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CTLA-4-Mediated Posttranslational Modifications Direct Cytotoxic T-Lymphocyte Differentiation

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CTLA-4-Mediated Posttranslational Modifications Direct Cytotoxic T-Lymphocyte Differentiation Cell Death and Differentiation (2017) 24, 1739–1749 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cdd CTLA-4-mediated posttranslational modifications direct cytotoxic T-lymphocyte differentiation Holger Lingel1, Josef Wissing2, Aditya Arra1, Denny Schanze3, Stefan Lienenklaus4, Frank Klawonn2,5, Mandy Pierau1, Martin Zenker3, Lothar Jänsch*,2 and Monika C Brunner-Weinzierl*,1 The blockade of inhibitory receptors such as CTLA-4 (CD152) is being used as immune-checkpoint therapy, offering a powerful strategy to restore effective immune responses against tumors. To determine signal components that are induced under the control of CTLA-4 we analyzed activated murine CD8+ T cells by quantitative proteomics. Accurate mass spectrometry revealed that CTLA-4 engagement led to central changes in the phosphorylation of proteins involved in T-cell differentiation. Beside other targets, we discovered a CTLA-4-mediated induction of the translational inhibitor programmed cell death-4 (PDCD4) as a result of FoxO1 nuclear re-localization. PDCD4 further bound a distinct set of mRNAs including Glutaminase, which points out a critical role for CTLA-4 in CD8+ T-cell metabolism. Consequently, PDCD4-deficient cytotoxic T-lymphocytes (CTLs) expressed increased amounts of otherwise repressed effector molecules and ultimately led to superior control of tumor growth in vivo. These findings reveal a novel CTLA-4-mediated pathway to attenuate CTLs and indicate the importance of post-transcriptional mechanisms in the regulation of anti-tumor immune responses. Cell Death and Differentiation (2017) 24, 1739–1749; doi:10.1038/cdd.2017.102; published online 23 June 2017 Cytotoxic T-lymphocytes (CTLs) are the effector cells of the mechanisms how CTLA-4 affects CD8+ T-cell differentiation. adaptive immune system that exclusively recognize MHC-I We confirmed that CTLA-4-mediated mechanisms abrogated presented antigens, thus having a central role in the the phosphorylation of the AP-1 family transcription factor Fos- recognition and clearance of malignant cells.1,2 A complex related antigen 2 (Fra-2) and led to a nuclear re-localization of interplay of stimulatory and inhibitory receptor-ligand interac- the central transcription factor FoxO1, which caused a strong tions, as well as inflammatory cytokines orchestrate the induction of the translational inhibitor programmed cell death-4 activation of CD8+ T cells and their differentiation into CTLs.3 (PDCD4). Strikingly, PDCD4-deficient CTLs showed enhanced To control the magnitude of T-cell responses, the inhibitory production of the otherwise repressed effector molecule IFN-γ surface receptor CTLA-4 has been identified as a primary and loss of PDCD4 ultimately resulted in superior control of attenuator of T cells.4,5 Furthermore, CTLA-4 is overex- tumor growth in vivo. This novel pathway delineates how pressed in exhausted CTLs during chronic diseases alongside CTLA-4 is able to regulate CD8+ T-cell differentiation and the other inhibitory receptors such as PD-1 (ref. 6). Its blockade identified mechanisms further provide new strategies to during immune-checkpoint therapy promisingly restores anti- improve anti-tumor immune responses. tumor immunity in mice and men.7–9 CTLA-4 has already been + 10,11 shown to be involved in the regulation of CD8 T cells; Results however, the mechanisms by which CTLA-4 controls CTL responses still remain incompletely understood. Because of CTLA-4 modulates central CD8+ T-cell processes. To the temporary delayed increase of receptor expression, identify novel proteins and signaling mechanisms exclusively CTLA-4 strongly impacts on highly activated cells with already targeted by CTLA-4 in CD8+ T cells, we performed a comp- established transcription profiles.12 We therefore hypothesize arative mass spectrometry analysis of phosphorylated proteins that CTLA-4 could exploit posttranscriptional or -translational from cells that were differentiated in vitro with or without CTLA-4 mechanisms to modulate CD8+ T-cell differentiation. engagement concomitant with α CD3 and α CD28 activation In this study we characterized the phosphoproteome (Supplementary Figure S1a upper).4 To control the effective- response that resulted from CTLA-4 engagement in activated ness of CTLA-4-mediated signals we monitored CD8+ T cells by CD8+ T cells by using iTRAQ quantitative mass spectrometry. flow cytometry. The cells showed equal activation on day 1 as The analysis identified previously unknown targets and controlled by proliferation, CD62L downregulation, CD44 and 1Department of Experimental Pediatrics, University Hospital, Health Campus Immunology, Infectiology and Inflammation, Otto-von-Guericke-University, Magdeburg 39120, Germany; 2Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig 38124, Germany; 3Department of Human Genetics, University Hospital, Otto-von-Guericke-University, Magdeburg 39120, Germany; 4Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig 38124, Germany and 5Department of Computer Science, Ostfalia University of Applied Sciences, Wolfenbüttel 38302, Germany *Corresponding author: MC Brunner-Weinzierl, Department of Experimental Pediatrics, University Hospital, Otto-von-Guericke-University, Leipziger Strasse 44, Magdeburg 39120, Germany. Tel: +49 391 6724003; Fax +49 391 6724202; E-mail: [email protected] or L Jänsch, Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, Braunschweig 38124, Germany. Tel: +49 531 61813030; Fax: +49 531 61817099; Email: [email protected] Received 28.9.16; revised 29.3.17; accepted 12.5.17; Edited by S Martin; published online 23.6.17 CTLA-4 regulates translation H Lingel et al 1740 T-bet expression; however, CD8+ T cells that received a CTLA-4 proved a strong impact of CTLA-4-mediated effects.13 Interest- stimulus had 55% less IFN-γ producers on day 2 and less than ingly, CTLA-4-triggered CTLs showed a pronounced re-exp- one-fifth on day 3 (Supplementary Figures S1b-d), which ression of CD62L on day 2 (Supplementary Figure S1d). E1 E2 E1 E2 E1 E2 S775+S778 Arhgap1 S51 CDK5RAP2 S485 S777+S778 RalGAPA1 S796 PCM-1 S1228 S1228 SRRM2 S1343 GAPVD1 S902 CENPC1 S1631 Niban S755 KNL2 T516 S2084 SNIP1 S18 MAP4 S517 T1836 CC2D1B S520 RPRC1 S460 ZNF265 S120 CARMA1 S930 CLASP2 S376 S574 SLAMF7 S328 NEK1 S997 SRRM1 S795+S797 SLY S26+27 ALDOA S46 SR-A1 S676+S682 S293 BAZ1B S1464 PAG S46 PININ S66 S295 STMN1 S100 CD43 S285 S1928 ELYS U2AF2 S79 PKD2 S211 S1928+S1931 RNH2A S299 NFAT1 S860 TXLNA S523 eIF3B S111 PKC-η S675 SLP-76 S210 RFC1 S365 VAV-1 Y826 JAML S370 S774 S94 RIC8A S435 DDX54 PDCD4 NCoA5 S381 S457 SIK3 T411 AKNA S302 BRCA1 S686 DEAF1 S212 SSRP1 S657 WRNIP1 S153 S58 S425 CDK12 S332+S333 RANBP3 SH3BP2 Trap150 S238+S243 S148 GRAMD1B S274 S95 NUP93 S180 TEX2 S195 γ S982 S19 HP1- S97 LRBA NUCKS S99 MIA3 S1566 S181 Ku70 S518 Treacle S1303 NHE1 S707 TOP2A S1211 SFRS7 S212 DEF6 S597 PTPN12 S434 hnRNPA1 S6 TNKS1BP1 S1657 PATL1 T178+S184 FAM65B S46 S120 WDR26 S101 Fra-2 S200 -1 1 ZC3H18 S530 R R E R R K P S K E D E P -7 -6 -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 +7 -7 -6 -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 +7 -7 -6 -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 +7 T-cell activation / Lyn cytokine production SHP-1 PAG SLP-76 SOCS1 CARMA1 FoxP3 VAV-1 SLY PKC-θPKC P38a RNA NFAT1 processing hnRNPK / Niban IL-4Il4 RPRC1 Fra-2 mRNA FasL STMN1 SRRM1 metabolic MAP4 ElysELYS PTPN12 SFRS7 process ZNF265 AICDA DDX58 IRF-4 SRRM2 RANBP3 DEF6 RARA hnRNPA1 IL-2Il2 TAL1 PDCD4 FBXO32 Smarca4SMARCA4 Jun eIF4A NPM1Npm1 SMARCA2 Regulation of Ku70 microtubule- eIF3B NHE1 based process UBC BRCA1 WRNIP1 BAZ1B NANOG SSRP1 AldoaALDOA E2A Iqcb1IQCB1 RalGAPA1 Treacle RIC8A LaminALmna Arrb1ARRB1 CDK12Cdk12 Oct4 CUL3 NUP93 PDK1 DNA replication NPHP4 Trap150 affected by CTLA-4 predicted interlink TOP2ATop2a Cell Death and Differentiation CTLA-4 regulates translation H Lingel et al 1741 After 48 h of stimulation, which marked the time-point of regulation is attributed to posttranslational effects, Fra-2 maximal CTLA-4 expression (Supplementary Figure S2a), the (Fosl2) mRNA was quantified and showed similar amounts phosphorylated proteins were isolated, digested and the in all stimulated samples (Figure 2b). Among the kinases that resulting phosphopeptides were measured for their abun- are able to phosphorylate Fra-2 we tested the involvement of dance in two independent biological replicates. These PKA. The application of the specific PKA inhibitor 14-22 amide analyses led to the detection of 89 phosphopeptides belong- led to a more than 30% decreased formation of the slow ing to 74 proteins that were differentially regulated upon (upper) and fast (lower) migrating Fra-2 phosphorylation forms CTLA-4 engagement. Sixty-three of 89 peptides showed in control cells, whereas increased PKA activity due to enhanced phosphorylated residues while 26 peptides were incubation with the cAMP elevator Forskolin specifically less phosphorylated. Among these proteins, PKC-η and intensified those phosphorylation forms more than six times VAV-1 have already been connected to CTLA-4.14,15 As in CTLA-4-triggered cells (Figure 2c). targets with multiple affected phosphopeptides, NUCKS and Collectively, these findings substantiate the significance of PDCD4 were found to be the most upregulated ones, whereas the mass spectrometry dataset for the identification of Fra-2 was the strongest dephosphorylated protein (Figure 1a CTLA-4-mediated signaling
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