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Journal of Virology, December 1998, p. 9441-9452, Vol. 72, No. 12
Department of Microbiology & Immunology,1
Department of
Pediatrics,3
UCLA AIDS
Institute,4 and
Jonsson
Comprehensive Cancer Center,5
UCLA
School of Medicine, Los Angeles, and The Scripps Research
Institute, La Jolla,2 California
Received 1 June 1998/Accepted 24 August 1998
Human thymocytes are readily infected with human immunodeficiency
virus type 1 (HIV-1) in vivo and in vitro. In this study, we found that
the kinetics of replication and cytopathic effects of two molecular
isolates, NL4-3 and JR-CSF, in postnatal thymocytes are best explained
by the distribution of chemokine receptors used for viral entry. CXCR4
was expressed at high levels on most thymocytes, whereas CCR5
expression was restricted to only 0.1 to 2% of thymocytes. The
difference in the amount of proviral DNA detected after infection of
fresh thymocytes with NL4-3 or JR-CSF correlated with the levels of
CXCR4 and CCR5 surface expression. Anti-CCR5 blocking studies showed
that low levels of CCR5 were necessary and sufficient for JR-CSF entry
in thymocytes. Interleukin-2 (IL-2), IL-4, and IL-7, cytokines normally
present in the thymus, influenced the expression of CXCR4 and CCR5
on thymocytes and thus increased the infectivity and spread of both
NL4-3 and JR-CSF in culture. NL4-3 was produced by both immature and
mature thymocytes, whereas JR-CSF production was restricted to the
mature CD1 Human immunodeficiency virus (HIV)
infection of the thymus leads to loss of thymocytes and eventual thymic
atrophy (8, 29, 50, 53). While the role of the thymus in
regeneration of the immune system of HIV-infected adults has not been
established, the thymus is required for T-cell generation in children
(18, 39). Therefore, HIV infection of thymocytes and thymic
emigrants may have an impact on disease progression in children. We and others have previously shown that NL4-3, a molecularly cloned highly
cytopathic CXCR4-tropic virus, as well as certain pediatric HIV type 1 (HIV-1) isolates, are able to replicate in immature and mature
thymocyte subsets, while JR-CSF, a relatively noncytopathic CCR5-tropic
isolate, and selected pediatric isolates have a more restricted tropism
for mature thymocyte subsets (27, 33, 64, 71, 73a). In
addition, interleukin-2 (IL-2), IL-4, and IL-7, cytokines implicated in
thymic subset expansion and maturation, have distinct effects on HIV-1
replication (69, 70, 72, 73, 78). NL4-3 and some pediatric
isolates from rapid disease progressors replicated faster in the
presence of IL-4 plus IL-7 than in the presence of IL-2 plus IL-4. In
contrast, JR-CSF and isolates obtained from pediatric patients with a
slow disease progression replicated faster in the presence of IL-2 plus
IL-4 (20, 71, 72, 73a).
Surface expression of CD4 and of specific chemokine coreceptors allows
HIV-1 entry into cells (13, 15, 17, 21). HIV-1 primary
isolates can use CXCR4, CCR5, both receptors (dualtropic isolates), or
a number of other reported seven-transmembrane, G-protein-coupled
chemokine receptors (3-5, 12, 14, 21, 34, 36, 60, 80). In
adults, the critical role of CCR5 in transmission and disease
progression has been suggested by genetic studies correlating
resistance or delay of HIV-1 infection with the presence of CCR5
mutations that result in no or low expression of CCR5 (25, 37, 55,
61). In children, the role of CCR5 in transmission and disease
progression has been assessed in a cross-sectional study of children
born to mothers seropositive for HIV-1. Heterozygosity for CCR5 In the thymus, where CD4 is expressed on more than 95% of the cells,
the distribution of HIV coreceptors would be expected to be an
important determinant of tropism. Wide distribution of CXCR4 surface
expression on fetal thymocytes has been recently reported
(31), while expression of the coreceptors CCR5, CCR8, and
STLR-33/GPR15 on total thymocytes has been reported at the mRNA level
(36, 46, 51, 54, 68). Other chemokine receptors, such as
CCR4 (49), not yet identified as HIV coreceptors, are also present in the thymus. Finally, three unique thymic orphan chemokines, macrophage-derived cytokine (MDC), thymus- and
activation-regulated cytokine (TARC), and thymus-expressed cytokine
(TECK), whose as yet unidentified receptors could potentially
support HIV-1 entry have been detected (19, 24, 77).
After viral entry, the activation state of the target cell determine
its ability to reverse transcribe, integrate, and support HIV
replication (63, 65, 82). In peripheral blood mononuclear cells (PBMC), for example, full reverse transcription requires at least
progression to the G1b phase of the cell cycle and
therefore is dependent on the activation state of the cell (32,
82). Thymocytes are a heterogeneous population of cells in terms
of differentiation and activation. In this study, we examined HIV replication in thymocyte subsets defined by the expression of surface
molecules that are commonly used as markers of T-cell development: CD1,
CD69, and CD45RA. The CD1 molecule is expressed at high levels in
CD3 We took advantage of the differential tropism of JR-CSF and NL4-3 for
thymocyte subsets to study the distribution and the usage of CCR5 and
CXCR4 as HIV coreceptors on freshly isolated postnatal thymocytes. The
chemokine receptors CCR5 and CXCR4 have been reported as coreceptors
for JR-CSF and NL4-3, respectively, in PBMC and transfected cell lines
(17, 60, 80). We found that postnatal thymocytes expressed
high levels of CXCR4 and low levels of CCR5. In postnatal thymocytes,
CXCR4 was broadly distributed on immature and mature subsets, as
previously reported for fetal thymocytes (31). Nevertheless,
CCR5 expression on a low percentage of thymocytes is necessary and
sufficient to support replication of a CCR5-tropic isolate. We also
demonstrate that both CXCR4 and CCR5 support viral entry into
CD69+ and CD69 Reagents and monoclonal antibodies.
Recombinant human IL-2
(1.5 × 106 U/ml) and IL-4 (0.7 mg/ml) were
provided by Amgen, Inc. (Thousand Oaks, Calif.). Recombinant human IL-7
(100 µg/ml) was a gift from Immunex Corp. (Seattle, Wash.).
7-Amino-actinomycin D (7-AAD) was obtained from Sigma (St. Louis, Mo.).
Actinomycin D (AD) was obtained from Boehringer Mannheim (Indianapolis,
Ind.). Normal mouse immunoglobulin G (IgG; 3 mg/ml) was obtained
from Caltag (Burlingame, Calif.). Monoclonal antibodies to CD8, CD4,
CD3, CD45RA, and CD69 conjugated with fluorescein (FITC), phycoerythrin
(PE), or peridinin chlorophyll protein (PerCP) and goat anti-mouse
IgG-FITC were obtained from Becton Dickinson Immunocytometry Systems
(BDIS; San Jose, Calif.). The antibodies KC57-FITC and KC57-PE, which
identify intracellular HIV p24gag antigen
expression (10, 40), CD1-PE, CD45RA-PE, and the unconjugated antibodies CD45RA and CD69, used for thymocyte subset separations, were
obtained from Coulter/Immunotech (Hialeah, Fla.). Unconjugated CXCR4
and CXCR4-PE (12G5) were obtained from Pharmingen (San Diego, Calif.).
Unconjugated monoclonal antibodies to the chemokine receptors CCR-3
(7B11) (21, 23) and CCR-5 (2D7) (79) were
obtained through the AIDS Research and Reference Reagent Program,
Division of AIDS, National Institute of Allergy and Infectious
Diseases, National Institutes of Health. CXCR-4 (12G5) was a gift from
James Hoxie (16). The monoclonal antibody to CCR-5 (3A9) was
a gift from LeukoSite, Inc. (80). CD4-IgG was a gift from
Genentech (San Francisco, Calif.).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Differential Tropism and Replication Kinetics of Human
Immunodeficiency Virus Type 1 Isolates in Thymocytes: Coreceptor
Expression Allows Viral Entry, but Productive Infection of Distinct
Subsets Is Determined at the Postentry Level
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/CD69+ population. Although CXCR4
and CCR5 distribution readily explained viral entry in mature
CD69+ and immature CD69 cells, and correlated
with proviral DNA distribution, we found that viral production was
favored in CD69+ cells. Therefore, while expression of
CD4 and appropriate coreceptors are essential
determinants of viral entry, factors related to activation and
stage-specific maturation contribute to HIV-1 replication in
thymocyte subsets. These results have direct implications for HIV-1
pathogenesis in pediatric patients.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
32
was not associated with transmission but was associated with a slower
development of HIV-related disease in children (42).
Consistent with reports of studies of HIV-1-infected adults
(12), viral isolates obtained from children at early disease
stages were CCR5 tropic, while those from later stages of disease used
CXCR4 as a coreceptor (56). Early acquisition of CXCR4 usage
by these viral isolates was associated with rapid disease progression
(12, 56).
/low thymocytes and therefore identifies immature
thymocytes (7). Downregulation of CD1 correlates with
acquisition of functional maturation of thymocytes (52).
During the process of positive selection, the activation marker CD69 is
expressed on 10% of CD4+/CD8+ double-positive
thymocytes and at high levels on mature single-positive CD4+ and CD8+ cells (67). However,
CD69 expression is absent on thymocytes that emigrate from the thymus
to the periphery (52, 76). By contrast, the CD45RA antigen,
a marker of naive cells in the periphery, is expressed only on mature
CD3+/high/CD1 thymocytes that are ready to
leave the thymus (66).
cells, whereas only the
CD69+ thymocyte subset sustained a highly productive
infection. These results help explain the reported HIV-1-induced
pathogenesis of the thymus by distinct HIV-1 tropic isolates.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
HIV infection and thymocyte cultures. Normal pediatric thymuses were obtained in the course of corrective cardiac surgery. Single-cell suspensions and nylon wool purification were done as previously described, and thymocytes were cultured at 1 × 107 to 2 × 107 cells/ml in serum-free medium (albumin-transferrin-IMDM [Iscove's modified Dulbecco's medium]; Irvine Scientific, Santa Ana, Calif.) supplemented with delipidated bovine serum albumin (BSA; Sigma) at 1,100 µg/ml, transferrin (Sigma) at 85 µg/ml, glutamine at 2 mM (0.3 mg/ml), and penicillin-streptomycin at 25 U/ml-25 µg/ml (73, 78). Thymocytes were cultured in the presence or absence of the cytokines IL-2 (20 U/ml), IL-4 (20 ng/ml), and IL-7 (200 U/ml).
Two molecular clones of HIV-1, the non-syncytium-inducing, CCR5-tropic clone JR-CSF (33) and the syncytium-inducing, CXCR-4-tropic hybrid clone NL-4-3, were used for these studies (1). Virus stocks of JR-CSF were prepared from 24-h harvests of supernatants from PBMC infected with the supernatant of COS cells electroporated with plasmid pYKJR-CSF. Virus stocks of NL4-3 were prepared from 24-h harvests of supernatants from CEM cells (CCRF-CEM) infected with the supernatant of COS cells electroporated with plasmid pNL4-3. Virus stocks were stored at
70°C and treated with DNase (2 µg/ml;
Worthington, Lakewood, N.J.) for 30 min at room temperature in the
presence of 0.01 M MgCl2 before infections.
Heat-inactivated controls were obtained by incubating DNase-treated
viruses at 65°C for 45 min. All infections were standardized by
determining infectious units (IU) in limiting dilution studies using
phytohemagglutinin (PHA)-stimulated PBMC (81, 82). For
thymocyte infections, JR-CSF was used at 10- to 20-fold higher
multiplicity of infection (MOI) than NL4-3 unless otherwise indicated.
Thymocytes were infected and cultured as previously described
(72). Briefly, virus infection was accomplished by
incubating thymocytes with 30 to 200 ng of viral p24/107
cells in the presence of Polybrene (10 µg/ml; Sigma) for 1 to 2 h at 37°C. Control thymocytes were sham infected in the
presence of Polybrene with supernatant from uninfected cells that were used for preparing the virus stocks. After infection, the cells were
washed extensively in A-IMDM and resuspended in serum-free medium in
the presence of cytokines. On day 1 postinfection and weekly
thereafter, the supernatant was removed and the cells were fed with
fresh medium and cytokines. Virus expression was assessed by measuring
p24 antigen in the supernatant by enzyme-linked immunosorbent assay
(Coulter, Hialeah, Fla.).
Blocking studies using antibodies to chemokine receptors. Thymocytes were preincubated with antibodies to CCR5 (2D7; 1 to 5 µg/107 cells) and/or CXCR4 (12G5; 5 to 10 µg/107 cells) or CD4-IgG (100 µg/107 cells) at 4°C for 1 to 2 h before infection. The antibodies and CD4-IgG were present during infection and throughout the duration of the experiment. On day 1 and weekly thereafter, the medium was removed and fresh medium containing the antibody was added, while CD4-IgG was added on days 1 and 7 only.
Isolation of thymocyte subsets.
Magnetic beads were used to
isolate thymocyte subsets. In initial experiments, magnetic beads
coated with goat anti-mouse IgG-plus-IgM antibody (Kirkegaard & Perry,
Gaithersburg, Md.) were used (73, 78). Since the Kirkegaard
& Perry beads are no longer available, Dynal (Lake Success, N.Y.) M280
magnetic beads coated with sheep anti-mouse IgG were used in later
experiments. Comparisons showed that subset purities were similar in
assays using the beads from the two manufacturers. CD45RA- and
CD69-positive and -negative subsets were obtained as follows. Magnetic
beads were preincubated at 108 beads/ml in A-IMDM
containing 1% BSA to prevent nonspecific binding to thymocytes and
then coated with the CD45RA or CD69 monoclonal antibody (1.25 tests of
the antibody as determined by the manufacturer/108
beads/ml) for at least 18 h at 4°C. Beads were washed once to remove excess unbound antibody immediately before use. For depletion of
CD45RA+ cells, thymocytes were combined with CD45RA-coated
beads at a bead-to-cell ratio of 1:2 and rotated at 4°C for 1 h.
Cells bound to beads were removed with a magnet (Collaborative
Research, Inc., Bedford, Mass.) and subjected to a second round of
depletion. The CD45RA-depleted cells were then combined with the
CD69-coated beads at a bead-to-cell ratio of 2:1 and rotated at 4°C
for 1 h. The CD45RA-depleted cells bound to CD69-coated beads
(CD69+ population) were magnetically removed, and unbound
cells (CD69 population) were subjected to a second round
of depletion. Following separation, the depleted subsets were
immunophenotyped and analyzed by flow cytometry. Both positively and
negatively immunoselected subsets were used for infection and culture
experiments. Their viability, as determined by trypan blue dye
exclusion, was >96%.
Immunofluorescent staining and flow cytometry. Surface and cytoplasmic immunophenotyping of thymocytes with directly conjugated antibodies were done as previously described (57, 58). When unconjugated antibodies were used, cells were washed in phosphate-buffered saline (PBS) containing 1% BSA (PBS-BSA). After blocking with 50 µl of human AB serum to prevent nonspecific protein binding, thymocytes (1 × 105 to 5 × 105) were incubated with optimal amounts of unconjugated monoclonal antibody for 20 min at 4°C in a total volume of 100 µl and then washed with 3 ml of PBS-BSA. Goat anti-mouse IgG-FITC antibody was added for 20 min at 4°C in the presence of 50 µl of human AB serum. Cells were washed with 3 ml of PBS-BSA and incubated for 10 min at 4°C with 50 µl of mouse IgG (3 mg/ml) diluted 1:15 in PBS-BSA to prevent nonspecific protein binding before incubation with directly conjugated PE- or PerCP-labeled antibodies for 20 min at 4°C. To exclude dead cells, the thymocytes were incubated in a solution of 2 µg of 7-AAD per ml in PBS for 20 min at 4°C protected from the light. The cells were washed in PBS and incubated in 1% paraformaldehyde solution in PBS containing 4 µg of AD per ml (57, 59). The samples were subjected to flow cytometric analysis in the paraformaldehyde-AD solution.
A FACScan flow cytometer equipped with a standard filter setup (BDIS) was used in these experiments. A minimum of 10,000 events was acquired on each sample. Multiparameter data acquisition and analysis were performed with Cell Quest software (BDIS).Quantitative DNA PCR.
At 16 to 20 h postinfection,
106 thymocytes were removed from the cultures, washed once
in PBS, lysed in urea lysis buffer (4.7 M urea, 1.3% [wt/vol] sodium
dodecyl sulfate, 0.23 M NaCl, 0.67 mM EDTA [pH 8.0], 6.7 mM
Tris-HCl), and then subjected to multiple phenol-chloroform extractions
and ethanol precipitation. Total nucleic acids obtained from thymocytes
were subjected to quantitative DNA PCR as described previously (2,
81, 82). HIV DNA was detected by using the
32P-end-labeled M667-AA55 primer pair specific for the R/U5
region of the viral long terminal repeat (LTR) (81, 82). For
detection of full-length reverse transcripts, the M667-M661 primer pair specific for the LTR/gag region was used (32,
82). Products obtained after 25 cycles of amplification were
resolved on a 6% polyacrylamide gel. Standard curves for HIV-1 DNA
were generated by using various dilutions of plasmid pYKJR-CSF
linearized with EcoRI, which does not digest viral
sequences. The dilutions were made into DNA from normal human PBMC (10 µg/ml). To normalize for cellular DNA, replicate samples were
analyzed for human 
-globin gene sequences (35, 81) by 25 cycles of amplification. Standard curves for human DNA were generated
from two- and fivefold dilutions of PBMC DNA. Values were obtained by
interpolation from the standard curves, using a radioanalytic imaging
system (Ambis, San Diego, Calif.).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cell surface expression of CCR5, CCR3, and CXCR4 on thymocytes from
children.
Postnatal thymus specimens obtained from 18 children
(both sexes, 15 days to 4 years old) were used for these studies.
Freshly isolated thymocytes were immunophenotyped with antibodies to
CCR5 (2D7 and/or 3A9) and CXCR4 (12G5) to determine the thymic
distribution of chemokine receptors that are reportedly the coreceptors
for JR-CSF and NL4-3, respectively, in transfected CD4+
cells and PBMC (60, 80). In all specimens analyzed, more than 95% of postnatal thymocytes expressed CXCR4, while the
percentages of CCR5+ cells ranged from 0.2 to 1%
(mean ± standard deviation = 0.45% ± 0.22%). A
representative experiment is shown in Fig.
1. The same coreceptor expression profile
was found in thymocyte single-cell suspensions before and after nylon
wool purification to enrich for T cells (data not shown). Figure 1
shows that high levels of CXCR4 expression were found in the immature
CD3 and CD3+/low subsets, while the mature
CD3+/high subset contained CXCR4+ and
CXCR4 cells, as previously reported for fetal thymocytes
(31). The determination of CCR5 expression on distinct
thymocyte subsets by immunofluorescence methods was hampered by the low
numbers of CCR5+ thymocytes. CCR3 surface expression was
not detectable with antibody 7B11 in any of the eight thymocyte samples
tested (not shown).
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Cytokines that favor HIV production by thymocytes upregulate CCR5 and CXCR4 surface expression. Expression of CCR5 and CXCR4 is tightly regulated on PBMC by stimulatory signals, mitogens, and cytokines such as IL-2 and IL-10 (6, 9, 38, 44, 45, 62, 80). We have previously shown that cytokines involved in thymocyte maturation distinctly regulate the expression of JR-CSF and NL4-3 in thymocyte subsets in vitro (72). To investigate the effect of these cytokines on chemokine receptor expression, cells were immunophenotyped at day 0 and cultured in serum-free medium in the presence of IL-2, IL-4, IL-7, IL-2 plus IL-4, or IL-4 plus IL-7 for 2 weeks. These cytokines affect proliferation and differentiation of different subpopulations, which results in different proportions of cells expressing high levels of CD3 (i.e., a CD3+/high population) (72, 73, 78). Cell surface phenotype was determined weekly, and the cursors were set in order to analyze coreceptor expression in the CD3+/high population (Fig. 2; Table 1).
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cells (Fig. 2A).
IL-4 alone was sufficient to increase the levels of CXCR4 expression
(Table 1). Interestingly, IL-4 increased the expression of CXCR4 in the
mature CD3+/high population that expresses low levels of
CXCR4 in freshly isolated thymocytes. In thymocytes cultured with IL-2
alone, there was a threefold increase in the fluorescence intensity and
in the percentage of CXCR4+/CD3+/high cells
(Table 1), but the mature CD3+/high population that did not
express CXCR4 was also expanded (data not shown). Thymocytes cultured
with IL-7 showed this same profile (Table 1).
In contrast, the percentage of cells expressing CCR5 increased after 2 weeks of culture with IL-2 plus IL-4 but not in the presence of IL-4
plus IL-7 (Fig. 2B). In six of seven thymocyte culture experiments,
IL-2 and IL-4 synergistically increased the percentages of
CCR5-expressing cells from 0.2 to 1% on day 0 to 1 to 6% after 2 weeks of culture. No effect of IL-4 or IL-7 alone on CCR5 expression
was seen, whereas upregulation of CCR5 expression in the
CD3+/high by IL-2 alone was observed in only one of seven
experiments. Further analysis of CCR5 distribution in thymocytes
cultured in IL-2 plus IL-4 showed that CCR5 was expressed on the
CD3+/high/CD4+/high/CD1 thymocyte
subset, in which we have previously observed JR-CSF expression (Fig.
2C) (71).
NL4-3 and JR-CSF replication kinetics in thymocytes correlate with the expression levels of CXCR4 and CCR5. The role of chemokine receptors in the different kinetics of replication of JR-CSF and NL4-3 in thymocytes was studied in vitro. Levels of CCR5 and CXCR4 expression were assessed before and after infection on freshly isolated thymocytes. Twenty-four hours after infection with JR-CSF (200 IU/104 cells) or NL4-3 (10 IU/104 cells), 106 cells were taken and analyzed by PCR for proviral DNA content as described previously (81). The level of proviral DNA in thymocytes infected with JR-CSF was significantly lower than the level of proviral DNA detected in thymocytes infected with NL4-3 (Fig. 3A), despite the 20-fold-higher MOI of JR-CSF, as determined in PHA-stimulated PBMC. This finding suggests that the difference observed between the replication kinetics of the two viruses is determined at the entry level. The copy number of NL4-3 proviral DNA in thymocytes (more than 50 copies/ng) correlated with the high numbers of cells expressing CD4 and CXCR4 in the thymus. The low level of JR-CSF proviral DNA in thymocytes (approximately 1 copy/ng) correlated with the low level of CCR5 surface expression (0.4%) on the specimen analyzed on day 0 (Fig. 1 and 3). Nevertheless, at 2 weeks postinfection p24 levels in the supernatant of JR-CSF infected cells reached 110 ng/ml. Notably, in all of eight infection experiments, thymus specimens containing at most 1% CCR5+ cells at the time of infection (day 0) were able to sustain JR-CSF production, as measured by p24 levels in the supernatant.
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populations (Fig. 3B). In subsequent experiments, at
later time points, JR-CSF expression was detected only in the
CCR5 population, in a manner reminiscent of the presence
of HIV expression in the CD4 thymocyte subpopulation at
late stages of infection (30).
Therefore, the slower replication of JR-CSF compared to that of NL4-3
in thymocytes correlated with lower levels of proviral DNA after
infection. This observation could be attributed at least in part to the
differences in the availability of cells expressing CD4 and the
appropriate coreceptor, presumably CCR5 and CXCR4, at the time of infection.
Low levels of CCR5 support replication of JR-CSF in
thymocytes.
The presence of JR-CSF in the CCR5
population could indicate downregulation of CCR5 on JR-CSF-infected
cells. However, JR-CSF adaptation to CXCR4 in culture and/or usage
of an alternative coreceptor could not be excluded in the experiments
described above. The antibody 2D7 was used to determine if JR-CSF
replication in thymocytes could be prevented by blocking the coreceptor
CCR5 (79). In all of four experiments, the p24 levels were
reduced up to 50-fold in JR-CSF-infected cells cultured in the
presence of 2D7, while in the presence of the 12G5 antibody to CXCR4
(16, 41) there was no reduction in p24 levels. As can be
seen in Fig. 4, thymocytes infected with
JR-CSF (30 IU/104 cells) produced high levels of p24 at 3 weeks postinfection. However, there was a delay in HIV expression in
the presence of 1 µg of 2D7 per ml, while with 5 µg/ml
the p24 levels were barely detectable up to 3 weeks
postinfection. Pretreatment and culture of thymocytes in the presence
of antibodies to both CXCR4 and CCR5 gave the same results as treatment
with antibody to CCR5 alone, indicating poor, if any, usage of CXCR4 by
JR-CSF in this system. Addition of 5 µg of 2D7 per ml 1 h after
infection of thymocytes with JR-CSF decreased p24 peak levels to 16 ng/ml, compared to 354 ng/ml in thymocytes cultured in the
absence of antibody.
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JR-CSF and NL4-3 production by different thymocyte subsets is
determined at the postentry level.
We used the CD69 and CD45RA
molecules as markers of thymocyte development to further characterize
the thymocyte subsets susceptible to JR-CSF and NL4-3 productive
infection. Thymocytes subsets at different stages of maturation were
obtained by using antibody-coated magnetic beads to negatively and
positively select specific subsets. As shown in Fig.
6A, thymocytes expressing CD69 are found
in the mature CD3+/high subset, which includes CD4 and CD8
single-positive cells and 5 to 10% of the
CD4+/CD8+ thymocytes as previously described
(67, 76). Mature CD3+/high/CD45RA+
cells were removed before CD69 depletion to eliminate the most mature
CD4 and CD8 single-positive thymocytes that are CD69 but
express CD45RA (7, 52, 66, 76). In the experiment shown in
Fig. 6, this procedure removed all but 0.8% of the CD69+
cells, which included single-positive CD4+ and
CD8+ cells, but did not remove the cells expressing CCR5 or
CXCR4 (Fig. 6A). In other experiments, CCR5 expression was very low or
below the detection level in CD69 cells while
present in low levels in the CD69+ population;
therefore CCR5 expression could not be ascribed to specific
thymocyte subsets defined by CD69 expression (data not shown).
Given the low levels of CCR5 surface expression on thymocytes and the
results obtained in the blocking experiments, we tried to determine
which thymocyte subsets expressed CCR5 on the basis of their
susceptibility to JR-CSF infection (see below).
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and CD69+ populations, suggesting that
viral entry occurred in both subsets. However, NL4-3 copy number in
each subset was at least 1,000-fold higher than JR-CSF copy number in
the same subset. The copy number of NL4-3 DNA was slightly higher in
the CD45RA/CD69 cells than in the
CD45RA/CD69+ cells, while the low copy number
of JR-CSF DNA did not permit a quantification of proviral levels in the
different subsets. These results indirectly suggest that CCR5 was
expressed in both populations, albeit at very low levels, confirming
the phenotype determined by flow cytometry (Fig. 6A). The ability of
the different subsets to complete reverse transcription after NL4-3
infection was assessed by amplifying the DNA samples with primers
detecting the LTR/gag region (32, 81, 82) as
shown in Fig. 6C. Full-length reverse transcripts were present in
the total population and in both CD69+ and
CD69 thymocyte subsets at relative levels (>50%) that
indicate completion of reverse transcription in all subsets (Fig. 6C).
Yet, in five of five experiments, the levels of p24 were higher in the
supernatant of CD69+ cells than in the supernatant of
CD69 cells after infection with JR-CSF or NL4-3 (Fig. 6D
and data not shown). This difference in viral expression was observed
in the presence of the appropriate coreceptors and of similar amounts of proviral DNA in both thymocyte subsets (Fig. 6A, B, and D).
These results suggest that postentry events determine the ability of
HIV to preferentially replicate in the more mature CD69+
thymocyte subset. Furthermore, full reverse transcription and low
levels of p24 expression were detected in CD69 cells
infected with NL4-3, indicating that late events in the virus cycle are
possibly involved in the differential tropism of HIV for different
thymocyte subsets. The expression level of a given virus isolate
(JR-CSF or NL4-3) in these different thymocyte subsets was not
determined at the entry level, although the differences between
expression of different virus isolates in a given subset (i.e.,
CD69-depleted cells) could be explained by availability of the
respective coreceptors.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study, we have demonstrated that the distribution of CXCR4
and CCR5 on thymocytes is a major determinant for NL4-3 and JR-CSF
tropism and determines the replication kinetics of these two isolates
(71). The majority of freshly isolated postnatal thymocytes
from uninfected children expressed moderate to high levels of CXCR4, in
comparison to CCR5 expression, which was present at low levels on 0.1 to 2% of the thymocyte population. Although we have shown that
expression of CXCR4 and CCR5 on thymocytes was necessary for viral
entry, additional host factors were required for a highly productive
infection in the CD69+ thymocyte subset. This was evident
in studies demonstrating that both the CD69+ and
CD69 cell populations allowed NL4-3 and JR-CSF entry,
whereas only the CD69+ population was identified as highly
susceptible to NL4-3 and JR-CSF productive infection.
CCR5 expression in fresh thymocytes, determined by both surface and
intracellular staining, was detected on few cells. Underestimation of
CCR5 expression could be occurring in our system due to downregulation of CCR5 in thymocytes by ligand occupation or virus binding. This is
unlikely because low levels of CCR5 mRNA were also detected by reverse
transcription-PCR (data not shown). In addition, Wu et al. reported
that 2D7 recognizes the chemokine binding site and does not
downregulate CCR5 expression (79). Furthermore, while low
levels of CCR5 could be detected on thymocytes with 2D7, this antibody
could block JR-CSF infection of thymocytes as previously reported for
other cell types (60, 79, 80). JR-CSF usage of alternative
coreceptors on thymocytes cannot be excluded by our studies (4,
54). However, an indirect effect of CCR5 blocking by 2D7 on such
putative receptors affecting JR-CSF and not NL4-3 replication would be
necessary to explain our data. For example, a link between mutations in
CCR2 and the level of expression of CCR5 has been proposed
(56). However, we favor the explanation that
CCR5 cells expressing HIV originated as CCR5+
cells that have either internalized CCR5 due to virus binding or
matured into CCR5 cells.
In the postnatal thymus, CXCR4 was present at high levels in immature
CD1+/CD3+/low thymocytes and at lower levels in
most but not all of the
CD3+/high/CD69/CD45RA+
thymocytes, cells that have the potential to leave the thymus (52,
76). Our results further suggest that there are fewer CCR5-expressing thymic emigrants than CXCR4-expressing thymic emigrants, which is consistent with reported studies demonstrating low
numbers of CCR5-expressing cells in the cord blood (48). This finding is also in agreement with the fact that in adults, CXCR4 expression in circulating T cells is detected mainly
in the naive
CD26low/CD45RA+/CD45RO
population, while CCR5 is expressed mostly in the effector/memory CD26high/CD45RAlow/CD45R0+
population that has previously undergone activation (6, 80).
In PBMC, CXCR4 is upregulated within 72 h upon stimulation with
PHA or anti-CD3, while increased CCR5 expression on stimulated T cells
requires addition of IL-2 for 2 to 3 weeks (6, 80). These
culture conditions form the basis of the slow/low versus rapid/high
biological phenotype of CCR5 and CXCR4 tropic primary isolates in PBMC
(5). In both PBMC and the SCID-hu mouse, the distribution of
thymocyte coreceptors described in this study is a major determinant of
the biological phenotype of NL4-3 and JR-CSF (27, 28, 64, 71,
74). The expression of CXCR4 on the immature
CD3/CD4+/low/CD8+ thymocytes may
lead to a rapid productive infection and destruction of this actively
proliferating cell population. We have found that cultures containing
IL-4 increased the level of CXCR4 expression in the mature
CD3+/high thymocyte subset, thereby increasing the number
of NL4-3 targets. The high levels of CXCR4 expression in
freshly isolated immature thymocytes, detected in all specimens
analyzed, may be related to the presence of IL-4 in the subcortical
area where immature thymocytes responding to IL-4 are found (22,
75). Consistent with this notion, Papiernik et al. reported that
pathological abnormalities in fetuses aborted from HIV-1-seropositive
women were present mainly in the cortex (47). Our
observations further suggest that immature thymocyte subsets from
children may be infected in vivo with CXCR4-tropic HIV isolates, as
observed in the SCID-hu model (27). Confirmation of a
similar effect of IL-4 on upregulation of CXCR4 expression in the
periphery might signify that the proposed shift from a Th1 to Th2
pattern of cytokine synthesis could favor the propagation of
CXCR4-tropic viruses in late stages of diseases (11).
Furthermore, a Th2-like cytokine pattern has been observed in
perinatally infected children progressing to AIDS (26).
Increased CCR5 expression in thymocytes was observed only in cultures containing IL-2 in combination with IL-4. As seen in stimulated PBMC, upregulation of CCR5 expression in thymocytes required the presence of IL-2 for at least 2 weeks (6, 80). The slower replication of JR-CSF in thymocytes was initially due to low availability of CCR5 and was reflected in the low levels of viral entry detected by PCR. The increase in JR-CSF production seen in IL-2 plus IL-4-supplemented cultures was presumably from upregulation of CCR5 on mature thymocytes and proliferation of these cells, thereby allowing viral spread. It is noteworthy that high levels of virus could be produced by very few infected cells, suggesting that a mature thymocyte population expressing CCR5 is highly permissive to JR-CSF replication.
We have found that both NL4-3 and JR-CSF replicate preferentially in
the CD69+ thymocyte population. This population includes
cells at various stages of maturation from the less mature
CD1+/CD4+/CD8+ cells through the
single-positive CD4+ or CD8+ populations
(52, 76). Since JR-CSF is not produced in immature CD1+ cells (71), we conclude that the thymocyte
subset producing high levels of JR-CSF is a mature subset that has
downregulated CD1, but not yet CD69, and therefore is not ready to
leave the thymus. In this CD1/CD69+ subset,
NL4-3 production is also highly favored, but the broad distribution of
CXCR4 expression allows NL4-3 entry into the immature CD1+/CD69 populations, thereby accounting for
the low level of NL4-3 production seen in the immature thymocyte
subset. Detection of full-length proviral DNA in all
populations confirms that while coreceptor expression is a major
determinant of tropism, cellular factors expressed at specific stages
of T-cell development affect postentry events and can
determine HIV replication in the thymus. In this regard, it should be
noted that in vivo, the CD69+ population consists of
thymocytes that are activated during the process of positive selection
(43, 76) and thus should be permissive for viral entry and replication.
We have previously proposed that pediatric isolates able to infect immature thymocytes might have a greater impact on disease progression (71). Here we show that a CXCR4-tropic isolate could produce this effect. We are now in the process of determining whether coreceptor use of isolates obtained from children with rapid and slow disease progression correlates with specific receptor use and subsequent loss of thymocytes. In this regard, the early acquisition of CXCR4 tropism in rapid progressors observed by Scarlatti et al. could be associated with CXCR4 targeting in the thymus (56).
It has been proposed that differences in the expression levels of CCR5
due to genetic factors can affect the rate of disease progression in
adults and children, where heterozygosity for the CCR532 deletion
substantially reduces disease progression (42, 61). It is
clear that in our in vitro conditions, at a low MOI, the threshold of
CCR5 expression required for replication in thymocytes is very low.
Although it takes longer, CD4 depletion occurs in SCID-hu mice infected
with JR-CSF (27). Since in our system the contribution of
stromal elements (potentially CCR5 positive) could not be evaluated, we
cannot determine the full contribution of CCR5 for HIV
pathogenesis on the thymus. Stanley et al. have shown that JR-CSF
causes a more pronounced disruption of stromal elements than a T-tropic
virus (64). The usage of coreceptors other than CCR5 and
CXCR4 by pediatric isolates in the thymus needs to be investigated.
In conclusion, our studies indicate that the ability of thymocyte subsets to support HIV productive infection is determined by the presence of the appropriate coreceptor and by cellular factors related to the state of maturation of the cells that affect postentry events in the virus replication cycle.
| |
ACKNOWLEDGMENTS |
|---|
The first two authors contributed equally to this work.
This work was supported by grants from the National Institutes of Health (HD 29341, HD 29341-S1, AI 28697, and DK49886), by UARP SRF01, and by student awards to K.B.G. from the Elizabeth Glaser Pediatric AIDS Foundation and the UCLA AIDS Institute (Esther Hays Graduate Student Award).
We thank Hillel Laks and his colleagues and staff for providing the thymus specimens; Jerome Zack and Irvin Chen for use of biocontainment facilities; Esther Hays, Beth Jamieson, John Ferbas, and Deborah Anisman-Posner for helpful discussions and critical reviews of the manuscript; and Deborah Anisman-Posner, Silvia Neagos, Kris Conners, and Prista Charuworn for excellent technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90095-1747. Phone: (310) 825-1982. Fax: (310) 206-1318. E-mail: uittenbo{at}ucla.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Virology, December 1998, p. 9441-9452, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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