PPARγ marks splenic precursors of multiple nonlymphoid-tissue Treg compartments

Abstract

Foxp3⁺CD4⁺ regulatory T cells (Tregs) regulate most types of immune response as well as several processes important for tissue homeostasis, for example, metabolism and repair. Dedicated Treg compartments-with distinct transcriptomes, T cell receptor repertoires, and growth/survival factor dependencies-have been identified in several nonlymphoid tissues. These Tregs are specifically adapted to function and operate in their home tissue-When, where, and how do they take on their specialized characteristics? We recently reported that a splenic Treg population expressing low levels of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) contains precursors of Tregs residing in visceral adipose tissue. This finding made sense given that PPARγ, the "master regulator" of adipocyte differentiation, is required for the accumulation and function of Tregs in visceral adipose tissue but not in lymphoid tissues. Here we use single-cell RNA sequencing, single-cell Tcra and Tcrb sequencing, and adoptive-transfer experiments to show that, unexpectedly, the splenic PPARγ^lo Treg population is transcriptionally heterogeneous and engenders Tregs in multiple nonlymphoid tissues beyond visceral adipose tissue, such as skin and liver. The existence of a general pool of splenic precursors for nonlymphoid-tissue Tregs opens possibilities for regulating their emergence experimentally or therapeutically.

Keywords: immunoregulation; precursor; single-cell RNA-seq; tissue Treg cell.

Fig. 1.

Heterogeneity of the splenic PPARγ^lo Treg population. scRNA-seq analysis of double-sorted PPARγ⁻ and PPARγ^lo Tregs from the spleen of 6- to 8-wk-old Pparg-Tdt.Foxp3-Gfp mice. (A) tSNE plot of the combined (Left), PPARγ⁻ Treg (Middle), and PPARγ^lo Treg (Right) single-cell datasets. Numbers indicate the frequency of each population. (B) Heatmap of the 10 most differentially expressed transcripts among the three clusters.

Fig. 2.

Transcriptionally assessed precursor potential of the splenic PPARγ^lo Treg subpopulations. (A) Violin plots of transcripts characteristically up-regulated as tissue-Treg precursors exit the spleen. Numbers refer to the percentage of cells in the designated cluster that express a given transcript. A.U., arbitrary units. (B) Violin plots of transcripts encoding proteins that need to be down-regulated for tissue-Treg precursors to exit the spleen. Numbers refer to the percentage of cells in the designated cluster expressing a given transcript. (C) Heatmaps depicting up to egress (Left) and down to egress (Right) scores, determined per SI Appendix, Materials and Methods. Intensity scales (Bottom). (D) Heatmap depicting expression of VAT (Left), liver (Middle), and skin (Right) Treg-specific transcripts, as per Dataset S1. Intensity scales (Bottom). (E) RNA-velocity analysis of the splenic PPARγ^lo and PPARγ⁻ scRNA-seq datasets. (F) Trajectory inference analysis of the splenic PPARγ^lo and PPARγ⁻ scRNA-seq datasets represented as a partition-based graph abstraction plot. Pie charts indicate the terminal cell fates averaged in each cluster. The edges show the direction of the inferred trajectory, and the thickness represents the transcriptional similarity between clusters.

Fig. 3.

No PPARγ requirement for accumulation of the putative tissue-Treg precursor population. (A) Frequencies of KLRG1⁺ and ST2⁺ cells among PPARγ⁻ and PPARγ^lo splenic Tregs of 6- to 8-wk-old Pparg-Tdt.Foxp3-Gfp mice. (A, Left) Representative dot plots. (A, Right) Summary plots. Mean ± SD; unpaired two-tailed t test; ****P < 0.0001. (B–D) Frequencies of Tregs among CD4⁺ T cells (B), KLRG1⁺ cells among Tregs (C), and ST2⁺ cells among Tregs (D) from spleens of 6- to 8-wk-old Pparg^+/+.Foxp3-Cre or Pparg^f/f.Foxp3-Cre mice.

Fig. 4.

scTcr-seq analysis of splenic Treg splits and tissue-Treg populations. (A) UMAP of scRNA-seq data from the PPARγ⁻ and PPARγ^lo splenic Treg populations and from the indicated tissue-Treg populations from a 20-wk-old Pparg-Tdt.Foxp3-Gfp male. (B) Pie charts were generated from corresponding scTcr-seq data. For each cell cluster, clones of cells sharing the exact same nucleotide sequence in the CDR3-encoding regions of the Tcra and Tcrb genes are shown as differently colored pie slices, the width of the slice reflecting the number of cells in that clone. Shared colors across cell clusters do not indicate shared clones in this representation. Values above show the total number of cells within that cluster with identified TCR-α and -β chains. Percentages and offset portions of the pies show the frequencies of clonally expanded cells, that is, cells that had a TCR sequence shared by two or more cells. (C) Linking scRNA-seq and scTcr-seq. UMAP space as in A. Cells are colored according to their clone size; color key (Bottom). Spl., spleen.

Fig. 5.

Sharing of TCR sequences between tissue-Tregs and PPARγ^lo putative precursors in the spleen. (A–C) Pie charts depicting cell clones shared between the various tissue-Treg populations and the splenic clusters. Offset pie slices and text below the pies indicate the number and frequency of splenic Tregs in the resting (Left), precursor X (Middle), and precursor Y (Right) clusters that share CDR3-encoding Tcra and Tcrb sequences with the VAT (A), liver (B), and skin (C) Treg populations. Shared clones are shown in individual colors; nonshared clones are colored in gray. Colors shared across the splenic subpopulations do not connote shared sequences. (D) Tissue-Treg signature scores for splenic PPARγ^lo Tregs and VAT (Top), liver (Middle), and skin (Bottom) Tregs. Plotted for cells with sharing (blue) or nonsharing (orange) CDR3-encoding Tcra and Tcrb nucleotide sequences between splenic PPARγ^lo Tregs and the indicated tissue-Tregs. Box plots indicate the interquartile range (IQR), and the line indicates the median. Whiskers indicate the range, and outliers outside this range (>1.5*IQR) are plotted. Wilcoxon rank-sum test; **P ≤ 0.01, ***P ≤ 0.001.

Fig. 6.

Adoptive-transfer studies. PPARγ^lo Tregs from spleen and lymph nodes of 6- to 8-wk-old CD45.1⁻2⁺ Pparg-Tdt.Foxp3-Gfp mice were sorted and transferred into 10-wk-old CD45.1⁺2⁺ Foxp3-Dtr recipients treated with DT before, on, and after the day of transfer. i.v., intravenously; LN, lymph nodes; w, week. (A) Experimental schema. (B) Representative dot plot showing frequencies of donor-derived cells among Tregs in the indicated organs. Values refer to the fraction of Tregs in the adjacent gate. (C) Summary plot. Mean ± SD. Representative of two independent experiments.