Received for publication, September 29, 2002, and in revised form, November 5, 2002
CD8+ T-cells are a major source
for the production of non-cytolytic factors that inhibit HIV-1
replication. In order to characterize further these factors, we
analyzed gene expression profiles of activated CD8+ T-cells
using a human cDNA expression array containing 588 human cDNAs.
mRNA for the chemokine I-309 (CCL1), the cytokines
granulocyte-macrophage colony-stimulating factor and interleukin-13,
and natural killer cell enhancing factors (NKEF) -A and -B were
up-regulated in bulk CD8+ T-cells from HIV-1 seropositive
individuals compared with seronegative individuals. Recombinant NKEF-A
and NKEF-B inhibited HIV-1 replication when exogenously added to
acutely infected T-cells at an ID50 (dose inhibiting HIV-1
replication by 50%) of ~130 nM (3 µg/ml). Additionally, inhibition against dual-tropic simian immunodeficiency virus and dual-tropic simian-human immunodeficiency virus was found. T-cells transfected with NKEF-A or NKEF-B cDNA were able to
inhibit 80-98% HIV-1 replication in vitro. Elevated
plasma levels of both NKEF-A and NKEF-B proteins were detected in 23% of HIV-infected non-treated individuals but not in persons treated with
highly active antiviral therapy or uninfected persons. These results indicate that the peroxiredoxin family members NKEF-A and
NKEF-B are up-regulated in activated CD8+ T-cells in HIV
infection, and suggest that these antioxidant proteins contribute to
the antiviral activity of CD8+ T-cells.
 |
INTRODUCTION |
CD8+ T-cells inhibit
HIV-11 replication by both
cytolytic and non-cytolytic mechanisms (1). The importance of
cell-mediated cytotoxic immunity for the partial control of human
immunodeficiency virus type 1 (HIV-1) replication in infected
individuals is now widely recognized (2-10). The direct killing of
virus-infected cells by antigen-specific cytotoxic T-lymphocytes is
considered to be the dominant mechanism of virus suppression.
Nevertheless, chemokines (MIP-1
, MIP-1
, and regulated
on activation normal T-cell
expressed and secreted (RANTES)) produced
by CD8+ T-cells have been shown to inhibit HIV-1
replication in vitro (11, 12) at the level of viral entry
(13, 14) and may play a critical role in vivo as an
antiviral host defense (15, 16). CD8+ T-lymphocytes can
suppress human immunodeficiency virus type I (HIV-1) replication
in vitro by secreting a soluble factor(s) that differs from
the chemokines in the mechanism of inhibition. These factors remain
undefined (17-26) and have been termed CD8+ T-lymphocyte
antiviral factors (CAF). Although the cytotoxic T-lymphocytes response
is major histocompatibility complex class I-restricted, this
restriction does not apply to inhibition of HIV-1 replication by CAF
(17, 25). Moreover, the production of CAF appears to be the property of
stimulated CD8+ T-cells and does not require HIV infection
(19, 20, 26, 27). One site of CAF action is the inhibition of HIV-1 RNA
transcription, particularly at the long terminal repeat (LTR) that is
assumed to function through down-regulation of the NF-
B pathway (19, 21-23). It seems probable that the antiviral action of CAF is achieved by more than one cytokine or chemokine secreted by CD8+
T-cells, perhaps acting in concert (28, 29).
Recently we showed that CAF consists at least two components (26), a
heparin-binding >50-kDa molecule, which we identified as a modified
form of antithrombin (30), and a smaller molecule, possibly the
-defensins (31). In this study, we used a gene array to perform a
more comprehensive analysis of gene expression by CD8+
T-cells from HIV-infected persons. We demonstrate here that the peroxiredoxin family proteins NKEF-A and NKEF-B are up-regulated by
CD8+ T-cells following activation. These proteins
exogenously added or intracellularly expressed have anti-HIV activity
and have been described to increase natural killer cell activity
(32-35), increase cell resistance to oxidative stress (36, 37), and
regulate transcription activator protein (AP-1) (see Ref. 38 and
reviewed in Ref. 39). We show that recombinant NKEF-A and NKEF-B
protein exogenously added to HIV-1 cultures can inhibit HIV-1
replication and that T-cells transfected with NKEF-A or NKEF-B cDNA
were resistant to HIV-1 infection.
 |
EXPERIMENTAL PROCEDURES |
Subjects--
Plasma was obtained from 13 long term
nonprogressors. Control plasma samples were obtained from 13 HIV-1
seronegative, healthy donors. Additional plasma samples from 6 asymptomatic, 5 symptomatic, and 2 AIDS patients, all under highly
active antiviral therapy treatment for progressive disease, were
investigated. Bulk CD8+ T-cells were purified, expanded,
and stimulated as described previously (26) by anti-CD3 cross-linking
from peripheral blood mononuclear cells, which were obtained from six
HIV-1-infected long term non-progessors (17393, 15188, CTS-02, NEW,
RK2000, and CX741) (26) and from seven HIV-1 seronegative individuals.
Bulk CD8+ T-cells for each individual were treated
separately. Long term non-progression was defined as being infected for
more than 10 years, having plasma HIV-1 loads <400 RNA copies per ml,
and CD4+ T-cell counts >500 per µl in the absence of therapy.
Assay for Inhibition of HIV-1 Replication--
For the
inhibition tests, HIV-1IIIB, a T-cell tropic strain of
HIV-1 (ATCC CRL-8543) was used. H9 cells (HLA Al, B6, Bw62, and Cw3)
were acutely infected with HIV-1IIIB at a multiplicity of
infection of 10
2 TCID50/ml and resuspended in
RPMI 1640 (Sigma) supplemented with 20% (v/v) heat-inactivated fetal
calf serum (Sigma; R20 medium) (26). The cells were then plated in 2 ml
of R20 medium at 5 × 105 cells/ml in a 24-well plate.
H9 cell supernatant (1 ml) was removed every 3 days and replaced with 1 ml of fresh R20 medium supplemented with recombinant NKEF-A or NKEF-B
protein. After 9 days the concentration of p24-antigen was measured
using an HIV-1 p24 ELISA kit (PerkinElmer Life Sciences). For the
dual-tropic SIV-239 or dual-tropic SHIVKU-1 studies,
the human T-cell line 174xCEM was infected at a multiplicity of
infection of 10
2 TCID50/ml and resuspended in
R20. The cells were then plated as described above. After 9 days the
concentration of SIV p27-antigen was measured by ELISA (Coulter, Miami,
FL). The medium controls demonstrated p24 or p27 antigen levels in
excess of 100 ng/ml at day 9 and were used to calculate percent virus inhibition.
Transfection with the NKEF-A and NKEF-B Gene--
Jurkat cells
were used for inhibition experiments following transfection with NKEF-A
and NKEF-B. NKEF-A- and NKEF-B-pBacPAK9 vectors (38) were digested with
BamHI and XhoI (New England Biolabs, Beverly,
MA). The digest was treated with T4 polymerase (Invitrogen) for
blunt-end ligation according to the manufacturer's instructions. The
NKEF-A and NKEF-B fragments were then inserted into the SmaI
cloning site of the pIRES2-EGFP expression vector (Clontech, Palo Alto, CA) and cultured in
Escherichia coli. Plasmid DNAs were purified, and the
direction of the inserts was confirmed by DNA sequencing. Jurkat cells
were then transfected with a DNA/liposome mixture (FuGENE, Roche
Diagnostics) and selected under G418 (Sigma) pressure (1.5 mg/ml).
Stable transfected cells were then used in the above described
inhibition test using 1.5 mg/ml G418. For days 1-9 the concentration
of p24 antigen was measured using an HIV-1 p24 ELISA kit (PerkinElmer
Life Sciences). The percentage of inhibition was calculated against a
control with the empty pIRES2-EGFP vector.
Total RNA Extraction and Northern Blot Analysis--
For total
RNA extraction cell pellets of 0 or 4 h, anti-CD3-activated bulk
CD8+ T-cells (107) were lysed with 1 ml of RNA
STAT60 (Tel-Test B, Friendswood, TX), and the cellular RNA was purified
using the RNA STAT60 protocol. To eliminate the DNA contaminant of the
RNA the CleanMessageTM Kit (Genhunter, Nashville, TN) was
used. 10 µg of total cellular RNA was fractionated on a 1.2%
agarose, 0.7% formaldehyde gel and transferred to a GeneScreen
membrane (DuPont). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)-specific DNA probes (Clontech) and
NKEF-A-specific DNA probes (33) were 32P-radiolabeled using
the DECAprimeTM Kit (Ambion, Austin, TX). Membranes were
sequentially hybridized with the 32P-radiolabeled cDNA
probes. Blots were washed at high stringency (0.2× SSC, 55 °C) and
were measured with Molecular PhosphorImager System GS-363 (Bio-Rad) for
an equal amount of time. Signal intensity calculations were performed
using the supporting software program Molecular AnalystTM
and calculated against the GAPDH signal.
RNA Expression Screening with the ATLASTM
Assay--
The ATLAS ArrayTM
(Clontech) is a nitrocellulose membrane with 588 spotted cDNAs. For the hybridization polyadenylated
(poly(A)+) mRNA was prepared from 100 µg of total RNA
of 4 h CD3-cross-linked or untreated bulk CD8+
T-cells. Bulk CD8+ T-cells for each individual were
examined separately. The mRNA was suspended in diethyl
pyrocarbonate-treated water and separated on poly(A) Quik® mRNA
Columns (Stratagene, La Jolla, CA) according to the manufacturer's
protocol. 1 µg (in 2 µl) of each poly(A)+ mRNA
sample was transcribed to radiolabeled cDNA using 1 µl of Moloney
murine leukemia virus-reverse transcriptase (50 units/ul; Stratagene)
and 3.5 µl [
-32P]dATP (3000 Ci/mmol, 10 mCi/ml)
according to the ATLAS assay protocol and used for hybridization. The
binding of radioactivity to membrane was measured with the Molecular
PhosphorImager System GS-363 (Bio-Rad) for an equal amount of time.
Signal intensity calculations were performed using the supporting
software program Molecular AnalystTM and calculated against
the GAPDH signal.
Expression in Sf21 Cells and Purification of NKEF-A and
NKEF-B from Sf21 Cells--
To produce the NKEF-A and NKEF-B
proteins for the above described inhibition test, the NKEF-A and NKEF-B
genes were cloned into the baculovirus expression vector pBacPAK9
(Clontech) and overexpressed in Spodoptera
frugiperda (Sf21 cells; Clontech) as
described (33). After transfections Sf21 cells were
harvested and lysed with insect lysis buffer (Pharmingen) at days 2-4.
Afterward recombinant protein was purified as described earlier
(35).
Characterization of Soluble Factors--
For the NKEF-A and
NKEF-B ELISAs 2 µg/ml of monoclonal mouse anti-NKEF-A or anti-NKEF-B
antibody (Pharmingen) was incubated overnight at 4 °C on protein
high-binding EIA/RIA plates (Costar, Cambridge, MA) in coating buffer
(0.05 M
CO
/HCO
buffer, pH 9.6). Plates were washed with PBST buffer
(phosphate-buffered saline (PBS), 0.05% (v/v) Triton X-100 (Sigma), pH
7.4) and blocked for 2 h at 37 °C with blocking buffer (PBS,
3% (v/v) goat serum, 3% (w/v) bovine serum albumin). Plates were
washed with PBST buffer. Standard protein or samples were incubated for
2 h at 37 °C. Plates were washed with PBST buffer. A rabbit
polyclonal NKEF-A/NKEF-B detection antibody (32), which recognizes both
forms of the NKEFs, was diluted 1:1000 in PBSBT buffer (PBS, 0.1%
(w/v) bovine serum albumin, 0.05% (v/v) Triton X-100) and was added at
37 °C for 30 min. After washing with PBST buffer a horseradish
peroxidase-coupled anti-rabbit antibody (Vector, Burlingame, CA) was
used at 1:50,000 dilution at room temperature for 20 min. After washing
with PBST buffer the ELISA was developed for 30 min at room temperature with a 1:1 dilution of peroxidase solution B and TMB peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and
stopped with 4 N H2SO4. SDS-PAGE
and Western blot was carried out as described previously (26) using
polyclonal NKEF-A- and NKEF-B-specific antibodies (32) at a dilution of
1:10,000. Protein concentration was determined by the BCA method (Pierce).
Statistical Analysis--
Fisher's exact test was used to
determine significance. Standard error is shown as error bars in
the figures.
 |
RESULTS |
Differential Gene Expression in CD8+ T-cells of HIV-1
Seronegative and Seropositive Individuals--
CD8+
T-cells are a major source for inhibitory non-cytolytic factors in
HIV-1-infected persons. We have shown previously (26) that secretion of
soluble antiviral factors is elevated in expanded CD8+
T-cells from HIV-infected persons. In order to assess potential differences in gene expression, which might also be responsible for the
antiviral activity, we evaluated mRNA derived from CD8+
T-cells using the ATLAS array, which contained 588 unique genes. Expanded CD8+ T-cell populations of HIV-1 seronegative and
the HIV-1 seropositive untreated individuals were evaluated prior to
stimulation and 4 h following stimulation with anti-CD3.
Although the ATLAS array includes a wide spectrum of genes, significant
differences were limited to the expression of only four genes. These
included significant differences (p < 0.001) in
mRNA levels for the chemokine I-309, the cytokines GM-CSF and IL-13, and the peroxiredoxin gene NKEF-B (Fig.
1A). The peroxiredoxin data
were confirmed by Northern blot analysis using NKEF-A cDNA, a
homologue of NKEF-B, which showed differences for this peroxiredoxin gene as well (Fig. 1B). Both proteins have antioxidant
function, and a natural killer cell activity was found for the NKEF-A
and NKEF-B complex or for recombinant NKEF-A (reviewed in Ref. 39). These mRNA results are consistent with previous studies showing higher secretion of I-309, GM-CSF, and IL-13 in stimulated
CD8+ T-cells from seropositive persons (26), and they
extend these prior studies by demonstrating the elevation in these
peroxiredoxin family mRNAs in CD8+ T-cells from HIV-1
seropositive persons.

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Fig. 1.
Differential gene expression (p < 0.001) between CD3 cross-linked CD8+ T-cells of
seronegative individuals and HIV-1-untreated patients using
the Atlas ArrayTM
(A) and Northern blot analysis of the NKEF-A
transcription (B) measured against GAPDH.
A, the ATLAS ArrayTM
(Clontech) used is a nitrocellulose membrane with
588 spotted cDNAs (see "Experimental Procedures"). Standard
errors for four independent experiments are shown, but some are too
small to show. B, Northern blot analysis of NKEF-A gene
expression shows a significantly higher expression before and 4 h
after anti-CD3 stimulation in CD8+ T-cells of
HIV-1-infected but untreated patients compared with CD8+
T-cells of seronegative individuals. Standard errors for three
independent experiments are shown with CD8+ T-cells of
three different individuals examined separately.
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Inhibition of HIV-1 Replication with Recombinant NKEF-A and NKEF-B
Protein--
By having demonstrated that GM-CSF, I-309, IL-13, and the
NKEFs were preferentially expressed in CD3-activated, HIV-1-infected but untreated individuals, we next evaluated whether some of these gene
products might contribute antiviral activity. Although GM-CSF, I-309,
and IL-13 have been shown to influence HIV-1 replication in some
in vitro systems (40), we found that these proteins did not
inhibit HIV-1IIIB replication in acutely infected T-cells when added up to 1 µg/ml, demonstrating that they do not contribute to the observed inhibition (see Ref. 26 and data not shown). In
contrast, we found that recombinant NKEF-A and NKEF-B proteins that we
expressed in Sf21 cells using a baculovirus expression system
and purified to homogeneity (Fig.
2A) inhibited
HIV-1IIIB replication at an ID50 of 130 nM (3 µg/ml), respectively (Fig. 2B).
Additionally, using the NKEF-B protein for the inhibition assay with
dual-tropic SHIV and dual-tropic SIV strains, we found nearly complete
suppression of these viruses at 3 µg/ml (Fig. 2, C and
D). Total protein lysate of untransfected Sf21 cells was used as a control and demonstrated no inhibition, excluding contaminating Sf21 protein as being responsible for inhibition (data not shown). The observed inhibition did not correlate with a
decrease in cell count as measured at log phase of cell growth from day
2 to 6 by trypan blue staining (data not shown). Our data indicate that
recombinant NKEFs can inhibit replication of X4 HIV-1, dual-tropic SIV,
and dual-tropic SHIV.

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Fig. 2.
SDS-PAGE of purified NKEF-A and NKEF-B
(A), NKEF-A and NKEF-B protein inhibition of T-cell
tropic HIV-1 replication (B), and inhibition of
dual-tropic SIV and dual-tropic SHIV strains (C and
D). A, Coomassie-stained SDS-PAGE
with NKEF-A (lane 1) and NKEF-B (lane 2) and
Western blot of NKEF-A (lane 3) and NKEF-B (lane
4) using the polyclonal NKEF-A/NKEF-B antibody. B,
dose-response curve of NKEF-A and NKEF-B. At a concentration of 1 µg/ml, NKEF-A appeared to inhibit HIV-1 replication in H9
CD4+ T-cells to a greater degree than NKEF-B. However, at
10 µg/ml or above, both NKEF-A and NKEF-B nearly eliminated HIV-1
infectivity (> 95% inhibition). C and D,
p27 protein measurements of SIV and SHIV after NKEF-B treatment in
three independent experiments after 9 days. Virus antigens in controls
were above 100 ng/ml.
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Secretion of NKEF-A and NKEF-B Proteins by CD8+
T-cells--
By having demonstrated that the NKEFs can inhibit HIV-1
replication, we next tested whether the NKEFs are secreted or released from stimulated CD8+ T-cells, and whether these proteins
contribute to the antiviral activity of these cells (26). Although
NKEF-A and NKEF-B proteins were originally described as endogenous
proteins (33, 37, 41), thioredoxin (also termed adult T-cell
leukemia-derived factor), another protein that serves as the electron
donor for most peroxiredoxins (reviewed in Ref. 42), has been shown to be secreted through a novel pathway despite having no signal sequence (43). A similar observation has been confirmed for peroxiredoxin IV
(44). We found that both NKEF-A and NKEF-B proteins were secreted after
4 h, regardless of whether the cells were stimulated. The
concentrations produced averaged 15-40 ng/ml (Fig.
3) or were at least 10-20 times higher
than seen for the chemokines and cytokines at this 4-h time point (26)
and were up to ~125 ng/ml at 16 h. The secretion was observed in
stimulated CD8+ T-cells from both infected and uninfected
individuals (Fig. 3). Thus, despite higher levels of NKEF mRNA in
cells from seropositive persons, supernatants of activated
CD8+ T-cells from both infected and uninfected individuals
contained comparable amounts of these proteins, and this might be the
result of the missing active protein secretion. This indicates that
although NKEFs are able to exert antiviral activity, they are not the
elusive CAF, which is produced in greater amounts from CD8+
T-cells of infected persons (26). Additionally, the concentrations of
secreted NKEFs at 4 h (15-40 ng/ml) are at levels below those causing significant inhibition (Fig. 2) at a time when significant inhibition by HIV-1-suppressive factor(s) was observed (26).

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Fig. 3.
Secretion profile of NKEF-A
(A) and NKEF-B (B) by
CD8+ T-cells of seropositive individuals. NKEF-A and
NKEF-B protein concentrations were measured by a specific ELISA without
(0 h) or with (4 and 16 h) anti-CD3 stimulation. Time 0 denotes
4-h supernatants without anti-CD3 stimulation. Medium was used as
control where no NKEFs were detectable. Standard errors for three
independent experiments are shown.
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Blood Plasma Levels of the NKEF-A and NKEF-B Proteins--
By
having demonstrated that the NKEFs are secreted, we next tested blood
plasma levels by a specific ELISA to evaluate if plasma concentrations
were sufficient to mediate inhibition of HIV-1 replication in
vivo. Additionally, we tested if blood plasma levels are dependent
on whether individuals are infected or not and treated or untreated. We
could not detect a significant difference in NKEF-A or NKEF-B levels in
plasma among the uninfected and the long term nonprogressor population.
Nevertheless, plasma levels of the NKEFs were elevated (up to 500 ng/ml) in 3 of 13 (~23%) HIV-1-infected but untreated persons
tested, with levels 2.5-8 times higher than the uninfected or treated
HIV patients (Fig. 4). For HIV-1-infected
but untreated persons NKEF-A and NKEF-B were found at levels up to 1 µg/ml. At this concentration HIV-1 inhibition was detectable in
vitro (Fig. 2B), and an increase of natural killer cell
activity in vitro has been noted (35), demonstrating that
NKEFs might have an antiviral influence in vivo. We
demonstrated that blood plasma of long term non-progressors have
significantly more NKEF-B compared with blood plasma of asymptomatic and symptomatic patients (Fig. 4). Our data indicate that elevated levels of NKEFs are seen in a small percentage of untreated
HIV-1-infected individuals and might be the result of a more active
CD8+ T-cell response. It was shown earlier that NKEF genes
up-regulated in a Th1 response (45) correlate with a more positive
disease outcome (46), showing an expansion of the TNF system, probably through an activation of the cytotoxic compartment (47).

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Fig. 4.
Box plots showing the 10th, 25th, 50th
(median), 75th, and 90th percentiles, of NKEF-A and NKEF-B plasma
concentrations of seronegative individuals, HIV-infected but untreated,
and highly active antiviral therapy-treated
asymptomatic, symptomatic, and AIDS patients.
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Inhibition of HIV-1 Replication through NKEF-A and NKEF-B
Transfection--
Another way to demonstrate inhibitory activity of
the NKEFs is to transfect the gene into target cells and determine
resistance of these transfected cells to HIV-1. This has been done for
peroxiredoxin IV, demonstrating resistance against HIV-1 replication
(48). We therefore transfected Jurkat CD4+ T-cells with
NKEF-A or NKEF-B expression plasmids, which increased intracellular
NKEF levels ~10-fold (Fig.
5A). These NKEF-A- and NKEF-B-transfected T-cells blocked 80-98% HIV-1IIIB
replication starting at day 6 post-infection, when compared with the
mock-transfected cells (Fig. 5B). Our data indicate that
enhanced expression of NKEFs in T-cells might inhibit HIV-1
replication, providing further evidence of the antiviral effect of
these compounds.

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Fig. 5.
NKEF-A and NKEF-B protein production by
transfected T-cells (A) and T-cell protection through
NKEF-A and NKEF-B gene transfection (B).
A, NKEF-A and NKEF-B protein expression of 2 × 105 mock-transfected (lane 1), NKEF-A
(lane 2), and NKEF-B-transfected (lane 3) Jurkat
cells compared with a Western blot with the polyclonal NKEF-A/NKEF-B
antibody that is recognizing both forms of the NKEFs.
B, NKEF-A- and NKEF-B-transfected Jurkat cells were
tested for p24 antigen, and percentage suppression was measured against
the mock-transfected T-cells. Virus antigen of mock control was above
100 ng/ml after day 8. Standard errors of three independent experiments
are shown.
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 |
DISCUSSION |
CD8+ T-cells are a major source for inhibitory
non-cytolytic factors. There are only few studies that compare gene
expression between bulk CD8+ T-cells of seropositive and
bulk CD8+ T-cells of seronegative individuals. Overall, a
Th1 cytokine response is noted as an implication for long term survival
(46). Another report shows that the expansion of the TNF system
positively correlated with higher expression of TNF-
,
interferon-
, and activation marker secretion of CD8+,
probably presenting the activation of the cytotoxic compartment (47).
Here we report that the NKEF-A and NKEF-B genes are expressed in
greater quantity in bulk CD8+ T-cells of HIV-1-infected
individuals compared with the bulk CD8+ T-cells of
seronegative individuals (Fig. 1). Furthermore, we show that these
genes, normally up-regulated through oxidative stress (49), can be
up-regulated by anti-CD3 stimulus in T-cells (Fig. 1). Additionally, we
show that these proteins are secreted (Fig. 2). They can inhibit HIV-1,
SIV, and SHIV (Fig. 3). They are present in elevated amounts in the
plasma of some but not all infected individuals in a way that blood
plasma from long term non-progressors have significantly more NKEF-B
than plasma of asymptomatic and symptomatic individuals (Fig. 4).
In vitro inhibition tests showed that HIV replication was
decreased when the NKEFs proteins were overexpressed in
CD4+ T-cells (Fig. 5).
It was shown earlier that some peroxiredoxins, e.g.
peroxiredoxin IV and its electron donor thioredoxin (42), have
antiviral activity against HIV-1, through decreasing free radical
concentrations with their antioxidant enzyme function (48, 50, 51) and down-regulating the NF-
B pathway, thereby blocking HIV-1 LTR transcription, a mechanism described for CAF (18, 23). For both NKEF-A
and NKEF-B, previously discovered as natural killer cell activity
enhancing factors, antioxidant functions were demonstrated (39).
Therefore, we hypothesize a similar mechanism for these proteins.
Our data indicate that NKEF-A and NKEF-B are secreted by
CD8+ T-cells at concentrations at least 10-20 times higher
than found for the chemokines and cytokines 4 h after anti-CD3
stimulation (26). Secretion was observed regardless of whether the
cells were stimulated or not or whether they originated from infected or uninfected individuals. These findings exclude the NKEFs from being
the elusive CAF for which enhanced secretion is found for CD8+ T-cells of HIV-infected individuals. Furthermore, the
concentrations released averaging 15-40 ng/ml at 4 h and ~125
ng/ml at 16 h (Fig. 3) are at levels below those suppressive
levels (range, 50-92%) described earlier (26) causing significant
inhibition (Fig. 2). CD8+ T-cells having a higher gene
expression level, but the same secretion level between infected and
uninfected individuals, can be a result of the fact that the NKEFs are
not secreted actively (43). Therefore, it is likely that most of the
protein might stay in the cytoplasm, and the differences in gene
expression may not be high enough to make a significant difference in
secretion over the period measured.
Our data indicate that NKEF-A and NKEF-B are elevated in plasma of some
but not all HIV-1-infected but untreated individuals. It was already
reported that HIV affects antioxidant factors through various
mechanisms, and a relationship between thioredoxin levels and the HIV-1
disease progression was already demonstrated (51). Additionally, it was
demonstrated that Th1 leukocytes, whose frequency is correlated with a
more favorable disease outcome (46), express higher levels of NKEFs
(45). Furthermore, elevated levels of thioredoxin correlate with a more
favorable disease outcome as long as they were not higher than 30 ng/ml
in AIDS patients with CD4+ T-cells counts below 200/µl
(52). It was already shown that restoring antioxidant capacity
paralleled immunologic and virologic improvements (53-55). The HIV Tat
protein was able to suppress antioxidant factor expression (56, 57).
Ongoing oxidative stress resulting from HIV infection stimulated
Fas-induced CD4+ T-lymphocyte apoptosis and mediated a Tat-
and gp160-induced functional impairment of uninfected T-lymphocytes and
enhanced NF-
B-dependent activation of virus
transcription (56, 58-60). We found no significant differences between
NKEF-A and NKEF-B plasma levels of seronegative and seropositive
treated and combined seropositive untreated individuals (Fig. 4).
Nevertheless, some untreated patients (3 of 13) have levels 2.5-8
times elevated than normal or found in seropositive treated
individuals. The plasma levels were up to 500-1000 ng/ml for NKEF-A
and NKEF-B when calculated together (Fig. 4), levels sufficient enough
to show in vitro inhibition (Fig. 2) or increasing in
vitro natural killer cell activity (35). The significant
differences found between the long term non-progressors and
asymptomatic and symptomatic patients might be the result of a higher
cytotoxic activity of the CD8+ T-cells of the long term
non-progressors. Because the amount of protein released is independent
from a secretion process, a higher rate of killing during the cytotoxic
response might be the reason for the higher amount of protein in the plasma.
Our data also indicate that T-cells overexpressing NKEF-A or NKEF-B
inhibited HIV-1 replication (Fig. 5, C and D). It
has been shown previously that overexpressing peroxiredoxin in T-cells inhibits HIV-1 transcription through inactivation of the
NF-
B-dependent initiation of HIV-1 LTR replication (48).
Additionally, this might be because of an inhibition of the AP-1
transcription factor-dependent HIV-1 replication, because
it was already shown that overexpression of NKEF-B can block the
TNF-
activation of AP-1 (38).
In summary, we report that NKEF-A and NKEF-B, members of the
peroxiredoxin gene family are released by activated CD8+
T-cells and are capable of inhibiting HIV-1 replication. Although chemokine-like features are described for this protein family, including heparin binding (44) and chemotaxis (61), the antiviral mechanism of NKEF-A and NKEF-B is likely to be very different from the
chemokines thus making their potential as a synergistic therapeutic
agent or a marker for disease progression attractive and warranting
further investigation.
We thank Charles Shih (Pharmingen) for
providing the monoclonal NKEF-A and NKEF-B antibodies and Paul Johnson
(New England Primate Center and Harvard Medical School) for providing
the T-cell line 174xCEM and the SIV239 strain. The
SHIVKU-1 reagent was obtained from Dr. Opendra Narayan and
Dr. Sanjay Joag of the AIDS Research and Reference Program, Division of
AIDS, NIADS, National Institutes of Health.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M209964200
The abbreviations used are:
HIV-1, human
immunodeficiency virus, type 1;
NKEF, natural killer cell enhancing
factor;
I-309, interleukin-309;
GM-CSF, granulocyte-macrophage
colony-stimulating factor;
IL-13, interleukin-13;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PBS, phosphate-buffered
saline;
LTR, long terminal repeat;
CAF, CD8+ T-lymphocyte
antiviral factors;
ELISA, enzyme-linked immunosorbent assay;
SIV, simian immunodeficiency virus;
SHIV, simian-human immunodeficiency
virus;
TNF, tumor necrosis factor.
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