A meta signature of murine Treg and Penk mRNA expression in lymphoid and non-lymphoid organs.

A) Top 25 genes enriched in Treg compared to Tconv from lymphoid tissue from at least 10 of the 11 datasets analyzed ranked by fold change (LogFC) of mean expression relative to Tconv. The Penk gene is highlighted in red. B) Correlation of Penk and Foxp3 expression in all the cell types listed in the legend according to the Immuno-navigator dataset. The Pearson correlation coefficient is indicated. Each dot is a sample, color coded as a subset according to the legend. C) Expression of Penk in Treg (blue) and Tconv (red) isolated from thymus, visceral adipose tissue (VAT) and muscle. The source of the data is indicated below each graph.

Characteristics of the datasets used for the Treg meta-signature

(NA = not available; LN = Lymph Nodes)

Penk expression is regulated by TNFR signaling and the BATF transcription factor.

A) A network of the genes most correlated to Penk in Treg is shown. The Pearson correlation values were extracted from the Immuno-Navigator database (selecting only Treg in the analysis) and integrated into Cytoscape v3.7 (Shannon et al., 2003). Each node is a gene linked by edges with width proportional to the Pearson correlation. (edge range: 0.538-0.758). B) Illustration of the correlation between expression of Penk and Batf in Treg. Each dot is a sample from the Immuno-Navigator database. The Pearson correlation coefficient is indicated on the figure. C) Penk and Batf mRNA expression after in vitro stimulation of purified Treg with the indicated TNFR agonists prior (0), and at 18 and 36 hrs after stimulation. Each dot is a biological replicate from a single experiment. D) GEO2R analysis of the GSE89656 dataset between wild type control Treg (WT) and BATF-KO Treg.

Penk is predominantly expressed by Treg at steady state.

A-B) (Left) UMAP representing all major cell types indicated in the figures determined by flow cytometry from lymph nodes (A) and colon (B). (Right) Projection of tdTomato expression on the UMAP of lymph nodes and colon. Subsets were manually gated as depicted in Figure S1A. C) Bubble plot displaying the average population size and frequencies of Penk+ cells for the listed cell populations and organs. Population size was calculated as the percentage out of total CD45+ single cells and represented on a log10 scale. (n = 3 mice for the VAT, spleen and bone marrow, n = 6 mice for the other groups; results cumulative of two independent experiments).

Immunosuppressive functions of Treg are unaffected by the lack of enkephalins.

A) In vitro suppression of WT Tconv proliferation by WT or Penk KO Tregs. B) In vitro suppression of WT Tconv proliferation by Penk WT Tregs in presence of Naloxone. C) Body weight and D) survival of Rag2-/- mice transferred with Tconv and Treg from Penk-WT or Penk-KO chimeric mice (2:1 ratio), as described in the methods. All data are cumulative of 2 independent experiments.

Heat hyperalgesia in mice deficient for Penk in Treg.

A) Withdrawal latency of WT and LOX mice before (baseline) and after administration of Tamoxifen (TMX). Each dot corresponds to the mean latency response (in seconds) of 4 measurements taken before TMX administration (baseline) and 4 measurements taken from day 7 onwards (TMX). Statistical modeling was performed using a non-parametric unpaired Mann-Whitney t-test with multiple corrections. The results shown in this figure are cumulative from two independent experiments with a total of 44 mice (26 WT and 18 LOX). Each dot is a mouse. B) Representative flow cytometry contour plot of CD25 and Foxp3 staining on pad skin CD45+CD3+CD4+ cells from WT or LOX mice 17 days after TMX gavage. C) Quantification of the frequency of Treg (Foxp3+CD25+) among CD4+ T cells in WT and LOX mice pad as shown in (B). The indicated p value was determined by a Mann-Whitney test. Each dot is a mouse from a single experiment. D) Immunofluorescence staining of CGRP neurons (red), Foxp3-GFP cells (green), and DAPI (blue) of footpad skin section of a female LOX mouse (scale bar represents 50µm). The right panel is the magnification of the area indicated on the left panel.