Regulatory T Cell-Derived TGF- b 1 Controls Multiple Checkpoints Governing Allergy and Autoimmunity

cells differentially control allergic and autoimmune responses remain unclear. We show that Treg cells in food allergy (FA) had decreased expression of transforming growth factor beta 1 (TGF- b 1) because of interleukin-4 (IL-4)- and signal transducer and activator of transcir-iption-6 (STAT6)-dependent inhibition of Tgfb1 transcription. These changes were modeled by Treg cell-spe-ciﬁc Tgfb1 monoallelic inactivation, which induced allergic dysregulation by impairing microbiota-dependent retinoic acid receptor-related orphan receptor gamma t (ROR- g t) + Treg cell differentiation. This dysregulation was rescued by treatment with Clostridiales species, which upregulated Tgfb1 expression in Treg cells. Biallelic deﬁciency precipitated fatal autoimmunity with intense autoantibody production and dysregulated T follicular helper and B cell responses. These results identify a privileged role of Treg cell-derived TGF- b 1 in regulating allergy and autoimmunity at distinct checkpoints in a Tgfb1 gene dose- and microbiota-depen-dent manner. and restraining autoimmunity. Tgfb1 Teff cell and autoantibody The latter phenotype associated with Tfh and germinal center B cell responses and dysregulated DC activation. These were largely absent in Foxp3 YFPcre Tgfb1 D /+ mice, consistent with the capacity of Tgfb1 haploinsufﬁcient Treg cells to restrain these pathogenic autoimmune responses. This dichotomy in the regulation of allergic versus autoimmune responses points to distinct checkpoints governing regulation by Treg cells of the immune responses in a Tgfb1 gene dose-dependent manner. Previous studies have linked deletion of Tgfbr2 in CD4 + T cells with Tfh cell dysregulation by a mechanism involving loss of Treg cell TGF- b 1-mediated suppression of Tfh cells (McCar-ron and Marie, 2014). Our results indicate that Treg cells are a critical source of TGF- b 1 mediating regulation of Tfh cell responses whose loss engenders dysregulated autoantibody production. m L of cold hypotonic lysis buffer Tris-HCl (pH NaCl, MgCl2 0.5% NP40], immediate centrifugation g re-suspension of pellet in m L of transposition reaction of Tagment DNA Enzyme and DNA Sample Prep L O]. which fragmentation tagging De-dupli-cation to tag visual-ization of into format bamCoverage fragmentLength 200 ﬁle Genomics Foxp3 YFPcre or Foxp3 YFPcre Il4ra F709 mice. The cells were then kept for 8 min in 1 mL of 1% para-formaldehyde (PFA; Sigma-Aldrich) at room temperature (RT). Next, the sample incubated with lysis buffer I (5mM PIPES pH 8, 85mM KCl, 0,5% NP40 (Igepal-CA630), Protease Inhibitor) for 20 min at RT, and then centrifuged for 5 min at 7,870 g at RT. The pellet was lysed with lysis buffer II (10mM Tris-HCl pH 7,5, 150mM NaCl, 1% NP40 (Igepal-CA630), 1% DOC (Natrium Deoxycholate), 0,1% SDS, 1mM EDTA, Protease Inhibitor). The chromatin was further sonicated to a length of 25-500 bp using bioruptor (Diagenode, USA) for 35 cycles (30 s on, 30 s off). The chromatin was then precipitated using either IgG mock control, STAT6 (Cell Signaling Tech-nology), H3K4me3 (Active Motif, USA) or H3k27me3 (Active Motif USA) histone methylation overnight. The chromatin was washed the the on the ﬂow cytometry Figure and the bottom for GF+Clostridia and GF+Proteobacteria Figure 3C switched late in the production process, after The correctly placed cytometry panels and plots, as described in the original ﬁgure legend, Figure 3B and 3C. The authors apologize for any