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Published as doi: 10.1096/fj.06-7883com.
 (The FASEB Journal. 2007;21:2323-2334.)
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HIV-1 Nef impairs the dynamic of DC/NK crosstalk: different outcome of CD56dim and CD56bright NK cell subsets

Maria Giovanna Quaranta*, Alfonso Napolitano*, Massimo Sanchez{dagger}, Luciana Giordani*, Benedetta Mattioli* and Marina Viora*,1

* Department of Drug Research and Evaluation,

{dagger} Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy

1Correspondence: Department of Drug Research and Evaluation, Istituto Superiore di Sanità; Viale Regina Elena, 299, 00161 Rome, Italy. E-mail: viora{at}iss.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a critical role in the first phase of host defense against infection. Interactions between DCs and NK cells have been demonstrated in a variety of settings, with evidence emerging of complex bidirectional crosstalk between the two cell types. The accessory HIV-1 Nef protein is a crucial determinant for viral replication and pathogenesis. We previously demonstrated that Nef, hijacking DC functional activity, subverts the DC arm of immune response to escape the adaptive immune attack. Here, we monitor the effect of Nef on the outcome of the innate immune response, focusing on the impact of Nef on DC/NK crosstalk. We demonstrate that Nef up-regulates the ability of DCs to stimulate the immunoregulatory NK cells (CD56bright) as assessed by the activated phenotype, up-regulation of their proliferative response and INF-{gamma} release. On the other hand, Nef-pulsed DCs inhibit cytotoxic NK cells (CD56dim), as assessed by the reduced HLA-DR surface expression, reduced proliferation and cytotoxic activity. Moreover, in the presence of Nef-pulsed DCs, we found a significant up-regulation of TNF-{alpha} secretion and a significant reduction of IL-10, GM-CSF, MIP-1{alpha} and RANTES secretion. Our findings suggest that the Nef-induced dysregulation in the DC/NK cell crosstalk may represent a potential mechanism through which HIV escapes innate immune surveillance.—Quaranta, M.G., Napolitano, A., Sanchez, M., Giordani, L., Mattioli, B., Viora, M. HIV-1 Nef impairs the dynamic of DC/NK crosstalk: different outcome of CD56dim and CD56bright NK cell subsets.


Key Words: AIDS • innate immunity • immune evasion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DENDRITIC CELLS (DCS) AND NATURAL KILLER (NK) cells are essential mediators and regulators of innate immunity, and both act as a link between innate and antigen-specific response. DCs and NK cells play a critical role in the first phase of HIV infection and are rapidly recruited from the blood to the mucosal surfaces, the primary site for HIV entry (1 2 3 4 5) .

Human NK cells can be divided into two distinct subsets based on their cell surface density of CD56 (6) . The majority (~90%) of NK cells have low-density expression of CD56 (CD56dim) and express high levels of CD16 (also known as Fc{gamma} receptor III), whereas ~10% of NK cells are CD56bright and lack or have low expression of CD16. The two subsets of NK cells differ also in terms of chemokine receptor and adhesion molecule expression, suggesting that they have different homing properties (7) . Indeed, CD56bright cells have been found to be the dominant NK cell subset in human lymph nodes, whereas in peripheral blood, CD56dim NK cells dominate. Functionally, CD56bright NK cells produce proinflammatory cytokines in response to cytokine costimulation, while CD56dim NK cells are inefficient cytokine producers yet efficient effectors of natural and antibody-dependent cellular cytotoxicity (ADCC) (8 , 9) . Contrary to reports suggesting that CD56bright NK cells represent a more immature state (10) , it has been recently demonstrated that CD56dim NK cells acquire a CD56bright NK cell phenotype in in vitro short-term culture with IL-12, indicative that CD56bright NK cells are terminally differentiated cells rather than an immature or a distinct specialized subset (11) .

DCs are instrumental in the development of pathogen-specific immune responses and are well equipped for activation of naive T cells (12) . Interactions between DCs and NK cells have been demonstrated in a variety of settings, with evidence emerging of complex bidirectional crosstalk between the two cell types. DCs and NK cells have been found in close proximity to each other in inflamed and tumor tissues, as well as in draining lymph nodes and therefore, they might interact in these tissues in vivo (4) . Contact with DCs activates NK cell cytolytic activity and IFN-{gamma} secretion, thus amplifying one of the mechanisms of primary host defense against pathogens (13 , 14) .

During HIV infection, the perturbation of the adaptive and innate immune responses contributes to the progressive immunosuppression. Several impairments of both DC and NK cell function and number have been observed in HIV-infected patients. HIV-1 takes advantage of DC biology to facilitate the onset of infection and its dissemination to surrounding permissive cells. HIV-1 can directly infect immature DCs (iDCs) and infected DCs might, in turn, infect and subsequently induce anergy or apoptosis of naive T cells, thereby contributing to early loss of HIV-specific CD4+ and CD8+ T cells (15 16 17 18) . NK cell function is also altered during HIV infection (19 20 21 22 23) , both in terms of cytotoxic activity and of the capacity to secrete CCR5-binding chemokines able to block virus entry (24) . However, studies assessing NK cell numbers and function in HIV-1 infection have resulted in conflicting results. An early work demonstrated that NK cell activity was seriously impaired in HIV-1 infected individuals despite the presence of a normal NK cell number (25) . More recent studies have demonstrated both reduced or enhanced cytolytic activity of NK cells in HIV-1 infection (23 , 24 , 26 , 27) . During HIV-1 infection, several different mechanisms can determine NK cell modulation, including increased cytokine production (e.g., increased IL-10 and TGF-ß) (26 27 28) ; decreased production of IL-2 and IFN-{alpha} (26 , 28 29 30 31 32) ; interaction with infected cells, particularly DCs, and with viral proteins, such as gp120, p17, Nef and Tat, released by infected cells (24 , 33 , 34) .

HIV-1 has developed several mechanisms to avoid NK cell-mediated lysis. Among these, the selective Nef-mediated down-regulation of MHC class I A and B expression on the surface of infected cells may represent for HIV-1 a balance between escape from cytotoxic T lymphocytes and maintenance of protection from NK cells (35 36 37) . In addition, Nef down-regulates the expression of the nonclassical MHC-I like CD1d molecule (38) . Thus, HIV-1 reduces the visibility of infected cells not only to MHC-I-restricted T cells but also to CD1d-restricted NKT cells. It is well known that the accessory HIV-1 Nef protein, a critical determinant of AIDS pathogenesis expressed early during infection, enhances virus replication and infectivity through a combination of different effects (39 40 41) . We previously demonstrated that Nef is efficiently taken up by iDCs inducing their activation (42 , 43) . This has a direct impact on CD4+ T lymphocyte bystander activation. On the other hand, we demonstrated that Nef targeting DCs induces anergy and apoptosis in CD8+ T cells (44) . Thus, Nef subverts the DC arm of immune response to escape adaptive immune attack (45) .

Because DC/NK interactions are likely to occur primarily during infection, this study was set out to monitor the effect of Nef in DC/NK crosstalk.

We found that Nef up-regulates the ability of DCs to stimulate the immunoregulatory NK cells (CD56bright), while Nef-pulsed DCs inhibit cytotoxic NK cells (CD56dim).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Nef protein
Recombinant HIV-1 Nef (strain ELI) from E. coli was obtained from Trinity Biotech (Carlsbad, CA, USA). Lyophilized protein was dissolved in sterile water as recommended by the manufacturer. Aliquots were prepared and stored at –70°C. In all experiments performed, Nef was used at 0.1 µg/ml. The Nef protein was monitored for endotoxin contamination using the Limulus assay (BioWhittaker, Verviers, Europe). Endotoxin levels were <0.03 endotoxin units/ml. To assess the specificity of the Nef-induced modulation, Nef was preincubated for 60 min at 37°C with 10 µg/ml anti-Nef monoclonal antibody (mAb) isotype immunoglobulin (Ig)G1 (Intracell), or with 10 µg/ml irrelevant anti-2,4,6-trinitrophenyl (TNP) Ab (Intracell, London, UK), displaying the same isotype of the anti-Nef mAb (data not shown).

Cell culture
DCs were generated from peripheral blood mononuclear cells (PBMCs) isolated by Ficoll-Hypaque (Flow Laboratories, Hornby, Ontario, Canada) gradient separation of buffy coats obtained from healthy volunteer blood donors by the Transfusion Center of Università Degli Studi "La Sapienza" Rome. Monocytes were purified from PBMCs by positive selection using magnetic cell separation columns and CD14 Microbeads (Milteny Biotec, Bergisch Gladbach, Germany). Highly enriched monocytes (>95% CD14+) were cultured at 6 x 105/ml in RPMI 1640 medium supplemented with 15% heat-inactivated fetal calf serum, L-glutamine and penicillin-streptomycin and 250 ng/ml granulocyte macrophage-colony stimulating factor (PeproTech, London, England) and 500 U/ml interleukin (IL)-4 (R&D System, Minneapolis, MN, USA) at 37°C for 5 days. Differentiation to DCs was assessed both by morphological observation and the detection of specific surface markers by flow cytometry. These cells were CD14–, CD1a+, HLA-DRintermediate, HLA-ABCintermediate, CD80low, CD86 low consistent with an immature DC phenotype (data not shown). After 5 days of culture, Nef was added to iDCs or to DCs induced to mature by 200 ng/ml LPS (E. coli serotype 0111:B4, Sigma, St Louis, MO) for 24 h. LPS-treated DCs were CD83+, HLA-DRhigh, HLA-ABChigh consistent with a mature DC (mDC) phenotype (data not shown).

CD56+/CD3– NK cells were purified from PBMCs by negative selection with the NK Cell Isolation Kit (Milteny Biotec). The resulting cell preparation was at least 99% viable by trypan blue dye exclusion. The purity of the CD56+/CD3– NK cells was verified as >95% by direct staining for membrane expression of CD56/CD3 (anti-CD56 and anti-CD3 mAbs purchased from BD, Biosciences, San Jose, CA, USA).

DC/NK cocultures were performed in RPMI 1640 supplemented with 10% fetal calf serum, L-glutamine, and penicillin/streptomycin in round-bottomed 48-well plates at a ratio of 1DC:2NK cells. In some experiments, blocking mAbs against IL-12, tumor necrosis factor (TNF)-{alpha} (R&D System), IL-15 (Genzyme, Boston, MA, USA), and IL-1ß (PeproTech) were added at the beginning of culture. Where indicated, NK cells and DCs were separated by porous membrane (pore size 0.4 µm) in a Transwell apparatus (Corning Costar Corp., Cambridge, MA, USA) in 24-well plates. In some experiments, purified NK cells were stimulated with 100 U/ml recombinant human IL-2 (R&D Systems, Minneapolis, MN, USA).

Flow cytometry and cell sorting
Analysis of cell surface markers was performed using the following mouse mAbs in direct immunofluorescence assays: phycoerythrin (PE)-conjugated or fluorescein isothiocyanate (FITC)-conjugated anti-CD56 (IgG1), FITC-conjugated anti-CD3 (IgG1), FITC-conjugated anti-HLA-DR (IgG2a), FITC-conjugated anti-CD25 (IgG1), FITC-conjugated anti-CD16 (IgG1) (all from BD Biosciences, San Jose, CA, USA), FITC-conjugated TNFR1 (CD120a) and FITC-conjugated TNFR2 (CD120b) (CALTAG Laboratories, Burlingame, CA, USA). For indirect immunofluorescence staining, anti-CCR7 mAb (BD Biosciences) was added, followed by FITC-conjugated isotype-specific goat anti-mouse Ab (BD Biosciences). Negative controls included directly labeled or unlabeled isotype-matched irrelevant mAbs. Cells were analyzed with a FACScan flow cytometer and CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA).

CD56bright and CD56dim NK cell subsets were purified based on CD56 cell surface density by FACS (FACSAria, Becton Dickinson) upon staining with the PE-conjugated anti-CD56. Cells labeled with fluorochrome-conjugated isotype antibodies (PharMingen, San Diego, CA, USA) were used to gate nonspecific fluorescence signals, while dead cells were excluded on the basis of propidium iodide (5 µg/ml, BD Biosciences) fluorescence intensity.

Proliferation assay
Autologous iDCs or mDCs were left untreated or treated for 24 h with Nef and, after extensive washing, suspended in RPMI 1640 supplemented with 10% human serum, L-glutamine, and penicillin/streptomycin and irradiated (3,000 rad from a 137Cs source). DCs were added in graded doses to 1 x 105 NK cells in 96 flat-bottom microplates (Falcon, Becton Dickinson, Meylan Cedex, France). After 5 days, cultures were pulsed for 18 h with 0.5 µCi/well of [3H]thymidine (Amersham International, Buckinghamshire, UK). Cells were then harvested onto glass fiber filters, and [3H]thymidine incorporation was measured by liquid scintillation spectroscopy. Results are expressed as mean counts per minutes (cpm).

Apoptosis detection
Annexin V assay was used to detect NK cell apoptosis. After 48 h of DC/NK coculture, cells were harvested and stained with PE-conjugate anti-CD56 mAb, for 30 min at 4°C. Cells were washed twice with ice-cold PBS and specific binding of FITC-conjugate annexin V was performed with an apoptosis detection kit (Pharmingen), according to the manufacturer’s instructions. The cells were then analyzed by flow cytometry to measure costaining of the CD56+ and annexin V+ population, gating on the living cells.

Cytokine and chemokine assay
Analysis of supernatant cytokine content was performed after 48 h of DC/NK coculture. Supernatants were collected and TNF-{alpha}, GM-CSF, macrophage inflammatory protein (MIP)-1 {alpha}, MIP-1ß, regulated on activation normal T cell expressed and secreted (RANTES) (all from R&D Systems), IL-10, and IFN-{gamma} (Pierce-Endogen, Rockford, IL, USA) contents were measured using a sandwich ELISA according to the manufacturer’s instructions.

Cytotoxicity assays
After 48 h of DC/NK coculture, the NK cell-mediated cytotoxic activity was determined using a 4 h 51Cr release assays. The DAUDI or K562 cell lines were used as target cells for antibody-dependent and -independent cytotoxicity, respectively. Moreover, autologous DCs were used as target cells in order to evaluate the cytolytic activity of NK cells against DCs. Target cells were incubated with 100 mCi of Na251Cr O4 for 60 min at 37°C and then extensively washed. Before the ADCC assay, the DAUDI cell line was incubated with 1 µg/106 cells anti-human CD27/TNFRSF7 Ab (R&D Systems) for 30 min at 4°C. Assays were performed in triplicate at the indicated effector:target ratios. After 4 h of effector/target coculture, supernatants were collected and radioactivity was counted on a gamma counter (Wallac, Turku, Finland). The percentage of cytotoxicity was determined as follows: [(experimental release-spontaneous release)/(maximal release-spontaneous release)] x 100. Maximal release was obtained by incubating 0.5% Triton-100 with target cells. Spontaneous release was determined through the incubation of culture medium with targets. Spontaneous chromium release was always less than 10% of the maximal release value.

Statistical analysis
Experiments were repeated at least four times, and P values were determined by using a two tailed Student’s t test. The statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nef-pulsed DCs modulate the expression of activation markers in NK cells
It has been recently demonstrated that DCs are capable of activating NK cells (4) . Therefore, we first investigated whether Nef was able to affect DC stimulatory capacity evaluating surface expression of the activation markers on cocultured NK cells. To this aim, iDCs or mDCs were pulsed with Nef for 24 h and then cocultured with autologous resting NK cells. As shown in Fig. 1 A,B, in the presence of Nef-pulsed iDCs the expression of HLA-DR is increased in the CD56bright NK cell subset, while is down-regulated on the CD56dim subset. In addition, increased expression of CD25 and CCR7 is detected on the CD56bright NK cell subset cocultured with Nef-pulsed iDCs (Fig. 1A, C, D ). As expected, the expression of these molecules is up-regulated in NK cells cocultured with mDCs. Nef-pulsed mDCs down-regulate HLA-DR expression on CD56dim NK cells (Fig. 1A, B ) and up-regulate CD25 and CCR7 expression on CD56bright NK cells (Fig. 1A,C,D ). IL-2, used as positive control, up-regulates HLA-DR, CD25, and CCR7 surface expression on NK cells (Fig. 1B-D ).


Figure 1
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  		Figure 1. Nef-pulsed DCs modulate the expression of activation markers in NK cells. A) NK cells were cocultured with untreated and Nef-treated iDCs or mDCs. Control cultures contain NK cells alone (medium) or Nef-treated NK cells (Nef). HLA-DR, CD25, and CCR7 surface expression was evaluated after 24 h on CD56+ NK cells by double staining. CD56bright and CD56dim NK cells were gated on the basis of their fluorescence intensity. Gates were placed based on isotype-matched mAbs. The percentages of HLA-DR positive cells in the CD56bright and CD56dim gated cells are shown. The percentages of CD25- and CCR7-positive cells in the CD56bright NK cells are shown. Flow cytometry dot plots from one representative experiment out of five are shown. B–D) Summary of the data (mean±SEM) from four different experiments done as in A. *P < 0.05

We found that NK cell activation by DCs is dependent on cell-to-cell contact as separation of the cells by a porous membrane (transwell) completely abrogates the up-regulation of HLA-DR, CD25, and CCR7 (Fig. 1B-D ).

A direct effect of Nef on resting and IL-2-activated NK cell is not observed (Fig. 1A-D ). Therefore, Nef modulates NK cell surface markers by an indirect mechanism involving DCs.

Nef-pulsed DCs modulate NK cell proliferation
We investigated whether Nef affects the ability of DCs to stimulate the proliferation of NK cells. To this aim, we set up an autologous coculture between NK cells and irradiated DCs. As expected, mDCs show a higher stimulatory capacity compared with iDCs. Nef-pulsed mDCs significantly down-regulate NK cell proliferation compared to untreated mDCs. iDCs weakly stimulate NK cell proliferation and Nef treatment further reduces this capacity (Fig. 2 A).


Figure 2
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  		Figure 2. Nef-pulsed DCs modulate NK cell proliferation. A) NK cell proliferation was evaluated by measuring the amount of [3H]thymidine incorporated culturing for 5 days resting NK cells in microwells containing different numbers of DCs, as indicated. Means cpm ± SEM from four different experiments are shown. *P < 0.05. B) Data represent the mean CD56bright/CD56+ cell ratio ± SEM of five separate experiments. C) Annexin V assay was used to detect apoptosis on CD56bright and CD56dim NK cells by double staining. A representative experiment out of three is shown. {square}, indicates CD56bright; {blacksquare} CD56dim.

We next investigate whether Nef-pulsed DCs affect the frequency of CD56bright and CD56dim NK cells. As expected, ~10% of NK cells in peripheral blood show a high-density expression of CD56 (Fig. 2B ). A selective enrichment of the CD56bright NK cell subset is observed in coculture with Nef-pulsed iDCs compared to coculture with untreated iDCs. However, the results obtained are not statistically significant, likely due to the high variability in the absolute NK cell number between different donors. As shown in Fig. 2C , we found that CD56dim NK cells are more prone to apoptosis compared to CD56bright NK cells. However, the enrichment in the CD56bright NK cells is not due to an increased susceptibility to apoptosis of CD56dim NK cell following coculture with Nef-pulsed iDCs. mDCs further increase the percentage of apoptosis of both NK cell subsets and Nef treatment does not exert any effect.

Nef-pulsed DCs modulate NK cell effector function
To better define the effect of Nef on DC/NK crosstalk, we monitored the effector function of the two NK cell subsets. First, we analyzed cytokine and chemokine secretion after 48 h of coculture. As shown in Fig. 3 , in the presence of Nef-pulsed iDCs, we found a significant up-regulation of IFN-{gamma} and TNF-{alpha} secretion, while IL-10 and GM-CSF are significantly down-modulated. IL-10 release is significantly down-regulated by mDCs compared to iDCs, and Nef treatment further reduces its release.


Figure 3
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  		Figure 3. Nef-pulsed DCs modulate cytokine and chemokine production. Analysis of cytokine and chemokine supernatant concentration in cocultures between NK and untreated and Nef-treated iDCs or mDCs was determined by ELISA. Control cultures contain NK cells alone (medium) or IL-2-activated NK cells (IL-2). Supernatants were harvested after 48 h of coculture and tested for cytokines (IFN-{gamma}, TNF-{alpha}, IL-10, GM-CSF) and chemokines (MIP-1{alpha}, MIP-1ß, RANTES). Results are expressed as picograms per milliliter and are the means ± SEM from three independent experiments. *P < 0.05 vs. iDC+NK.

Because ß-chemokines may suppress HIV replication (24) , we analyzed the pattern of the production of these mediators in DC/NK coculture. As shown in Fig. 3 , in the presence of Nef-pulsed iDCs, we found a significant down-regulation of MIP-1{alpha} and RANTES secretion, while MIP-1ß is not affected. Notably, mDCs significantly up-regulate RANTES production, and Nef treatment reduces its release.

Nef does not modulate cytokine and chemokine secretion when NK cells and DCs are separated in transwell (data not shown).

We next checked the effector function typical of the CD56dim subset. We tested the cytolytic activity in terms of natural cytotoxicity against K562 cell line and ADCC against DAUDI cell line. As expected, cytotoxic activity of NK cells is acquired on contact with DCs and mDCs induce the strongest cytotoxicity in cocultured NK cells. No difference is observed between untreated or Nef-treated DCs (Fig. 4 A). On the other hand, Nef-pulsed iDCs and mDCs induce a significant (P<0.05 at 25:1 E/T ratio) down-regulation of ADCC (Fig. 4B ). Inhibition of ADCC is not accompanied by a reduced expression of CD16 (Fc{gamma}-receptor), while intracellular perforins are significantly (P<0.05) down-regulated on CD56dim NK cells cocultured with Nef-pulsed iDCs (Fig. 4C ). Resting and Nef-treated NK cells do not express intracellular perforin (Fig. 4C ).


Figure 4
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  		Figure 4. Nef-pulsed DCs modulate NK cell-mediated cytotoxic activity. NK cells were cocultured with untreated and Nef-treated iDCs or mDCs for 24 h. A) NK cells (effector cells, E) cytotoxic activity was evaluated against K562 cell line (target cells, T). B) ADCC was evaluated against DAUDI cell line (T) preincubated with anti-human CD27/TNFRSF7 Ab. Assays were performed in triplicate at the indicated E:T ratios. A representative experiment out of four performed in triplicate is shown. C) CD16 surface expression and intracellular perforins were evaluated on CD56dim NK cells by double staining. The percentages of double-positive cells are shown. Control cultures contain NK cells alone (medium) or Nef-treated NK cells (Nef). Flow cytometry dot plots from one representative experiment out of four are shown. D) NK cells (E) were tested for their cytolytic activity against untreated or Nef-treated iDC or mDC (T). Assays were performed in triplicate at the indicated E:T ratios. A representative experiment out of two is shown.

The susceptibility to lysis of DCs by autologous NK cells has been widely described (14) . Therefore, we investigated whether Nef challenge could interfere with DC survival. As shown in Fig. 4D , we confirm the ability of NK cells to lyse iDCs, although they are less efficient against Nef-pulsed iDCs and mDCs.

Nef-pulsed DCs do not induce CD56dim NK cell terminal differentiation
The improved functional activity of the CD56bright NK cell subset in coculture with Nef-pulsed DCs, mainly consisting in activated phenotype and INF-{gamma} release, may be due to their higher proliferation or to the terminal differentiation of the CD56dim NK cells in CD56bright NK cells (11) . To test this hypothesis CD56dim and CD56bright NK cells were sorted and then independently cocultured with Nef-pulsed DCs at 1:2 DC/NK cell ratio. The two NK cell subsets are clearly identified by CD56 surface density expression where CD56bright NK cells represent a cell population with a fourfold higher mean fluorescence intensity compared to CD56dim NK cells (Fig. 5 A).


Figure 5
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  		Figure 5. Nef-pulsed DCs do not induce CD56dim NK cell terminal differentiation. A) CD56bright and CD56dim NK cell subsets were purified based on CD56 cell surface density by FACS upon staining with the anti-CD56-PE. B) CD56bright and CD56dim NK cells were independently cocultured with untreated or Nef-treated iDC or mDC for 24 h. The percentages of HLA-DR+ cells in the CD56bright and CD56dim NK cells are shown. C) CD56bright and CD56dim NK cell proliferation was evaluated by measuring the amount of [3H]thymidine incorporated culturing for 5-day resting sorted NK cells in microwells at different DC/NK cell ratio, as indicated. Means cpm ± SEM from three different experiments are shown. *P < 0.05. D) Supernatants were harvested after 48 h of coculture and tested for IFN-{gamma} by ELISA. Control cultures contain IL-2-activated NK cells. A representative experiment out of two is shown. n.d., not detected.

We do not observe the development of NK cells expressing high CD56 levels (CD56bright) in coculture between CD56dim NK cells and Nef-pulsed iDCs (Fig. 5B ) or Nef-pulsed mDCs (data not shown), indicating that CD56dim NK cells do not differentiate in CD56bright NK cells. Moreover, Nef-pulsed iDCs up-regulate HLA-DR expression (Fig. 5B ) and IFN-{gamma} release by cocultured CD56bright NK cells (Fig. 5D ), while down-regulate HLA-DR expression on CD56dim NK cell surface (Fig. 5B ). We next evaluated whether Nef-pulsed DCs selectively modulate the proliferation of one of the two NK cell subset. As shown in Fig. 5C , Nef-pulsed mDCs significantly up-regulate CD56bright NK cell proliferation. On the other hand, Nef-pulsed mDCs down-regulate CD56dim NK cell proliferation compared to untreated mDCs, although statistical significance has not been reached (P=0.07). This may be due to the high susceptibility to apoptosis of CD56 dim NK cells (see Fig. 2C ) that further increases after sorting and 5 days of coculture (data not shown). Nevertheless, considering that CD56dim NK cells represent ~90% of total peripheral NK cells, the significant inhibition of proliferative response of unseparated NK cells showed in Fig. 2A is likely ascribable to CD56dim NK cells. iDCs weakly stimulate both CD56dim and CD56bright NK cell proliferation (data not shown). Therefore, Nef-pulsed DCs exert a different and specific effect on the two NK cell subsets.

From these data, we can exclude that the divergent effects on the two NK cell subsets is due to the lower percentage of CD56bright NK cells (~10%) and the higher percentage of CD56dim NK cells (~90%) in coculture between DCs and total CD56+ NK cells.

Nef-pulsed DCs modulate NK cell function through cytokine production
We have previously demonstrated that Nef up-regulates IL-12, IL-15, IL-1ß, and TNF-{alpha} production by iDCs (42) . Because these cytokines are critical in NK cell activation, we evaluated their involvement in NK cell modulation induced by Nef-pulsed DCs.

The results obtained indicate that IL-12 and IL-15 could be involved in the activation of the CD56bright NK cell subset since the addition of anti-IL-12 or anti-IL-15 mAbs abrogates the enhancement of HLA-DR (Fig. 6 A). Similarly, the addition of anti-IL-12 and anti-IL-15 abrogates the enhancement of CD25 and CCR7 expression induced by Nef-pulsed iDCs on CD56bright NK cells (data not shown). On the other hand, anti-IL-1ß and anti-TNF-{alpha} mAbs do not exert any effect (Fig. 6A ). In addition, TNF-{alpha} could be involved in the inhibition of the CD56dim NK cell subset, since the addition of anti-TNF-{alpha} mAbs abolish the down-modulation of HLA-DR surface expression (Fig. 6A ).


Figure 6
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  		Figure 6. Nef-pulsed DCs modulate NK cell function through cytokine production. NK cells were cocultured with Nef-pulsed iDCs and, where indicated, with blocking mAbs. A) HLA-DR surface expression was evaluated after 24 h on CD56bright and CD56dim NK cells by double staining. Means ± SEM from three independent experiments are shown. {square} indicates CD56bright; {blacksquare}, CD56dim. B) Supernatants were harvested after 48 h of coculture and tested for IFN-{gamma} by ELISA. Results are expressed as fold increase over the basal level of IFN-{gamma} release of NK cells cocultured with untreated iDCs. *P < 0.05 vs. medium. C) TNFR1 and TNFR2 surface expression on resting or activated NK cells. CD56bright and CD56dim NK cells were gated on the basis of their fluorescence intensity. A representative experiment of two is shown.

Moreover, IL-12, IL-15, and IL-1ß could be the factors involved in the up-regulation of IFN-{gamma} production observed in coculture between Nef-pulsed iDCs and NK cells, as assessed by adding of anti-IL-12, anti-IL-15, or anti-IL-1ß mAbs (Fig. 6B ).

As well known, NK cells constitutively express receptors for monocyte-derived cytokines (46) , and these cytokines are able to induce CD56dim NK cell-mediated cytotoxic activity and cytokine production by CD56bright NK cells. Because we found that TNF-{alpha} inhibits CD56dim NK cell but not CD56bright NK cell activation, we wondered whether its receptors are differently expressed by the two NK cell subsets. Interestingly, we found that TNFR1 and TNFR2 are constitutively expressed on CD56dim NK cells while CD56bright NK cells do not express TNFRs. Moreover, neither IL-2 or DC stimulation induces TNFR expression on CD56bright NK cells (Fig. 6C ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we outline a novel mechanism by which Nef, targeting DCs, modulates NK cell functions. We found that Nef up-regulates the capacity of DCs to stimulate the immunoregulatory NK cells (CD56bright), as assessed by the up-regulation of activation marker surface expression, the up-regulation of their proliferative response, as well as of INF-{gamma} release. On the other hand, Nef-pulsed DCs inhibit cytotoxic NK cells (CD56dim), as assessed by the reduced HLA-DR surface expression, the reduced proliferation, as well as reduced cytotoxic activity. Therefore, Nef may both favor HIV replication via cytokine secretion and may contribute to the immune evasion hindering NK cell cytotoxic activity.

The role of DC/NK cells interactions in the pathogenesis of HIV infection is an area of active investigation. A recent study shows an aberrant interaction between DCs and the CD56neg subset of NK cells from HIV-1-infected viremic individuals (47) .

Recently, we demonstrated that Nef is efficiently internalized by iDCs inducing their morphological, phenotypical, and functional differentiation (42 , 43) . This has a direct impact on CD4+ T lymphocyte bystander activation, thus favoring viral spread. On the other hand, we demonstrated that Nef targeting iDCs impairs functional competence and survival of CD8+ T cells (44) . In addition, we previously demonstrated that Nef down-regulates antibody response (48) . Thus, Nef subverts host immune functions to escape both cellular and humoral adaptive immune responses. Here, we demonstrate that Nef, targeting DCs, may also impair innate immune response favoring evasion.

Nef-pulsed DCs have a different impact on CD56dim and CD56bright NK cells, and this is relevant since both NK cell subsets exert a role in antiviral defense. In accordance with the quantitative imbalance of the CD56bright and CD56dim subset within the total NK cell population observed in HIV+ patients (49) , we found that Nef-pulsed DCs are much stronger inducers of CD56bright NK cell activation and proliferation than untreated DCs. Our finding closely fits with in vivo studies showing a decreased number of CD16+CD56dim NK cell subset with disease progression, whereas the CD16–CD56bright NK cell subset is usually conserved and proportionally increased (50) .

The up-regulation of CD25, a component of the IL-2 receptor complex, may be functionally relevant since its expression renders NK cells highly responsive to IL-2. In addition, the up-regulation of CCR7 surface expression may promote CD56bright NK cell recruitment in lymph node. Notably, this may be crucial in AIDS pathogenesis since the decreased pool of peripheral NK cells may reduce the extent of innate response.

Activation of CD56bright NK cells is also observed during Mycobacterium tuberculosis infection. It has been reported that the enrichment in the CD56bright NK cell subset in tuberculosis pleurisy is due to an increased susceptibility to apoptosis of CD56dim NK cell subset (51) . In the present study, the enrichment in the CD56bright NK cell subset is due to the lower proliferation rate of CD56dim NK cells instead of an increased susceptibility to apoptosis of CD56dim NK cell following coculture with Nef-pulsed DCs.

Recently, it has been suggested that CD56bright NK cells are terminally differentiated, activated NK cells (11) . Thus, to test this event in DC/NK cell coculture, CD56dim and CD56bright NK cells were sorted and then independently cocultured with Nef-pulsed DCs. In coculture between CD56dim NK cells and Nef-pulsed DCs, we do not observe the development of NK cells expressing high CD56 levels, confirming a different and specific effect on the two NK cell subsets.

Since innate immunity play a central role in host defense against HIV infection by cytolytic and noncytolytic mechanisms, we evaluated the functional competence of NK cells cocultured with Nef-pulsed DCs. In these experimental conditions, we found a significant up-regulation of INF-{gamma} production. These findings fit well with the activated phenotype of the immunoregulatory CD56bright NK cell subset. In the presence of Nef-pulsed DCs, we also found a significant up-regulation of TNF-{alpha} secretion. As we previously demonstrated, Nef up-regulates TNF-{alpha} secretion by iDCs (42) . Nevertheless, it is unlike that TNF-{alpha} measured in DC/NK coculture is instead secreted by DCs on interaction with NK cells, since after Nef starvation, they produce low cytokine and chemokine levels. Notably, we found that NK cell modulation by DCs is dependent on cell-to-cell contact confirming the relevance of surface expressed cytokines rather than their soluble form.

Cytokine release, as well as the down-regulation of IL-10 production, might favor activation of T cells, rendering them permissive for the replication of the virus (52) and activating HIV replication in latently infected cells (53 , 54) . Moreover, the down-modulation of RANTES and MIP-1{alpha} production, two ß-chemokines licensed for the recruitment of other effector cells and for the inhibition of HIV entry, may represent a viral tool to escape host immune response. Since we previously demonstrated that Nef up-regulates chemokine production by iDCs (42) , it is unlike that chemokines measured in DC/NK coculture are instead secreted by DCs on interaction with NK cells. Overall, these results indicate that Nef-pulsed DCs in coculture with NK cells generate a microenvironment that may contribute to viral spreading.

Besides noncytolytic mechanisms, ADCC and natural cytotoxicity play an important role in protective immunity against viral infection. The protective nature of ADCC in HIV infection has been documented in a number of human and animal studies and strategies employing NK cells prearmed with ADCC-directing Ab has been extensively investigated (55) . Here, we found that Nef inhibits the ability of DCs to induce ADCC, without affecting CD16 surface expression. It has been reported that factors other than absolute HIV-1 specific Ab titer were responsible for decreased ADCC observed during disease progression (56) . Our finding suggests that Nef could be an active player in the alteration of ADCC and could impact on the accumulation of an anergic subset of effector cells of innate immune system.

We have demonstrated that Nef-pulsed DCs activate CD56bright NK cells through an up-regulation of IL-12, IL-15, and IL-1ß production. On the other hand, we found that TNF-{alpha} plays a critical role in the induction of CD56dim NK cell inactivation. Similarly, we previously demonstrated that TNF-{alpha} is in charge of the Nef-induced CD8+ T cell anergy and apoptosis (44) .

CD56bright NK cells have been shown to be generally better responders to various cytokine stimulations, as compared with the CD56dim NK subset, due to preferential expression of a number of membrane-associated receptors (57) . Moreover, several proteins expressed at higher levels on the CD56bright NK cell subset could also contribute to the enhanced cytokine responsiveness of this subset, rendering them "metabolically ready" to engage in the protein synthesis process (46) . It has been reported that differential expression of SHIP1 in CD56dim and CD56bright NK cells provides a molecular basis for distinct functional responses to IL-1ß (58 , 59) .

Recent reports indicate that IL-15, which is essential for development and survival of NK cells, decreases surface expression of CX3CR1, a chemokine receptor that modulates adhesion, migration, and killing. Thus, IL-15 may act as a negative regulator of innate immune response that depends on CX3CR1. Since CX3CR1 is expressed on almost all CD56dim NK cell subset, while CD56bright NK cells express very low levels of CX3CR1 (60 , 61) , it can be hypothesized that IL-15 differently modulates the two NK cell subsets.

Interestingly, we found for the first time that TNFRs are constitutively expressed only on CD56dim NK cells, while CD56bright NK cells do not express TNFRs and this may account for the observed functional inactivation of the CD56dim subset. A previous study reported that TNF-{alpha} plays a pivotal role in the activation of NK cells by IL-2 (62) . Thus, TNF-{alpha} may either activate or inhibit the function of NK cells depending on the nature of the stimulus used. Such dual roles for TNF-{alpha} are consistent with other findings demonstrating that different TNF-{alpha} signaling pathways have been reported to lead either to anergy and cell death by apoptosis or to activate NF-{kappa}B and inhibit cell death (63) . Moreover, different effects of membrane vs. soluble TNF have been reported (64) . Here, we found that a direct cell-to-cell contact is mandatory for NK cell modulation by DCs, indicating that surface expressed cytokines but not their soluble form are relevant for NK cell activation or inactivation.

There are several in vivo implications relevant to our present findings. The model system used in this study, i.e., Nef-pulsed DC interactions with NK cells, recapitulates several of the functional defects that are seen in NK cells isolated from chronically HIV-infected individuals in vivo (65) .

Our observations suggest a scenario in which Nef-pulsed DCs are especially suited to activate NK cells in lymph nodes where CD56bright NK cells are enriched and where high levels of HIV replication are associated with abundant extracellular Nef. Qiao et al. (66) found that uninfected DCs and B cells might accumulate Nef in the germinal centers of infected lymphoid follicles not as a result of endogenous synthesis, but instead as a result of internalization from the extracellular environment. Moreover, they reported that Nef penetrates in B cells in vitro, and we previously demonstrated that Nef enters CD14+ monocytes, U937 promonocytic cell line (67) , and iDCs (43) . HIV-1-infected cells would release Nef through a nonclassical secretory pathway, or after lysis (68) . Gould et al. (69) proposed the "Trojan horse" hypothesis, which suggests that the exosomal secretion of viral proteins not packaged in viral particles, includes Nef.

Another critical checkpoint where Nef-pulsed DC and NK cell interaction could take place is peripheral tissue, since it has been reported that a high percentage of sera from HIV-1 infected individuals contains soluble Nef (from 1 to 10 ng/ml), while the patient’s sera in which Nef is not detectable, contain high titers of anti-Nef Abs (68) .

Our present data add new evidence for a role of extracellular Nef as a viral tool to escape a first line of immune response and may support the discovery of suitable anti-Nef drugs. Moreover, knowledge of the distinct functional attributes of CD56bright and CD56dim NK cell subsets and the factors involved in their development and expansion may enable us to design strategies that preferentially activate the subset with the greatest therapeutic potential for HIV infection.


   ACKNOWLEDGMENTS
 
This research has been funded by a grant to M. V. from the National Research Project on AIDS.

Received for publication December 6, 2006. Accepted for publication March 1, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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