Single cell characterization of blood and expanded regulatory T cells in autoimmune polyendocrine syndrome type 1

Summary Immune tolerance fails in autoimmune polyendocrine syndrome type 1 (APS-1) because of AIRE mutations. We have used single cell transcriptomics to characterize regulatory T cells (Tregs) sorted directly from blood and from in vitro expanded Tregs in APS-1 patients compared to healthy controls. We revealed only CD52 and LTB (down) and TXNIP (up) as consistently differentially expressed genes in the datasets. There were furthermore no large differences of the TCR-repertoire of expanded Tregs between the cohorts, but unique patients showed a more restricted use of specific clonotypes. We also found that in vitro expanded Tregs from APS-1 patients had similar suppressive capacity as controls in co-culture assays, despite expanding faster and having more exhausted cells. Our results suggest that APS-1 patients do not have intrinsic defects in their Treg functionality, and that their Tregs can be expanded ex vivo for potential therapeutic applications.


Figure S2 .
Figure S2.Flow cytometry expression of FOXP3 after the expansion of Tregs.Related to main Figure 2.

Figure S5 .
Figure S5.Heat map of the top 15 most differentially expressed genes in expanded Tregs between APS-1 patients (N=8) and healthy controls (N=8).Related to main Figure 2.

Figure S7 .
Figure S7.Differentially expressed genes in both freshly sorted and expanded Tregs.Related to main Figure 1 and 2.

Figure
Figure S9.V, D and J gene usage per sample for expanded Tregs from APS-1 patients (N=7).Related to main Figure 3.

Figure
Figure S10.V, D and J gene usage per sample for expanded Tregs from healthy controls (N=9).Related to main Figure 3.

Figure S13 .
Figure S13.The gating strategy for flow cytometry characterization of expanded Tregs.Related to main Figure 4.

Figure S16 .
Figure S16.The gating strategy for CyTOF characterisation of expanded Tregs.Related to main Figure 5.

Figure S17 .
Figure S17.Heat map showing the expression of all CyTOF panel markers for expanded Tregs (N=17 APS-1 patients and 17 healthy controls).Related to main Figure 5.

Figure S20 .
Figure S20.Proliferation and expansion indices in flow cytometry experiments of responder cell with/without co-culture with Tregs.Related to main Figure 6.

Figure S21 .
Figure S21.Measurement of Treg-specific cytokines in expanded Treg suppression assay supernatant.Related to main Figure 6.

Table S9B : Overview of healthy controls and experiments. Control Sex a Age (years) Single-cell sequencing Suppression assay CyTOF characterization Flow characterization
a F, female; M, male.