BATF is Required for Treg Homeostasis and Stability to Prevent Autoimmune Pathology

Abstract Regulatory T (Treg) cells are inevitable to prevent deleterious immune responses to self and commensal microorganisms. Treg function requires continuous expression of the transcription factor (TF) FOXP3 and is divided into two major subsets: resting (rTregs) and activated (aTregs). Continuous T cell receptor (TCR) signaling plays a vital role in the differentiation of aTregs from their resting state, and in their immune homeostasis. The process by which Tregs differentiate, adapt tissue specificity, and maintain stable phenotypic expression at the transcriptional level is still inconclusivei. In this work, the role of BATF is investigated, which is induced in response to TCR stimulation in naïve T cells and during aTreg differentiation. Mice lacking BATF in Tregs developed multiorgan autoimmune pathology. As a transcriptional regulator, BATF is required for Treg differentiation, homeostasis, and stabilization of FOXP3 expression in different lymphoid and non‐lymphoid tissues. Epigenetically, BATF showed direct regulation of Treg‐specific genes involved in differentiation, maturation, and tissue accumulation. Most importantly, FOXP3 expression and Treg stability require continuous BATF expression in Tregs, as it regulates demethylation and accessibility of the CNS2 region of the Foxp3 locus. Considering its role in Treg stability, BATF should be considered an important therapeutic target in autoimmune disease.


Figure S2 .Figure
Figure S2.BATF in Tregs is required for homeostatic regulation involving both adaptive and innate immunity.Related to Figure 1. A. Dot plots showing the frequency of lymphocytes in the inguinal lymph nodes from Foxp3Cre BatfF/F mice compared to control Foxp3Cre Batf+/+ mice.B. Representative flow plots (left) and quantified dot plots (right) showing frequency of activated CD4 T cells producing IFNγ, IL17, and TNFα in Foxp3Cre BatfF/F mice compared to control (Foxp3Cre Batf+/+) mice.C. Representative flow plots (left) and quantified dot plots (right) showing frequency of activated CD8 T cells producing IFNγ and TNFα in Foxp3Cre BatfF/F mice compared to control mice.D. Representative flow plots (left) and quantified dot plots (right) showing frequency of innate immune cells macrophages and neutrophils in Foxp3Cre BatfF/F mice compared to control mice.These data were from mice around 8 to 11 weeks of age.All data are pooled from at least 3 individual repeats with a minimum of 3-5 mice per group.Data here are shown ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (two tailed unpaired t test)

Figure S4 .
Figure S4.Loss of BATF promotes exTreg phenotype in large intestine.Related to Figure 3. A. Flow cytometry plot (left) and bar plot (right) showing viability of total lymphocytes from large intestine of WLT and TBKLT mice B. Flow cytometry plot (up) and bar plot (down) showing per cell expression (gMFI) of ICOS and IL6ra in Foxp3 + Tregs from large intestine of WLT and TBKLT mice.Foxp3 + expression in Tregs is conferred by direct staining with and detection of intranuclear fluorophore conjugated antibody.These data were from male mice around 9 weeks of age.All data are pooled from at least one experiment with a minimum of 2-3 mice per group.Data here are shown ± SEM.

FigureDFigure S6 .
Figure S5.BATF is needed for iTreg differentiation.Related to Figure 3. A. Experimental design for in vitro iTreg differentiation using naïve CD4 T cells from spleens of WLT and TBKLT mice.B. Frequency of Foxp3+ Tregs in CD4 T cells from WLT and TBKLT mice cultured for 5 days in Treg skewing conditions.C and D. Representative histograms (left) and quantified dot plots (right) showing expression of FOXP3 (C) and Ki67 (D) in FOXP3+ Tregs.The data represented here were pulled from two individual repeats (A-D) with 1-3 mice per group.Data here are shown ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (two tailed unpaired t test).

Figure S7 .
Figure S7.BATF regulated accessibility of genes involved in Treg stability.Related to Figure 5. Representative tracks of WLT and TBKLT Treg ATAC-seq data and published Treg BATF ChIP-seq data in the enhancer regions of Gata3 (A) and Ets1 (B).

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Figure S8: Deletion of BATF in Tregs results in significantly reduced Foxp3 expression.Related to Figure 6. A. Schematic showing experimental design for the treatment of ex vivo sorted (CD4 + CD25+) Tregs with tamoxifen in an in vitro culture system.B. Sorting strategy and post sorting purity of ex vivo sorted Tregs from spleens of ERCreBatfF/F mT/mG mice.C. Representative flow plots (left) and quantified frequencies (right) of CD4+ CD25+ ex vivo sorted Tregs treated with PBS (control) or 1 μM of tamoxifen.D. Similar to C, showing frequency of GFP+ Tregs after treatment with PBS or tamoxifen.E-F.MFI of BATF (E) and FOXP3 (F) in ex vivo Tregs treated with PBS or tamoxifen.Data are pooled from two independent experiments analyzed on day 12 or day 15 post-treatment with 1-2 mice per group.Data here are shown ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (two tailed unpaired t test).