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Journal of Virology, February 2009, p. 1193-1200, Vol. 83, No. 3
0022-538X/09/$08.00+0     doi:10.1128/JVI.01023-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Engagement of the CD4 Receptor Affects the Redistribution of Lck to the Immunological Synapse in Primary T Cells: Implications for T-Cell Activation during Human Immunodeficiency Virus Type 1 Infection {triangledown}

Alice M. Nyakeriga,1 Carl J. Fichtenbaum,2 Jens Goebel,3 Stella A. Nicolaou,4 Laura Conforti,4 and Claire A. Chougnet1*

Divisions of Molecular Immunology,1 Nephrology, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio 45229,3 Divisions of Infectious Diseases,2 Nephrology and Hypertension, University of Cincinnati Medical Center, Cincinnati, Ohio 452674

Received 15 May 2008/ Accepted 9 November 2008


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ABSTRACT
 
Understanding the molecular mechanisms underlying dysregulated immune responses in human immunodeficiency virus type 1 (HIV-1) infection is crucial for the control of HIV/AIDS. Despite the postulate that HIV envelope glycoprotein gp120-CD4 interactions lead to impaired T-cell responses, the precise mechanisms underlying such association are not clear. To address this, we analyzed Lck and F-actin redistribution into the immunological synapse in stimulated human primary CD4+ T cells from HIV-1-infected donors. Similar experiments were performed with CD4+ T cells from HIV-uninfected donors, which were exposed to anti-CD4 domain 1 antibodies, as an in vitro model of gp120-CD4 interactions, or aldithriol-inactivated HIV-1 virions before stimulation. CD4+ T cells from HIV-infected patients exhibited a two- to threefold inhibition of both Lck and F-actin recruitment into the synapse, compared to cells from uninfected donors. Interestingly, defective recruitment of Lck was ameliorated following suppressive highly active antiretroviral therapy. Engagement of the CD4 receptor on T cells from HIV-uninfected donors before anti-CD3/CD28 stimulation led to similar defects. Furthermore, the redistribution of Lck into lipid rafts was abrogated by CD4 preengagement. Our results suggest that the engagement of CD4 by HIV gp120 prior to T-cell receptor stimulation leads to dysregulation of early signaling events and could consequently play an important role in impaired CD4+ T-cell function.


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INTRODUCTION
 
Human immunodeficiency virus (HIV) infection is characterized by impaired cell-mediated immune responses, which are primarily manifested by a progressive decline in the number and function of CD4+ T cells. Of note, the qualitative loss in the CD4+ T-cell function begins well in advance of its quantitative decline during HIV infection (31, 59, 68), although there is a selective loss of memory/effector CD4+ CCR5+ cells occurring early after HIV/simian immunodeficiency virus infection, particularly in the mucosal areas (reviewed in references 8, 52, and 55). This implies that the impairment in immune response could be due to mechanisms other than direct viral destruction. HIV-1 binds through its envelope glycoprotein, gp120, to the CD4 receptor molecule (13). The CD4 receptor plays a critical role during antigenic stimulation by major histocompatibility complex (MHC)-bearing cells. It is important in subsequent signal transduction through activation of the CD4-associated tyrosine kinase, p56Lck (Lck) (22), which in turn modulates T-cell differentiation as well as T-cell receptor (TCR)-induced signaling (67). This phenomenon could involve an up- or a downregulation of signaling effects depending on the timing of CD4 engagement relative to TCR-induced stimulation (26). Indeed, in vitro studies have demonstrated that CD4-induced stimulation, in the absence of or prior to TCR engagement, leads to apoptosis of the cell (1, 5) or inhibition of subsequent TCR-induced T-cell activation (6), which underscores the possible role of CD4 engagement in HIV pathogenesis.

Lck is a member of the Src family of non-receptor tyrosine kinases expressed primarily in thymocytes and lymphocytes and predominantly in T cells. In MHC II-restricted T cells, approximately 75 to 95% of cellular Lck is found associated with the cytoplasmic portion of CD4, involving about 85 to 95% of CD4 molecules (7). Lck interacts specifically with CD4 molecule through a dicysteine motif which binds to a corresponding motif in the cytoplasmic domain of the CD4 molecule (62). TCR-induced signaling involves the activation of lck, which, in turn, phosphorylates the immuno-receptor tyrosine activation motifs (ITAMs) within the TCR complex, as well as the tyrosine kinase ZAP-70 that docks onto the phosphorylated ITAMs. In T lymphocytes, following stimulation, Lck redistributes into lipid rafts and accumulates at the stable region between T cells and antigen-presenting cells, the immunological synapse (IS) (20, 43, 60). Together with cytoskeletal reorganization (15, 27, 34, 66), the membrane lipid raft domains are thought to orchestrate protein interactions in space and time by regulating raft coalescence and/or to control the recruitment of proteins to these domains. The temporal and spatial control of protein interactions at the plasma membrane regulates cell signaling and pathogen infection of cells. CD4 molecules take part in these two processes, as previous studies have reported that binding of CD4 to MHC aids in the lateral recruitment of cytoplasmic associated Lck to the membrane rafts (16) and that recruitment of Lck to the immunological synapse is dependent on the CD4 molecule (60). Similarly, other studies have shown that CD4 and the HIV coreceptors interact with the actin-binding protein filamin A, whose binding to HIV-1 receptors regulates their clustering on the cell surface (36). Besides, an intact cytoskeleton is important for the HIV-1 viral synapse formation and subsequent infectivity (37, 38, 40). Thus, disruption of the interaction of CD4 with MHC or Lck or actin would disrupt normal signaling.

While there is consensus on the integration and functioning of Lck in TCR-induced signaling, much remains to be elucidated with respect to the precise role of Lck in dysregulated T-cell function as evident in HIV infection. Previous studies have reported that the interaction between CD4 and gp120 mediates dual signaling activities. Indeed, it participates into the formation of the virus synapse, which aids in the virus entry (17, 35, 37, 39, 40). Conversely, the interaction is associated with defective immune responses in subsequent TCR-induced stimulation (9, 47). However, the exact mechanisms underlying these events are not well characterized in primary cells.

Recently, Thoulouze and others (61) showed that HIV-1-infected T cells exhibit impaired IS formation, and they attributed the defects to the effects of HIV Nef protein. Similarly, another study reported that nef-associated activity inhibited actin polymerization and Lck recruitment to the IS (30). However, we previously observed that TCR-induced stimulation of CD4+ T cells from HIV-1-infected persons or cells from HIV-uninfected donors whose CD4 molecule is preligated in vitro with either noninfectious HIV-1 virions or anti-CD4 domain 1 antibodies was accompanied by diminished expression of CD40 ligand (CD40L) (69), one of the key players in T-cell activation and subsequent effector functions. This data suggested that the engagement of CD4 receptor alone or its interaction with envelope glycoprotein could be sufficient to induce dysfunctional CD4+ T cells in the absence of virus integration. Furthermore, the blunted CD40L expression was overcome by using phorbol myristate acetate or ionomycin, implying that the defects must be in the proximal signaling events. In this study, we investigated the effects of CD4 ligation prior to TCR-induced stimulation on T-cell activation, in particular on the IS formation with a focus on the effects on Lck redistribution. Lck or F-actin recruitment to the IS was inhibited in CD4+ T cells from HIV-1-infected patients. Moreover, the interaction between CD4 and anti-CD4 domain 1 antibodies, which bind the same domain as HIV-1 gp120, inhibited the recruitment of Lck to lipid rafts and prevented the formation of the IS. In vitro exposure of CD4+ T cells to aldithriol (AT-2)-treated HIV-1 virions prior to TCR-induced stimulation also inhibited subsequent Lck recruitment to the IS. Furthermore, reduction of viral load through highly active antiretroviral therapy (HAART) ameliorated recruitment of Lck to the IS. These data imply that engagement of CD4 by HIV virions or free gp120 may contribute to the dysregulated CD4+ T-cell responses during chronic HIV infection.


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MATERIALS AND METHODS
 
Patients. Heparinized blood samples were obtained from HIV-1-infected adult patients, from the Center of Infectious Diseases, University of Cincinnati, Cincinnati, OH, and samples were processed within 2 h of blood collection. Patients had CD4 counts of less than 750 CD4 cells/mm3 with viral loads greater than 1,000 copies/ml (ultrasensitive reverse transcription-PCR 1.0 with a detection limit of 50 copies/ml; Roche Diagnostic Systems, Indianapolis, IN). Two of the patients were treatment naïve, while the others had been on various antiretroviral therapies but had been off treatment for at least 6 months before the first blood samples were obtained. Four of the patients started HAART and had a second blood draw more than 6 months after HAART initiation. Patients did not have hepatitis B virus infection, opportunistic infections, or cancer at the time of blood sampling. One patient was coinfected with hepatitis C virus. Buffy coats were obtained from HIV-uninfected donors from the Hoxworth blood bank center (Cincinnati, OH). Informed consent was obtained from each donor, and the study was approved by the Institutional Review Committees at the University of Cincinnati and Cincinnati Children's Hospital Medical Center.

Cell preparation. (i) Peripheral blood mononuclear cells. Peripheral blood mononuclear cells were separated by density gradient centrifugation on Ficoll (Amersham Biosciences, Piscataway, NJ). Cells were washed in phosphate-buffered saline (PBS) and suspended in complete RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 5 mM HEPES (all reagents from Life Technologies, Gaithersburg, MD).

(ii) CD4+ T cells. CD4+ T cells were purified by negative selection. After the first step of monocyte adherence (1 h at 37°C), CD4+ T cells were purified from nonadherent cells by negative selection according to the manufacturer's instructions (CD4-negative selection kit; Miltenyi Biotech, Inc., Auburn, CA). Purified CD4+ T cells were >96% pure as assessed by fluorescence-activated cell sorting.

Reagents. The following reagents were purchased: anti-CD3 or -CD28 monoclonal antibodies (MAbs) (eBioscience, San Diego, CA), mouse anti-Lck MAb (Santa Cruz, CA), rabbit polyclonal anti-Lck (Upstate, Billerica, MA), Alexa Fluor 546-phalloidin (Invitrogen, Eugene, OR), fluorescein isothiocyanate-conjugated anti-HIV p24Gag antibody (Beckman-Coulter, Miami, FL), goat anti-mouse, and Cy-5-conjugated goat anti-rabbit immunoglobulin (Ig), Fc{gamma} fragment (Jackson, ImmunoResearch West Grove, PA). Anti-CD4 domain 1 (clone QS4120) or anti-domain 2 (clone MT441) and their isotype-matched control antibody (mouse IgG1) were from Ancell (Bayport, MN). AT-2-inactivated viruses and microvesicles were obtained from J. Lifson (AIDS Vaccine Program, Science Applications International Corporation—Frederick, National Cancer Institute, Frederick, MD).

Detection of Lck distribution into membrane lipid rafts. CD4+ T cells from HIV-uninfected donors were pretreated with anti-CD4 domain 1 antibodies or an isotype control (all antibodies at 10 µg/ml). After 30 min at 4°C, the CD4+ T cells were incubated with anti-CD3/CD28 antibodies for 30 min (herein referred to as TCR-induced stimulation), followed by cross-linking with goat antimouse antibody for 10 min at 37°C. Then, T-cell lysates were prepared and analyzed by Western blotting as previously described (24). Briefly, the cells were lysed in the presence of protease and phosphatase inhibitors and 0.5% Triton X-100 and overlaid on a sucrose gradient at 4°C. This procedure allows for efficient separation of rafts, which are detergent insoluble and of low density due to their high lipid content. The distribution of Lck was determined by Western blotting of protein content-adjusted aliquots of each fraction, using mouse anti-Lck antibody, followed by chemiluminescence detection.

IS formation, immunostaining, and image analysis. (i) T-cell activation. CD4+ T cells obtained from HIV-uninfected donors were incubated for 20 min at 4°C with either MAb against domain 1 or domain 2 of the CD4 molecule or the isotype control (all antibodies at 10 µg/ml) or were left untreated. In some experiments, CD4+ T cells from HIV-uninfected donors were incubated at 37°C for 2 h with either AT-2-treated HIVMN virions (1 µg HIV Gag equivalent/106 cells) or the microvesicle controls (added at a concentration that provides an equal amount of total protein to the equivalent AT-2-treated virus), as described earlier (56). After washing, cells were stimulated with anti-CD3/CD28-coated beads (Dynal Biotech, Lake Success, NY) and resuspended in preheated PBS, at a bead/cell ratio of 1:1, followed by incubation at 37°C for 5 or 30 min. Similarly, CD4+ T cells from HIV-1-infected donors were stimulated with anti-CD3/CD28-coated beads. Slide preparation, imaging, and analysis were all blinded.

(ii) Immunostaining and imaging. Stimulated cells were adhered onto poly-L-lysine-coated coverslips for approximately 5 min. The cells were then fixed in 4% formaldehyde for 20 min at room temperature followed by permeabilization/blocking using 10% goat serum-0.2% Triton X-100-PBS buffer. The cells were then incubated overnight with rabbit anti-Lck polyclonal antibody followed by 1 h of incubation with goat Alexa Fluor 488- or Cy-5 conjugated anti-rabbit antibody as indicated. For double staining, the cells were stained with either labeled phalloidin for F-actin staining or fluorescein isothiocyanate-labeled antibody for anti-HIV p24 for 30 min. Each stage was followed by three or four PBS washes of 5 min each. Then, cells were mounted onto glass slides using Fluoromount antifade (Sigma-Aldrich, St Louis, MO) and stored until microscopy was done. Images were obtained using a fluorescent microscope, Nikon Microphot FXA (Melville, NY), fitted with a spot II charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI). In some experiments, confocal microscopy was carried out using an inverted confocal microscope using the Zeiss LSM 510 software (Axioscope; Carl Zeiss, Micro imaging, Inc.) with a x63 oil objective. Green, red, and far red were sequentially acquired, taking care not to allow any fluorochrome overlapping. To determine localization of Lck and F-actin, Z series images were acquired with pinholes opened to obtain sections 0.8 µm thick.

(iii) Fluorescent intensity quantification. To assess redistribution of proteins into the IS and/or their colocalization, fluorescent intensities of unprocessed images were quantified using the Metamorph imaging system (Molecular Devices, Downington, PA), as illustrated in Fig. 1A. Regions of the same size and number were drawn at the area of cell-bead contact (herein referred to as IS) or at the cell membrane outside of the cell-bead contact (60), as shown on Fig. 1A. After background subtraction, the relative mean fluorescent intensities (rMFIs) were calculated: rMFI = average (MFI for each region – background MFI) of five regions at IS/average (MFI for each region – background MFI) of five regions on cell membrane. Recruitment was defined as rMFI ≥ 1.5, based on our previous studies (50). A minimum of 40 cell-bead conjugates/condition were randomly chosen and analyzed.


Figure 1
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  		FIG. 1. Lck and F-actin colocalized at the IS following TCR-induced stimulation. CD4+ T cells were pretreated with anti-CD4 antibodies or its isotype control followed by TCR-induced stimulation. The cells were plated onto poly-L-lysine-coated coverslips, fixed, permeabilized, and stained for microscopic imaging. (A) Illustration of quantification of relative fluorescent intensity. Light intensity ratios were scored as detailed in Materials and Methods. DIC, differential interference contrast. (B) Representative example of a cell-bead conjugate with Lck and F-actin recruitment at the IS. (C) Representative example of a cell-bead conjugate without Lck or F-actin recruitment at the IS.

Statistical analysis. Protein recruitment at the IS was compared between HIV-1-infected and HIV-uninfected donors using the Mann-Whitney test. The effect of treatment with anti-CD4 MAbs or AT-2 virus was analyzed using the Wilcoxon sign rank test or paired t tests following log transformation of the data. Similarly, the effect of HAART was analyzed using paired t tests following log transformation of the data. P values of <0.05 were considered to be significant.


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RESULTS
 
HIV-1 infection impaired Lck and F-actin recruitment to the IS. Based on our previous finding that PMA and/or ionomycin stimulation that bypasses early TCR-induced signaling events overcame the blunted CD40L expression on T cells from HIV-1-infected patients (69), we postulated that the defect might be due to defective early signaling events. Given the significance of IS formation in TCR-induced signaling and activation, we investigated the recruitment of Lck and F-actin to the IS following TCR-induced stimulation using anti-CD3/CD28-coated beads (a typical image for recruitment or absence of recruitment is shown in Fig. 1B and C, respectively).

The percentage of cell-bead conjugates with IS recruitment of Lck or F-actin was significantly reduced in CD4+ T cells from HIV-1-infected individuals (Fig. 2). After 5 min of stimulation, 7 to 25% (median, 15%) of cell-bead conjugates from HIV-1-infected patients showed Lck recruitment to the IS, in contrast to 21 to 48% (median, 33%) in cells from HIV-uninfected subjects (P = 0.002, Mann-Whitney analysis). Similarly, 0 to 19% (median, 13%) of cell-bead conjugates from HIV-1-infected donors showed F-actin recruitment to IS, compared to 11 to 48% (median, 39%) in HIV-uninfected donors (P = 0.02, Mann-Whitney analysis). These differences tended to persist after 30 min of stimulation (Fig. 2A and B). As expected, there was minimal recruitment of Lck or F-actin in unstimulated cells, and no difference in recruitment for either molecule was seen between unstimulated cells from HIV-1-infected and uninfected donors (P = 0.73 and 0.236 for Lck and F-actin, respectively).


Figure 2
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  		FIG. 2. HIV infection impaired Lck and F-actin recruitment to the IS. CD4+ T cells were obtained from either HIV-uninfected (n = 8) or HIV-1-infected (n = 9) subjects. The cells were stimulated with beads coated with anti-CD3/28 antibodies for 5 or 30 min or left unstimulated. The cells were then fixed, permeabilized, stained for Lck or F-actin, and imaged to determine relative fluorescent intensities, as described in Materials and Methods. The percentage of cells exhibiting IS recruitment for Lck (A) or F-actin (B) is reported as relative to the number of cell-bead conjugates analyzed. Each point represents the data obtained in one individual. Solid lines represent the medians. P values represent the comparison between HIV-uninfected and -infected donors (Mann-Whitney tests).

To determine whether decreased recruitment was characteristic of HIV-infected cells, we costained cells for Lck and HIV p24 in two chronically infected untreated HIV-infected subjects. As expected, the percentage of CD4+ p24+ cells detected by immunohistochemistry was low, 2.8 and 3.2%, respectively, which was in the range of the percentage of p24+ cells (2.7% ± 1.5%) found by flow cytometry in a similar cohort (data not shown). Interestingly, most p24+ cells were not found in contact with beads, excluding them from the analysis. Analysis of the IS formation performed in p24– CD4+ T cells showed a low Lck recruitment 5 min postactivation in those two subjects (14.1% and 17.7%, respectively). Therefore, defects in Lck/F-actin redistribution do not appear to mainly occur in productively infected CD4+ T cells.

HAART ameliorated Lck recruitment to the IS. We speculated that constant exposure to HIV virions/HIV gp120 contributed to the CD4 receptor-associated signaling defects. Thus, viral control through HAART should ameliorate defective T-cell activation. We therefore evaluated Lck and F-actin IS recruitment in four patients before and after HAART initiation. All subjects had a greater than 2-log drop of their viral load following HAART and exhibited undetectable viral loads (<50 copies/ml) at the time of the second draw. A general rise in CD4 counts also occurred in all patients (average increase of 126 ± 24 cells/mm3; P < 0.05). As shown in Fig. 3A, after 5 min of anti-CD3/CD28 stimulation, Lck recruitment to the IS significantly improved following HAART (P = 0.016 by paired t test using log-transformed values). Furthermore, the mean Lck recruitment (30.9%) became comparable to that measured in cells from HIV-uninfected donors (P > 0.05). Similarly, F-actin recruitment to the IS improved following HAART, although to a lesser extent than what was observed for Lck (Fig. 3B; P = 0.021 by paired t test using log-transformed values). Stimulation of the cells for 30 min provided similar results (data not shown).


Figure 3
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  		FIG. 3. Control of viral replication through HAART led to improved Lck recruitment to the IS. CD4+ T cells from four HIV-infected donors, denoted as patients (Pt) 1, 2, 3, and 4, were obtained before and after HAART initiation. They were stimulated with anti-CD3/28 antibody-coated beads for 5 min, fixed and stained for Lck (left) and F-actin (right), and analyzed as described in the legends to Fig. 1 and 2.

CD4 preengagement decreased the recruitment of Lck and F-actin to the IS. While our ex vivo observations showed a significant reduction in Lck or F-actin recruitment to the IS, we could not dissociate effects due to HIV Nef, as previously described (61), from possible consequences of the chronic interaction between HIV gp120/virions and CD4 molecules. Thus, we investigated if CD4 engagement prior to TCR-induced stimulation could affect the recruitment of Lck and/or F-actin to the IS. To this end, we used the in vitro model that we previously used: e.g., CD4 engagement by anti-CD4 domain-1 antibodies in T cells from HIV-uninfected donors (69). Importantly, incubation of CD4+ T cells with anti-CD4 domain 1 antibodies up to 30 min did not reveal significant CD4 receptor endocytosis, as measured by flow cytometry analysis of surface CD4 expression at 5-min intervals (data not shown).

The cells that were treated with anti-CD4 antibodies before TCR-induced stimulation exhibited a significantly lower percentage of Lck or F-actin recruitment to the IS compared with those treated with the isotype control antibody, at both 5 and 30 min of stimulation (Fig. 4A and B; Wilcoxon signed rank analysis, all P < 0.05). It is also possible that, in cells where Lck and F-actin were recruited, the level of protein recruitment might have been affected by CD4 preengagement. Thus, we estimated the level of recruitment of Lck or F-actin to the IS as an index of absolute light intensity for each protein. No significant difference was found (data not shown). We then wanted to determine if CD4 engagement was affecting F-actin polymerization and Lck recruitment to the same extent. Consistent with the previous single-protein analysis, the percentages of cell-bead conjugates that had both Lck and F-actin recruitment to the IS were significantly lower in cells treated with anti-CD4 MAbs before stimulation than in those that were treated with isotype control (Table 1; P = 0.008, Wilcoxon sign rank test). Confocal microscopy confirmed the colocalization of Lck and actin in those cells (data not shown).


Figure 4
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  		FIG. 4. Preengagement of CD4 decreased recruitment of Lck and F-actin to the IS. CD4+ T cells from HIV-uninfected donors (n = 8) were treated with anti-CD4 domain 1 antibody or its isotype-matched control before stimulation with anti-CD3/28 antibody-coated beads for 5 min or 30 min. The cells were analyzed as described in the legends to Fig. 1 and 2. The percentages of cell-bead conjugates that showed recruitment of either Lck (A) or F-actin (B) at the IS are shown, with each triangle representing data from one donor. The solid lines represent the group medians. Unstim, unstimulated. The P values represent the difference between the anti-CD4 and isotype control-treated cells (Wilcoxon sign rank analysis).


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  		TABLE 1. Percentage of cell-bead conjugates showing recruitment of Lck, actin, or both to the IS following TCR-induced stimulation

Pretreatment of CD4 cells with AT-2-treated HIV-1 virions inhibited Lck recruitment to the IS. We then investigated the effect of CD4 engagement by HIV-1 virions on Lck recruitment to the IS. We used AT-2-treated virions, which are replication deficient while harboring a conformationally intact gp120 on their surface (56). These noninfectious virions thus provide a powerful tool to dissect mechanisms of HIV pathogenesis that do not require viral replication. As a control, we used microvesicles prepared from the AT-2-treated uninfected cell line CEMX174. AT-2-treated HIVMN-infected cells exhibited a significantly lower percentage of cell-bead conjugates with Lck recruitment to the IS compared to the vesicle-treated control cells (Fig. 5; P = 0.0001, paired t test on log-transformed values). AT-2-treated HIVMN had an inhibitory effect on IS similar to the anti-domain 1 MAb (P = 0.74), which suggests that CD4 engagement represents a major mechanism involved in HIV-mediated inhibition of IS formation. Similarly, microvesicles did not appear to affect IS formation, because the percentage of cell conjugates exhibiting Lck recruitment in microvesicle-treated cultures was similar to that of cells treated with the isotype control.


Figure 5
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  		FIG. 5. Treatment of CD4 cells with AT-2 HIV prior to TCR-induced stimulation impaired Lck recruitment to IS. Purified CD4 cells from three HIV-uninfected donors were pretreated with anti-CD4 domain 1 or domain 2 antibodies or their isotype control for 20 min or cultured with AT-2-treated-HIV virions or microvesicle controls for 2 h, followed by stimulation with anti-CD3/28 antibody-coated beads for 5 min, fixation, and staining for Lck, as described in the legend to Fig. 1. Logarithmic transformed values were compared using Student's t test.

Similarly, we wanted to determine if another anti-CD4 MAb that binds to another domain of CD4 than HIV gp120, the anti-domain 2 antibody, had similar effects to those mediated by the anti-domain-1 MAb. Thus, CD4+ cells from four HIV-uninfected individuals were treated in parallel with anti-domain 1, anti-domain 2, and isotype control MAbs. Interestingly, the anti-domain 2 antibody also decreased Lck recruitment, compared to isotype control-treated cells (Fig. 5; P = 0.030, paired t test on log-transformed values). The effects of anti-CD4 domain 2 and anti-domain 1 MAbs were similar, although responses to anti-domain 2 binding were more variable between individuals.

CD4 preengagement prevented Lck redistribution into the lipid rafts. During IS formation, various molecules, including coreceptors, adhesion molecules, and signaling molecules, translocate into membrane lipid rafts. Lck activity is important for the translocation of most of these molecules (16, 28). However, it is not clear if CD4 is important for Lck translocation into membrane lipid rafts. Thus, using human primary CD4+ T cells, we determined whether CD4 preengagement prevents Lck redistribution into membrane rafts. As expected, Lck was not detectable in the raft fraction in the absence of TCR stimulation (Fig. 6A, lane 1). Stimulation of CD4+ T cells by cross-linked anti-CD3/CD28 antibodies induced an approximately threefold increase in the quantity of Lck present in the lipid rafts (see Fig. 6A, lane 2, for a representative example, and B), compared to unstimulated cells. Such increase was abolished when cells where stimulated after pretreatment by anti-CD4 antibodies (see Fig. 6A, lane 3, for a representative example, and B). Isotype control-treated cells (Fig. 6A, lane 4) did not exhibit the same defect after TCR stimulation. Treatment with anti-CD4 antibodies alone (without further stimulation) did not induce substantial Lck translocation in the rafts (Fig. 6A, lane 5). However, most if not all cellular proteins are differentially distributed across these fractions, and the overall protein content of these fractions can vary up to 100-fold, with raft fractions having much lower protein content per volume than nonraft fractions (49). Nonetheless, these results suggest that in vitro CD4 engagement, similar to what HIV gp120 can do in vivo, prevents Lck redistribution into the membrane lipid rafts upon subsequent TCR-mediated activation.


Figure 6
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  		FIG. 6. Preengagement of CD4 prevented Lck redistribution into the lipid rafts. CD4+ T cells were treated with nothing, anti-CD4, or its isotype control antibodies before stimulation with cross-linked anti-CD3/28 antibodies. The cells were then extracted with Triton X-100, and extracts were separated into six fractions by sucrose gradient centrifugation, with fractions 1 and 2, 3 and 4, and 5 and 6 representing the raft, membrane, and cytosol, respectively (20). Aliquots from each fraction containing equal amounts of protein were immunoblotted for Lck determination. (A) One representative experiment is shown. (B) Average Lck levels in rafts after anti-CD3/28 stimulation, in the absence or presence of anti-CD4 antibodies. We performed protein assays on each fraction and then loaded equal amounts of protein from each fraction into their respective lanes. Density of the Lck band was determined in the raft fractions (fractions 1 and 2) of cells stimulated with cross-linked anti-CD3/CD28 (TCR alone) or cross-linked anti-CD3/CD28 after anti-CD4 treatment (anti-CD4 + TCR), as well as in unstimulated cells. Results are expressed as the increases (fold) in the Lck levels in rafts of stimulated cells (TCR alone or after anti-CD4+ treatment [anti-CD4 + TCR]) over unstimulated cells, for each individual. Averaged data for TCR alone (five individuals) and anti-CD4 plus TCR stimulation (two individuals) are shown.


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DISCUSSION
 
The precise mechanisms of the downregulation of CD4+ T-cell function during chronic HIV infection, before progression into AIDS, are still not completely understood. While there is a general consensus that HIV envelope glycoproteins can exert powerful biological effects on the immune system (57, 58), the precise signaling cascade resulting from gp120-CD4 interactions has not yet been fully elucidated in primary CD4+ T cells. Only a small number of CD4+ T cells, at least in the periphery, appear to be productively infected (44). This implies that, in addition to direct viral effects, other mechanisms are involved in dysregulated T-cell responses during HIV infection.

We observed that primary CD4+ T cells from HIV-1-infected donors exhibited defective recruitment of Lck and F-actin to the IS compared to those obtained from HIV-uninfected donors. The role played by chronic exposure to HIV virions in defective T-cell activation is strongly suggested by the fact that these defective responses were ameliorated following HAART initiation. The lesser restoration of F-actin recruitment compared to Lck recruitment might be due to differential modulatory effects of the virus and/or HAART on actin. A previous report has shown that viral Nef also downregulates RhoA GTPase, with subsequent disruption of the cytoskeleton (45). Alternatively, Wang and others (64) have shown that some protease inhibitors, in particular ritonavir, disrupt the osteoclast cytoskeleton, as well as the recruitment of tumor necrosis factor receptor-associated factor 6-c-Src complex to lipid rafts. A longer follow-up of patients on HAART will be necessary to tease out the mechanisms underlying the incomplete restoration of F-actin recruitment to the synapse. Nonetheless, our observations suggest that the amelioration of T-cell responses could be due to decreased exposure to virions/gp120. Here, the patient with the lowest viral load after HAART had the greatest increase (twofold) in Lck recruitment to the IS. However, it should be noted that HIV-induced immune defect is multifactorial, and redistribution of CD4+ T-cell subsets in the periphery may also have played in a role in our data. Indeed, in untreated chronically infected patients, poorly responsive naive T cells are found in the periphery and HAART induces the recirculation of memory/effector CD4+ T cells (reviewed in reference 48). Those cells being more likely to form a functional IS upon activation, this additional mechanism is likely to have also played a role in the reduced IS defect we observed following HAART initiation.

Importantly, impaired recruitment of Lck and F-actin to the IS could be induced in primary cells by the ligation of CD4 with anti-CD4 domain 1 MAbs prior to TCR-induced stimulation. Furthermore, we confirmed these data in CD4+ T cells exposed to AT-2-treated HIV-1 virions prior to TCR-induced stimulation. These data confirm in primary cells the findings of previous studies using mainly cell lines and gp120 or anti-CD4 antibodies (25, 32, 42), which have shown that HIV-1 impairs the formation of IS and early TCR signaling, including the inhibition of F-actin cytoskeleton reorganization and Lck recruitment to the IS. Chronic exposure to HIV gp120 is expected to occur in vivo. Indeed, HIV gp120 can be detected in tissues (41, 53) and circulates in the blood of HIV-infected donors on the surface of virions (both infectious and noninfectious) and as a free protein (51). Because a limited fraction (<0.1%) of circulating virions are demonstrably infectious (14, 54), exposure of CD4+ T cells to viral molecules engaging the CD4 receptor is expected to occur more frequently than productive infection. Our results suggest that the ligation of CD4 receptor by either anti-domain 1 MAb or AT2-treated HIV-1 virions is sufficient to trigger this defect, which could then be sustained in vivo by Nef-associated activities.

The mechanisms by which engagement of CD4 by HIV affects CD4+ T-cell responses are not completely elucidated. CD4 receptor endocytosis, which is prominent during HIV infection, is thought to partly underlie the dysregulated CD4 immune responses (10). However, in our in vitro model, incubation of CD4 with anti-CD4 domain 1 antibodies up to 30 min did not reveal significant CD4 receptor endocytosis (data not shown). Other mechanisms, including dissociation of Lck from CD4 (42) and steric hindrance of CD4 participation during TCR-induced stimulation, have also been implicated (29, 46, 47, 65). Epitope masking of CD4 molecules by gp120 or gp120-anti-gp120 complexes, thus blocking the interaction of CD4 with MHC in vivo, has also been alluded (2). Recently, it was shown that CD4+ T cells exposed to HIV gp120 phosphorylated AKT and mitogen-activated protein kinase to a comparable extent as control-treated cells after anti-CD3 activation (19). However, the gp120-treated cells exhibited defective proliferation and failed to upregulate activation markers such as HLA-DR and CD25 (33). While the two anti-CD4 clones we tested significantly inhibited Lck recruitment to the IS, we have previously shown that only anti-domain 1 MAb or AT-2-treated HIV-1 virions had a significant impact on downstream signaling events such as CD40L upregulation by activated CD4+ T cells (11, 18, 69). Previous studies have shown that different anti-CD4 MAbs or gp120 could trigger activation of Lck, but they differed in their ability to activate NF-AT (4), proliferation, or interleukin-2 (IL-2) production (11, 18, 69). Of note, IL-16, which binds to CD4 domain 4, was shown to inhibit CD3-dependent lymphocyte activation and desensitize chemokine receptors (12, 63), but IL-16-induced defects were not all dependent on Lck (63). Taken together, these observations suggest that different types of CD4 engagement affect differently CD4+ T-cell functions.

The role of CD4 in Lck redistribution into membrane lipid rafts remains controversial. On one hand, Tavano and others (60), using the Jurkat cell line, suggested that CD28 but not CD4 engagement induced Lck recruitment to the lipid rafts and accumulation of the kinase at the IS. On the other hand, using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS exclusively depended on CD4, whereas CD28 sustained Lck activation (32). Similarly, Ehrlich and others (16) using cell lines showed that the synapse-localized Lck was CD4 associated and that these complexes remained associated throughout synapse formation. In support of the latter observations, we have demonstrated that preengagement of CD4 not only inhibited the Lck redistribution into IS but also its recruitment into the lipid rafts, in human primary CD4+ T cells. The different results observed with Jurkat cell lines might be due to inherent differences between cell lines and primary cells such as levels of surface or intracellular molecules, including the amount of rafts (23), CD4 receptor itself, and other surface markers such as coreceptor expression or the different effects of the detergents used to extract raft fractions, Brij versus Triton X-100 (3, 21).

Taken together, our data underscore the importance of defective IS formation in dysfunctional T-cell activation during HIV-1 infection, including in those cells that are not directly infected. Such mechanisms shed light on why interactions between CD4 and gp120 contribute to the defective immune responses manifested in HIV-1-infected individuals. Importantly, our findings also show that successful HAART not only increases CD4+ T-cell numbers but also ameliorates the intrinsic functionality of these cells.


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ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grants AI-05692 (to C.A.C.), AI-06953 (to C.J.F.), AI-049241 (to J.G.), and CA-95286 (to L.C.).

We thank David Hildeman for helpful discussions and Matthew Kofron for assistance with confocal imaging, as well as Barbara Logan and Kris Orsborn for expert assistance. We also thank Tammy Mansfield and Diane Daria for assistance with collection of samples. Microscopy was conducted at the Molecular Biology Core Unit, University of Cincinnati.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Molecular Immunology, Cincinnati Children's Hospital Medical Research Center, 3333 Burnet Avenue, Mail location code 7021, Cincinnati, OH 45229. Phone: (513) 636-8847. Fax: (513) 636-5355. E-mail: Claire.chougnet{at}cchmc.org Back

{triangledown} Published ahead of print on 19 November 2008. Back


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Journal of Virology, February 2009, p. 1193-1200, Vol. 83, No. 3
0022-538X/09/$08.00+0     doi:10.1128/JVI.01023-08
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