DOI: 10.5061/dryad.9s4mw6msg
title: Meningeal regulatory T cells inhibit nociception in female mice

description: T cells have emerged as orchestrators of pain chronification, but the
 mechanism by which T cells control pain processing is unresolved. Here, we
 demonstrate an influence of regulatory T cells (Tregs) on nociception that
 is distinct from their canonical functions of immune regulation and tissue
 repair. Site-specific depletion or expansion of meningeal Tregs (mTregs)
 leads to profound female-specific and sex hormone-dependent modulation of
 mechanical sensitivity. Specifically, mTregs produce the endogenous opioid
 enkephalin that exerts an anti-nociceptive action through the delta opioid
 receptor expressed by MrgprD+ sensory neurons. Although enkephalin
 restrains nociceptive processing, it is dispensable for Treg-mediated
 immunomodulation. Together, our findings uncover a fundamental sexually
 dimorphic immunological circuit that restrains nociception and establish
 Tregs as sentinels of pain homeostasis.
description: # **# Regulatory T cell-derived enkephalin gates nociception**
 \[[https://doi.org/10.5061/dryad.9s4mw6msg](https://doi.org/10.5061/dryad.9s4mw6msg)]([https://doi.org/10.5061/dryad.9s4mw6msg](https://doi.org/10.5061/dryad.9s4mw6msg)) ## ## Description of the data and file structure We provide raw data of main figures 1 to 4 organized by figure number. ### Figure 1 Fig 1C: Number of tissue Tregs across organs, in both sexes combined. Treg numbers were normalized to organ weight (g). Fig 1D: Number of tissue Tregs from male and female mice across organs. Treg numbers were normalized to female Treg numbers. Fig 1F: Number of tissue Tregs 2 days after a single IT pegDT injection across organs in female mice. Treg numbers were normalized to vehicle-injected Treg numbers. 15 represents mean number of tissue Tregs in IT vehicle-injected mice per organ. Fig 1G: Nociceptive thresholds following mTreg depletion in female and male mice. Fig 1H: Behavioral differences between control and mTreg depleted mice in female and male mice. ### **Figure 2** Fig 2C: Percent response to 0.008 g von Frey filament in mTreg depleted female mice with SNI. Fig 2D: Percent response to 0.008 g von Frey filament in mTreg depleted male mice with SNI. Fig 2F: Number of tissue Tregs in the spinal cord meninges following mTreg expansion using IL-2, in both sexes combined. Treg numbers were normalized to organ weight (g). Fig 2G: Nociceptive thresholds following mTreg expansion in female mice with SNI. Fig 2E: Nociceptive thresholds following mTreg expansion in male mice with SNI. Fig 2J: Nociceptive thresholds following mTreg depletion in uninjured Four Core Genotype female and male mice. Fig 2K: Percent baseline nociceptive thresholds of the data in Fig. 2J. Fig 2L: Nociceptive thresholds following mTreg expansion in Four Core Genotype female mice with SNI. Fig 2M: Nociceptive thresholds following mTreg expansion in Four Core Genotype male mice with SNI. Fig 2N: Anti-nociceptive efficacy of the data in Fig. 2L and M. Fig 2P: Nociceptive thresholds following mTreg expansion in ovariectomized female mice with SNI injected with estrogen or progesterone or estrogen antagonist. Fig 2Q: Anti-nociceptive efficacy of the data in Fig. 2P. ### **Figure 3** Fig 3F: Number of tissue Tregs across organs in the spinal cord meninges in female and male mice following mTreg expansion using IL-2. Treg numbers were normalized to organ weight (g). Fig 3G: Enkephalin levels in mouse CSF following mTreg depletion or exapansion.Enkephalin levels were normalized to CSF volume collected (ml). Fig 3H: Nociceptive thresholds following mTreg depletion in uninjured female PenkCreRosa26DTR bone marrow chimera mice. Fig 3I: Nociceptive thresholds following mTreg depletion in uninjured male PenkCreRosa26DTR bone marrow chimera mice. Fig 3J: Nociceptive thresholds following mTreg depletion in nerve injured female PenkCreRosa26DTR bone marrow chimera mice. Fig 3K: Nociceptive thresholds following mTreg depletion in nerve injured male PenkCreRosa26DTR bone marrow chimera mice. Fig 3L: Nociceptive thresholds following Treg specific enkephalin depletion in uninjured female Foxp3CreERT2;Penkfl mice. Fig 3M: Nociceptive thresholds following Treg specific enkephalin depletion in nerve injured female Foxp3CreERT2;Penkfl mice. Fig 3N: Nociceptive thresholds following mTreg specific enkephalin depletion in uninjured female Foxp3-DTR:PenkCreRosa26DTR mixed marrow chimera mice. Fig 3O: Nociceptive thresholds following mTreg specific enkephalin depletion in nerve injured female Foxp3-DTR:PenkCreRosa26DTR mixed marrow chimera mice. ### **Figure 4** Fig 4b: Anti-nociceptive efficacy of mTreg expansion following naltrindole IT injection in nerve injured female mice. Fig 4C: Nociceptive thresholds and anti-nociceptive efficacy following mTreg expansion in Oprd1 flox female mice with SNI injected with AAV-Cre targeting the PNS. Fig 4D: Nociceptive thresholds following mTreg expansion in Oprd1 flox female mice with SNI injected with AAV-Cre targeting the CNS. Fig 4E: Expression from DRG sensory neurons clusters from combined GSE139088 and GSE201653. Expression was normalized by row. Fig 4K: Area under the curve of DRG neuron calcium imaging following von Frey stimulation following mTreg expansion in nerve injured MrgprdDCre-ERT2;GCaMP6f female mice. Fig 4L: Percent response to 0.008 g von Frey fiber stimulation after SNI in MrgprD-CreERT2;Oprd1fl female mice. Fig 4M: Nocifensive behaviors following 0.008 g von Frey fiber stimulation after SNI in MrgprD-CreERT2;Oprd1fl female mice. Fig 4N: Anti-nociceptive efficacy of Deltorphin IT injection in nerve injured MrgprD-CreERT2;Oprd1fl female mice. LMC: Littermate controls Fig 4O: Nociceptive thresholds following mTreg expansion in nerve injured MrgprD-CreERT2;Oprd1fl female mice. Fig 4P: Anti-nociceptive efficacy of mTreg expansion in MrgprD-CreERT2;Oprd1fl female mice.LMC: Littermate controls Experimental data files of main figures. Each .xls contains main figure files delineated by individual sheets corresponding to a subpanel.  ### Analyzed data file DRG data.xls includes analyzed data set showing the number of neurons with* Oprd1* expression of &gt;1 from Figure 4E-G.  There is an analyzed normalization of RNA expression amongst activated, resting Tconv and Treg from Figure 3A-B.  There are 4 ATAC-seq bedwig files of concocted samples of activated, resting Tconv and Treg from Figure 3C.  ## ## Acknowledgements We would like to acknowledge the following prior work and publicly available datasets from: J. van der Veeken, A. Glasner, Y. Zhong, W. Hu, Z.-M. Wang, R. Bou-Puerto, L.-M. Charbonnier, T. A. Chatila, C. S. Leslie, A. 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