PIP4Ks impact on PI3K, FOXP3, and UHRF1 signaling and modulate human regulatory T cell proliferation and immunosuppressive activity

Abstract

Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P₂ They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linked with susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.

Keywords: Phosphatidylinositol 5-phosphate 4-kinase; T-regulatory cells; UHRF1; immunosuppression; phosphoinositide kinases.

Fig. 1.

Silencing of PIP4K2B or PIP4K2C impairs FOXP3 expression in TCR-stimulated naïve T cells and Treg differentiation. (A) Graphical representation of isolation/stimulation and transduction of CD4+ naïve T cells. (B) PIP4Ks were silenced using different shRNAs targeting PIP4Ks isoforms (sh_2A, sh_2B, sh_2C). mRNA expression was analyzed using qRT-PCR and compared to control (sh_Ctrl). (C) qRT-PCR analysis of expression of transcription factors for Th1 (Tbx21/T-bet), Th2 (GATA3), and Treg (FOXP3) in cells depleted of PIP4Ks (n ≥ 3). (D) FACS analyses of T-bet, GATA3, and FOXP3 protein levels after PIP4K2B and PIP4K2C silencing (representative of n = 5). (E) PIP4Ks were silenced in naïve T cells differentiated into Tregs, and FOXP3 gene expression was assessed by qRT-PCR (n = 4). (F) Protein levels of FOXP3 in differentiated Tregs were analyzed by FACS. (G) Mean fluorescence intensity (MFI) quantitation of FOXP3 staining from independent experiments shown in F) (n ≥ 7). The charts represent means with SD. Statistical analyses were performed using unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. The number of replicates (n) refers to individual healthy donors.

Fig. 2.

PIP4K2B and PIP4K2C regulates FOXP3 expression and suppressive capacity of human Treg cells. (A) Graphical representation of isolation/stimulation and lentiviral transduction of Treg cells. (B) FACS analysis of FOXP3 expression in Tregs compared to Tconv cells. (C) Western blotting analysis of PIP4K2B and PIP4K2C silencing in Treg cells using indicated shRNAs. Empty pLKO_1 was used as control (sh_Ctrl). (D) FACS analyses of FOXP3 levels in Treg cells after silencing of PIP4K2B or PIP4K2C obtained as in C and Treg where PIP4Ks expression was rescued in the cells (PIP4K2B: sh_2B/OV_2B and PIP4K2C: sh_2C/OV_2C). (E) Mean fluorescence intensity (MFI) quantitation of FOXP3 staining of experiments in D (n ≥ 6 for sh_2B and sh_2C, n = 4 for sh_2B/OV_2B and sh_2C/OV_2C). (F) Suppression assay showing Tconv proliferation upon coculture with Treg cells depleted of PIP4K2B or PIP4K2C at different Treg/Tconv ratios. Tconv not stimulated (NS) or TCR stimulated (ST) were employed as negative and positive controls to gate proliferating cells. (G) Suppression index (diminished proliferation of Tconv cells) quantitation of experiments represented in F (n ≥ 4). The charts represent means with SD. Statistical analyses were performed using unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. The number of replicates (n) refers to individual healthy donors.

Fig. 3.

PIP4K2B and PIP4K2C regulate Treg cells transcriptomic profile. (A) Volcano plots (log fold change against the −log of the adjusted P value) of expressed genes in Tregs after depletion of PIP4K2B (Left) or PIP4K2C (Right). The numbers and dots in blue and red refer to genes that are significantly down-regulated or up-regulated, respectively (P < 0.05 and absolute log2fold change > 0.6). (B) Same as A, except PIP4K depletions were in Tconv cells. In both A and B, the x- and y-axis have the same minimum and maximum to illustrate the difference in effects of PIP4K depletions in Tregs compared to Tconv. (C) Scatter plots to show the changes in the expression of genes from the Treg_shPIP4K-DEG gene set after depletion of PIP4K in either Tregs or Tconvs as indicated. (Left) Depletion of PIP4K2B (log(sh_2B/sh_Ctrl) plotted on the x-axis and PIP4K2C depletion (log(sh_2C/sh_Ctrl) on the y-axis. (Middle) Depletion of PIP4K2B in Tregs against depletion of PIP4K2B in Tconv cells. (Right) Depletion of PIP4K2C in Tregs against depletion of PIP4K2C in Tconv cells. The data depict the average of three RNA-seq data sets for each knockdown in Treg and two in Tconvs. (D) The Treg_shPIP4K-DEG gene set was used for PCA. (E, Left) The Treg_shPIP4K-DEG (P-adjusted < 0.05) was used to generate a heat map with scaled rows and three hierarchical clusters. (Right) Gene sets from each cluster were extracted, and the average expression of genes within each cluster in Tregs and Tconv before and after depletion of PIP4Ks were quantitated. (F) Genes from cluster 3 (E) were used to interrogate their expression in an unrelated dataset comprising Tconv and Tregs before (Tconv_NS, Treg_NS) and after CD3/CD28 activation (Tconv_ST, Treg_ST) isolated from four separate donors. The heat map shows the average expression of each gene within cluster 3, while the graph depicts the average expression of all genes from cluster 3. The data illustrate that cluster 3 genes are up-regulated upon T cell activation. (G) Gene ontology enrichment analysis showing that cluster 3 genes are highly enriched for genes related to cell cycle, mitosis, and chromosome segregation. Box plots follow standard Tukey representation. Statistics are derived using Krushkal–Wallis hypothesis testing with post hoc–paired testing using Benjamini–Hochberg probability adjustments.

Fig. 4.

PIP4K2B and PIP4K2C silencing specifically inhibits TCR-dependent signaling and activation of Tregs. (A) PIP4K2B and PIP4K2C were silenced in Tregs (Top) and in Tconv (Bottom) and stimulated or not (CD3/CD28 stimulation) for 1 h. The activation of PI3K, MAPK, and mTORC1 signaling was assessed by Western blotting using the indicated antibodies. (B) Quantitation of experiments shown in A in Tregs (Top) and in Tconv (Bottom) upon PIP4Ks depletion and presented after normalization to sh_Ctrl NS and Histone 3A (n = 4). (C) Proliferation analysis (Cell Trace dye dilution) of Tregs (Top) and Tconv (Bottom) upon PIP4K silencing as indicated. (D) Quantitative data for experiments in G are presented (n = 5). (E) Cell apoptosis (Annexin V staining) in Tregs (Top) and Tconv (Bottom) upon PIP4Ks silencing. (F) Quantitative data are presented for the experiments in E (n = 5). (G and H) PI3K inhibition impacts on FOXP3 levels. Expanded Treg cells (Top) and in vitro–differentiated Treg cells (Bottom) were treated with rapamycin (100 nm) or CAL-101 (10 μM), and the levels of FOXP3 were analyzed by FACS. DMSO was used as vehicle control (n = 3). The charts represent means with SD. Statistical analyses were performed using unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. The number of replicates (n) refers to individual healthy donors.

Fig. 5.

PIP4K depletion decreases UHRF1 levels in Tregs altering immunosuppressive capacity and cell viability. (A) Treg cells were TCR stimulated for the times indicated, and the expression of UHRF1 and FOXP3 was measured by qRT-PCR. (B) Expression levels of UHRF1 in Tregs (Left) and Tconv (Right) cells upon PIP4K silencing as indicated was analyzed using qRT-PCR. The empty pLKO_1 was used as control (sh_Ctrl) (n = 3). (C) Western blotting analysis of the expression levels of UHRF1 in Treg cells depleted of PIP4K or UHRF1 as indicated compared to control cells (sh_Ctrl). (D) RT-qPCR analysis of mRNA levels of UHRF1 (Left) or FOXP3 (Right) in Tregs treated with p110δ inhibitor (CAL-101, 10 μM) for 24 or 48 h (n = 3). DMSO was used as vehicle control. (E) UHRF1 was silenced in Tregs (sh_UHRF1), and the expression of UHRF1 (Left) and of FOXP3 (Right) were measured using qRT-PCR (n = 3). (F–I) UHRF1 was silenced, and protein levels of FOXP3 (F) or CTLA4 (H) were measured by FACS. (G and I) Quantitation of mean fluorescence intensity (MFI) values of FOXP3 and CTLA4 (n = 3). (J) UHRF1 was silenced in Tregs, and their immunosuppressive activity toward T conv cells was assessed (representative of n = 3). (K) UHRF1 was silenced in Tregs, and apoptosis was assessed by Annexin V staining (representative of n = 3). The charts represent means with SD. Statistical analyses were performed using unpaired Student’s t test, *P < 0.05, **P < 0.01. The number of replicates (n) refers to individual healthy donors.

Fig. 6.

Pharmacological inhibition of PIP4K2C with NIH-12848 phenocopies PIP4K2C silencing in Treg cells. (A) Treg cells were treated with DMSO or NIH-12848 (20 μM) for 48 h and then TCR stimulated (+) or maintained as controls (−). Lysates were probed with the indicated antibodies by Western blotting (representative of n = 3). Tregs were treated with DMSO or NIH-12848 for 48 h at the concentrations indicated, and cell proliferation (B), apoptosis (C), or FOXP3 levels (D) were measured by FACS (representative of n = 3). (E) Inhibition of PIP4K2C affects Treg cells capacity to suppress Tconv proliferation. Quantitative data for suppressive experiments are presented (n = 3). The charts represent means with SD. Statistical analyses were performed using unpaired Student’s t test, *P < 0.05. The number of replicates (n) refers to individual healthy donors.