| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy;
Department of Experimental Medicine, Tor Vergata University, Rome, Italy; and
Department of Biochemistry and Molecular Biology, Ferrara University, Ferrara, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and 
chemokines, which drive Th1-type
immune response. In contrast, these effects are lost by oxidation and
inactivation of the protein. Finally, native Tat enhances Ag
presentation by MDDC, increasing Ag-specific T cell responses. These
data indicate that native Tat selectively targets MDDC, is taken up by
these cells via specialized pathways, and promotes their maturation and
Ag-presenting functions, driving Th1-type immune responses. Thus, Tat
can act as both Ag and adjuvant, capable of driving T cell-mediated
immune responses. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chemokine receptors
CCR5 and CXCR4, favoring transmission of both macrophage and T
lymphocyte-tropic HIV-1 strains (9, 10). Evidence suggests that the immune response to Tat has a key role in the control of HIV infection. In fact, a Tat-specific immune response is present in HIV-1-infected subjects and SIV-infected monkeys, and correlates inversely with progression to the symptomatic stage of the infection (11, 12, 13, 14, 15, 16, 17, 18). Moreover, a recent study in macaques experimentally infected with SIV indicates that the immune response to Tat, namely anti-Tat CTL, is key to control early virus replication after primary infection and exerts a selective immune pressure on the virus, leading to the appearance of slowly replicating and apparently less pathogenic escape mutants (19). Furthermore, vaccination with the biologically active Tat protein or tat DNA induces protection against SHIV89.6P virus replication and disease onset, which correlates with the presence of Th1 responses, including specific CTLs (20, 21, 22 and our unpublished observations). Similar protection data have been observed with a tat-rev vaccine delivered with viral vectors in macaques (23). In contrast, a limited containment of the infection has been observed in monkeys vaccinated with inactivated Tat or Tat peptides, in which Ab and Th responses, but no CTL, had been induced (24, 25). In contrast, the repeated intradermal (i.d.)3 inoculation of monkeys with native and active Tat protein alone (in the absence of any adjuvant) at low doses (5–6 µg) selectively induced a Th1 response and specific CTLs in the absence of any significant Ab production (Ref. 20 and our unpublished observations).
Soluble Tat protein or specific Tat peptides have been shown to enter different cell types (4, 6, 7, 8, 26, 27, 28), to be presented with MHC class I Ag (27, 28), and to induce CTL in vivo (20). This is a property of soluble proteins called penetratins, capable of entering APC and of delivering hydrophilic compounds (29). However, little or nothing is known on the uptake and effects of native Tat, as opposed to inactivated Tat, on APC.
Monocyte-derived dendritic cells (MDDC), T cell blasts (TCB), and B
lymphoblastoid cell lines (BLCL) are among the most commonly used APC.
In particular, dendritic cells (DC) are the most efficient APC and are
key to the induction of immune responses against viral infections
(30, 31). Their function is associated with a high
expression of MHC and costimulatory (CD40, CD80, CD86) molecules and
with the production of cytokines (IL-1, TNF-, IL-12, IL-15),
known to activate T lymphocytes, and chemokines
(macrophage-inflammatory protein (MIP)-1, MIP-1, RANTES). Upon
encountering the Ag, DC undergo a maturation process characterized by
an increase of costimulatory molecule expression and by a reduction of
their phagocytic and pinocytic capability (30, 31).
Furthermore, due to the up-regulation of the homing receptor CCR7 and
to the down-regulation of CCR5, mature DC migrate to lymph nodes, where
they present Ag to T lymphocytes (30, 31).
In this study, we show that biologically active, but not
oxidized/inactivated, Tat protein is very efficiently and selectively
taken up by MDDC in a dose-, time-, maturation-, and
temperature-dependent fashion. Upon uptake, Tat induces in MDDC an
increase of the expression of MHC and costimulatory molecules, and
production of Th1 cytokines and chemokines. All these effects are
lost when Tat is oxidized and inactivated. Furthermore, native Tat
enhances both allogeneic and Ag-specific presentation by MDDC, thus
increasing T cell-specific immune responses. Thus, due to its capacity
to efficiently enter MDDC, to enhance their functions, and to drive
Th1-specific immune responses, native Tat may favor its own
presentation and the induction of specific immune responses, but may
also adjuvate T cell responses to other Ag.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HIV-1 Tat from the human T lymphotropic virus type IIIB-BH-10 (subtype B) was expressed in Escherichia coli and purified to homogeneity by heparin-affinity chromatography and HPLC, as described previously (7, 20, 32, 33), as a good laboratory practice (GLP) manufactured product. The purified Tat protein was fully biologically active, as tested by the rescue assays on the HLM-1 cell line carrying a Tat-defective HIV provirus, as previously published (7, 8), or by the induction of chloramphenicol acetyl transferase activity with HL3T1 cells containing the HIV long terminal repeat chloramphenicol acetyl transferase construct (34), and by several other assays with the same protein lots on endothelial cells or mice (5, 32, 33, 35). To prevent oxidation that occurs easily because Tat contains seven cysteines, the Tat protein was stored lyophilized at -80°C and reconstituted in degassed buffer before use, as described (7, 8, 35). To prevent attachment of the protein to surfaces, plastic tips and vials were previously rinsed in 0.1% PBS-BSA or in RPMI 1640 supplemented with 20 mM HEPES (Sigma-Aldrich, St. Louis, MO), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (Life Technologies, Paisley, U.K.), and 15% FBS (HyClone Laboratories, Logan, UT) (complete medium). In addition, because Tat is also photo- and thermosensitive (7, 8), the handling of the protein was always performed in the dark and on ice. Experiments were also performed with Tat oxidized by exposure to the light and air for 18 h. By this procedure, Tat loses all biological activity due to conformational changes, including multimerization and aggregation of the protein with loss of the monomeric active form (7, 8, 32, 33). Different GLP lots of Tat were used with reproducible results, and in all cases endotoxin concentration was below the detection limit (<0.05 EU/µg), as determined by the Limulus Amoebocyte Lysate analysis (Pyrochrome, Associates of Cape Cod, Falmouth, MA).
Cell preparation and culture
MDDC were obtained from peripheral blood monocytes of 14 different healthy human donors, according to established methods (36). Briefly, PBMC were isolated by density gradient separation (Ficoll-Paque Research Grade; Pharmacia Biotech, Uppsala, Sweden). Monocytes were further purified by incubation with anti-CD14-coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by sorting with a magnetic device (MiniMacs Separation Unit; Miltenyi Biotec), according to the manufacturer’s instructions. The purity of monocytes was always >95%, as assessed by flow cytometry (FACScan; BD Biosciences, San Jose, CA).
Monocytes were induced to differentiate to DC (MDDC) by 6 days of culture in complete medium in the presence of GM-CSF (200 ng/ml; Leucomax; Novartis, Origgio, Italy) and IL-4 (100 ng/ml; PeproTech, London, U.K.). Differentiation to DC was assessed by morphologic observation and by the detection of specific surface markers (HLA-DR, CD86, CD83, CD40, CD80) by flow cytometry.
BLCL were generated by culturing human PBMC from two healthy donors for 2 h in the presence of supernatants from the EBV producer B95-8 marmoset cell line, and further expansion for at least 4 wk, as described earlier (37). TCB were obtained by stimulation of human PBMC from four different donors with 1 µg/ml PHA (Murex Diagnostics, Chatillon, France) for 3 days and further expansion for 2 wk in complete medium supplemented with 10 IU/ml rIL-2 (BD Labware, Bedford, MA), as described earlier (37).
Cellular uptake of the Tat protein
MDDC from 14 healthy donors were cultured at the density of 2 x 105/ml in complete medium in the presence of native or oxidized HIV-1 Tat or with reconstitution buffer or medium alone (negative controls) for 5, 10, 30, or 60 min at 37°C in the dark. Cells were then washed with cold medium and treated for 10 min at 37°C with trypsin-EDTA (Life Technologies) to remove any externally bound protein. After fixation and permeabilization, MDDC were stained with affinity-purified rabbit polyclonal anti-Tat IgG Ab (7, 8, 35) or rabbit IgG control Ab (ICN Biomedicals, Opera, Italy), followed by FITC-conjugated anti-rabbit Ig (Pierce, Rockford, IL). Fluorescence was analyzed by flow cytometry, and results were expressed as the percentage of positive cells as compared with isotype-stained samples. To demonstrate the specific intracytoplasmatic localization of the protein, staining with anti-Tat Ab was always performed also with nonpermeabilized MDDC. In some experiments, uptake of Tat by MDDC was also performed at both 4 and 37°C and at both 10 and 30 min of incubation. To this goal, all the steps described above, including the treatment of the cells with trypsin-EDTA, were performed with cells and solutions at 4°C.
BLCL from two healthy donors and TCB from four healthy donors were also cultured at 5 x 105/ml in complete medium in the presence of native Tat at concentration ranging from 100 to 10,000 ng/ml, reconstitution buffer, or medium alone for 30 or 60 min at 37°C in the dark, and processed as described for MDDC.
Analysis of MDDC maturation and cytokine and chemokine
production
MDDC from 10 donors were cultured at the density of 2 x
105/ml in complete medium in the absence or
presence of serial concentrations of native or oxidized Tat or with
reconstitution buffer. LPS from E. coli, serotype 055:B5 (10
µg/ml) (Sigma-Aldrich), was used as the positive control. After
18 h, the cell supernatants (from 8 of the 10 donors) were
collected and stored at -70°C in small aliquots and assayed for the
presence of TNF- and IL-12 and the chemokines RANTES, MIP-1,
and MIP-1 with commercially available kits, according to the
manufacturers’ instructions (Cytoscreen TNF- and IL-12 ELISA kits,
Biosource Europe, Nivelle, Belgium; Quantikine RANTES, MIP-1,
and MIP-1, R&D Systems, Abingdon, U.K.). The cellular fraction was
washed twice and analyzed for the expression of surface molecules by
flow cytometry. The following mAb were used: FITC- or PE-conjugated IgG
isotypes, FITC-conjugated anti-CD14 and
anti-HLA-DR (BD Biosciences), FITC-conjugated anti-CD40,
anti-CD80, anti-CD83, and PE-conjugated anti-CD86 (BD
PharMingen, San Diego, CA).
Mixed allogeneic cultures
MDDC (2 x 105 cells/ml) were incubated for 18 h with LPS (positive control), native Tat protein (10 µg/ml), or reconstitution buffer. Then they were washed and cultured in complete medium (containing 5% FBS) in 96-well plates together with monocyte-depleted allogeneic PBL (2 x 105/well) at ratios ranging from 1:10 to 1:640. After 6 days, [3H]thymidine was added for additional 16 h, and samples were harvested onto glass fiber filters (Printed Filtermat A; Wallac, Turku, Finland) and counted with a Betaplate (Wallac), and the values were expressed in cpm.
Ag-specific presentation assays
MDDC (2 x 105 cells/ml) from three healthy donors were incubated for 18 h with active Tat protein (10 µg/ml) or reconstitution buffer and then cultured in complete medium in the presence of 5% FBS, together with autologous PBL (2 x 105/well), at a ratio of 1:20 in the presence of 5 µg/ml tetanus toxoid (TT; Pasteur Merieux Connaught, Willowdale, Canada). After 6 days, [3H]thymidine was added for an additional 16 h, samples were harvested, and cpm were counted, as reported above. The stimulation indexes (SI) were the ratios between counts from DC-PBL cocultures and those from PBL alone.
Statistical analysis
Statistical analysis was performed by the two-tailed Student t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The uptake of active Tat by MDDC, TCB, and BLCL was evaluated by
intracellular immunofluorescence in flow cytometry, using a specific
affinity-purified polyclonal Ab on permeabilized cells. Fig. 1
shows the results of a representative
donor whose levels of Tat uptake represented the median of the values
obtained from 14 donors tested. Tat uptake by MDDC was very efficient
and occurred in a dose- and time-dependent fashion (Fig. 1A). Uptake was already evident with the lowest dose of Tat
utilized (0.1 ng/ml). Regardless of the Tat concentration tested, the
level of staining always peaked after 5 min of incubation and was
reduced after 60 min, most likely due to the processing of the protein.
However, uptake of Tat remained high (98%) up to 60 min of incubation
at the highest dose of Tat (10 µg/ml) used. No staining was observed
with cells incubated in medium alone or reconstitution buffer (Fig. 1A). In addition, the Tat detected was almost entirely
intracellular because no staining was observed after 10 or 30 min of
incubation of Tat with nonpermeabilized cells (Fig. 1B).
Similar dose and time kinetic of Tat uptake by MDDC was reproducibly
observed with different protein lots.
|
C). Thus,
efficient uptake of active Tat is a selective feature of MDDC. Tat uptake by MDDC enhances with cell maturation and is lost by oxidation and inactivation of the protein or by low temperatures
Immature MDDC take up Ag by phagocytosis and pinocytosis
(30, 31). Mature DC lose these activities while acquiring
strong Ag presentation capability. To verify whether cell maturation
affects the uptake of native Tat, MDDC were induced to maturate with
LPS. Mature MDDC expressed higher levels of HLA-DR, CD83, and CD86
surface markers (data not shown). Both immature or mature cells were
then used for the uptake experiments. Tat uptake was highly increased
by MDDC maturation at all protein concentrations tested (Fig. 2A). In fact, incubation of
mature MDDC with low Tat concentrations gave levels of intracellular
staining similar to those observed in immature cells with the highest
doses of Tat (Figs. 1A and 2A).
|
), and active or inactive Tat protein
was then compared in the uptake experiments. As shown in Fig. 2B, no staining was observed with oxidized Tat up to 1
µg/ml of the protein, and, at this concentration, only a very low
Tat-specific staining was detected as compared with native Tat. Thus,
Tat must have native conformation and biological activity for efficient
uptake by MDDC.
|
C). At 4°C, Tat uptake by MDDC, both at
10 and 30 min, resulted greatly reduced at all doses of the protein
used, as compared with 37°C and as both the percentage of stained
cells and the mean fluorescence intensity (MFI). This was particularly
evident at the lower doses of Tat (0.1–100 ng/ml) at which MDDC
staining was extremely reduced or abolished at both 10 and 30 min, as
compared with 37°C (Fig. 2C). Because MDDC maturation that
is associated with a reduced pino/phagocytic activity increases Tat
uptake, whereas oxidation/inactivation of the protein or low
temperatures greatly reduces it to levels comparable with those
observed with BLCL and TCB, it is conceivable that uptake of native Tat
by MDDC is mediated by specific receptors and entry mechanisms that are
not related to the pino/phagocytic activity of the cells. Native, but not oxidized, Tat induces MDDC maturation
To evaluate the effect of the Tat protein on MDDC maturation, the
surface expression of MHC, HLA-ABC, and HLA-DR, and of the
costimulatory molecules CD40, CD80, CD86, and CD83 was analyzed by flow
cytometry on cells cultured for 18 h in the presence of the
protein, complete medium, reconstitution buffer, or LPS (positive
control). Experimental data obtained with 10 different
donors indicated that Tat induces a
dose-dependent enhancement of the expression of MHC and costimulatory
molecules in the absence of any cell toxicity (Table II). A
marked increase of the MFI was observed for HLA-ABC (3 of 6 donors,
average 37%), for HLA-DR (10 of 10, average 53%), for CD40 (6 of 10,
average 35%), for CD80 (8 of 8, average 54%), for CD83 (9 of 10,
average 177%), and for CD86 (10 of 10, average 146%). Reconstitution
buffer or medium alone did not change the expression levels of the
molecules analyzed. Of note, oxidation of Tat markedly reduced the
capacity of the protein to up-regulate MHC and costimulatory molecules
on MDDC (Table III). Thus, only native
Tat promotes the maturation of MDDC.
|
|
chemokines by MDDC
To evaluate the effects of Tat on DC activation, the production of
the cytokines IL-12 and TNF-, known to activate immune cells and to
induce Th1-type responses (38), and of the chemokines
RANTES, MIP-1, and MIP-1 that are known mediators of immune
responses (39), was assessed by ELISA in the supernatants
of cells cultured for 18 h with the protein, reconstitution buffer
(negative control), or LPS (positive control) (Fig. 3). Incubation with native Tat induced a
dose-dependent increase of the levels of IL-12 and TNF-, reaching,
at the highest dose of Tat, an increase of 23-fold
(p < 0.02) for IL-12 and 20-fold
(p < 0.03) for TNF- as compared with cells
treated with buffer alone. Similarly, Tat markedly enhanced, in a
dose-dependent fashion, the production of RANTES (10-fold,
p < 0.02), MIP-1 (97-fold, p <
0.005), and MIP-1 (15-fold, p < 0.01). The
reconstitution buffer had no effects, whereas LPS markedly enhanced the
production of both cytokines and chemokines.
|
(data not shown). Thus,
only native Tat increases the production and secretion of Th1 cytokines
and chemokines by MDDC. Native Tat increases allogeneic presentation by MDDC
To evaluate the effect of Tat on the Ag-presenting capacity of
MDDC, cells were exposed to the Tat protein, reconstitution buffer,
complete medium or LPS (positive control), and cultured with allogeneic
PBL at serial cell:cell ratios (Fig. 4).
This assay was chosen because, although not specific for a given Ag, it
provides adequate information on the overall Ag-presenting function of
DC. Untreated MDDC induced some levels of proliferation of allogeneic
lymphocytes, depending on the number of APC used. However, the
proliferative response of allogeneic PBL was significantly enhanced by
MDDC pulsed with Tat (3.3-fold, p < 0.01, at the
highest DC/PBL ratio), reaching levels similar to those induced by LPS
(3.8-fold, p < 0.005) at the same cell to cell ratio.
In contrast, no enhancement of allogeneic lymphocyte proliferation was
observed by treatment of MDDC with reconstitution buffer (Fig. 4).
Thus, native Tat increases DC presentation function.
|
The effect of Tat on the Ag-specific presenting capacity of MDDC
was evaluated by transiently treating the cells with the protein,
reconstitution buffer, or complete medium, and culturing them together
with autologous lymphocytes in the presence of the recall Ag TT. As
shown in Fig. 5, untreated MDDC induced
TT-specific proliferation of autologous lymphocytes in two of the three
donors analyzed (SI 15.4 and 7.3, respectively), and this effect was
enhanced by their treatment with Tat (SI 27.9 and 12.5, respectively),
but not with reconstitution buffer (SI 13 and 5.9, respectively).
Tat-treated MDDC did not induce lymphocyte proliferation to TT in the
subject who did not respond to the recall Ag. Thus, Tat can boost
specific T cell responses to other Ag.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This suggested that the active Tat protein may target APC and drive
Th1-type cellular responses, as occurs for an intracellularly expressed
protein delivered by a DNA plasmid. This was also suggested by the
known properties of active Tat protein or peptides to 1) enter cells of
various types (4, 6, 7, 8, 26, 27, 28), 2) localize in the nuclei
and trans activate virus or cellular gene expression upon
cellular uptake (4, 6, 7, 8, 40), 3) bind via its RGD region
to the v3 and
51 integrins and via
its basic region to heparan sulfate proteoglycans of activated
endothelial cells that also function as APC (6, 32, 33, 41, 42), 4) be very efficiently taken up by activated endothelial
cells expressing integrin receptors and much less efficiently by
nonactivated cells (6, 7), 5) interact with DC and inhibit
apoptotic bodies engulfment via the binding to the
v3 integrin
(43, 44), 6) interact with monocytes, inducing an increase
of proinflammatory cytokines and promoting their chemotactic function
(45, 46, 47), and 7) be processed and presented by APC with
MHC class I molecules (27, 28) and, therefore, induce a
CTL response as other penetratins can do (29). Notably, a
biologically active Tat was required to observe these effects.
Altogether these in vitro and in vivo data suggested that the effect of Tat on APC might vary according to its conformation and biological activity. Therefore, we analyzed the uptake of native or oxidized/inactivated Tat by different types of APC, including MDDC, BLCL, and TCB. Furthermore, we evaluated the effects of both native and oxidized/inactivated Tat on the function of MDDC, which are the most efficient among the APC, and key for the induction of antiviral immune responses (30, 31).
We first studied the capability of MDDC of specifically taking up soluble active Tat and found that this is very efficient, since most of the Tat is taken up by the cells very rapidly with a peak after 5–10 min, depending on the concentration of Tat given to the cells. In contrast, the uptake of active Tat by TCB or BLCL is very poor, requiring very high protein concentrations (10 µg/ml) and much longer time of incubation and, even under these conditions, most Tat remains bound to the cell surface and does not enter cells. Furthermore, active Tat is also rapidly processed by MDDC, as indicated by the reduction of the intracellular staining after 30 min of incubation.
Mature MDDC are able to take up Tat 10- to 100-fold more efficiently
than immature cells, as indicated by the values of intracellular
staining observed with low concentrations of Tat (100 ng/ml) as
compared with those observed with 1 or 10 µg/ml Tat with immature
cells. In addition, Tat uptake requires a native conformation and full
biological activity of the protein. Oxidation of Tat by exposure to
light and air abolishes or markedly reduces (by 
100-fold) the
uptake observed with Tat by MDDC. Interestingly, the type of uptake
by MDDC observed with oxidized Tat is similar or identical to that of
native Tat with TCB and BLCL. Furthermore, at a temperature of 4°C,
uptake of Tat by MDDC is markedly reduced at all the doses of the
protein, and this is particularly evident at low Tat concentrations
(0.1–100 ng/ml), suggesting that Tat uptake is an energy-dependent
process.
Taken together, these data indicate that Tat targets MDDC, and they suggest that the selective and efficient uptake of Tat by MDDC is not mediated by the high pino/phagocytic activity of these cells, but it requires specialized uptake pathways that are selectively expressed by immature MDDC and at higher levels by mature MDDC. Furthermore, the different uptake observed at low vs high concentrations of Tat with MDDC indicates the presence of at least two different uptake pathways, the first one occurring at low Tat concentrations (up to 100 ng/ml) and the other one observed at higher Tat concentrations (>100 ng/ml). In this regard, the low temperature (4°C) reduced or abolished particularly the uptake of low doses of Tat (0.1–100 ng/ml), suggesting the involvement of a receptor-mediated endocytosis.
Of note, endothelial cells activated by IFN-
, IL-1, and TNF-,
but not nonactivated cells, bind and take up native Tat very
efficiently and in fashion similar to MDDC (Refs. 7, 32 ,
and 33 , and data not shown). Tat uptake by endothelial
cells occurs via the RGD domain of Tat that binds the
v3 and
51 integrins (the
classical receptors for vitronectin and fibronectin) and via the basic
region of Tat that binds heparan sulfate proteoglycans of the cell
surface and extracellular matrix (8, 32, 33, 35, 41, 42).
In this respect, integrin antagonists block endothelial cell uptake of
low (up to 100 ng/ml), but not high Tat concentrations (>1 µg/ml).
Of interest, binding to the same integrins also mediates the effects of
Tat on activated endothelial cells, and these effects are abolished by
oxidation/inactivation of the protein, or by specific Ab, or by
competitor peptides or ligands (6, 7, 32, 33, 35, 41).
Preliminary data indicate that also for MDDC, the uptake of low
concentrations of Tat is blocked by specific anti-integrin mAb or
competitor ligands, while that of high Tat concentrations is only
partially reduced. This suggests that integrins mediate Tat entry into
MDDC through a receptor-mediated endocytic pathway.
To investigate the effects of Tat on MDDC phenotype and function, cell
viability, surface molecule expression, cytokine and chemokine
production, and Ag-presenting function were evaluated after 18 h
of incubation of MDDC with Tat. A dose-dependent enhancement of the
surface expression of HLA-ABC, HLA-DR, CD40, CD80, CD86, and CD83 on
MDDC was found in most of the donors examined. However, this effect was
observed with native, but not with oxidized, Tat. In addition, Tat
induced a dose-dependent increase of the production of both IL-12 and
TNF-, cytokines essential for driving a Th1-type response
(38), and of the chemokines RANTES, MIP-1, and
MIP-1, which are key players in the effector phase of the lymphocyte
response (39). Importantly, in both cases, the levels were
comparable with those induced by the known activator LPS. Again,
oxidized Tat did not induce these effects.
Active Tat also enhanced the Ag-presenting function of MDDC, increasing the proliferative response of T cells to allogeneic and recall Ag.
Thus, active Tat is selectively and efficiently taken up and processed by MDDC, induces their maturation, and promotes their capacity of presenting Ag, eliciting immune responses toward a Th1 pattern, and increasing T cell responses to other Ag. Taken together, these properties indicate that active Tat is not only an Ag but also a potent T cell adjuvant.
Our data are in apparent contrast with reports showing that Tat exerts toxic and immunosuppressive effects (48, 49, 50, 51). However, in most of these studies, high amounts of Tat were used, and quality controls of Tat composition, endotoxin content, biological activity, and specificity were not shown, hampering both interpretation and comparison of the data. In our hands, different lots of a GLP product of a monomeric form, fully active at picomolar concentrations and endotoxin free, have never exerted toxic and/or immunosuppressive effects in vitro (with primary endothelial cells, PBMC, MDDC, CD4 and CD8 T cells, T and B cell lines, fibroblasts) (5, 6, 7, 8, 20, 22, 32, 33, 35, 41), nor in vivo after inoculation in nude mice or guinea pigs (33, 35), or after immunization in mice or monkeys (Refs. 20, 21 , and 52 , and B. Ensoli, unpublished observations). Similarly, no toxic effects have ever been observed in animals with AIDS (our unpublished observations) or after vaccination with tat DNA in monkeys or in HIV-1-infected humans (22, 23, 53), indicating that vaccination with Tat is safe. Moreover, no toxicity or immunosuppressive effects were observed by mucosal or systemic immunization of mice with Tat and heterologous Ag (B. Ensoli, unpublished observations).
The promoting effects of Tat on DC functions and its capability of being presented in association with MHC class I molecules may explain the type of immune response and the protective role exerted by vaccination with the soluble active Tat protein. Because the induction of Th1 responses and CTL correlates with reduced viral load also with Env-based vaccines, the data presented suggest that native Tat protein or tat DNA should be exploited to drive or to increase Th1 immune responses and CTL activity also against other HIV Ag to support an effective and long-lasting antiviral immunity capable of controlling virus replication and blocking disease onset.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Barbara Ensoli, Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail address: ensoli{at}iss.it
3 Abbreviations used in this paper: i.d., intradermal; DC, dendritic cell; MDDC, monocyte-derived DC; BLCL, B lymphoblastoid cell line; GLP, good laboratory practice; MFI, mean fluorescence intensity; MIP, macrophage-inflammatory protein; SI, stimulation index; TCB, T cell blast; TT, tetanus toxoid.
Received for publication June 7, 2001. Accepted for publication October 30, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
51 and v3 integrins by mobilizing sequestered basic fibroblast growth factor. Blood 94:663.v3 integrin that are required for Tat activity. J. Immunol. 163:1929.v3 integrin and requires intracellular and extracellular calcium. Eur. J. Immunol. 27:1893.[Medline]
and Tat involvement in the immunosuppression of uninfected T cells and C-C chemokine decline in AIDS. Proc. Natl. Acad. Sci. USA 95:3851.This article has been cited by other articles:
|
|
 |
|
  W. Ma, S. Mishra, N. Gajanayaka, J. B. Angel, and A. Kumar HIV-1 Nef Inhibits Lipopolysaccharide-induced IL-12p40 Expression by Inhibiting JNK-activated NF{kappa}B in Human Monocytic Cells J. Biol. Chem., March 20, 2009; 284(12): 7578 - 7587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Fanales-Belasio, S. Moretti, V. Fiorelli, A. Tripiciano, M. R. Pavone Cossut, A. Scoglio, B. Collacchi, F. Nappi, I. Macchia, S. Bellino, et al. HIV-1 Tat Addresses Dendritic Cells to Induce a Predominant Th1-Type Adaptive Immune Response That Appears Prevalent in the Asymptomatic Stage of Infection J. Immunol., March 1, 2009; 182(5): 2888 - 2897. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gupta, R. Boppana, G. C. Mishra, B. Saha, and D. Mitra HIV-1 Tat Suppresses gp120-Specific T Cell Response in IL-10-Dependent Manner J. Immunol., January 1, 2008; 180(1): 79 - 88. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Jain, J. Ahuja, Z. K. Khan, S. Shimizu, O. Meucci, S. R. Jennings, and B. Wigdahl Modulation of dendritic cell maturation and function by the Tax protein of human T cell leukemia virus type 1 J. Leukoc. Biol., July 1, 2007; 82(1): 44 - 56. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Demberg, R. H. Florese, M. J. Heath, K. Larsen, I. Kalisz, V. S. Kalyanaraman, E. M. Lee, R. Pal, D. Venzon, R. Grant, et al. A Replication-Competent Adenovirus-Human Immunodeficiency Virus (Ad-HIV) tat and Ad-HIV env Priming/Tat and Envelope Protein Boosting Regimen Elicits Enhanced Protective Efficacy against Simian/Human Immunodeficiency Virus SHIV89.6P Challenge in Rhesus Macaques J. Virol., April 1, 2007; 81(7): 3414 - 3427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ranjbar, R. Rajsbaum, and A. E. Goldfeld Transactivator of Transcription from HIV Type 1 Subtype E Selectively Inhibits TNF Gene Expression via Interference with Chromatin Remodeling of the TNF Locus J. Immunol., April 1, 2006; 176(7): 4182 - 4190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kittiworakarn, A. Lecoq, G. Moine, R. Thai, E. Lajeunesse, P. Drevet, C. Vidaud, A. Menez, and M. Leonetti HIV-1 Tat Raises an Adjuvant-free Humoral Immune Response Controlled by Its Core Region and Its Ability to Form Cysteine-mediated Oligomers J. Biol. Chem., February 10, 2006; 281(6): 3105 - 3115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Mascarell, C. Fayolle, C. Bauche, D. Ladant, and C. Leclerc Induction of Neutralizing Antibodies and Th1-Polarized and CD4-Independent CD8+ T-Cell Responses following Delivery of Human Immunodeficiency Virus Type 1 Tat Protein by Recombinant Adenylate Cyclase of Bordetella pertussis J. Virol., August 1, 2005; 79(15): 9872 - 9884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. S. Carbonell, S.-I. Murase, A. F. Horwitz, and J. W. Mandell Migration of Perilesional Microglia after Focal Brain Injury and Modulation by CC Chemokine Receptor 5: An In Situ Time-Lapse Confocal Imaging Study J. Neurosci., July 27, 2005; 25(30): 7040 - 7047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Majumder, M. L. Janket, E. A. Schafer, K. Schaubert, X.-L. Huang, J. Kan-Mitchell, C. R. Rinaldo Jr., and V. Ayyavoo Human Immunodeficiency Virus Type 1 Vpr Impairs Dendritic Cell Maturation and T-Cell Activation: Implications for Viral Immune Escape J. Virol., July 1, 2005; 79(13): 7990 - 8003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. N. Nair, S. D. Mahajan, S. A. Schwartz, J. Reynolds, R. Whitney, Z. Bernstein, R. P. Chawda, D. Sykes, R. Hewitt, and C. B. Hsiao Cocaine Modulates Dendritic Cell-Specific C Type Intercellular Adhesion Molecule-3-Grabbing Nonintegrin Expression by Dendritic Cells in HIV-1 Patients J. Immunol., June 1, 2005; 174(11): 6617 - 6626. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Fantuzzi, C. Purificato, K. Donato, F. Belardelli, and S. Gessani Human Immunodeficiency Virus Type 1 gp120 Induces Abnormal Maturation and Functional Alterations of Dendritic Cells: a Novel Mechanism for AIDS Pathogenesis J. Virol., September 15, 2004; 78(18): 9763 - 9772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Gavioli, E. Gallerani, C. Fortini, M. Fabris, A. Bottoni, A. Canella, A. Bonaccorsi, M. Marastoni, F. Micheletti, A. Cafaro, et al. HIV-1 Tat Protein Modulates the Generation of Cytotoxic T Cell Epitopes by Modifying Proteasome Composition and Enzymatic Activity J. Immunol., September 15, 2004; 173(6): 3838 - 3843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Ramakrishna, K. K. Anand, K. M. Mohankumar, and U. Ranga Codon Optimization of the Tat Antigen of Human Immunodeficiency Virus Type 1 Generates Strong Immune Responses in Mice following Genetic Immunization J. Virol., September 1, 2004; 78(17): 9174 - 9189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sugaya, K. Lore, R. A. Koup, D. C. Douek, and A. Blauvelt HIV-Infected Langerhans Cells Preferentially Transmit Virus to Proliferating Autologous CD4+ Memory T Cells Located within Langerhans Cell-T Cell Clusters J. Immunol., February 15, 2004; 172(4): 2219 - 2224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Petersen, C. R. Morris, and J. C. Solheim Virus Evasion of MHC Class I Molecule Presentation J. Immunol., November 1, 2003; 171(9): 4473 - 4478. [Full Text] [PDF]
|
|
|
|
|
|
Y. Yang, I. Tikhonov, T. J. Ruckwardt, M. Djavani, J. C. Zapata, C. D. Pauza, and M. S. Salvato Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4+ Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism J. Virol., June 15, 2003; 77(12): 6700 - 6708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Dolganiuc, K. Kodys, A. Kopasz, C. Marshall, T. Do, L. Romics Jr., P. Mandrekar, M. Zapp, and G. Szabo Hepatitis C Virus Core and Nonstructural Protein 3 Proteins Induce Pro- and Anti-inflammatory Cytokines and Inhibit Dendritic Cell Differentiation J. Immunol., June 1, 2003; 170(11): 5615 - 5624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Agwale, M. T. Shata, M. S. Reitz Jr., V. S. Kalyanaraman, R. C. Gallo, M. Popovic, and D. M. Hone A Tat subunit vaccine confers protective immunity against the immune-modulating activity of the human immunodeficiency virus type-1 Tat protein in mice PNAS, July 23, 2002; 99(15): 10037 - 10041. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), LPS (•),
reconstitution buffer (
), or native Tat (10 µg/ml) (
), and
cultured together with allogeneic PBL. [3H]Thymidine
uptake was measured after 6 days of culture to evaluate lymphocyte
proliferation. Data are from a representative experiment and have been
reproduced with other three different donors. Means and SEM are
reported.