An amphiregulin reporter mouse enables transcriptional and clonal expansion analysis of reparative lung Tregs
Lucas F. Loffredo, Katherine A. Kaiser, Adam Kornberg, Samhita Rao, Kenia de los Santos-Alexis, Arnold Han, Nicholas Arpaia
Lucas F. Loffredo, Katherine A. Kaiser, Adam Kornberg, Samhita Rao, Kenia de los Santos-Alexis, Arnold Han, Nicholas Arpaia
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Research Article Cell biology Immunology

An amphiregulin reporter mouse enables transcriptional and clonal expansion analysis of reparative lung Tregs

Abstract

Regulatory T cells (Tregs) are known to play critical roles in tissue repair via provision of growth factors, such as amphiregulin (Areg). Areg-producing Tregs have previously been difficult to study because of an inability to isolate live Areg-producing cells. In this report, we created a reporter mouse to detect Areg expression in live cells (AregThy1.1). We employed influenza A and bleomycin models of lung damage to sort Areg-producing and non-Areg-producing Tregs for transcriptomic analyses. Single-cell RNA-Seq revealed distinct subpopulations of Tregs and allowed transcriptomic comparisons of damage-induced populations. Single-cell TCR sequencing showed that Treg clonal expansion was biased toward Areg-producing Tregs and largely occurred within damage-induced subgroups. Gene module analysis revealed functional divergence of Tregs into immunosuppression-oriented and tissue repair–oriented groups, leading to identification of candidate receptors for induction of repair activity in Tregs. We tested these using an ex vivo assay for Treg-mediated tissue repair, identifying 4-1BB agonism as a mechanism for reparative activity induction. Overall, we demonstrate that the AregThy1.1 mouse is a promising tool for investigating tissue repair activity in leukocytes.

Authors

Lucas F. Loffredo, Katherine A. Kaiser, Adam Kornberg, Samhita Rao, Kenia de los Santos-Alexis, Arnold Han, Nicholas Arpaia

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Figure 3

scRNA-Seq of Areg-producing and non-Areg-producing lung Tregs from IAV- or bleomycin-treated mice.

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scRNA-Seq of Areg-producing and non-Areg-producing lung Tregs from IAV- ...
(A) Schematic of scRNA-Seq experiments using Thy1.1+ vs. Thy1.1– lung Tregs from IAV and bleomycin models (and control saline-treated, all Thy1.1–). (B) UMAP of clustered cells from sc gene expression analysis. (C) UMAP from B, split by treatment status of Tregs. Circles highlight groups that are largely specific to each type of tissue damage model (IAV or bleomycin). (D) Left: Feature plots of select genes uncovered by marker gene analysis of groups from B. Using these genes and/or by presence in a specific model (IAV or bleomycin), identities were assigned to groups of cells (reassigned UMAP on right). (E) Left: Volcano plot of DEGs between induced Treg subgroups from the bleomycin model (“Bleo. Rorc” and “Bleo. Ccr8” combined) vs. from the IAV model (“IAV Rorc” and “IAV Ccr8” combined). Red dots: significantly differentially expressed (FDR adj. P value < 0.05). No fold-change cutoff. Numbers of significantly upregulated and downregulated genes indicated on plots. Right: Enrichment plots of the top 2 significant pathways (FDR q value < 0.05) from GSEA of this gene signature, using Hallmark curated gene sets. (F) Proportions of each assigned subgroup in Thy1.1+ vs. Thy1.1– Tregs in each model (Supplemental Figure 2E, separate reclustering from each model, assignment to subgroups in these plots). (G) Volcano plots of DEGs from Thy1.1+ vs. Thy1.1– induced Tregs (Ccr8 and Rorc subgroups combined) from IAV and bleomycin models. Red dots: significant DEGs (FDR adj. P value < 0.05). No fold-change cutoff. Numbers of significantly upregulated and downregulated genes indicated on plots. (H) Pathway analysis using GSEA, in full gene signatures from IAV or bleomycin datasets depicted in G (from Hallmark curated gene sets). All pathways displayed are significant at FDR q value < 0.05.