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Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration;
Laboratory of Clinical Investigation and
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimentally, thymocytes can be infected with HIV-1 both in vitro and in vivo in the SCID-hu mouse model (10, 11, 12, 13, 14, 15, 16). Although thymocytes can be productively infected by diverse HIV-1 strains, including primary isolates, the so-called T-tropic or syncytium-inducing strains mediate faster depletion of thymocytes than M-tropic nonsyncytium-inducing strains (17, 18, 19). In these experiments, disruption of thymopoiesis was associated with the ability of viral isolates to infect immature thymic populations, including CD4-CD8- double-negative (DN) thymocytes (which express very low levels of CD4) and CD4+CD8+ double-positive (DP) thymocytes. The primary HIV-1 receptor CD4 is expressed by both immature and mature thymocytes. However, the HIV-1 coreceptors used for thymocyte infection have not been established.
The major HIV-1 coreceptors appear to be the chemokine receptors CXCR4 and CCR5, which mediate infection with T-tropic and M-tropic HIV-1 strains, respectively (20, 21, 22, 23, 24, 25). Normally, CXCR4 and CCR5 function as chemoattractant receptors for specific types of leukocytes (26). Several other chemokine receptors with HIV-1 coreceptor activity have been identified, but their importance for infection of primary targets of HIV-1 is not well established (24, 25).
Our laboratory produced a panel of polyclonal Abs against the extracellular regions of CXCR4 and CCR5 capable of staining primary human cells and blocking HIV-1 envelope glycoprotein-mediated cell fusion (27, 28). In the present study, we have used these antisera and specific functional assays to examine the signaling potential of CXCR4 and CCR5 in response to chemokines and their ability to support HIV-1 infection of primary human thymocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fresh thymus fragments were obtained during cardiac surgery from children (aged 1 mo to 3 yr) with congenital valvular malformations. The tissue was minced, large aggregates were removed by passing through a nylon mesh, and thymocytes were separated by centrifugation on a Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, Sweden).
Thymic fibroblasts were propagated by growing thymic cell suspensions
in 
-MEM (Life Technologies, Gaithersburg, MD) with 20% FBS for 4
wk. After the first 4 days of culture, the medium was changed.
Fibroblasts were passaged using 0.05% trypsin-EDTA solution (Life
Technologies). Before harvesting, cells were inspected for typical
fibroblast morphology.
To enrich for CD34+ cells, 2 x 108 thymocytes were incubated with anti-human CD4 and anti-human CD8 Abs conjugated with biotin (PharMingen, San Diego, CA) for 15 min on ice, followed by a 20-min incubation with streptavidin-conjugated magnetic microbeads (Miltenyi Biotec, Auburn, CA) on ice. Depletion of double-positive and single-positive (SP) thymocytes was performed using VarioMACS magnet separator and a CS column according to the manufacturer’s instructions (Milenyi Biotec). Unbound thymocytes (1.5 x 106) were collected and analyzed for expression of CD3, CD34, CXCR4, and CCR5.
Umbilical cord blood was collected during normal deliveries and was
obtained from Advanced Biotechnologies (Columbia, MD). Heparinized
peripheral blood was drawn from healthy donors at the National
Institutes of Health Blood Bank. The interphase cells from Ficoll-Paque
gradient centrifugation were collected. These PBMCs were passed through
a nylon wool column, and the nonadherent cell population, enriched for
T cells (
80%), was used for flow cytometry.
HIV Env-dependent cell fusion assay
CD4- 12E1 cells were infected with recombinant vaccinia viruses encoding envelopes (Env) from HIV-1 IIIB (T-tropic) (29) or JR-FL, ADA, and Ba-L (M-tropic) (30) strains at 10 plaque-forming units/cell. Thymocytes were mixed with Env-expressing 12E1 cells at a 1:1 ratio (105 cells each) in triplicate and cocultured for 4 to 5 h (T-tropic Env) or for 5 to 18 h (M-tropic Envs). Cell fusion activity was quantified by counting syncytia. Where indicated, rabbit antiserum against CXCR4 or CCR5, preimmune rabbit IgG, or the SDF-1 or RANTES (PeproTech, Rocky Hill, NJ) were added to the thymocytes for 1 h at 37°C before the addition of Env-expressing 12E1 cells.
Flow cytometry
The following Abs were used: FITC-labeled mouse anti-human
CD4 mAb (Becton Dickinson, San Jose, CA), anti-CD34 mAb
(PharMingen, San Diego, CA), anti-CCR5 mAb (clone 2D7, PharMingen),
and PE-labeled anti-CD8 mAb (Becton Dickinson). TCR levels were
determined using PE-labeled anti-
mAb (anti-CD3; Becton
Dickinson). IgG fractions of sera from rabbits immunized with CXCR4 and
CCR5 N-terminal peptides or from preimmune rabbits were prepared as
previously described (27, 28).
Cells isolated from the thymus, cord blood, or peripheral blood were incubated with 10 µg/ml of rabbit anti-CXCR4 or anti-CCR5 or with preimmune IgG (NRIgG) for 1 h at 4°C followed by incubation with biotin-conjugated goat anti-rabbit F(ab)2 (1 h at 4°C) and a subsequent incubation with TC-streptavidin (1 h at 4°C; both reagents from Caltag, South San Francisco, CA). The last incubation was performed in the presence of mAbs specific for other surface markers: CD4, CD8, CD34, or CD3. In some experiments, the total thymocyte suspension was stained with FITC-labeled mouse anti-CCR5 mAb (2D7). Thirty thousand cells were collected per sample and were analyzed using FL-1 (for FITC), FL-2 (for PE), and FL-3 (for TC) channels on a FACScan (Becton Dickinson) with CellQuest Software. Spectral overlap between cells stained with specific Abs and those incubated with PE-, TC-, and FITC-conjugated isotype controls was electronically compensated using analogue subtraction. The differences in the number of mean fluorescence channels between experimental and control samples above 200 were considered to be high levels of expression.
For intracellular staining, thymic-derived fibroblasts were permeabilized using the Fix and Perm kit (Caltag) according to the manufacturer’s instructions and were stained with rabbit polyclonal serum against human collagen III (provided by L. Fisher) (31). The presence of intracellular collagen III is a marker of fibroblasts (32, 33).
Calcium flux assay using fluorometer
Thymocytes (107) were loaded with 2 µM fura-2/AM
(Molecular Probes, Eugene, OR) in 1 ml of PBS for 30 to 60 min at
37°C and washed twice in HBSS (BioWhittaker, Walkersville, MD).
Chemokines were added at the indicated times to 106 cells
in 2 ml of HBSS in a continuously stirred cuvette at 37°C in an
MS-III fluorometer (Photon Technology, South Brunswick, NJ). The
relative ratio of fluorescence emitted at 510 nm following sequential
excitation at 340 and 380 nm was recorded every 200 ms. MIP-1,
SDF-1, and MIP-1ß were purchased from PeproTech; all chemokines were
used at a final concentration of 50 nM.
Calcium flux assay by multiparameter flow cytometry
Simultaneous evaluation of calcium mobilization in response to chemokine binding and phenotypic analysis by flow cytometry were performed as previously described (see footnote 5). In brief, total unseparated thymocytes were incubated in a 10-µM solution of the fluorescent calcium probe indo-1 in the presence of pleuronic detergent (300 µg/ml; both reagents purchased from Molecular Probes) in HBSS buffer with calcium and magnesium, 10 mM HEPES, and 1% FBS for 45 min at 30°C. Thymocytes were washed twice and incubated with PE-conjugated anti-CD8 mAb (clone HIT8a, PharMingen), Cy-5-PE-conjugated anti-CD4 (Sigma), and FITC-conjugated anti-CD34 mAb for 15 min at room temperature; washed; and resuspended in HBSS. Labeled thymocytes were analyzed on a FACSVantage (Becton Dickinson) equipped with a Time Zero injection module (Cytek, Fremont, CA) and with a ratio offset (Becton Dickinson), which allowed us to keep the baseline signal in the lower channels. Indo-1 fluorescence was analyzed at 390/20 (violet) and 530/20 (blue) for bound and unbound indo-1, respectively. For each stimulation, an aliquot of thymocyte suspension was warmed at 37°C for 3 min before beginning the collection on the flow cytometer. After 30 s of collection, 50 µl of HBSS was injected, and after 60 s, 50 µl of chemokine was injected to a final concentration of 50 nM. Cells were collected at a rate of 4000/s. The percentage of thymocytes within the subset of interest that mobilized calcium in response to chemokines was determined using Multitime software for analysis of kinetic flow cytometry data (Phoenix Flow Systems, San Diego, CA) as previously described.5 The percentage of cells above the threshold was calculated for sequential 6-s intervals, and the percentage of responding cells was determined from the maximal value after the chemokine addition minus the value after buffer addition.
Chemotaxis
Thymocytes (300,000 in 25 µl of serum-free RPMI 1640 medium) were loaded into the upper compartment of a microchemotaxis chamber (Neuroprobe, Cabin John, MD). Thirty-one microliters of chemokines were added to the lower compartment at the indicated concentrations. The two compartments were separated by a polyvinylpyrolidone-free polycarbonate filter with 5-µm pores. The chemotaxis chamber was incubated overnight at 37°C with 100% humidity and 5% CO2. The filter was then removed, and the number of cells migrating into each bottom compartment was counted. The mean and SD of triplicate chambers were calculated.
In vitro infection of thymocytes with HIV-1
Stocks of HIV-1LAI were prepared by infection of PM1 cells (35), and HIV-1AD and HIV-1Ba-L were prepared in Jurkat-CCR5 cells (23). Viruses were harvested at about the peak of RT activity and were clarified by centrifugation at 2000 x g for 5 min. The amount of virus was determined by RT assay (36). DP and SP thymocytes from normal pediatric donors were positively selected using the VarioMACS cell separation system according to the manufacturer’s instructions. Thymocytes (3.5 x 106) were inoculated with HIV-1LAI or HIV-1Ba-L at a multiplicity of infection (MOI) of 0.01 for both viruses and allowed to adsorb for 1 h before extensive washing and addition of fresh medium. DNA was extracted from thymocytes immediately after washing (time zero) or after culture for 24 h in the presence of IL-2 (100 U/ml; R&D Systems, Minneapolis, MN). Cell pellets from 2 million thymocytes/group were lysed in 0.5 ml of DNA lysis buffer as previously described (37). Fifty microliters of DNA lysate was amplified by PCR with gag-specific primers (SK38 and SK39) and 2 U of Taq DNA polymerase (Perkin-Elmer/Cetus, Norwalk., CT) for 25 cycles with an annealing temperature of 55°C. Ten percent of the amplified products (equivalent of 2 x 105 cells) were subjected to electrophoresis on 10% polyacrylamide gels and hybridized to a [32P]ATP end-labeled SK19 probe. The signals on the autoradiographs were compared with those from simultaneously amplified DNAs from serially diluted ACH-2 T cells, which contain one provirus per cell. Video images of the film were taken using the GDS 5000 system (UVP, San Gabriel, CA), and densitometry was performed with SW 5000 software (UVP, Cambridge, UK) as previously described (38). A standard curve was generated using absorption values derived from amplified DNA extracted from serial dilutions of ACH-2 cells. An estimate of the number of infected thymocytes (assuming one proviral copy per cell) was obtained by plotting signals derived from experimental DNA samples against a standard curve.
p24 ELISA
Sorted CD4+ thymocytes were inoculated with
HIV-1LAI or HIV-1Ba-L at a MOI of 0.01 for
1 h, washed extensively, and cultured in the presence of IL-2 (100
U/ml) and PHA (1 µg/ml; Sigma) for 6 days. In some
experiments, thymocytes were incubated with MIP-1ß or SDF-1 (at
various concentrations) for 4 h before infection and during the
culture. Aliquots of supernatants from infected cells were collected on
days 0, 4, and 6 postinfection and analyzed for p24 by ELISA (DuPont,
Wilmington, DE).
SDF-1 RT-PCR
Total RNA was isolated from human thymic fibroblasts or from DN, DP, and SP thymocyte subsets using RNAzol B solution (Tel-Test, Friendswood, TX). cDNA was prepared from total RNA using oligo(dT) primers (Perkin-Elmer/Cetus) and MMLV RT enzyme (Life Technologies) according to the manufacturer’s instructions. Aliquots of cDNA were amplified by PCR using Taq polymerase (Perkin-Elmer/Cetus) and primer pairs specific for SDF-1 and ß-actin. The SDF-1 mRNA-specific primers were designed to span the first three exons (39): upstream, 5'-TCC TCG TGC TGA CCG CGC TCT G; and downstream, 5'-TGC ACA CTT GTC TGT TGT TGT TCT TC. Amplification was performed for 35 cycles with an annealing temperature of 54°C for 1 min. The amplified product had a predicted size of 160 bp. The ß-actin mRNA-specific primers and PCR conditions were reported previously (38).
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Results |
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Rabbit polyclonal IgGs specific for the N-terminal regions
of CXCR4 and CCR5 were generated and were shown to bind specifically to
cells expressing these receptors and to block their fusion with HIV-1
envelope-expressing cells in a coreceptor-specific manner (28). These
reagents were used in two-color flow cytometry to determine the
expression levels of CXCR4 and CCR5 on human thymocyte subsets. Using
anti-CD3 and rabbit polyclonal anti-CXCR4 Abs, an inverse
correlation was found between the levels of expression of TCR and CXCR4
(Fig. 1
A). The highest density
of CXCR4 was found on the TCRneg and TCRlow
cells, while expression was lowest on the most mature,
TCRhigh, thymocytes. The distribution of CCR5 had a
different pattern, in that similar low level expression was found on
most thymocytes, with a significant reduction on TCRhigh
thymocytes (Fig. 1, A and B). Similar results
were obtained with FITC-conjugated anti-CCR5 Ab (mAb 2D7) and with
a polyclonal Ab directed against the second extracellular loop of CCR5.
These results were also supported by the presence of low levels of CCR5
mRNA in all thymocyte subsets (data not shown). Thus, the most mature
TCRhigh thymocytes displayed the lowest density of both
chemokine receptors.
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The most immature thymocytes (TCRneg/low) were
found to express the highest density of CXCR4. It was of interest to
define the phenotype of the thymic progenitor cells that express this
chemokine receptor. Cells with a phenotype resembling that of
multipotent hemopoietic progenitors in the bone marrow are considered
to be the earliest intrathymic T cell precursors that have the
potential to develop into T cells (40, 41, 42). To enrich for
CD34+ progenitor cells, DN thymocytes were isolated by
negative selection using CD4/CD8-magnetic bead depletion. The resultant
DN cells contained 60% CD34+ cells (Fig. 3). The
CD34+TCR- thymocytes expressed the highest
levels of CXCR4 and CCR5 compared with other thymocyte subsets (Figs. 1
and 3). The CD34-TCR- DN thymocytes
expressed CXCR4 and CCR5 at lower levels (Fig. 3). Since the mean
channel numbers for CXCR4 and CCR5 expression on
CD34+TCR- thymocytes were higher than those of
all the TCRneg/low thymocytes in the total thymocyte
analysis (Fig. 1B), it is conceivable that the
CD34+TCR- thymocytes represent the thymic
population with the highest levels of CXCR4 and CCR5. In addition, we
compared the levels of CXCR4 and CCR5 expressed on thymic
CD34+ cells with those of circulating CD34+
stem cells, which represent 2% of the total mononuclear cells in
cord blood. The mean channel numbers for CXCR4 and CCR5 on cord blood
CD34+ cells were significantly lower than the values for
intrathymic CD34+ cells (Fig. 3).
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We next tested whether thymocyte CXCR4 and CCR5 were functional.
Interaction of a chemokine receptor with its ligand induces rapid
mobilization of intracellular calcium and chemotaxis (24). CXCR4 is the
only known receptor for the chemokine SDF-1 (43, 44), while CCR5 is
a receptor for the ß chemokines MIP-1, MIP1-ß, and RANTES (23).
SDF-1 induced chemotaxis of total thymocytes over a wide concentration
range, plateauing around 500 nM. In contrast, MIP-1, MIP-1ß, and
RANTES were all relatively ineffective in the chemotaxis assay (Fig. 4A and data not shown). The
SDF-1-mediated thymocyte migration was characterized as chemotaxis
(direct movement) and not chemokinesis (increased random movement),
since in the absence of a concentration gradient, the number of
migrated thymocytes was the same as that in medium alone (Fig. 4B). In contrast, MIP-1, MIP-1ß, and RANTES were all
relatively ineffective in the chemotaxis assay. Low levels of
chemotaxis were seen only with the highest doses of chemokines (1000
nM; Fig. 4A and data not shown). Similarly, only SDF-1 (but
not MIP-1, MIP-1ß, and RANTES) at 50 nM was able to generate a
calcium flux response in total thymocytes (Fig. 4C).
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did not induce
significant calcium flux in any of the thymocyte subsets (Fig. 5A). In contrast, SDF-1 (at 50
nM) induced similar levels of calcium mobilization in DP and
CD4+ SP thymocytes (13 and 15% responding cells,
respectively), with little response in CD8+ SP (2.0%; Fig. 5B). To decipher which of the CD4+ SP thymocytes
mediate calcium flux in response to SDF-1, we gated separately on
CD4bright and CD4dull cells (the latter
representing thymocyte precursors; 40 . SDF-1 induced calcium flux
in 42% of the CD4dull thymocytes compared with only 12%
of mature CD4bright thymocytes (similar to DP thymocytes;
Fig. 5C). Thus, calcium flux in CD4+ SP
thymocytes in response to SDF-1 is mediated mainly by the immature
thymocyte precursors with low levels of CD4 expression. No
Ca2+ flux was induced by MIP-1 (at 50 nM) in any of the
CD4+ subsets (data not shown).
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D). Further analysis of the responding population employing
CD34 surface staining revealed that among the immature DN thymocytes,
88% of CD34bright and 22% of CD34-
thymocytes mobilized calcium in response to SDF-1. Altogether,
simultaneous evaluation of calcium flux with surface staining confirmed
our earlier findings with total thymocytes (Fig. 4) that the proportion
of cells mobilizing calcium in response to SDF-1 was significantly
higher in the precursor CD34+ population than in all other
thymocyte subsets. SDF-1 mRNA is expressed in thymic stromal cells
In vitro chemotaxis of thymocytes in response to SDF-1 suggested
that SDF-1 could be locally produced in the thymus. To test this
hypothesis, thymic stromal cell monolayers were propagated in vitro.
These cultures were highly enriched for fibroblasts, as demonstrated by
strong intracellular staining with rabbit anti-collagen III serum
LF-71 (Fig. 6A). SDF-1 mRNA
was highly expressed in thymic-derived fibroblasts, while thymocyte
subsets did not express SDF-1 mRNA (Fig. 6B).
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To test whether CXCR4 or CCR5 function as HIV-1 coreceptors
on thymocytes, total thymocyte target cells were mixed with 12E1
effector cells infected with recombinant vaccinia viruses expressing
either T-tropic (IIIB) or M-tropic (JR-FL, ADA, Ba-L) Envs, and
syncytium formation was quantified in the presence and in the absence
of chemokines and antisera against CXCR4 and CCR5. Syncytium formation
was seen in cultures containing effector cells expressing the
prototypic T-tropic IIIB Env (Table I).
The involvement of CXCR4 in fusion was confirmed by specific blocking
(50–70%) in the presence of SDF-1 (1 µg/ml), but not RANTES (Table I). In addition, rabbit CXCR4-specific polyclonal IgG specifically
inhibited the fusion activity by 55%. Preimmune antiserum and
anti-CCR5 antiserum had no effect.
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To determine whether thymocytes are susceptible to infection with
T-tropic and M-tropic viral strains, the DP and SP thymocytes from
normal pediatric donors were infected in vitro with
HIV-1LAI and HIV-1Ba-L (MOI of 0.01), and
24 h later were analyzed for the presence of viral DNA as a
measurement of viral entry. When DNA was extracted from thymocytes
immediately after infection (time zero), no signal was detected,
demonstrating that no residual proviral DNA was present in the virus
stock preparations (Fig. 7). On the other
hand, proviral DNA was detected for both viruses in thymocytes after
24 h (Fig. 7A). The increase in signal was reduced by
carrying on the infection in the presence of 3'-azido-3-deoxythymidine
(AZT) (data not shown). To estimate the efficiency of viral
entry, DNA was extracted from serially diluted ACH-2 cells (containing
one provirus copy per cell), PCR amplified, and used to generate a
standard curve (Fig. 7, B and C). LAI proviral
DNA was detected in DP and SP thymocytes (Fig. 7A). However,
DP cells contained a 10-fold higher number of cells with proviral DNA
than the CD4+ SP population (0.1 and 0.01% of infected
cells, respectively; Fig. 7D). In contrast, infection with
the M-tropic strain Ba-L was detected at similar levels (0.001–0.002%
of cells) in the subsets of SP and DP thymocytes (Fig. 7, A
and D). Two additional M-tropic strains (AD and JR-CSF)
infected SP CD4+ thymocytes with even lower efficiency
(data not shown). These data demonstrate that the efficiency of T- and
M-tropic viral entry may correlate with co-receptor expression levels.
The mature SP CD4+ thymocytes are similarly susceptible to
infection with T- and M-tropic viruses, but immature DP thymocytes
(which form the largest pool of thymocytes) are 10-fold more
susceptible to infection with T-tropic than to infection with M-tropic
strains.
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). The levels of p24 produced increased
dramatically by day 6 and reached 23 ± 5 and 29 ± 1 ng/ml
for HIV-1LAI and HIV-1Ba-L, respectively (Fig. 8).
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Since CD4+ SP thymocytes were productively infected
with HIV-1Ba-L, it was of interest to determine whether
CCR5 plays a role in M-tropic virus infection of thymocytes. To address
this question, CD4+ SP thymocytes were incubated with
chemokines 4 h before viral adsorption and in the course of
infection. MIP-1ß inhibited p24 production by
HIV-1Ba-L-infected thymocytes in a dose-dependent manner
(Table II). At the same time, no
reduction in the levels of p24 in the cultures of
HIV-1Ba-L-infected thymocytes was observed in the presence
of SDF-1 at 100 ng/ml (Table II). These data provide direct evidence
for the role of CCR5 in the infection of thymocytes with M-tropic
HIV-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induced
Ca2+ mobilization and chemotaxis in total thymocytes. In
contrast, no Ca2+ mobilization and only minimal chemotaxis
were observed in response to several ß-chemokines known to bind to
CCR5. However, despite these differences in signaling, both CXCR4 and
CCR5 supported HIV-1 infection of human thymocytes. High surface expression of CXCR4 was found on immature thymocytes and was significantly reduced on the mature SP CD4+ and CD8+ subsets. Consistent with this, calcium mobilization in response to SDF-1 was detected in immature DN and DP subsets, but was greatly diminished in mature SP CD4+ thymocytes. Although all immature thymocytes expressed similar levels of CXCR4, a significantly higher percentage of the DN subset compared with the DP subset demonstrated calcium mobilization in response to SDF-1. This indicates that the level of expression of this receptor does not necessarily correlate with its ability to mobilize Ca2+ in response to its ligand. It also suggests that during thymocyte maturation, shutdown of CXCR4 signaling may precede the reduction of surface CXCR4 expression. Our finding of the inverse correlation between the level of thymocyte maturity and CXCR4 surface expression is in agreement with a recent report by Kitchen and Zack (45).
CD34+ cells appear to be the earliest intrathymic precursors, and low levels of CD4 were detected on a subset of thymic CD34+ cells (40). As demonstrated in the present study, the CD34+ thymocyte progenitors expressed the highest density of CXCR4, which correlated with the high proportion of CD34+ cells (90%) mobilizing Ca2+ in response to SDF-1. In our study, the CD4dull, but not the CD4bright, SP thymocytes demonstrated calcium flux in response to SDF-1 and expressed high levels of CXCR4. Thus, CD4lowCD34+CXCR4high thymocyte precursors may represent a vulnerable target population for infection with HIV-1 primary isolates that can use CXCR4 for cell entry. Infection of this particular subset is expected to curtail thymopoiesis and to result in the depletion of thymic output.
A previous report demonstrated a potent chemoattraction of SDF-1 for bone marrow-derived CD34+ progenitor cells, but not for peripheral blood-derived CD34+ progenitor cells (46). We found that CD34+ stem cells in cord blood expressed much lower levels of CXCR4 than thymus-derived CD34+ cells. Together, the data suggest that CD34+ stem cells are heterogeneous in their surface expression of CXCR4, and that thymic CD34+ progenitor cells may be more similar to bone marrow-derived CD34+ cells than to circulating CD34+ stem cells in terms of their surface CXCR4 expression and its activity.
The finding of SDF-1 mRNA expression in thymic-derived stromal cells supports and extends the report by Shirozu et al. that SDF-1 mRNA is present in the thymic tissue but is not detected in PBL (39). Studies in knockout mice demonstrated that SDF-1 plays a critical role in B cell development and suggested that it does not affect thymopoiesis in an embryo (47). However, since SDF-1-/- mice die prenatally (days 15–18 after conception), no data on thymic cellularity in SDF-1-/- mice after birth are available. It is possible that SDF-1 plays a role in attracting thymocyte progenitors from the bone marrow to the subcortical zone in the thymus after birth. It remains to be determined whether SDF-1/CXCR4 interactions are important factors in intrathymic development. The gradual decrease in CXCR4 signaling and expression along with thymocyte maturation into SP TCRhigh cells might be required to allow their exit from the thymus via the afferent lymphatics.
Unlike CXCR4, CCR5 was expressed at relatively low levels on the
majority of thymocytes (with significant reduction on mature SP
subset). Moreover, no evidence of signaling through CCR5 was detected
in either of the two calcium mobilization assays using 50 nM
ß-chemokines. Similarly, only limited chemotaxis in response to
ß-chemokines was observed in response to a high dose of MIP-1
(1000 nM), suggesting that high concentrations of ß-chemokines may
induce signaling in thymocytes.
To determine whether expression of coreceptors by thymocytes
correlates with their sensitivity to HIV-1 infection in vitro, three
assays were employed. In the cell fusion assay, large numbers of
syncytia were formed between thymocytes and T-tropic Env-expressing
effector cells. Importantly, a significant reduction (50–70%) in the
number of syncytia was observed in the presence of SDF-1 or our
rabbit anti-CXCR4 IgG, demonstrating the direct involvement of
surface CXCR4 in the fusion process. The lack of complete blocking
suggests that receptors other than CXCR4 may also be involved in
thymocyte fusion with T-tropic viruses. In the PCR-based viral entry
assay, both DP and CD4+ SP thymocytes became infected with
the T-tropic HIV-1LAI, but entry into the DP population was
10-fold more efficient than that into the SP subset, consistent with
the relative levels of their CXCR4 surface expression. In the same
viral entry assay, HIV-1LAI and HIV-1Ba-L
proviral DNA were detected in the SP subset 24 h after viral
exposure. The p24 assay confirmed the finding that CD4+ SP
thymocytes can support a productive infection with both T-tropic and
M-tropic strains.
To determine whether M-tropic HIV-1 uses CCR5 on human thymocytes,
CD4+ SP thymocytes were infected with HIV-1Ba-L
in the absence or the presence of ß-chemokines (or SDF-1) as
inhibitors. It was found that MIP-1ß, but not SDF-1, inhibited
productive infection of SP thymocytes with the M-tropic
HIV-1Ba-L viral strain, suggesting that CCR5 functions as
an HIV-1 coreceptor on mature thymocytes. The dissociation between CCR5
signaling and HIV-1 coreceptor function found in our study is in
agreement with previous reports that CCR5 HIV-1 coreceptor function is
separate from CCR5-mediated signal transduction (48).
Studies from several laboratories demonstrated that thymocytes at various stages of differentiation are susceptible to HIV-1 infection (11, 12, 13, 14, 15, 16, 18, 49, 50). However, the effects on thymopoiesis of infection with T- or M-tropic viral strains are significantly different. This difference could be in part explained by the pattern of coreceptor expression on thymocyte subsets and on the ability of the coreceptors to support HIV-1 infection. Our results are in agreement with studies that have demonstrated that T-tropic isolates infect predominantly immature TCRneg/low thymocytes both in vitro (11, 49) and in vivo in the SCID-hu mouse model (17, 18, 19). Destruction of these subsets was suggested to play a critical role in the interruption of thymopoiesis. Unlike infection with T-tropic viral isolates, infection with M-tropic isolates (JR-CSF, JR-FL, and Ba-L), as reported by Kollmann et al. (19), or with primary isolates (EW and JD), as reported by Su et al. (18), caused delayed depletion of thymic cellularity in the SCID-hu models. These viral strains were recently shown to use primarily CCR5 and to a lesser degree other ß-chemokine receptors (CCR2b and CCR3) for cell entry (25, 51). In studies by others, the kinetics of in vitro infection with JR-CSF were much slower than those of the T-tropic isolate NL4–3 (13, 50). These results suggest that mature thymocytes are the primary targets for M-tropic infection. It was also shown that thymus-derived dendritic cells are susceptible to infection with M-tropic, but not T-tropic, HIV-1 strains (52), suggesting that M-tropic viruses may initially enter the thymus through dendritic cells, which could then transmit virus to the mature CD4+ thymocytes.
In addition to CCR5 and CXCR4, other recently described chemokine receptors, STRL-33, CCR8, gpr1, and gpr15 (53, 54, 55, 56), may also support infection of thymocytes with different viral strains.
In conclusion, our data may provide an explanation for the rapid progression of HIV-1 infection to AIDS in some pediatric patients (3, 4, 5). The vertically transmitted M-tropic viruses may enter thymuses in infants by infected dendritic cells and/or mature CD4+ thymocytes without inducing interruption of thymopoiesis. However, once fast replicating, CXCR4-using, T-tropic viruses emerge, they can infect immature thymocytes (including CD34+CD4low thymocyte progenitors), which express high levels of CXCR4, leading to a rapid depletion of thymic cellularity, which results in severe impairment of export of naive T cells from the thymus to the periphery. The role of the thymus in adults is not as critical as it is in children, although thymic tissue may remain at least partially functional (57). The thymus of the HIV-1-infected adult may be able to seed the lymphoid organs with T-cell precursors during the early phases of infection with M-tropic variants (34, 58), which are unlikely to interfere with thymopoiesis. Thus, early intervention with effective antiviral therapies may allow preservation and/or reconstitution of the immune system, in part through effects on the thymus.
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Acknowledgments |
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Footnotes |
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2 M.B.Z and S.L. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Marina Zaitseva and Dr. Shirley Lee, Division of Viral Products, Food and Drug Administration, Center for Biologics Evaluation and Research, Building 29B, 8800 Rockville Pike, Bethesda, MD 20892.
4 Abbreviations used in this paper: M-tropic, macrophage-tropic; T-tropic, T cell line-tropic; DN, double negative; DP, double postive; SP, single positive; SDF-1, stroma-derived factor-1; PE, phycoerythrin; Tc, tri-color; MIP-1ß, macrophage inflammatory protein-1ß; MOI, multiplicity of infection; TCRneg, no T cell receptor; TCRlow, low level of T cell receptor; TCRhigh, high level of T cell receptor; CD4bright, bright staining for CD4; CD4dull, dull staining for CD4.
5 R. L. Rabin, M. K. Prk, F. Liao, R. Swofford, D. Stephany, and J. M. Farber. CC and CXC chemokines target distinct lymphoid subsets. Submitted for publication.
Received for publication December 9, 1997. Accepted for publication May 11, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+ and TCR
+ to human immunodeficiency virus infection: a mechanism for CD4+ (T4) lymphocyte depletion. Proc. Natl. Acad. Sci. USA 87:7727., MIP-1ß receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955.[Abstract]
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