Download Reduce Formation of SLP-76 Linker of Activated T Cells and SLP

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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
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Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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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).
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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-
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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
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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).
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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-
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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.
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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
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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).
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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.
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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
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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
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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.
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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
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