Regulatory T (Treg) cells have an essential role in maintaining immune homeostasis, in part by suppressing effector T cell functions. Phosphoinositide-dependent kinase 1 (PDK1) is a pleiotropic kinase that acts as a key effector downstream of PI3K in many cell types. In T cells, PDK1 has been shown to be critical for activation of NF-κB and AKT signaling upon TCR ligation and is therefore essential for effector T cell activation, proliferation, and cytokine production. Using Treg cell–specific conditional deletion, we now demonstrate that PDK1 is also essential for Treg cell suppressive activity in vivo. Ablation of Pdk1 specifically in Treg cells led to systemic, lethal, scurfy-like inflammation in mice. Genome-wide analysis confirmed that PDK1 is essential for the regulation of key Treg cell signature gene expression and, further, suggested that PDK1 acts primarily to control Treg cell gene expression through regulation of the canonical NF-κB pathway. Consistent with these results, the scurfy-like phenotype of mice lacking PDK1 in Treg cells was rescued by enforced activation of NF-κB downstream of PDK1. Therefore, PDK1-mediated activation of the NF-κB signaling pathway is essential for regulation of Treg cell signature gene expression and suppressor function.

Regulatory T (Treg) cells maintain immune homeostasis and peripheral tolerance by suppressing immune responses by conventional effector T (Teff) cells and other immune cells. Treg and Teff cells develop from the same progenitors in the thymus, and both require the engagement of the TCR complex for their development and function. However, the genetic programs activated by TCR conjugation in these two cells types and the function of these cells in the immune response are diametrically opposed. Given the importance of restoring and manipulating immune homeostasis in a variety of clinical settings, it is vital to understand how the components of the TCR signaling pathway function in regulatory and Teff cells.

Upon TCR and CD28 costimulation in conventional T cells (Tconv), activation of PI3K leads to accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) and translocation of phosphoinositide-dependent kinase 1 (PDK1) to the plasma membrane. Activation of PDK1 leads to AKT phosphorylation (14), as well as activation of the protein kinase A–protein kinase G–protein kinase C (AGC) family of kinases (5). In T cells, the AGC kinase PKCθ is a crucial signaling intermediate downstream of TCR/CD28 stimulation and is required for full activation of the NF-κB pathway (68). PDK1 phosphorylates PKCθ and brings together PKCθ and the CARMA1-Bcl10-Malt1 (CBM) complex (9, 10). Recruitment of phosphorylated PKCθ to the CBM leads to phosphorylation of CARMA1 (11, 12) and subsequent activation of NF-κB through IKK. Hence, in the absence of PDK1, NF-κB is not activated by TCR/CD28 costimulation (10). Despite these defects in CD4 Tconv activation, Cd4crePdk1f/f mice spontaneously developed colitis (13). This colitis resulted from activation of γδT cells because of defective Treg cell function in the absence of PDK1. These results strongly suggested that PDK1 was required for either Treg cell development or suppressive activity, although it was not clear exactly how PDK1-dependent signaling contributed to Treg cell function. Given the pleiotropic activities of PDK1 in Teff cells, we suspected that PDK1 likely affected multiple aspects of Treg biology.

In the current study, we set out to examine the role of PDK1 in Treg cells independent of thymic development, which could have been impacted in our previous studies that used Cd4cre (13). Conditional deletion of Pdk1 in lineage-committed Treg cells using Foxp3-driven Cre recombinase expression resulted in systemic lethal inflammation due to complete loss of Treg cell function. There was significant overlap in genes whose expression was dysregulated upon deletion of Pdk1 in Treg cells and upon deletion of NF-κB Rela and Rel (14). Concordant gene expression changes and Treg cell functional similarities strongly suggested that the loss of PDK1 and canonical NF-κB influenced Treg cell function through the same mechanism. Remarkably, and in support of this hypothesis, the phenotype of mice lacking PDK1 in Treg cells was rescued by constitutively activating NF-κB specifically in Treg cells. Taken together, these results demonstrate that PDK1 plays a critical role in the expression of the Treg cell transcriptome through its effect on NF-κB activation in Treg cells and hence in Treg cell function.

Pdk1-floxed mice (15) were crossed to Foxp3YFP-cre (The Jackson Laboratory). R26-StopFLikk2ca mice were purchased from The Jackson Laboratory and crossed to Foxp3YFP-cre and Foxp3YFP-crePdk1f/f mice.

Mouse tissues were fixed in 10% formalin and stored in 70% ethanol until staining. All fixed tissues were embedded in paraffin, sliced, and stained with H&E by the Histology Core at the Columbia University Irving Medical Center. The disease severity score was determined by severity of immune cell infiltration: >4 (most severe), 2–3 (severe), 1 (mild), and 0. For ears, thickness of the epidermis was measured in AxioVision software.

Isolated cells were stained with CD4, CD8, CD3, TCRβ, CD44, GITR, CTLA4, Klrg1, CD25, CD62L, Foxp3, Ki67, p–mammalian target of rapamycin (mTOR), p-S6, IL-10, IL-17, and IFN-γ Abs (eBioscience, BioLegend, BD Biosciences, or Tonbo Biosciences). For Foxp3, p-mTOR, p-S6, CTLA4, or Ki67 staining, cells were stained with the Foxp3 Transcription Factor Staining Buffer Set according to the manufacturer’s protocol (eBioscience). For cytokine staining, cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin in the presence of GolgiPlug (BD Biosciences) for 4 h at 37°C and stained for surface markers, followed by fixation with 4% paraformaldehyde or fixation buffer from the eBioscience kit. The cells were then permeabilized and stained for cytokines. Stained cells were acquired with LSR II or LSRFortessa cytometers; data were analyzed using FlowJo (Tree Star) (BD Biosciences). Magnetically enriched (Miltenyi Biotec) CD4+ T cells were sorted into Treg cell (CD4+ YFP+) and Tconv cell (CD4+ YFP) populations using a FACSAria (BD Biosciences).

Tconv cells were sorted from wild-type (WT) mice, and Treg cells were sorted from WT (Foxp3YFP-cre) or conditional knockout mice. Tconv and Treg cells were cocultured at different ratios in the presence of αCD3 and αCD28 Abs. Cells were cultured for 3 d and [3H]thymidine was added to each well 8 h before harvest. T cell proliferation was observed by measuring the incorporation of [3H]thymidine by the scintillation counter.

Tconv cells were sorted from WT mice, and Treg cells were sorted from WT or conditional knockout mice. A total of 4 × 105 Tconv cells were transferred to Rag1–/– mice by i.v. injection alone or together with WT or knockout Treg cells (1 × 105). Weight was monitored weekly after adoptive transfer to observe progression of disease. Mice were euthanized when they reached 20% reduction of initial weight, and colons and other lymphoid organs were collected for H&E staining and flow cytometric analyses.

Treg cells were sorted, and RNA samples were prepared by using the RNeasy Mini Kit (QIAGEN). The quantity and quality of RNA samples were measured by Bioanalyzer (Molecular Pathology Shared Resource at Herbert Irving Comprehensive Cancer Center, Columbia University). Library construction and sequencing of Treg cells from Foxp3YFP-crePdk1+/+ or Foxp3YFP-crePdk1f/f mice were done by the Columbia Genome Center. The poly-A pull-down was used to enrich mRNAs from total RNA samples, and libraries were prepared by using an Illumina TruSeq RNA Sample Prep Kit. Libraries were then sequenced using an Illumina HiSeq 2000. Library construction of CD4+YFP+ Treg cells from Foxp3YFP-cre/+Pdk1+/+ or Foxp3YFP-cre/+Pdk1f/f mice was performed using NEBNext Ultra II RNA Library Prep Kit for Illumina in our laboratory and then sequenced by Hiseq 2500. Upon sequencing, raw FASTQ files were aligned on the mm10 genome using the STAR aligner with default parameters (16). Normalization and differential expression analysis were completed with the Bioconductor package DESeq2 after removing the batch effect using the ComBat function of the sva package v3.18.0.

The development of colitis in aged Cd4crePdk1f/f mice suggested that PDK1 might have an important role in Treg cells (10). However, as Pdk1 was deleted early in thymopoiesis, it was not clear whether the functional impairment was due to a defect in the development of Treg cells within the thymus or, potentially, extrathymic, peripheral generation of Treg cells. To investigate the role of PDK1 in mature Treg cells, we crossed Pdk1-floxed mice with Foxp3YFP-cre mice (17) to delete Pdk1 in Treg cells but not Tconv cells (Fig. 1A). Although loss of Pdk1 did not affect the development of Treg cells in the thymus or the proliferation of Treg cells in the noninflammatory lymph nodes (Supplemental Fig. 1A–C), Foxp3YFP-cre/YFP-cre (referred to as Foxp3YFP-cre in the rest of the manuscript) Pdk1f/f mice died 2–4 wk after birth. Foxp3YFP-crePdk1f/f mice were severely runted compared with their WT littermates (Fig. 1B) and showed gross evidence of severe dermatitis (Fig. 1C), splenomegaly, and lymphadenopathy (Fig. 1D). The length of the colon was shortened, consistent with severe colitis (Fig. 1D). The large size of the spleen and lymph nodes was accompanied by significantly increased cell numbers, indicative of lymphoproliferative diseases (Fig. 1E). Lung and liver histology revealed significantly increased immune infiltration (Fig. 1F). To further assess the systemic inflammatory phenotype of Foxp3YFP-crePdk1f/f mice, we analyzed the expression of activation markers on CD4+ and CD8+ T cells in the spleen and lymph nodes. Effector (CD44hiCD62Llo) and central (CD44hiCD62Lhi) memory CD4+ T cells were drastically increased compared with WT littermates (Fig. 1G, 1H). Consistent with their inflammatory phenotype, production of IFN-γ by CD4+ T cells was significantly increased in spleen and lymph nodes (Fig. 1I, 1J). A similar trend was detected in CD4+ and CD8+ T cells from the lung and colon lamina propria (Fig. 1I and data not shown). Moreover, we detected a trend toward increased IL-17A expression in the lymphoid tissues of Pdk1-deficient mice (Fig. 1J). Taken together, these results demonstrate a requirement for PDK1 in maintaining immune tolerance.

The early mortality and the systemic inflammatory phenotype of Foxp3YFP-crePdk1f/f mice resembled the phenotype of scurfy mice, which lack functional Foxp3+ Treg cells. However, when we analyzed Foxp3YFP-crePdk1f/f mice we observed normal numbers of Treg cells in most tissues, including the thymus and peripheral lymph node (PLN), but a slightly decreased percentage in the spleen (Fig. 2A, 2B). Given this disconnect between Treg cell numbers and the systemic lymphoproliferative and inflammatory phenotype, we next examined the expression of Treg cell signature gene products, such as Cd44, Gitr, Ctla4, and Klrg1 (Supplemental Fig. 2A, 2B). The expression of all of these proteins were downregulated in PDK1-deficient Foxp3YFP+ Treg cells from Foxp3YFP-cre/+Pdk1f/f mice, compared with WT Foxp3YFP+ Treg cells from Foxp3YFP-cre/+Pdk1+/+ control mice, whereas CD62L and CD25 were highly expressed. Moreover, Treg cells from Foxp3YFP-crePdk1f/f mice expressed reduced levels of the suppressive cytokine IL-10 (Supplemental Fig. 2C). Next, we tested the function of PDK1-deficient Treg cells by in vitro or in vivo suppression assay. Surprisingly, in in vitro suppression assays, Treg cells from Foxp3YFP-crePdk1f/f mice inhibited proliferation of conventional CD4+ T cells (Fig. 2C). We then investigated the ability of PDK1-deficient Treg cells to prevent weight loss in Rag1–/– mice following adoptive transfer of Tconv cells (Fig. 2D). Colon histology confirmed that Pdk1–/– Treg cells were incapable of suppressing the development of colitis (Fig. 2E). Taken together with the scurfy-like phenotype of Foxp3YFP-crePdk1f/f mice, these results demonstrate that PDK1 is essential for the suppressive function of Treg cells in vivo.

PDK1 regulates multiple signaling pathways in T cells, including the canonical NF-κB pathway and Akt/mTOR pathways. To investigate the contribution of these and other PDK1-regulated pathways to the phenotype observed in PDK1-deleted Treg cells, we analyzed their gene expression profiles. We performed RNA sequencing (RNA-seq) using Treg cells isolated from Foxp3YFP-cre (WT) and Foxp3YFP-crePdk1f/f mice. This analysis revealed widespread dysregulation of gene expression in the absence of PDK1 (Fig. 3A). In Teff cells, PDK1 plays a key role in integrating CD28 coreceptor and TCR signaling. Consistent with this established role, pathway analysis (Gene Set Enrichment Analysis; GSEA) revealed significant effects of PDK1 deletion in Treg cells on the expression of genes associated with the CD28 pathway (Fig. 3B). GSEA also revealed effects on the expression of genes involved in glucose transport (Fig. 3C), consistent with the previously described role of PDK1 in T cell metabolic reprogramming. Finally, and as predicted based on the loss of in vivo suppressive function of Treg cells lacking PDK1, we also observed significant dysregulation of Treg cell signature genes in the absence of PDK1 (Fig. 3D). Taken together, these results are consistent with an essential role of PDK1 in TCR-associated signaling in Treg cells. When we performed motif analysis with DiRE (18) to identify transcription factors that may have altered activity upon loss of PDK1, we found that NF-κB was the most significant transcription factor associated with the differential gene expression in Treg cells lacking PDK1 (Fig. 3E). Together with the phenotypic similarities resulting from loss of PDK1 and loss of canonical NF-κB in Treg cells [Oh et al. (14)], these results suggest that PDK1 may have an important role in regulating canonical NF-κB in Treg cells.

We therefore compared the Pdk1–/– Treg cell RNA-seq data to RNA-seq data from Treg cells isolated from Foxp3YFP-creRelaf/fRelf/f mice (Rela–/–Rel–/– Treg). Hierarchical clustering suggested that PDK1-deficient and c-Rel/p65-deficient Treg cells shared common transcriptional changes when compared with WT Treg cells (Fig. 3F). Furthermore, we observed that the majority of genes that were significantly dysregulated in PDK1-deficient Treg cells (up- or downregulated) were similarly impaired in Treg cells lacking canonical NF-κB (Fig. 3G, 3H). Pdk1–/– and Rela–/−Rel–/– Treg cells had decreased expression of genes implicated in Treg suppressive function (Fig. 3I), including Id3 (19, 20), Nrn1 (encodes neuritin) (21), and Foxo1 (22, 23). Furthermore, expression of Treg cell signature genes, including Ikzf4, Hdac9, and P2rx7, were significantly reduced (Fig. 3I). Genes encoding cell adhesion molecules, such as Itgb8 and Selp, were also downregulated in both Pdk1–/– and Rela–/–Rel–/–Tregs in addition to chemokine receptors (e.g., Cxcr5 and Ccr6), suggesting that defects in cell migration in the absence of PDK1 or canonical NF-κB may contribute to the defective suppressive function of PDK1-deficient Treg cell in vivo (Fig. 3I). Of note, at odds with Rela–/−Rel–/– Treg cells, expression of inflammatory cytokines was not increased in Pdk1–/– cells (data not shown). Finally, both Pdk1–/– and Rela–/–Rel–/– Treg cells displayed increased expression of genes involved in cell cycle progression and cell division (Fig. 3J), which was likely a consequence of surrounding inflammatory mediators that are potent stimulators of Treg cell division (24). Considering that PDK1 is also involved in the mTOR signaling pathway, we analyzed phosphorylated mTOR and its downstream key player phosphorylated S6 (Supplemental Fig. 1D, 1E). The results showed that there is no significant difference between WT and PDK1-deficient (KO) Treg cells. Overall, our genome-wide analysis of gene expression suggests that PDK1 signaling in Treg cells controls a pattern of gene sets involved in cell metabolism and Treg cell function that largely overlap with canonical NF-κB–regulated genes.

FIGURE 1.

Early lethal autoimmune syndrome upon Treg cell–specific deletion of Pdk1. (A) Treg and Tconv cells were sorted from the spleen and lymph nodes of WT (Foxp3YFP-crePdk1+/+) and Foxp3YFP-crePdk1f/f, and Pdk1 expression was measured by quantitative RT-PCR. (B and C) Representative photographs of 3-wk-old mice. (D) Representative spleen, PLN, and colon of 3-wk-old mice. (E) Total cell count in spleen and PLN. (F) H&E staining of lung and liver sections from 3-wk-old mice. Scale bar, 100 μm; original magnification ×100. (GJ) Lymphoid and nonlymphoid tissues of 3-wk-old mice were analyzed by flow cytometry. (G) Representative CD44 and CD62L expression in spleen T cells. (H) Percentage of naive (CD44lowCD62Lhi), central memory (TCM) (CD44hiCD62Lhi) and effector memory (TEM) (CD44hiCD62Llow) cells in spleen CD4+Foxp3 (Tconv) and CD8+ T cells. (I) Representative IL-17A and IFN-γ expression in CD4+ Tconv in the indicated organs. Numbers indicate the percentage in quadrants. (J) Cumulative percentage of IFN-γ+ and IL-17A+ in CD4+ Tconv. Mean ± SD is shown. Data are representative or cumulative of three to six mice per group from at least 3 experiments. *p < 0.05, **p < 0.005. NS, not significant.

FIGURE 1.

Early lethal autoimmune syndrome upon Treg cell–specific deletion of Pdk1. (A) Treg and Tconv cells were sorted from the spleen and lymph nodes of WT (Foxp3YFP-crePdk1+/+) and Foxp3YFP-crePdk1f/f, and Pdk1 expression was measured by quantitative RT-PCR. (B and C) Representative photographs of 3-wk-old mice. (D) Representative spleen, PLN, and colon of 3-wk-old mice. (E) Total cell count in spleen and PLN. (F) H&E staining of lung and liver sections from 3-wk-old mice. Scale bar, 100 μm; original magnification ×100. (GJ) Lymphoid and nonlymphoid tissues of 3-wk-old mice were analyzed by flow cytometry. (G) Representative CD44 and CD62L expression in spleen T cells. (H) Percentage of naive (CD44lowCD62Lhi), central memory (TCM) (CD44hiCD62Lhi) and effector memory (TEM) (CD44hiCD62Llow) cells in spleen CD4+Foxp3 (Tconv) and CD8+ T cells. (I) Representative IL-17A and IFN-γ expression in CD4+ Tconv in the indicated organs. Numbers indicate the percentage in quadrants. (J) Cumulative percentage of IFN-γ+ and IL-17A+ in CD4+ Tconv. Mean ± SD is shown. Data are representative or cumulative of three to six mice per group from at least 3 experiments. *p < 0.05, **p < 0.005. NS, not significant.

Close modal
FIGURE 3.

Dysregulation of the Treg cell transcriptome in the absence of PDK1. CD4+YFP+ Treg cells from spleen and PLN of Foxp3YFP-crePdk1+/+ or Foxp3YFP-crePdk1f/f mice were FACS sorted and submitted to deep RNA-seq analysis. (A) Volcano plot showing DEGs (downregulated genes [log2(fold change) <−1] are in blue; upregulated genes [log2(fold change) >1] are in red). (BD) Datasets were analyzed for signature enrichment using GSEA packages C2, C5, and C7. Representative Enplot graphs and expression heatmaps with selected genes are shown. (E) Transcription factor binding motif enrichment analysis using the Distant Regulatory Elements in DEGs in Pdk1-deficient versus WT Treg cells. (F) Heatmap showing the expression of DEGs from (A) in WT, Pdk1–/–, and Rela–/–Rel–/– Treg cells [data from Oh et al. (14)] upon batch correction and hierarchical clustering. (G and H) Plots showing the correlation of changes in gene expression between Pdk1–/– and Rela–/–Rel–/– Treg cells. (G) Genes significantly downregulated in Pdk1–/– cells. (H) Genes significantly upregulated in Pdk1–/– cells. (I and J) Expression heatmaps with selected Treg cell signature genes (I) and genes involved in cell cycle (J) are shown.

FIGURE 3.

Dysregulation of the Treg cell transcriptome in the absence of PDK1. CD4+YFP+ Treg cells from spleen and PLN of Foxp3YFP-crePdk1+/+ or Foxp3YFP-crePdk1f/f mice were FACS sorted and submitted to deep RNA-seq analysis. (A) Volcano plot showing DEGs (downregulated genes [log2(fold change) <−1] are in blue; upregulated genes [log2(fold change) >1] are in red). (BD) Datasets were analyzed for signature enrichment using GSEA packages C2, C5, and C7. Representative Enplot graphs and expression heatmaps with selected genes are shown. (E) Transcription factor binding motif enrichment analysis using the Distant Regulatory Elements in DEGs in Pdk1-deficient versus WT Treg cells. (F) Heatmap showing the expression of DEGs from (A) in WT, Pdk1–/–, and Rela–/–Rel–/– Treg cells [data from Oh et al. (14)] upon batch correction and hierarchical clustering. (G and H) Plots showing the correlation of changes in gene expression between Pdk1–/– and Rela–/–Rel–/– Treg cells. (G) Genes significantly downregulated in Pdk1–/– cells. (H) Genes significantly upregulated in Pdk1–/– cells. (I and J) Expression heatmaps with selected Treg cell signature genes (I) and genes involved in cell cycle (J) are shown.

Close modal

To eliminate any confounding effects of the spontaneous inflammation observed in Foxp3YFP-crePdk1f/f mice, we performed RNA-seq using CD4+Foxp3YFP+Treg cells isolated from Foxp3YFP-cre/+ (WT) and Foxp3YFP-cre/+Pdk1f/f (KO) mice. Because of X chromosome inactivation, these mice do not develop an inflammatory phenotype, as only half of the Treg cells express Cre and are thus PDK1-deficient, whereas the remaining Treg cells remain intact and functional. There were 774 differentially expressed genes (DEGs), among which 524 genes were downregulated, whereas 250 genes were upregulated in PDK1-deficient Treg cells (Supplemental Fig. 3A, 3B, Supplemental Table I). Among the DEGs were 66 Treg cell signature genes, confirming that PDK1 expression is essential for normal Treg cell function (Supplemental Fig. 3C). Expression of Ctla4, Klrg1, Cd44, IL-10, and Gitr were downregulated in the RNA-seq data set, which we confirmed by flow cytometry (Supplemental Fig. 2). Importantly, GSEA identified an enrichment of known NF-κB binding sites in the DEG set (Supplemental Fig. 3D–G). These data further corroborate the notion that PDK1 deficiency disrupts Treg cell function, at least partly due to impaired NF-κB activation.

FIGURE 2.

Defective Treg cell homeostasis in Foxp3YFP-crePdk1f/f animals. (A and B) Lymphoid and nonlymphoid tissues of 3-wk-old mice were analyzed by flow cytometry. (A) Representative Foxp3 expression in CD4+ T cells. Numbers indicate the percentage in the gate. (B) Cumulative percentage and number of Treg cells in the indicated tissues. (C) In vitro Treg cell suppression assay. (D and E) In vivo colitis suppression assay. (D) Weight curves, shown as percentage of original weight. (E) Representative colon histology 7 wk after transfer. Scale bar, 100 μm; original magnification ×100. Mean ± SD is shown. Data are representative or cumulative of five mice per group from at least three experiments. *p < 0.05. NS, not significant.

FIGURE 2.

Defective Treg cell homeostasis in Foxp3YFP-crePdk1f/f animals. (A and B) Lymphoid and nonlymphoid tissues of 3-wk-old mice were analyzed by flow cytometry. (A) Representative Foxp3 expression in CD4+ T cells. Numbers indicate the percentage in the gate. (B) Cumulative percentage and number of Treg cells in the indicated tissues. (C) In vitro Treg cell suppression assay. (D and E) In vivo colitis suppression assay. (D) Weight curves, shown as percentage of original weight. (E) Representative colon histology 7 wk after transfer. Scale bar, 100 μm; original magnification ×100. Mean ± SD is shown. Data are representative or cumulative of five mice per group from at least three experiments. *p < 0.05. NS, not significant.

Close modal

We have found that canonical NF-κB activity is essential for the suppressive function of Treg cells (14). In contrast, several reports suggest AKT signaling may oppose Treg cell suppressive function and Treg cell signature genes (25, 26). In this context, the results of our RNA-seq analysis strongly suggest that the loss of suppressive function in PDK1-deficient Tregs is due to loss of canonical NF-κB activation. If NF-κB is the key downstream effector of PDK1 in Treg cells, then loss of PDK1 in Treg cells would be overcome by activating NF-κB in a PDK1-independent manner. Therefore, we crossed Foxp3YFP-crePdk1f/f mice to R26-StopFLikk2ca, a transgenic strain that conditionally expresses a constitutively active form of IκB kinase 2 (IKKβ). IKKβCA would function downstream of PDK1 to directly drive activation of the canonical NF-κB pathway. Strikingly, the lethal inflammatory phenotype seen in the Foxp3YFP-crePdk1f/f mice was rescued by activation of NF-κB in Treg cells. Histological evidence of inflammatory infiltrates in the lung and liver (Fig. 4A) and epidermal thickening of ears seen in Foxp3YFP-crePdk1f/f mice were both dramatically reduced upon conditional expression of constitutively active IKKβCA (Fig. 4B, 4C). Quantification of disease severity in the lung, liver, and skin shows that the inflammatory infiltrate resulting from Treg cell PDK1 deficiency is significantly reversed by constitutive activation of NF-κB (Fig. 4D). These results therefore demonstrate that loss of suppressive function in PDK1-deficient Treg cells in vivo can be rescued by downstream activation of the canonical NF-κB pathway. These studies, placed in the context of previous analyses of NF-κB and AKT signaling in Treg cells, strongly support a model in which the primary function of PDK1 in Treg cells, in contrast to Tconv cells, is to mediate TCR-induced activation of NF-κB and thereby help maintain the Treg cell signature transcriptome.

PDK1 is a pleiotropic kinase that is responsible for the regulation of multiple signaling pathways downstream of the numerous events that influence PI3K activity (27). In lymphocytes, there has been significant interest in dissecting the contributions of PDK1 to the multiple pathways activated upon Ag receptor signaling. Using a variety of genetic models and tools, PDK1 has been definitively shown to have a key role in both B and T cell development (2832). However, the contributions of PDK1 and the downstream signaling pathways regulated by PDK1 to the functional response of B and T cells following Ag receptor ligation have remained more controversial. We previously demonstrated that PDK1 is required for the activation of NF-κB by TCR/CD28 ligation in CD4+ T cells and that PDK1 is therefore important for T cell activation and cytokine production (10). Other studies demonstrated that PDK1 was also required for AKT activation downstream of the TCR and that this signaling was essential for regulation of mTOR (1), Foxo1 (33), and NOTCH (34) signaling pathways in T cells. In CD8+ T cells, it has been shown that, in CTLs, PDK1 maintains IL-2–driven proliferation and controls glucose metabolism and glycolysis through an mTOCR1–HIF1 pathway-regulated transcriptional program (35, 36). Hence, loss of PDK1 results in substantive disruption of Tconv cell functions.

Despite the established role of PDK1 in T cell responses, we previously observed that Cd4crePdk1f/f mice, which have defective Tconv cells (10), develop colitis (13). TCRγδ+ T cells, which are not affected by CD4-mediated deletion of PDK1, were found to be driving colitis in this model (13). In this model, PDK1 expression was lost in all TCRαβ+ T cells, including Tconv and Treg cells. As PDK1 is essential for TCR signaling and thereby Tconv function, Tconv cells could not have contributed to the development of spontaneous colitis in Cd4crePdk1f/f mice. These results therefore suggested that PDK1 might have an important role in the suppressive function of Treg cells, which possess the ability to suppress activation of TCRγδ+ T cells in intestines (13). However, the exact role of PDK1 in Treg cells had remained unclear. For example, PKCθ, a well described downstream target of PDK1, is involved in Treg cell development (37), whereas inhibition of PKCθ surprisingly augments Treg cell function (38). A recent study showed that Treg cells lacking PKCθ are normally suppressive in vitro (37, 39), but the activity of PKCθ-deficient Treg cells in vivo (e.g., in the colitis transfer model) was not tested. Of note, we recently established that the canonical NF-κB subunits p65 and c-Rel play critical roles in the maintenance of Treg cell identity and function [Oh et al. (14)]. However, despite the NF-κB–deficient Treg cells being completely defective in in vivo suppression of transfer colitis, they were fully active in in vitro assays of suppression. Therefore, it is possible that absence of PKCθ, similar to the absence of PDK1, leads to functionally inactive Treg cells.

To definitively determine the role of PDK1 in mature Treg cells, we deleted Pdk1 specifically in Treg cells using Foxp3YFP-cre. The resulting severe, systemic inflammatory phenotype in Foxp3YFP-crePdk1f/f mice resembles the scurfy phenotype, which is observed in the absence of Foxp3+ Treg cells, suggesting that PDK1 is critical for the suppressive function of Treg cells in vivo (Figs. 1, 2). These results are consistent with the phenotype of mice lacking the canonical NF-κB subunits p65 and c-Rel (14) or those lacking IKKβ (40) in Treg cells. Given that the roles of AKT and mTOR in Treg cell function are far less definitive and are unlikely to account for the observed phenotype (41, 42), it is more likely that it a lack of PDK1-induced NF-κB activation is responsible for the observed effects on Treg cell function. Consistent with this hypothesis, genome-wide gene expression analysis of Pdk1–/– Treg cells demonstrated enrichment of genes with NF-κB motifs in their promoters among the DEGs and showed a significant overlap between Pdk1–/– and Rela–/–Rel–/– Treg cells (Fig. 3). In contrast, gene signatures related to cholesterol/lipid metabolism, which are the accepted targets for AKT/mTOR pathways, were not significantly perturbed in Pdk1–/– Treg cells. Taken together, both the phenotypic, and genomic data suggest that NF-κB is the primary downstream target of PDK1 in Treg cell function. To clearly demonstrate that loss of canonical NF-κB accounts for the scurfy phenotype of Cd4crePdk1f/f mice, we used conditional expression of a constitutively active form of IKKβ to activate NF-κB downstream of PDK1 (Fig. 4). Strikingly, these mice no longer exhibited the scurfy-like phenotype, demonstrating that PDK1-mediated activation of NF-κB is essential for Treg cell function. These results definitively establish that PDK1 is essential for Treg cell suppressive function in vivo and that this function is likely manifested through signaling to NF-κB. These results also suggest the need for further efforts to understand the signaling pathways leading to NF-κB activation in Treg cells. The severe, scurfy-like phenotype exhibited by mice lacking canonical NF-κB (14, 40) or PDK1 in Treg cells is far more dramatic than that observed upon loss of CD28 (43) or TCR (44) in Treg cells. Taken together with the still unclear role of PKCθ in Treg cells, these results suggest that PDK1-mediated NF-κB activation may occur downstream of additional Treg cell signaling pathways. Complete elucidation of the signals that regulate PDK1 and NF-κB in Treg cells will therefore be essential for the continued development of immunomodulatory therapies in autoimmune diseases and cancer.

FIGURE 4.

Constitutive activation of canonical NF-κB in Treg cells rescues the inflammatory phenotype in Pdk1-deficient mice. Foxp3YFP-crePdk1f/f mice were crossed to R26-StopFLikk2ca (Ikk2ca) mice. (A) H&E staining of lung and liver sections from 3-wk-old mice. Scale bar, 100 μm; original magnification ×100. (B and C) Representative H&E staining (B) and cumulative ear thickness of 3-wk-old mice of the indicated genotypes; (C) mean ± SD is shown. (D) Inflammatory features in the lung, skin, and liver of each genotype, as described in the Materials and Methods section. Data are representative or cumulative of five mice per group from five experiments. **p < 0.005.

FIGURE 4.

Constitutive activation of canonical NF-κB in Treg cells rescues the inflammatory phenotype in Pdk1-deficient mice. Foxp3YFP-crePdk1f/f mice were crossed to R26-StopFLikk2ca (Ikk2ca) mice. (A) H&E staining of lung and liver sections from 3-wk-old mice. Scale bar, 100 μm; original magnification ×100. (B and C) Representative H&E staining (B) and cumulative ear thickness of 3-wk-old mice of the indicated genotypes; (C) mean ± SD is shown. (D) Inflammatory features in the lung, skin, and liver of each genotype, as described in the Materials and Methods section. Data are representative or cumulative of five mice per group from five experiments. **p < 0.005.

Close modal

This work was supported by National Institutes of Health, National Institute of Allergy and Infectious Diseases grant R01AI068977 (to S.G.).

The data presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE163663.

The online version of this article contains supplemental material.

Abbreviations used in this article:

DEG

differentially expressed gene

GSEA

Gene Set Enrichment Analysis

IKKβ

constitutively active form of IκB kinase 2

KO

knockout

mTOR

mammalian target of rapamycin

PDK1

phosphoinositide-dependent kinase 1

PLN

peripheral lymph node

Rela–/–Rel–/– Treg

Treg cell isolated from Foxp3YFP-creRelaf/fRelf/f mice

RNA-seq

RNA sequencing

Tconv

conventional T cell

Teff

effector T

Treg

regulatory T

WT

wild-type.

1
Calleja
,
V.
,
D.
Alcor
,
M.
Laguerre
,
J.
Park
,
B.
Vojnovic
,
B. A.
Hemmings
,
J.
Downward
,
P. J.
Parker
,
B.
Larijani
.
2007
.
Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo.
PLoS Biol.
5
:
e95
.
2
Costello
,
P. S.
,
M.
Gallagher
,
D. A.
Cantrell
.
2002
.
Sustained and dynamic inositol lipid metabolism inside and outside the immunological synapse.
Nat. Immunol.
3
:
1082
1089
.
3
Garçon
,
F.
,
D. T.
Patton
,
J. L.
Emery
,
E.
Hirsch
,
R.
Rottapel
,
T.
Sasaki
,
K.
Okkenhaug
.
2008
.
CD28 provides T-cell costimulation and enhances PI3K activity at the immune synapse independently of its capacity to interact with the p85/p110 heterodimer.
Blood
111
:
1464
1471
.
4
Bayascas
,
J. R.
,
S.
Wullschleger
,
K.
Sakamoto
,
J. M.
García-Martínez
,
C.
Clacher
,
D.
Komander
,
D. M.
van Aalten
,
K. M.
Boini
,
F.
Lang
,
C.
Lipina
, et al
.
2008
.
Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance.
Mol. Cell. Biol.
28
:
3258
3272
.
5
Mora
,
A.
,
D.
Komander
,
D. M.
van Aalten
,
D. R.
Alessi
.
2004
.
PDK1, the master regulator of AGC kinase signal transduction.
Semin. Cell Dev. Biol.
15
:
161
170
.
6
Sun
,
Z.
,
C. W.
Arendt
,
W.
Ellmeier
,
E. M.
Schaeffer
,
M. J.
Sunshine
,
L.
Gandhi
,
J.
Annes
,
D.
Petrzilka
,
A.
Kupfer
,
P. L.
Schwartzberg
,
D. R.
Littman
.
2000
.
PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes.
Nature
404
:
402
407
.
7
Lin
,
X.
,
A.
O’Mahony
,
Y.
Mu
,
R.
Geleziunas
,
W. C.
Greene
.
2000
.
Protein kinase C-theta participates in NF-kappaB activation induced by CD3-CD28 costimulation through selective activation of IkappaB kinase beta.
Mol. Cell. Biol.
20
:
2933
2940
.
8
Coudronniere
,
N.
,
M.
Villalba
,
N.
Englund
,
A.
Altman
.
2000
.
NF-kappa B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-theta.
Proc. Natl. Acad. Sci. USA
97
:
3394
3399
.
9
Lee
,
K. Y.
,
F.
D’Acquisto
,
M. S.
Hayden
,
J. H.
Shim
,
S.
Ghosh
.
2005
.
PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation.
Science
308
:
114
118
.
10
Park
,
S. G.
,
J.
Schulze-Luehrman
,
M. S.
Hayden
,
N.
Hashimoto
,
W.
Ogawa
,
M.
Kasuga
,
S.
Ghosh
.
2009
.
The kinase PDK1 integrates T cell antigen receptor and CD28 coreceptor signaling to induce NF-kappaB and activate T cells.
Nat. Immunol.
10
:
158
166
.
11
Matsumoto
,
R.
,
D.
Wang
,
M.
Blonska
,
H.
Li
,
M.
Kobayashi
,
B.
Pappu
,
Y.
Chen
,
D.
Wang
,
X.
Lin
.
2005
.
Phosphorylation of CARMA1 plays a critical role in T Cell receptor-mediated NF-kappaB activation.
Immunity
23
:
575
585
.
12
Sommer
,
K.
,
B.
Guo
,
J. L.
Pomerantz
,
A. D.
Bandaranayake
,
M. E.
Moreno-García
,
Y. L.
Ovechkina
,
D. J.
Rawlings
.
2005
.
Phosphorylation of the CARMA1 linker controls NF-kappaB activation.
Immunity
23
:
561
574
.
13
Park
,
S. G.
,
R.
Mathur
,
M.
Long
,
N.
Hosh
,
L.
Hao
,
M. S.
Hayden
,
S.
Ghosh
.
2010
.
T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells.
Immunity
33
:
791
803
.
14
Oh
,
H.
,
Y.
Grinberg-Bleyer
,
W.
Liao
,
D.
Maloney
,
P.
Wang
,
Z.
Wu
,
J.
Wang
,
D. M.
Bhatt
,
N.
Heise
,
R. M.
Schmid
, et al
.
2017
.
An NF-κB transcription-factor-dependent lineage-specific transcriptional program promotes regulatory T cell identity and function.
Immunity
47
:
450
465.e5
.
15
Hashimoto
,
N.
,
Y.
Kido
,
T.
Uchida
,
S.
Asahara
,
Y.
Shigeyama
,
T.
Matsuda
,
A.
Takeda
,
D.
Tsuchihashi
,
A.
Nishizawa
,
W.
Ogawa
, et al
.
2006
.
Ablation of PDK1 in pancreatic beta cells induces diabetes as a result of loss of beta cell mass.
Nat. Genet.
38
:
589
593
.
16
Dobin
,
A.
,
C. A.
Davis
,
F.
Schlesinger
,
J.
Drenkow
,
C.
Zaleski
,
S.
Jha
,
P.
Batut
,
M.
Chaisson
,
T. R.
Gingeras
.
2013
.
STAR: ultrafast universal RNA-seq aligner.
Bioinformatics
29
:
15
21
.
17
Rubtsov
,
Y. P.
,
J. P.
Rasmussen
,
E. Y.
Chi
,
J.
Fontenot
,
L.
Castelli
,
X.
Ye
,
P.
Treuting
,
L.
Siewe
,
A.
Roers
,
W. R.
Henderson
Jr.
, et al
.
2008
.
Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces.
Immunity
28
:
546
558
.
18
Gotea
,
V.
,
I.
Ovcharenko
.
2008
.
DiRE: identifying distant regulatory elements of co-expressed genes.
Nucleic Acids Res.
36
:
W133
W139
.
19
Miyazaki
,
M.
,
K.
Miyazaki
,
S.
Chen
,
M.
Itoi
,
M.
Miller
,
L. F.
Lu
,
N.
Varki
,
A. N.
Chang
,
D. H.
Broide
,
C.
Murre
.
2014
.
Id2 and Id3 maintain the regulatory T cell pool to suppress inflammatory disease.
Nat. Immunol.
15
:
767
776
.
20
Rauch
,
K. S.
,
M.
Hils
,
E.
Lupar
,
S.
Minguet
,
M.
Sigvardsson
,
M. E.
Rottenberg
,
A.
Izcue
,
C.
Schachtrup
,
K.
Schachtrup
.
2016
.
Id3 maintains Foxp3 expression in regulatory T cells by controlling a transcriptional network of E47, Spi-B, and SOCS3.
Cell Rep.
17
:
2827
2836
.
21
Barbi
,
J. J.
,
P. D. A.
Vignali
,
H.
Yu
,
F.
Pan
,
D.
Pardoll
.
2016
.
The neurotrophic factor neuritin maintains and promotes the function of regulatory T cells in autoimmunity and cancer.
J. Immunol.
196
:
58.12
.
22
Ouyang
,
W.
,
W.
Liao
,
C. T.
Luo
,
N.
Yin
,
M.
Huse
,
M. V.
Kim
,
M.
Peng
,
P.
Chan
,
Q.
Ma
,
Y.
Mo
, et al
.
2012
.
Novel Foxo1-dependent transcriptional programs control T(reg) cell function.
Nature
491
:
554
559
.
23
Kerdiles
,
Y. M.
,
E. L.
Stone
,
D. R.
Beisner
,
M. A.
McGargill
,
I. L.
Ch’en
,
C.
Stockmann
,
C. D.
Katayama
,
S. M.
Hedrick
.
2010
.
Foxo transcription factors control regulatory T cell development and function. [Published erratum appears in 2011 Immunity 34: 135.]
Immunity
33
:
890
904
.
24
Grinberg-Bleyer
,
Y.
,
D.
Saadoun
,
A.
Baeyens
,
F.
Billiard
,
J. D.
Goldstein
,
S.
Grégoire
,
G. H.
Martin
,
R.
Elhage
,
N.
Derian
,
W.
Carpentier
, et al
.
2010
.
Pathogenic T cells have a paradoxical protective effect in murine autoimmune diabetes by boosting Tregs.
J. Clin. Invest.
120
:
4558
4568
.
25
Kitz
,
A.
,
M.
de Marcken
,
A. S.
Gautron
,
M.
Mitrovic
,
D. A.
Hafler
,
M.
Dominguez-Villar
.
2016
.
AKT isoforms modulate Th1-like Treg generation and function in human autoimmune disease.
EMBO Rep.
17
:
1169
1183
.
26
Sauer
,
S.
,
L.
Bruno
,
A.
Hertweck
,
D.
Finlay
,
M.
Leleu
,
M.
Spivakov
,
Z. A.
Knight
,
B. S.
Cobb
,
D.
Cantrell
,
E.
O’Connor
, et al
.
2008
.
T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR.
Proc. Natl. Acad. Sci. USA
105
:
7797
7802
.
27
Pearce
,
L. R.
,
D.
Komander
,
D. R.
Alessi
.
2010
.
The nuts and bolts of AGC protein kinases.
Nat. Rev. Mol. Cell Biol.
11
:
9
22
.
28
Kelly
,
A. P.
,
H. J.
Hinton
,
R. G.
Clarke
,
D. A.
Cantrell
.
2006
.
Phosphoinositide-dependent kinase l (PDK1) haplo-insufficiency inhibits production of alpha/beta (alpha/beta) but not gamma delta (gamma/delta) T lymphocytes.
FEBS Lett.
580
:
2135
2140
.
29
Hinton
,
H. J.
,
D. R.
Alessi
,
D. A.
Cantrell
.
2004
.
The serine kinase phosphoinositide-dependent kinase 1 (PDK1) regulates T cell development.
Nat. Immunol.
5
:
539
545
.
30
Baracho
,
G. V.
,
M. H.
Cato
,
Z.
Zhu
,
O. R.
Jaren
,
E.
Hobeika
,
M.
Reth
,
R. C.
Rickert
.
2014
.
PDK1 regulates B cell differentiation and homeostasis.
Proc. Natl. Acad. Sci. USA
111
:
9573
9578
.
31
Park
,
S. G.
,
M.
Long
,
J. A.
Kang
,
W. S.
Kim
,
C. R.
Lee
,
S. H.
Im
,
I.
Strickland
,
J.
Schulze-Luehrmann
,
M. S.
Hayden
,
S.
Ghosh
.
2013
.
The kinase PDK1 is essential for B-cell receptor mediated survival signaling.
PLoS One
8
:
e55378
.
32
Venigalla
,
R. K.
,
V. A.
McGuire
,
R.
Clarke
,
J. C.
Patterson-Kane
,
A.
Najafov
,
R.
Toth
,
P. C.
McCarthy
,
F.
Simeons
,
L.
Stojanovski
,
J. S.
Arthur
.
2013
.
PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development.
EMBO J.
32
:
1008
1022
.
33
Finlay
,
D. K.
,
L. V.
Sinclair
,
C.
Feijoo
,
C. M.
Waugh
,
T. J.
Hagenbeek
,
H.
Spits
,
D. A.
Cantrell
.
2009
.
Phosphoinositide-dependent kinase 1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes.
J. Exp. Med.
206
:
2441
2454
.
34
Kelly
,
A. P.
,
D. K.
Finlay
,
H. J.
Hinton
,
R. G.
Clarke
,
E.
Fiorini
,
F.
Radtke
,
D. A.
Cantrell
.
2007
.
Notch-induced T cell development requires phosphoinositide-dependent kinase 1.
EMBO J.
26
:
3441
3450
.
35
Macintyre
,
A. N.
,
D.
Finlay
,
G.
Preston
,
L. V.
Sinclair
,
C. M.
Waugh
,
P.
Tamas
,
C.
Feijoo
,
K.
Okkenhaug
,
D. A.
Cantrell
.
2011
.
Protein kinase B controls transcriptional programs that direct cytotoxic T cell fate but is dispensable for T cell metabolism.
Immunity
34
:
224
236
.
36
Finlay
,
D. K.
,
E.
Rosenzweig
,
L. V.
Sinclair
,
C.
Feijoo-Carnero
,
J. L.
Hukelmann
,
J.
Rolf
,
A. A.
Panteleyev
,
K.
Okkenhaug
,
D. A.
Cantrell
.
2012
.
PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells.
J. Exp. Med.
209
:
2441
2453
.
37
Gupta
,
S.
,
S.
Manicassamy
,
C.
Vasu
,
A.
Kumar
,
W.
Shang
,
Z.
Sun
.
2008
.
Differential requirement of PKC-theta in the development and function of natural regulatory T cells.
Mol. Immunol.
46
:
213
224
.
38
Zanin-Zhorov
,
A.
,
Y.
Ding
,
S.
Kumari
,
M.
Attur
,
K. L.
Hippen
,
M.
Brown
,
B. R.
Blazar
,
S. B.
Abramson
,
J. J.
Lafaille
,
M. L.
Dustin
.
2010
.
Protein kinase C-theta mediates negative feedback on regulatory T cell function.
Science
328
:
372
376
.
39
Siegmund
,
K.
,
N.
Thuille
,
K.
Wachowicz
,
N.
Hermann-Kleiter
,
G.
Baier
.
2017
.
Protein kinase C theta is dispensable for suppression mediated by CD25+CD4+ regulatory T cells.
PLoS One
12
:
e0175463
.
40
Heuser
,
C.
,
J.
Gotot
,
E. C.
Piotrowski
,
M. S.
Philipp
,
C. J. F.
Courrèges
,
M. S.
Otte
,
L.
Guo
,
J. L.
Schmid-Burgk
,
V.
Hornung
,
A.
Heine
, et al
.
2017
.
Prolonged IKKβ inhibition improves ongoing CTL antitumor responses by incapacitating regulatory T cells.
Cell Rep.
21
:
578
586
.
41
Haxhinasto
,
S.
,
D.
Mathis
,
C.
Benoist
.
2008
.
The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells.
J. Exp. Med.
205
:
565
574
.
42
Zeng
,
H.
,
K.
Yang
,
C.
Cloer
,
G.
Neale
,
P.
Vogel
,
H.
Chi
.
2013
.
mTORC1 couples immune signals and metabolic programming to establish T(reg)-cell function.
Nature
499
:
485
490
.
43
Zhang
,
R.
,
A.
Huynh
,
G.
Whitcher
,
J.
Chang
,
J. S.
Maltzman
,
L. A.
Turka
.
2013
.
An obligate cell-intrinsic function for CD28 in Tregs.
J. Clin. Invest.
123
:
580
593
.
44
Levine
,
A. G.
,
A.
Arvey
,
W.
Jin
,
A. Y.
Rudensky
.
2014
.
Continuous requirement for the TCR in regulatory T cell function.
Nat. Immunol.
15
:
1070
1078
.

The authors have no financial conflicts of interest.

Supplementary data