céline Serazin1,2,3, Léa Flippe1,2,3, Dimitri Meistermann1,2,3, Séverine Bézie1,2,3, Cynthia Fourgeux1,2,3, Laurent David1,2,3, Carole Guillonneau1,2,3.
1Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, University of Nantes, Nantes, France; 2Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France; 3LabEx IGO “Immunotherapy, Graft, Oncology”, University of Nantes, Nantes, France
One major goal in transplantation is to develop novel specific and non-toxic anti-rejection immunotherapies. Strategies based on regulatory T cells (Tregs) are promising to prevent graft rejection. Our group has shown that rat and human CD8+CD45RClow/- Tregs display significant suppressive function. The team has also shown that cell therapy using human CD8+CD45RClow/- Tregs was efficient to prevent graft rejection and GVHD in humanized NSG mice models. However, the heterogeneity of the CD8+CD45RClow/- Tregs population is important from a phenotypic point of view, suggesting that either a fraction of the population is tolerogenic, or the induction of tolerance is due to a combination of cells forming an "immunological niche". In order to be able to discriminate between these two hypotheses, we explored heterogeneity inside the CD8+CD45RClow/- T cell population.
We sorted CD8+CD45RClow/- Tregs from blood of healthy volunteers and sequenced their transcriptomes by single cell RNA sequencing methods using two technologies: one based on microfluidic systems (Fluidigm C1) and one based on droplets (10x Genomics). To our knowledge those are the first single cell RNA-seq datasets of human CD8+ Tregs. The cell transcriptomes analysis with Seurat package in R highlighted the heterogeneity inside the population with the identification of 4 distinct clusters with a specific signature. We identified one cluster of particular interest based on its immune and regulatory gene signature. However, other genes such as TGFβ were expressed by all clusters inside CD8+CD45RClow/- T cells. We then focused our analysis on genes that encode for membrane proteins that have never been used for CD8+ Tregs isolation yet such as TNFR2. We confirmed their expression at protein level on human PBMC from healthy volunteers by a subset of CD8+CD45RClow/- T cells. Finally, we sorted cell subsets according to the expression of membrane markers to test their suppressive function in vitro.
In conclusion, this project characterized the heterogeneity inside CD8+CD45RClow/- Tregs both at single cell level and proteomic level. This project will help us to better define a consensus phenotype for CD8+ Tregs and will also improve the use of human CD8+ Tregs as a cell therapy for treating transplanted patients.
Key words: single cell RNA sequencing, CD8+ regulatory T cells, cell therapy.
