Figure 5. Treg-derived enkephalin is dispensable for suppressing inflammation.

(A) Baseline nociceptive thresholds of uninjured Rag2^{+/+, +/− or −/−} female mice. (B) Nociceptive thresholds of female Foxp3-DTR mice injected with pegDT IT + IV clodronate (pink) or control (white) liposomes showing peripheral macrophages do not mediate the nociception induced by mTreg depletion, n=5 per group. (C) Representative flow cytometry histograms of proliferated conventional T cells (Tconv) alone (pink) or 4:1 with WT Tregs (gray), Penk^−/− Tregs (yellow), or unstimulated, un-proliferated cell trace violet-stained control (blue). Histogram shows cells that have not proliferated. (D) Suppression of Tconv cell proliferation by different concentrations of WT Tregs (white) or Penk^−/− Tregs (yellow). (E) Schematic representation of competition experiment showing 1:1 transfer of WT or Penk^−/− T cells into Rag2^−/− mice. SNI surgery was performed and organs were harvested 4 weeks later for F-H. (F) Equal competition of Penk sufficient CD45.1 and Penk deficient CD45.2 CD4⁺ T cells in the meninges represented as a concatenated flow cytometry plot, n=4 per group. Representative flow cytometric plots. (G) Pooled proportion of Penk sufficient CD45.1 and Penk deficient CD45.2 CD4⁺ T cells in different organs, n=4 per genotype. (H) Percent of FoxP3⁺, IFN-ɣ ⁺ and IL-17A⁺ CD4⁺ T cells from G. (I) Representative flow cytometric plots of cytokine secretion by CD4⁺ T cells after GVHD induced by transfer of pre-activated Tconv alone or combined with Penk^+/+ or Penk^−/− Tregs. (J) Weight curves of GVHD mice, n=3–4 per group. ns = not significant, *p<0.05, **p<0.01,***p<0.001. Related to Figure S5.