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. 2016 Jul 12;16(2):405-418.
doi: 10.1016/j.celrep.2016.05.083. Epub 2016 Jun 23.

IκB Kinase ε Is an NFATc1 Kinase That Inhibits T Cell Immune Response

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Free PMC article

IκB Kinase ε Is an NFATc1 Kinase That Inhibits T Cell Immune Response

Junjie Zhang et al. Cell Rep. .
Free PMC article

Abstract

Activation of nuclear factor of activated T cells (NFAT) is crucial for immune responses. IKKε is an IκB kinase (IKK)-related kinase, and the function of IKKε remains obscure in T cells, despite its abundant expression. We report that IKKε inhibits NFAT activation and T cell responses by promoting NFATc1 phosphorylation. During T cell activation, IKKε was transiently activated to phosphorylate NFATc1. Loss of IKKε elevated T cell antitumor and antiviral immunity and, therefore, reduced tumor development and persistent viral infection. IKKε was activated in CD8(+) T cells of mice bearing melanoma or persistently infected with a model herpesvirus. These results collectively show that IKKε promotes NFATc1 phosphorylation and inhibits T cell responses, identifying IKKε as a crucial negative regulator of T cell activation and a potential target for immunotherapy.

Figures

Figure 1
Figure 1. Loss of IKKε Reduces the Latent Infection of γHV68 and Boost Antiviral T Cell Immunity
Age- and gender-matched mice were infected with γHV68 via intranasal (A, 40 PFU) or intraperitoneal (B–J, 1 × 106 PFU) route. (A) Viral lytic replication in the lung was determined by plaque assay at indicated days postinfection (dpi). (B) Viral genome frequency at 42 dpi was determined by limiting-dilution PCR. (C) Representative tetramer staining (ORF61) at 13 dpi. (D) Tetramer staining was performed at indicated dpi. (E) Splenocytes were isolated from γHV68-infected Ikbke+/+ and Ikbke−/− mice at 13 dpi and stimulated with viral antigenic peptide (ORF61). IFN-γ was determined by intracellular staining. (F) Splenocytes labeled with CFSE were pulsed with or without viral peptide and transferred into γHV68 infected Ikbke+/+ and Ikbke−/− mice. The specific killing was quantified. (G) Ikbke+/+ and Ikbke−/− mice were mock treated or depleted with anti-CD8 antibody starting at 16 dpi. Splenocytes were harvested at 42 dpi, and viral genome frequency was determined as in (B). (H and I) CD8+ splenocytes of Ikbke+/+ or Ikbke−/− mice were transplanted into CD45.1 immune-competent mice infected with γHV68 for 16 days. CD8+ T cell response and viral latent infection in the splenocytes were analyzed by tetramer staining (H) and limiting-dilution PCR (I), respectively. (J) Ikbke+/+ and Ikbke−/− CD8+ splenocytes were mixed and transferred into Rag-2−/− mice that were infected with γHV68. At 13 dpi, splenocytes were harvested and analyzed for CD45.1 (Ikbke+/+) and CD45.2 (Ikbke−/−) within the CD8+ T cell subset. Error bars denote SD. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 2
Figure 2. Knockdown or Pharmacological Inhibition of IKKε Promotes T Cell Activation
(A) Jurkat T cells transduced with control shRNA (shCTL) or shRNA against IKKε (shIKKε) were treated with DMSO or PMA plus ionomycin for 3 hr. Total RNA was extracted and analyzed by microarray. Top differentially expressed genes were shown. Color intensity denotes log2 normalized raw data. (B and C) Jurkat T cells were transduced with lentiviral shRNA and whole-cell lysates were analyzed by immunoblotting (B, top) and total RNA was analyzed by reverse transcription qPCR (B, bottom). IL-2 protein expression was examined by intracellular staining (C). (D) Isolated mouse CD3+ primary T cells were stimulated with PMA and ionomycin and IL-2 mRNA level was analyzed by real-time q-PCR. (E) Jurkat T cells were stimulated with PMA and ionomycin (Iono), with increasing concentrations of amlexanox (Amlex) and analyzed by IL-2 intracellular staining. (F) Jurkat T cells stably expressing NFATc1 were activated with PMA plus ionomycin without (DMSO) or with amlexanox (50 μM). Precipitated NFATc1 was analyzed by two-dimensional gel electrophoresis and immunoblotting. Error bars denote SD.
Figure 3
Figure 3. IKKε Inhibits NFAT Activation in a Kinase-Dependent Manner
(A) 293T cells were transfected with a NFAT reporter plasmid cocktail and plasmids containing the indicated genes. NFAT activation was determined by luciferase assay. WCL, whole-cell lysate; IB, immunoblotting. (B) Reporter assay was performed as in (A), except that increasing amounts of plasmid containing WT IKKε or IKKεK38A were used. (C and D) 293T cells were transfected with EGFP-NFATc1 alone or together with IKKε or IKKεK38A. Cells were treated with ionomycin (1 μM) for 1 hr. NFATc1 subcellular localization was analyzed by fluorescence microscopy. Representative images were shown (C). Around 300 cells were counted for nuclear NFATc1 localization, and percentage was calculated (D). Error bars denote SD.
Figure 4
Figure 4. IKKε Phosphorylates NFATc1
(A) 293T cells were transfected with plasmids containing EGFP-NFATc1 and the indicated IKK. Whole-cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies. (B) In vitro kinase assay was performed using purified GST fusion proteins containing the N-terminal regulatory domain (1–319) of NFATc1. IKKε and IKKεK38A were purified from 293T cells (silver staining), while GST and GST-NFATc1-N were purified from bacteria. (C) Summary of the IKKε phosphorylation sites. (D) 293T cells were transfected with plasmids containing the indicated genes. NFATc1 was precipitated with anti-FLAG antibody and, along with whole-cell lysates (WCL), analyzed by immunoblotting. (E) 293T cells were transfected with plasmids containing the indicated genes, and whole-cell lysates were analyzed by immunoblotting. (F) NFAT activation in 293T cells by NFATc1-4A mutant was determined by luciferase assay. (G) Jurkat T cells were infected with control lentivirus or lentivirus containing WT NFATc1 or NFATc1-4A mutant. NFATc1 was precipitated and analyzed by immunoblotting (top). After stimulation, the cells were analyzed by IL-2 intracellular staining (bottom). Error bars denote SD.
Figure 5
Figure 5. T Cell Activation Upregulates the Kinase Activity of IKKε
(A) Jurkat T cells were mock treated or stimulated with PMA plus ionomycin for 1 hr. Cell extracts were analyzed by gel filtration chromatography with superose 6. Fractions were analyzed by immunoblotting with indicated antibodies. V0, void volume. Numbers at the top indicate the size of protein complex in kilodaltons. (B) Jurkat T cells were treated with antibodies against CD3 and CD28 (left panels) or PMA plus ionomycin. WCLs were analyzed by immunoblotting. WCL, whole-cell lysate. (C) Jurkat T cells expressing control shRNA or IKKε-shRNA were stimulated and analyzed by immunoblotting. P, phosphorylation. (D) Jurkat T cells stably expressing FLAG-NFATc1 were stimulated with vehicle (DMSO) or ionomycin, with or without Amlexanox (50 μM) for 2 hr. NFATc1 was affinity purified and analyzed by mass spectrometry for quantitative measurement of phosphorylation of peptides containing S117, S151, S161, and S324. (E) Jurkat T cells were stimulated and fractionated into the cytoplasmic and nuclear fractions for immunoblotting analysis. P+I, PMA plus ionomycin. (F) Jukat T cells were stimulated for 1 hr, and chromatin immunoprecipitation (ChIP) was performed using NFATc1 or NFATc2 antibodies. The enrichment of IL-2 promoter was determined by qPCR. (G) Purified mouse CD8+ T cells were stimulated with PMA and ionomycin, and WCLs were analyzed by immunoblotting. Error bars denote SD. *p < 0.05.
Figure 6
Figure 6. Loss of IKKε Elevates T Cell Antitumor Immunity and Reduces Tumor Development
(A) Gender- and age (9- to 10-month-old)-matched Ikbke+/+ and Ikbke−/− mice were inoculated with B16 MO5 cells via tail-vein injection. Mouse survival was showed by Kaplan-Meyer survival curves. (B–F) Gender- and age (10- to 12-week-old)-matched Ikbke+/+ and Ikbke−/− mice were inoculated with B16 MO5 cells via tail-vein injection. Mice were euthanized at 32 days after inoculation. Representative lungs were photographed (B) and tumors were enumerated (C). The isolated lung cells were analyzed by staining of CD8 (D), CD44 (E), and OVA tetramer (F). Error bars denote SD. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7
Figure 7. Chronic Activation of IKKε in Persistent Viral Infection and Tumor Development
Age- (12-week-old) and gender-matched mice were infected with γHV68 via intraperitoneal injection, the splenocytes were harvested, and CD8+ T cells were purified. (A) Whole-cell lysates (WCL) of CD8+ T cells were analyzed by immunoblotting with indicated antibodies. (B and C) The relative levels of phosphorylated NFATc1 (B), IKKε, and phosphorylated IKKε (C) were determined by densitometry analysis. (D) Age (12-week-old) and gender-matched mice were inoculated with B16 melanoma cells. Mice were euthanized at 28 dpi, lung CD8+ T cells were collected, and whole-cell lysates were analyzed by immunoblotting. Error bars denote SD. *p < 0.05; **p < 0.01.

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