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

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

LIVIA PEDROZA-MARTINS,

1

KEVIN B. GURNEY,

1

BRUCE E. TORBETT,

2 AND

CHRISTEL H. UITTENBOGAART

1,3,4,5

*

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

ex-pression 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

produc-tion was restricted to the mature CD1

2

/CD69

1

population. Although CXCR4 and CCR5 distribution readily

explained viral entry in mature CD69

1

and immature CD69

2

cells, and correlated with proviral DNA

distri-bution, we found that viral production was favored in CD69

1

cells. Therefore, while expression of CD4 and

ap-propriate 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.

Human immunodeficiency virus (HIV) infection of the

thy-mus 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

es-tablished, the thymus is required for T-cell generation in

chil-dren (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

core-ceptors allows HIV-1 entry into cells (13, 15, 17, 21). HIV-1

primary isolates can use CXCR4, CCR5, both receptors

(du-altropic isolates), or a number of other reported

seven-trans-membrane, 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

infec-tion with the presence of CCR5 mutainfec-tions 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

D

32 was not

associated with transmission but was associated with a slower

development of HIV-related disease in children (42).

Consis-tent 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).

In the thymus, where CD4 is expressed on more than 95% of

the cells, the distribution of HIV coreceptors would be

ex-pected to be an important determinant of tropism. Wide

dis-tribution of CXCR4 surface expression on fetal thymocytes has

been recently reported (31), while expression of the

corecep-tors CCR5, CCR8, and STLR-33/GPR15 on total thymocytes

has been reported at the mRNA level (36, 46, 51, 54, 68).

* Corresponding author. Mailing address: Department of

Microbi-ology and ImmunMicrobi-ology, UCLA School of Medicine, Los Angeles, CA

90095-1747. Phone: (310) 825-1982. Fax: (310) 206-1318. E-mail:

[email protected].

9441

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Other chemokine receptors, such as CCR4 (49), not yet

iden-tified 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

de-termine 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 G

1b

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

exam-ined HIV replication in thymocyte subsets defexam-ined by the

ex-pression of surface molecules that are commonly used as

mark-ers of T-cell development: CD1, CD69, and CD45RA. The

CD1 molecule is expressed at high levels in CD3

2/low

thymo-cytes and therefore identifies immature thymothymo-cytes (7).

Down-regulation 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

1

/CD8

1

double-positive thymocytes and at high levels on

mature single-positive CD4

1

and CD8

1

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

1/high

/CD1

2

thymocytes that are ready to

leave the thymus (66).

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

1

and CD69

2

cells,

whereas only the CD69

1

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

Reagents and monoclonal antibodies.Recombinant human IL-2 (1.53106

U/ml) and IL-4 (0.7 mg/ml) were provided by Amgen, Inc. (Thousand Oaks, Calif.). Recombinant human IL-7 (100mg/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 intracel-lular HIV p24gag_{antigen expression (10, 40), CD1-PE, CD45RA-PE, and the}

unconjugated antibodies CD45RA and CD69, used for thymocyte subset sepa-rations, 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 Re-search 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.).

Freshly isolated, nonstimulated PBMC were used as the positive control for CCR5 detection. In the same PBMC adult donors, CCR5 was present in 14 to 20% of the lymphocytes stained with 2D7 but only 2 to 3% of cells stained with 3A9. However, staining with antibodies 3A9 and 2D7 gave similar results on monocytes from these donors and in freshly isolated postnatal thymocytes. To rule out the possibility that the epitopes recognized by these antibodies were not exposed on the surface of thymocytes, cells were permeabilized and stained intracellularly with antibody 2D7-PE (58). The intracellular level of CCR5 was below the detection level on thymocytes but was detectable in PBMC, although at lower levels than on the cell surface.

HIV infection and thymocyte cultures.Normal pediatric thymuses were ob-tained in the course of corrective cardiac surgery. Single-cell suspensions and nylon wool purification were done as previously described, and thymocytes were cultured at 13107_{to 2310}7_{cells/ml in serum-free medium}

(albumin-trans-ferrin-IMDM [Iscove’s modified Dulbecco’s medium]; Irvine Scientific, Santa Ana, Calif.) supplemented with delipidated bovine serum albumin (BSA; Sigma) at 1,100mg/ml, transferrin (Sigma) at 85mg/ml, glutamine at 2 mM (0.3 mg/ml), and penicillin-streptomycin at 25 U/ml–25mg/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 at270°C and treated with DNase (2mg/ml; Worthing-ton, Lakewood, N.J.) for 30 min at room temperature in the presence of 0.01 M MgCl2before 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 phytohemag-glutinin (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 (10mg/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 5mg/107_{cells) and/or CXCR4}

(12G5; 5 to 10mg/107_{cells) or CD4-IgG (100mg/10}7_{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}

non-specific binding to thymocytes and then coated with the CD45RA or CD69 monoclonal antibody (1.25 tests of the antibody as determined by the manufac-turer/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 CD45RA1 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 (CD691 popu-lation) were magnetically removed, and unbound cells (CD692_{population) were} subjected to a second round of depletion. Following separation, the depleted subsets were immunophenotyped and analyzed by flow cytometry. Both posi-tively and negaposi-tively immunoselected subsets were used for infection and culture experiments. Their viability, as determined by trypan blue dye exclusion, was .96%.

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Immunofluorescent staining and flow cytometry.Surface and cytoplasmic im-munophenotyping 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 50ml of human AB serum to prevent nonspecific protein binding, thymocytes (13105_{to 5310}5_{) were incubated with optimal}

amounts of unconjugated monoclonal antibody for 20 min at 4°C in a total volume of 100ml 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 50ml of human AB serum. Cells were washed with 3 ml of PBS-BSA and incubated for 10 min at 4°C with 50ml 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 2mg 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 4mg of AD per ml (57, 59). The samples were subjected to flow cytometric analysis in the paraformalde-hyde-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 ex-tractions 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 the32_{P-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 (10mg/ml). To normalize for cellular DNA, replicate samples were analyzed for humanb-globin gene se-quences (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.).

RESULTS

Cell surface expression of CCR5, CCR3, and CXCR4 on

thymocytes from children.

Postnatal thymus specimens

ob-tained 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

1

cells

and PBMC (60, 80). In all specimens analyzed, more than 95%

of postnatal thymocytes expressed CXCR4, while the

percent-ages of CCR5

1

cells ranged from 0.2 to 1% (mean

6

standard

deviation

5

0.45%

6

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

2

and CD3

1/low

subsets, while the

mature CD3

1/high

subset contained CXCR4

1

and CXCR4

2

cells, as previously reported for fetal thymocytes (31). The

determination of CCR5 expression on distinct thymocyte

sub-sets by immunofluorescence methods was hampered by the low

numbers of CCR5

1

thymocytes. CCR3 surface expression was

not detectable with antibody 7B11 in any of the eight

thymo-cyte samples tested (not shown).

Cytokines that favor HIV production by thymocytes

upregu-late CCR5 and CXCR4 surface expression.

Expression of

CCR5 and CXCR4 is tightly regulated on PBMC by

stimula-tory 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

ex-pression 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

1/high

population)

(72, 73, 78). Cell surface phenotype was determined weekly,

and the cursors were set in order to analyze coreceptor

expres-sion in the CD3

1/high

population (Fig. 2; Table 1).

Thymocytes cultured in the presence of IL-2 plus IL-4 or

IL-4 plus IL-7 showed increased levels of CXCR4 expression

as measured by fluorescence intensity and by the absence of

CXCR4

2

cells (Fig. 2A). IL-4 alone was sufficient to increase

[image:3.612.109.491.73.228.2]

the levels of CXCR4 expression (Table 1). Interestingly, IL-4

increased the expression of CXCR4 in the mature CD3

1/high

population that expresses low levels of CXCR4 in freshly

iso-lated thymocytes. In thymocytes cultured with IL-2 alone,

there was a threefold increase in the fluorescence intensity and

in the percentage of CXCR4

1

/CD3

1/high

cells (Table 1), but

the mature CD3

1/high

population that did not express CXCR4

FIG. 1. Distribution of chemokine receptor expression on freshly isolated human thymocytes. Thymocytes were isolated by nylon wool separation and phenotyped with CD3-PE and nonlabeled antibodies to the chemokine receptors CXCR-4 (antibody 12G5) and CCR5 (antibody 2D7), followed by goat anti-mouse IgG-FITC (GAM-FITC). Appropriate isotype control antibodies (IgG2a and IgG1) followed by goat anti-mouse IgG-FITC were used to set the cursors. The percentage of cells staining with the isotype control antibodies followed by goat anti-mouse IgG-FITC was 0%. Dead cells were excluded from the analysis by using 7-AAD.

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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

in-creased 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

thy-mocyte culture experiments, IL-2 and IL-4 synergistically

in-creased 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

1/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

1/high

/

CD4

1/high

/CD1

2

thymocyte subset, in which we have

previ-ously observed JR-CSF expression (Fig. 2C) (71).

NL4-3 and JR-CSF replication kinetics in thymocytes

cor-relate with the expression levels of CXCR4 and CCR5.

The

role of chemokine receptors in the different kinetics of

repli-cation 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/10

4

cells) or NL4-3 (10 IU/10

4

cells), 10

6

cells were taken and

analyzed by PCR for proviral DNA content as described

pre-viously (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

differ-ence observed between the replication kinetics of the two

vi-ruses 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

ex-periments, thymus specimens containing at most 1% CCR5

1 [image:4.612.59.547.67.428.2]

cells at the time of infection (day 0) were able to sustain

FIG. 2. Effects of cytokines on chemokine receptor expression. Thymocytes were cultured for 2 weeks in serum-free medium alone or supplemented with IL-2 plus IL-4 or IL-4 plus IL-7. Before culture (day 0) and on days 7 and 15, cells were removed for immunophenotyping to examine expression of chemokine receptors by flow cytometry. Appropriate isotype control antibodies and single-color staining with CD3-FITC were used to set the cursors defining the CD31/high_{population (A and B).}

Dead cells were excluded from the analysis by using 7-AAD. (A and B) CXCR4 and CCR5 expression on thymocyte subsets was determined by using the antibodies CD3-FITC, CXCR4-PE (12G5), and CCR5-PE (2D7). (C) In a different experiment, immunophenotyping was performed on day 12 of culture to identify the distribution of CCR5 on thymocyte subsets that respond to IL-2 plus IL-4. Thymocytes were stained with CD3-PE, CD4-PE, or CD1-PE in combination with nonlabeled antibody to CCR5 (2D7) and then with goat anti-mouse IgG-FITC (GAM-FITC). The percentage of cells staining with the IgG1 isotype control antibody for CCR5 followed by goat anti-mouse IgG-FITC was 0%.

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JR-CSF production, as measured by p24 levels in the

super-natant.

To determine if JR-CSF was produced by the small subset of

CCR5

1

thymocytes, cell surface staining was combined with

intracellular staining for HIV Gag proteins with the KC57

antibody (10, 40, 71). As observed for uninfected thymocytes,

expression levels of both CXCR4 and CCR5 increased on

mature thymocytes during culture of infected cells with IL-2

plus IL-4, although the percentage of CCR5

1

positive cells

detected remained below 10% (Fig. 3B). At 2 weeks after

infection with JR-CSF, KC57

1

cells were detected in both the

CCR5

1

and CCR5

2

populations (Fig. 3B). In subsequent

ex-periments, at later time points, JR-CSF expression was

de-tected only in the CCR5

2

population, in a manner reminiscent

of the presence of HIV expression in the CD4

2

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

attrib-uted 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

thy-mocytes.

The presence of JR-CSF in the CCR5

2

population

[image:5.612.66.535.73.572.2]

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

FIG. 2—Continued.

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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/10

4

cells) produced high levels of

p24 at 3 weeks postinfection. However, there was a delay in

HIV expression in the presence of 1

m

g of 2D7 per ml, while

with 5

m

g/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,

indi-cating poor, if any, usage of CXCR4 by JR-CSF in this system.

Addition of 5

m

g of 2D7 per ml 1 h after infection of

thymo-cytes with JR-CSF decreased p24 peak levels to 16 ng/ml,

compared to 354 ng/ml in thymocytes cultured in the absence

of antibody.

To determine if the effect of 2D7 on HIV replication on

thymocytes was due to blocking of CCR5 coreceptor function

and not to another effect of 2D7 on the cells which prevented

them from producing virus, thymocytes were pretreated with 5

m

g of 2D7 per ml and infected with either JR-CSF or NL4-3.

Production of NL4-3 was not blocked by antibody to CCR5 but

could be partially blocked by antibody to CXCR4 at 10

m

g/ml

(data not shown). The p24 data were confirmed by intracellular

staining with the KC57 antibody 2 and 3 weeks after infection

(Fig. 5). After infection with JR-CSF, KC57 expression was

detected in 3% of the untreated thymocytes, as opposed to

0.05% of the cells in the presence of 5

m

g of CCR5 antibody

per ml. In thymocytes infected with NL4-3, percentages of

KC57

1

cells were similar in the nontreated thymocytes and in

the thymocytes treated with antibody to 2D7 at 2 weeks

postin-fection (Fig. 5A). In addition, when 2D7 was present, a

pro-found depletion of CD4

1

thymocytes, a hallmark of NL4-3

infection, had already taken place (Fig. 5B). Cells infected with

JR-CSF in the presence of 2D7 did not have a CD4/CD8

profile significantly different from that of mock infected cells

cultured with 2D7 (Fig. 5B).

Taken together, these results suggest that JR-CSF uses

CCR5 as a coreceptor in thymocytes. Furthermore, they

indi-cate that very low levels of CCR5 surface expression can

sup-port replication of CCR5-tropic viruses in the thymus.

JR-CSF and NL4-3 production by different thymocyte

sub-sets 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

anti-body-coated magnetic beads to negatively and positively select

specific subsets. As shown in Fig. 6A, thymocytes expressing

CD69 are found in the mature CD3

1/high

subset, which

in-cludes CD4 and CD8 single-positive cells and 5 to 10% of the

CD4

1

/CD8

1

thymocytes as previously described (67, 76).

Ma-ture CD3

1/high

/CD45RA

1

cells were removed before CD69

depletion to eliminate the most mature CD4 and CD8

single-positive thymocytes that are CD69

2

but express CD45RA (7,

52, 66, 76). In the experiment shown in Fig. 6, this procedure

removed all but 0.8% of the CD69

1

cells, which included

[image:6.612.50.289.90.185.2]

single-positive CD4

1

and CD8

1

cells, but did not remove the

FIG. 3. CXCR4 and CCR5 expression levels correlate with the amount of NL4-3 and JR-CSF proviral DNA after infection. Thymocytes were infected with JR-CSF (200 IU/104_{cells) or NL-4-3 (10 IU/10}4_{cells) and cultured with IL-2}

plus IL-4. CCR5 expression on day 0 is shown in Fig. 1. (A) Twenty-four hours postinfection, 106_{cells were removed and analyzed by using primers (R/U5)}

[image:6.612.310.543.262.637.2]

specific for the LTR region of HIV-1 to detect the presence of proviral DNA. To normalize for the amount of cellular DNA, PCR was performed in parallel for sequences in theb-globin gene. (B) JR-CSF-infected thymocytes were cultured with IL-2 plus IL-4. At 13 days postinfection, cells were subjected to surface staining with CCR5-PE/CD3-PerCP followed by intracellular staining with KC57-FITC.

TABLE 1. Effects of cytokines on CXCR4 expression

in thymocytes

a

Cytokine

Mean fluorescence intensity of CXCR4 on CD31/high

cells (% CD31/high_/CXCR41_cells)b

Expt 1 Expt 2 Expt 3 Expt 4

None

47 (3)

38 (2)

37 (6)

37 (5)

IL-2

60 (9)

118 (21)

139 (28)

ND

IL-4

444 (22)

354 (24)

359 (29)

256 (15)

IL-7

ND

70 (25)

ND

160 (24)

IL-2

1

IL-4

431 (29)

489 (33)

361 (35)

ND

IL-4

1

IL-7

ND

398 (33)

ND

326 (17)

a_{Freshly isolated thymocytes were cultured for 2 weeks in serum-free medium}

in the presence or absence of cytokines. Surface expression of CXCR4 was determined by flow cytometry using directly conjugated antibodies specific for CD3 (FITC) and CXCR4 (PE), with 7-AAD used to exclude dead cells.

b_{The geometric mean of the fluorescence intensity of CXCR4 in the CD3}1/

highpopulation and the percentage of these cells in the gated population were

calculated with the Cell Quest software. Cursors were set by using isotype controls for all cytokine conditions within an individual experiment. Single-color staining with CD3-FITC was used to identify the CD31/high_{population. Note}

that the percentage of total CD31/high_{cells depended on the cytokine used (Fig.}

2A). ND, not determined.

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cells expressing CCR5 or CXCR4 (Fig. 6A). In other

experi-ments, CCR5 expression was very low or below the detection

level in CD69

2

cells while present in low levels in the CD69

1

population; therefore CCR5 expression could not be

as-cribed to specific thymocyte subsets defined by CD69

ex-pression (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

thymo-cyte subsets expressed CCR5 on the basis of their susceptibility

to JR-CSF infection (see below).

The total population and the immunoselected thymocyte

subsets were infected with either JR-CSF (100 IU/10

4

cells) or

NL4-3 (10 IU/10

4

cells), and virus production was monitored

by measuring p24 levels in the culture supernatants for up to 3

weeks. Proviral DNA levels in the distinct thymocyte subsets

were assessed by using PCR primers detecting partial and

full-length reverse transcription. Figure 6B shows that the amount

of proviral DNA in the total population infected by NL4-3 or

JR-CSF correlated with the expression levels of the respective

coreceptors as shown in Fig. 3A. JR-CSF and NL4-3 proviral

DNA could be detected in both CD69

2

and CD69

1

popula-tions, 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

sub-set. The copy number of NL4-3 DNA was slightly higher in the

CD45RA

2

/CD69

2

cells than in the CD45RA

2

/CD69

1

cells,

[image:7.612.51.290.68.287.2]

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

1

and CD69

2

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

1

cells than in the supernatant of CD69

2

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

pro-viral 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

1

thymocyte subset. Furthermore, full reverse

transcrip-tion and low levels of p24 expression were detected in CD69

2

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

thy-mocyte 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.

DISCUSSION

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

ex-pressed 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

1

thymocyte subset.

This was evident in studies demonstrating that both the CD69

1

and CD69

2

cell populations allowed NL4-3 and JR-CSF entry,

whereas only the CD69

1

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

transcrip-tion-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

pre-viously 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

2

cells expressing HIV

originated as CCR5

1

cells that have either internalized CCR5

due to virus binding or matured into CCR5

2

cells.

In the postnatal thymus, CXCR4 was present at high levels

in immature CD1

1

/CD3

1/low

thymocytes and at lower levels in

most but not all of the CD3

1/high

/CD69

2

/CD45RA

1

thymo-cytes, cells that have the potential to leave the thymus (52, 76).

Our results further suggest that there are fewer

CCR5-express-FIG. 4. The antibody (2D7) to CCR5 is able to block productive infection of thymocytes by JR-CSF. Thymocytes were preincubated in the presence or ab-sence of antibody to CCR5 (2D7) or CXCR4 (12G5) for 2 h before infection with JR-CSF (30 IU/104_{cells). The antibodies were present during the infection and}

throughout the culture with IL-2 plus IL-4. HIV replication was detected by measuring p24 antigen in the culture supernatants on days 8, 15, and 22 postin-fection. Preincubation conditions: no antibody (gray bars), 1mg of 2D7 (verti-cally striped bars), 5mg of 2D7 (diagonally striped bars), 10mg of 12G5 (hori-zontally striped bars), 1mg of 2D7 plus 10mg of 12G5 (checkered bars), 5mg of 2D7 plus 10mg of 12G5 (black bars), and 100mg of CD4-IgG (white bars).

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ing thymic emigrants than CXCR4-expressing thymic

emi-grants, 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 CD26

low

/CD45RA

1

/CD45RO

2

population, while

CCR5 is expressed mostly in the effector/memory CD26

high

/

CD45RA

low

/CD45R0

1

population that has previously

under-gone activation (6, 80).

In PBMC, CXCR4 is upregulated within 72 h upon

stimu-FIG. 5. The antibody to CCR5 (2D7) specifically blocks expression of JR-CSF in thymocytes. Thymocytes were preincubated in the presence or absence of 5mg of CCR5 antibody per ml for 2 h before infection with JR-CSF (30 IU/104_{cells) or NL4-3 (1.5 IU/10}4_{cells). The antibody was present during infection and throughout}

the culture with IL-2 plus IL-4. (A) At 2 weeks postinfection, cells were subjected to surface staining with CD3-PerCP followed by intracellular staining with KC57-FITC. (B) To determine the effect of CCR5 antibody on thymocytes, uninfected and infected cells were immunophenotyped with CD4-PE and CD8-PerCP and intracellularly stained with KC57-FITC 2 weeks postinfection. CD4-PE/CD8-PerCP expression is shown.

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[image:8.612.76.518.68.613.2]

[image:9.612.51.535.75.706.2]

FIG. 6. Infection of total thymocytes and CD691_{and CD69}2_thymocyte sub-sets by JR-CSF and NL4-3. CD691_{and CD69}2_{subsets were obtained by using} antibody-coated immunomagnetic beads from the CD45RA2_{thymocytes and} immunophenotyped to determine the purity of the isolation. The resulting CD45RA2_/CD691_{cells bound to beads, and CD45RA}2_/CD692_{cells were used} for infection. (A) Immunophenotype of thymocytes before and after depletion of CD45RA- and CD69-expressing cells. (B) The total thymocyte population and the CD691_{and CD69}2_{thymocyte subsets were infected with JR-CSF (100} IU/104_{cells) or NL4-3 (10 IU/10}4_{cells) and cultured for 2 weeks in the presence}

of IL-2 plus IL-4. Twenty-four hours postinfection 106_{cells were removed and}

analyzed by using primers (R/U5) specific for the LTR region of HIV-1 to detect the presence of proviral DNA. To normalize for the amount of cellular DNA, PCR was performed in parallel for sequences in theb-globin gene. Heat-inactivated virus (2) were run in parallel with the live-virus-treated samples (1) as controls for DNA contamination from the inoculum. (C) PCR amplification of proviral DNA was performed on diluted DNA samples from the NL4-3-infected cells described above to detect the presence of fully reverse transcribed (RT) proviral DNA (LTR/gag) in parallel with the partially reverse transcribed (R/U5) proviral DNA. (D) HIV replication was detected by measuring p24 antigen in the culture supernatants of JR-CSF-infected CD691_{cells (vertically striped bars), JR-CSF-infected CD69}2 cells (white bars), NL4-3-infected CD691_{cells (black bars), and NL4-3-infected} CD692_{cells (gray bars) on days 1, 5, and 12 postinfection.}

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lation with PHA or anti-CD3, while increased CCR5

expres-sion 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

core-ceptors 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

2

/CD4

1/low

/

CD8

1

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

1/high

thymocyte subset,

thereby increasing the number of NL4-3 targets. The high

lev-els of CXCR4 expression in freshly isolated immature

thymo-cytes, detected in all specimens analyzed, may be related to the

presence of IL-4 in the subcortical area where immature

thy-mocytes responding to IL-4 are found (22, 75). Consistent with

this notion, Papiernik et al. reported that pathological

abnor-malities in fetuses aborted from HIV-1-seropositive women

were present mainly in the cortex (47). Our observations

fur-ther 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).

Fur-thermore, 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

thy-mocytes required the presence of IL-2 for at least 2 weeks (6,

80). The slower replication of JR-CSF in thymocytes was

ini-tially 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

thy-mocytes 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

pref-erentially in the CD69

1

thymocyte population. This

popula-tion includes cells at various stages of maturapopula-tion from the less

mature CD1

1

/CD4

1

/CD8

1

cells through the single-positive

CD4

1

or CD8

1

populations (52, 76). Since JR-CSF is not

produced in immature CD1

1

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

2

/

CD69

1

subset, NL4-3 production is also highly favored, but

the broad distribution of CXCR4 expression allows NL4-3

entry into the immature CD1

1

/CD69

2

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

expres-sion is a major determinant of tropism, cellular factors

ex-pressed at specific stages of T-cell development affect

post-entry events and can determine HIV replication in the thymus.

In this regard, it should be noted that in vivo, the CD69

1

pop-ulation consists of thymocytes that are activated during the

process of positive selection (43, 76) and thus should be

per-missive 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 CCR5

D

32 deletion substantially reduces disease

progres-sion (42, 61). It is clear that in our in vitro conditions, at a low

MOI, the threshold of CCR5 expression required for

replica-tion 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

(po-tentially CCR5 positive) could not be evaluated, we cannot

de-termine 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

thymo-cyte 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

biocontain-ment facilities; Esther Hays, Beth Jamieson, John Ferbas, and

Debo-rah Anisman-Posner for helpful discussions and critical reviews of the

manuscript; and Deborah Anisman-Posner, Silvia Neagos, Kris

Con-ners, and Prista Charuworn for excellent technical assistance.

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