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*
Experimental Immunology Branch and
Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892;
Department of Immunology/Microbiology and Medicine, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, IL 60617; and
Immunex Corp., Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine production were similar by DC from
HIV- donors infected in vitro with the CCR5-using HIV Ba-L
strain (n = 8) compared with uninfected DC from the
same donors. Production of -chemokines, but not of cytokines, was
increased by a CXCR4-using IIIB strain-infected DC
(n = 7). Stimulation of HIV-infected DC with CD40LT
decreased infection in Ba-L-infected DC, but had no effect on
IIIB-infected DC. Consistent with this finding, CD40LT
down-regulated CCR5 and up-regulated CXCR4 expression on DC.
Monocyte-derived DC were also propagated from 15 HIV+ and
13 HIV- donors. They exhibited similar expression of
costimulatory molecules and produced similar amounts of IL-12, IL-10,
and -chemokines, following stimulation. By contrast, stimulated PBMC
from HIV+ patients exhibited decreased IL-12 and increased
IL-10 production. In summary, phenotype, cytokine secretion, and
-chemokine production by DC from HIV+ individuals were
normal. These cells may prove useful in boosting cellular immune
responses in HIV+ individuals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the role of DC in the transmission of HIV to T cells has been demonstrated in vitro by several groups (7, 8, 9, 10, 11), the extent to which DC remain phenotypically and functionally unaltered during HIV infection is not clear. Given the efficiency of DC for stimulating T cell-dependent immune responses, alterations in DC number and/or function could be an important factor in the development of T cell functional impairment in HIV disease, as previously suggested (12). To address this question, our laboratories and others have studied the capacity of Langerhans cells or blood-derived DC to present recall Ag to CD4+ or CD8+ T cells and have detected no clear defect (13, 14, 15, 16, 17). Similarly, no decrease in levels of HLA-DR or costimulatory molecule expression were reported either on DC obtained from HIV-infected (HIV+) patients or after in vitro infection (14, 18, 19).
In vitro techniques are now available that permit the differentiation and expansion of DC from blood nonproliferating precursors using GM-CSF and IL-4. These monocyte-derived DC have been suggested to be good candidates for clinical use directed at enhancing immune responses in humans (2, 20). Although this approach is efficient and convenient in that it allows for experimentation on relatively large numbers of purified DC, caution must be exercised in drawing parallels between these in vitro, cytokine-driven DC and natural DC, found in nonlymphoid as well as lymphoid tissues. Mindful of this caveat, we studied cytokine and chemokine secretion of both DC from HIV- donors following in vitro infection with HIV-1 and DC obtained from HIV+ donors that were at different clinical stages of HIV disease.
Production of several cytokines by monocyte/macrophages (M/M) obtained from HIV+ donors or after in vitro infection is severely dysregulated (21, 22, 23, 24, 25, 26, 27, 28). Of particular interest is the finding that IL-12 production by activated PBMC obtained from HIV+ patients or after in vitro infection of PBMC from HIV-uninfected (HIV-) donors was decreased (25, 26, 27). In contrast, IL-10 production was reported to be increased in some studies (26, 28), but not in other (25). IL-12/IL-10 imbalance was also described in the plasma of HIV+ patients compared with HIV- donors (29). Furthermore, an elevated IL-10/IL-12 ratio predicted the loss of type 1 cellular immunity in the feline immunodeficiency virus model (30). Therefore, we also compared cytokine and chemokine production between DC and PBMC from which they were derived to investigate whether DC obtained from HIV+ donors would exhibit the same cytokine imbalance that was reported for PBMC and M/M.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood samples were obtained from 15 HIV+ donors from Rush-Presbyterian-St. Luke’s Medical Center (Chicago, IL). They were shipped by overnight express to our National Cancer Institute laboratory in Bethesda, MD, where they were processed within 24 h of collection. These patients were receiving stable antiretroviral therapy, and their CD4 counts ranged from 0–1,297 cells/mm3 (four patients, <300; nine patients, 300–1,000; two patients, >1,000). Their viral loads ranged from undetectable (<500) to 273,100 copies/ml. Blood from 24 healthy adult HIV- donors was obtained from the Department of Transfusion Medicine, National Institutes of Health (Bethesda, MD), and processed using the same conditions as the blood from HIV+ donors. The protocols were approved by the institutional review boards of all participating centers.
Reagents
Soluble trimeric recombinant human CD40L (CD40LT) was provided by Immunex (Seattle, WA) (31). Recombinant human IL-4 (rhIL-4) and rhGM-CSF were obtained from PeproTech (Rocky Hill, NJ). All reagents were screened for low endotoxin levels using the Limulus amebocyte lysate assay (E-Toxate, Sigma, St. Louis, MO). Staphylococcus aureus Cowan (SAC) was purchased from Calbiochem-Behring (Pansorbin, La Jolla, CA).
Cell preparation
PBMC. PBMC were separated on lymphocyte separation medium (Lymphoprep, Organon Teknika, Rockville, MD) and resuspended at 3 x 106/ml in complete medium (RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, 5 mM HEPES, and 2 mM glutamine).
Blood-derived DC. DC were propagated from adult peripheral blood using a previously described protocol (11). Briefly, PBMC were suspended in DC medium at 5–8 x 106 cells/ml in 35-mm tissue culture plates (Costar, Cambridge, MA) for 2 h at 37°C. DC medium is composed of complete medium supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT) and 5 x 10-5 M 2-ME (Life Technologies, Gaithersburg, MD). After this incubation, nonadherent cells were removed, and fresh DC medium was added to culture wells, supplemented with 1000 U/ml rhGM-CSF and 1000 U/ml rhIL-4. Cells were cultured for 7 days at 37°C at 7% C02. Half the volume of the medium was removed on alternate days and replaced with fresh medium supplemented with the two above mentioned cytokines.
On day 7 nonadherent cells were harvested, washed, resuspended at 108 cells/ml in washing medium (PBS containing 10% heat-inactivated FCS), and purified by negative selection. Cells were incubated with a mixture containing the following mouse anti-human Ab (each at 5 µg/ml): anti-CD3, -CD19, -CD16, and -CD14 (PharMingen, San Diego, CA), for 30 min on ice with gentle agitation. After this incubation, cells were washed three times in washing medium and incubated with magnetic beads coated with sheep anti-mouse IgG Abs (10 beads/cell; Dynal, Great Neck, NY), for 30 min on ice with gentle agitation. Unbound cells were separated through a series of washes using a magnetic particle concentrator (MCP-6, Dynal).
Detection of surface markers on DC
DC were washed twice in FACS buffer (balanced salt solution,
0.1% BSA, and 0.01% sodium azide), incubated with human IgG (20
µg/ml) for 10 min at 4°C to block Fc receptors, and stained with Ab
that recognize the following cell surface markers: anti-CD1a-FITC,
-CD40-FITC, -CD80-PE, -CD86-PE, -CD95-PE, -CXCR4-PE (all from
PharMingen), and anti-CCR5 (Coulter/Immunotech, Miami, FL) or with
isotype-matched Ab (
1-FITC and 2-PE, PharMingen), for 30 min at
4°C. The purity of the preparation was checked by staining with T
cell, B cell, or M/M markers (CD3-PE, CD19-PE, and CD14-PE,
PharMingen). The cells were then washed twice and resuspended in FACS
buffer, and surface expression was determined by FACS analysis, using a
Becton Dickinson FACScan and CellQuest software (Becton Dickinson,
Mountain View, CA). Results are presented as the percentage of cells
expressing a given marker compared with the isotype staining or as the
mean fluorescence intensity (MFI).
In vitro infection of DC
Stocks of HIV-1 BaL, a CCR5-using strain, and HIV-1 IIIB, a CXCR4-using strain (Advanced Biotechnologies, Columbia, MD), were used in these experiments. DC were resuspended in DC medium supplemented with rhIL-4 and rhGM-CSF, at 106 cells/ml, and HIV-1 was added to cultures for overnight incubation. On day 0 (after overnight coincubation with HIV), DC were harvested, washed three times in washing medium, resuspended in DC medium supplemented with cytokines at the same cellular concentration, and cultured for 10–14 additional days (time point when maximum p24 production is measured). In addition, DC obtained from three different donors were infected and cultured for only 5 days, when p24 Ag could be first detected. These different time points were chosen according to previous kinetics experiments (11). On alternate days, half the medium was removed and replaced by fresh medium and cytokines. To assess DC infection by HIV, aliquots of these supernatants were inactivated with Triton X-100 (1% final concentration; Sigma, St. Louis, MO) and kept frozen for measurement of HIV-1 p24 protein levels by ELISA (Coulter, Miami, FL; detection limit, 8 pg/ml).
Production of cytokines and chemokines
PBMC were stimulated with SAC (0.01% final concentration) at
37°C for 48 h at 1.5 x 106/ml in
48-well plates (Costar). DC were stimulated with SAC (0.01% final
concentration) or CD40LT (10 µg/ml) at 37°C for 48 h at
0.4 x 106/ml in 48-well plates, in DC
medium supplemented with rhIL-4 and rhGM-CSF. In vitro HIV-infected DC
on days 10–14 postinfection were stimulated with CD40LT using the
above mentioned conditions. After 48 h supernatants were harvested
and frozen at -80°C before cytokine and chemokine analysis. IL-12
p70 and -chemokines (RANTES, MIP-1
, and MIP-1) were measured
by ELISA, using R&D Systems kits (Minneapolis, MN). IL-10 was measured
using PharMingen reagents. The detection limits of these ELISA are 4,
31.2, and 20 pg/ml for IL-12, chemokines, and IL-10, respectively. For
relevant statistical processing of the data, all values below the
detection limits were assigned an arbitrary value of half the detection
limit.
Immunostimulatory properties of DC toward allogeneic T lymphocytes
To assess immunostimulatory functions, DC were tested for their ability to stimulate proliferation of purified allogeneic T cells. T cells were prepared from PBMC obtained from a healthy blood donor by negative selection, using the same protocol as that described for DC with a different mixture of mouse anti-human Ab: anti-CD14, -CD16, -CD19, and -CD1a (PharMingen), and were >90% CD3+. T cells were resuspended in freezing medium (FCS containing 10% DMSO; Sigma) and frozen in liquid nitrogen. At the time of testing DC for allogeneic stimulating activity, T cells were thawed and washed twice in DC medium. T cells (2 x 105) were resuspended in DC medium and cocultured with varying numbers of irradiated DC (3000 cGy, Cs137 source). Cultures were performed in triplicate in flat-bottom 96-well plates (Costar) and incubated at 37°C for 5 days. Cultures were pulsed with 1 µCi of [3H]thymidine overnight, and thymidine incorporation was detected using a beta counter. Results are expressed as the stimulation index = cpm (cultures containing DC + T cells)/cpm (cultures containing T cells alone).
Statistical analysis
Data were compared using nonparametric tests (Mann-Whitney test for unpaired samples or Wilcoxon signed rank test for paired samples). A p value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC from HIV- donors were infected in vitro
with a CCR5-using (R5-using) strain of HIV-1 (Ba-L; n =
8) or a CXCR4-using (X4-using) strain (IIIB;
n = 7) (32). For these infection
experiments, DC were >95% CD1a+ (results not
shown). As previously reported, secreted HIV-1 p24 protein could be
detected in the supernatants of infected cells, starting 4–5 days
after infection, with increasing amounts occurring between days 10 and
14 postinfection (11). According to previous experiments,
p24 Ag could be detected in 2–3% of these DC (11). Four
Ba-L and two IIIB infections induced detectable,
but relatively low, levels of p24 (<10 ng/ml). The remaining nine
infections (four Ba-L and five IIIB) induced high
levels of p24 (>10 ng/ml; cf., Fig. 1
).
As controls, uninfected cells from the same donors were cultured under
the same conditions.
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). To exclude that the lack of
differences observed between uninfected and infected cells was due to
low levels of HIV infection achieved in some experiments, we performed
the same analysis taking into account only infections inducing p24
production >10 ng/ml. Results were identical for both IL-12 and IL-10
and for both Ba-L and IIIB (all p
> 0.07, by Wilcoxon signed rank test). Ba-L-infected DC also produced
amounts of chemokines similar to uninfected DC (all p
> 0.06; cf., Table I). By contrast, after CD40LT stimulation,
production of MIP-1 and MIP-1 by
IIIB-infected DC was significantly higher
compared with that by uninfected DC (both p < 0.03;
cf., Table I). Unstimulated IIIB-infected DC also
tended to produce more MIP-1 and MIP-1 than unstimulated
uninfected controls (MIP-1, 0.6 vs 0.3 ng/ml; MIP-1, 2.4 vs 0.4
ng/ml), although differences were not significant (both
p = 0.14).
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-chemokines was not increased with BaL infection compared with that
in uninfected controls (median MIP-1 production for uninfected and
Ba-L-infected, respectively: 17.5 and 11.5 ng/ml;
MIP-1 production: 10.2 and 9.9 ng/ml; RANTES production: 7.7 and
10.9 ng/ml; all p > 0.6). By contrast, the increased
production of chemokines by IIIB-infected DC at
days 10–14 was not observed at an earlier time point (median MIP-1
production for uninfected and IIIB-infected,
respectively: 17.5 and 15.2 ng/ml; MIP-1 production: 10.2 and 9.2
ng/ml; RANTES production: 7.7 and 5.8 ng/ml; all p
> 0.6).
DC infected for 10–14 days were stimulated with CD40LT for 48 h
or were left unstimulated, and p24 production in supernatants from DC
infected with Ba-L (n = 7) or
IIIB (n = 7) was measured and
compared with that in unstimulated HIV-infected DC. Production of p24
after CD40LT stimulation was significantly decreased in Ba-L-infected
DC compared with unstimulated cells (cf., Fig. 2; p = 0.018, by Wilcoxon
test). Interestingly, stimulation with CD40LT did not induce the same
decrease in IIIB-infected DC (cf., Fig. 2;
p > 0.99, by Wilcoxon test). A possible explanation
for the differential effect of CD40LT stimulation on p24 release by DC
infected with Ba-L or IIIB virus is that
stimulation through the CD40-CD154 pathway differentially regulates
chemokine receptor expression at the surface of DC. Therefore, we
stimulated DC obtained from HIV- donors for
48 h with CD40LT and analyzed CCR5 and CXCR4 expression as well as
CD86 expression as a marker of CD40LT-induced maturation of DC. As
shown in Fig. 3, expression of CCR5 was
down-regulated on CD40LT-stimulated DC (
8-fold decrease), whereas
expression of CXCR4 was up-regulated (14-fold increase). As
expected, expression of CD86 was strongly up-regulated on
CD40LT-stimulated DC (28-fold increase).
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Because in vitro HIV infection of DC did not alter their capacity
to produce cytokines upon stimulation (our results), their phenotypic
characteristics (18), or their capacity to induce an
allogeneic response (11), we investigated whether DC
derived from PBMC from HIV+ patients would also
have similar characteristics to DC derived from PBMC from
HIV+ donors. Therefore, DC were obtained by
culture of plastic-adherent PBMC from HIV+ and
HIV- donors for 7 days in the presence of
rhGM-CSF and rhIL-4. In the case of HIV+ donors,
samples of 50–100 ml of peripheral blood were drawn, and 0.2–3.8
x 106 DC were obtained after culture and
negative selection. The median yield of DC per unit of PBMC was not
significantly different between HIV+ and
HIV- donors (2 x 106
and 1.8 x 106 DC/100 x
106 PBMC for HIV+ and
HIV- donors, respectively; p =
0.89, by Mann-Whitney test). Cells were >75%
CD1a+ bright (results not shown), and,
importantly, the rest of the cells did not express T, B, or M/M lineage
markers (<3% CD3+ and
CD19+ cells, and <1%
CD14+), and were CD1a+
dull. In five different experiments, DC were derived in parallel from
PBMC obtained from one HIV+ and one
HIV- donor, and expression of several surface
markers was analyzed. DC presented with a low level of CD80 and high
levels of CD86, CD40, and CD95 expression as previously described
(11). No differences in phenotype were observed between
cells derived from HIV+ and
HIV- donors (cf., Fig. 4; all p > 0.27, by
Wilcoxon signed rank test). To test for Ag-presenting function, we
measured the capacity of DC to induce an allogeneic response. DC from
both HIV+ and HIV- donors
induced a strong dose-dependent stimulation of allogeneic T cells (cf.,
Fig. 5); this result is in agreement with
the recent study of APC function of monocyte-derived DC obtained from
HIV+ patients (17).
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Monocyte-derived DC produce IL-12 and IL-10 following
stimulation through the CD40-CD154 pathway or by bacterial products
(33, 34). Therefore, we investigated whether cytokine
production by DC from 12 HIV+ patients would be
defective compared with that by cells obtained from 13
HIV- donors. DC were cultured without
stimulation, with CD40LT, or with SAC. Unstimulated DC from
HIV+ and HIV- donors did
not produce detectable amounts of IL-12 and produced low levels of
IL-10. After stimulation, DC from all HIV+ donors
produced large amounts of IL-12 (cf., Fig. 6). This production was similar to that
by cells from HIV- donors (both
p > 0.33, by Mann-Whitney test). Production of IL-10
by DC from the HIV+ and
HIV- donors was also similar after CD40LT or SAC
stimulation (cf., Fig. 6; both p > 0.21).
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, and MIP-1. After
stimulation, DC from all HIV+ donors produced
large amounts of all three chemokines (cf., Fig. 7). Production of chemokines was similar
to that by cells from HIV- donors after CD40LT
or SAC stimulation (cf., Fig. 7; all p >
0.1).
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Comparison of cytokine and chemokine production by DC and PBMC from the same donors
Production of cytokines by SAC-stimulated PBMC from nine
HIV+ donors and eight HIV-
donors, whose cells were used to derive DC, was analyzed in parallel.
In contrast to the results obtained with DC, IL-12 production by PBMC
was decreased in HIV+ donors compared with
HIV- donors (cf., Fig. 6
; p =
0.006, by Mann-Whitney test). IL-10 production by PBMC was also
significantly increased compared with that in
HIV- donors (cf., Fig. 6; p =
0.05). Interestingly, SAC-stimulated IL-12 and IL-10 production was
greater by DC than by PBMC of both HIV+ and
HIV-donors. -Chemokine production by PBMC
after SAC stimulation was also measured. In contrast to what was
observed for cytokine production, chemokine production was similar
between HIV+ and HIV-
donors (cf., Fig. 7; all p > 0.14, by Mann-Whitney
test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokines, even in the case of DC obtained from AIDS patients. To
our knowledge this is the first study investigating the cytokine and
chemokine secretion profile of DC obtained from
HIV+ patients or DC infected in vitro with
different HIV-1 strains. In addition, the cells from
HIV+ patients were morphologically and
phenotypically comparable to DC from healthy donors, expressing similar
high levels of costimulatory molecules. The DC obtained from
HIV+ donors also had potent allogeneic
stimulating capacity, a defining characteristic of DC (2).
Furthermore, DC obtained from healthy donors and infected in vitro with
HIV-1 had characteristics similar to those of uninfected DC obtained
from the same donors. The extent to which DC remain phenotypically and functionally intact during HIV infection is still controversial. The various methods of isolation and maturation of DC may result in the study of different cell populations, and this variability may lead to discrepancies in the reports of their numbers, infection levels, and functional capacities. No decrease in numbers of HLA-DR+ cells or in levels of surface expression of HLA-DR or costimulatory molecules was reported on Langerhans cells or spleen DC obtained from HIV+ patients or after in vitro infection of blood-derived DC (14, 18, 19). In addition, using samples from monozygotic twins discordant for HIV infection or sequential samples from the same patients at different stages of disease progression, no defect was observed in the ability of Langerhans cells or blood-derived DC to present recall Ag to CD4+ or CD8+ T cells (15, 16). In contrast, a reduction in the number and function of blood DC, which was particularly severe in AIDS patients, has been reported (35). These deficient parameters were observed to improve in zidovudine-treated patients (36). Thus, differences in duration and efficiency of antiviral drug therapy of patients whose DC functions are analyzed could also contribute to discrepant results.
Monocyte-derived DC generated from cells of HIV+
patients may serve as vehicles for enhancing the reactivity of
secondary memory T cells to HIV-1 (16). Therefore, our
results showing that those cells have a normal cytokine profile and, in
particular that they can produce large amounts of IL-12 have
significant potential implications. In parallel to our DC studies, PBMC
were obtained from the same HIV+ and
HIV- donors whose cells were used to derive DC.
In contrast to DC, PBMC from HIV+ patients
demonstrated cytokine imbalance that we and others have described.
Specifically, decreased production of IL-12 and increased production of
IL-10 were observed after stimulation (26, 27, 28, 37, 38, 39).
Several studies have shown that IL-12 production by PBMC can be
restored in vitro in most HIV+ donors to a level
similar to that in HIV- donors (27, 39). Of particular interest, was the observation that
pretreatment of PBMC from HIV+ patients with IL-4
or IL-13 primed these cells for enhanced production of IL-12 in
response to SAC (40). Thus, the phase of differentiation
of monocytes into DC, by culturing cells in the presence of high doses
of hIL-4 and hGM-CSF could have primed DC from
HIV+ donors to produce normal IL-12 levels. In
vitro infection of M/M with HIV leads to decreased IL-12 production
(25, 26), increased IL-10 production (41),
and increased expression of Fas ligand molecules (42). In
contrast, in vitro infection of DC does not induce any of these changes
(our results, (42)), highlighting the differential effects
of HIV infection on DC and M/M. A similar discrepancy between DC and
M/M has been reported in the murine model of Leishmania
major infection; infection of M/M with amastigotes inhibited IL-12
production (43, 44, 45). In contrast, amastigotes, in the
presence of IFN-, induced fetal skin-derived DC to release IL-12
(44).
In addition, no changes in the capacity of DC obtained from
HIV+ donors to produce -chemokines (RANTES,
MIP-1, or MIP-1) was detected. Interestingly, the amount of
-chemokines produced by stimulated DC (10–100 ng/ml, depending on
the chemokine) was similar to that produced by activated M/M. These
-chemokines have been reported to inhibit T cell infection by
R5-using HIV-1 (46). Those results suggest that in
addition to their potential role to induce or enhance T cell responses,
activated DC might represent a major source of the HIV-1-suppressive
-chemokines. Ba-L-infected DC produced the same amount of chemokines
as uninfected controls. In contrast,
IIIB-infected DC produced significantly more
MIP-1 or MIP-1 than uninfected controls. Similar results have
been reported after ex vivo HIV infection of tonsils; endogenous
MIP-1, MIP-1, and RANTES production is up-regulated following
infection with X4-using, but not with R5-using, HIV (47).
However, the cellular sources of these chemokines were not
characterized in the tonsil model.
Interestingly, CD40LT stimulation of infected DC had a differential effect on p24 release by these cells depending on the type of virus. It significantly decreased p24 release from Ba-L-infected DC, while it had no effect on IIIB-infected DC. One explanation of this differential effect of CD40LT is that stimulation through the CD40-CD154 pathway differentially regulates chemokine receptor expression at the surface of DC. Accordingly, our results showed that expression of CCR5 was down-regulated on CD40LT-stimulated DC, whereas expression of CXCR4 was up-regulated. These results are in agreement with recent studies of chemokine receptor expression on DC and M/M (48, 49). An alternative, but nonexclusive, explanation is that chemokines produced following CD40LT stimulation were able to block DC infection with an R5-using virus, whereas they had no effect on an X4-using virus, similar to stimulated M/M (46, 50).
These results suggested that stimulated APC could limit transmission of R5-using virus to CD4+ T cells, in contrast to earlier work in which CD40/CD154 stimulation enhanced replication of X4-using virus in DC cocultures with CD4+ T cells (8). However, it was recently reported that addition of CD40LT to cocultures of naive CD4+ T cells and autologous DCs infected with an R5-using isolate caused a reduction in RT activity, whereas its addition to cocultures of T cells and DC infected with an X4-using virus caused the inverse result, i.e., an increase in RT activity (51). Therefore, activation of APC could create a more favorable environment for X4-using strains and a less favorable one for R5-using viruses, and thus accelerate the transition from R5- to X4-using virus. However, caution should be exercised in patients with advanced disease, who might harbor predominantly X4-using viruses. No detectable p24 production could be measured in the DC preparations we analyzed, and a recent study showed the absence of HIV-1 DNA in cytokine-derived DC obtained from 13 asymptomatic patients (17). Nevertheless, the existence of a small number of DC infected with X4-using virus cannot be completely ruled out in the monocyte-derived DC, and activation of those DC could lead to an enhanced transmission of virus to T cells.
Monocyte-derived DC could constitute a source of potent APC and be used to stimulate naive CD4 and CD8 T cell-mediated responses in HIV+ patients or to enhance the reactivity of secondary memory T cells to HIV and non-HIV Ag (16, 52, 53). Moreover, recent studies conducted in the mouse showed that CD40L-stimulated DC can prime CTL, even in the absence of CD4 lymphocytes (54, 55, 56). Procedures recently introduced permit the efficient generation of DC from a limited amount of peripheral blood (20). Our results show that this approach can be applied to obtain DC from HIV+ patients who have a wide range of CD4+ T cell counts, including AIDS patients. These monocyte-derived patient DC have a normal cytokine and chemokine profile, express normal and high levels of costimulatory molecules, and do not produce p24 viral Ag. Because a recent study has shown that cytokine-derived DC obtained from HIV+ donors have strong APC function (17), our findings of normal cytokine and chemokine production by these cells raise the possibility of ex vivo differentiation and expansion of DC from precursors present in the blood of HIV+ patients receiving effective anti-retroviral therapy. These autologous DC might then be pulsed with specific Ag and introduced back into the patients as effective adjuvants for active immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cells; M/M, monocytes/macrophages; CD40LT, recombinant trimeric human CD40 ligand; rhIL-4, recombinant human IL-4; SAC, Staphylococcus aureus Cowan; MFI, mean fluorescence index; MIP-1, macrophage inflammatory protein-1.
Received for publication January 19, 1999. Accepted for publication May 13, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H. Donaghy, J. Wilkinson, and A. L. Cunningham HIV interactions with dendritic cells: has our focus been too narrow? J. Leukoc. Biol., November 1, 2006; 80(5): 1001 - 1012. [Abstract] [Full Text] [PDF]
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S. Patterson, H. Donaghy, P. Amjadi, B. Gazzard, F. Gotch, and P. Kelleher Human BDCA-1-Positive Blood Dendritic Cells Differentiate into Phenotypically Distinct Immature and Mature Populations in the Absence of Exogenous Maturational Stimuli: Differentiation Failure in HIV Infection J. Immunol., June 15, 2005; 174(12): 8200 - 8209. [Abstract] [Full Text] [PDF]
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P. Beuria, H. Chen, M. Timoney, and K. Sperber Impaired Accessory Cell Function in a Human Dendritic Cell Line after Human Immunodeficiency Virus Infection Clin. Vaccine Immunol., March 1, 2005; 12(3): 453 - 464. [Abstract] [Full Text] [PDF]
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 A. Smed-Sorensen, K. Lore, L. Walther-Jallow, J. Andersson, and A.-L. Spetz HIV-1-infected dendritic cells up-regulate cell surface markers but fail to produce IL-12 p70 in response to CD40 ligand stimulation Blood, November 1, 2004; 104(9): 2810 - 2817. [Abstract] [Full Text] [PDF]
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C. Carbonneil, V. Donkova-Petrini, A. Aouba, and L. Weiss Defective Dendritic Cell Function in HIV-Infected Patients Receiving Effective Highly Active Antiretroviral Therapy: Neutralization of IL-10 Production and Depletion of CD4+CD25+ T Cells Restore High Levels of HIV-Specific CD4+ T Cell Responses Induced by Dendritic Cells Generated in the Presence of IFN-{alpha} J. Immunol., June 15, 2004; 172(12): 7832 - 7840. [Abstract] [Full Text] [PDF]
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R. Zhang, C. J. Fichtenbaum, D. A. Hildeman, J. D. Lifson, and C. Chougnet CD40 Ligand Dysregulation in HIV Infection: HIV Glycoprotein 120 Inhibits Signaling Cascades Upstream of CD40 Ligand Transcription J. Immunol., February 15, 2004; 172(4): 2678 - 2686. [Abstract] [Full Text] [PDF]
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J. Poudrier, X. Weng, D. G. Kay, Z. Hanna, and P. Jolicoeur The AIDS-Like Disease of CD4C/Human Immunodeficiency Virus Transgenic Mice Is Associated with Accumulation of Immature CD11bHi Dendritic Cells J. Virol., November 1, 2003; 77(21): 11733 - 11744. [Abstract] [Full Text] [PDF]
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C. Chougnet Role of CD40 Ligand dysregulation in HIV-associated dysfunction of antigen-presenting cells J. Leukoc. Biol., November 1, 2003; 74(5): 702 - 709. [Abstract] [Full Text] [PDF]
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H. Donaghy, B. Gazzard, F. Gotch, and S. Patterson Dysfunction and infection of freshly isolated blood myeloid and plasmacytoid dendritic cells in patients infected with HIV-1 Blood, June 1, 2003; 101(11): 4505 - 4511. [Abstract] [Full Text] [PDF]
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T. Kawamura, H. Gatanaga, D. L. Borris, M. Connors, H. Mitsuya, and A. Blauvelt Decreased Stimulation of CD4+ T Cell Proliferation and IL-2 Production by Highly Enriched Populations of HIV-Infected Dendritic Cells J. Immunol., April 15, 2003; 170(8): 4260 - 4266. [Abstract] [Full Text] [PDF]
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J. Pacanowski, S. Kahi, M. Baillet, P. Lebon, C. Deveau, C. Goujard, L. Meyer, E. Oksenhendler, M. Sinet, and A. Hosmalin Reduced blood CD123+ (lymphoid) and CD11c+ (myeloid) dendritic cell numbers in primary HIV-1 infection Blood, November 15, 2001; 98(10): 3016 - 3021. [Abstract] [Full Text] [PDF]
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J. Lieberman, P. Shankar, N. Manjunath, and J. Andersson Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection Blood, September 15, 2001; 98(6): 1667 - 1677. [Abstract] [Full Text] [PDF]
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M. C. Braun, J. M. Wang, E. Lahey, R. L. Rabin, and B. L. Kelsall Activation of the formyl peptide receptor by the HIV-derived peptide T-20 suppresses interleukin-12 p70 production by human monocytes Blood, June 1, 2001; 97(11): 3531 - 3536. [Abstract] [Full Text] [PDF]
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C. Chougnet, C. Freitag, M. Schito, E. K. Thomas, A. Sher, and G. M. Shearer In Vivo CD40-CD154 (CD40 Ligand) Interaction Induces Integrated HIV Expression by APC in an HIV-1-Transgenic Mouse Model J. Immunol., March 1, 2001; 166(5): 3210 - 3217. [Abstract] [Full Text] [PDF]
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R. S. Kornbluth The emerging role of CD40 ligand in HIV infection J. Leukoc. Biol., September 1, 2000; 68(3): 373 - 382. [Abstract] [Full Text] [PDF]
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X. Ma and L. J. Montaner Proinflammatory response and IL-12 expression in HIV-1 infection J. Leukoc. Biol., September 1, 2000; 68(3): 383 - 390. [Abstract] [Full Text] [PDF]
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MFI), defined
as 
