Bach2 regulates homeostasis of Foxp3+ regulatory T cells and protects against fatal lung disease in mice

Abstract

Variants of the Bach2 gene are linked to vitiligo, celiac disease, and type 1 diabetes, but the underlying immunological mechanisms are unknown. In this study, we demonstrate that Bach2 plays crucial roles in maintaining T cell quiescence and governing the differentiation, activation, and survival of Foxp3(+) regulatory T (Treg) cells. Bach2-deficient T cells display spontaneous activation and produce elevated levels of Th1/Th2-type cytokines. Without Bach2, Treg cells exhibit diminished Foxp3 expression, depleted numbers, hyperactivation, enhanced proliferation, and profound loss of competitive fitness in vivo. Mechanistically, reduced survival of Bach2-deficient Treg cells was associated with reduced Bcl-2 and Mcl-1 levels and elevated Bim/Bcl-2 ratio. Additionally, Bach2 deficiency induced selective loss of Helios(-)Foxp3(+) Treg cells and a Treg cell transcriptome skewed toward the Th1/Th2 effector program at the expense of the Treg program. In vitro experiments confirmed that Bach2: 1) is indispensable for TCR/TGF-β-induced Foxp3 expression; and 2) mitigates aberrant differentiation of Treg cells by repression of the competing Gata3-driven Th2 effector program. Importantly, perturbations in the differentiation of induced Treg cells was linked to a fatal Th2-type chronic inflammatory lung disease in Bach2-deficient mice. Thus, Bach2 enforces T cell quiescence, promotes the development and survival of Treg lineage, restrains aberrant differentiation of Treg cells, and protects against immune-mediated diseases.

Figure 1. Bach2 regulates peripheral T cell homeostasis

Splenocytes were collected from naïve WT and Bach2 KO mice. (A–C) Activation of T cells was analyzed by staining with anti-CD44 and anti-CD62L. (A) Frequencies of naïve versus activated CD4 and CD8 T cells. (B) Numbers of naïve versus activated CD4 and CD8 T cells were calculated per spleen. (C) Median fluorescence intensity (MFI) of CD44 for each subset is plotted. (D) Splenocytes were stimulated for 5 hrs in vitro with PMA and ionomycin in the presence of Brefeldin A, and cytokine production was measured by intracellular staining. (E) Expression of transcription factors such as T-bet and Gata3 was assessed by flow cytometry following intracellular staining. Data are representative of two independent experiments. *p < 0.05

Figure 2. Bach2 controls the homeostasis of regulatory T cells

Spleens and thymus were collected from naïve WT and Bach2 KO mice (over 9 weeks old), and analyzed for Treg cells by flow cytometry. (A) Frequency and number of thymic Treg cells. (B) Phenotypic analysis of thymic foxp3⁺ Treg cells. (C) Frequency and numbers of splenic Treg cells. (D) Phenotypic analysis of splenic foxp3⁺ Treg cells. (E) Surface expression of KLRG1 and intracellular expression of granzyme B were analyzed for foxp3⁺ Treg cells by flow cytometry. (F) Bcl-2 and Bim expression was assessed by intracellular staining in splenic foxp3⁺ Treg cells and foxp3⁻ conventional CD4 T cells; numbers indicates MFI of Bcl-2. (G) Treg (CD4⁺ CD25⁺ GITR⁺) cell apoptosis was measured by Annexin V staining directly ex vivo. (H) Ki67 staining on thymic and splenic foxp3⁺ Treg cells. (I) Expression levels of foxp3, blimp1 and Tcf1 on thymic (upper panel) and splenic (lower panel) foxp3⁺ Treg cells were assessed by flow cytometry. (J) Treg cells from mLN nodes and small intestinal LP were quantified by flow cytometry. Data are representative of two independent experiments. *p < 0.05

Figure 3. Bach2 regulates the balance of the effector and Treg transcription programs

Whole genome transcriptional profiles from WT and Bach2 KO CD4⁺ CD25⁺ GITR⁺ splenic Tregs were analyzed by DNA microarray. (A) Scatter plot represents comparison of normalized expression values in WT versus Bach2 KO. (B and C) Expression of genes associated with T_H1, T_H2, T_H17 or Treg functions in WT and Bach2 KO mice is presented as Box Whisker plots (B) and heat maps(C).

Figure 4. Bach2 regulates the homeostasis of Treg cells by cell-intrinsic mechanisms

Mixed bone marrow chimeras were generated by the transfer of a mixture of bone marrow cells from WT/Ly5.1 and WT/Ly5.2 or Bach2 KO/Ly5.2 into lethally irradiated WT/Ly5.1 mice. While control chimeras were reconstituted by a mixture of WT/Ly5.2 and WT/Ly5.1 BM cells, experimental chimeras were reconstituted by the mixture of Bach2 KO/Ly5.2 and WT/Ly5.1 BM cells. At least eight weeks after reconstitution, thymus and spleens were harvested, and analyzed for Treg cells. (A) Spleens were harvested, and T cells were analyzed for their activation status. Cells are gated on WT/Ly5.2 (control chimera) and Bach2 KO/Ly5.2 (experimental chimera) for direct comparison. Frequencies and numbers of naïve versus activated CD4 and CD8 T cells were assessed and calculated per spleen. (B) Frequencies of foxp3⁺ Treg cells among the gated cells in both thymus and spleen were measured by flow cytometry. (B and C) Surface markers and Treg cell markers on WT/Ly5.2 (control chimera) and Bach2 KO/Ly5.2 (experimental chimera) Treg cells were measured by flow cytometry. (C) Frequency of Ly5.2⁺ Treg cells positive for CD44, CD69 and Ki67 and Blimp1 MFI. (D) MFIs of foxp3 for Ly5.2⁺ WT and Bach2 KO Treg cells. Data are representative of two independent experiments.* p < 0.05.

Figure 5. Bach2 is required for the induction of foxp3 and differentiation of iTregs

(A) Splenocytes from WT and Bach2 KO mice were stained with anti-CD4, anti-foxp3 and anti-Helios. Frequencies and numbers of Helios⁺ and Helios⁻ Tregs are displayed. (B–F) Conventional CD4 T cells (CD25⁻ GITR⁻) from WT and Bach2 KO mice were purified by FACS, and stimulated with anti-CD3 in the presence or absence of recombinant human TGF-β for 72 hr. (B) Representative contour plots depict foxp3 expression following in vitro Treg differentiation. Bar graph displays frequencies of foxp3⁺ iTregs with or without anti-CD3 in the presence of indicated concentrations of TGF-β. (C) MFIs of foxp3 and GITR for foxp3⁺ iTregs from WT and Bach2 KO are plotted. (D–E) RNA from cells cultured for iTreg cell differentiation was reverse transcribed. Relative levels of Treg signature transcription factors (D), CD4 T cell lineage-specific transcription factors (E) and cytokines (F) were quantified by quantitative RT-PCR. Data are representative of two independent experiments. *p < 0.05

Figure 6. Bach2 KO mice develop lung pathology

B6 WT and Bach2 KO mice were sacrificed and examined by H&E stain for lung immune-pathology. (A) WT mouse, 15x original magnification. Normal lung with large caliber bronchioles and pulmonary vessels located centrally (stars), and well-defined small-caliber and terminal airways and alveoli located peripherally (arrowheads). (B) Bach2 KO mouse, 15x original magnification. Lung with eosinophilic crystalline pneumonia (ECP) exhibiting regional loss of alveolar spaces resulting from densely cellular alveolar exudates and interstitial infiltrates (arrowheads). (C) Bach2 KO mouse, 200x original magnification. Lung with ECP: Multiple alveolar spaces are filled with aggregates of hypereosinophilic inflammatory cells (stars). Infiltrates of eosinophils, histiocytes, and plasma cells expand the alveolar walls (arrows). Plasma cells and eosinophils encircle a pulmonary vein (arrowhead) (D) Bach2 KO mouse, 600x original magnification. Lung with ECP, affected alveolus: The densely cellular alveolar exudate is primarily composed of tightly packed variably sized macrophages containing intracytoplasmic hypereosinophilic acicular crystals(star). Eosinophils expand the adjacent interstitum (arrow). (E) Bach2 KO mouse, 600x original magnification. Lung with ECP: A medium-caliber pulmonary vein (star) is encircled by a cuff of plasma cells (arrowheads) admixed with fewer eosinophils and small lymphocytes. (F) Bach2 KO mouse, 600x original magnification. Densely cellular aggregates of eosinophils, plasma cells, and lymphocytes distrupt and fill an airway in a 7 month-old mouse with severe concurrent multisystemic eosinophilic and plasmacytic inflammation. (G) mRNAs of indicated cytokines in WT and Bach2 KO lung lysates were quantitated by qPCR.