A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes Treg cell activation and homeostasis

Abstract

The molecular programs involved in regulatory T (T_reg) cell activation and homeostasis remain incompletely understood. Here, we show that T cell receptor (TCR) signaling in T_reg cells induces the nuclear translocation of serine/threonine kinase 4 (Stk4), leading to the formation of an Stk4-NF-κB p65-Foxp3 complex that regulates Foxp3- and p65-dependent transcriptional programs. This complex was stabilized by Stk4-dependent phosphorylation of Foxp3 on serine-418. Stk4 deficiency in T_reg cells, either alone or in combination with its homolog Stk3, precipitated a fatal autoimmune lymphoproliferative disease in mice characterized by decreased T_reg cell p65 expression and nuclear translocation, impaired NF-κB p65-Foxp3 complex formation, and defective T_reg cell activation. In an adoptive immunotherapy model, overexpression of p65 or the phosphomimetic Foxp3^S418E in Stk3/4-deficient T_reg cells ameliorated their immune regulatory defects. Our studies identify Stk4 as an essential TCR-responsive regulator of p65-Foxp3-dependent transcription that promotes T_reg cell-mediated immune tolerance.

Figure 1.. STK3/4 deficiency leads to activation defects of Treg cells.

(A) Flow cytometric analysis of CD4⁺ Foxp3⁺Treg cells from spleen, liver and lung of Foxp3^YFPCre/+ and Foxp3^YFPCre/+Stk3^∆/∆Stk4^∆/∆ mice and (B) quantification of frequencies (n=10–13 for each group) and numbers (n=8 for Foxp3^YFPCre/+ group; n=9 for Foxp3^YFPCre/+Stk3^∆/∆Stk4^∆/∆ group) (scatter plots with means ± S.E.M.) of each contour plot in (A). (C and D) Representative flow cytometric analysis (C) and frequencies (D, scatter plots with means ± S.E.M.) of IFNγ⁺ and IL-17⁺ CD4⁺Foxp3⁻Teff cells (n=6–7 per group). The results represent one of three independent experiments. (E) Representative flow cytometric analysis and frequencies (scatter plots with means ± S.E.M.) of CD62L^loCD44^hi CD4⁺YFP⁺Treg cells (n=9 per group). The results represent pool of three independent experiments. (F) Representative flow cytometric analysis and frequencies (scatter plots with means ± S.E.M.) of CD4⁺YFP⁺CD69⁺ Treg cells (n=5 per group). (G and H) AnnexinV⁺ Viability dye⁺ splenic Treg cells (G) (n=9 for each group) and Ki67⁺ Treg cells (G) (n=11 for each group). (I) Flow cytometric analysis and scatter plot representation of relative Foxp3 mean fluorescence intensity (MFI), relative CD25 MFI and relative CD73 MFI of CD4⁺YFP⁺ Treg cells from spleen of Foxp3^YFPCre/+ and Foxp3^YFPCre/+Stk3^∆/∆Stk4^∆/∆ mice (n=5 per group). Each point represents one mouse. Error bars indicate the standard error of the means (s.e.m). The results are representative of three independent experiments. Statistical tests: ***, P<0.005, ****, P<0.0001 by two-way ANOVA with Sidak’s multiple comparisons test (B) or Student’s unpaired two tailed t test (B to I).

Fig 2.. Stk3/4-deficient Treg showed transcriptome defects on TCR/p65/Foxp3 regulated genes.

(A) Volcano plot of gene expression and (B) pathway analysis of gene transcripts using the KEGG database in YFP⁺ CD4⁺ Treg cells from Foxp3^YFPCre/+Stk3^∆/∆Stk4^∆/∆ versus Foxp3^YFPCre mice. (C to E) Gene set enrichment of TCR/p65/Foxp3 dependent genes. (F) Heatmap of gene transcripts of YFP⁺ CD4⁺ Treg cells isolated from Foxp3^YFPCre (n=7) and Foxp3^YFPCre/+Stk3^∆/∆Stk4^∆/∆ (n=6) mice. Counts were normalized using DESeq2-VST for heatmap visualization. (G) Heatmap showing the binding profile of p65 ChIP-seq signal for Foxp3^YFPCre, Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg, pooled input and IgG isotype control samples at TSS and flanking +/− 2kb region of the expressed genes. The Y-axis indicates the number of counts per million mapped to denoted regions. (H) Venn diagram on the number of p65 pulldown ChIP-Seq enriched genes overlapping between WT Treg cells, Stk3/4-deficient Treg cells and Foxp3 binding genes (I) Arrows indicate the scatter plots of related gene transcripts expression in RNA-seq (n=7 for Foxp3^YFPCre group; n=5 for Foxp3^YFPCre/+Stk3^∆/∆Stk4^∆/∆ group). (J) Example genes Foxp3 and Mknk2 from the set of genes enriched by p65 Treg ChIP-Seq analysis. Coverage tracks were visualized in UCSC genome browser and display normalized p65 sequencing signal. The black bars indicate the p65 ChIP-seq peaks enriched in Foxp3^YFPCre Treg cells group. Each point represents one mouse. Error bars indicate the standard error of the means (s.e.m). Statistical tests: for transcript analysis (A to F) was called at adjusted p values <0.05, fold-changes over ±1.5 and false discovery rate (FDR) were below 0.1. *, P<0.05 by Mann-Whitney test (panel I).

Figure 3.. TCR activation stimulates Stk4 nucleus translocation and colocalization with Foxp3.

(A and B) Confocal microscopic analysis of CD4, Foxp3, STK4, DAPI and Merge (A), and ratios of nuclear/total Stk4 and frequencies of Stk4 and Foxp3 co-localization (B) in Foxp3^YFPCre Treg cell cultures either unstimulated or stimulated with anti-CD3+anti-CD28 mAbs as indicated (n=10 per group). (C) Immunoblot analysis of Stk4 and Foxp3 association with p65 in Foxp3^YFPCre Treg cells that were stimulated overnight with anti-CD3/CD28. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (D and E) Immunoblot analysis of Stk4 and Foxp3 association with p65 in HEK293T cells transfected with the respective plasmids. Cell lysates were immunoprecipitated with anti-V5 mAb (specific for V5-tagged Foxp3) (D) or anti-c-Myc mAb (E), then immunoblotted with the indicated antibodies. (F) densitometric analysis of immunoblots of p65 (upper panel) and Foxp3 (lower panel) shown in (E). (G and H) Confocal microscopic analysis (G) and frequencies (H) of CD4, Foxp3, STK4, DAPI and Merge, ratios of nuclear/total Stk4 and frequencies of Stk4 and Foxp3 co-localization in Foxp3^YFPCre Treg cells either unstimulated or stimulated with anti-CD3+anti-CD28 mAbs without or with the Stk4 kinase inhibitor XMU-MP-1(n=19 per group). (I) Stk4-p65 association is Stk4 kinase activity-dependent. Immunoblot analysis of Stk4-p65 association in HEK293T cells transfected with plasmids encoding either Stk4 kinase-competent (Stk4) or deficient (Stk4^K59R) proteins. Each point represents one cell for confocal studies and one blot for immunoblot analyses. Error bars indicate the standard error of the means (s.e.m). Statistical tests: one-way ANOVA with post-test analysis (B and F), two-way ANOVA with post- test analysis (H) and Student’s unpaired two tailed t test (C,D,I). *, P<0.05,**, P<0.01, ***, P<0.005, ****, P<0.0001.

Figure 4.. Foxp3 S418 phosphorylation is Stk4-dependent and stabilizes the Stk4-Foxp3-p65 complex.

(A,B) Immunoblot analysis (upper panel) and densitometry (lower panel) of phospho-serine/threonine Foxp3 (A) and Foxp3 phospho-S418 Foxp3 (B) in Foxp3^YFPCre and Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg cells stimulated with anti-CD3/ CD28 beads. Cell lysates were immunoprecipitated with anti-Foxp3 antibody and then immunoblotted with the indicated antibody. (C,D) Immunoblot analysis (upper panel) and densitometry (lower panel) of phospho-S418 Foxp3 in Jurkat cells transfected with the indicated plasmids then stimulated with CD3/CD28 beads. Cell lysates were immunoprecipitated with an anti-V5 mAb (C) or with anti-Flag mAb (D) and then immunoblotted with the indicated antibodies. (E,F) immunoblot analysis (E) and densitometry (F) of p65 and Foxp3 association in HEK293T cells transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag mAb, then immunoblotted with the indicated antibodies. (G) Scatter plot representation of CD25 and Thy1.1 MFI in Foxp3^YFPCre or in transfected Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg with the indicated retrovirus (n=9 for each group). The results represent a pool of three independent experiments. Each point represents one blot for immunoblot analysis and one mouse. Error bars indicate S.E.M. Statistical tests: Student’s unpaired two tailed t test (A,B,D), one-way ANOVA with post-test analysis (C,F,G), ns: Not significant. *, P<0.05, **, P<0.01, ***, P<0.005, ****, P<0.0001.

Figure 5.. Impaired p65 nuclear translocation and enhanced degradation in Stk3/4 deficient Treg cells.

(A) Confocal microscopic analysis of CD4, Foxp3, p65, DAPI and Merge (B) ratios of nuclear/total Stk4 and p65/Foxp3 colocalization in Foxp3^YFPCre Treg cells either unstimulated or stimulated with anti-CD3+anti-CD28 mAbs at the indicated time points (n=12 per group). (C to D) Immunoblot analysis (C) and densitometry (D) of p-p65, p65 and β-actin in Foxp3^YFPCre and Foxp3^YFPcreStk3^∆/∆Stk4^∆/∆ Treg cells. (E) Representative flow cytometric analysis and scatter plot representation of p65 (n=10 per group) and p-p65 (n=7 per group) MFI in Treg cells from Foxp3^YFPCre/+ or Foxp3^YFPCre/+Stk3^∆/∆Stk4^∆/∆. (F) Scatter plot representation of p65 MFI in Foxp3^YFPCre or in Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg cells either untreated or treated with MG132. (G) Volcano plot of phosphorylated proteins identified by phosphoproteomics in Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ versus Foxp3^YFPCre Treg cells. (H) Pathway analysis of phosphorylated protein in Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ versus Foxp3^YFPCre Treg cells. (I) Increased or decreased phosphorylated proteins identified in Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ versus Foxp3^YFPCre Treg. (J) Predicted upstream regulators deduced from phosphoproteomic analysis of Stk3/4 deficient versus WT Treg cells. Each point represents one cell in the confocal analysis, one blot for immunoblot analysis and one mouse for the flow analysis. Error bars indicate S.E.M. Statistical tests: Student’s unpaired two tailed t test (E), two-way ANOVA with post-test analysis (B,D,F), ns: Not significant. P<0.05, **, P<0.01, ***, P<0.005, ****, P<0.0001.

Figure 6.. Overexpression of p65 and Foxp3^S418E in Stk3/4-deficient Treg cells restores their regulatory functions.

(A) Flow cytometric analysis and scatter plot representation of MFI of p65, CD25 and Foxp3 in Foxp3^YFPCre and Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg cells transfected with either empty vector or with p65-encoding retrovirus, as indicated (n=5 per group). (B) Survival of Foxp3^∆EGFPiCre mice injected with empty vector-transfected (n=8), p65-transfected (n=5) or Foxp3^S418E transfected Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg cells (n=5). (C) Flow cytometric analysis and frequencies of CD62L^loCD44^hi CD4⁺Foxp3⁻ and CD62L^hiCD44^lo CD4⁺Foxp3⁻ Teff cells from spleens of Foxp3^∆EGFPiCre mice injected with Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg cells that had been transfected with either empty vector (n=7), p65 (n=7), Foxp3^S418A (n=6) or Foxp3^S418E encoding vectors (n=5). (D, E) Flow cytometric analysis and frequencies of IFNγ⁺ CD4⁺Foxp3⁻ T cells from spleens (D) and lungs (E) of the respective groups (n=7 for empty vector; n=7 for p65; n=6 for Foxp3^S418A; n=5 for Foxp3^S418E). The results represent three pooled independent experiments. Each point represents one mouse. Error bars indicate S.E.M. Statistical tests: log-rank-test (B), one-way ANOVA with post-test analysis (A,C,D,E), ns: Not significant, *P<0.05, **P<0.01, ***,P<0.005, ****P<0.0001.

Fig. 7.. Immune regulatory abnormalities in STK4-deficient patients.

(A) Schematic representation of STK4 illustrating its encoding exons, the protein domains, and mapped mutations of four patients. The kinase domain, the inhibitory domain and the SARAH (Sav/Rassf/Hpo) domain are indicated. (B and C) Flow cytometric analysis and cell frequencies of CD45RA and CCR7 expression on circulating Treg cells (B) and Teff cells (C) of control subjects (n=4) and STK4 deficient patients (n=5). (D) Cell frequencies of AnnexinV and Ki67 expression on circulating Treg and Teff cells of control subjects (n=4) and STK4 deficient patients (n=5 for AnnexinV and n=4 for Ki67). (E) Cell frequencies and MFI of CD69 and CD25 expression on circulating Treg cells of control and STK4 deficient patients (n=4/group). (F) Flow cytometric analysis and MFI of P65 expression on circulating Treg cells of control and STK4 deficient patients (n=4/group). (G) Representative histograms of MFI of phospho-S418 Foxp3 in circulating Treg cells of healthy control (HC) and STK4 deficient patients that were either unstimulated (US) or stimulated with anti-CD3 +IL-2 (n=4/group). Each dot represents one patient. Error bars indicate S.E.M. Statistical tests: Student’s two tailed t test (B to F) or one-way ANOVA with post-test analysis (G). *p<0.05, **,P<0.01, ***,P<0.001, ****,P<0.0001.