IL-2 and IL-15 drive intrathymic differentiation of functionally distinct regulatory T lymphocyte-clusters (CITEseq)
Purpose: Intrathymic development of Foxp3-expressing regulatory T lymphocytes (Treg) depends on signals from the TCR, cytokine-receptors, and other cell-surface receptors. The cytokines IL-2 and IL-15 have been suggested to quantitatively modulate Treg-development. However, recent evidence showed that very large proportions of thymic Treg are cells that had migrated from the periphery back to the thymus. It remained therefore unclear if IL-2 and IL-15 quantitatively control the development of Treg. Moreover, these cytokines may also contribute to the differentiation of functionally distinct Treg subsets. Using an experimental setting in which we unambiguously distinguish newly developing from recirculating Treg, we here address the quantitative and qualitative roles of IL-2 and IL-15 in Treg-development, selection, and differentiation. Our results definitively demonstrate that in absence of IL-2 or IL-15 the thymus produces substantially less Treg. Importantly, IL-2 and IL-15 drive the differentiation of transcriptionally and phenotypically distinct Treg clusters, the number of which is substantially higher than previously appreciated. These cytokines also differentially modulate the TCR-repertoire expressed by newly developing Treg. Adoptive transfer of Treg from wt, IL-2- or IL-15-deficient animals into Treg-deficient new-born Scurfy mice, revealed differences in the in vivo function of these cells. Combined, our results indicate that IL-2 and IL-15 drive the development of functionally complementary Treg clusters. Methods: Sample preparation and staining were performed using standard procedures. Labelled cells were acquired using an LSRII or a Fortessa flow cytometer and the data analysed using FlowJo software. Doublets and dead cells were excluded from the analysis by using appropriate FSC/SSC gates. 13-color flow-cytometry acquisition was performed on a LSRII SORP platform. For the tSNE analysis on 13-color cytometry panels, the Fortessa flow-cytometer was standardized allowing perfect reproducibility between samples analysed on different days. The “Phenograph” plugin of the FlowJo software was used to determine different Treg-subsets in a non-overseen manner. Thymocytes from three-week-old Rag2-Gfp Foxp3-Thy1a B6 mice were stained with the oligonucleotide-tag-labelled antibodies, GFP+CD4+CD8-S1P1+Thy1.1+ (for CITEseq) cells were FACS-sorted and processed using the 10X Genomics platform according to the manufacturer's protocols. Libraries were sequenced using the Illumina hiSeq platform. Results: To investigate in more detail the respective roles of IL-2 and IL-15 in the differentiation of Treg-subsets, we first searched for cell-surface markers allowing their identification. We verified that thus identified markers were expressed at varying levels on newly developing Treg and then used them to identify Treg-clusters. We thus found five CD25high and six CD25-/low clusters among the Treg ready to leave the thymus (i.e. S1P1+CD69low). Given their very mature phenotype, it appears quite unlikely that these CD25-/low clusters are precursors of CD25+ Treg. Among RTE we identified six CD25-/low Treg-clusters, indicating the reality of the thymic production of multiple distinct CD25-/low Treg-clusters. CITEseq analysis suggested that the identified distinct Treg clusters have functionally distinct potentials: They differed in expression of gene-sets involved, most notably, in T cell-differentiation in the periphery and in immune-tolerance. Further work will be required to investigate in greater detail the functional differences between the Treg-clusters. Conclusions: Based on the CITEseq antibody-signals, we performed Phenograph analysis and thus identified 11 Treg-clusters. One of these clusters expressed high levels of CD69, should therefore be considered not totally mature, and was excluded from further analysis. To assess differences in functional potential between the thus identified Treg clusters, we performed a gene ontology analysis of the genes differentially expressed between them. Overall design: Gene expression and cell surface protein exploring the thymocyte populations.