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. 2018 Apr 1;200(7):2362-2371.
doi: 10.4049/jimmunol.1800042. Epub 2018 Feb 19.

The Alternative NF-κB Pathway in Regulatory T Cell Homeostasis and Suppressive Function

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Free PMC article

The Alternative NF-κB Pathway in Regulatory T Cell Homeostasis and Suppressive Function

Yenkel Grinberg-Bleyer et al. J Immunol. .
Free PMC article

Abstract

CD4+Foxp3+ regulatory T cells (Tregs) are essential regulators of immune responses. Perturbation of Treg homeostasis or function can lead to uncontrolled inflammation and autoimmunity. Therefore, understanding the molecular mechanisms involved in Treg biology remains an active area of investigation. It has been shown previously that the NF-κB family of transcription factors, in particular, the canonical pathway subunits, c-Rel and p65, are crucial for the development, maintenance, and function of Tregs. However, the role of the alternative NF-κB pathway components, p100 and RelB, in Treg biology remains unclear. In this article, we show that conditional deletion of the p100 gene, nfkb2, in Tregs, resulted in massive inflammation because of impaired suppressive function of nfkb2-deficient Tregs. Surprisingly, mice lacking RelB in Tregs did not exhibit the same phenotype. Instead, deletion of both relb and nfkb2 rescued the inflammatory phenotype, demonstrating an essential role for p100 as an inhibitor of RelB in Tregs. Our data therefore illustrate a new role for the alternative NF-κB signaling pathway in Tregs that has implications for the understanding of molecular pathways driving tolerance and immunity.

Figures

Figure 1
Figure 1. T-cell specific ablation of nfkb2 drives peripheral Treg cell expansion
Thymus, spleen and peripheral lymph nodes of 6–8 weeks-old CD4cre and CD4crenfkb2F/F mice were stained for FACS. (A) Dot plot showing CD4 and CD8 expression in gated total thymus (top) and spleen (bottom) live cells. Numbers indicate the % in each quadrant. (B, C) Proportion (B) and absolute numbers (C) of live TCR-β+CD4+ and CD8+ in the indicated tissues. (D) Expression of Foxp3 in gated spleen TCR-β+CD4+ live cells. Numbers indicate the percentage in gate. (E) % in TCR-β+CD4+ and absolute numbers of Treg cells. Data is representative (A, D) or the pool (B–C, E) of 3 experiments. In B, C and D, each dot represents a mouse. *p<0.05, ***p<0.001, n.s.: non-significant.
Figure 2
Figure 2. Intrinsic NF-κB2 p100/p52 signaling restricts the peripheral Treg cell pool
(A–C) Spleen and peripheral lymph nodes of 6–8 weeks-old Foxp3cre and Foxp3crenfkb2F/F mice were stained for FACS. (A) Expression of Foxp3 in gated spleen TCR-β+CD4+ live cells. Numbers indicate the percentage in gate. (B) % in TCR-β+CD4+ and absolute numbers of Treg cells. (C) Expression of Foxp3 and pooled MFI in gated spleen TCR-B+CD4+Foxp3+ Treg cells. (D–F) The cell-intrinsic role of nfkb2 in Treg cells was evaluated in mixed BM chimeras. (D) Experimental design. (E) % of Treg cells in TCR-β+CD4+CD45.2+ cells. (F) Representative Ki67 expression in gated spleen in TCR-β+CD4+CD45.2+Foxp3+ Treg cells, and pooled % of Ki67+ in Treg cells. Numbers indicate the percentage in gate. (G) Expression of Nrp-1 and Helios in gated spleen in TCR-β+CD4+Foxp3+ Treg cells from Foxp3cre and Foxp3crenfkb2 F/F animals. (G) Naïve T cells from CD4cre and CD4crenfkb2 F/F LN were cultured under iTreg polarization conditions for 4 days. The % of Treg in each genotype in 3 experiments is shown. Data is representative (A, C, D–G) or the pool (B, H) of 2–3 experiments. In B, E and F, each dot represents a mouse. *p<0.05,** p<0.005, ***p<0.001.
Figure 3
Figure 3. Uncontrolled inflammation in aged Foxp3crenfkb2F/F mice
Tissues of 12-month-old Foxp3cre and Foxp3crenfkb2F/F mice were analyzed by histology and FACS. (A) (left) Representative section of colon stained with H/E. Bars: 200μm; original magnification 10X. (right) Quantification of abscesses (immune infiltrates) in each colon section. (B) Gross live cell count in spleen and LN. (C) CD44 and CD62L expression in gated spleen TCR-β+CD8+ cells. Numbers indicate the % in each quadrant. (D) % (in TCR-β+CD4+Foxp3 (CD4) and TCR-β+CD8+ (CD8)) and absolute numbers of CD44high T cells. (E) % of Ki67+ cells in spleen CD4 and CD8 cells. (F) Expression of IFN-γ upon PMA-ionomycin restimulation in gated spleen TCR-β+CD8+ cells. Numbers indicate the percentage in gate. (G) % (in CD4 and CD8) and absolute numbers of IFN-γ+ T cells. (H) % of IL-17A+ cells in CD4 cells. Data is representative (A, C, F) or the pool (A, B, D, E, G, H) of 3 experiments. In A, B, D, E, G, H, each dot represents a mouse. *p<0.05,** p<0.005, ***p<0.001, n.s.: non-significant.
Figure 4
Figure 4. Impaired suppressive function of nfkb2-deficient Treg cells
(A) Spleen and peripheral lymph nodes of 12-month-old Foxp3cre and Foxp3crenfkb2F/F mice were analyzed by FACS. The % of Treg cells among CD4 T cells and their absolute numbers is shown. (B) In vitro suppressive function of Treg sorted from Foxp3cre (WT) and Foxp3crenfkb2F/F (nfkb2 −/−) animals was assessed as described in the Material and Methods section. (left) representative proliferation of responder T cells at D4; (right) Mean +/− SEM of suppression of responder T cells proliferation by Treg cells at different ratios. (C–E) In vivo suppression assay, as described in the Material and Methods section. (C) Mean +/− SEM % of original weight. (D) Representative (left) and pooled (right) expression of IFN-γ in gated CD45.1+CD4+ T cells at D50. (E) % of Foxp3+ Treg cells among live CD45.2+CD4+ cells. In A, C, E, data is the pool of 3 pooled experiments. In B, data is from 1 out of 3 experiments. *p<0.05,** p<0.005, ***p<0.001, n.s.: non-significant.
Figure 5
Figure 5. Overactivation of RelB in the absence of NF-κB2 drives uncontrolled Treg expansion and inflammation
(A) Treg cells were sorted from Foxp3cre and Foxp3crenfkb2F/F spleen and LN and activated overnight with anti-CD3/CD28 Abs and IL-2. Cytosolic and nuclear lysates were separated by SDS-PAGE and membranes were blotted with the indicated Ab. (B) Thymus, spleen and peripheral lymph nodes of 6–8 weeks-old CD4cre and CD4crenfkb2F/FrelbF/F mice were stained for FACS. The % in TCR-β+CD4+ and absolute numbers of Treg cells is shown (C–G) Tissues of 12-month-old Foxp3cre and Foxp3crenfkb2F/FrelbF/F mice were analyzed by histology and FACS. (C) Representative section of colon stained with H/E. Bars: 200μm; original magnification 10X. (D) Quantification of abscesses (immune infiltrates) in each colon section. (E) Gross live cell count in spleen and LN. (F) % in TCR-β+CD4+ and absolute numbers of Treg cells. (G–I) Cells were restimulated with PMA-ionomycin prior to FACS analysis. (G) CD44 and IFN-γ expression in gated spleen TCR-β+CD8+ cells. Numbers indicate the % in each quadrant. (H, I) % (in CD4 and CD8 cells) and absolute numbers of CD44high and IFN-γ+ T cells, respectively. Each dot represents a mouse. Data is representative (A, C, G) or pooled (B, D–G, H–I) from 2 to 4 experiments. *p<0.05, n.s.: non-significant.
Figure 6
Figure 6. NF-κB2 represses aberrant expression of inflammatory and homing genes in Treg cells
Treg cells were sorted from Foxp3cre, Foxp3crenfkb2F/F, Foxp3crerelbF/F mice and Foxp3crenfkb2F/FrelbF/F mice (3 mice/genotype), activated overnight and processed for RNA-seq. (A) Venn diagrams showing genes with a fold-change <0.6 and >1.5 and a p-value< 0.05 compared to WT Treg cells. (B) Unsupervised hierarchical row clustering. Only genes with changed expression in at least 1 genotype is shown. The biological functions indicate selected enriched pathways upon GSEA and Metacore analysis. (C) Heatmaps showing the relative expression of selected genes enriched in different biological processes. (D) qRT-PCR quantification of selected genes. Mean +/− SEM of expression relative to GAPDH is shown. (E) Spleens of 12-month-old mice were analyzed by FACS. (left) relative expression of IL-17A in gated Treg cells; numbers indicate the % in the gate. (right) Mean +/− SEM of IL-17A expression in spleen Treg cells. Data is from two (A–C) or 3 (D–E) experiments.

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