A novel model to study deficiency of RORγt+ Tregs reveals net suppressive function and dependency on the CCR6/CCL20 axis for expansion and activation

Background: Recently, Tregs expressing the Th17 characteristic transcription factor RORγt were identified (biTregs). Both, pro- and anti-inflammatory functions have been described and the sum effect of biTreg deficiency in inflammation remains unknown. Furthermore, it is unclear, whether biTregs are specialized for the control of a defined type of immune response, as in particular Th2 or Th17 immunity. Methods: FACsorted CD4+ T-cells from fluorescence reporter mice, containing or lacking biTregs, were transferred into RAG1-/- recipients and the nephrotoxic nephritis model of glomerulonephritis was studied. Interactions of biTregs with various leukocyte populations were assessed by multicolor immunofluorescence. Furthermore, biTregs were compared to other Treg subtypes including suppression assays and RNAseq analyses. Finally, the role of the CCR6/CCL20 axis for biTreg biology was characterized. Results: Lack of endogenous biTregs resulted in aggravation of glomerulonephritis, demonstrating net immunosuppressive functions. However, contrary to our hypothesis, biTreg deficiency did not result in selectively overshooting Th2 or Th17 responses. Comprehensive analysis instead revealed a unique functional and transcriptional biTreg profile, including high production of IL-10 and dependency on IL-6R, cMAF and IKZF3 activation. Interestingly, we found that expression of the chemokine receptor CCR6 marked a particularly activated biTreg subset, whose absence significantly worsened glomerulonephritis. Unexpectedly, we found that signaling via the CCR6/CCL20 axis mediates biTreg expansion, rather than trafficking. Our data indicate net immunosuppressive effects of endogenous biTregs, with no preference for regulation of Th2 or Th17 responses. Surprisingly, signaling via the Conclusion: CCR6/CCL20 axis was integral for biTreg expansion and activation, which offers novel therapeutic options.