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* Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021;
University of Massachusetts Medical Center, Program in Molecular Medicine, Worchester, MA 01605;
Center for Biomedical Research, The Population Council, New York, NY 10021;
Aaron Diamond AIDS Research Center, Rockefeller University, New York, NY 10016; and
¶ Tulane Regional Primate Research Center, Tulane University, Covington, LA 70433
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, CXCL8, CCL3, and
CCL4 release, but without up-regulating costimulatory and other
molecules characteristic of mature DCs. Coincident with this,
nef-expressing immature DCs stimulated stronger
autologous CD4+ T cell responses. Both SIV and HIV
nef-expressing DCs complemented defective SIVmac239
delta nef, driving replication in autologous immature
DC-T cell cultures. In contrast, if DCs were activated after capturing
delta nef, virus growth was not exacerbated. This
highlights one way in which nef-defective virus-bearing
immature DCs that mature while migrating to draining lymph nodes could
induce stronger immune responses in the absence of overwhelming
productive infection (unlike nef-containing wild-type
virus). Therefore, Nef expressed in immature DCs signals a distinct
activation program that promotes virus replication and T cell
recruitment but without complete DC maturation, thereby lessening the
likelihood that wild-type virus-infected immature DCs would activate
virus-specific immunity, but facilitating virus
dissemination. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the context of immunodeficiency viruses, however, DCs effectively promote virus amplification and spread especially upon encountering CD4+ T cells (reviewed in Ref. 9). Furthermore, immature DCs may be one of the first leukocytes entrapping and possibly replicating immunodeficiency virus crossing a mucosal surface (10, 11) and then (most notably as a more mature cell) transmitting infection to nearby CD4+ T cells for amplification (12, 13, 14). Thus, a paradox exists between the ability of DCs to present virus for activation of anti-viral immunity vs their ability to exacerbate virus growth. Understanding what drives virus replication in the DC-T cell milieu is critical to identify ways to limit this and favor immune activation.
Nef proteins of SIV and HIV are critical for viral pathogenesis and contain motifs that have been implicated in modulating cellular signaling as well as the trafficking of molecules between the outside and the inside of the cells in ways to facilitate virus dissemination (reviewed in Ref. 15, 16, 17). However, the exact mechanism of enhanced virus replication mediated through nef is not understood. Nef has been shown to exert both positive (15, 16, 17, 18) and negative (19, 20, 21) effects on T cell activation that might contribute to virus spread. In addition, soluble factors secreted from nef-expressing macrophages are sufficient to recruit and activate resting T cells, rendering them permissive to HIV infection (22). Recent studies have provided the first insight into how nef may also modify immature DCs to promote virus spread rather than Ag presentation. One report suggests that nef modulates DC-specific, ICAM-3-grabbing nonintegrin (DC-SIGN, CD209) expression on the DC surface to favor DC-T cell interactions and subsequent virus spread (23). Other work revealed that nef (introduced via a vaccinia recombinant vector) down-modulated class I MHC expression in immature DCs (24), reducing their ability to stimulate class I MHC-restricted CD8+ T cell responses. However, this was not supported by a separate study using an adenoviral vector to introduce nef (25). Exogenously added recombinant Nef was also shown to activate immature DCs while decreasing class I MHC (26).
SIV (27) and HIV (28) replication in immature
DC-T cell cocultures is dependent on the presence of nef.
Yet mature DCs readily promote SIV delta nef (delta
nef) growth (27), suggesting that when a mature
DC encounters the virus it somehow overrides the need for
nef. To investigate more closely how nef in a
wild-type virus could influence immature DC functions to foster virus
dissemination, we introduced nef into immature DCs using
recombinant adenovirus (Adeno). Several groups, including our own,
found that Adeno infection had little if any impact on DC membrane
phenotype (Fig. 2
) (25, 29, 30, 31, 32, 33, 34, 35), making it a reliable way
to introduce genes of interest. Expression of nef in an
immature DC triggers the DC to secrete inflammatory cytokines and
chemokines much like mature DCs, yet in the absence of the membrane
phenotypic changes that are typical of maturation. Furthermore,
nef-expressing DCs exhibited increased ability to activate
autologous CD4+ T cells and were able to enhance
replication of delta nef in immature macaque DC-T cell
cultures. However, maturation of the DCs within the virus-loaded
cultures was not sufficient to rescue delta nef replication.
HIV nef and SIV nef were interchangeable between
human and macaque DCs in their ability to trigger these functions.
These data suggest that nef induces selective or different
pathways of the DC maturation signaling network, creating an
environment encouraging T cell recruitment and generalized activation
of T cells by phenotypically immature DCs. This milieu readily
amplifies virus replication, but would less efficiently induce
Ag-specific T cell responses, accenting how wild-type virus growth may
be driven in the absence of an effective anti-viral immune
response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 (Cellgro; Fisher Scientific, Springfield, NJ) was supplemented with 2 mM L-glutamine (Life Technologies, Grand Island, NY), 50 µM 2-ME (Sigma-Aldrich, St. Louis, MO), 10 mM HEPES (Life Technologies), penicillin (100 U/ml)-streptomycin (100 µg/ml; Life Technologies), and 1% human plasma (heparinized).
Animals
Adult macaques (Macaca mulatta) were housed in the Tulane Regional Primate Research Center. Animal care operations were in compliance with the regulations detailed under the animal welfare act, and in the Guide for the Care and Use of Laboratory Animals. Before use, all animals used in this study tested negative for Abs to SIV, type D retroviruses, and simian T cell leukemia virus type 1. Male and female adult macaques were used for this study.
SIV isolates
The cloned viruses SIVmac239 (wild type) and SIVmac239 delta nef (delta nef) (36) were grown as previously described (37).
Generation of DCs
DCs were generated from PBMCs isolated from healthy macaques or HIV seronegative human donors. Donors were not screened for anti-Adeno immunity. Buffy coat units were purchased from the New York Blood Center to generate human DCs. Peripheral blood was collected by standard venipuncture using heparinized Vacutainers (BD Bioscience, San Jose, CA) from healthy SIV-seronegative rhesus macaques (anesthetized with 10 mg/kg ketamine HCl). The mononuclear cell fraction was isolated by Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient (38). DCs were generated from either adherent PBMCs as previously described (27) or CD14+ monocytes isolated using the anti-human CD14 MACS system (Miltenyi Biotech, Auburn, CA) (6) and were plated at 3 x 106 cells/well in a six-well tray (3 ml/well) in the presence of 100 U/ml recombinant human IL-4 (R&D Systems, Minneapolis, MN) and 1000 U/ml recombinant human GM-CSF (Immunex, Seattle, WA). Cytokines were added to the cultures on days 0, 2, 4, and 6 in culture. After 6–7 days in culture, immature DCs were harvested for infection.
Analysis of DC phenotype
The DC phenotype was monitored by flow cytometry for each experiment. At least 1 x 104 DCs were resuspended in PBS/5% FCS/0.1% sodium azide (staining buffer; 100 µl/well of a V-bottom 96-well tray (ICN Biomedicals, Aurora, OH)). The cultures were stained with the appropriate PE-conjugated IgGs (BD Biosciences; Ancell, Bayport, MN; R&D Systems) or with PE-labeled mAbs against HLA-DR, CD25, CD58, CD80, (BDIS), CD4, CD40, CD86, CD206, CXCR4, CCR5 (BD PharMingen, San Diego, CA), CD83 (Immunotech, Marseilles, France), HLA-ABC (DAKO, Carpinteria, CA), CD74 (Ancell), CD209 (used for both human and macaque cells; R&D Systems), or CCR5 (used for macaque cells; R&D Systems). Indirect staining with goat F(ab')2 anti-mouse Ig (BioSource, Camarillo, CA) was used to detect CD205 expression using the mouse anti-human CD205 clone 38-2 (39), CD209 staining using either mAb 507 (provided by Dr. V. KewalRamani) (40) for macaque cells or DC28 for human cells (AIDS Research and Reference Reagent Program, National Institutes of Health, Bethesda, MD). The necessary unconjugated IgGs were included as negative controls for the indirect staining. The cells were incubated with the Abs for at least 20 min at 4°C, then were washed four times with staining buffer and fixed in 10% formalin in PBS (pH 7.2–7.4). For the indirect stains, the cells were washed after the primary Ab staining, incubated with the secondary goat-anti-mouse Ig, and washed again before fixation. Stained cells were examined by flow cytometry using a FACScan (BD Biosciences), and the data were analyzed with CellQuest software (BD Biosciences).
Isolation and phenotyping of CD4+ T cells
The CD14-negative fractions of the macaque and human PBMCs were
cultured at 0.5–1 x 107/ml for 6–7 days
with the DCs, with additional fresh medium being added to the cultures
every 2 days. On the day of the assay, the CD4+ T
cells were further purified by negative selection using anti-CD8
and anti-HLA-DR MACS beads (Miltenyi Biotech) (6). The
resulting cell preparations were at least 99% viable by trypan blue
dye exclusion. Comparative infection studies (using macaque cells that
are usually more fragile than human cells) confirmed that such cultured
T cells were functional and behaved just like T cells freshly isolated
from PBMCs on the day of the experiment, avoiding the need to re-bleed
the donors (data not shown). The purity of the
CD3+CD4+ T cells was
verified as >95% by direct staining flow cytometry for membrane
expression of CD8 and HLA-DR (vs the simultest isotype control). The
activation state of the isolated CD4+ T cells was
also monitored each time before performing the experiments by direct
staining with FITC-conjugated anti-CD3 (BD PharMingen) combined
with PE-conjugated anti-CD69, -CD25, or -HLA-DR (BD
Biosciences, Fig. 5F).
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Adenovirus expressing the HIV-1 SF2 nef allele (Adeno-nefHIV), a mutant in the PxxP region of SF-2 nef (Adeno-nefHIVPA), or Adeno expressing the green fluorescent protein (Adeno-gfp) were previously described by Swingler et al. (22). Adeno expressing gfp and the HIV-1 SF2 nef allele was constructed using the Adeno-quest kit from Quantum Biotech (Qbiogene, Carlsbad, CA) and their pQBI-AdCMV5GFP shuttle vector. This virus was used for the extensive FACS analyses, allowing us to gate on gfp+ cells in both the control and nef-loaded populations. Recombinant Adeno encoding the SIVmac239 nef allele (Adeno-nefSIV) was derived from Adeno type 5, deleted of E1 and E3 regions to generate a replication-deficient Adeno (29), and purchased from Quantum Biotech (Durham, NC). For Adeno infection, 105 immature DCs were infected in 50 µl medium with 100-1000 PFU of virus in a 96-well, round-bottom tray (ICN Biomedicals) for 2 h at 37°C. After 2 h fresh medium containing 1% human plasma was added. For T cell activation experiments 102–104 immature DCs were infected in 50 µl medium, and after 2 h 105 autologous T cells were added to the DCs. Adeno-infected DCs or DCs mixed with T cells were cultured for various lengths of time before the indicated parameters were measured. Adeno stocks were monitored for endotoxin contamination using the Single Test Limulus Amebocyte Lysate Assay (N289-06; BioWhittaker, Walkersville, MD). Endotoxin levels in the Adeno preparations were routinely <0.06 endotoxin units/ml.
Immunoperoxidase staining of cells for Nef
Cytospins of DCs were prepared using a cytocentrifuge (Shandon,
Pittsburgh, PA), loading 
2 x 104
cells/slide (41). Slides were air-dried for 1 h at
room temperature and subsequently fixed in absolute acetone for 10 min
at room temperature. The slides were incubated with a mouse mAb
recognizing SIVmac251 Nef (17.2, AIDS Research and Reference Reagent
Program) for 30 min at room temperature, washed five times with
staining buffer, and incubated for 30 min with HRP-conjugated donkey
anti-mouse Ig (DAM-HRP; 1/300 dilution; Jackson ImmunoResearch,
West Grove, PA) for 30 min at room temperature. Non bound
HRP-conjugated donkey anti-mouse Ig was washed off, and bound HRP
was detected with stable 3,3'-diaminobenzidine tetrahydrochloride
dihydrate (Research Genetics, Huntsville, AL). The slides were mounted
in PBS/glycerol and analyzed using an Olympus AX70 microscope
(Melville, NY).
Determination of T cell activation
To assess levels of T cell activation and proliferation, 102–104 immature DCs (infected or not with Adeno) were mixed with 1 x 105 CD4+ T cells and cultured in a round-bottom, 96-well tray for up to 6 days. Tritiated thymidine ([3H]TdR; 1 µCi/well; NEN, Boston, MA) was added to the cells for the final 8 h of culture, and the [3H]TdR incorporated by proliferating cells was measured using a Wallac 1205 Betaplate liquid scintillation counter (Gaithersburg, MD). Responses are reported as mean counts per minute of [3H]TdR incorporated by duplicate or triplicate cultures (±SEM). T cell activation was also monitored by flow cytometry, where additional wells containing replicate DC-T cell mixtures were collected after 3 days and stained for T cell activation markers (PE-CD25, CD69, or HLA-DR) in combination with CD3 (FITC). The T cells were analyzed by gating on small and large activated lymphocytes by forward scatter excluding the DCs and DC-T cell conjugates. The gate marker was set relative to the isotype controls for each condition, and the percentage of cells stained above this cut-off point is given. Expression of the nuclear activation Ag Ki-67 was also monitored as a measure of T cell activation after 1–3 days of DC-T cell coculture by immunoperoxide staining of acetone-fixed cells (above) using the anti-Ki-67 mAb (MIB-1, AMAC, Westbrook, ME) (41). The percentage of Ki-67+ cells was calculated by counting the numbers of Ki-67+ cells in five independent fields (averaging 100 total cells in each) and is expressed as mean ± SEM over the five fields.
Measurement of chemokines and cytokines
The supernatants of Adeno-infected DCs (vs mock controls) were
collected, by taking 50 µl from the 200-µl cultures and replacing
it with 50 µl fresh medium at the indicated time points. Aliquots
were stored at -20°C before analysis of the chemokine and cytokine
content by ELISA. Recombinant anti-human CCL3 (macrophage
inflammatory protein-1 (MIP-1)), CCL4 (MIP-1
), CCL5 (RANTES),
CXCL8 (IL-8), IL-10, and IL-6 ELISAs were obtained from R&D Systems.
CCL3, CCL4, CCL5, CXCL8, and IL-6 ELISAs cross-reacted with macaque
chemokines and cytokines. Anti-human IL-12 ELISA was purchased from
Endogen (Woburn, MA) and cross-reacted with macaque IL-12. Recombinant
anti-monkey ELISAs for TNF-, IL-10, and IFN-
were purchased
from U-CyTech (Utrecht, The Netherlands).
In vitro SIV infection of DC-T cell cultures
After infection of immature DCs with Adeno as described above,
105 T cells were added to
104 DCs (per well) in a 96-well, round-bottom
tray. The cocultures were subsequently pulsed with 5 x
103 50% T cell infectious dose
of SIVmac239 wild type or delta
nef/105 cells for 1.5 h at 37°C
as previously described (27). Every 2 days of a 15-day
coculture, 50-µl aliquots were collected and stored at -20°C
before the p27 content was analyzed by ELISA (Zeptometrix, Buffalo,
NY). To determine whether the addition of maturation stimuli would
rescue delta nef replication in immature DC-T cell cultures,
the cultures were infected (27), after which the indicated
maturation stimuli were added. These included 50% monocyte-conditioned
medium (MCM) (42), soluble CD40 ligand (CD40L; 1/100 of a
baculovirus stock) (6), or a combination of
PGE2 (10-7 M,
PGE2; Sigma P6532) with TNF- (50 ng/ml; R&D
Systems). Two or 3 days postinfection, DC maturation in the infected
cultures was verified by FACS, measuring the expression of CD25, CD86,
CD83, and HLA-DR. In some experiments 5 ng/ml staphylococcal
enterotoxin B (SEB) or allogeneic CD4+ T cells
were added to provide an activated T cell environment.
Statistical analysis
The statistical significance of the nef-induced chemokine and cytokine production was analyzed by comparing the peak values of chemokine or cytokine production in Adeno-nef- vs Adeno-gfp-infected DCs. The nonparametric SIGN test was used to directly compare the two groups. There were no statistically significant differences between the mock and Adeno-gfp controls, and p values (to three decimal places) are shown for the Adeno-gfp vs Adeno-nef cultures. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To measure nef-induced changes in immature DCs, Adeno
vectors were used to express nef in the DCs
(Adeno-nef). Infection of immature DCs with Adeno expressing
the green fluorescent protein (gfp; Adeno-gfp) or
Adeno expressing gfp and the HIV-1 SF2 nef allele
(Adeno-gfp-nefHIV) enabled measurement of
infection efficiency by FACS by monitoring the percentage of
gfp-positive DCs (Fig. 1A). The infection efficiency
in different donors ranged from 5–90% (>10 experiments), averaging
40% for human DCs and 53% for macaque DCs. Similarly, the levels of
gfp expression varied between donors and the various Adeno
preparations, with the Adeno-gfp infections sometimes
appearing more efficient than the
Adeno-gfp-nefHIV infections, as shown in Fig. 1A. However, this did not alter the nef effects
observed (below). Both the levels of expression and the numbers of
expressing cells typically peaked around 48 h postinfection with
either Adeno-gfp-nefHIV or Adeno-gfp,
and therefore gfp expression was routinely measured at this
time. The levels of Nef expression (immunoperoxidase staining; Fig. 1B) were variable between cells, with some Nef-positive
cells staining very strongly for the Nef protein (dark reaction
product) and others quite faintly, but above background (asterisks).
The amount of Nef-expressing DCs and that of gfp-expressing
DCs correlated closely. Nef expression in
Adeno-nefHIV-infected DCs was also confirmed by Western blot
(data not shown).
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) up-regulated the
expression of common maturation markers (HLA-DR, CD25, CD83, CD86). The
same was true when SIVmac239 nef was tested in both human
and macaque DCs (data not shown), but more detailed analyses have been
performed with the HIV construct that we have available in house.
Advances in the DC field highlight many molecules that are expressed by
DCs at different stages of activation, some of which may be important
in HIV/SIV replication and/or DC-T cell communication (reviewed in Ref.
9). Thus, a wide panel of markers was monitored to more
extensively assess the effects of nef on immature DC
membrane phenotype (Fig. 2). Uninfected controls run in parallel (not
shown) with those cells in Fig. 2 confirmed that Adeno infection has no
impact on the expression of these markers by DCs (35).
Notably, the DC phenotype was identical in the Adeno-gfp-
and Adeno-gfp-nef-infected DC cultures (Fig. 2). Despite
some variability between both human and macaque donors (Fig. 2B), there was no consistent up- or down-modulation of any
markers in response to nef, even when the
gfp-positive fractions (Adeno-infected cells) were directly
compared (Fig. 2). Importantly there were no changes in the expression
of known HIV-binding molecules such as CD4, CCR5, CXCR4, CD206
(macrophage mannose receptor), or CD209 (DC-SIGN) in response to
nef. Nef induces chemokine and inflammatory cytokine production in immature DCs
Another characteristic feature of DC maturation is the secretion
of chemokines and inflammatory cytokines (6, 43, 44). It
has been shown that nef triggers macrophages to secrete CCL3
(MIP-1) and CCL4 (MIP-1) (22) and that exogenous
recombinant Nef induces human DCs to secrete several cytokines and
chemokines (26). We specifically investigated whether
nef expressed in an immature DC could induce changes in
chemokine or cytokine expression. Human and macaque DCs were
analyzed for HIV nef-induced
production of chemokines (Figs. 3 and 4).
Nef expression in DCs induced the secretion of
-chemokines CCL3, CCL4, and CCL5 (RANTES). The secretion of CCL3 and
CCL4 in human immature DCs peaked at around 6–18 h. CCL5 secretion
tended to peak later (18–48 h) and at consistently lower levels. When
immature macaque DCs were infected with Adeno-nefHIV,
chemokine production was induced, but was delayed compared with that of
human DCs, with the peak production occurring after 48–72 h (Fig. 3A). The peak chemokine levels from independent experiments
using DCs from 6–12 different human (upper row) or macaque
(lower row) donors are summarized in Fig. 4A. The p values indicate the significance of the
nef-induced secretion in each case (Adeno-nef vs
Adeno-gfp populations). While nef-dependent CCL3
and CCL4 secretion was statistically significant, CCL5 production was
not; it was generally secreted at lower levels and less reproducibly
between donors. CCL3 secretion was induced by both HIV and SIV
nef in macaque (Fig. 3B) and human DCs (data not
shown). The kinetics of CCL4 production by HIV or SIV
nef-bearing DCs were similar to those of CCL3 (data not
shown).
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C), CCL4, and
CCL5 (data not shown) production in immature DCs. Therefore, the
proline-rich motif is not required for nef-induced chemokine
production by DCs.
We also observed that nef triggered statistically
significant secretion of IL-12, TNF-, CXCL8 (IL-8), and IL-6 in
immature human (Fig. 4) and macaque (Fig. 4A and data not
shown) DCs. On the average, cytokine release by macaque DCs was lower
than that produced by human DCs (e.g., IL-12 (Fig. 4A) and
TNF- (data not shown)) and in the case of TNF- did not reach
statistical significance. There was almost no background cytokine
secretion in uninfected or Adeno-gfp-infected immature DCs
of TNF-, IL-6, or IL-12. In contrast, high levels of CXCL8 secretion
were observed in uninfected immature DCs, as previously described
(6). However, the nef-induced CXCL8 production
was statistically significant. No nef-induced IL-1,
IL-10, or IFN- production was detected (data not shown).
Adeno-nef-infected DCs activate autologous T cells in coculture
The secretion of inflammatory cytokines and chemokines by
nef-expressing DCs (Figs. 3 and 4) is a characteristic
feature of DC maturation, yet the membrane phenotype of
nef-expressing DCs remains that of an immature DC (Fig. 2).
Since mature DCs are more potent in T cell stimulation than immature
DCs, and this usually coincides with elevated costimulatory molecule
expression (1), we were interested to ascertain what
impact nef had (if any) on the ability of immature DCs to
activate autologous CD4+ T cells. Cocultures of
nef-expressing DCs (vs control DCs) and autologous T cells
were analyzed for T cell activation by several methods. T cell
proliferation was monitored by [3H]TdR
incorporation (Fig. 5, A–D),
expression of the cell cycle nuclear Ag Ki-67 (Fig. 5E), and
up-regulation of activation markers by FACS (Fig. 5F).
The negligible expression of CD69 and CD25 by the purified T cells
before culture with nef-bearing vs control DCs is evident in
Fig. 5F (top row).
Infection of immature DCs with either Adeno-nefHIV or
Adeno-nefSIV provoked CD4+ T cell
proliferation in human and macaque autologous DC-T cell cultures (Fig. 5, A and C). In the human system T cells cultured
with Adeno-nefSIV-infected DCs showed lower
[3H]TdR uptake compared with
Adeno-nefHIV-infected DCs. This was not apparent, however,
in the macaque system. These levels of increased autologous T cell
proliferation stimulated by the nef-expressing immature DCs
reflected responses induced by mature DCs, but were significantly lower
than responses seen in allogeneic systems (Fig. 5B). The
increased autologous T cell proliferation observed in the presence of
nef-expressing DCs was dependent on the numbers of DCs added
to the cocultures (Fig. 5C). Differences in T cell
activation were still evident at a dose of 1 DC/30 T cells, but not at
the 1/100 ratio. The background autologous MLRs induced by the mock and
gfp-loaded DCs similarly decreased with lower DC numbers.
Further evidence that DCs are needed to augment T cell activation was
provided by the fact that addition of nef-triggered DC
supernatants to purified CD4+ T cells resulted in
minimal T cell proliferation (<500 cpm [3H]TdR
uptake compared with 100–200 cpm for the untreated, mock, or
gfp-DC supernatant controls vs the stronger responses seen
in PMA/ionomycin-positive controls; Fig. 5D). Thus, soluble
factors released by nef-triggered DCs alone are not
sufficient to drive the T cell proliferation seen in the
nef-DC-T cell cultures.
Expression of the nuclear activation Ag Ki-67 was also increased in
nef-bearing DC-T cell cocultures (Fig. 5E). In
addition, FACS analysis revealed an increase in the expression of the
activation markers CD69 and CD25 by T cells cocultured with
nef-bearing autologous DCs (Fig. 5F). The
percentage of CD69+ T cells went from 26–29%,
and that of CD25+ T cells from 14–18% in the
mock or gfp-infected DC-T cell cultures to 45 and 26%,
respectively, in the nef-loaded DC-T cell mixtures (Fig. 5F). Peak T cell activation levels are shown (Fig. 5).
Hence, paralleling the elevated secretion of chemokines and cytokines
induced by nef, nef-loaded immature DCs also
stimulate greater autologous MLRs.
Nef, but not activation with maturation stimuli, drives delta nef replication in immature DC-T cell cultures
Mature, but not immature, DCs facilitate delta nef amplification in concert with autologous CD4+ T cells, whereas wild-type virus replicates strongly in the presence of either DC subset (27). We have hypothesized that nef in the wild-type virus can modulate immature DCs to act more like their mature counterparts to drive virus growth. While we detected nef-induced secretion of soluble factors by immature DCs as well as a nef-dependent augmentation of their ability to stimulate autologous MLRs, this did not coincide with changes in DC membrane phenotype. Thus, we set out to delineate whether activating DCs via traditional maturation stimuli vs nef would promote delta nef replication in the immature DC-T cell milieu.
Immature macaque DC-T cell cocultures were pulsed with delta
nef (27), and the maturation stimulus, MCM, was
added after the infection to mature the DCs in the culture. After 2–3
days the DCs had matured (FACS confirmed up-regulated CD25, CD80, CD83,
and CD86; data not shown); however, replication of delta nef
was consistently lower than that of wild-type virus (Fig. 6A). Since MCM contains
chemokines (50) that may interfere with the spread of
infection in the DC-T cell mixtures, other maturation stimuli that are
known to activate macaque DCs (PGE2/TNF- or
CD40L) (6) were examined. Just as with MCM-activated DCs,
maturation of the DC with either PGE2/TNF- or
CD40L (confirmed by FACS analysis after 2–3 days; data not shown) did
not rescue theimpaired replication of the delta nef
virus (Fig. 6B). As expected, wild-type virus grew normally
in the immature DC-T cell mixtures, and activation of T cells with SEB
rescued the delta nef replicative capacity. The slight
decrease in virus production upon addition of the maturation stimuli
was seen at most time points in all experiments, but was not
significant.
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B)
(27). Addition of SEB to delta nef-infected
cocultures immediately or 2 days later rapidly amplified delta
nef in these cultures (Fig. 6C). Similarly,
adding delta nef-pulsed DCs to allogeneic
CD4+ T cell immediately or 1 day after the virus
pulse also resulted in robust delta nef replication and
underscored how DCs retain infectious delta nef virus for
considerable lengths of time (Fig. 6D). Therefore,
infectious virus persists for up to 2 days and can be rescued upon
encountering activated CD4+ T cells, but not
simply by maturation of virus-carrying DCs within the cultures.
Since nef appears to have unique influence over immature
DCs, triggering certain pathways of DC activation, but not others, we
were interested in determining whether nef-bearing DCs could
drive virus growth in the immature DC-T cell milieu. Immature DCs
carrying nef (or not) were mixed with autologous
CD4+ T cells and immediately exposed to delta
nef. The expression of either SIV (Fig. 6, E and
F) or HIV (Fig. 6F) nef in DCs
facilitated SIV delta nef replication in immature DC-T cell
cultures (Fig. 6, E and F). Taken together these
results stress how HIV and SIV nef are interchangeable and
signal selective pathways in immature DCs that drive virus replication
in immature DC-T cell cultures.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of nef in human and macaque DCs induces
significant production of inflammatory cytokines and chemokines (Figs. 3 and 4). This probably requires infection and actual expression of the
Nef protein, since loading immature DCs with large amounts of
inactivated, noninfectious virus (51) does not induce
chemokine or cytokine secretion (our unpublished observations).
Exogenously applied recombinant Nef was recently shown to trigger
similar patterns of cytokine and chemokine release by immature human
DCs (26). We similarly saw chemokine release by immature
DCs exposed to recombinant Nef (our unpublished observations). However,
the levels of cytokine and chemokine production appear to be greater in
response to endogenous DC-derived Nef (Fig. 3 and 4), while we
similarly observed negligible secretion of IL-1 and IL-10
(26). How exogenous or endogenous Nef signal DCs still
remains to be determined and is being examined in ongoing studies.
While macrophages were induced to secrete CCL3 and CCL4 by
nef (22), immature DCs secreted a much broader
panel of factors: CCL3, CCL4, CCL5, TNF-, CXCL8, IL-6, and
IL-12.
Chemokine and cytokine production by DCs normally correlates with
complete DC maturation when stimulated with bacteria, CD40L, LPS,
dsRNA, or mixtures of cytokines (5, 6, 43, 44, 52).
The soluble factors secreted during these responses differ
depending on the stimulus (6, 43), suggesting that
specific stimuli trigger DCs differently. Notably, nef
induced the production of a wide range of chemokines and cytokines, but
this did not parallel membrane phenotypic changes typical of DC
activation (Fig. 2) that are critical for DCs to acquire potent
immunostimulatory activity (reviewed in Ref. 2). This
contrasts what was observed when recombinant Nef was provided
exogenously (26). Our data suggest that endogenously
derived Nef uncouples the secretion of inflammatory cytokines and
chemokines from triggering membrane phenotypic maturation in immature
primate DCs.
Credence for one stimulus activating distinct pathways in DCs was first
provided by Rescigno et al. (53) when they described how
LPS regulated two separate pathways in murine DCs: one inducing
membrane phenotypic maturation, and the second preventing cell death.
Subsequent studies examining LPS triggering through TLR4
(54) or dsRNA via TLR3 (55) further highlight
how one stimulus-receptor interaction can signal distinct pathways
within DCs, specifically MyD88 adaptor protein-dependent cytokine
production vs MyD88-independent membrane phenotypic maturation.
Interestingly, we observed nef-mediated Stat3 activation in
immature DCs (35) that may favor MyD88-dependent cytokine
secretion (56). The fact that nef, but not
maturation stimuli, could rescue the defective replication of SIV delta
nef in the immature DC-T cell mixtures (Fig. 6), further
emphasizes how nef exploits DC biology and uniquely triggers
immature DCs to favor virus amplification rather than activation of
anti-viral immunity. Similarly, membrane phenotypic changes
indicative of low level DC activation were comparable in the immature
DC-T cell cocultures exposed to delta nef or wild-type SIV
where only wild-type virus grew effectively (27). Certain
DC stimuli have been suggested to protect DCs from viral infection
(44), while nef probably favors pathways to
promote virus amplification.
Unlike earlier reports using primary DCs and transfected cell lines, we
observed no consistent down-modulation of class I MHC molecules
(24, 26) or up-regulation of CD209 (23) or
CD74 (57). While we observed up-regulation of CD74 and
CD209 in response to nef in two donors, there was no
change or even down-regulation of these molecules in all other donors
tested (Fig. 2B). This underscores the importance of
studying primary cells from multiple donors. It is postulated that the
down-modulation of MHC class I or II molecules would reduce immune
function in infected settings, while elevated CD209 would foster DC-T
cell contact, and each would drive virus growth. The low level CD209
expression present on macaque DCs (Fig. 2) (40) makes it
even less likely that this is the sole mechanism at work here, agreeing
with the indication that CD209-ICAM-3 interactions are not essential
for HIV replication (58).
The reason for the discrepancies in nef-mediated effects on immature DCs is not immediately apparent, but may reflect the different viral vectors used to introduce nef, the use of recombinant Nef, and/or the nef alleles examined in primary cells vs transfected cell lines. The different methodologies used to generate the DCs in each study also cannot be excluded. However, many of our observations were made using both HIV and SIV nef expressed in primary macaque and human DCs and not cells lines often used by others. The level of Nef expression in primary DCs vs the variously tested cell lines or the amount of recombinant Nef added may also influence these events. This has been described for T cells, where the intracellular concentrations of Nef dictated whether CD4 vs class I MHC were down-regulated or T cell activation was affected (59). Interestingly, nef-induced chemokine production was routinely detected in immature DCs independent of the Adeno infection frequency or the intensity of the gfp (and so Nef) expression (data not shown), suggesting that sufficient Nef was being expressed under these conditions. Accurate comparative measurements of DC-associated Nef were not possible in these primary cell cultures. Studies are needed (e.g., using systems in which the nef gene is under the control of inducible promoters) to better define how different levels of Nef may impact primary DC biology.
The nef gene can directly activate
CD4+ T cells (15, 60). HIV or SIV
nef-expressing immature DCs augmented autologous
CD4+ T cell activation (much like a mature DC) in
both human and macaque systems (Fig. 5), but required fewer
nef-expressing DCs compared with human DCs exposed to
recombinant Nef (26). At this stage it is not clear how
DC-derived Nef exactly influenced the T cells in these cultures and
whether it was mediated directly or indirectly via the
nef-carrying DCs. Although we do not see a
nef-induced increase in CD209 expression that may favor DC-T
cell binding (23), it is possible that
nef-bearing DCs up-regulate other T cell-binding molecules
and/or that the T cells become more adhesive as a result of chemokine
exposure (reviewed in Refs. 61 and 62),
resulting in elevated autologous reactivity (63, 64).
In our previous studies we did not observe T cell activation in autologous immature DC-T cell cultures infected with wild-type SIV (27) or HIV (41, 65). It is quite possible, however, that much smaller subsets of T cells were activated, making them difficult to detect in the infected cultures. This may have been further confounded by preferential infection (and ultimately death) of the activated cells in these cultures. Similarly, significant chemokine or cytokine production was not detected in wild-type vs delta nef SIV-infected cultures (our unpublished observations). This is probably largely due to the relatively low frequency of virus-producing DCs in these cultures (9). Even if secreted at lower levels in wild type-infected cultures, it is possible that cells in the cultures use up the factors being secreted, rendering them even more difficult to detect. Counteractions by other lentiviral determinants cannot be ruled out at this point. As appreciated by others (23, 24), introducing nef in a viral vector enables the provision of larger amounts of nef into more DCs in the absence of an infectious immunodeficiency virus, allowing us to reveal otherwise subtle nef-induced changes (in DCs and T cells) that probably go undetected in wild-type virus infection.
Although the mechanism(s) needs to be elucidated, we have demonstrated
that DC-borne nef overtly modulates the immature DC-T cell
milieu, fostering virus spread. Chemokines have been shown to prevent
in vitro infections when added to cultures in recombinant forms
(66). While immature DCs (Figs. 3 and 4) (26)
and macrophages (22) secrete -chemokines in response to
Nef, HIV (22) and SIV (Fig. 6) still replicate normally.
It is possible that macrophage or DC-derived chemokines (secreted in
response to Nef) exhibit different biological activities to the
recombinant forms and/or that the presence of a variety of factors
within such a cellular milieu overrides any blocking effect that a
solitary chemokine might otherwise have. Furthermore, the concentration
of chemokine(s) used may have quite different effects on the outcome of
the infection, since, for example, high dose RANTES has been shown to
actually enhance infection (67). Thus, the amounts and
combinations of chemokines secreted in these cultures may actually
create a more conducive environment for virus growth.
One function of nef in a wild-type virus infection may be to
modify macrophages (22) and immature DCs (Figs. 3 and 4)
(26) to secrete chemokines that would recruit T cells
(especially memory T cells (reviewed in Refs. 61, 68 , and
69) to the initial site of infection, driving cell-to-cell
spread of the virus. T cells in a DC-T cell milieu do not need to enter
the cell cycle for virus to replicate in vitro (41, 65) or
in vivo (70, 71, 72), and cytokines (73) secreted
by the DCs (Figs. 3 and 4) and/or DCs themselves (27)
might be sufficient to signal the recruited T cells (much like in the
autologous MLR) to amplify virus replication. Preferential recruitment
of memory T cell subsets would further favor virus amplification in the
presence of immature DCs (9).
Recent findings support the idea that both soluble factors and cell contact are essential to drive virus expansion in resting CD4+ T cells. Specifically, the factor(s) present in the supernatants of Nef-stimulated macrophages that renders resting CD4+ T cells permissive to HIV infection requires contact between the target T cells and B cells (M. Stevenson, unpublished observations). We propose that similar events take place in our DC-T cell cocultures, where Nef-triggered immature DCs are able to provide the necessary soluble and cellular determinants to drive virus growth in the presence of resting CD4+ T cells. This is supported by observations that the levels of T cell activation diminished with decreasing DC numbers and that simply adding the supernatants from Nef-expressing immature DCs was insufficient to induce the same levels of T cell proliferation. Studies to elucidate these complex mechanisms are ongoing.
The presence of wild-type virus (nef)-carrying immature DCs
in this locale would be unlikely to stimulate active anti-viral
immunity, since the DCs have not up-regulated MHC and costimulatory
molecules needed to appropriately activate strong Ag-specific immune
responses. While wild-type virus-bearing DCs (as well as
Adeno-nef-infected DCs) are responsive to maturation stimuli
(our unpublished observations), the robust replication of virus
already established before virus-specific T cell activation would be
exacerbated by subsequently matured DCs (e.g., Fig. 6A)
triggering the T cells and probably simply overwhelm the immune system.
In contrast, when confronted with delta nef infection,
immature DCs could capture virus and begin to process viral Ags in the
absence of an all-consuming infection (27). Our in vitro
evidence that delta nef virus growth was not augmented after
maturing the DCs in this milieu (Fig. 6, A and B)
suggests that these matured cells could then go on to more effectively
activate anti-viral immune responses that might contribute to the
lessened viral loads seen during acute delta nef infection
(74, 75). Once T cells are activated, virus will
ultimately begin to spread between the permissive cells. This
underscores how without nef to exploit the APCs, the immune
system can initially cope to somewhat restrict virus dissemination.
Recombinant adenoviral vectors are also being used in HIV vaccine studies (76). Since in vitro studies have indicated that adenoviruses tend not to directly activate DCs (25, 29, 30, 31, 32, 33, 34, 35), optimal vaccine efficacy would require that the DCs be additionally activated for maximal presentation of introduced Ags (2). In light of the findings reported herein, we speculate that if an adenoviral vaccine construct contained nef, the initial triggering of immature DCs by Nef may favor the recruitment of T cells to the site of immunization, but additional stimuli would be necessary to fully activate the DCs for induction of strong anti-viral immunity.
Encompassing primary DCs from macaques and humans, these studies highlight how the immunodeficiency virus nef can manipulate immature DCs, exploiting unique aspects of DC biology to forge virus dissemination while avoiding the activation of virus-specific T cells. A better understanding of how these and potentially other virus-mediated modifications of DCs are manifest will be critical to identify strategies to bias the DC system toward activation of anti-viral immunity instead of facilitating virus spread.
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Acknowledgments |
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Footnotes |
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2 Current address: North Shore-LIJ Research Institute, 350 Community Drive, Manhasset, NY 11030.
3 Current address: Department of Pathology and Immunology, Washington University, 660 South Euclid Avenue, Box 8118, St. Louis, MO 63110.
4 Address correspondence and reprint requests to Dr. Melissa Pope, Center for Biomedical Research, The Population Council, 1230 York Avenue, New York, NY 10021. E-mail address: mpope{at}popcbr.rockefeller.edu
5 Abbreviations used in this paper: DC, dendritic cell; Adeno, adenovirus; CD40L, CD40 ligand; gfp, green fluorescent protein; MCM, monocyte-conditioned medium; MIP, macrophage inflammatory protein; SEB, staphylococcal enterotoxin B; TLR, Toll-like receptor; SIGN, specific ICAM-3-grabbing nonintegrin.
Received for publication June 5, 2002. Accepted for publication August 7, 2002.
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, 
, and •, Three or
four individual macaques; 
, 
, and 
, three human donors.
Staining equivalent to the isotype control was assigned a value of 0.1
to accommodate log-scale analysis.
