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Cyclin-Dependent Kinase 7 Promotes Th17/Th1 Cell Differentiation in Psoriasis by Modulating Glycolytic Metabolism

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    4 These authors contributed equally to this work.
    Yiting Lin
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    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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    4 These authors contributed equally to this work.
    Ke Xue
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    4 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China

    PLA Institute of State Key Laboratory of Cancer Biology, Department of Biopharmaceutics, Fourth Military Medical University, Xi'an, China
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    4 These authors contributed equally to this work.
    Qingyang Li
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    4 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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  • Zhenhua Liu
    Affiliations
    Department of Physiology and Pathophysiology, Fourth Military Medical University, Xi'an, China
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  • Zhenlai Zhu
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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  • Jiaoling Chen
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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  • Erle Dang
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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  • Lei Wang
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    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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  • Weigang Zhang
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    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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    5 These authors contributed equally to this work.
    Gang Wang
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    5 These authors contributed equally to this work.
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    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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    5 These authors contributed equally to this work.
    Bing Li
    Correspondence
    Correspondence: Bing Li, Department of Dermatology, Xijing Hospital, Fourth Military Medical University, 127 Changlexi Road, Xi’an 710032, China.
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    5 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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    5 These authors contributed equally to this work.
Open ArchivePublished:May 15, 2021DOI:https://doi.org/10.1016/j.jid.2021.04.018
      Excessive activation of CD4+ T cells and T helper type (Th) 17/Th1 cell differentiation are critical events in psoriasis pathogenesis, but the associated molecular mechanism is still unclear. Here, using quantitative proteomics analysis, we found that cyclin-dependent kinase 7 (CDK7) expression was markedly increased in CD4+ T cells from patients with psoriasis compared with healthy controls and was positively correlated with psoriasis severity. Meanwhile, genetic or pharmacological inhibition of CDK7 ameliorated the severity of psoriasis in the imiquimod-induced psoriasis-like mouse model and suppressed CD4+ T-cell activation as well as Th17/Th1 cell differentiation in vivo and in vitro. Furthermore, the CDK7 inhibitor also reduced the enhanced glycolysis of CD4+ T cells from patients with psoriasis. Proinflammatory cytokine IL-23 induced increased CDK7 expression in CD4+ T cells and activated the protein kinase B/mTOR/HIF-1α signaling pathway, enhancing glycolytic metabolism. Correspondingly, CDK7 inhibition significantly impaired IL-23–induced glycolysis via the protein kinase B/mTOR/HIF-1α pathway. In summary, this study shows that CDK7 promotes CD4+ T-cell activation and Th17/Th1 cell differentiation by regulating glycolysis, thus contributing to the pathogenesis of psoriasis. Targeting CDK7 might be a promising immunosuppressive strategy to control skin inflammation mediated by IL-23.

      Graphical abstract

      Abbreviations:

      Akt (protein kinase B), CDK7 (cyclin-dependent kinase 7), IMQ (imiquimod), Th (T helper type)

      Introduction

      Psoriasis is a T-cell–mediated chronic inflammatory skin disease (
      • Greb J.E.
      • Goldminz A.M.
      • Elder J.T.
      • Lebwohl M.G.
      • Gladman D.D.
      • Wu J.J.
      • et al.
      Psoriasis.
      ), the development of which is significantly affected by excessive activation of CD4+ T cells (
      • Boehncke W.H.
      • Schön M.P.
      Psoriasis.
      ). After exposure of genetically susceptible individuals to risk factors, such as infection and stress, the initial activation of innate immune response induces the proliferation and differentiation of CD4+ T cells (
      • Lowes M.A.
      • Suárez-Fariñas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ), from which T helper type (Th) 17 and Th1 cells are polarized and serve as the primary pathogenic cells in psoriasis (
      • Boehncke W.H.
      • Schön M.P.
      Psoriasis.
      ;
      • de Masson A.
      • Bouaziz J.D.
      • Battistella M.
      • Bagot M.
      • Bensussan A.
      Immunopathologie du psoriasis – from bench to bedside [Immunopathology of psoriasis: from bench to bedside].
      ;
      • Frischknecht L.
      • Vecellio M.
      • Selmi C.
      The role of epigenetics and immunological imbalance in the etiopathogenesis of psoriasis and psoriatic arthritis.
      ;
      • Lowes M.A.
      • Suárez-Fariñas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ;
      • Schön M.P.
      Adaptive and innate immunity in psoriasis and other inflammatory disorders.
      ). However, the mechanisms underlying T-cell activation and Th17/Th1 cell differentiation are poorly understood.
      T cells are dependent on metabolic reprogramming during activation (
      • Freitag J.
      • Berod L.
      • Kamradt T.
      • Sparwasser T.
      Immunometabolism and autoimmunity.
      ;
      • Geltink R.I.K.
      • Kyle R.L.
      • Pearce E.L.
      Unraveling the complex interplay between T cell metabolism and function.
      ;
      • Hu Z.
      • Zou Q.
      • Su B.
      Regulation of T cell immunity by cellular metabolism.
      ;
      • Pearce E.L.
      • Pearce E.J.
      Metabolic pathways in immune cell activation and quiescence.
      ). Naive T cells preferentially use oxidative phosphorylation and fatty acid oxidation for energy metabolic requirements (
      • Geltink R.I.K.
      • Kyle R.L.
      • Pearce E.L.
      Unraveling the complex interplay between T cell metabolism and function.
      ). However, following TCR stimulation, naive T cells engage in glycolytic metabolism to facilitate differentiation into effector lineages (
      • Geltink R.I.K.
      • Kyle R.L.
      • Pearce E.L.
      Unraveling the complex interplay between T cell metabolism and function.
      ;
      • O'Neill L.A.
      • Kishton R.J.
      • Rathmell J.
      A guide to immunometabolism for immunologists.
      ;
      • Pearce E.L.
      • Pearce E.J.
      Metabolic pathways in immune cell activation and quiescence.
      ). Notably, Th1, Th2, and Th17 cells primarily depend on glycolysis, whereas memory T cells and regulatory T cells preferentially use oxidative phosphorylation and fatty acid oxidation (
      • Dumitru C.
      • Kabat A.M.
      • Maloy K.J.
      Metabolic adaptations of CD4+ T cells in inflammatory disease.
      ;
      • Geltink R.I.K.
      • Kyle R.L.
      • Pearce E.L.
      Unraveling the complex interplay between T cell metabolism and function.
      ). Hence, the dynamic regulation of metabolic profiles governs the phenotype of T-cell subsets (
      • Geltink R.I.K.
      • Kyle R.L.
      • Pearce E.L.
      Unraveling the complex interplay between T cell metabolism and function.
      ;
      • Hu Z.
      • Zou Q.
      • Su B.
      Regulation of T cell immunity by cellular metabolism.
      ). Therefore, altering T-cell metabolic pathways represents an attractive treatment for inflammation (
      • Bettencourt I.A.
      • Powell J.D.
      Targeting metabolism as a novel therapeutic approach to autoimmunity, inflammation, and transplantation.
      ;
      • Freitag J.
      • Berod L.
      • Kamradt T.
      • Sparwasser T.
      Immunometabolism and autoimmunity.
      ;
      • O'Neill L.A.
      • Kishton R.J.
      • Rathmell J.
      A guide to immunometabolism for immunologists.
      ;
      • von Meyenn L.
      • Bertschi N.L.
      • Schlapbach C.
      Targeting T cell metabolism in inflammatory skin disease.
      ).
      Cyclin-dependent kinase 7 (CDK7), a member of the CDK family, regulates the cell cycle and is a component of the general transcription factor that modulates transcription (
      • Fisher R.P.
      Secrets of a double agent: CDK7 in cell-cycle control and transcription.
      ). Indeed, CDK7 reportedly regulates the proliferation of tumor cells in various cancers (
      • Diab S.
      • Yu M.
      • Wang S.
      CDK7 inhibitors in cancer therapy: the sweet smell of success?.
      ;
      • Fisher R.P.
      Cdk7: a kinase at the core of transcription and in the crosshairs of cancer drug discovery.
      ;
      • Wang J.
      • Zhang R.
      • Lin Z.
      • Zhang S.
      • Chen Y.
      • Tang J.
      • et al.
      CDK7 inhibitor THZ1 enhances antiPD-1 therapy efficacy via the p38α/MYC/PD-L1 signaling in non-small cell lung cancer.
      ). It also activates glucose consumption and glycolysis in lung cancer (
      • Cheng Z.J.
      • Miao D.L.
      • Su Q.Y.
      • Tang X.L.
      • Wang X.L.
      • Deng L.B.
      • et al.
      THZ1 suppresses human non-small-cell lung cancer cells in vitro through interference with cancer metabolism.
      ;
      • Ghezzi C.
      • Wong A.
      • Chen B.Y.
      • Ribalet B.
      • Damoiseaux R.
      • Clark P.M.
      A high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells.
      ). Recently, a variety of CDKs have been reported to exert a remarkable regulatory role in influencing proinflammatory responses, especially in T-cell differentiation. Of these, CDK7 has been shown to control neutrophil survival (
      • Leitch A.E.
      • Lucas C.D.
      • Marwick J.A.
      • Duffin R.
      • Haslett C.
      • Rossi A.G.
      Cyclin-dependent kinases 7 and 9 specifically regulate neutrophil transcription and their inhibition drives apoptosis to promote resolution of inflammation.
      ), Th17 cell response (
      • Xia Y.
      • Lin L.Y.
      • Liu M.L.
      • Wang Z.
      • Hong H.H.
      • Guo X.G.
      • et al.
      Selective inhibition of CDK7 ameliorates experimental arthritis in mice.
      ), and the process of inflammation (
      • Hoodless L.J.
      • Robb C.T.
      • Felton J.M.
      • Tucker C.S.
      • Rossi A.G.
      Models for the study of the cross talk between inflammation and cell cycle.
      ;
      • Leitch A.E.
      • Haslett C.
      • Rossi A.G.
      Cyclin-dependent kinase inhibitor drugs as potential novel anti-inflammatory and pro-resolution agents.
      ;
      • Srikumar T.
      • Padmanabhan J.
      Potential use of flavopiridol in treatment of chronic diseases.
      ). However, the precise function of CDK7 in T cells, particularly in the context of inflammatory diseases, remains ambiguous.
      In this study, we investigated the role of CDK7 in CD4+ T-cell activation and differentiation in psoriasis and the underlying mechanism. We demonstrated an increase of CDK7 in psoriatic CD4+ T cells, which was induced by proinflammatory factors, especially IL-23. We also found that genetic or pharmacological inhibition of CDK7 attenuated inflammation in an imiquimod (IMQ)-induced psoriasiform mouse model and limited Th17 and Th1 cell development in vitro and in vivo through glycolytic disturbance through the protein kinase B (Akt)/mTOR/HIF-1α axis. Our findings provide an unreported insight into the pathogenesis of psoriasis and highlight targeting CDK7 as a potential immunosuppressive strategy for the treatment of psoriasis.

      Results

      The expression of CDK7 is increased in CD4+ T cells of patients with psoriasis

      We initially performed an isobaric tag for relative and absolute quantitation quantitative proteomics assay (Supplementary Table S1) and identified 79 differentially expressed proteins in peripheral CD4+ T cells of patients with psoriasis compared with healthy controls (Supplementary Figure S1). The results confirmed that CDK7 was not only markedly upregulated with 75 upregulated proteins, but also the only member of the CDK family that is dramatically upregulated in psoriatic CD4+ T cells (Figure 1a).
      Figure thumbnail gr1
      Figure 1CDK7 expression is upregulated in psoriatic CD4+ T cells from peripheral blood and lesions. (a) Heat map showing gene expression profiles of differentially expressed proteins in circulating CD4+ T cells from Pso (Psoriasis) compared with age- and sex-matched HC (Healthy) (n = 3 for each group). Colors represent high (red) and low (green) intensity. (b) Representative flow cytometry data showing CDK7 protein expression in circulating CD4+ T cells from Pso and HC. The MFI for CDK7 protein expression was analyzed by flow cytometry (n = 21 for each group, mean ± SD, two-tailed Student’s t-test, ∗∗∗∗P < 0.0001). (c) Correlation of CDK7 protein levels in circulating CD4+ T cells with PASI in Pso (n = 21). The adjusted R2 and P-values are plotted in each graph. P-values were calculated by linear regression. (d) Representative images of immunofluorescence were analyzed for the expression of CD4 (green) and CDK7 (red) in healthy skin and psoriatic lesions. Arrowheads indicate double-positive CD4/CDK7 cells. Nuclei were counterstained with DAPI (blue). Bar = 50 μm (n = 3 for each group). HC, healthy control; MFI, mean fluorescence intensity; HC, healthy control; Pso, patients with psoriasis.
      To further confirm the increased level of CDK7 in psoriatic CD4+ T cells, its expression was assessed by flow cytometry and immunofluorescence. Consistent with the proteomics data, CDK7 was excessively expressed in circulating CD4+ T cells and lesional CD4+ T cells from patients with psoriasis (Figure 1b and d). Moreover, the mean fluorescence intensity of CDK7 calculated from the flow cytometry data was positively correlated with PASI scores in patients with psoriasis (Figure 1c). Together, these results suggest increased expression of CDK7 in CD4+ T cells of patients with psoriasis, which may be involved in the pathogenesis of psoriasis.

      Deletion of CDK7 in CD4+ T cells suppresses psoriatic inflammatory responses in the IMQ-induced psoriasis model

      To determine whether CDK7 contributes to the development of psoriasis, THZ1, a selective and potent covalent CDK7 inhibitor, was intraperitoneally administered to IMQ-induced psoriasis-like mice (Supplementary Figure S2a). The pharmacological inhibition of CDK7 significantly mitigated the psoriasiform phenotype (Supplementary Figure S2b), with dramatically decreased PASI scores, reduced lesional epidermis thickness, and less lesional T-cell infiltration (Supplementary Figure S2c–e). The lesional mRNA expressions of Ifng and Il17a were also downregulated in THZ1-treated mice (Supplementary Figure S2f). We found that THZ1 alleviated IMQ-induced splenomegaly (Supplementary Figure S3a) without weight loss (Supplementary Figure S3b). Furthermore, the proportion of IL-17A– and IFN-γ–producing CD4+ T cells was decreased in the spleen of psoriasis-like mice after THZ1 treatment (Supplementary Figure S3c and d). Together, these results suggest that improvement of IMQ-induced psoriasis-like phenotype by CDK7 inhibitor may be dependent on blocking the over Th17/Th1 response in CD4+ T cells.
      Therefore, we further employed adoptive transfer of CD4+ T cells with genetic ablation of CDK7 into Rag2–/– mice to exclude other cell types influenced by THZ1 treatment. Because Rag2–/– mice only could exhibit psoriasiform dermatitis after being transferred with the CD4+ T cells compared with nontransferred mice in the IMQ-induced mouse model (Supplementary Figure S4a and b), which suggested that the psoriasis phenotypes of IMQ-induced Rag2–/– mice depend on the intradermal injection of CD4+ T cells. Cdk7 knockdown in CD4+ T cells was achieved by CDK7 short hairpin RNA lentivirus infection and verified by flow cytometry and western blot (Supplementary Figure S5a and b). The mouse was injected intradermally with CD4+ T cells transfected with CDK7 short hairpin RNA lentivirus in the right ear and CD4+ T cells transfected with normal control lentivirus on the left ear on the third day of IMQ application. PBS injection was set as a negative control. Thereafter, IMQ was applied locally once a day for 9 days consecutively (Figure 2a). H&E staining showed that psoriasiform dermatitis was markedly alleviated, and the thickness of the epidermis was significantly reduced in mice receiving the injection of CD4+ T cells with Cdk7 knockdown as compared with the normal control groups (Figure 2b and c), suggesting that Cdk7 knockdown in CD4+ T cells prevents the development of psoriasis. In addition, in the mice treated with CD4+ T cells with Cdk7 knockdown, the number of Th1 and Th17 cells decreased significantly (Figure 2d), and mRNA expression of Il17a and Ifng also decreased in lesions compared with normal control groups (Figure 2e and f). Together, these in vivo results suggest that CDK7 is essential for Th1 and Th17 cells in the development of psoriasis, and CDK7 inactivation–mediated Th1 and Th17 dysplasia hinders the development of psoriasis.
      Figure thumbnail gr2
      Figure 2Cdk7 knockdown of CD4+ T cells ameliorates psoriasiform symptoms and reduces Th17/Th1 cell differentiation in the IMQ-induced psoriasis-like mouse model. (a) Schematic diagram of mouse experimental protocol. (b) H&E staining of the lesions. Bar = 200 μm. (c) Mean thickness of the epidermis. (d) Representative images of immunofluorescence of IFN-γ (green) or IL-17A (green) in the lesions. Bar = 200 μm. (e) qRT-PCR for mRNA expression of Il17a and (f) Ifng in lesional skin. Data are mean ± SEM. ∗P < 0.05; ∗∗∗∗P < 0.0001. P-values were calculated by one-way ANOVA with Tukey’s post hoc test. n = 5 mice per group. IMQ, imiquimod; NC, normal control; shCDK7, CDK7 short hairpin RNA.

      CDK7 regulates CD4+ T-cell activation and Th17/Th1 cell differentiation in vitro

      To further explore the role of CDK7 in the pathogenicity of CD4+ T cells, we established an in vitro model of CD4+ T-cell activation and differentiation. CD3/CD28 antibodies were used to activate naive CD4+ T cells from healthy controls, and IL-1β/IL-23 (
      • Annunziato F.
      • Cosmi L.
      • Romagnani S.
      Human and murine Th17.
      ;
      • Sallusto F.
      • Zielinski C.E.
      • Lanzavecchia A.
      Human Th17 subsets.
      ) or IL-12 were separately added to induce the differentiation of Th17/Th1 cells in vitro. On T-cell activation, the mRNA level of CDK7 was significantly upregulated (Figure 3a). It was also increased in Th1 and Th17 cells compared with Th0 cells, and Th17 cells showed higher CDK7 expression than Th1 cells (Figure 3b). Moreover, THZ1-treated naive CD4+ T cells expressed much less CD69, IL-2, and CD25 in a dose-dependent manner after activation (Figure 3c and Supplementary Figure S6a and b), together with decreasing proliferation index (Supplementary Figure S6c), but no cellular toxicity for CD4+ T cells (Supplementary Figure S6d), suggesting that CDK7 can regulate T-cell activation and proliferation activity. Meanwhile, exposure to THZ1 also downregulated the mRNA levels of IL17A and IFNG in psoriatic CD4+ T cells in a dose-dependent manner (Figure 3d). THZ1 also inhibited the differentiation of the IL-17A+ROR-γt+Th17 subset in a dose-dependent manner (Figure 3e). Similar results were observed in IFN-γ+T-bet+Th1 cells (Figure 3f).
      Figure thumbnail gr3
      Figure 3Inhibiting CDK7 suppresses CD4+ T-cell activation and constrains Th17 and Th1 cell polarization in vitro. (a) Relative mRNA expression of CDK7 gene was measured by qRT-PCR in resting human CD4+ T cells and activated CD4+ T cells from healthy controls stimulated by CD3 and CD28 antibodies for 24 hours (n = 3 for each group). (b) CDK7 gene expression was quantified by qRT-PCR in Th17 and Th1 subsets induced by IL-1β and IL-23 or IL-12 acquired by in vitro differentiation for 5 days (n = 3 for each group). (c) The percentage of CD69+ cells in the CD4+ T-cell population was evaluated by flow cytometry in human naive CD4+ T cells stimulated with different concentrations of THZ1 (0 nM, 2.5 nM, 5 nM, 10 nM) in the presence of CD3/CD28 antibodies for 24 hours (n = 5 for each group). (d) mRNA expression of IL17A and IFNG (n = 4 for each group). (e) The frequency of IL-17A+ ROR-γt+ Th17 cells and (f) IFN-γ+ T-bet+ Th1 cells evaluated by flow cytometry (n = 5 for each group). (g) Correlation of the percentage of ROR-γt+ cells and (h) T-bet+ cells in the CD4+ T-cell population with CDK7 MFI (n = 13 for each group). Data are mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. P-values were calculated by one-way ANOVA with Tukey’s post hoc test (a, b, c, d, e, f) or linear regression (g, h). MFI, mean fluorescence intensity; ns, not significant; Th, T helper type.
      Furthermore, we found that CDK7 levels were positively correlated with the frequencies of circulating Th17 and Th1 cells in patients with psoriasis (Figure 3g and h). Thus, our results suggest that CDK7 can regulate CD4+ T-cell activation and Th17/Th1 cell differentiation in psoriasis.

      CDK7 regulates CD4+ T-cell glycolysis in patients with psoriasis

      T cells have different metabolic requirements depending on their activation or differentiation states (
      • Geltink R.I.K.
      • Kyle R.L.
      • Pearce E.L.
      Unraveling the complex interplay between T cell metabolism and function.
      ;
      • Hu Z.
      • Zou Q.
      • Su B.
      Regulation of T cell immunity by cellular metabolism.
      ). Glycolysis determines T-cell activation and effector T-cell differentiation, especially Th17 and Th1 cells (
      • Geltink R.I.K.
      • Kyle R.L.
      • Pearce E.L.
      Unraveling the complex interplay between T cell metabolism and function.
      ;
      • Hu Z.
      • Zou Q.
      • Su B.
      Regulation of T cell immunity by cellular metabolism.
      ;
      • Pearce E.L.
      • Pearce E.J.
      Metabolic pathways in immune cell activation and quiescence.
      ). To investigate whether CDK7 mediates CD4+ T-cell dysfunction via the modulation of cellular glycolysis in psoriasis, we investigated the metabolic profile of circulating CD4+ T cells by analyzing previous proteomics assays for gene set enrichment analysis and identified glycolysis as the most significantly enriched metabolism in psoriasis (Figure 4a and Supplementary Figure S7). In addition, extracellular acidification rate was assessed using a Seahorse XF24e Extracellular Flux Analyzer (Agilent, Santa Clara, CA) to investigate the glycolysis profile of psoriatic CD4+ T cells, which revealed that both the glycolytic flux and glycolytic capacity of CD4+ T cells were significantly higher in patients with psoriasis than healthy controls (Figure 4b and c). Moreover, psoriatic CD4+ T cells displayed markedly enhanced glucose uptake and lactate production compared with healthy controls (Figure 4d and e), indicating heightened glycolysis in psoriatic CD4+ T cells, which was confirmed by increased mRNA expression of key glycolysis genes (Figure 4f). In conclusion, circulating CD4+ T cells from patients with psoriasis possessed increased glycolytic metabolism.
      Figure thumbnail gr4
      Figure 4CD4+ T cells from patients with psoriasis have enhanced glycolytic metabolism activity. (a) Gene set enrichment analysis of proteomic data, CD4+ T cells of Pso versus HC CD4+ T cells (n = 3 for each group). (b) ECAR in CD4+ T cells from Pso and HCs was assessed by the Seahorse system. (c) Statistical analysis of the glycolytic flux and glycolytic capacity from ECAR (n = 3 for each group). (d) The uptake of 2-NBDG in CD4+ T cells from Pso and HCs (n = 5 for each group). (e) Extracellular lactate production in CD4+ T cells from Pso and HCs (n = 5 for each group). (f) mRNA expression of the key genes associated with glycolysis in CD4+ T cells from Pso and HC (n = 5 for each group). Data were mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. P-values were calculated by unpaired Student’s t-test. 2-DG, 2-deoxyglucose; ECAR, extracellular acidification rate; ES, enrichment score; FDR, false discovery rate; HC, healthy control; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; Pso, patients with psoriasis.
      Then, we explored the influence of CDK7 on glycolysis metabolism in psoriatic CD4+ T cells by performing global gene expression analysis by RNA sequencing, comparing THZ1-treated psoriatic CD4+ T cells with DMSO-treated psoriatic CD4+ T cells (Supplementary Table S2). Gene Ontology enrichment analysis showed that the metabolic process was the most significantly enriched biological process, and among all common metabolic processes, glycolysis was the most significantly enriched one (Figure 5a). Furthermore, gene set enrichment analysis showed inhibition of glycolysis by THZ1 in psoriatic CD4+ T cells (Figure 5b). Gene expression analysis and qRT–PCR experiments confirmed that THZ1 treatment significantly inhibited several glycolytic genes in psoriatic CD4+ T cells, including some key glycolytic genes that are essential for aerobic glycolysis in T cells (Figure 5c and Supplementary Figure S8). Extracellular acidification rate data were measured in psoriatic CD4+ T cells treated with THZ1 or DMSO for 24 hours in vitro and showed that THZ1 significantly repressed the abnormally high-level glycolysis in psoriatic CD4+ T cells (Figure 5d) as well as glycolytic flux and glycolytic capacity (Figure 5e). As expected, glucose uptake and lactate production were also decreased in psoriatic CD4+ T cells treated with THZ1 compared with the DMSO-treated group (Figure 5f and g). In summary, CDK7 inhibition could suppress key glycolysis steps, leading to a decrease in the glycolytic process and reduced energy supply for psoriatic CD4+ T cells, which indicates CDK7 as a potential regulator of glucose metabolism for CD4+ T cells in psoriasis.
      Figure thumbnail gr5
      Figure 5THZ1 inhibits glycolysis of psoriatic CD4+ T cells. Psoriatic CD4+ T cells were stimulated with CD3/CD28 antibodies for 24 hours in the presence of THZ1 (10 nM) (Pso + THZ1) or DMSO (Pso + DMSO). (a) The top 30 GO terms associated with biological process and GO metabolism enrichment analysis of RNA-seq (n = 2 for each group). (b) Glycolysis pathway analysis of RNA-seq using GSEA (n = 2 for each group). (c) Heat map of expression levels of genes in glycolytic pathways by RNA-seq (n = 2 for each group). (d) ECAR was assessed by the Seahorse system (n = 3 for each group). (e) Statistical analysis of the glycolytic flux and glycolytic capacity from ECAR (n = 3 for each group). (f) The uptake of glucose by measuring 2-NBDG using flow cytometry (n = 5 for each group). (g) Extracellular lactate production was measured by lactate concentrations in the supernatant (n = 5 for each group). Data were mean ± SEM. ∗P < 0.05; ∗∗P < 0.01. P-values were calculated by unpaired Student’s t-test. 2-DG, 2-deoxyglucose; ECAR, extracellular acidification rate; GO, Gene Ontology; GSEA, gene set enrichment analysis; max, maximum; min, minimum; Pso, patients with psoriasis; RNA-seq, RNA sequencing.

      IL-23 induces CDK7 upregulation that modulates glycolysis metabolism of CD4+ T cells via the Akt/mTOR/HIF1α axis

      Next, to explore the upstream cause for excessive expression of CDK7 in psoriatic CD4+ T cells, we investigated the effect of IL-23, IL-1β, and IL-12, the cytokines that induce Th17/Th1 cell differentiation, on the expression of CDK7 of CD4+ T cells. Only IL-23 and IL-12 upregulated CDK7 expression in CD4+ T cells of healthy controls, and IL-23 induced higher levels of CDK7 than IL-12 (Figure 6a). CDK7 expression in psoriatic CD4+ T cells was positively correlated with the level of serum IL-23 in patients with psoriasis (Figure 6b), indicating that IL-23 is probably the inducer of CDK7 expression in CD4+ T cells.
      Figure thumbnail gr6
      Figure 6IL-23–induced CDK7 activates the Akt/mTOR/HIF1-α signaling pathway to promote glycolytic metabolism in psoriatic CD4+ T cells. (a) Human CD4+ T cells were activated for 2 days in the presence of PBS (normal control) or IL-23 (50 nM)/IL-12 (2.5 ng/ml)/IL-1β (50 nM), respectively, and analyzed for CDK7 expression by flow cytometry. Quantification of MFI of CDK7 is shown (n = 4 for each group). (b) Correlation of CDK7 protein levels in circulating CD4+ T cells with serum IL-23 levels in patients with psoriasis (n = 13 for each group). (c) Heatmap analysis of expression levels of phosphatidylinositol 3 kinase/Akt/mTOR signaling genes, mTORC1-regulated genes, and HIF-1α–regulated genes by RNA-seq in psoriatic CD4+ T cells treated with or without THZ1 in the presence of CD3/CD28 antibody activation (n = 2 for each group). (d, e) Human CD4+ T cells were activated with IL-23 (50 nM) for 2 days in the presence of THZ1 (10 nM). (d) The expressions of p-Akt (s473), 4EBP1 (pT36/pT45), and HIF-1α were measured by flow cytometry. Quantification MFI is shown (n = 4 for each group). (e) mRNA expression of key genes associated with glycolysis (n = 4 for each group). Data were mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. P-values were calculated by one-way ANOVA with Tukey’s post hoc test (a, d, e) or linear regression (b). Akt, protein kinase B; max, maximum; MFI, mean fluorescence intensity; min, minimum; ns, not significant; p-Akt, phosphorylated protein kinase B; RNA-seq, RNA sequencing.
      Then, we tried to investigate which pathway was involved in CDK7-regulated glycolytic metabolism in psoriatic CD4+ T cells. Recent studies have revealed that THZ1 inhibits the phosphorylation of Akt at Ser473, the downstream activator of mTORC1 and its target proteins in non–small cell lung cancer cells (
      • Cheng Z.J.
      • Miao D.L.
      • Su Q.Y.
      • Tang X.L.
      • Wang X.L.
      • Deng L.B.
      • et al.
      THZ1 suppresses human non-small-cell lung cancer cells in vitro through interference with cancer metabolism.
      ). Furthermore, the Akt/mTOR/HIF-1α axis has been shown to regulate glycolytic metabolism and energy balance (
      • Hu Z.
      • Zou Q.
      • Su B.
      Regulation of T cell immunity by cellular metabolism.
      ;
      • Huang H.
      • Long L.
      • Zhou P.
      • Chapman N.M.
      • Chi H.
      mTOR signaling at the crossroads of environmental signals and T-cell fate decisions.
      ). To evaluate the impact of CDK7 on the Akt/mTOR/HIF-1α pathway of CD4+ T cells in psoriasis, we performed global gene expression analysis by RNA sequencing, comparing THZ1-treated psoriatic CD4+ T cells with DMSO-treated psoriatic CD4+ T cells. Gene expression analysis confirmed that THZ1 treatment severely impaired phosphatidylinositol 3 kinase/Akt/mTOR signaling genes and HIF-1α– and mTOR-induced genes in psoriatic CD4+ T cells in vitro (Figure 6c). Consistently, the upstream cytokine IL-23 was found to upregulate the expression of phosphorylated Akt (Ser473); phosphorylated 4EBP1 (Thr36/Thr45), which is the downstream protein of activated mTORC1; and HIF-1α, respectively, in CD4+ T cells; however, this could be blocked by the treatment with THZ1 (Figure 6d and Supplementary Figure S9a and b). As expected, THZ1 also markedly decreased the mRNA expressions of key genes critical for glycolysis enhanced by IL-23 (Figure 6e). Collectively, these results indicate that IL-23–induced CDK7 regulates glycolytic metabolism in psoriatic CD4+ T cells via the Akt/mTOR/HIF-1α axis.

      Discussion

      Here, we demonstrate that CDK7 is excessively expressed in psoriatic CD4+ T cells, and CDK7 inhibition impairs Th1/Th17 differentiation and corrects the higher glycolysis metabolism signature of psoriatic CD4+ T cells via the Akt/mTOR/HIF-1α axis, alleviating the severity of psoriasis in an IMQ-induced psoriasis-like mouse model. Thus, our results suggest that CDK7 contributes to psoriasis pathogenesis by regulating the glycolysis metabolism signature of CD4+ T cells.
      Psoriasis is a T-cell–mediated inflammatory disorder characterized by excessive activation of CD4+ T cells and expansion of Th17/Th1 cells (
      • Lowes M.A.
      • Suárez-Fariñas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ). In the past decades, proinflammatory cytokines have been considered as the key mechanism causing excessive activation and differentiation of CD4+ T cells (
      • Saravia J.
      • Chapman N.M.
      • Chi H.
      Helper T cell differentiation.
      ). Recently, increasing evidence suggests that, on CD4+ T-cell activation and differentiation to Th17/Th1 cells, the metabolic reprogramming is preferentially directed toward glycolysis (
      • Hu Z.
      • Zou Q.
      • Su B.
      Regulation of T cell immunity by cellular metabolism.
      ). Regulating metabolic reprogramming can modulate the activation and differentiation of CD4+ T cells (
      • Bettencourt I.A.
      • Powell J.D.
      Targeting metabolism as a novel therapeutic approach to autoimmunity, inflammation, and transplantation.
      ). Expectedly, regulating glycolysis in T cells reportedly alleviates autoimmune conditions (
      • Angiari S.
      • Runtsch M.C.
      • Sutton C.E.
      • Palsson-McDermott E.M.
      • Kelly B.
      • Rana N.
      • et al.
      Pharmacological activation of pyruvate kinase M2 inhibits CD4+ T cell pathogenicity and suppresses autoimmunity.
      ;
      • Kornberg M.D.
      The immunologic Warburg effect: evidence and therapeutic opportunities in autoimmunity.
      ), including systemic lupus erythematosus (
      • Bettencourt I.A.
      • Powell J.D.
      Targeting metabolism as a novel therapeutic approach to autoimmunity, inflammation, and transplantation.
      ;
      • Yin Y.
      • Choi S.C.
      • Xu Z.
      • Perry D.J.
      • Seay H.
      • Croker B.P.
      • et al.
      Normalization of CD4+ T cell metabolism reverses lupus.
      ), rheumatoid arthritis (
      • Abboud G.
      • Choi S.C.
      • Kanda N.
      • Zeumer-Spataro L.
      • Roopenian D.C.
      • Morel L.
      Inhibition of glycolysis reduces disease severity in an autoimmune model of rheumatoid arthritis.
      ;
      • Okano T.
      • Saegusa J.
      • Nishimura K.
      • Takahashi S.
      • Sendo S.
      • Ueda Y.
      • et al.
      3-bromopyruvate ameliorate autoimmune arthritis by modulating Th17/Treg cell differentiation and suppressing dendritic cell activation.
      ), multiple sclerosis (
      • Chen Z.
      • Liu M.
      • Li L.
      • Chen L.
      Involvement of the Warburg effect in non-tumor diseases processes.
      ;
      • Stathopoulou C.
      • Nikoleri D.
      • Bertsias G.
      Immunometabolism: an overview and therapeutic prospects in autoimmune diseases.
      ), and autoimmune encephalomyelitis (
      • Gerriets V.A.
      • Kishton R.J.
      • Nichols A.G.
      • Macintyre A.N.
      • Inoue M.
      • Ilkayeva O.
      • et al.
      Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation.
      ). Consistent with these findings, our study showed that CD4+ T cells from patients with psoriasis exhibited elevated glycolysis metabolic activity compared with healthy controls. Furthermore, we found that CDK7 was crucial for regulating the metabolic activities of CD4+ T cells in psoriasis.
      CDKs have been traditionally described as key cell cycle regulators (
      • Besson A.
      • Dowdy S.F.
      • Roberts J.M.
      CDK inhibitors: cell cycle regulators and beyond.
      ), and they have recently been found to modulate transcription in response to several extra- and intracellular cues (
      • Malumbres M.
      Cyclin-dependent kinases.
      ;
      • Palmer N.
      • Kaldis P.
      Less-well known functions of cyclin/CDK complexes.
      ;
      • Wells A.D.
      • Morawski P.A.
      New roles for cyclin-dependent kinases in T cell biology: linking cell division and differentiation.
      ). Previous studies have primarily focused on the role of CDKs in tumor pathogenesis (
      • Malumbres M.
      Cyclin-dependent kinases.
      ), with some CDKs identified as therapeutic targets to prevent or limit tumor progression (
      • Sánchez-Martínez C.
      • Lallena M.J.
      • Sanfeliciano S.G.
      • de Dios A.
      Cyclin dependent kinase (CDK) inhibitors as anticancer drugs: recent advances (2015–2019).
      ). Similar to the process of tumor progression, the activation of T cells also requires metabolic reprogramming to support their proliferation and differentiation (
      • Hu Z.
      • Zou Q.
      • Su B.
      Regulation of T cell immunity by cellular metabolism.
      ). However, little is known regarding the role of CDKs in T-cell function. Recent studies have discovered that CDKs provide a potential link between T-cell division and differentiation (
      • Wells A.D.
      • Morawski P.A.
      New roles for cyclin-dependent kinases in T cell biology: linking cell division and differentiation.
      ). CDK8/CDK19 inhibition promoted regulatory T-cell differentiation (
      • Guo Z.
      • Wang G.
      • Lv Y.
      • Wan Y.Y.
      • Zheng J.
      Inhibition of Cdk8/Cdk19 activity promotes Treg cell differentiation and suppresses autoimmune diseases.
      ). The inhibition of CDK8/CDK19 enabled the conversion of effector/memory T cells into FOXP3-expressing regulatory T cells (
      • Akamatsu M.
      • Mikami N.
      • Ohkura N.
      • Kawakami R.
      • Kitagawa Y.
      • Sugimoto A.
      • et al.
      Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8/19.
      ). Moreover, selective inhibition of CDK7 suppressed the Th17 response in an arthritis mouse model (
      • Xia Y.
      • Lin L.Y.
      • Liu M.L.
      • Wang Z.
      • Hong H.H.
      • Guo X.G.
      • et al.
      Selective inhibition of CDK7 ameliorates experimental arthritis in mice.
      ). A recent study showed that the inhibition of CDK4/6 in skin abrogated psoriasis-related proinflammatory gene expression, indicating CDK4/6 as potential therapeutic targets for psoriasis (
      • Müller A.
      • Dickmanns A.
      • Resch C.
      • Schäkel K.
      • Hailfinger S.
      • Dobbelstein M.
      • et al.
      The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis.
      ). In our study, by comparing the protein expression profile of psoriatic CD4+ T cells with that of healthy CD4+ T cells, CDK7 was significantly upregulated in psoriatic CD4+ T cells, rather than CDK8/CDK19 or CDK4/CDK6. Furthermore, CDK7 modulated the differentiation of Th17/Th1 cells in psoriasis. Hence, CDK7 plays a regulatory role in CD4+ T-cell function, which may be associated with the development of autoimmune diseases.
      In addition to modulating cell proliferation and differentiation, several studies suggest that CDK7 can regulate glycolytic metabolism (
      • Cheng Z.J.
      • Miao D.L.
      • Su Q.Y.
      • Tang X.L.
      • Wang X.L.
      • Deng L.B.
      • et al.
      THZ1 suppresses human non-small-cell lung cancer cells in vitro through interference with cancer metabolism.
      ;
      • Ghezzi C.
      • Wong A.
      • Chen B.Y.
      • Ribalet B.
      • Damoiseaux R.
      • Clark P.M.
      A high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells.
      ). CDK7 activated glucose consumption in lung cancer cells (
      • Ghezzi C.
      • Wong A.
      • Chen B.Y.
      • Ribalet B.
      • Damoiseaux R.
      • Clark P.M.
      A high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells.
      ). Moreover, CDK7 inhibition could block the glycolysis pathway to inhibit the growth of human non–small cell lung cancer cells (
      • Cheng Z.J.
      • Miao D.L.
      • Su Q.Y.
      • Tang X.L.
      • Wang X.L.
      • Deng L.B.
      • et al.
      THZ1 suppresses human non-small-cell lung cancer cells in vitro through interference with cancer metabolism.
      ). However, the effect of CDK7 on glycolytic metabolism in other cells, especially T cells, is unknown. In our study, a remarkable increase in glycolytic metabolism was observed in psoriatic CD4+ T cells. More importantly, CDK7 inhibition significantly downregulated enhanced glycolysis in psoriatic CD4+ T cells and reduced the expression of key genes associated with glycolysis in CD4+ T cells.
      We further investigated the mechanism by which CDK7 regulated Th17/Th1 cell differentiation. We found that, rather than IL-1β, IL-23 and IL-12 induced CDK7 expression in CD4+ T cells, and IL-23 induced CDK7 expression more significantly than IL-12. The serum IL-23 level was also positively correlated with CDK7 expression in CD4+ T cells from patients with psoriasis, supporting that IL-23 is an important upstream effector of CDK7 in CD4+ T cells.
      Additionally, CDK8/CDK19 modulate regulatory T-cell differentiation via TGFβ–SMAD signaling and signal transducer and activator of transcription 5 pathways (
      • Akamatsu M.
      • Mikami N.
      • Ohkura N.
      • Kawakami R.
      • Kitagawa Y.
      • Sugimoto A.
      • et al.
      Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8/19.
      ;
      • Guo Z.
      • Wang G.
      • Lv Y.
      • Wan Y.Y.
      • Zheng J.
      Inhibition of Cdk8/Cdk19 activity promotes Treg cell differentiation and suppresses autoimmune diseases.
      ). Moreover, the Akt/mTOR/HIF-1α pathway is a critical signaling pathway involved in both glycolysis and differentiation of T cells (
      • Pearce E.L.
      • Pearce E.J.
      Metabolic pathways in immune cell activation and quiescence.
      ). By analyzing the gene expression profile, we found that compared with psoriatic CD4+ T cells, mRNA expression of genes associated with the Akt/mTOR/HIF-1α pathway and its downstream targets were dramatically reduced in psoriatic CD4+ T cells treated with the CDK7 inhibitor. CDK7 inhibition also downregulated IL-23–induced activation of the Akt/mTOR/HIF-1α pathway and IL-23–induced expression of key genes associated with glycolysis in CD4+ T cells. Thus, CDK7 regulates glycolysis in psoriatic CD4+ T cells through the Akt/mTOR/HIF-1α pathway, which accounts for the regulatory effect of CDK7 on CD4+ T-cell activation and Th17/Th1 cell differentiation in psoriasis.
      Collectively, the CDK family members, as the key regulators of the cell cycle and as transcriptional modulators, have been found to contribute to the pathogenesis of various tumors (
      • Leitch A.E.
      • Haslett C.
      • Rossi A.G.
      Cyclin-dependent kinase inhibitor drugs as potential novel anti-inflammatory and pro-resolution agents.
      ) and are gradually discovered to affect the function of immune cells (
      • Hong H.
      • Zeng Y.
      • Jian W.
      • Li L.
      • Lin L.
      • Mo Y.
      • et al.
      CDK7 inhibition suppresses rheumatoid arthritis inflammation via blockage of NF-κB activation and IL-1β/IL-6 secretion.
      ;
      • Rossi A.G.
      • Sawatzky D.A.
      • Walker A.
      • Ward C.
      • Sheldrake T.A.
      • Riley N.A.
      • et al.
      Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis [published correction appears in Nat Med 2006;12:1434].
      ), especially T-cell differentiation (
      • Akamatsu M.
      • Mikami N.
      • Ohkura N.
      • Kawakami R.
      • Kitagawa Y.
      • Sugimoto A.
      • et al.
      Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8/19.
      ;
      • Guo Z.
      • Wang G.
      • Lv Y.
      • Wan Y.Y.
      • Zheng J.
      Inhibition of Cdk8/Cdk19 activity promotes Treg cell differentiation and suppresses autoimmune diseases.
      ;
      • Wells A.D.
      • Morawski P.A.
      New roles for cyclin-dependent kinases in T cell biology: linking cell division and differentiation.
      ). This study demonstrates that CDK7 is excessively expressed in CD4+ T cells of patients with psoriasis because of TCR activation and the stimulation of IL-23 and IL-12. Moreover, CDK7 can modulate CD4+ T-cell activation and Th17/Th1 cell differentiation by regulating glycolytic metabolism as a consequence of the activated Akt/mTOR/HIF-1α pathway, thus contributing to the pathogenesis of psoriasis.

      Materials and Methods

      Patients and clinical samples

      Peripheral blood samples were obtained from 59 patients with psoriasis vulgaris who visited our department at Xijing Hospital. Xi’an, China. All patients were unmedicated for at least 1 month. Peripheral blood samples from 39 healthy volunteers with a similar age distribution to the patients served as controls. Individuals with respiratory tract infections or other infectious diseases in the previous month were excluded. Skin samples were obtained from healthy donors undergoing plastic surgery (n = 3) and patients with psoriasis vulgaris (n = 3). The study was approved by the Ethics Committee of the Fourth Military Medical University, Xi’an, China, and written informed consent was obtained from all participants. Information for all participants is provided in Supplementary Table S3.

      Mice

      All animal studies were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Review Committee for the Use of Animals of the Fourth Military Medical University. Male BALB/c mice and male C57BL/6 mice aged 8–9 weeks were purchased from the Animal Experimental Center of the Fourth Military Medical University. Rag2–/– mice (C57BL/6 background) were purchased from HFK Biosciences (Beijing, China).
      Methods for short hairpin RNA knockdown, THZ1 treatment in the IMQ-induced psoriasis model, adoptive transfer of CD4+ T cells into Rag2–/– mice in the IMQ-induced psoriasis model, western blot, quantitative proteomics analysis, cell isolation and culture, skin histopathology and epidermis thickness measurements, skin immunofluorescence, ELISA, intracellular CDK7 staining, flow cytometry, qRT–PCR, cell proliferation assays, cell apoptosis assays, RNA sequencing, and transcriptomics analysis and glycolytic activity analysis are provided in the Supplementary Materials and Methods (Supplementary Table S4). The sequences of the oligonucleotides are showed in Supplementary Table S5. The primer sequences are listed in Supplementary Table S6. Information of the antibodies are listed.

      Statistical analysis

      Statistical analyses were carried out using Student’s t-test or one-way ANOVA with GraphPad Prism version 8.0 software (GraphPad Software, San Diego, CA). Differences were considered significant if P-values were <0.05.

      Data availability statement

      Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171676, hosted at National Center for Biotechnology Information, Cene Expression Omnibus DataSets.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by the National Natural Science Foundation of China (number 82030096, number 82073435, number 81972958, andnumber 81903207). We would like to thank our patients and healthy volunteers for their enthusiastic participation. We would also like to thank Wenjing Li for her assistance with the immunofluorescence staining.

      Author Contributions

      Conceptualization: YL, BL, GW; Data Curation: KX, ZL, YL; Formal Analysis: YL, QL, ZL; Funding Acquisition: GW, BL, KX, WZ; Investigation: YL, ZZ, JC; Methodology: ED, YL, BL; Project Administration: GW; Supervision: GW, BL; Validation: LW; Visualization: QL; Writing - Original Draft Preparation: YL; Writing - Review and Editing: KX, QL, WZ, BL, GW

      Supplementary Material

      Supplementary Materials and Methods

      Short hairpin RNA knockdown

      Short hairpin RNA targeting mouse CDK7 was carried by lentiviral vectors (Hanbio Biotechnology, Shanghai, China). The sequences of the oligonucleotides are shown in Supplementary Table S5. CD4+ T cells were cultured in 96-well U-bottom plates coated with CD3 (10 μg/ml; BioLegend, San Diego, CA). Recombinant IL-2 (80 units/ml; R&D Systems, Minneapolis, MN) and CD28 antibodies (1 μg/ml; BioLegend) were then added to the cultures, and cells were incubated for 72 hours. Lentivirus was then added to the cultures (multiplicity of infection = 100). Three days later, red fluorescence protein–positive lentiviral-transduced cells were identified using flow cytometry. Knockdown efficiency was also examined using western blotting.

      THZ1 treatment in the imiquimod-induced psoriasis model

      Male BALB/c mice (8–9 weeks) were randomly subdivided into a vehicle group, imiquimod (IMQ) group, and THZ1 (CDK7 inhibitor) treatment group. Mice with shaved backs were locally administered IMQ cream (Inova, Chatswood, Australia) as previously described (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ) or vaseline in combination with an intraperitoneal injection of THZ1 (10 mg/kg, MedChemExpress, Monmouth Junction, NJ) or vehicle daily for seven consecutive days. Body weight and disease severity score was assessed daily. Mice were euthanized on day 7 with an overdose of 10% chloral hydrate, and skin and spleen tissues were harvested. After collection and weighing, spleen tissues were minced and homogenized with PBS. Single-cell suspensions were prepared by passing through a 70-μm cell strainer (BD Falcon, Durham, NC). Splenocytes were collected by centrifuging single-cell suspensions over a Ficoll-Paque gradient (Thermo Fisher Scientific, Waltham, MA). After washing with PBS, cells were counted and stimulated with eBioscience Cell Stimulation Cocktail (Thermo Fisher Scientific) for 6 hours at 37 °C. The animal studies were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Review Committee for the Use of Animals of the Fourth Military Medical University (Xi’an, China).

      Adoptive transfer of CD4+ T cells into Rag2–/– mice in an IMQ-induced psoriasis model

      CD4+ T cells were obtained from male C57BL/6 mice aged 8–9 weeks and infected with lentivirus to achieve Cdk7 knockdown. Psoriasis-like models were established in Rag2–/– mice by the adoptive transfer of CD4+ T cells combined with the local application of IMQ cream (Inova). First, IMQ was locally administered to both ears of Rag2–/– mice on 2 consecutive days. Then, CD4+ T cells with Cdk7 knockdown (5 × 104/μl) were injected intradermally into the right ear of Rag2–/– mice (50 μl per ear), and control CD4+ T cells were injected intradermally into the left ear of Rag2–/– mice (50 μl per ear) as a positive control, and PBS was used as the negative control. After the cell transfer, mice were treated with IMQ locally on each day for 9 consecutive days. Mice were killed on day 12 with an overdose of 10% chloral hydrate, and skin lesions were harvested.

      Western blot

      Western blot analysis was performed as previously described (
      • Yang G.
      • Liang Y.
      • Zheng T.
      • Song R.
      • Wang J.
      • Shi H.
      • et al.
      FCN2 inhibits epithelial-mesenchymal transition-induced metastasis of hepatocellular carcinoma via TGF-β/Smad signaling.
      ). The cells were prepared using radioimmunoprecipitation assay buffer. After electrophoresis, proteins were electroeluted at 120 volts onto a polyvinylidene difluoride membrane (Invitrogen, Carlsbad, CA). The antibodies against CDK7 (Santa Cruz Biotechnology, Dallas, TX), tubulin (Abcam, Cambridge, United Kingdom), and anti-mouse IgG (Cell Signaling Technology, Danvers, MA) were used. Protein bands were visualized by an enhanced chemiluminescence assay kit (Super Signal Pierce Biotechnology, Rockford, IL).

      Quantitative proteomics analysis

      The whole cells extracted from peripheral CD4+ T cells of patients with psoriasis and age- and sex-matched healthy controls (n = 3 for each group) were lysed in an extraction buffer and quantified using the BCA assay (Thermo Fisher Scientific). Proteins for the isobaric tag for relative and absolute quantitation quantitative analysis were prepared according to the manufacturer’s instructions (ABSciex, Framingham, MA) at CapitalBio Technology (Beijing, China). Liquid chromatography–tandem mass spectrometry analysis was performed by CapitalBio Technology using a Q Exactive mass spectrometer (Thermo Fisher Scientific). A two-fold increase or reduction in abundance with significance (P < 0.05) constituted differentially expressed proteins. The metabolism pathways (Molecular Signatures Database [MSigDB] gene set: M11521, M19540, M699, M6999, M4377, M7934, M15902, M10357, M11551, M12883, M24355, M13851, M15416, and M13431) were assessed using Gene Set Enrichment Analysis (version 4.0.3, Broad Institute, Cambridge MA; I Massachusetts Institute of Technology, Cambridge, MA; and Regents of the University of California, Oakland, CA).

      Cell isolation and culture

      PBMCs were isolated from whole blood using lymphoprep (Dakewe, Shenzhen, China). Human CD4+ T cells were obtained from PBMCs by magnetic cell sorting with the human CD4 Microbead kit (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s instructions, and the purity of CD4+ T cells was determined by flow cytometry (Supplementary Figure S1). Human naive CD4+ T cells were obtained from PBMCs by magnetic cell sorting with the human naive CD4+ T-cell isolation kit Ⅱ (Miltenyi Biotec) according to the manufacturer’s instructions. Mouse CD4+ T cells were obtained from mouse spleens by magnetic cell sorting with the CD4+ T-cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were stimulated with plate-bound CD3 and CD28 antibodies (2 μg/ml and 1 μg/ml, respectively; BioLegend) in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco). To induce T-cell proliferation, naive CD4+ T cells were activated in the presence of hIL-2 (50 ng/ml). To induce T-cell differentiation, naive CD4+ T cells were activated in the presence of the following: for T helper type (Th) 17, hIL-1β (50 nM) and hIL-23 (50 nM); for Th1, hIL-12 (2.5 ng/ml); for control cells (Th0), without cytokines. To detect the effect of cytokines on CDK7 expression, cells were activated in the presence of rhIL-1β (50 nM), hIL-23 (50 nM), or hIL-12 (2.5 ng/ml). All cytokines were purchased from Sino Biological (Beijing, China). Cells were also cocultured with THZ1 (MedChemExpress), a CDK7 inhibitor, or equal amounts of DMSO during CD3/CD28 activation. Cells were cultured for 1 day (T-cell activation), 2 days (CDK7 expression), or 5 days (cell differentiation) after activation. The harvested differential cells were restimulated with eBioscience Cell Stimulation Cocktail (Thermo Fisher Scientific) for 6 hours at 37 °C for intracellular inflammatory cytokine staining.

      Skin histopathology and epidermis thickness measurements

      Skin tissues from psoriasis-like mice were fixed overnight in buffered formalin, embedded in paraffin, cut into 4-μm sections, and stained using H&E for histological analysis. Epidermis thickness was measured using NanoZoomer Digital Pathology software.

      Skin immunofluorescence

      Paraffin skin cross-sections (4 μm) were pretreated for deparaffinization, dehydration, and antigen retrieval. Sections were blocked with 5% goat serum in PBS for 30 minutes. Human samples were incubated with rabbit anti-CD4 (Abcam) and mouse anti-CDK7 (Santa Cruz Biotechnology) at 4 °C overnight. After washing in PBS, the sections were incubated with appropriate secondary antibodies (goat anti-mouse IgG, Cy3-conjugated and goat anti-rabbit IgG, FITC-conjugated; Abcam) for 1 hour. Mouse samples were incubated with rabbit anti-CD3 (Abcam) overnight at 4 °C, followed by a 1-hour incubation with secondary antibody goat anti-rabbit IgG, Cy3–conjugated (Abcam). DAPI was used for nuclear counterstaining. Skin sections were analyzed by confocal microscopy (FV-1000, Olympus, Tokyo, Japan).

      ELISA

      Serum from patients with psoriasis vulgaris was processed using a human IL-23 ELISA kit (Elabscience, Wuhan, China), according to the manufacturer’s instructions.

      Intracellular CDK7 staining

      Peripheral CD4+ T cells from PBMCs of healthy controls and patients with psoriasis treated with or without inflammatory cytokines (IL-23, IL-12, and IL-1β) were collected and incubated with live/dead stain (Fixable Viability Stain 620, BD, San Jose, CA) for 10 minutes, followed by incubation with Fcγ block (BD) for 10 minutes. Cells were stained with PECY7 rat anti-human CD4 antibody (BioLegend, San Diego, CA) for 30 minutes at 4 °C, fixed and permeabilized with FOXP3 staining buffer kit (eBioscience, San Diego, CA), and incubated with mouse anti-CDK7 primary antibody (Santa Cruz Biotechnology) for 50 minutes at 4 °C. The cells were then washed and stained with secondary FITC anti-mouse IgG antibody (BioLegend) for 30 minutes at 4 °C. Gating was performed on single, live, CD4+ T cells. All samples were analyzed using a Cytomics FC500 flow cytometer (Beckman Coulter, Indianapolis, IN). Data were analyzed using FlowJo software.

      Flow cytometry

      To detect the Th1/Th17 cell proportion of peripheral CD4+ T cells from patients with psoriasis or splenocytes from IMQ-induced psoriasis-like mice, eBioscience Cell Stimulation Cocktail (Thermo Fisher Scientific) was added for 6 hours. After incubation with live/dead stain (Fixable Viability Stain 620, BD) for 10 minutes followed by incubation with Fcγ block (BD) for 10 minutes, the cells were stained with PECY7 anti-human/mouse CD4 antibody (BioLegend). Cells were fixed and permeabilized with the FOXP3 staining buffer kit (eBioscience) and stained with Alexa Fluor 488 anti-human ROR-γt (BD) and PE anti-human IL-17A antibody for human Th17 cells (PE anti-mouse IL-17A for mouse Th17 cells, all from BioLegend), or PE anti–T-bet antibody and Alexa Fluor 488 anti-human IFN-γ antibody for human Th1 cells (PE anti-mouse IFN-γ for mouse Th1 cells, all from BioLegend). To detect T-cell activation, cells were stained with PE anti-human CD69 antibody (BioLegend). To evaluate the activity of the protein kinase B/mTOR/HIF-1α pathway, cells were stained with PE mouse anti–protein kinase B (pS473) (BD), PE mouse anti-4EBP1 (pT36/pT45) (BD), or PE anti-human HIF-1α antibody (BioLegend). Cells were then collected/run using a Cytomics FC500 flow cytometer (Beckman Coulter), and data were analyzed using FlowJo software.

      qRT-PCR

      Total RNA was extracted from mouse skin tissues or cultured cells using TRIzol reagent (Takara, Kyoto, Japan), and cDNA was synthesized with PrimeScript RT reagent kit (Takara) according to the manufacturer’s protocols. The primers were purchased from Sangon (Shanghai, China), and primer sequences are listed in Supplementary Table S5. qRT-PCR was performed using SYBR Green Master (Takara) on a Chromo4 continuous fluorescence detector with a PTC-200 DNA Engine Cycler (Bio-Rad, Hercules, CA). Gene expression was normalized to Rplp0.

      Cell proliferation assays

      CD4+ T cells were labeled with carboxyfluorescein succinimidyl ester (Thermo Fisher Scientific) at a final concentration of 5 μM for 20 minutes and protected from light, then stimulated with dynabeads human T-activator CD3/CD28 (Thermo Fisher Scientific) and hIL-2 (50 ng/ml) for 5 days. After 5 days, cells were analyzed using flow cytometry. Cell proliferation was calculated using the Proliferation Wizard Model of Modifit LT software (Verity Software House, Topsham, ME).

      Cell apoptosis assays

      Cell apoptosis was measured by Annexin Ⅴ and propidium iodide using the dead cell apoptosis kit with Annexin Ⅴ-FITC/propidium iodide (Thermo Fisher Scientific). CD4+ T cells were harvested after treatment, washed in cold PBS, and the supernatant discarded. The cells were resuspended in 195 μl binding buffer and incubated with 5 μl Annexin Ⅴ-FITC in the dark at room temperature for 10 minutes. The cells were centrifuged, and the supernatant was discarded. The cells were resuspended in 190 μl binding buffer and incubated with 5 μl propidium iodide in the dark at room temperature for 5 minutes. Apoptosis was quantified using Flow cytometry.

      RNA sequencing and transcriptomics analysis

      Total RNA of THZ1-treated and DMSO-treated psoriatic CD4+ T cells was extracted with TRIzol (Invitrogen), analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), and then quantified using Qubit 2.0 (Thermo Fisher Scientific). Sequencing libraries were generated and sequenced by CapitalBio Technology (Beijing, China). Gene Ontology enrichment analyses were performed using the Gene Ontology database and Wego website. The glycolysis pathway was assessed using Gene Set Enrichment Analysis (version 4.0.3, Broad Institute, Massachusetts Institute of Technology, and Regents of the University of California). A heatmap was drawn to show the expressions of genes involved in glycolysis, phosphatidylinositol 3 kinase/protein kinase B/mTOR, MTORC1, and HIF1A signaling, which were obtained from MSigDB (v7.2, Broad Institute, Massachusetts Institute of Technology, and Regents of the University of California). The glycolysis gene list was obtained from MSigDB gene set M15109 ‘‘BIOCARTA_GLYCOLYSIS_PATHWAY’’. The phosphatidylinositol 3 kinase/protein kinase B/MTOR gene list was obtained from MSigDB gene set M5923 ‘‘HALLMARK_PI3K_AKT_MTOR _SIGNALING’’. The MTORC1 gene list was obtained from MSigDB gene set M5924 ‘‘HALLMARK_MTORC1_SIGNALING’’. The HIF1A gene list was obtained from MSigDB gene set M12299 ‘‘SEMENZA_HIF1_TARGETS’’. Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171676, hosted at National Center for Biotechnology Information Gene Expression Omnibus datasets.

      Glycolytic activity analysis

      The supernatant was collected after 24-hour cell culture, and lactate was assessed using a lactate assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. For glucose uptake detection, cultured cells were incubated with 100 μM 2-NBDG (Invitrogen) for 2 hours before measuring fluorescence by flow cytometry. The extracellular acidification rate was measured using an XF24 Extracellular Flux Analyzer (Agilent Technologies) in basal conditions and response to glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose (50 mM) (all from Sigma Aldrich, St. Louis, MO) to determine the glycolytic rate and glycolytic capacity of the tested cells.
      Figure thumbnail fx2
      Supplementary Figure S1CD4+ T cells reach over 95% purity after magnetic bead sorting. PBMCs were isolated by gradient centrifugation, then CD4+ T cells were negatively selected by magnetic bead sorting. The purity of each sorted population was confirmed by flow cytometry analysis. MACS, magnetic cell sorting.
      Figure thumbnail fx3
      Supplementary Figure S2CDK7 inhibitor THZ1 ameliorates psoriasiform symptoms in an IMQ-induced psoriasis-like mouse model. (a) Schematic diagram of mouse experimental protocol. (b) Phenotype (upper panel) and H&E staining (lower panel) of the lesions in different groups on day 7 after IMQ application. Bar = 200 μm. (c) Dorsal skin thickness, erythema, and scaling were recorded daily with a score from 0 to 4. Additionally, the cumulative PASI score was depicted. (d) Quantified epidermal thickness of psoriasiform lesions by H&E staining. (e) Immunofluorescence staining of CD4 (red) in skin lesions. Nuclei were counterstained with DAPI (blue) (bar = 200 μm) and counts of infiltrated CD4+ T-cell numbers in skin lesions. (f) qRT-PCR for mRNA expression of Ifng and Il17a in lesional skin. Data are mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. P-values were calculated by one-way ANOVA with Tukey’s post hoc test. n = 9 mice per group. IMQ, imiquimod.
      Figure thumbnail fx4
      Supplementary Figure S3CDK7 inhibitor THZ1 attenuates inflammatory profiles and reduces Th17 and Th1 cell differentiation in an IMQ-induced psoriasis-like mouse model. (a) Representative pictures and weight of spleens on day 7. Bar = 1 cm. (b) Time course of the body weight of the mice for 7 days. (c) The percentage of CD4+ IL-17A+ cells and (d) CD4+ IFN-γ+ cells in the CD4+ T-cell population from the mouse spleens was measured by flow cytometry. Data are mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. P-values was calculated by one-way ANOVA with Tukey’s post hoc test. n = 9 mice per group. IMQ, imiquimod; ns, not significant.
      Figure thumbnail fx5
      Supplementary Figure S4The adoptive transfer model of CD4+ T cells. IMQ was applied locally to both ears of Rag2–/– mice for 2 consecutive days. Then, WT-derived CD4+ T cells were injected intradermally into the right ears of the mice. Meanwhile, PBS was injected into the left ears as the vehicle group, and no injection was the negative control. IMQ was then applied locally daily for 9 consecutive days. (a) H&E staining. (b) Mean thickness of the epidermis. Data are mean ± SEM. ∗∗∗∗P < 0.0001. P-values were calculated by one-way ANOVA with Tukey’s post hoc test. n = 5 mice per group. IMQ, imiquimod; ns, not significant; WT, wild type.
      Figure thumbnail fx6
      Supplementary Figure S5Cdk7 knockdown in CD4+ T cells. CD4+ T cells isolated from WT mice were transduced with lentiviral vectors containing shRNA targeting CDK7. The lentiviral vector also expresses RFP that was used to monitor the efficiency of transduction. (a) Flow cytometric analysis of RFP expression in lentiviral-transduced CD4+ T cells. (b) Western blot analysis of CDK7 expression in WT, NC, and lentiviral-transduced CD4+ T cells. Tubulin was used as a loading control. NC, normal control; shRNA, short hairpin RNA; WT, wild type.
      Figure thumbnail fx7
      Supplementary Figure S6CD4+ T-cell response treated with THZ1. Human naive CD4+ T cells stimulated with different concentrations of THZ1 (0 nM, 2.5 nM, 5 nM, 10 nM) in the presence of CD3/CD28 antibodies for 24 hours. (a) The percentage of IL-2+ cells and (b) CD25+ cells in the CD4+ T-cell population was evaluated by flow cytometry. (c) Proliferation index analysis of CD4+ T cells was evaluated by flow cytometry. (d) Apoptosis detected by Annexin Ⅴ FITC/PI staining. Data are mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. P-values were calculated by one-way ANOVA with Tukey’s post hoc test. n = 5 for each group. CFSE, carboxyfluorescein succinimidyl ester; ns, not significant; PI, propidium iodide.
      Figure thumbnail fx8
      Supplementary Figure S7Amino sugar and nucleotide sugar metabolism are also significantly enriched, although to a lesser degree than glycolysis. Gene set enrichment analysis of proteomic data, CD4+ T cells in Pso versus CD4+ T cells in HC (n = 3 for each group). ES, enrichment score; FDR, false discovery rate; HC, healthy control; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; Pso, patients with psoriasis.
      Figure thumbnail fx9
      Supplementary Figure S8THZ1 treatment decreases the mRNA expression of key genes involved in glycolysis. The mRNA expression level of the key genes involved in glycolysis were measured by qRT-PCR (n = 5). Data were mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. P-values were calculated by unpaired Student’s t-test. Pso, patients with psoriasis.
      Figure thumbnail fx10
      Supplementary Figure S9Effect of IL-23 and THZ1 on the Akt/mTOR/HIF-1α signaling pathway of CD4+ T cells. Human CD4+ T cells were activated with IL-23 (50 nM) or THZ1 (10 nM) for 2 days. (a) The expressions of p-Akt (s473), 4EBP1 (pT36/pT45), and HIF-1α following treatment with IL-23 or (b) THZ1 were measured by flow cytometry. Quantification MFI is shown. Data were mean ± SEM. ∗P < 0.05; ∗∗P < 0.01. P-values were calculated by unpaired Student’s t-test. n = 4 for each group. Akt, protein kinase B; MFI, mean fluorescence intensity; NC, normal control; p-Akt, phosphorylated protein kinase B.
      Supplementary Table S3Information of Patients with Psoriasis and Healthy Volunteers
      PASI Score
      GroupSexNumberAge (Years, Mean ± SD)0–1010–20≥20
      Contributed the skin
      PatientsMale341.7 ± 3.1210
      Healthy ControlMale342.0 ± 4.0
      Contributed the blood
      PatientsMale3433.4 ± 11.62572
      Female2534.4 ± 10.11951
      Healthy ControlMale3231.8 ± 7.0
      Female732.3 ± 9.4
      Supplementary Table S4Antibody
      AntibodyCompanyArt Number
      Ultra-LEAF purified anti-human CD3 AntibodyBioLegend300334
      Ultra-LEAF purified anti-human CD28 AntibodyBioLegend302960
      Anti-CD4 antibodyAbcamab133616
      CDK7(C-4) antibodySanta Cruz Biotechnologysc-7344
      Goat anti-mouse IgG H&L (Cy3) preadsorbedAbcamab97035
      Goat anti-rabbit IgG H&L (FITC)Abcamab6717
      Anti-CD3 antibodyAbcamab16669
      Human BD Fc BlockBD564219
      PE/Cyanine7 anti-human CD4 AntibodyBioLegend357410
      FITC anti-mouse IgG AntibodyBioLegend406001
      PE/Cyanine7 anti-mouse CD4 AntibodyBioLegend116015
      Alexa Fluor 488 mouse anti-human RORγtBD563621
      PE anti-human IL-17A antibodyBioLegend512306
      PE anti-mouse IL-17A antibodyBioLegend506904
      PE anti–T-bet antibodyBioLegend644809
      Alexa Fluor 488 anti-human IFN-γ antibodyBioLegend502515
      PE anti-mouse IFN-γ antibodyBioLegend505808
      PE anti-human CD69 antibodyBioLegend310906
      PE mouse anti-Akt (pS473)BD561671
      PE mouse anti-4EBP1 (pT36/pT45)BD560285
      PE anti-human HIF1α antibodyBioLegend359704
      APC anti-human IL-2 antibodyBioLegend500310
      PE anti-human CD25 antibodyBioLegend302604
      Anti-tubulin antibodyAbcamAb6046
      Rabbit anti-mouse IgGCST58802
      Abbreviations: BD, BD Biosciences; CST, Cell Signaling Technology.
      Supplementary Table S5Sequence of CDK7 shRNA
      NameSequence
      CDK7 shRNAGAGCGAAGCGCTATGAGAAACTGGA
      Abbreviation: shRNA, short hairpin RNA.
      Supplementary Table S6qRT-PCR Primer Sequences
      NameOrientationSequence 5′ →3′
      IL17AfwAGATTACTACAACCGATCCACCT
      revGGGGACAGAGTTCATGTGGTA
      IFNGfwGAGTGTGGAGACCATCAAGGA
      revGTATTGCTTTGCGTTGGACA
      CDK7fwATGGCTCTGGACGTGAAGTCT
      revGCGACAATTTGGTTGGTGTTC
      HK2fwTTGACCAGGAGATTGACATGGG
      revCAACCGCATCAGGACCTCA
      PFKLfwGGCTTCGACACCCGTGTAA
      revCGTCAAACCTCTTGTCATCCA
      PFKMfwAGCTGCCTACAACCTGGTGA
      revTCCACTCAGAACGGAAGGTGT
      PFKPfwGACCTTCGTTCTGGAGGTGAT
      revCACGGTTCTCCGAGAGTTTG
      PGK1fwTGGACGTTAAAGGGAAGCGG
      revGCTCATAAGGACTACCGACTTGG
      PKM2fwATGTCGAAGCCCCATAGTGAA
      revTGGGTGGTGAATCAATGTCCA
      LDHAfwTTGACCTACGTGGCTTGGAAG
      revGGTAACGGAATCGGGCTGAAT
      SLC2A1fwATTGGCTCCGGTATCGTCAAC
      revGCTCAGATAGGACATCCAGGGTA
      Abbreviations: fw, forward; rev, reverse.

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