Normal Treg homeostasis and suppressive function require both FOXP1 and FOXP4

Abstract

FOXP3+ Treg cells are critical for immune tolerance. Genetic deletion of the Forkhead domain-containing proteins of the FOXP-subfamily member FOXP1 from Tregs results in impaired function associated with reduced CD25 expression and IL-2 signaling, but to date the only other FOXP family member expressed in Tregs, FOXP4, has been minimally studied. To investigate the potential functional interactions among FOXP family members in Tregs, we specifically deleted Foxp1, Foxp4, or both in FOXP3+ committed Tregs in mice. Our findings show that mice with combined, but not individual, deficiency in FOXP1 and FOXP4 exhibit lymphoproliferation, inflammation, autoimmunity, and early lethality. The combined absence of FOXP1 and FOXP4 in Tregs results in an activated/effector-like phenotype with compromised suppressive function in peripheral lymphoid organs, an enhanced germinal center response, and proinflammatory cytokine production. We further show that FOXP1 and FOXP4 bind to Il2ra promoter regions to regulate CD25 expression in Tregs. Through pairwise comparison among mouse strains with Treg-specific deletion of Foxp1, Foxp4, or both, our findings indicate a nonredundant but insufficient role of FOXP4 in Treg function.

Keywords: Adaptive immunity; Autoimmunity; Immunology; Tregs.

Figure 1. Combined absence of FOXP1 and FOXP4 induces lymphoproliferation and fatal autoimmunity.

(A) Representative images of spleens (left) and peripheral lymph nodes (pLNs, right) for each mouse strain at the age of 8 to 10 weeks. Scale bar: 1 cm. (B) The absolute number of total lymphocytes in spleen or pLN. (C) Kaplan-Meier survival curve of the respective mouse strains. (D) Representative H&E-stained tissue sections of liver, pancreas, kidney, and salivary gland from 2- and 6-month-old Cre^Pos and cDKO mice showing lymphocytic infiltrates. Scale bar: 100 μm. n = 2 mice per time point in each group. (E) Samples were collected from cDKO mice at a moribund state and Cre^Pos at matched time points. Hemoglobin (left) and hematocrit (middle) of the peripheral blood, and anti-erythrocyte IgM antibodies (right) in the serum of cDKO at euthanasia with age-matched Cre^Pos littermates. Each symbol represents an individual sample. Data are presented as mean ± SD. Statistical tests: 1-way ANOVA with Tukey’s post hoc test (B) and Mann-Whitney U test (E). NS, not significant. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2. Combined FOXP1 and FOXP4 loss in Tregs results in unrestrained GC response, hyperimmunoglobulinemia, and Tfh cell expansion.

Total serum levels of (A) IgG, IgG1, IgG2b/c, IgG3, and (B) IgM and IgA were assessed by ELISA. (C) Representative spleen H&E staining with GCs (outlined with by a dotted lines) from 2-month-old Cre^Pos and cDKO mice spleen. Scale bar: 100 μm. (D) Representative plots showing flow cytometry analysis of splenic CD19⁺ B cells. CD95⁺GL7⁺ GC B cells are gated. Absolute number of splenic (E) GC B and (F) CD4^–CD8^–GR-1^–F4/80^–IgD^loCD138⁺ plasma cells in Cre^Pos and cDKO mice. (G) Representative flow cytometry analysis of CD4⁺TCRβ⁺ gated cells; the gated area indicates CXCR5⁺PD-1⁺ Tfh cells. (H) Absolute numbers of Tfh (CD4⁺CXCR5⁺PD-1⁺FOXP3^–), (I) Tfr (CD4⁺CXCR5⁺PD-1⁺FOXP3⁺) (left), and Tfh/Tfr ratio (right) in Cre^Pos and cDKO mice. Numbers indicate the percentage of cells in gates (D and G). Data are representative of 3 independent experiments. Each symbol represents an individual sample. Data are presented as mean ± SD. Unpaired, nonparametric Mann-Whitney U test was performed for statistical analysis. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Enhanced turnover and expansion of FOXP3⁺ Tregs in peripheral lymphoid organs of cDKO mice.

(A) Representative flow cytometry gating strategy used to identify splenic CD4⁺FOXP3⁺ Treg cells from each KO mouse. The percentage of FOXP3⁺ in CD4⁺ T cells and the FOXP3 median fluorescence intensity (MFI) are indicated. (B) Total number of FOXP3⁺ Treg cells in spleens (left) and pLNs (right) at the age of 2 months. (C) Flow cytometry analysis of Ki-67⁺ cell expression within CD4⁺FOXP3⁺ Treg cells in Cre^Pos and cDKO mice. (D) Treg frequency and number. Numbers indicate the percentage of cells in gates (A and C). Data are representative of 3 independent experiments (A–D). Each symbol represents an individual mouse. Data are presented as mean ± SD. Statistical tests: 1-way ANOVA with Tukey’s post hoc test (B) and Mann-Whitney U test (D). *P < 0.05, ****P < 0.0001.

Figure 4. Loss of FOXP1 and FOXP4 changes the phenotype and subsets of Treg cells.

(A) Flow cytometry analysis of CD69, KLRG1, CXCR3, PD-1, GITR, and CTLA4 surface expression on CD4⁺FOXP3⁺ splenic Treg cells. Median fluorescence intensity (MFI) indicated on plots. (B) Representative flow cytometry analysis of cTreg and eTreg subsets in peripheral lymphoid organs and in blood. Gated cell frequency indicated on the plots. (C) Absolute number of splenic CD62L^hi and CD62L^lo Treg cells in Cre^Pos and cDKO mice. (D) Representative flow cytometry analysis of ICOS expression in YFP⁺ Treg subsets. MFI indicated on plots. (E) Flow cytometry analysis of Helios^hi subpopulation in CD4⁺FOXP3⁺ Treg cells. Gated proportion indicated on the plot. (F) Flow cytometry analysis of frequency and (G) quantification of IL-17 and INF-γ production by CD4⁺FOXP3⁺ cells upon PMA/ionomycin stimulation. Data are presented from at least 3 independent experiments. Each symbol in C represents an individual mouse. Data are presented as mean ± SD. Significance was assessed with Mann-Whitney U test. ****P < 0.0001.

Figure 5. FOXP1 and FOXP4 deficiency cause spontaneous T cell activation and increased production of proinflammatory cytokines.

(A) Flow cytometry analysis of the frequency of naive and effector/memory subsets in CD4⁺FOXP3^– (top) and CD8⁺ cells (bottom) in the spleen from respective mouse strains. (B) Number of activated subsets in CD4⁺FOXP3^– cells from respective mouse strains. (C) Number of activated subsets in CD8⁺ cells from respective mouse strains. (D) Flow cytometry analysis of frequency and (E) quantification of IL-17 and INF-γ production by CD4⁺FOXP3^– cells upon PMA/ionomycin stimulation. (F) Quantification of INF-γ and IL-17a production by CD8⁺ cells. Numbers indicate the percentage of cells in gates (A and D). Data are representative of at least 3 independent experiments. Each symbol represents an individual mouse. Data are presented as mean ± SD. Statistical tests: 1-way ANOVA with Tukey’s post hoc test (B and C) and Mann-Whitney U test (D and E). *P < 0.05; ***P < 0.001; ****P < 0.0001.

Figure 6. FOXP1 and FOXP4 are essential for Treg suppressive function.

(A) Representative flow cytometry analysis of CTV-labeled naive T (CD45.1⁺CD4⁺CD25^–CD44^lo) cells after 4-day coculture with Tregs (CD45.2⁺CD4⁺YFP⁺) from Cre^Pos or cDKO. Representative of 3 independent experiments using a total of 7 cDKO and 7 control mice as the Treg source. (B) Absolute number of adoptively transferred CD45.1⁺CD4⁺ cells in the pLNs from TCR^–/– recipients on day 7 after transfer is shown. Data are representative of at least 2 independent experiments. (C) Changes in body weights of Rag-KO recipient mice after adoptive transfer of CD4⁺ (CD45.1⁺CD4⁺CD25^–) cells with or without sorted Tregs (CD45.2⁺CD4⁺YFP⁺) from Cre^Pos or cDKO. Statistical differences among respective groups were quantified in the 10th week of reconstitution (right), n = 5 mice in each group. (D) Representative frequency of CD45.1⁺ naive T cells (left) and absolute number of CD45.2⁺ Tregs (right) in mesenteric lymph nodes (mLN) from recipient mice at the end of the study. (E) Colitis scores of the respective groups were evaluated in the 10th week of reconstitution. Each symbol represents an individual mouse (except the left panel in C). Data are presented as mean ± SD. Statistical tests: 1-way ANOVA with Tukey’s post hoc test (B–E) and Mann-Whitney U test (D, right panel). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7. FOXP1 and FOXP4 trans regulates Il2ra expression in Tregs.

(A) Volcano plot of the RNA-seq analysis showing 1545 differentially expressed genes in Cre^Pos and cDKO YFP⁺ Treg cells. (B) Expression heatmap of Treg-associated genes in Cre^Pos and cDKO YFP⁺ Treg cells. (C) GSEA of TGF-β and IL-2/STAT5 pathways in Treg cells, shown in cDKO/Cre^Pos. (D) Flow cytometry analysis of CD25 expression in CD4⁺FOXP3⁺ Treg cells from the spleen of respective mouse strains. Median fluorescence intensity (MFI) indicated on the plot. (E) Flow cytometry analysis of CD25 expression in total Treg, eTreg, and cTreg cells from the spleen of Cre^Pos and cDKO mice. MFI indicated on the plots. (F) Relative Il2ra mRNA expression in sorted Treg cells from Cre^Pos and cDKO mice. (G) Quantitation of Il2ra promoter fragments following immunoprecipitation of DNA from Cre^Pos and cDKO Tregs. Data are representative of at least 2 independent experiments and presented as mean ± SD. Each symbol represents an individual sample. Nonparametric Mann-Whitney U test was performed for statistical analysis. **P < 0.01.

Figure 8. Proposed interaction pattern of FOXP TF members in Treg cells.

A schematic view of a potential model of how FOXP1, FOXP3, and FOXP4 interact with each other to regulate gene expression in Treg cells. CD25 was used as an example; the dotted line represents the expression level of CD25 in Cre^Pos control mice.