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Secretion in Suppressed HIV-Infected Patients Despite Mature NK Cell Recovery: Evidence for a Defective Reconstitution of Innate Immunity1
,
,
* Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, and
Philadelphia Field Initiating Group for HIV-1 Trials, Philadelphia, PA 19107;
HIV-1 Immunopathogensis Laboratory, The Wistar Institute, and
Division of Infections Diseases, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
-producing NK and T cells in
viremic or therapy-suppressed HIV-infected subjects, and noninfected
control donors. Viremic HIV+ individuals had significantly
lower proportions of mature
CD3-/CD161+/CD56+ NK cells and of
IFN--producing NK cells compared with noninfected donors,
independent of CD4+ T cell counts. HIV-infected subjects
with undetectable viral load recovered mature
CD3-/CD161+/CD56+ NK cells and
cytotoxicity against tumor (K562) and HSV-infected target cells to
percentages comparable with those of uninfected individuals, but their
NK cells remained impaired in their ability to produce IFN-. In
parallel to these ex vivo findings, in vitro NK cell differentiation of
CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1
resulted in the production of NK cells with a normal mature phenotype,
but lacking the ability to produce IFN-, whereas coculture of
uninfected PBMC with HIV failed to affect mature NK cell properties or
IFN- secretion. Altogether, our findings support the hypothesis that
mature NK cell phenotype may be uncoupled from some mature functions
following highly active antiretroviral therapy-mediated suppression of
HIV-1, and indicate that relevant innate immune functions of NK cell
subsets may remain altered despite effective viral suppression
following antiretroviral treatment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which may
directly contribute to HIV control and lack of disease progression.
IFN- secretion by NK cells also contributes to adaptive type 1
responses, via IFN- priming for IL-12 production by APC
(4) associated with type 1 T cell differentiation
(19, 20, 21, 22). Although a decrease in type 1 responses in AIDS
patients is associated with reduced serological levels of IFN-
(23) and decreased IFN- secretion by activated NK and T
cells (24, 25), it remains undetermined to what extent
IFN--secreting NK cell subsets decrease in frequency, and whether or
not they increase following suppressive therapy, as shown for T cell
IFN- responses (25). Studies that have addressed
recovery of NK subsets following antiretroviral therapy have largely
focused on changes of frequency of mature NK phenotypes and
cytotoxicity (26, 27, 28, 29, 30, 31, 32).
To define, on a cell-specific basis, the distribution of
IFN--secreting NK cell subpopulations in uninfected and infected
(viremic or suppressed) subjects, we have analyzed phenotype and
intracellular cytokine expression of proinflammatory (TNF-) and type
1 (IFN-) cytokines in peripheral blood-derived NK cells after an
acute 6-h stimulation. Our results show a sustained impairment of
IFN- production in viremic and highly active antiretroviral therapy
(HAART)4-suppressed
HIV-infected subjects, regardless of a normalization of the proportion
of NK cells with mature phenotype and restored cytotoxic function
following viral suppression, indicating that a persistent dysfunctional
subset of mature NK cells results in a sustained impairment of innate
immune function. The association between HIV-1 infection and the
presence of a dysfunctional mature NK subset(s) observed ex vivo is
further supported by the observed in vitro differentiation, in the
presence of HIV-1, of phenotypically mature NK cells with impaired
ability to produce IFN-.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood samples from 56 HIV-infected individuals were
provided by the Philadelphia Field Initiating Group for HIV Trials
(FIGHT, Philadelphia, PA). The clinical profile of the patients is
summarized in Table I
. Peripheral blood
samples from healthy volunteers (three females, six males, age 27–50)
were used as controls. Informed consent was obtained from all donors in
accordance with protocols approved by the Institutional Review Boards
of the Wistar Institute of Anatomy and Biology (Philadelphia, PA),
Philadelphia FIGHT, and Thomas Jefferson University (Philadelphia, PA).
Umbilical cord blood samples from HIV-1-noninfected placentas were
provided by R. Wapner (Department of Obstetrics and Gynecology, Thomas
Jefferson University Hospital, Philadelphia, PA).
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HIV-1 strains NL4.3 (T-tropic, provided by A. Srinivasan, Thomas Jefferson University), BA-L, and Ada-M (M-tropic) were used. Viral stocks were prepared in CEMx174 cells. The infectivity of the viral stocks was titrated assessing 7-day syncytia formation in CEMx174 (minimum infectious multiplicity of infection = 2 x 10-5). Concentrations corresponding to 4 x 10-4 multiplicity of infection were used in all experiments (respectively 50 ng/ml p24/p27 for NL4.3, 28 ng/ml for Ada-M, and 32 ng/ml for BA-L).
Cell culture
PBMC were obtained from peripheral or umbilical cord blood after density gradient centrifugation (Histopaque-1077; Sigma-Aldrich, St. Louis, MO). To support mature NK cell viability in short-term infection experiments with or without the indicated viral strains, PBMC were cultured with IL-2 (100 U/ml) or with 50 Gy-irradiated lymphoblastoid B cell line RPMI 8866 (33) for 15 days.
Surface phenotype and intracellular cytokine detection
Based on prior time course analysis, PBMC or cells from
long-term cultures were stimulated for 6 or 18 h, as indicated,
with the indicated combinations of IL-2 (100 U/ml) and IL-12 (5 ng/ml,
sp. act. 4.5 x 106 U/mg, provided by S.
Wolf, Genetics Institute, Andover, MA). A combination of PMA
(10-9 M; Sigma-Aldrich),
Ca2+ ionophore (A23187, 0.1 µg/ml;
Sigma-Aldrich) was used as a lineage-independent control stimulus to
assess cytokine detection; T cell-specific stimuli (plastic-immobilized
anti-CD3 mAb (OKT3), 10 µg/ml in 0.05 M Tris (pH 9.5) alone or in
combination with PMA) were used to control for NK cell-specific
effects. Surface phenotype was detected in multiple color
immunofluorescence with FITC-labeled anti-CD56 or anti-CD3 mAb
(Caltag Laboratories, Burlingame, CA), biotin-labeled anti-CD161
mAb (B199.2) (34), and CyChrome streptavidin (BD
PharMingen, San Diego, CA). Results are percentage of
CD3- cells expressing the indicated markers
(result range is lower than expected >50% range due to use of PBMC
rather than PBL in the assays). Intracellular cytokines were detected
in cells treated with 10 µg/ml brefeldin A during the last 3 h
of culture; fixed in 3.7% formaldehyde; permeabilized with a solution
of 0.5% saponin, 0.02% FBS, and 0.005% Tween 20; and stained with
PE-labeled anti-IFN- or anti-TNF- (Caltag Laboratories).
Nonreactive isotype-matched mAb were used as negative controls.
The samples were analyzed using an EPICS Profile cytofluorometer (Coulter, Fullerton, CA) and WinMDI version 2.8 (J. Trotter, The Scripps Research Institute, La Jolla, CA; http://www.scripps.edu). The percentage of cytokine-producing cells upon stimulation was calculated by subtracting the percentage of background production in parallel nonstimulated samples; this was similarly negligible in both HIV+ and control donors.
Target cells and NK cell-mediated cytotoxic assays
The 51Cr release cytotoxic assays with K562 and FS4-HSV-infected fibroblasts. The erythroleukemia cell line K562 was maintained in RPMI 1640 medium, supplemented with 10% FBS. Human neonate foreskin fibroblasts (FS4, kindly provided by J. Vilcek, New York Medical Center, New York, NY) were maintained in Eagle’s modified MEM (Flow Laboratories, McLean, VA) supplemented with 10% heat-inactivated FBS, and used at passage 16–24. FS4 cells were infected with HSV-1-NS (kindly provided by H. Friedman, University of Pennsylvania) and cryopreserved, as previously described. Target cells (K562 1 x 106 viable cells) were labeled with Na251CrO4 (100 µCi) for 1 h (K562) or 3 h (HSV-1-FS4) at 37°C, washed, and resuspended at a concentration of 5 x 104 cells/ml in culture medium. Effectors and labeled targets were incubated in triplicates in 0.2-ml vol at different E:T ratios (50:1, 25:1, 12.5:1, and 6.25:1) in round-bottom 96-well plates and incubated for 4 h for K562 and HIV-infected targets and 18 h for the HSV-FS4. Percentage of lysis was determined by the following formula: ((experimental cpm - spontaneous released counts)/(total cpm - spontaneous released cpm)) x 100 (35, 36).
IFN- ELISA
Cell-free supernatants were collected from cells cultured and
stimulated for 18 h, as described above. IFN- ELISA was
performed with anti-IFN- mAb B133.1 for capture and
biotin-conjugated anti-IFN- mAb B133.5 and HRP-conjugated
streptavidin for detection, as previously described (37).
Sensitivity of the assay was 0.05 ng/ml.
NK cell differentiation culture
The in vitro differentiation model used has been described in
detail (38) (see also Refs. 39 and
40). CD34+ cells were obtained from
umbilical cord blood lymphocytes by indirect panning using
anti-CD34 mAb My10 (American Type Culture Collection, Manassas,
VA); this procedure routinely resulted in cell preparations of >90%
CD34+ cells, expressing no detectable CD16, CD56,
or CD161. CD34+ cells were cultured for 30 days
on monolayers of SL/SL4/hSCF220, a
murine stromal cell line expressing the p220 membrane-bound form of
human stem cell factor (provided by D. Williams, University of Indiana,
Indianapolis, IN) (41), with 50 U/ml IL-2
(Hoffman-LaRoche, Nutley, NJ; obtained through the Biological Response
Modifier Program, National Cancer Institute, Bethesda, MD), or 10 ng/ml
IL-15 (sp. act. 2.95 x 108 U/mg; Immunex,
Seattle, WA), and with or without the indicated viral strains. Fresh
viral inoculum was provided every week. This culture system has been
studied extensively, and allows the differentiation of distinct
populations of immature
(CD3-/CD161+/CD56-/low,
exert cytotoxicity via TNF-related apoptosis-inducing ligand, but not
Fas ligand or granule release; unable to produce IFN-) and mature NK
cells
(CD3-/CD161+/CD56high,
exert cytotoxicity via Fas ligand and granule release, but not
TNF-related apoptosis-inducing ligand; able to produce IFN-).
Statistical analysis
Results (median, 25th and 75th percentile) are expressed as
background-subtracted percentage of positive cells. Where indicated,
the HIV+ cohort was stratified based on
CD4+ T cell counts (< or 
500/mm3) or serum HIV RNA levels (<50; 50–5000,
>5000 copies/ml, as detected by PCR by an independent clinical
laboratory). We did not assume Gaussian distributions and used
nonparametric tests (Mann-Whitney and Spearman’s) for analysis.
Two-tailed p values <0.05 were considered significant.
To analyze interstrata differences, we used nonparametric ANOVA (Kruskal-Wallis test), followed by Dunn’s postrun analysis on runs with p < 0.05. In selected experiments, in which data were found to follow a normal distribution based on Kolmogorov-Smirnoff analysis, we also performed classical parametric ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD56 is expressed at later differentiation stages than CD161 in NK
cells, and its expression is associated with functional maturation
(38, 42, 43). Because CD161 is the earliest marker of the
NK lineage, and allows the recognition of both mature and immature NK
cells (40–50% of CD3- PBMC in control or
HIV+ donors), we will henceforth define total NK
cells as CD3-/CD161+
cells. To determine whether HIV infection results in alteration of the
relative proportions of NK cell subpopulations, we investigated CD161
and CD56 expression on CD3- PBMC from 36
HIV+ patients and 9 control donors. The
percentage of
CD3-/CD161+/CD56+
NK cells was significantly lower (p = 0.0095)
in the HIV+ donors (median = 15.2% (9.2;
23.8), n = 39) than control HIV-
individuals (median = 27.8% (18.8; 43.9), n = 9),
in accordance with previous reports (5, 6, 7, 8, 9). However,
similar percentages of mature
CD3-/CD161+/CD56+
NK subsets were observed in HAART-treated HIV+
individuals with <50 RNA copies/ml and control donors (Fig. 1), whereas viremic donors had
significantly lower percentages of the same cells. Consistent with
percent-based findings, analysis of a representative subset of
HIV+ individuals (n = 37)
demonstrated that also absolute
CD3-/CD161+/CD56+
cell numbers were significantly lower (median = 31.2 cells/µl
(19.1; 44.9)) in viremic patients with viral load >5000 RNA copies/ml,
as compared with patients with viral load <50 (median = 88.3
(73.4; 140.6); p < 0.05). A negative correlation
between numbers of
CD3-/CD161+/CD56+
cells and serum viral load (Spearman r = -0.38,
p < 0.05) further suggests that a reconstitution of
the CD161+/CD56+ mature NK
cell subset may result from viral suppression.
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Assessment of cytotoxic activity of PBMC from
HIV+ donors (Fig. 1
B) demonstrated
that cytotoxicity to the erythroleukemia cell line K562 was
significantly reduced in donors with viral load of 50–5000
(median = 23% (22;31)) and >5000 (median = 14% (12.5;17))
as compared with normal control donors (median = 41% (39;50)) or
virally suppressed donors (median = 32% (25;40)). However,
no significant difference was found between suppressed and uninfected
donors; comparable results were also obtained using HSV-infected
fibroblast FS4 as targets (not shown), suggesting the possibility of a
reconstitution of NK cell function(s) upon viral suppression; in
support of this contention, cytotoxicity was inversely correlated to
the log viral load (Spearman r = -0.4285;
p = 0.0008, and r = -0.8346;
p < 0.0001, respectively).
Impairment of IFN- secretion irrespective of mature NK cell
frequency in HIV+ donors
The ability of NK cells to produce type 1 and/or proinflammatory
cytokines was analyzed detecting IFN- and TNF- accumulation at
the single cell level, using total PBMC from HIV+
or control individuals treated with IL-2 in combination with
Ig-sensitized bovine erythrocytes or IL-12, the latter
combination being known to optimally activate production of these
cytokines in NK cells (44, 45). Upon 6-h IL-2/IL-12
stimulation, the percentage of IFN--producing
CD3-/CD161+ NK cells
(total NK cells) was significantly lower (p =
0.038) in HIV+ patients (median = 2.9 (0.5;
6), n = 36) as compared with uninfected controls
(median = 23.8 (2.1; 45.4), n = 9) (Fig. 2A, representative
experiment). Furthermore, analysis of absolute numbers of
IL-2/IL-12-induced
CD3+/CD161+/IFN-+
cells in a representative subset of HIV+
individuals (n = 20) showed no significant difference
between patients with viral load <50 RNA copies/ml (median = 2.39
cells/µl (0.96;3.62)), 50–5000 RNA copies/ml (median = 2.43
(1.11; 2.9)), or >5000 RNA copies/ml (median = 0.92 (0.85;
1.57)). No correlation was detected between percentage or absolute
numbers of IFN--producing
CD3-/CD161+ cells and
serum viral load. Stratification of HIV+ donors
based on CD4+ T cell counts or viral load (see
Materials and Methods) did not reveal significant
differences between strata. Taken together, these data indicate that,
in contrast to the changes in phenotype and cytotoxicity described
above, IL-2/IL-12-induced cytokine secretion remains impaired in
HAART-suppressed subjects (Table II).
Consistent with an impaired cytokine response, following IL-2/IL-12 the
percentage of TNF- NK cells was also decreased in
HIV+ individuals. Furthermore, the differences in
percentage or absolute numbers of TNF--producing
CD3-/CD161+ NK cells
between HIV RNA-stratified groups were not significantly different,
supporting the hypothesis that TNF- production may remain affected
in HIV+ patients, regardless of viral
suppression. No positive correlation was detected between percentages
or absolute numbers of IFN--producing NK cells and mature
CD3-/CD161+/CD56+
NK cells, indicating that a loss of IFN--producing capability was
not directly attributable to loss of the mature NK cell subset. As
expected, CD3-mediated control stimulations (see Materials and
Methods) did not elicit responses in
CD3-/CD161+ NK cells, but
resulted in percentages of IFN--producing
CD161+ and CD161- T cells
that were not significantly different between
HIV+ and control HIV-
donors. Similar results were observed comparing 6- and 18-h
stimulations, confirming that prolonged cytokine treatment did not lead
to a recovery of the cytokine-producing ability of NK cells from
HIV+ individuals to the same levels of
HIV- donors, albeit higher cytokine-producing
frequencies were detected in all conditions following prolonged
stimulation (Fig. 2B).
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secretion in NK cells differentiated in vitro in
the presence of HIV-1
The observed decrease in IFN--producing NK cells in
HIV+ donors suggested that chronic HIV infection
may inhibit mature NK cell cytokine secretion or affect their
differentiation. To address these two possibilities, we cultured: 1)
PBMC-derived mature NK cells, and 2) CD34+ cord
blood precursor cells with R5 or X4 HIV-1 isolates, and measured the
effects of viral particles on NK cell cytokine secretion and in vitro
NK differentiation.
To model in vitro the effects of chronic viremia on mature NK cells, we
analyzed cytokine production in mature NK cells from
HIV- PBMC cultured for 15 days (in the presence
of IL-2 or the lymphoblastoid B cell line RPMI 8866 to support NK cell
viability) with X4 or R5 HIV-1 strains (same concentrations used in the
differentiation experiments). Consistent with HIV affecting NK cell
during their differentiation rather than directly inhibiting cytokine
production in functional mature subsets, the presence of HIV in mature
PBMC cultures did not affect NK cell phenotype as defined by CD161/CD56
expression on CD3- lymphocytes, or the
percentage of IFN--producing
CD3-/CD161+ NK cells
detected by immunofluorescence after IL-2/IL-12 stimulation (not
shown).
Using an in vitro model, we have previously demonstrated that NK
differentiation to mature
CD161+/CD56+ cells capable
of cytotoxicity and IFN- production involves an intermediate
CD3-/CD161+/CD56-
stage, at which they do not exert granule release-dependent
cytotoxicity nor produce IFN- (38). Phenotypically
mature NK cells generated in the presence of X4 (NL4.3)
infectious HIV viral particles were tested for production of IFN-
following an 18-h stimulation with IL-2/IL-12, as compared with
controls that cultures differentiated without HIV-1. Cell numbers and
viability were comparable in HIV-treated and control cultures. IFN-
secretion was evaluated by ELISA, measuring the cytokine levels in
cell-free culture supernatants from IL-2/IL-12/PMA-stimulated cells
(Fig. 3a). Supernatant from
cultures without virus showed increased (mean = 475%) IFN-
upon stimulation with IL-2/IL-12, as compared with those from cells
differentiated in the presence of HIV (mean = 81%). The observed
lack of IFN- production was confirmed analyzing its
intracellular expression in gated
CD3-/CD161+ NK cells
(38): low intensity accumulation of intracellular IFN-
was detectable (Fig. 3b) in control cells upon stimulation
(nonstimulated = 0.8, IL-2/IL-12/PMA = 1.53; mean
fluorescence intensity values, average of three experiments), but was
absent in NL4.3-treated cultures (nonstimulated = 1.07,
IL-2/IL-12/PMA = 0.72). Analysis of the phenotype of NK cells
differentiated in vitro in the presence of X4 or R5 HIV, as described
above, revealed that both immature
CD3-/CD161+/CD56-/low
and mature
CD3-/CD161+/CD56+
cells were produced in similar numbers and proportions (data not
shown), indicating that the presence of virus did not alter the
production of phenotypically mature NK cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
despite the presence of
CD161+/CD56+ mature NK
cells in proportions similar to these observed in uninfected control
donors. The observation that this cell subset could sustain a competent
cytotoxic response to tumor and viral-infected targets suggests that
viral suppression may result in reconstitution of a mature NK cell
subset with selective functional impairment of cytokine production. We
also expand previous population-based NK cell analyses by showing that
a decreased IFN- secretion by NK cells represents a loss of
cytokine-producing NK cell subsets, in accordance with previous reports
showing a loss of mature CD56+ NK cells in HIV
infection (5, 6, 7, 8, 9). Unlike previous reports addressing NK
cell cytokine production in HIV infection, which have been limited to
the measurement of secreted cytokines in supernatants from cultures of
purified NK cell populations, we have defined the activation-induced
cytokine production of NK cells obtained ex vivo without prior
purification or extended culture, thus reducing the possibility of in
vitro culture artifacts. The use of CD161, the only known marker
expressed in the majority of NK cells that encompasses both immature
and mature subsets, allowed us to study cytokine production in the
total NK cell population, avoiding the potential underestimation of
relative variations in cytokine-producing cells due to decreased
percentages of CD56+ cells in
HIV+ donors. Although a
CD56bright NK cell subset responsible for high
levels of IFN- production has been previously described
(46) without description of the amount of CD161 expressed
in this subset, we ruled out a segregation of cytokine secreting in
regard to CD161 expression by demonstrating that a CD56 was expressed
at comparable fluorescence intensity in
CD3-/CD161+ and
CD3-/CD161- cells.
Interestingly, the depletion of IFN--producing NK cells observed
does not correlate with viral load or CD4+ T cell
counts, suggesting that the onset of NK cell alterations may represent
an early event following infection. This is in agreement with
observations by Hu et al. (6), reporting a decrease in NK
cells despite sustained CD4+ T cell percentages
in HIV-1-infected PBMC. However, our data do not rule out that studies
of an expanded number of end-stage patients with CD4 counts below 200
might reveal CD4-associated differences.
In determining the effects of HIV infection on NK cytokine secretion,
our results, if confirmed with studies based on direct analysis of NK
cell differentiation of precursors derived from HIV-infected
individuals, support the hypothesis that HIV selectively affects some
aspects of the differentiation of NK cell precursors from
CD34+ precursors, as the ability of NK cells to
produce IFN- in response to IL-2/IL-12 is greatly reduced when their
differentiation takes place in vitro in the presence of HIV. The
surprising finding that HIV-1 did not affect the in vitro development
of mature CD161+/CD56+ NK
cells when analyzed by phenotype rather than cytokine secretion
suggests an uncoupling of these otherwise associated properties of
mature NK cells. The potential existence of this uncoupling in vivo is
supported by the same observation in suppressed subjects, in which
phenotypically mature subsets of NK cells are also impaired in IFN-
secretion. Of particular interest is the observation that, parallel to
mature phenotype, NK cell-dependent cytotoxic activity is not
significantly different in suppressed patients and control donors, but
is significantly reduced in viremic patients, suggesting that selected
NK cell functions are restored upon viral suppression and
reconstitution of a mature phenotype.
Importantly, our data also suggest that, while in viremic patients the
loss of the CD56+ NK cell subset may contribute
to the loss of IFN- secretion, the latter represent a distinct
impairment that, unlike mature phenotype and cytotoxic activity, might
not be recovered following effective antiretroviral therapy. Further
investigation on the possible replication of HIV-1 in bone marrow sites
of NK cell differentiation despite plasma viral levels of <50
copies/ml is needed to define whether or not HIV replication is
inhibited within these compartments. Recent reports
(29, 30, 31) have demonstrated the enhancing effects of G-CSF
and GM-CSF over HAART alone in restoring CD56+ NK
cell numbers, indicating that bone marrow stimulation may directly
result in increased production of mature NK cells in vivo. Although a
comprehensive study of the innate immune function in these patients has
not been published to date, and will require longitudinal studies
following HIV+ patients throughout HAART-mediated
viral suppression and immune reconstitution, it is tempting to
speculate whether approaches that also target the bone marrow
compartment may result in a more complete reconstitution of NK cell
functions as compared with approaches directly targeting circulating
mature NK cells such as those using IL-2 or IL-12 (26, 27, 32). The alternative hypothesis that a loss of IFN-
production by NK cells depends on a direct effect of the virus on
mature NK cells is not supported by our data, as IFN- secretion was
sustained by mature PBMC-derived NK cells exposed to R5 or X4 HIV-1 in
vitro. Because in our in vitro developmental model the virus was added
to the cultures from day 0, rather than just before the experimental
tests as in prior reports of NK inhibition of function following acute
exposure to soluble gp120 (47), our experiments do not
directly address acute effects of exposure to viral particles, but
rather suggest the presence of a sustained inhibitory effect. Lack of
cytokine-induced IFN- production could also be explained by a
reduced response to cytokine-receptor signaling (e.g., through IL-2R or
IL-12R chains) in NK cells from HIV+ individuals,
as described for the response to IFN- (48). However,
our observations are most consistent with an intrinsic defect in the
transcriptional activation or synthesis of IFN- within mature NK
cells, as independent induction by IL-2 or IL-12 did enhance TNF-
secretion and cytotoxic function (not shown). It will be important to
expand this study to other NK-tropic cytokines (e.g., IL-18) to
determine whether any alternative NK cell stimulation (direct or
indirect) can overcome the impaired IFN--producing response in
HIV+ individuals. The potential mechanisms
associated with intrinsic impairment, such as methylation of the
regulatory region of the IFN- gene (49), or indirect
mechanisms, such as depletion of HLA-DR+ cells
accessory to NK cell functions (50, 51, 52, 53), remain to be
addressed; further studies of transcriptional events correlated to
cellular activation in NK cells from HIV-infected individuals may help
unravel the biochemical underlying the suppression of cytokine
production. Importantly, the impairment of IFN- production response
to IL-2/IL-12 was restricted to cells of NK lineage, as indicated by
the fact that HIV infection did not result in significantly reduced
proportions of IFN--producing cells in
CD3+/CD161- T cells or
CD3+/CD161+ NK-like T cells
from the same samples. These observations, together with a lack of
association with CD4+ T cell counts, raise the
hypothesis that an early impairment of NK cell function may precede the
onset of T cell dysfunction and act to increase susceptibility to
infection, inflammation, and HIV replication (19, 20, 21, 22).
Although novel longitudinal population studies, presently underway,
will allow us to directly address the issues of immune reconstitution
of the NK cell subsets and function upon HAART, the observations
reported in this study strongly suggest that a decrease in the viral
load, such as observed during HAART, may not result in a recovery of NK
cell IFN- production, which may bear on overall immune control and
disease resistance.
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Acknowledgments |
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Footnotes |
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2 Current address: HIV-1 Immunopathogenesis Laboratory, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104.
3 Address correspondence and reprint requests to Dr. Luis J. Montaner, HIV-1 Immunopathogenesis Laboratory, Immunology Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: montaner{at}mail.wistar.upenn.edu
4 Abbreviation used in this paper: HAART, highly active antiretroviral therapy.
Received for publication January 16, 2002. Accepted for publication March 20, 2002.
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  S. J. Conry, K. A. Milkovich, N. L. Yonkers, B. Rodriguez, H. B. Bernstein, R. Asaad, F. P. Heinzel, M. Tary-Lehmann, M. M. Lederman, and D. D. Anthony Impaired Plasmacytoid Dendritic Cell (PDC)-NK Cell Activity in Viremic Human Immunodeficiency Virus Infection Attributable to Impairments in both PDC and NK Cell Function J. Virol., November 1, 2009; 83(21): 11175 - 11187. [Abstract] [Full Text] [PDF]
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Q. Liu, Y. Sun, S. Rihn, A. Nolting, P. N. Tsoukas, S. Jost, K. Cohen, B. Walker, and G. Alter Matrix Metalloprotease Inhibitors Restore Impaired NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Human Immunodeficiency Virus Type 1 Infection J. Virol., September 1, 2009; 83(17): 8705 - 8712. [Abstract] [Full Text] [PDF]
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 P. Bostik, J. Kobkitjaroen, W. Tang, F. Villinger, L. E. Pereira, D. M. Little, S. T. Stephenson, M. Bouzyk, and A. A. Ansari Decreased NK Cell Frequency and Function Is Associated with Increased Risk of KIR3DL Allele Polymorphism in Simian Immunodeficiency Virus-Infected Rhesus Macaques with High Viral Loads J. Immunol., March 15, 2009; 182(6): 3638 - 3649. [Abstract] [Full Text] [PDF]
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B. Majumder, N. J. Venkatachari, S. O'Leary, and V. Ayyavoo Infection with Vpr-Positive Human Immunodeficiency Virus Type 1 Impairs NK Cell Function Indirectly through Cytokine Dysregulation of Infected Target Cells J. Virol., July 15, 2008; 82(14): 7189 - 7200. [Abstract] [Full Text] [PDF]
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W. M. Ballan, B.-A. N. Vu, B. R. Long, C. P. Loo, J. Michaelsson, J. D. Barbour, L. L. Lanier, A. A. Wiznia, J. Abadi, G. J. Fennelly, et al. Natural Killer Cells in Perinatally HIV-1-Infected Children Exhibit Less Degranulation Compared to HIV-1-Exposed Uninfected Children and Their Expression of KIR2DL3, NKG2C, and NKp46 Correlates with Disease Severity J. Immunol., September 1, 2007; 179(5): 3362 - 3370. [Abstract] [Full Text] [PDF]
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 S. Vasan, M. A. Poles, A. Horowitz, E. E. Siladji, M. Markowitz, and M. Tsuji Function of NKT cells, potential anti-HIV effector cells, are improved by beginning HAART during acute HIV-1 infection Int. Immunol., August 16, 2007; (2007) dxm055v1. [Abstract] [Full Text] [PDF]
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C. Tomescu, J. Chehimi, V. C. Maino, and L. J. Montaner NK Cell Lysis of HIV-1-Infected Autologous CD4 Primary T Cells: Requirement for IFN-Mediated NK Activation by Plasmacytoid Dendritic Cells J. Immunol., August 15, 2007; 179(4): 2097 - 2104. [Abstract] [Full Text] [PDF]
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G. Alter, T. J. Suscovich, N. Teigen, A. Meier, H. Streeck, C. Brander, and M. Altfeld Single-Stranded RNA Derived from HIV-1 Serves as a Potent Activator of NK Cells J. Immunol., June 15, 2007; 178(12): 7658 - 7666. [Abstract] [Full Text] [PDF]
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 J.-Y. Zhang, Z. Zhang, X. Wang, J.-L. Fu, J. Yao, Y. Jiao, L. Chen, H. Zhang, J. Wei, L. Jin, et al. PD-1 up-regulation is correlated with HIV-specific memory CD8+ T-cell exhaustion in typical progressors but not in long-term nonprogressors Blood, June 1, 2007; 109(11): 4671 - 4678. [Abstract] [Full Text] [PDF]
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 A. R. Rodriguez, B. P. Arulanandam, V. L. Hodara, H. M. McClure, E. K. Cobb, M. T. Salas, R. White, and K. K. Murthy Influence of interleukin-15 on CD8+ natural killer cells in human immunodeficiency virus type 1-infected chimpanzees J. Gen. Virol., February 1, 2007; 88(2): 641 - 651. [Abstract] [Full Text] [PDF]
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 Q. Wei, J. W. Stallworth, P. J. Vance, J. A. Hoxie, and P. N. Fultz Simian Immunodeficiency Virus (SIV)/Immunoglobulin G Immune Complexes in SIV-Infected Macaques Block Detection of CD16 but Not Cytolytic Activity of Natural Killer Cells. Clin. Vaccine Immunol., July 1, 2006; 13(7): 768 - 778. [Abstract] [Full Text] [PDF]
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 W. Droge Oxidative stress and ageing: is ageing a cysteine deficiency syndrome? Phil Trans R Soc B, December 29, 2005; 360(1464): 2355 - 2372. [Abstract] [Full Text] [PDF]
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G. Alter, N. Teigen, B. T. Davis, M. M. Addo, T. J. Suscovich, M. T. Waring, H. Streeck, M. N. Johnston, K. D. Staller, M. T. Zaman, et al. Sequential deregulation of NK cell subset distribution and function starting in acute HIV-1 infection Blood, November 15, 2005; 106(10): 3366 - 3369. [Abstract] [Full Text] [PDF]
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Y. Jiang, H. Shang, Z. Zhang, Y. Diao, D. Dai, W. Geng, M. Zhang, X. Han, Y. Wang, and J. Liu Alterations of Natural Killer Cell and T-Lymphocyte Counts in Adults Infected with Human Immunodeficiency Virus through Blood and Plasma Sold in the Past in China and in Whom Infection Has Progressed Slowly over a Long Period Clin. Vaccine Immunol., November 1, 2005; 12(11): 1275 - 1279. [Abstract] [Full Text] [PDF]
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 D. Mavilio, G. Lombardo, J. Benjamin, D. Kim, D. Follman, E. Marcenaro, M. A. O'Shea, A. Kinter, C. Kovacs, A. Moretta, et al. Characterization of CD56-/CD16+ natural killer (NK) cells: A highly dysfunctional NK subset expanded in HIV-infected viremic individuals PNAS, February 22, 2005; 102(8): 2886 - 2891. [Abstract] [Full Text] [PDF]
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G. Alter, J. M. Malenfant, R. M. Delabre, N. C. Burgett, X. G. Yu, M. Lichterfeld, J. Zaunders, and M. Altfeld Increased Natural Killer Cell Activity in Viremic HIV-1 Infection J. Immunol., October 15, 2004; 173(8): 5305 - 5311. [Abstract] [Full Text] [PDF]
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 A. Kobayashi, R. M. Greenblatt, K. Anastos, H. Minkoff, L. S. Massad, M. Young, A. M. Levine, T. M. Darragh, V. Weinberg, and K. K. Smith-McCune Functional Attributes of Mucosal Immunity in Cervical Intraepithelial Neoplasia and Effects of HIV Infection Cancer Res., September 15, 2004; 64(18): 6766 - 6774. [Abstract] [Full Text] [PDF]
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J. J. Lum, D. J. Schnepple, Z. Nie, J. Sanchez-Dardon, G. L. Mbisa, J. Mihowich, N. Hawley, S. Narayan, J. E. Kim, D. H. Lynch, et al. Differential Effects of Interleukin-7 and Interleukin-15 on NK Cell Anti-Human Immunodeficiency Virus Activity J. Virol., June 1, 2004; 78(11): 6033 - 6042. [Abstract] [Full Text] [PDF]
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J. Xu, R. Lucas, M. Schuchmann, S. Kuhnle, T. Meergans, A. P. Barreiros, A. W. Lohse, G. Otto, and A. Wendel GM-CSF Restores Innate, But Not Adaptive, Immune Responses in Glucocorticoid-Immunosuppressed Human Blood In Vitro J. Immunol., July 15, 2003; 171(2): 938 - 947. [Abstract] [Full Text] [PDF]
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 G. M. Bahr, X. De La Tribonniere, E. Darcissac, F. Ajana, L. Bocket, D. Sissoko, Y. Yazdanpanah, J. Dewulf, C. Amiel, and Y. Mouton Clinical and immunological effects of a 6 week immunotherapy cycle with murabutide in HIV-1 patients with unsuccessful long-term antiretroviral treatment J. Antimicrob. Chemother., June 1, 2003; 51(6): 1377 - 1388. [Abstract] [Full Text] [PDF]
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D. Verthelyi, M. Gursel, R. T. Kenney, J. D. Lifson, S. Liu, J. Mican, and D. M. Klinman CpG Oligodeoxynucleotides Protect Normal and SIV-Infected Macaques from Leishmania Infection J. Immunol., May 1, 2003; 170(9): 4717 - 4723. [Abstract] [Full Text] [PDF]
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G. M. Bahr Non-specific immunotherapy of HIV-1 infection: potential use of the synthetic immunodulator murabutide J. Antimicrob. Chemother., January 1, 2003; 51(1): 5 - 8. [Full Text] [PDF]
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