An amphiregulin reporter mouse enables transcriptional and clonal expansion analysis of reparative lung Tregs
Lucas F. Loffredo, … , Arnold Han, Nicholas Arpaia
Lucas F. Loffredo, … , Arnold Han, Nicholas Arpaia
Published July 8, 2025
Citation Information: JCI Insight. 2025;10(13):e187245. https://doi.org/10.1172/jci.insight.187245.
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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 4

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

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scTCR-Seq of Areg-producing and non-Areg-producing lung Tregs from IAV- ...
(A) Chao1 diversity scores for lung Treg TCR repertoires (separate analysis for CDR3α and CDR3β nucleotide-level sequences) of individual mice across different datasets. (B) Pie charts representing the clonal expansion status of Tregs within each dataset using nucleotide-level CDR3α/CDR3β sequences. (C) Pie charts as in A, broken down by Thy1.1– vs. Thy1.1+ status of Tregs (control mice not included, due to their being all Thy1.1–). (D) Stacked bar plots representing the clonal expansion status of Tregs within each dataset using nucleotide-level CDR3α/CDR3β sequences, subdivided by subgroup as determined from previous clustering (Supplemental Figure 4E and Supplemental Figure 5D). Cycling and undefined/intermediate subgroups not included. (E) UpSet plot depicting sharing of nucleotide-level CDR3α/CDR3β sequences between subgroups of Tregs from bleomycin 21 dpi. Connections between dots indicate sharing between subgroups. (F) Sharing of amino acid–level CDR3α/CDR3β sequences between mice in each treatment group. Numbers over bars indicate total number of TCRs shared by n mice. Whether graphs consider any clones (including unexpanded, single clones), expanded clones (≥2 clones in individual mice), or highly expanded clones (≥10 clones in individual mice) is indicated. Inset/table: specific CDR3α/CDR3β sequences that are expanded (≥2 clones in each mouse) in at least 3 mice from bleomycin 21 dpi, sharing with any clones from the other treatment datasets, and generation probabilities (Pgen) (calculated using the OLGA algorithm). Red: TCR clones found in separate reports (see Results). (G) Volcano plot of DEGs between highly expanded Tregs (≥10 clones in any individual mouse at the nucleotide CDR3α/CDR3β level) vs. unexpanded Tregs, in the bleomycin 21 dpi Ccr8 subgroup. Red dots: significantly differentially expressed (FDR adj. P < 0.05). No fold-change cutoff. Numbers of significantly upregulated and downregulated genes indicated on plots. Mean ± SEM displayed on graphs. Statistical analysis was done using Bonferroni’s multiple comparisons test. *: 0.01 < P < 0.05, **: 0.001 < P < 0.01, ***: 0.0001 < P < 0.001.