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This information is current as of June 14, 2017. HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76−Signaling Microclusters following TCR Stimulation Libin Abraham, Peter Bankhead, Xiaoyu Pan, Ulrike Engel and Oliver T. Fackler J Immunol published online 16 July 2012 http://www.jimmunol.org/content/early/2012/07/16/jimmun ol.1200652 http://www.jimmunol.org/content/suppl/2012/07/16/jimmunol.120065 2.DC1 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Email Alerts Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 Supplementary Material Published July 16, 2012, doi:10.4049/jimmunol.1200652 The Journal of Immunology HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76–Signaling Microclusters following TCR Stimulation Libin Abraham,*,† Peter Bankhead,‡ Xiaoyu Pan,* Ulrike Engel,‡ and Oliver T. Fackler* D4+ T lymphocytes and macrophages represent the major target cells of HIV type 1 (HIV-1), the causative agent of AIDS. In primary human T lymphocytes, the efficacy of HIV replication is tightly coupled to their activation state. Whereas HIV-1 undergoes early replication events in quiescent CD4+ T lymphocytes, subsequent steps in the viral life cycle require cell activation (1). HIV-1 infection of resting T lymphocytes is thus often nonproductive and leads to the establishment of latent reservoirs from which the virus can be reactivated upon cell activation. T cell activation is primarily governed by signaling through the TCR complex upon engagement in a tight contact with APCs referred to as immunological synapse (IS). TCR engagement by specific MHC-presented peptides launches highly dynamic and coordinated transport events that recruit specific factors to the IS and exclude others from it. This signal initiation triggers a broad cascade of downstream signaling that includes dynamic F-actin remodeling at the IS, tyrosine phosphorylation, and release of C *Department of Infectious Diseases, Virology, University Hospital Heidelberg, 69120 Heidelberg, Germany; †Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, 69120 Heidelberg, Germany; and ‡Nikon Imaging Center, University of Heidelberg, 69120 Heidelberg, Germany Received for publication February 27, 2012. Accepted for publication June 13, 2012. This work was supported by Deutsche Forschungsgemeinschaft Grants SFB638 and TRR83 (to O.T.F.), a Cluster of Excellence EXO81:Ph.D. fellowship (to L.A.), and Heidelberg Molecular Life Science (to P.B.). Address correspondence and reprint requests to Dr. Oliver T. Fackler, Department of Infectious Diseases, Virology, University Hospital Heidelberg, INF 324, 69120 Heidelberg, Germany. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: IS, immunological synapse; LAT, linker of activated T cells; MC, microcluster; SLP-76, Src homology-2 domain-containing leukocyte protein of 76 kDa; TGN, trans Golgi network; WT, wild type. Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200652 calcium flux, which are coordinated to trigger transcriptional profiles, including induced expression of the T cell survival cytokine IL-2 (2). Early TCR signaling events are commonly studied in the context of the IS. However, at these cell contacts, signaling events can only be analyzed with limited optical and temporal resolution. Surfacemediated TCR stimulation is thus widely used as an alternative experimental system that reflects key aspects of physiological T cell activation and allows for enhanced visualization of dynamic signaling events and facilitates quantitative single-cell analysis (3). Upon such TCR engagement, cells rapidly spread on the stimulatory substrate and generate highly dynamic membrane-associated multiprotein aggregates that are constantly subject to change in composition and phosphorylation state. These so-called signaling microclusters (MCs) are viewed as the minimal unit of T cell activation, and continuous spatial-temporal generation of MCs provides sustained signals required for T cell activation (4–6). Upon engagement, TCR-CD3 itself is organized into MCs and rapidly recruits active forms of the Src kinase family members Lck and Fyn that phosphorylate the z-chain of the TCR (7, 8). This activation step triggers the recruitment of ZAP70 (9), which itself is activated by phosphorylation by the TCR-proximal Src family kinase Lck (8). Active ZAP70 subsequently phosphorylates two critically important adaptor proteins, linker of activated T cells (LAT) and Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) (10). LAT facilitates the formation of several multiprotein complexes and acts as nucleation center for T cell signal transduction (11). Upon phosphorylation at Tyr191 by ZAP70 (12), LAT recruits SLP-76 into MCs via an additional adaptor protein, GADS (13). Together, the LAT–SLP-76 complex nucleates multiple important downstream signaling events such as calcium flux, MAPK activation, integrin activation, and cytoskeletal organization (14). Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCRCD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nefexpressing and HIV-1–infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef’s ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76–signaling MCs. The Journal of Immunology, 2012, 189: 000–000. 2 Materials and Methods Expression constructs, reagents, and Abs The plasmid encoding the Nef protein of HIV-1SF2 with a C-terminal Myc epitope tag and the corresponding control plasmid encoding just the myc epitope tag were described previously (40). The expression plasmid for Nef F195I.myc was generated by subcloning the nef orf from an expression plasmid for a Nef F195I.GFP fusion protein (38). Expression plasmids for TCR z.GFP or Unc119.GFP and Unc119.RFP were gifts of M. Davis (Stanford University) or described previously (40), respectively. The proviral constructs used are based on HIV-1NL4-3 that lack Nef expression (HIV ΔNef) or encode Nef from HIV-1SF2 (HIV-wild type [WT]) or its F195I mutant (HIV Nef F195I), as described (41). Poly-L-lysine was purchased from Sigma-Aldrich. For F-actin staining, tetramethylrhodamine isothiocyanate-conjugated phalloidin was obtained from Sigma-Aldrich and Cytoskeleton. The following Abs were used: anti- CD3 (clone HIT3a against CD3ε; BD Pharmingen), rabbit anti–c-Myc (Sigma-Aldrich), rabbit anti–phospho-ZAP70 (Tyr319), rabbit anti– phospho-LAT (Tyr191) (both from Cell Signaling Technology), and sheep anti–HIV-1 capsid (provided by H.-G. Kräusslich, University Hospital, Heidelberg, Germany) (41). The ARP444 sheep anti-Nef antiserum was a gift of M. Harris (University of Leeds). Secondary goat anti-rabbit and anti-sheep Alexa Fluor 350/488/568-conjugated Abs were purchased from Invitrogen. Cell culture and transfection Human PBMC from healthy donors were obtained from the Heidelberg University Hospital Blood Bank and purified by Ficoll gradient centrifugation, as described (40). HEK 293T and Jurkat T cell lines or primary T lymphocytes were cultivated in DMEM and RPMI 1640 medium plus GlutaMAX-I, respectively, supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin-streptomycin (all from Invitrogen). For live-cell imaging, cells were resuspended in CO2 independent medium (from Life Technologies) before imaging. Jurkat E6.1 cells stably expressing ZAP70. GFP or LAT.GFP were gifts of L. Samelson (National Institutes of Health) (42). J14 cells stably expressing SLP-76.YFP (J14SLP-76.YFP) were provided by S. Bunnell (Tufts University) (43, 44). For transfection, 1 3 107 Jurkat T lymphocytes were electroporated using 25–50 mg plasmid DNA (850 mF, 250 V; Bio-Rad Genepulser) and analyzed 24 h posttransfection. HIV-1 production and infection Virus stocks were generated by transfection of proviral HIV plasmids into 293T cells, as previously described (41). Two days after transfection, culture supernatants were harvested and the HIV-1 p24 Ag concentration was determined by a p24 Ag ELISA. Isolation, activation, and HIV-1 infection of PBL were carried out, as described (37). A total of 1000 ng p24 was used to spin-infect 5 3 106 Jurkat cells or PBLs. Virus was removed 24 h postinfection, and new medium containing 10 ng/ml IL-2 was added. Two days postinfection, cells were analyzed, as described below, except that the cells were stained additionally for p24CA to identify productively infected cells. Analysis of expression levels To determine the effect of Nef on expression levels of TCR and other TCRproximal signaling molecules, all cell lines used in our study were transfected with expression plasmids for either RFP or Nef.RFP. Twenty-four hours posttransfection, RFP or Nef.RFP-positive cells were analyzed by flow cytometry to check the expression levels of various GFP/YFP-tagged fusion proteins (also see Supplemental Fig. 4D). To determine the Nef expression levels following transfection and infection, Jurkat T cells were tranfected and infected with expression plasmid coding for Nef.GFP for 24 h and HIV-WT virus for 48 h, respectively. The percentage of transfected and infected cells was determined by GFP expression and p24 staining, respectively, by flow cytometry. Equal numbers of Nef-positive cells were analyzed by Western blotting. Quantification revealed that per cell Nef expression was ∼3 times higher in tranfected relative to infected cells (data not shown). T cell spreading and actin ring assay T cell activation using anti-CD3ε–coated stimulatory surfaces for TCRmediated cell spreading was essentially carried out as described (37, 45). Briefly, microscope cover glasses (Marienfeld) were placed on a parafilmcoated petri dish and incubated with a 0.01% poly-lysine (Sigma-Aldrich) solution at room temperature for 10 min. The solution was aspirated and dried at 60˚C for 3 min. For Ab coating, dried cover glasses were then covered with anti-CD3ε Ab diluted in TBS to a final concentration of 10 mg/ml, incubated at 37˚C for 3 h, washed with TBS, and stored in the same at 4˚C. To analyze cell spreading and formation of F-actin–rich circumferential rings or MCs, cells were plated on anti-CD3ε–coated cover glasses at densities that facilitate free spreading without formation of cell aggregates and incubated typically for 5 min at 37˚C, except in experiments in which the kinetics of MC formation was analyzed and cells were fixed every 5 min in a time frame between 0 and 30 min. Subsequently, cells were fixed for 20 min (Jurkat T lymphocytes) or 90 min (infected PBL) by adding TBS–3% paraformaldehyde. After permeabilization with TBS–0.1% Triton X-100 for 1–5 min, the cells were blocked with TBS–1% BSA for 30 min and stained, as previously described (37). Cover glasses were mounted in LinMount (Linaris). For live-cell imaging, glass-bottom dishes (MatTek) were treated and functionalized with anti-CD3ε Ab similar to coverslips, as described above, washed with TBS, and stored at 4˚C. Live-cell imaging was conducted on cells 24 h posttransfection. Cells were resus- Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 T cell activation can be viewed as beneficial for HIV-1, as it allows transcriptional activation of latent provirus and progression of the life cycle. However, activation-induced cell death after TCR engagement runs the risk of limiting the life span of productively infected cells and thus the amount of viral progeny produced. Consequently, HIV-1 encodes gene products, such as Nef, to finetune activation states of infected T lymphocytes (15, 16). Nef is a 25- to 34-kDa myristoylated accessory protein encoded by HIV1, HIV-2, and SIV. Ex vivo, Nef enhances the single-round infectivity of virus particles and moderately accelerates virus spread over multiple rounds (17). In vivo, Nef strongly boosts virus replication particularly during primary infection and is critical for rapid disease progression (18–20). This role of Nef as a pathogenicity factor is also revealed in transgenic mice in which Nef expression induces AIDS-like depletion of CD4+ T lymphocytes (21). In the absence of intrinsic enzymatic activity, Nef’s effects are thought to be mediated by a wealth of interactions with host cell proteins that trigger alterations in intracellular transport and signaling pathways (22, 23). This includes modulating exposure of cell surface receptors, such as MHC-I and II, CD4, and chemokine receptors to evade immune recognition and prevent superinfection of infected cells, respectively (reviewed in Ref. 17). In addition, Nef affects the basal states of T cell activation and the responsiveness of T lymphocytes to TCR signaling (24–27) with the majority of studies that addressed effects of Nef on distal responses to exogenous TCR stimulation by mitogens or anti-TCR Abs reporting a moderate enhancement by HIV-1 Nef (16, 28–34). In contrast, HIV-1 Nef severely impairs the formation and organization of IS structures between Nef-expressing T lymphocytes and APCs by reducing the frequency of IS formation, blocking Factin polarization at cell-cell contacts, and inducing mislocalization of the TCR itself as well as its effector kinase Lck (28, 35– 39). These morphological alterations at the IS were accompanied by interference with early TCR signaling such as induction of tyrosine phosphorylation (36, 37, 39). We recently reported that Nef achieves enhanced T cell activation responses despite this disruption of early signaling events by the assembly of active signaling complexes at the trans Golgi network (TGN) (40). This mechanism largely uncouples TCR signaling from the plasma membrane and appears to tailor T cell activation outputs in a manner that particularly favors HIV-1 replication. However, the mechanisms by which Nef disrupts early TCR signaling events remain to be elucidated. To address this question, we employed in this study quantitative single-cell analysis on formation, composition, and dynamics of MCs following surfacesupported TCR engagement. Our results define a two-pronged mechanism by which Nef disrupts the formation of MCs containing the signaling adaptor SLP-76 as the central event in interference with early TCR signaling by the viral protein. DISRUPTION OF EARLY TCR SIGNALING BY HIV-1 Nef The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 FIGURE 1. HIV-Nef does not interfere with formation of TCR MCs, but reduces the efficacy of TCR recruitment to MCs. (A) Representative micrographs of Jurkat T lymphocytes transiently cotransfected with the indicated expression plasmids. Twenty-four hours posttransfection, cells were stimulated on anti-CD3ε–coated coverslips for 5 min, fixed, and stained for F-actin and myc. Right panel, Displays binary images of the GFP channel produced by our spot detection algorithm, in which pixels belonging to MCs detected at the cell-cover glass interface are shown in white (see details in Materials and Methods and Supplemental Fig. 1). Gray lines indicate cell boundaries. Scale bar, 10 mm. (B) Frequency of cells shown in (A) with pronounced TCR MCs at the stimulatory contact site. Depicted are mean values from three independent experiments 6 SD with at least 100 cells analyzed per condition each, with cells scored as containing TCR MCs if they exhibited TCR MCs comparable to those of untransfected neighboring cells. Values of p were determined using the Student t test. Single-cell quantifications indicating TCR MC density (C), mean TCR MC size per cell (D), and magnitude of recruitment into MC in the contact plane (E) were calculated using the spot analysis algorithm described above (see details in Materials and Methods). Depicted are values from .25 cells analyzed per condition in which each symbol designates the value for an individual cell and gray bars indicate the mean values of all cells analyzed. The p values were determined using the Mann-Whitney U test (*p , 0.01, **p , 0.001, ***p , 0.0001). 4 DISRUPTION OF EARLY TCR SIGNALING BY HIV-1 Nef detected in channel 2 were computed, along with the percentages of those in channel 2 also found in channel 1. These values are similar to Manders’ colocalization coefficients M1 and M2, except that in this study fluorescence intensities were ignored (47). Image acquisition, analysis, and quantification Statistical evaluation Images were acquired using an Olympus IX81 S1F-3 microscope equipped with Olympus UPlanSApo objectives (3100/1.40 oil) and an Olympus U-CMAD3 F-View camera of cells that were identified as transfected or infected by the GFP/YFP/RFP fluorescence or their positivity for p24CA. Images were acquired with identical microscope settings using the cell^M 3.1 software and exported as 16-bit TIFF files. MCs were analyzed using Fiji (see below and Supplemental Fig. 1) to measure MC density (number of clusters/area of the cell), mean MC size per cell (area occupied by all MCs/number of MCs), and magnitude of recruitment into MCs in the stimulatory contact plane (integrated density of MC signal/integrated density of total signal at the contact site). For quantification of frequencies of observed phenotypes, transfected or infected cells were judged microscopically for presence or absence of the respective phenotypes. More than 100 positive cells that were randomly chosen among all positive cells were counted per experiment by two independent members of the laboratory without disclosing the identity of the samples. For single-cell intensity quantification of LAT accumulation and subcellular localization, wide-field z-stacks were taken under identical conditions and deconvolved and background subtracted using Huygens software (Scientific Volume Imaging, Hilversum, The Netherlands). Sum intensity projections were made and sum pixel intensities were determined using Fiji, as recently described for Lck (40). Signal intensities in areas of interest were plotted relative to the total per cell signal. Statistical significance was calculated by performing a Student t test or Mann-Whitney U test (***p , 0.0001, **p , 0.001, *p , 0.01). Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 pended in CO2 independent medium and dropped into functionalized glass-bottom dishes, and image acquisition was started immediately once the initial MCs were visible at contact site (t = 0). Frames were acquired every 5 s, and imaging was performed for 5 min. MC detection We developed a strategy to detect and quantify microclusters in Fiji (http:// www.fiji.sc), which we applied to the YFP/GFP channels of images acquired using identical microscope settings. After initially marking cell boundaries manually using Fiji’s polygon region of interest tool, as shown in Supplemental Fig. 1A, all subsequent steps were automated using a Beanshell script. The detection of MCs involved four main steps, as follows: 1) subtracting the camera offset, 2) replacing each pixel with the square root of its value, 3) applying difference of Gaussians filtering, and 4) thresholding based upon an estimate of the SD of nonspot pixels in the filtered image (see Supplemental Fig. 1). The camera offset is a constant value added to each pixel and was identified from dark images. The second step served to reduce the signal dependence of the photon noise present in fluorescence images, which is Poisson distributed and therefore has a SD that varies with the square root of the signal. Replacing pixels with the square root of their values has the effect of making the noise approximately Gaussian distributed with the same SD everywhere (46). This is useful because the probability of falsely detecting clusters when applying a global threshold to the image depends upon the local noise SD, which in the case of Poisson noise would be higher in brighter cells. Difference of Gaussians filtering consists of applying two different Gaussian filters to duplicates of the image (in this study, Gaussian sigma values of 1.5 and 2.4 pixels were used) and subtracting the second filtered image from the first. The smaller filter reduces noise, whereas the larger filter suppresses both noise and other small structures. The result of the subtraction is then an image in which the overall mean of pixel values is zero, and the largest positive values correspond primarily to the clusters we wish to detect (Supplemental Fig. 1E). Other pixels belonging to flat or slowly-varying background regions have smaller absolute values due to noise. We estimated the SD of these values by dividing the median absolute deviation of all pixels by a normalizing factor of 0.6745, before setting a threshold at 6 times this estimated SD to detect potential clusters. This made it possible to detect dim clusters, provided these were sufficiently elevated above the local noise (Supplemental Fig. 1F). The “Find Maxima” command in Fiji was then used to identify individual clusters as local intensity maxima rising above the threshold in the difference of Gaussians image, and to separate these into individual regions using a watershed algorithm (Supplemental Fig. 1G). Finally, the number, area, and integrated densities of clusters within each cell were computed. The filtering and noise estimation were implemented as a separate ImageJ plugin, which is available on request. The script implementing the entire algorithm was applied to all images with identical settings (i.e., filter sizes and thresholds) to reduce the chance of introducing bias when quantifying MCs. Colocalization analysis As a measure of colocalization, clusters were detected using the algorithm described above, and the percentages of cluster pixels in channel 1 also FIGURE 2. HIV-Nef does not interfere with the formation of ZAP70containing MCs. (A) Representative micrographs of Jurkat T lymphocytes stably expressing ZAP70.GFP were transiently transfected with the indicated expression plasmids. Cells were stimulated on anti-CD3ε–coated coverslips for 5 min, fixed, and stained for F-actin and myc. Gray lines indicate cell boundaries. Scale bar, 10 mm. (B) Frequency of cells shown in (A) with pronounced ZAP70 MCs at stimulatory contact sites. Depicted are mean values from three independent experiments 6 SD with at least 100 cells analyzed per condition each, with cells scored as containing ZAP70 MCs if they exhibited ZAP70 MCs comparable to those of untransfected neighboring cells. Values of p were determined using the Student t test. Single-cell quantifications for ZAP70 MC density (C), mean MC size per cell (D), and magnitude of recruitment into MC in the contact plane (E). Depicted are values from .25 cells analyzed per condition in which each symbol designates the value for an individual cell and gray bars indicate the mean values of all cells analyzed. The p values were determined using the Mann-Whitney U test. The Journal of Immunology Results Early TCR MC formation is undisturbed in the presence of Nef To dissect the effects of Nef on early TCR signaling, we adopted a quantitative microscopy-based single-cell analysis of signaling MCs induced upon TCR engagement. To this end, Jurkat T lym- 5 phocytes were transfected with expression constructs for Nef from HIV-1 strain SF2 carrying a myc epitope tag (Nef.myc) or an empty control plasmid (control). Twenty-four hours posttransfection, the cells were placed on cover glasses coated with anti- Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 FIGURE 3. HIV-1 Nef potently inhibits formation of SLP-76–containing MCs. (A) Representative micrographs of J14SLP-76.YFP cells transiently expressing the indicated proteins. Twenty-four hours posttransfection, cells were stimulated on anti-CD3ε–coated coverslips for 5 min, fixed, and stained for F-actin and myc. Right panel, Displays binary images of detected MCs for the YFP channel. Gray lines indicate cell boundaries. Scale bar, 10 mm. (B) Frequency of cells shown in (A) with pronounced SLP-76 MCs at the stimulatory contact site. Depicted are mean values from three independent experiments 6 SD with at least 100 cells analyzed per transfection. Cells were scored as containing SLP-76 MCs if they exhibited comparable MCs relative to that of untransfected neighboring cells. The p values were determined using Student t test. Single-cell quantification indicating SLP-76 MC density (C), mean MC size per cell (D), and magnitude of recruitment into MC in the contact plane (E) is shown. Depicted are values from .25 cells analyzed per condition in which each symbol designates the value for an individual cell. Gray bars indicate the mean values of all cells analyzed. The p values were determined using the Mann-Whitney U test (*p , 0.01, **p , 0.001, ***p , 0.0001). FIGURE 4. Nef potently inhibits formation of SLP-76–containing MCs in HIV-infected J14SLP-76.YFP cells. (A) Representative micrographs of J14SLP-76.YFP cells following infection with WT HIV-1 (HIV-1 WT), its nef-deleted counterpart HIV-1ΔNef, or the isogenic virus HIV-1 Nef F195I. Forty-eight hours postinfection, cells were stimulated on antiCD3ε–coated cover glasses for 5 min, fixed, and stained for intracellular p24CA and F-actin. Gray lines indicate cell boundaries. Scale bar, 10 mm. (B) Frequency of cells shown in (A) with pronounced SLP-76 MCs at stimulatory contact site. Depicted are mean values from three independent experiments 6 SD with at least 100 infected cells analyzed per each condition. Cells were scored as positive for SLP-76 MCs if they exhibited MCs comparable to those of uninfected neighboring cells. The p values were determined using the Student t test. Single-cell quantification indicating SLP-76 MC density (C), mean MC size per cell (D), and magnitude of recruitment into MC in the contact plane (E) was calculated using the SD plugin. Depicted are values from .10 cells analyzed per condition in which each symbol designates the value for an individual cell. Gray bars indicate the mean values of all cells analyzed. The p values were determined using the Mann-Whitney U test (*p , 0.01, **p , 0.001, ***p , 0.0001). 6 To allow thorough quantification of such MCs in single cells, we implemented a semiautomated analysis in the Fiji software that enabled us to compute MC number, area, mean signal intensity, and integrated density from which we quantified MC density, mean MC size per cell, and the efficiency of recruitment of individual components into MCs at stimulatory contact sites (see Materials and Methods, Supplemental Fig. 1). Plotting the density of TCR MCs (number of MCs per unit area) revealed that Nef or Nef F195I moderately or not at all reduced TCR MC density, respectively (Fig. 1C; 1.0 6 0.2 per mm2 for control, 0.8 6 0.2 per mm2 for Nef, and 0.9 6 0.2 per mm2 for Nef F195I). A slight Nefdependent reduction was observed in the mean TCR MC size per cell (Fig. 1D; 0.07 6 0.02 mm2 for control, 0.05 6 0.01 mm2 for Nef, and 0.06 6 0.02 mm2 for Nef F195I) and, in line with results by Thoulouze et al. (36), Nef diminished the magnitude of recruitment of TCR molecules into MCs at stimulatory contacts (Fig. 1E; 14.6 6 6.2%, 7.8 6 3.5%, 10.2 6 5.5% for control, Nef, and Nef F195I, respectively). In addition to these effects on the TCR, Nef potently retargets Lck to the TGN and thus markedly reduces its availability at sites of TCR engagement (40). To estimate the direct functional consequence of these alterations in recruitment of TCR and Lck to sites of stimulation, the distribution and MC incorporation of the downstream kinase ZAP70 were investigated (Fig. 2). Surprisingly, ZAP70-positive MCs were observed with comparable frequency in the presence or absence of Nef (Fig. 2A, 2B; 80.7 6 6.1% for control, 73.7 6 5.4% for Nef, and 77.2 6 10.4% for Nef F195I). Nef also did not significantly affect ZAP70 MC density (Fig. 2C; 0.7 6 0.1 per mm2 for control, 0.6 6 0.1 per mm2 for Nef, and 0.7 6 0.1 per mm2 for Nef F195I) or mean MC size/cell (Fig. 2D; 0.07 6 0.01 mm2 for control, 0.06 6 0.01 mm2 for Nef, and 0.07 6 0.01 mm2 for Nef F195I) and did FIGURE 5. Nef disrupts proliferation and translocation of SLP-76 MCs. (A) Still images of time-lapse Supplemental Videos 1–3 with the time points indicated in seconds. Analyses were conducted with J14 SLP-76.YFP cells transiently expressing the indicated RFP fusion proteins. Twenty-four hours posttransfection, cells were dropped on anti-CD3ε–coated glass-bottom dishes. Image acquisition was started upon appearance of first MCs (t = 0) and continued over 300 s. Upper and lower panels, Depict original and binary images of the YFP channel, respectively. The RFP panel on the right depicts the RFP channel at the end of the video. Original magnification 320. Time series plot indicating SLP-76 MC number (B) and total MC area (C) for cells shown in (A). The images and time series plots presented are representative for three independent experiments with at least three cells analyzed per condition. Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 CD3 Ab to trigger TCR signaling, fixed 5 min later, and subjected to immunostaining against the myc epitope tag to identify Nef-positive cells. At this time point, control cells displayed hallmarks of early T cell activation, including cell spreading and formation of circumferential F-actin rings, both of which were potently inhibited by the presence of Nef, as reported earlier (Fig. 1A) (37–39, 48). Coexpression of the TCR z-chain fused to GFP (TCR.GFP) in these cells revealed the formation of numerous MCs at cell-cover glass stimulatory contacts that were distributed across the entire cell diameter, including its periphery (see schematic in Supplemental Fig. 1). As described previously (37, 39, 40, 49), Nef localized to the plasma membrane, diffusely in the cytoplasm and at a perinuclear compartment, but was not detected in MCs. Quantification of the frequency of cells that displayed TCR MCs revealed that Nef did not significantly affect their overall occurrence (Fig. 1B; 78.5 6 7.4%, 68.5 6 9.5%, and 78.7 6 8.2% for control, Nef, and Nef F195I, respectively). The Nef mutant F195I, which specifically fails to associate with PAK2 and thus does not alter host cell actin remodeling and cell spreading, but exerts all other know activities similarly to Nef WT (50–52), was included as additional control. Nef F195I displayed a subcellular localization similar to that of Nef and had also no effect on the frequency of TCR MC formation. In addition to this analysis at 5 min post-TCR engagement, MC formation was also assessed for up to 30 min following plating of cells on TCR stimulatory surfaces (Supplemental Fig. 2A). This kinetic analysis revealed that Nef induced an intracellular accumulation of TCR. GFP prior to T cell activation, as reported (36). Nevertheless, formation of TCR MCs and disassembly toward the end of the observation period were indistinguishable between control and Nef-expressing cells. DISRUPTION OF EARLY TCR SIGNALING BY HIV-1 Nef The Journal of Immunology not influence the magnitude of ZAP70 recruitment to TCR stimulatory contacts (Fig. 2E; 7.7 6 1.8% for control, 6.2 6 2.6% for Nef, and 8.9 6 3.9% for Nef F195I). To test the functionality of such ZAP70 MCs, we used a phospho-specific Ab to ZAP70 that detects the active form of ZAP70 following autophosphorylation of Tyr319 (pZAP70). Similar to bulk ZAP70, Nef failed to alter the frequency and density of MCs containing pZAP70; however, it slightly reduced the mean size of pZAP70-containing MC/cell and the overall recruitment of pZAP70 to stimulatory contacts (Supplemental Fig. 3). Consistent with our previous findings (37) and presumably reflecting the low magnitude of Nef’s direct effects on TCR-CD3 and a functional substitution of Lck by Fyn in Nef-expressing cells (40), HIV-1 Nef does not cause a major disruption of the earliest stages of TCR signaling involving the formation of TCR-ZAP70 MCs. Nef potently disrupts the formation of SLP-76 MC following TCR engagement a potent block that Nef imposes on the formation and persistence of SLP-76 MC following TCR engagement. Colocalization of activated ZAP70 with SLP-76 is minimized in the presence of Nef Because efficient communication between the TCR and SLP-76 relies on the transient colocalization of active ZAP70 MCs with SLP-76 MCs (42), we next addressed how the Nef-mediated disruption of formation of SLP-76 MCs impacts on the colocalization of pZAP70 with SLP-76 (Fig. 6). Following exposure to TCR stimulatory surfaces, J14SLP-76.YFP cells were stained for pZAP70 (Tyr319), and rendered binary images from both SLP-76 and pZAP70 channels were employed to determine Manders’ colocalization coefficients. Expectedly (44), significantly more MCs positive for pZAP70 than for SLP-76 were observed in the absence of Nef. Of these, almost all SLP-76 MCs contained active pZAP70 (86.3 6 9.1%; M2) and a substantial fraction of pZAP70 Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 We next addressed the effect of Nef on the behavior of SLP-76, a signaling adaptor that is recruited into MCs following TCR engagement in a ZAP70-dependent manner (12). J14SLP-76.YFP cells (Jurkat T lymphocytes stably expressing SLP-76 fused to YFP) were used for these analyses as established model of SLP-76 MC formation (43, 44). Whereas control cells displayed a large number of SLP-76–positive MCs across the cover glass-cell interface, including the cell periphery, only few such MCs could be detected in cells expressing Nef with most cells virtually lacking detectable SLP-76 MCs (Fig. 3A, 3B; 86.0 6 3.6% for control, 27.2 6 7.2% for Nef, and 46.1 6 2.5% for Nef F195I). Nef also exerted potent negative effects on SLP-76 MC density (Fig. 3C; 0.6 6 0.1 per mm2 for control, 0.2 6 0.1 per mm2 for Nef, and 0.3 6 0.1 per mm2 for Nef F195I), the mean SLP-76 MC size per cell (Fig. 3D; 0.11 6 0.1 mm2 for control, 0.06 6 0.02 mm2 for Nef, and 0.06 6 0.01 mm2 for Nef F195I), and the overall recruitment of SLP-76 to MCs at stimulatory contacts (Fig. 3E; 16.2 6 4.1%, 3.3 6 3.6%, and 3.9 6 2.7% for control, Nef, and Nef F195I, respectively). Similar disruption of SLP-76 MCs by Nef was also observed when cells were costimulated using anti-CD3 and anti-CD28 Abs (data not shown). Importantly, these effects were not a direct consequence of Nef-induced inhibition of actin remodeling and cell spreading, as Nef F195I, which lacks these inhibitory activities, disrupted SLP-76 MC formation and organization as efficiently as Nef. F195I-independent inhibition of SLP-76 MC formation to similar frequency and magnitude by Nef was also observed at physiological levels of expression in the context of HIV-1 infection of J14SLP-76.YFP cells (Fig. 4; productively infected cells identified by p24CA immunostaining). Real-time imaging of SLP-76 MC formation revealed that in the absence of Nef, few MCs were formed at the initial contact site of cells with the stimulatory surface (t = 0). The number of MCs then rapidly increased concomitant with cell spreading, and MCs subsequently persisted across the entire surface area of the cell (Fig. 5; RFP). Initial MCs were also observed in the presence of Nef; however, no subsequent MC proliferation and translocation occurred (Fig. 5; Nef.RFP). Nef F195I prevented MC proliferation in a manner similar to Nef; however, only few MCs were detected at the cell periphery at later time points, presumably reflecting the cells’ ability to spread in the presence of this mutant Nef protein (Fig. 5; Nef F195I.RFP). No effect of Nef or a palmitoylated Nef mutant with enhanced membrane association (53) on SLP-76 localization was found in unstimulated cells prior to TCR activation (Supplemental Fig. 3F), and SLP-76–containing MCs were also not observed at late time points following TCR stimulation (Supplemental Fig. 2B). Together these results identify 7 FIGURE 6. HIV-1 Nef minimizes the colocalization of SLP-76 with active ZAP70 in MCs. Twenty-four hours posttransfection with the indicated plasmids, J14 SLP-76.YFP cells were stimulated on anti-CD3ε–coated cover glasses for 5 min, fixed, and stained for phospho-ZAP70. (A) Representative binary images were generated for each channel using identical settings, and the degree of overlap of MC pixels was determined to compute the coefficients M1 and M2 (as described in Materials and Methods). Red lines indicate cell boundaries. Scale bar, 10 mm. Single-cell quantification indicating the percentage of overlap of pZAP70 with SLP-76 MCs (M1) (B) and the percentage of overlap of SLP-76 with pZAP70 MCs (M2) (C). Depicted are values from .25 cells analyzed per condition in which each symbol designates the value for an individual cell. Red bars indicate the mean values of all cells analyzed. The p values were determined using the Mann-Whitney U test (*p , 0.01, **p , 0.001, ***p , 0.0001). 8 MCs was positive for SLP-76 (20.82 6 6.53%; M1). As before, expression of Nef or Nef F195I markedly reduced the number of SLP-76 MCs, resulting in a minimal fraction of pZAP70 MCs that contained SLP-76 (Fig. 6B; 4.2 6 2.7%, M1 for Nef and 4.0 6 2.4%, M1 for Nef F195I). The few remaining SLP-76 MCs, however, contained pZAP70 as frequently as in the absence of Nef (Fig. 6C; 82.6 6 18.2%, M2 for Nef and 80.0 6 7.2%, M2 for Nef F195I). Thus, Nef potently decreases the functional interaction between active ZAP70 and SLP-76 by reducing the abundance of SLP-76–containing MCs. Nef affects the subcellular localization of the adaptor protein LAT the plasma membrane, but also diffusely in the cytoplasm and intracellular compartments. In contrast, expression of Nef or Nef F195I induced a marked enrichment of LAT.GFP at a prominent intracellular membrane compartment (Fig. 7A). This effect was reminiscent of the Nef-mediated targeting of Lck to the TGN (36, 38, 40); however, in preliminary colocalization analyses, we failed to detect substantial overlap between the LAT.GFP-positive compartment and subcellular markers for the TGN or early and late endosomes, leaving the nature of this compartment undefined (data not shown). Such intracellular accumulation of LAT.GFP was frequently observed in cells expressing Nef or Nef F195I (55.7 6 4.04% and 42.3 6 7.4%, respectively), but not in control cells (7.7 6 2.6%). Notably, a subpopulation of Nef and NefF195I also appeared to reside at this intracellular compartment. Singlecell quantification of the LAT.GFP signal detected in the intracellular accumulation relative to total LAT.GFP signal per cell revealed that Nef and Nef F195I induced a ∼3-fold enrichment of LAT.GFP in this compartment (Fig. 7C; total LAT per cell in intracellular accumulation: control, 7.3 6 2.8%; Nef, 23.6 6 5.8%; Nef F195I, 24.8 6 6.4%). F195-independent intracellular FIGURE 7. Nef alters the subcellular localization of LAT. (A) Representative sum intensity projections of wide-field z-stacks of Jurkat E6.1 cells stably expressing LAT.GFP transiently expressing the indicated proteins. Cells were dropped on polylysine-coated cover glasses for 5 min, fixed, and stained for F-actin and myc. Scale bars, 10 mm. (B) Frequency of cells with pronounced LAT accumulation. Depicted are mean values from three independent experiments 6 SD with at least 100 cells analyzed per condition. Cells were scored as positive for intracellular LAT accumulation if they exhibited a pronounced enrichment of LAT in a large intracellular compartment not detected in untransfected neighboring cells. The p values were determined using the Student t test. (C) Quantification of LAT distribution in single cells. Presented are the percentages of the LAT signal detected in intracellular accumulation versus the total LAT signal in the cell [similar to the recently described quantification of Lck TGN targeting shown in (40)]. Depicted are values from .25 cells analyzed per condition in which each symbol designates the value for an individual cell. Gray bars indicate the mean values of all cells analyzed. The p values were determined using the Mann-Whitney U test. (D) Representative micrographs of Jurkat E6.1 cells stably expressing LAT.GFP infected with WT HIV-1 (HIV-1 WT), HIV-1ΔNef, or HIV-1 Nef F195I. Forty-eight hours postinfection, cells adhered to polylysine-coated cover glasses for 5 min, fixed, and stained for intracellular p24CA and F-actin. Scale bars, 10 mm. (E) Frequency of the infected cells shown in (A) with pronounced intracellular LAT accumulation. Depicted are mean values from three independent experiments 6 SD with at least 100 cells analyzed per each condition. Cells were scored positive for intracellular LAT accumulation if they exhibited a pronounced intracellular enrichment of LAT relative to that of uninfected neighboring cells. The p values were determined using the Student t test (*p , 0.01, **p , 0.001, ***p , 0.0001). Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 Formation of SLP-76 MCs critically depends on the adaptor protein LAT (11, 54, 55). In search for a mechanism by which Nef prevents the formation of SLP-76 MCs, we therefore analyzed next whether the viral protein affects the subcellular localization of LAT using Jurkat E6.1 cells stably expressing LAT.GFP that had been FACS sorted for medium levels of LAT.GFP expression. In the absence of TCR stimulation, LAT.GFP was detected mainly at DISRUPTION OF EARLY TCR SIGNALING BY HIV-1 Nef The Journal of Immunology enrichment of LAT.GFP by Nef was also observed with comparable frequency in the context of HIV-1 infection (Fig. 7D, 7E; HIV-1 Δ Nef, 14 6 4%; HIV-1 WT, 55.3 6 6.1%; HIV-1 Nef F195I, 39 6 4.5%; infected cells identified by p24CA immunostaining). Despite this altered subcellular localization of LAT, Nef or Nef F195I only exerted subtle effects on the frequency of formation of LAT MCs, LAT MC density, or recruitment of the signaling adaptor to TCR stimulatory contact sites (Fig. 8). Nef interferes with the induction of pLAT and its recruitment to SLP-76 MC remaining SLP-76 MCs were mostly positive for pLAT (Supplemental Fig. 4B, 4C). Inhibition of pLAT-containing MCs persisted .30 min post-TCR activation, and, similar to total LAT, a slight intracellular enrichment of pLAT was observed prior to TCR activation in the presence of Nef or NefF195I (Supplemental Fig. 2C). Comparable Nef-dependent reduction in the frequency of cells that displayed pLAT MCs post-TCR stimulation was also observed upon HIV-1 infection of primary human T lymphocytes (Fig. 9F, 9G). In cells that displayed pLAT MCs despite the expression of Nef or Nef F195I, the mean size of pLAT MCs per cell was significantly reduced relative to control cells (Fig. 9D; 0.07 6 0.01 mm2 for Nef, 0.06 6 0.01 mm2 for Nef F195I, 0.12 6 0.01 mm2 for control cells). However, the number of these smaller MCs per cell was comparable to that of large MCs observed in control cells, resulting in similar MC densities in these cell populations (Fig. 9C). This reduction in mean pLAT MC size most likely reflected the Nef-mediated decrease in pLAT MC recruitment to stimulatory contact sites (Fig. 9E; 33.2 6 4.5%, 12.3 6 4.1%, and 10.3 6 3.9% for control, Nef, and Nef F195I, respectively). Similar to the disruption of SLP-76 MCs by Nef, costimulation with anti-CD3 and anti-CD28 Abs did not facilitate induction of pLAT MCs (data not shown). These results revealed that Nef impairs the recruitment of active LAT to TCR activation sites and its organization in MCs in a manner that does partially depend on the F195 interaction motif. Disruption of SLP-76 MCs by Nef is a combined effect of reduced actin dynamics and altered intracellular transport FIGURE 8. Nef does not affect LAT MC formation. (A) Representative micrographs of Jurkat T lymphocytes stably expressing LAT.YFP transiently expressing the indicated proteins following 5-min stimulation on anti-CD3–coated cover glasses. Scale bars, 10 mm. Single-cell quantification indicating LAT MC density (B) and magnitude of recruitment into MC in the contact plane (C) is shown. The p values were determined using the Mann-Whitney U test (*p , 0.01, **p , 0.001, ***p , 0.0001). Trapping of Lck at the TGN in the presence of Nef can be overcome by coexpression of the plasma membrane transport adaptor Unc119 (40). Because the alterations induced by Nef to the subcellular localization of LAT resembled its effects on Lck, we tested whether coexpression of Unc119 could also restore an undisturbed subcellular localization for LAT in the presence of Nef. Whereas Nef caused the intracellular accumulation of LAT.GFP in almost 60% of RFP-expressing control cells, coexpression of Unc119. RFP significantly reduced the frequency of cells that displayed such LAT.GFP accumulation to levels only slightly above those observed in the absence of Nef (Fig. 10A, 10B; control cells, 15.0 6 5.0%; Nef, 54 6 8.5%; Nef + Unc119.RFP, 25.0 6 5.0%; Unc119.RFP, 15.0 6 3.0%). We thus tested whether coexpression of Unc119 was also sufficient to restore the TCR-induced formation of signaling MCs in Nef-expressing cells. As before, in GFPexpressing control cells, Nef potently disrupted the formation of MCs containing pLAT (Fig. 10C, 10E) or SLP-76 (Fig. 10D, 10F), and this activity partially depended on Nef’s ability to prevent actin remodeling and cell spreading (see Nef F195I). Coexpression of Unc119 with Nef did not reduce the suppression of MC formation by the viral protein. In sharp contrast, formation of MCs positive for pLAT as well as for SLP-76 was indistinguishable from Nef-negative cells when Nef F195I was coexpressed with Unc119 (Fig. 10E, control, 73.3 6 1.8%; Nef + Unc119, 26.6 6 2.3%; Nef F195I + Unc119, 70.8 6 7.7% for pLAT, and Fig. 10F, control, 68.0 6 2.6%; Nef + Unc119, 28.0 6 5.5%; Nef F195I + Unc119, 69.6 6 4.5% for SLP-76). Importantly, under these conditions, SLP-76 MC density, mean MC size per cell, and magnitude of MC localization were essentially restored (Supplemental Fig. 4E–G). Together these results reveal that Nef employs a combination of interference with host cell actin remodeling and intracellular transport to disrupt early TCR signaling. The combination of these effects disrupts communication between ZAP70 and SLP-76, which prevents the formation of pLAT/SLP-76– containing signaling MCs critical to mediate downstream signaling following TCR stimulation. Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 Because the above analyses focused on bulk LAT, we next used a phospho-specific Ab to specifically analyze the subpopulation of LAT that is able to associate with SLP-76 due to the phosphorylation at tyrosine 191 (pLAT) (Fig. 9). pLAT MCs were readily observed in 70% of control cells on anti-CD3–coated cover glasses. Similar to pZAP70, more MCs per cell positive for pLAT than for SLP-76 were observed and most SLP-76 MCs contained pLAT (Supplemental Fig. 4A–C). Paralleled by the potent disruption of SLP-76 MCs, expression of Nef reduced the frequency of cells displaying clearly detectable pLAT MCs, whereas the inhibitory activity of Nef F195I was slightly reduced (Fig. 9B; control, 70.3 6 2.8%; Nef, 28.9 6 3.0%; Nef F195I, 47.6 6 3.0%). Consistently, both Nef proteins markedly reduced the occurrence of pLAT MCs that contained SLP-76, whereas the 9 10 DISRUPTION OF EARLY TCR SIGNALING BY HIV-1 Nef Discussion Aiming at dissecting the steps in early TCR signaling that are affected by the HIV-1 pathogenesis factor Nef, we carried out a quantitative analysis of formation and composition of signaling MCs induced by surface-supported TCR stimulation. These analyses revealed that the predominant action of Nef in the TCR signaling cascade is below the initial events mediated by TCR-CD3 and the proximal kinase ZAP70. Rather, Nef, upon isolated expression or in the context of HIV-1 infection, potently disrupts the formation of MCs in which the signaling adaptors SLP-76 and active LAT reside in close proximity to elicit and sustain downstream signaling. Based on our mechanistic analysis, this defect reflects the synergy between two independent mechanisms by which Nef 1) limits the availability of LAT for de novo MC recruitment by retargeting the adaptor to an intracellular compartment, and 2) prevents cell spreading and MC proliferation by disrupting dynamic actin remodeling triggered upon TCR engagement. The quantitative microscopy approach employed in this study enabled us for the first time to appreciate the effects of Nef at the level of individual signaling MCs. The results obtained significantly expand previous observations on the inhibitory action of Nef on early TCR signaling. In line with a previous report (36), the recruitment of TCR-CD3 to stimulatory contacts was slightly reduced by Nef. However, the undisturbed organization of MCs containing ZAP70, an immediate kinase downstream of TCR-CD3 that is activated by Lck, indicated that direct effects of HIV-1 Nef on TCR-CD3 and Lck do not account for the disruption of subsequent signaling events. Consistent with our previous observation that Nef reduces the extend of LAT phosphorylation following TCR stimulation (37), the results presented rather demonstrate that Nef predominantly affects the TCR signaling cascade downstream of ZAP70 and define the potent inhibition of recruitment of adaptor-competent, phosphorylated LAT and SLP-76 into signaling MCs as the central underlying mechanism. Mechanistically, our results suggest that this defect in early TCR signaling in the presence of Nef is determined by alterations in the subcellular localization of LAT. The ability of LAT to serve as signaling adaptor is initiated by ZAP70 via phosphorylation when LAT is in close proximity to sites of TCR engagement (56). This Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 FIGURE 9. Nef prevents recruitment of phosphorylated LAT to stimulatory contact sites. (A) Representative micrographs of J14SLP-76.YFP cells transiently expressing the indicated proteins. Twenty-four hours posttransfection, cells were stimulated on anti-CD3ε–coated coverslips for 5 min, fixed, and stained for phospho-LAT. Scale bars, 10 mm. (B) Frequency of cells shown in (A) with pronounced pLAT MCs at stimulatory contact sites. Depicted are mean values from three independent experiments 6 SD with at least 100 cells analyzed per condition. Cells were scored positive for pLAT- and SLP-76– containing MCs if they exhibited prominent MCs comparable to those of untransfected neighboring cells. The p values were determined using the Student t test. Single-cell quantification indicating pLAT MC density (C), mean MC size per cell (D), and magnitude of recruitment into MC in the contact plane (E) was calculated using the spot detection algorithm. Depicted are values from .25 cells analyzed per condition in which each symbol designates the value for an individual cell. Red bars indicate the mean values of all cells analyzed. The p values were determined using the Mann-Whitney U test. (F) Representative micrographs of primary human T lymphocytes infected with WT HIV-1 (HIV-1 WT), HIV-1ΔNef, or HIV-1 Nef F195I. Forty-eight hours postinfection, cells were stimulated on anti-CD3ε–coated cover glasses for 5 min, fixed, and stained for pLAT and p24 capsid. Scale bar, 10 mm. (G) Frequency of the cells shown in (F) with pLAT-containing MCs at the contact site. Depicted are mean values from duplicate infections 6 SD for two independent donors with at least 100 cells analyzed per condition. *p , 0.01, **p , 0.001, ***p , 0.0001. The Journal of Immunology 11 phosphorylation event enables pLAT to form complexes with GADS and SLP-76 that are organized in signaling MCs essential for phospholipase Cg1 activation and subsequent RAS signaling. Conceivably, the retargeting of LAT to intracellular compartments and thus away from sites of TCR engagement at the plasma membrane in Nef-expressing cells significantly limits its availability for LAT phosphorylation by ZAP70, causing the prominent defect in assembly of SLP-76 MCs. According to an emerging concept, signaling competent MCs depend on the de novo recruitment of LAT from intracellular pools to TCR-proximal sites, where it is activated by phosphorylation, whereas pre-existing LAT MCs at the plasma membrane are largely devoid of pLAT and thus signaling incompetent (57, 58). The LAT MCs observed in Nef- or Nef F195I-expressing cells most likely represent such pre-existing, inactive MCs. In this scenario, the intracellular pool of LAT in Nef-expressing cells would thus not be available for recruitment to the plasma membrane as well as subsequent phosphorylation and incorporation into signaling competent MCs. The use of Nef mutants and overexpression of Unc119 allowed us to define that Nef mediates this block in MC formation via two independent molecular mechanisms. First, the partial requirement of the interaction motif surrounding F195 indicated that the Nef-mediated block to the formation of pLAT MCs involves the disruption of critical dynamic actin remodeling events. Notably, it was recently demonstrated that SLP-76 MC organization after TCR engagement depends on dynamic actin remodeling (56). Second, the sensitivity of the block in MC formation by overexpression of Unc119 indicated that specific anterograde transport processes of LAT are inhibited in the presence of Nef. Unc119 acts as adaptor protein in the transport of Lck to the plasma membrane (59) and may facilitate intracellular trafficking of LAT via similar mechanisms. Nef might interfere with these processes by, for example, prevention of the association of transport cargo with the Unc119 transport machinery or by modulating the activity of the associated Rab11 GTPase (40, 59, 60). In this scenario, the differential destination of Lck (TGN) and LAT (undefined) upon disruption of their anterograde transport in the presence of Nef might reflect their specific topology as peripheral and integral membrane protein, respectively. Despite this similarity in transport routes used by LAT and Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 FIGURE 10. Nef-mediated inhibition of actin remodeling and relocalization of LAT synergize to the disruption of SLP-76 MC formation. (A) Representative micrographs of Jurkat E6.1 cells stably expressing LAT.GFP transiently expressing the indicated proteins. Following adherence on polylysine-coated cover glasses for 5 min, cells were fixed and stained for Myc. Scale bars, 10 mm. (B) Frequency of cells with pronounced LAT accumulation. Depicted are mean values from three independent experiments 6 SD with at least 100 cells analyzed per condition. Cells were scored as positive for intracellular LAT accumulation if they exhibited a pronounced enrichment of LAT in intracellular compartments relative to untransfected neighboring cells. The p values were determined using the Student t test. (C) Representative micrographs of Jurkat T cells transiently expressing the indicated proteins. Twenty-four hours posttransfection, cells were stimulated on anti-CD3ε–coated cover glasses for 5 min, fixed, and stained for phospho-LAT. (D) Representative micrographs of J14SLP-76.YFP cells transiently expressing the indicated proteins. Cells were stimulated on anti-CD3ε–coated cover glasses for 5 min, fixed, and stained for Myc. Binary images of YFP channels were shown in lower panel, and gray lines indicate cell boundaries. Scale bars, 10 mm. Frequency of cells shown in (C, E) and (D, F) with pronounced pLAT and SLP-76 MCs, respectively, at stimulatory contact site. Depicted are mean values from three independent experiments 6 SD with at least 100 cells analyzed per condition. Cells were scored positive for pLAT or SLP-76 MCs if they exhibited MCs comparable to those of untransfected neighboring cells. The p values were determined using the Student t test (*p , 0.01, **p , 0.001, ***p , 0.0001). 12 Acknowledgments We are grateful to Drs. Stephen Bunnell, Lawrence Samelson, and Mark Davis for the gift of reagents, Nadine Tibroni for expert technical help, and Bettina Stolp, Jochen Rudolph, and Oliver T. Keppler for stimulating discussion. Disclosures The authors have no financial conflicts of interest. References 1. Stevenson, M. 2003. HIV-1 pathogenesis. Nat. Med. 9: 853–860. 2. Billadeau, D. D., J. C. Nolz, and T. S. Gomez. 2007. Regulation of T-cell activation by the cytoskeleton. Nat. Rev. Immunol. 7: 131–143. 3. Balagopalan, L., E. Sherman, V. A. Barr, and L. E. Samelson. 2011. Imaging techniques for assaying lymphocyte activation in action. Nat. Rev. Immunol. 11: 21–33. 4. Saito, T., and T. Yokosuka. 2006. Immunological synapse and microclusters: the site for recognition and activation of T cells. Curr. Opin. Immunol. 18: 305–313. 5. Seminario, M. C., and S. C. Bunnell. 2008. Signal initiation in T-cell receptor microclusters. Immunol. Rev. 221: 90–106. 6. Yokosuka, T., and T. Saito. 2009. Dynamic regulation of T-cell costimulation through TCR-CD28 microclusters. Immunol. Rev. 229: 27–40. 7. Denny, M. F., B. Patai, and D. B. Straus. 2000. Differential T-cell antigen receptor signaling mediated by the Src family kinases Lck and Fyn. Mol. Cell. Biol. 20: 1426–1435. 8. Iwashima, M., B. A. Irving, N. S. van Oers, A. C. Chan, and A. Weiss. 1994. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263: 1136–1139. 9. Chan, A. C., M. Iwashima, C. W. Turck, and A. Weiss. 1992. ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Cell 71: 649– 662. 10. Au-Yeung, B. B., S. Deindl, L. Y. Hsu, E. H. Palacios, S. E. Levin, J. Kuriyan, and A. Weiss. 2009. The structure, regulation, and function of ZAP-70. Immunol. Rev. 228: 41–57. 11. Balagopalan, L., N. P. Coussens, E. Sherman, L. E. Samelson, and C. L. Sommers. 2010. The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harb. Perspect. Biol. 2: a005512. 12. Paz, P. E., S. Wang, H. Clarke, X. Lu, D. Stokoe, and A. Abo. 2001. Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells. Biochem. J. 356: 461–471. 13. Liu, S. K., N. Fang, G. A. Koretzky, and C. J. McGlade. 1999. The hematopoietic-specific adaptor protein gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors. Curr. Biol. 9: 67-75. 14. Koretzky, G. A., F. Abtahian, and M. A. Silverman. 2006. SLP76 and SLP65: complex regulation of signalling in lymphocytes and beyond. Nat. Rev. Immunol. 6: 67–78. 15. Fackler, O. T., A. Alcover, and O. Schwartz. 2007. Modulation of the immunological synapse: a key to HIV-1 pathogenesis? Nat. Rev. Immunol. 7: 310–317. 16. Schindler, M., J. Münch, O. Kutsch, H. Li, M. L. Santiago, F. Bibollet-Ruche, M. C. Müller-Trutwin, F. J. Novembre, M. Peeters, V. Courgnaud, et al. 2006. Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell 125: 1055–1067. 17. Laguette, N., C. Brégnard, S. Benichou, and S. Basmaciogullari. 2010. Human immunodeficiency virus (HIV) type-1, HIV-2 and simian immunodeficiency virus Nef proteins. Mol. Aspects Med. 31: 418–433. 18. Kestler, H. W., III, D. J. Ringler, K. Mori, D. L. Panicali, P. K. Sehgal, M. D. Daniel, and R. C. Desrosiers. 1991. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65: 651– 662. 19. Deacon, N. J., A. Tsykin, A. Solomon, K. Smith, M. Ludford-Menting, D. J. Hooker, D. A. McPhee, A. L. Greenway, A. Ellett, C. Chatfield, et al. 1995. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270: 988–991. 20. Kirchhoff, F., T. C. Greenough, D. B. Brettler, J. L. Sullivan, and R. C. Desrosiers. 1995. Brief report: absence of intact nef sequences in a longterm survivor with nonprogressive HIV-1 infection. N. Engl. J. Med. 332: 228– 232. 21. Hanna, Z., D. G. Kay, N. Rebai, A. Guimond, S. Jothy, and P. Jolicoeur. 1998. Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95: 163–175. 22. Geyer, M., O. T. Fackler, and B. M. Peterlin. 2001. Structure-function relationships in HIV-1 Nef. EMBO Rep. 2: 580–585. 23. Kirchhoff, F., M. Schindler, A. Specht, N. Arhel, and J. Münch. 2008. Role of Nef in primate lentiviral immunopathogenesis. Cell. Mol. Life Sci. 65: 2621– 2636. 24. Baur, A. S., E. T. Sawai, P. Dazin, W. J. Fantl, C. Cheng-Mayer, and B. M. Peterlin. 1994. HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization. Immunity 1: 373–384. 25. Fackler, O. T., and A. S. Baur. 2002. Live and let die: Nef functions beyond HIV replication. Immunity 16: 493–497. 26. Simmons, A., V. Aluvihare, and A. McMichael. 2001. Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity 14: 763–777. 27. Simmons, A., B. Gangadharan, A. Hodges, K. Sharrocks, S. Prabhakar, A. Garcı́a, R. Dwek, N. Zitzmann, and A. McMichael. 2005. Nef-mediated lipid raft exclusion of UbcH7 inhibits Cbl activity in T cells to positively regulate signaling. Immunity 23: 621–634. 28. Fenard, D., W. Yonemoto, C. de Noronha, M. Cavrois, S. A. Williams, and W. C. Greene. 2005. Nef is physically recruited into the immunological synapse and potentiates T cell activation early after TCR engagement. J. Immunol. 175: 6050–6057. 29. Fortin, J. F., C. Barat, Y. Beauséjour, B. Barbeau, and M. J. Tremblay. 2004. Hyper-responsiveness to stimulation of human immunodeficiency virus-infected CD4+ T cells requires Nef and Tat virus gene products and results from higher NFAT, NF-kappaB, and AP-1 induction. J. Biol. Chem. 279: 39520–39531. 30. Schrager, J. A., V. Der Minassian, and J. W. Marsh. 2002. HIV Nef increases T cell ERK MAP kinase activity. J. Biol. Chem. 277: 6137–6142. 31. Schrager, J. A., and J. W. Marsh. 1999. HIV-1 Nef increases T cell activation in a stimulus-dependent manner. Proc. Natl. Acad. Sci. USA 96: 8167–8172. 32. Manninen, A., G. H. Renkema, and K. Saksela. 2000. Synergistic activation of NFAT by HIV-1 nef and the Ras/MAPK pathway. J. Biol. Chem. 275: 16513–16517. 33. Wu, Y., and J. W. Marsh. 2001. Selective transcription and modulation of resting T cell activity by preintegrated HIV DNA. Science 293: 1503–1506. 34. Keppler, O. T., N. Tibroni, S. Venzke, S. Rauch, and O. T. Fackler. 2006. Modulation of specific surface receptors and activation sensitization in primary resting CD4+ T lymphocytes by the Nef protein of HIV-1. J. Leukoc. Biol. 79: 616–627. 35. Arhel, N., M. Lehmann, K. Clauss, G. U. Nienhaus, V. Piguet, and F. Kirchhoff. 2009. The inability to disrupt the immunological synapse between infected Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 Lck, the analysis of various Nef mutants revealed that TGN targeting of Lck is not sufficient to block formation of SLP-76 MCs (data not shown), thus further underscoring the critical role of LAT in this process. The need to overcome simultaneously Nef’s effects on actin dynamics and vesicular transport governing the subcellular localization of LAT suggests that vesicular LAT is driven to TCR activation sites for phosphorylation by cytoskeletal remodeling. The two mechanisms used by Nef would thus act on individual steps of the same process. With the identification of Lck and LAT as cargo of this Nef-sensitive trafficking pathway, it is beginning to emerge that Nef acts selectively on this general transport route. Defining how this specificity is achieved represents an important goal of future studies. Together with results from previous studies, our current findings can be integrated in a comprehensive view on how Nef manipulates TCR signaling in infected T lymphocytes. Early TCR signaling events are markedly decreased by the viral protein, reflecting its ability to reduce the frequency of stimulatory cell-cell contacts (39) and to limit LAT–SLP-76–mediated signaling. Possibly reflecting a high selective pressure on this Nef activity, disruption of TCRproximal events is mediated by two synergistic mechanisms affecting host cell actin dynamics and vesicular transport. The relative quantitative impact of these individual changes as well as the more subtle effects of Nef on TCR recruitment to stimulatory contacts on strength and breadth of signal initiation in individual MCs as well as TCR downstream signaling remains currently unknown. Addressing this issue will require quantitative and timeresolved visualization of signaling processes, which will provide important insight in the input-output relationship of TCR signaling at the level of single cells or even individual MCs. In the context of HIV-1 infection, however, this negative regulation of TCRproximal signaling is converted into a selective enhancement of Ras-Erk signaling downstream of the TCR via the rerouting of active Lck to the TGN (40). Disruption of the formation of TCRproximal signaling MCs and induction of intracellular TCR signaling is thus achieved by hijacking the same vesicular transport pathway as a remarkably efficient strategy of host cell manipulation. Our recent results suggest that these mechanisms enable Nef to promote survival of infected T lymphocytes and to facilitate HIV spread following antigenic stimulation (40). Future studies will address the relevance of this mechanism for AIDS pathogenesis and attempt the pharmacological inhibition of this conserved Nef activity. DISRUPTION OF EARLY TCR SIGNALING BY HIV-1 Nef The Journal of Immunology 36. 37. 38. 39. 40. 41. 42. 43. 45. 46. 47. 48. 49. Wolf, D., S. I. Giese, V. Witte, E. Krautkrämer, S. Trapp, G. Sass, C. Haller, K. Blume, O. T. Fackler, and A. S. Baur. 2008. Novel (n)PKC kinases phosphorylate Nef for increased HIV transcription, replication and perinuclear targeting. Virology 370: 45–54. 50. Rauch, S., K. Pulkkinen, K. Saksela, and O. T. Fackler. 2008. Human immunodeficiency virus type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface into plasma membrane microdomains for association with p21-activated kinase 2 activity. J. Virol. 82: 2918–2929. 51. Agopian, K., B. L. Wei, J. V. Garcia, and D. Gabuzda. 2006. A hydrophobic binding surface on the human immunodeficiency virus type 1 Nef core is critical for association with p21-activated kinase 2. J. Virol. 80: 3050–3061. 52. Schindler, M., D. Rajan, A. Specht, C. Ritter, K. Pulkkinen, K. Saksela, and F. Kirchhoff. 2007. Association of Nef with p21-activated kinase 2 is dispensable for efficient human immunodeficiency virus type 1 replication and cytopathicity in ex vivo-infected human lymphoid tissue. J. Virol. 81: 13005–13014. 53. Krautkrämer, E., S. I. Giese, J. E. Gasteier, W. Muranyi, and O. T. Fackler. 2004. Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts. J. Virol. 78: 4085–4097. 54. Zhang, W., J. Sloan-Lancaster, J. Kitchen, R. P. Trible, and L. E. Samelson. 1998. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92: 83–92. 55. Wange, R. L. 2000. LAT, the linker for activation of T cells: a bridge between T cell-specific and general signaling pathways. Sci. STKE 2000: re1. 56. Sherman, E., V. Barr, S. Manley, G. Patterson, L. Balagopalan, I. Akpan, C. K. Regan, R. K. Merrill, C. L. Sommers, J. Lippincott-Schwartz, and L. E. Samelson. 2011. Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor. Immunity 35: 705–720. 57. Williamson, D. J., D. M. Owen, J. Rossy, A. Magenau, M. Wehrmann, J. J. Gooding, and K. Gaus. 2011. Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol. 12: 655–662. 58. Purbhoo, M. A., H. Liu, S. Oddos, D. M. Owen, M. A. Neil, S. V. Pageon, P. M. French, C. E. Rudd, and D. M. Davis. 2010. Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse. Sci. Signal. 3: ra36. 59. Gorska, M. M., Q. Liang, Z. Karim, and R. Alam. 2009. Uncoordinated 119 protein controls trafficking of Lck via the Rab11 endosome and is critical for immunological synapse formation. J. Immunol. 183: 1675–1684. 60. Madrid, R., K. Janvier, D. Hitchin, J. Day, S. Coleman, C. Noviello, J. Bouchet, A. Benmerah, J. Guatelli, and S. Benichou. 2005. Nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of HIV-1 infectivity. J. Biol. Chem. 280: 5032–5044. Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 44. human T cells and APCs distinguishes HIV-1 from most other primate lentiviruses. J. Clin. Invest. 119: 2965–2975. Thoulouze, M. I., N. Sol-Foulon, F. Blanchet, A. Dautry-Varsat, O. Schwartz, and A. Alcover. 2006. Human immunodeficiency virus type-1 infection impairs the formation of the immunological synapse. Immunity 24: 547–561. Haller, C., S. Rauch, N. Michel, S. Hannemann, M. J. Lehmann, O. T. Keppler, and O. T. Fackler. 2006. The HIV-1 pathogenicity factor Nef interferes with maturation of stimulatory T-lymphocyte contacts by modulation of N-Wasp activity. J. Biol. Chem. 281: 19618–19630. Haller, C., S. Rauch, and O. T. Fackler. 2007. HIV-1 Nef employs two distinct mechanisms to modulate Lck subcellular localization and TCR induced actin remodeling. PLoS One 2: e1212. Rudolph, J. M., N. Eickel, C. Haller, M. Schindler, and O. T. Fackler. 2009. Inhibition of T-cell receptor-induced actin remodeling and relocalization of Lck are evolutionarily conserved activities of lentiviral Nef proteins. J. Virol. 83: 11528–11539. Pan, X., J. M. Rudolph, L. Abraham, A. Habermann, C. Haller, J. KrijnseLocker, and O. T. Fackler. 2012. HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-Golgi network-associated Lck signaling. Blood 119: 786–797. Fackler, O. T., A. Moris, N. Tibroni, S. I. Giese, B. Glass, O. Schwartz, and H. G. Kräusslich. 2006. Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology 351: 322–339. Bunnell, S. C., D. I. Hong, J. R. Kardon, T. Yamazaki, C. J. McGlade, V. A. Barr, and L. E. Samelson. 2002. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158: 1263–1275. Nguyen, K., N. R. Sylvain, and S. C. Bunnell. 2008. T cell costimulation via the integrin VLA-4 inhibits the actin-dependent centralization of signaling microclusters containing the adaptor SLP-76. Immunity 28: 810–821. Sylvain, N. R., K. Nguyen, and S. C. Bunnell. 2011. Vav1-mediated scaffolding interactions stabilize SLP-76 microclusters and contribute to antigen-dependent T cell responses. Sci. Signal. 4: ra14. Bunnell, S. C., V. Kapoor, R. P. Trible, W. Zhang, and L. E. Samelson. 2001. Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14: 315–329. Anscombe, F. J. 1948. The transformation of poisson, binomial and negativebinomial data. Biometrika 35: 8. Manders, E. M. M., F. J. Verbeek, and J. A. Aten. 1993. Measurement of colocalization of objects in dual-color confocal images. J. Microsc. 169: 7. Stolp, B., M. Reichman-Fried, L. Abraham, X. Pan, S. I. Giese, S. Hannemann, P. Goulimari, E. Raz, R. Grosse, and O. T. Fackler. 2009. HIV1 Nef interferes with host cell motility by deregulation of Cofilin. Cell Host Microbe 6: 174–186. 13