LZTFL1 Upregulated by All-Trans Retinoic Acid during CD4 + Activation Enhances IL-5 Production

This information is current as Hong Jiang, Kanyarat Promchan, Bor-Ruei Lin, Stephen of September 25, 2021. Lockett, De Chen, Heather Marshall, Yunden Badralmaa and Ven Natarajan J Immunol 2016; 196:1081-1090; Prepublished online 23 December 2015;

doi: 10.4049/jimmunol.1500719 Downloaded from http://www.jimmunol.org/content/196/3/1081

Supplementary http://www.jimmunol.org/content/suppl/2015/12/23/jimmunol.150071 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 64 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/196/3/1081.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

LZTFL1 Upregulated by All-Trans Retinoic Acid during CD4+ T Cell Activation Enhances IL-5 Production

Hong Jiang,*,1 Kanyarat Promchan,* Bor-Ruei Lin,* Stephen Lockett,† De Chen,† Heather Marshall,* Yunden Badralmaa,* and Ven Natarajan*

Retinoic acids, which are metabolites of vitamin A, have been shown to be involved in multiple T cell effector responses through their binding to the retinoic acid receptor, a ligand-activated factor. Because the molecular mechanism of regulation by retinoic acid is still not fully uncovered, we investigated the gene expression profile of all-trans retinoic acid (ATRA)–treated human CD4+ T cells. Leucine zipper -like 1 (LZTFL1) was upregulated by ATRA in a dose- and time-dependent manner. The expression of LZTFL1 depended on both ATRA and TCR signaling. LZTFL1 accumulated in the plasma membrane compartment of human CD4+ T cells, and, during immunological synapse formation, it transiently redistributed to the T cell and

APC contact zone, indicating its role in T cell activation. Live-cell imaging demonstrates that at the initial stage of immunological Downloaded from synapse formation, LZTFL1 is concentrated at the APC contact site, and, during later stages, it relocates to the distal pole. Knockdown of LZTFL1 reduced the basal- and ATRA-induced levels of IL-5 in CD4+ T cells, and overexpression of LZTFL1 enhanced the TCR-mediated NFAT signaling, suggesting that LZTFL1 is an important regulator of ATRA-induced T cell re- sponse. Together, these data indicate that LZTFL1 modulates T cell activation and IL-5 levels. The Journal of Immunology, 2016, 196: 1081–1090. http://www.jimmunol.org/ etinoic acids (RAs), especially all-trans RA (ATRA), the mice given a vitamin A or RA supplement showed decreased active metabolite of vitamin A, are known to regulate production of the Th1 cytokine IFN-g and increased production of R cell differentiation, proliferation, and in a va- Th2 cytokines IL-4, -IL-5, and -IL-13 (15). Even though the riety of cell types through their binding to the RA receptor (RAR), mechanism of RAs’ impact on Th2 cell development is still not a ligand-activated transcription factor (1, 2). Vitamin A and RAs fully understood, the direct and indirect effects of RAs have been influence T cell function in many ways, including peripheral T cell suggested. By inhibiting IL-12 production in activated macro- differentiation, gut-homing capacity, and effector T cell activity phages, RA pretreatment of macrophages reduced IFN-g pro- (3–8). RAs are known to favor Th2 cell development (8–14). duction and increased IL-4 production in Ag-primed CD4+ T cells Vitamin A deficiency causes immune dysfunction, including IFN-g (18), and stimulating Ab-primed human PBMCs and purified by guest on September 25, 2021 overproduction and impaired Ab responses, which is the result of T cells with RAs in vitro directly increased the mRNA and protein excess Th1 and insufficient Th2 function (15, 16). Vitamin A– levels of IL-4, IL-5, and IL-13, and decreased the levels of IFN-g, deficient mice showed reduced Th2 cytokine production and bone IL-2, IL-12, and TNF-a (8, 11). marrow eosinophilia with parasitic helminth infection (17), and The differentiation of naive CD4+ T cells into Th2 cells is in- duced by APCs and also requires TCR-mediated signaling (19, 20). In vivo, gut dendritic cells and macrophages process vitamin *Laboratory of Molecular Cell Biology, Leidos Biomedical Research, Frederick A to generate RAs and present them to T cells during Ag pre- National Laboratory for Cancer Research, Frederick, MD 21702; and †Optical Mi- croscopy and Analysis Laboratory, Leidos Biomedical Research, Frederick National sentation and T cell activation (16, 21), indicating the important Laboratory for Cancer Research, Frederick, MD 21702 role of RAs in T cell activation and differentiation. Even though 1Current address: Graduate Center for Toxicology, Markey Cancer Center, University little is known about the mechanism of RAs’ regulation in this of Kentucky, Lexington, KY. process, their influence on T cell activation is suggested. T cell ORCID: 0000-0002-4502-233X (H.J.). activation markers CD69 and CD38 are upregulated by ATRA, Received for publication March 26, 2015. Accepted for publication November 17, indicating the engagement of RA-RAR signaling in T cell acti- 2015. vation (10). Moreover, RAs also upregulate transcriptional factors This work was supported in whole or in part by National Cancer Institute, National for Th2 differentiation, including cMAF, GATA-3, and STAT-6, Institutes of Health Contract HHSN261200800001E. This work was also supported by the National Institute of Allergy and Infectious Diseases. with a concomitant downregulation of the Th1 factor T-bet (11). All these observations indicate that RA-RAR signaling is engaged The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, com- in the induction of Th2 differentiation by RA. mercial products, or organizations imply endorsement by the U.S. Government. Leucine zipper transcription factor-like 1 (LZTFL1) was first Address correspondence and reprint requests to Dr. Ven Natarajan, Building 550/ identified as a tumor suppressor. The gene encoding LZTFL1 is Room 120, Leidos Biomedical Research, Frederick National Laboratory for Cancer located on human chromosome 3p21.3 and is found to be deleted in Research, Frederick, MD 21702. E-mail address: [email protected] several types of cancer (22). LZTFL1 overexpression in cervical The online version of this article contains supplemental material. cancer cell line HeLa cells inhibited anchorage-independent cell Abbreviations used in this article: ATRA, all-trans RA; BBS, Bardet–Biedl syn- drome; cSMAC, central supramolecular activation cluster; DIG, digoxigenin; DP, growth and cell migration in vitro and repressed tumor growth distal pole; IS, immunological synapse; LZTFL1, leucine-zipper transcription in vivo (23). Recently, a deletion mutant of LZTFL1 was also factor–like 1; RA, retinoic acid; RAR, RA receptor; SEE, staphylococcal entero- found in a family with Bardet–Biedl syndrome (BBS), which toxin E; siRNA, small interfering RNA. suggests that LZTFL1 is involved in BBS (24). Seo et al. (25) Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 further showed that LZTFL1 interacts with a BBS protein com- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500719 1082 T CELL ACTIVATION–INDUCED LZTFL1 ENHANCES IL-5 PRODUCTION plex, known as the BBSome, and regulates its primary ciliary CA) and the following primers: LZTFL1, forward, 59- GGCCTAAATGAG- trafficking. A role for LZTFL1 in hedgehog signaling is also CACCATCA-39 and reverse, 59-ATCCACTTCTCAGCTTGTGC-39; pre- suggested (24, 25). developed FAM- and TAMRA-labeled internal oligonucleotide probes and + primers for IL-5 and GAPDH (Life Technologies). The quantity of LZTFL1 In our effort to understand the role that ATRA plays in CD4 and IL-5 mRNAs was normalized by the levels of GAPDH mRNA. T cell development, we have found that both LZTFL1 mRNA and protein production are upregulated by ATRA treatment in human Western blot for LZTFL1 + + CD4 T cells. During CD4 T cell activation in contact with APC, Whole-cell proteins were extracted using M-PER mammalian protein LZTFL1 transiently localizes to the immunological synapse (IS). extraction reagent with protease inhibitor cocktails (Thermo Scientific, Overexpression of LZTFL1 in CD4+ T cells further enhanced the Waltham, MA). Protein extracts were electrophoresed in a 4–12% gradient NuPAGE Bis Tris Gel (Life Technologies), transferred to polyvinylidene T cell activation signal, as indicated by increased NFAT activity. difluoride membrane, and detected with fluorophore-labeled secondary Ab Moreover, LZTFL1 knockdown decreased Th2 cytokine produc- using Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE). tion, especially IL-5 mRNA and protein production, and further suppressed ATRA-induced IL-5 production. Our data suggest that Northern blotting LZTFL1 is involved in ATRA-regulated Th2 cytokine production, Total RNA from activated CD4+ T cells treated with DMSO or ATRA was possibly through LZTFL1-induced TCR–NFAT signaling. purified using RNAqueous-4PCR Kit (Life Technologies), according to the manufacturer’s protocol, and treated with DNase to remove residual contaminations with DNA. Digoxigenin (DIG)-labeled probes to LZTFL1 Materials and Methods coding region were generated using the DIG Northern Starter Kit (Roche Reagents Diagnostics, Indianapolis, IN). For Northern blot analysis, 20 mg total

RNA was separated on a denaturing formaldehyde 1% agarose gel. The Downloaded from ATRA, actinomycin D, and latrunculin B were purchased from Sigma- RNA was transferred onto a positively charged nylon filter by capillary Aldrich (St. Louis, MO). The following Abs were used: mouse anti- transfer and hybridized with DIG-labeled DNA probes directed against LZTFL1 and rabbit anti–TCR-b (Santa Cruz Biotechnology, Santa Cruz, human LZTFL1 and b-actin, according to the manufacturer’s protocol CA); mouse anti-Flag M2 (Sigma-Aldrich, St. Louis, MO); and mouse (DIG Northern Starter Kit; Roche Diagnostics). The hybridization signal anti-GAPDH (Abcam, Cambridge, MA). was detected by chemiluminescence with an anti-DIG alkaline phospha- Plasmids tase conjugate and CDP-Star. http://www.jimmunol.org/ NFAT and NF-kB luciferase reporter plasmids were described earlier (26). Nuclear run-on assay pRL-TK and pHTN HaloTag CMV-neo plasmids were purchased from A nuclear run-on assay was performed following the method reported Promega (Madison, WI). The vector encoding Myc-DDK–tagged human previously with some modification (28). Briefly, primed CD4+ T cells were LZTFL1 (accession number NM_020347) was purchased from OriGene treated with DMSO or ATRA for 24 h. Cells were washed with cold PBS (Rockville, MD). LZTFL1 was cloned in-frame downstream of the and lysed in lysis buffer (10 mm Tris-HCl [pH 7.4], 3 mm MgCl2,10mm HaloTag sequence of pHTN HaloTag plasmid, and a fragment contain- NaCl, and 0.5% Nonidet P-40) for 5 min on ice. Nuclei were pelleted and ing HaloTag-LZTFL1 from the resulting plasmid was cloned into pENTR washed once with lysis buffer without Nonidet P-40 and resuspended in vector (Life Technologies, Grand Island, NY) to obtain pENTRHalo- freezing buffer (50 mM Tris-HCl [pH 8.3], 40% glycerol, 5 mM MgCl2, LZTFL1. A termination codon after the HaloTag coding sequence was in- and 0.1 mM EDTA) at 280˚C until use. For in vitro transcription, isolated troduced to obtain pENTRHalo-LZSTOP. Lentiviral vectors containing ei- nuclei were incubated in the transcription buffer (100 mM KCl; 10 mM ther Halo-LZTFL1 or Halo-LZSTOP were generated by transfecting HEK- by guest on September 25, 2021 Tris-HCl [pH 8.0]; 2.5 mM MgCl2; 2 mM DTT; 2 mM each of ATP, GTP, 293 cells, as suggested by manufacturer (Life Technologies). and CTP; 100 mM sucrose; and 10% glycerol) in the presence of UTP or biotin-16-UTP (Roche Molecular Biochemicals) for 30 min at 29˚C. In vitro transcription was stopped by DNase I treatment at 37˚C for 20 min. Synthe- Primary human CD4+ T cells were isolated from PBMCs of healthy do- sized RNAs were extracted using Master Pure RNA purification kit (Epicentre nors using Dynabeads Untouched Human CD4 T cells isolation kit (Life Biotechnology, Madison, WI). Biotin-labeled RNA was isolated with M-280 Technologies) following the manufacturer’s instruction. Cells were cul- streptavidin magnetic beads (Life Technologies), according to the manufac- tured in RPMI 1640 supplemented with 10% dialyzed FBS, 100 U/ml turer’s recommendations, and used for reverse transcription and real-time PCR. penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 50 U/ml IL-2 (PeproTech, Rocky Hill, NJ). To activate CD4+ T cells, cells were Confocal microscopy primed with anti-CD3 and anti-CD28 Abs using Dynabeads CD3/CD28 For LZTFL1 staining, cells were allowed to rest in 0.1% poly-l-lysine– T cell expander (Life Technologies). Jurkat E6.1 cell line, a CD4+ human coated 8-well chamber slide for 5 min before a short spin, fixed with 4% T cell lymphoblast-like cell line, was cultured, as described (27). D10.G4.1 formaldehyde (Thermo Scientific) for 30 min at room temperature, and cell line, a mouse Th2 lymphoblast, was purchased from American Type permeabilized for 5 min at room temperature with 0.2% Triton X-100 in Culture Collection and cultured in RPMI 1640 medium supplemented with PBS. Cells were then stained with indicated primary Ab and visualized by 10% T-STIM with Con A (BD Biosciences, Franklin Lakes, NJ), 10% Alexa-Fluor 488– or 568–labeled secondary Ab. Lipid raft was stained FBS, 0.05 mM 2-ME, and 10 pg/ml mouse IL-1a (R&D Systems, Min- with Alexa-Fluor 594–labeled cholera toxin b. F-actin was stained with neapolis, MN). Jurkat T cells stably expressing Halo-LZTFL1 (Halo- Alexa-Fluor 594 phalloidin (Life Technologies). Coverslips were mounted LZTFL1) or Halo-LZSTOP were generated by infecting Jurkat cells onto the slides with Prolong Gold Antifade reagent with DAPI (Life with lentivirus Halo-LZTFL1 or Halo-LZSTOP, and growing the cells Technologies) to stain the nucleus, and fluorescent images were captured positive for Halo-Tag expression by FACS analysis. by confocal microscope (FV-1000; Olympus). For latrunculin B treatment, Halo-LZTFL1 Jurkat T cells were plated on For conjugation analysis, Raji B cells were stained with CellTracker poly-l-lysine–coated chamber slides and immediately treated with 1 mM Violet BMQC (Life Technologies) and pulsed with or without 5 mg/ml latrunculin B (Sigma-Aldrich) for 30 min at 37˚C and 5% CO . Cells were 2 staphylococcal enterotoxin E (SEE; Toxin Technology, Sarasota, FL) for then fixed and incubated with phalloidin-conjugated Alexa 488 at room 30 min at 37˚C. Then Raji B cells were mixed with an equal number of temperature for 1 h and then with anti-Halo Ab (Promega) at 4˚C for Jurkat T cells and plated onto poly-l-lysine–precoated slide. After brief overnight. After washing, cells were incubated with goat anti-rabbit at centrifugation, cells were incubated for 1.5, 5, and 15 min at 37˚C; fixed room temperature for 1 h. The ratio of fluorescent intensities of membrane- for 30 min at room temperature with 4% formaldehyde in PBS; per- localized LZTFL1 to that of total cellular LZTFL1 was calculated by meabilized for 5 min at room temperature with 0.2% Triton X-100 in PBS; Matlab software (Mathwork) after imaging with a confocal microscope. stained with indicated primary Ab; and visualized by Alexa-Fluor 568– or Reverse transcription and real-time PCR Alexa-Fluor 488–labeled secondary Ab. Coverslips were mounted onto the slides with Prolong Gold Antifade reagent (Life Technologies), and fluo- Total cellular RNA was extracted using RNAqueous-4PCR Kit (Life rescent images were captured by confocal microscope (FV-1000; Olympus). Technologies). To quantitatively analyze gene expression, 200 ng total Conjugates formed between Jurkat T cells and Raji B cells were scored in RNA was used to synthesize the first-strand DNA with random primers 10 fields chosen at random at indicated time points. The percentage of (SuperScript II Reverse Transcriptase; Life Technologies). The real-time conjugates was calculated as T cells forming interface with Raji B cells to PCR was performed by using SYBR Green Master Mix (Qiagen, Valencia, total T cells in the field. The percentage of conjugates with accumulation of The Journal of Immunology 1083

LZTFL1 at the T cell–B cell contact site, IS and distal pole (DP), was also Statistical analysis quantified. Statistical analysis was assessed by Student t test. Results are shown as Live-cell imaging for LZTFL1 localization at the IS mean 6 SD. A p value ,0.05 was considered statistically significant. Halo-LZTFL1– or Halo-LZSTOP–expressing Jurkat cells were stained with TMR ligand (Promega) at 37˚C and 5% CO2 for 40 min and washed Results with complete media four times, followed by incubation at 37˚C and 5% ATRA upregulates LZTFL1 in human CD4+ T cells CO2 for 1 h. After washing cells once with complete media, cells were then incubated with 5 mg/ml SEE superantigen-loaded Raji B cells prestained RAs have been shown to influence the function of T cells, with Hoechst as APCs on poly-L-lysine–coated glass-bottom dishes. Cells although their effects on T cells are not fully understood. To assess were maintained at 37˚C and 5% CO2 during the entire period of imaging. the effect of ATRA on gene expressions in CD4+ T cells, we treated Halo-tagged T cell images were acquired by fast wide field imaging using + an Olympus TIRF 3 system with a 360 oil objective (1.49 NA), under non- anti-CD3/CD28 Ab-primed CD4 T cells from healthy donors + TIRF mode. The wide field illumination was achieved by focusing the laser with or without ATRA, and we isolated RNAs from the CD4 beams into the back focal plane of the objective. Samples labeled with T cells and used them for gene array analysis (30). LZTFL1 is one HaloTag and Hoechst for nucleus were excited with 561 nm and 405 nm of the genes that are most upregulated by ATRA treatment (http:// lasers, respectively, and the fluorescence signals were extracted with filters LF561-A-OMF for red (Semrock, Rochester, NY) and LF405-A-OMF for www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE74280). To blue and then recorded with an EMCCD camera (Hamamatsu). Images of confirm this result, LZTFL1 mRNA levels, in response to ATRA the two color channels were acquired in a time course of 10-s interval treatment, were assessed using quantitative real-time PCR, fol- without interruption. The acquired images were first processed to remove lowing reverse transcription. Consistent with the gene array result, background and then combined into a video with home Matlab codes on a LZTFL1 mRNA levels were dramatically upregulated by ATRA Downloaded from Matlab programming environment (Mathwork). treatment in cells from all donors tested (Fig. 1A). Northern Transfection blotting also indicated the elevation of the LZTFL1 transcript upon For luciferase assay, Jurkat T cells were washed twice with serum-free ATRA treatment (Fig. 1B). The induction of LZTFL1 expression RPMI 1640 and 1.0 3 107 cells were resuspended in 250 ml serum-free was both time and dose dependent. ATRA upregulated LZTFL1 RPMI 1640 containing LZTFL1 expression plasmid, NFAT, and NF-kB RNA expression at concentrations as low as 1 nM, and, at phys- reporter luciferase plasmid, and pRL-TK (Renilla luciferase control plas- iological concentrations of 10 nM, it could induce LZTFL1 RNA http://www.jimmunol.org/ mid for normalization). Cells were electroporated at 750V and 1000 mFin expression .100-fold (Fig. 1C). As early as 4 h after ATRA a 0.4-cm–gap cuvette using Gene Pulser (Bio-Rad Laboratories, Hercules, CA) and allowed to recover for 24 h before stimulation. To transfect CD4+ treatment at concentration of 1 mM, LZTFL1 mRNA expression T cells from healthy donors and D10.G4.1 cells, cells were suspended in increased by almost 3-fold, and, by 72 h of treatment, the stimu- the buffer for T cells and electroporated with Neon Electroporation System lation of LZTFL1 expression reached the maximum level (Fig. 1D). (Life Technologies). The effect of ATRA on LZTFL1 protein expression was also ana- RNA synthesis and transfection lyzed by Western blotting. As shown in Fig. 1E, the basal expres- sion of LZTFL1 is barely detectable in primary human CD4+ A plasmid DNA with N-terminal flag-tagged LZTFL1 cDNA cloned down- stream of T7 promoter was linearized and used for in vitro synthesis of T cells. In response to stimulation with ATRA, LZTFL1 level sig- LZTFL1 mRNA with the T7 mMessage mMachine Kit (Ambion, Austin, nificantly increased after 8 h of treatment and reached maximum by by guest on September 25, 2021 TX). CD4+ T cells were isolated from healthy donor, activated, and ex- 48 h, which is parallel to the finding of induced LZTFL1 mRNA panded 10- to 20-fold over 1 wk using T cell expander beads (Life production. Alternative mechanisms could contribute to elevated Technologies) in presence of IL-2 and dialyzed FCS. LZTFL1 mRNA levels during ATRA treatment, including tran- For mRNA transfection, 30–40 3 106 expanded CD4 T cells were mixed with 40 mg flag-tagged LZTFL1 mRNA and electroporated, as scription initiation rate and RNA degradation rate. To evaluate described by Li et al. (29). Electroporated cells were treated with DMSO whether the ATRA-induced increase in LZTFL1 mRNA levels or ATRA for 72 h, and cellular RNA and proteins were analyzed. resulted from increased mRNA stability, we incubated the DMSO- or ATRA-treated CD4+ T cells with actinomycin D (5 mg/ml) for RNA interference 10 h. Treatment with actinomycin D inhibits RNA synthesis by Small interfering RNA (siRNA) SMARTpools targeting human and mouse blocking the function of the transcriptional machinery. As shown LZTFL1 were from predesigned siGenome collection from Dharmacon in Fig. 1F, ATRA treatment did not change the LZTFL1 mRNA (Lafayette, CO). siGenome Non-Targeting siRNA was used as control. For siRNA transfection, 200 pmol siRNA was mixed with 2.0 3 106 cells and turnover rate significantly. Next, we analyzed the effect of ATRA on electroporated with either Nuleofector (Lonza) for Jurkat cells or Neon for LZTFL1 transcription initiation using the nuclear run-on assay. primary CD4+ T cells and D10.G4.1 cells, according to the manufacturer’s Newly synthesized RNAs were labeled with biotin, captured by instruction. Cells were allowed to recover for 24 h before stimulation. magnetic beads, and analyzed by RT-PCR, as described in Mate- Luciferase reporter assays rials and Methods. This semiquantitative technique revealed that newly synthesized LZTFL1 mRNA was markedly increased in Cells were transfected and stimulated with or without anti-CD3/CD28– ATRA-treated CD4+ T cells (Fig. 1G). These data clearly indicate coated beads (Life Technologies) for 10 h. Luciferase activities were an- alyzed using the Promega dual luciferase assay system and measured in a that ATRA treatment increased LZTFL1 transcription. luminometer. Renilla luciferase activities were used to normalize trans- fection efficiency. Normalized luciferase activities were determined in LZTFL1 induction by ATRA is dependent on TCR signaling triplicate and expressed as fold increase relative to the basal activity ob- ATRA has no effect on LZTFL1 expression in resting CD4+ tained in unstimulated mock-transfected cells. T cells. Upon T cell activation with anti-CD3 and CD28 Abs, the Cytokine ELISA expression of LZTFL1 increased ∼100-fold in response to ATRA Human Th1/Th2/Th17 Cytokines Multi-Analyte ELISArray Kits treatment. PMA and PHA treatment, together with ATRA, also (SABiosciences, Frederick, MD) were used to examine the following cy- dramatically increased LZTFL1 expression (Fig. 1H), whereas, tokines: IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-g, TNF-a, without ATRA, T cell activation alone had no effect. These results G-CSF, and TGF-b1. The IL-4 and IL-5 ELISA was obtained from indicate that both ATRA and TCR signaling are required for eBioscience (San Diego, CA). All of the ELISAs and multiplex assays were performed according to the manufacturer’s instructions. The results LZTFL1 expression, and TCR signaling is essential for ATRA’s are expressed as fold increase relative to the basal activity obtained in effect on LZTFL1 upregulation. Together with the result that unstimulated mock-transfected cells. All assays were run in triplicates. ATRA upregulates LZTFL1 expression at physiological concen- 1084 T CELL ACTIVATION–INDUCED LZTFL1 ENHANCES IL-5 PRODUCTION Downloaded from http://www.jimmunol.org/

FIGURE 1. ATRA induces LZTFL1 expression in human primary CD4+ T cells. (A) Human CD4+ T cells isolated from healthy donors were primed with anti-CD3 and anti-CD28 Abs (anti-CD3/CD28) and treated with DMSO or 1 mM ATRA for 3 d. RNAs were extracted from these cells, and LZTFL1 RNA was quantitated by reverse transcription, followed by real-time PCR. LZTFL1 RNA expression was normalized to GAPDH RNA expression, and the by guest on September 25, 2021 relative RNA fold changes compared with those from DMSO treatment were plotted (mean 6 SD). (B) LZTFL1 and actin mRNAs (indicated by arrows) were detected by Northern blotting. Original magnification 360. (C and D) LZTFL1 RNA expression levels in response to different concentrations of ATRA for 72 h (C), and at 1 mM ATRA for various lengths of time (D), were analyzed. (E) LZTFL1 and GAPDH proteins were detected by Western blotting. The vertical line indicates where parts of the image were joined. (F) CD4+ T cells treated with DMSO or ATRA for 24 h were incubated with or without actinomycin D (5 mg/ml) for 10 h, and the levels of LZTFL1 and GAPDH RNAs were analyzed by real-time PCR. (G) Nuclear run-on assay was used to analyze the effect of ATRA on LZTFL1 transcription initiation. CD4+ T cells were treated with DMSO or ATRA for 24 h. Nuclei were isolated, and biotinylated transcripts were synthesized in vitro, captured, and analyzed by real-time PCR. (H) The effect of different T cell activators on LZTFL1 RNA expression was analyzed. Results are representative of three experiments with cells from three different donors. *p , 0.05, **p , 0.01. trations (Fig. 1D), our data suggest a possible function of LZTFL1 expected, latrunculin B–treated cells had very low levels of F-actin, in ATRA-induced T cell response. causing LZTFL1 to lose most of its membrane localization and relocate to the cytoplasmic compartment (Fig. 2C). This result in- LZTFL1 localizes to the T cell membrane in an F-actin– dicates that LZTFL1 depends on F-actin for membrane localization. dependent manner To investigate the function of LZTFL1, we first analyzed its lo- LZTFL1 is transiently recruited to the T cell–APC contact site calization in CD4+ T cells. Cells expressing Flag-tagged LZTFL1 during T cell activation were fixed for immunological staining. We detected the enrichment Next, we performed immunoprecipitation, followed by mass spec- of LZTFL1 in the CD4+ T cell membrane compartment, with some trometry, to identify LZTFL1-interacting proteins. HEK-293 cells distribution in the cytoplasm (Fig. 2A). To further confirm the were used. Our results showed that LZTFL1 interacts with BBS membrane localization of LZTFL1, cholera toxin B was used to proteins BBS-2 and BBS-7. Similarly, in recent publications, stain the lipid raft. The staining for LZTFL1 and the lipid raft LZTFL1 is shown to be involved in BBS (24, 25). A subset of showed some overlap between the two, even though most of the LZTFL1 interacts with the BBS-9 through its C-terminal half. LZTFL1 was located outside the lipid raft. We also stained en- Even though there is no enrichment of LZTFL1 in cilia or basal dogenous LZTFL1 in Jurkat T cells using anti-LZTFL1 Ab, and the bodies, it influenced BBSome trafficking in primary cilium and results confirmed membrane localization of LZTFL1 (Fig. 2B). In a hedgehog signaling (25). Primary cilium is a specialized cell previous publication, it was suggested that LZTFL1 binds to actin surface projection in almost all vertebrate cells. It plays important in vitro (23). To verify the effect of actin on LZTFL1 membrane roles in sight, smell, mechanosensation, and intercellular signaling distribution, Jurkat cells stably expressing Halo-tagged LZTFL1 (31). Lymphocytes are among the very few types of cells that do were treated with latrunculin B to disturb the polymerization of not form cilium. Instead, during T cell activation, lymphocytes actin. Cells were fixed and stained for LZTFL1 and F-actin. As and APCs form a highly organized interface in their interaction The Journal of Immunology 1085 Downloaded from http://www.jimmunol.org/

FIGURE 2. LZTFL1 localizes on the cell membrane in an F-actin–dependent manner. (A) Human CD4+ T cells isolated from healthy donors were transiently transfected with LZTFL1-Flag expression DNA. Cells were fixed and stained with anti-Flag Ab for LZTFL1-Flag (green). Cholera toxin B and DAPI were used to stain the cytoplasmic membrane marker GM1 ganglioside (red) and the nucleus (blue), respectively, and the staining was analyzed by confocal microscopy. Original magnification 3100. (B) Fluorescent images of Jurkat T cells stained with anti-LZTFL1 Ab (green) and DAPI (blue). Original magnification 360. (C) Fluorescent images of Halo-LZTFL1–expressing Jurkat T cells treated with or without latrunculin B (1 mM) for 30 min. by guest on September 25, 2021 Cells were stained with phalloidin for F-actin (green), anti-halo Ab for LZTFL1 (red), and DAPI for the nucleus (blue). Arrows indicate LZTFL1 relocates to cytoplasmic compartment. Original magnification 360. area: IS (32, 33). The IS is considered to be a homolog of primary Similar to fixed cells, live-cell imaging of Jurkat T cells stably cilium based on the common features between IS and primary expressing a halo-tagged LZTFL1 also showed the movement cilium (34, 35). The IS plays an important role in lymphocyte of LZTFL1 toward the site of the IS as a result of SEE-pulsed activation and allows for the polarized delivery of cytokines or Raji B cells (Fig. 3D, 3E). Jurkat T cells stably expressing lytic granules to target cells. To investigate whether LZTFL1 has a Halo-LZTFL1 were imaged during migration and interaction with role in IS formation or T cell activation, superantigen-pulsed Raji SEE superantigen-loaded Raji B cells. Before contact with the B cells were used. Raji B cells were labeled with CellTracker APC, Halo-LZTFL1 in the motile T cells was evenly distributed in Violet BMQC to distinguish them from T cells. Raji B cells pulsed the cytoplasm and cell membrane. After contact with the APC, with superantigen SEE were incubated with Jurkat T cells for the Halo-LZTFL1 first moves toward, and concentrates at, the IS, and indicated time to allow for the formation of conjugates (Fig. 3A). then it moves to the DP; the localization of Halo-LZSTOP does The distribution of LZTFL1 was detected by immunostaining, not change (Supplemental Video 1). These data demonstrate that followed by confocal microscope analysis. TCR-b was used as an LZTFL1 undergoes dynamic trafficking during IS formation, indicator for the formation of the IS. During T cell activation, the raising the possibility that LZTFL1 plays a role in T cell TCR cluster at the center of the T cell/APC interface formed a key activation. component of the IS (36). After 15 min of incubation with SEE- pulsed Raji cells, ∼40% of Jurkat T cells formed conjugates with RNA interference of LZTFL1 inhibits ATRA’s function on Th2 Raji B cells (Fig. 3B). In the absence of the superantigen, LZTFL1 cytokine production was homogeneously distributed in the membrane of Jurkat cells RAs are known to play a role in Th2 T cell differentiation and Th2 (Fig. 3A). Following superantigen stimulation, a time-dependent cytokine production (9–11). Because ATRA induced a dramatic relocalization of LZTFL1 was observed. At the early stages of increase in LZTFL1 production in primed human CD4+ T cells, conjugate formation, LZTFL1 was relocated to the Jurkat/Raji cell we reasoned that the expression of LZTFL1 could have a role in contact zone in 90% of the conjugates. With the maturation of the Th2 cytokine production. To test this hypothesis, we screened for IS, as indicated by TCR-b aggregation to the center of the contact cytokine expression in primed and ATRA-treated CD4+ T cells zone, the percentage of cells containing LZTFL1 in the contact from three healthy donors. As shown in Fig. 4, ATRA specifically zone decreased. After 15 min of incubation, LZTFL1 was ex- stimulated the production of Th2 cytokines, including IL-4 and cluded from the center of the contact zone to the DP of Jurkat IL-5, in all three donors, with minor stimulation of IL-13 pro- T cells in 60% of the conjugates (Fig. 3A, 3C). duction. IL-10 was found induced in one of the donors. The 1086 T CELL ACTIVATION–INDUCED LZTFL1 ENHANCES IL-5 PRODUCTION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. LZTFL1 transiently redistributes to the contact zone between T cells and APCs. (A) Jurkat T cells (denoted by T) were copelleted with BMQC-stained, SEE-loaded Raji B cells (blue; denoted by B) and incubated at 37˚C for 1.5-, 5-, and 15-min increments to induce IS formation. Cell conjugates were fixed and costained with Abs for LZTFL1 (green) and TCR-b (red). SEE-, no SEE-loading control, incubated for 15 min. Arrows indicate the T cell and B cell contact zone. (B) The percentage of conjugated T cells was calculated for all T cells in 10 fields chosen at random at indicated time points (mean 6 SD). Results are representative of three independent experiments. *p , 0.05. (C) LZTFL1 accumulation at the IS and the DP was quantified, and the percentage in total conjugates was plotted for both (mean 6 SD). Results are representative of three independent experiments. *p , 0.05 (comparison with LZTFL1 located at the IS after 1.5 min of incubation), #p , 0.05 (comparison with LZTFL1 located at the DP after 1.5 min of in- cubation); (D and E) live-cell imaging of LZTFL1 localization at the IS. Jurkat T cells (red; denoted by T) stably expressing Halo-STOP (D) or Halo- LZTFL1 (E) interacting with SEE superantigen-loaded Raji B cells (blue; denoted by B) and forming the IS at indicated times on live-cell imaging, as analyzed by wide field microscope (Supplemental Video 1). Data are representative of two independent experiments. Scale bar, 10 mm. production of IL-5 was affected the most by ATRA. Negative inhibited the upregulation of IL-5 production by ATRA (Fig. 5B). effects of ATRA on IL-6 and IL-17 were also detected in all the We also tested the effect of LZTFL1 in the D10.G4.1 cells of a donors. ATRA has little effect on other cytokines tested. Knock- mouse Th2 cell line. ATRA upregulated LZTFL1 protein ex- down of LZTFL1 with RNA interference significantly suppressed pression in D10.G4.1 cells, just as it did in human CD4+ T cells IL-4 and IL-5 production. IL-13 production was also inhibited to a from PBMCs (Fig. 5D). Activation of these cells with bead-bound lesser degree. Contrary to the Th2 cytokines, there was a small anti-CD3/CD28 Ab further increased the level of LZTFL1. To increase in the production of IL-6. No effect was detected for investigate the effect of LZTFL1, D10.G4.1 cells were transfected other cytokines tested. Next, we investigated the involvement of with LZTFL1 siRNA, followed by activation with bead-bound LZTFL1 and ATRA in the regulation of Th2 cytokine production. anti-CD3/CD28 Ab and ATRA treatment. Western blot analysis Due to a stronger IL-5 production response to ATRA treatment, shows that siRNA against LZTFL1 mRNA reduced the basal and IL-5 was used as an indicator of ATRA’s effect. Human CD4+ ATRA-induced LZTFL1 levels (Fig. 5D). Results in Fig. 5E show T cells transfected with LZTFL1 siRNA were primed with bead- that ATRA induced IL-5 production in both resting and activated bound anti-CD3/CD28 Ab and incubated in the medium with D10.G4.1 cells. As expected, LZTFL1 knockdown significantly 1 mM ATRA. The knockdown of LZTFL1 was first confirmed by suppressed IL-5 production in both resting and activated D10.G4.1 Western blotting (Fig. 5A). Knockdown of LZTFL1 significantly cells. More interestingly, in activated D10.G4.1 cells transfected The Journal of Immunology 1087

ment further increased IL-5 RNA levels. LZTFL1 knockdown decreased IL-5 RNA levels by .50% in both resting and activated D10.G4.1 cells. Also, in cells transfected with LZTFL1 siRNA, compared with the DMSO control, ATRA treatment did not in- crease the IL-5 RNA as much as it did in control siRNA-treated cells. Furthermore, when LZTFL1 was ectopically expressed in expanded CD4+ T cells, it significantly enhanced ATRA-induced IL-5 mRNA expression, even though overexpressed LZTFL1 by itself has little effect (Fig. 5G, 5H). These results confirm a role for LZTFL1 in ATRA-mediated IL-5 expression. LZTFL1 enhances the TCR signal to NFAT TCR engagement activates a series of proximal signaling molecule cascades, leading to transcriptional activation of cytokine genes. There are three major signaling pathways downstream of TCR signaling that are responsible for TCR-mediated gene expression, as follows: the calcium-NFAT pathway, NF-kBpathway,and Ras-MAPK-AP-1 pathway (38). These pathways are important

for TCR activation-driven cytokine gene transcription. NFATc1, a Downloaded from downstream signal of the TCR-signaling pathway, plays an essential role in regulating Th2 cytokine production and Th2 cell differen- tiation, and NFATc12/2 mice showed impaired Th2 responses (39). NFAT regulates Th2 cytokine expression by binding to the NFAT response elements on Th2 cytokine gene promoter. To investigate

the role of LZTFL1 on these signaling pathways, Jurkat T cells http://www.jimmunol.org/ were transfected with reporter vectors containing the responsive elements to NFAT, NF-kB, and AP-1 upstream of the firefly lucif- erase gene, along with a plasmid expressing LZTFL1. Cells were activated by incubation with bead-bound anti-CD3/CD28 Abs, and FIGURE 4. Involvement of LZTFL1 in ATRA-induced Th2 cytokine luciferase activity was estimated to quantitate the promoter activity. + expression. Human CD4 T cells isolated from healthy donors were As shown in Fig. 6A, TCR engagement in Jurkat cells induced both transfected with control siRNA (siControl) or siRNA targeting human NFAT and NF-kB reporter activities, with a preference to NFAT. LZTFL1 (siLZTFL1). Cells were then activated with anti-CD3 and anti- ∼ CD28 Ab-bound beads (anti-CD3/CD28) and treated with DMSO or 1 mM LZTFL1 further enhanced NFAT reporter activity by 2-fold and ATRA for 3 d. Th2 (A), Th1, and Th17 (B) cytokine expressions in the did not have any effect on NF-kB activity. LZTFL1 did not show by guest on September 25, 2021 culture supernatants were quantitated by ELISA. n =3.*p , 0.05 any effect on AP-1 reporter activity (data not shown). The regula- (comparison with cells transfected with siControl and treated with tion of LZTFL1 on NFAT reporter activity is dose dependent, in- DMSO); #p , 0.05 (comparison with cells transfected with siControl and dicating that this effect is specific (Fig. 6B). Taken together, all treated with ATRA). these data suggest that LZTFL1 is an important regulator of ATRA induction on Th2 cytokine production. Increased TCR–NFAT sig- with LZTFL1 siRNA, compared with the DMSO control, ATRA naling may contribute to this effect. failed to further induce IL-5 production. All these data indicate that LZTFL1 plays an important role in ATRA-induced IL-5 production. Discussion RAs favor Th2 cell differentiation and could directly induce the LZTFL1 stimulates IL-5 RNA expression production of Th2 cytokines IL-4, IL-5, and IL-13 in Ab-primed Th2 cytokines were regulated at the promoter level by Th2 cell human PBMCs (11). In this study, we identified LZTFL1 as an differentiation transcriptional factors during T cell activation. essential regulator of ATRA-induced Th2 cytokine expression, IL-4, IL-5, and IL-13 are coordinately expressed in Th2 cells (37). especially IL-5 expression in CD4+ T cells. LZTFL1 was mark- The genes of these cytokines are clustered on chromosomal locus edly and rapidly upregulated at physiological concentrations of 5q31 in humans and chromosome 11 in mice. The genes encoding ATRA in primed CD4+ T cells (Fig. 1D). LZTFL1 knockdown IL-4 and IL-13 are linked and transcribed in the same direction, using siRNA particularly reduced IL-5 production in primed hu- whereas the gene encoding IL-5 is separated by the rad50 gene man CD4+ T cells from PBMCs and in a mouse Th2 cell line, and and is transcribed in the opposite direction (37). Next, we asked it significantly suppressed ATRA-induced IL-5 production in these whether LZTFL1 regulates Th2 cytokine gene expression. To cells (Fig. 5). Given the fact that LZTFL1 is upregulated by ATRA answer this question, CD4+ T cells from healthy donors were in activated T cells, we reason that LZTFL1 is a positive regulator transfected with LZTFL1 siRNA, followed by T cell activation of ATRA-induced T cell response. with bead-bound anti-CD3/CD28 Ab priming. Changes in IL-5 How does LZTFL1 regulate IL-5 expression? LZTFL1 knock- mRNA levels in response to LZTFL1 knockdown were assessed down using siRNA suppressed ATRA-induced IL-5 expression at by quantitative real-time PCR. As shown in Fig. 5C, in line with both protein and RNA levels (Figs. 4, 5). To date, no RAR or the upregulation of IL-5 protein levels by ATRA, IL-5 mRNA retinoid X receptor response element has been located in the 59 expression increased ∼3-fold in response to ATRA treatment. promoter region of the Th2 cytokine genes. Unlike Th1 cells, Th2 LZTFL1 knockdown decreased IL-5 mRNA levels by 40% and polarization requires prolonged TCR stimulation (40). Th2 cyto- ATRA-induced IL-5 mRNA expression by 30%. Similar results kine genes are regulated in two steps (41–44). In the first step, were also observed in D10.G4.1 cells (Fig. 5F). IL-5 mRNA levels naive T cells were differentiated to mature effector Th2 cells increased ∼3-fold following T cell activation, and ATRA treat- triggered by Ag presenting, IL-4, and STAT6. In this stage, DNase 1088 T CELL ACTIVATION–INDUCED LZTFL1 ENHANCES IL-5 PRODUCTION Downloaded from http://www.jimmunol.org/

FIGURE 5. LZTFL1 enhances ATRA-induced IL-5 protein and RNA levels. (A) Human CD4+ T cells isolated from healthy donors were transfected with control siRNA (siControl) or siRNA targeting human LZTFL1 (siLZTFL1). Cells were then activated with anti-CD3/CD28 and treated with DMSO or 1 mM ATRA for 1 d. LZTFL1 expression was detected by Western blotting. (B) The level of the IL-5 protein in the culture supernatants was quantitated by ELISA. (C) IL-5 RNA expression was detected by reverse transcription, followed by real-time PCR, as described in Materials and Methods. IL-5 RNA by guest on September 25, 2021 expression was normalized to GAPDH RNA expression, and relative RNA fold changes compared with those from DMSO treatment were plotted (mean 6 SD). (D) D10.G4.1 cells, a mouse Th2 cell line, were transfected with siControl or siLZTFL1 targeting mouse LZTFL1. Cells were activated and treated with 1 mM ATRA for 1 d. LZTFL1 expression was detected by Western blotting. (E) Expression of the IL-5 protein was quantitated by ELISA. (F) IL-5 RNA expression was analyzed. (G and H) CD3/CD28 activation bead-expanded human CD4+ T cells were transfected with LZTFL1 mRNA, and, 72 h later, cells were used to prepare protein lysates for Western blot analysis (G) and RNA for real-time PCR measurement of GAPDH and IL-5 RNA (H). The vertical line in (G) indicates where parts of the image were joined. Results are representatives of five independent experiments for (A–F) and three for (G and H). *p , 0.05 (comparison with each corresponding cell transfected with siControl or control RNA); #p , 0.05 (comparison with control and cells transfected with LZTFL1 mRNA and treated with ATRA).

I–hypersensitive regions are detected in the IL-4/IL-5/IL-13 gene cluded to the DP of T cells when the IS matured, and over- cluster. DNA methylation status is also changed. The second step expression of LZTFL1 in CD4+ T cells enhanced TCR–NFAT is the stage that Th2 cytokine gene expression is induced. Many signaling (Fig. 6). These data indicate that LZTFL1 plays a role in transcription factors are involved in this stage, including NFAT, immune response. LZTFL1 was induced by T cell activation, and AP-1, Est-1, Maf, and GATA3. Among them, Maf and GATA3 are its protein expression could be detected after 8 h. It is possible Th2 specific (45, 46). NFAT, a downstream signal of the TCR- LZTFL1 could be induced during the prolonged period of APC– signaling pathway, although not specific for Th2 cells, plays an T cell interaction (51), which in turn could enhance TCR signaling essential role in the regulation of Th2 cytokine production and and facilitate ATRA-induced IL-5 production. Th2 cell differentiation (39). Mice with either NFATc1 or NFATc2 During T cell activation, a rearrangement of membrane and knockout showed impaired Th2 responses. NFAT binds the IL-4 cytosolic molecules occurs at the T cell and APC contact zone, and IL-5 promoters only in stimulated Th2 cells, not Th1 cells (47, resulting in the formation of a highly organized interface known 48). NFATc1 is also required to recruit GATA-3 to IL-5 promoter as the IS. The IS provides a platform for signal molecule assembly in cAMP-mediated IL-5 expression in activated Th2 cells (48). and accumulation at the interface, and promotes polarized exo- RAs affect Th2 differentiation through regulating the expression cytosis (32, 33). Similar to the IS, primary cilium is also a highly of Th2-specific transcription factors, including Ets-1, cMAF, organized structure. It is characterized as the site for directional GATA-3, and STAT-6 (11, 49, 50). RA also participates in T cell movement of structural and regulatory molecules (31). Both the activation, supported by upregulation of the expression of T cell IS and primary cilium show reorientation of the microtubule- activation markers CD38 and CD69 (10). In this work, we show a organizing center. Most of the vertebrate cells have primary cil- link between LZTFL1 and TCR–NFAT signaling. LZTFL1 ac- ium. Lymphocytes are among a very few types of cells that do cumulates in the plasma membrane of human CD4+ T cells. not form cilium. Based on the molecular similarity, the IS is During T cell activation, it transiently redistributed to the T cell considered to be a homolog to primary cilium (33–35). Besides and APC contact zone at the beginning of IS formation, and ex- the reorientation of the microtubule-organizing center in both The Journal of Immunology 1089

The mechanism by which LZTFL1 relocates during IS for- mation and impacts NFAT activity remains to be elucidated. One possibility is that LZTFL1 associates with polymerized actin or F-actin. Actin is particularly important for T cell activation. Actin or myosin interference affects surface protein trafficking in the IS more in T cell side than in the B cell side (58–60). Upon TCR engagement, the actin cytoskeleton in T cells starts to reorganize. It polymerizes beneath the area of the T cell and APC cell contact zone. When TCR microclusters move to the center of the contact, and the central supramolecular activation cluster (cSMAC) forms, F-actin forms a ring structure around the cSMAC and stabilizes integrin-dependent adhesive interactions between T cells and APCs (61). F-actin also relocates to the DP, the site that is op- posite to the IS, and facilitates the formation of a DP complex (61, 62), although the exact picture of the dynamic change of F-actin localization is not fully established yet. The IS is a fine-tuned process; like F-actin, actin-binding proteins also traffic in T cells in a bipolar manner during IS formation. Actin-binding protein 1,

HIP55, has been found to translocate to the IS and regulate TCR Downloaded from internalization (63), whereas ezrin-radixin-moesin proteins, from a protein family that binds to F-actin through the C-terminal domain, are excluded from the mature IS (64). Upon TCR engagement, moesin is depleted from the IS, whereas ezrin transit locates to the IS and then redistributes to the DP complex. Ezrin-radixin-moesin

proteins mediate the exclusion of CD43 from the IS, which is be- http://www.jimmunol.org/ lieved to be important for T cell activation (64–66). Wei et al. (23) have reported that LZTFL1 binds to actin in vitro, and because both actin and LZTFL1 show similar relocation patterns during IS for- mation, it is possible that LZTFL1 relocates along the actin cyto- skeleton. Blocking the polymerization of F-actin with latrunculin B abolished LZTFL1 membrane localization, providing additional support for this hypothesis (Fig. 2C). However, we cannot rule out other possibilities. Alternatively, LZTFL1 may be required to se- FIGURE 6. LZTFL1 enhances T cell activation by specifically upreg- quester a negative regulator from the cSMAC. In primary cilia, by guest on September 25, 2021 A ulating NFAT activity. ( ) Jurkat cells were cotransfected with DNAs LZTFL1 was found to interact with BBS-9 and to sequester it from encoding LZTFL1-Flag and NFAT-Luc or LZTFL1-Flag and NF-kB-Luc its ciliary localization, which negatively regulates BBSome ciliary for 2 d. Renilla luciferase, under the control of the thymidine kinase trafficking and hedgehog signaling (25). The plasma membrane and promoter, was used as a transfection control. Cells were activated with anti-CD3 and anti-CD28 Ab-bound beads for 10 h. The firefly luciferase to cytoplasmic localization of LZTFL1 in T cells makes it a good Renilla luciferase ratio was calculated, and levels relative to those of the candidate as an adaptor. Precisely how LZTFL1 regulates TCR– pCMV6 empty vector–transfected and unactivated cells were plotted NFAT signaling is under further investigation. (mean 6 SD). n =5.*p , 0.05 (comparison with cells transfected with # pCMV6 empty vector and unactivated); p , 0.05 (comparison with cells Acknowledgments transfected with pCMV6 empty vector and activated). (B) Jurkat cells We thank Drs. J. Jiang, M. Ishaq, and H. Young for helpful discussions. cotransfected with reporter NFAT-Luc and an increasing amount of LZTFL1 expression DNA. Cells were activated, and the relative firefly luciferase to Renilla luciferase ratio was plotted as in (A) (mean 6 SD). Disclosures n =5.*p , 0.05 (comparison with cells transfected with pCMV6 empty The authors have no financial conflicts of interest. vector and unactivated); #p , 0.05 (comparison with cells transfected with pCMV6 empty vector and activated). The expression of endogenous and transfected LZTFL1 was analyzed by Western blotting. References 1. De Luca, L. M. 1991. Retinoids and their receptors in differentiation, embryo- genesis, and neoplasia. FASEB J. 5: 2924–2933. processes, some proteins functioning in the primary cilium are also 2. Collins, S. J. 2002. The role of retinoids and retinoic acid receptors in normal hematopoiesis. 16: 1896–1905. foundtoplayaroleintheIS.GTPase rat brain 11 (Rab11), an es- 3. Hall, J. A., J. L. Cannons, J. R. Grainger, L. M. Dos Santos, T. W. Hand, S. Naik, sential protein for primary ciliogenesis (52), is involved in traf- E. A. Wohlfert, D. B. Chou, G. Oldenhove, M. Robinson, et al. 2011. Essential role for retinoic acid in the promotion of CD4(+) T cell effector responses via ficking to the IS (53). SNARE proteins regulate cilia exocytosis retinoic acid receptor alpha. Immunity 34: 435–447. as well as TCR recycling to the IS (54, 55). Intraflagellar transport 20 4. Hall, J. A., J. R. Grainger, S. P. Spencer, and Y. Belkaid. 2011. The role of (IFT20), an IFT component essential for ciliary assembly, was also retinoic acid in tolerance and immunity. Immunity 35: 13–22. 5. Iwata, M., A. Hirakiyama, Y. Eshima, H. Kagechika, C. Kato, and S. Y. Song. found to be expressed in lymphocytes (56) and was required for 2004. Retinoic acid imprints gut-homing specificity on T cells. Immunity 21: polarized recycling of TCR to the IS. LZTFL1 was recently identified 527–538. as a member of the BBsome (24). It affects BBsome ciliary traffic 6. Elias, K. M., A. Laurence, T. S. Davidson, G. Stephens, Y. Kanno, E. M. Shevach, and J. J. O’Shea. 2008. Retinoic acid inhibits Th17 polarization and hedgehog signaling (25). In the current study, we have shown that and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling LZTFL1 is upregulated by ATRA in activated T cells. It transiently pathway. Blood 111: 1013–1020. 7. Mucida, D., Y. Park, G. Kim, O. Turovskaya, I. Scott, M. Kronenberg, and located to the IS and enhanced NFAT activity. LZTFL1 regulation H. Cheroutre. 2007. Reciprocal TH17 and regulatory T cell differentiation me- by RA was also shown by Kang et al. (57). diated by retinoic acid. Science 317: 256–260. 1090 T CELL ACTIVATION–INDUCED LZTFL1 ENHANCES IL-5 PRODUCTION

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Legend for video1.mov.

Live cell imaging for LZTFL1 localization at the IS. Movement of LZTFL1 during IS

was captured as described in Materials and Methods. Jurkat T cells (red) stably

expressing Halo-STOP or Halo-LZTFL1 interacting with SEE superantigen–loaded Raji

B cells (blue) and forming the IS.