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Divisions of
*
Immunology and
Pathology, New England Regional Primate Research Center, Harvard Medical School, Southborough, MA 01772; and
AIDS Research Center and Infectious Disease Unit, Massachusetts General Hospital, Charlestown, MA 02129
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Abstract |
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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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Evidence documents that the thymus is a major target of HIV and SIV infection and that infection may involve both thymocytes and thymic epithelial cells (1, 2). In vitro studies have shown that HIV-1 can infect thymocytes from uninfected subjects, and in vivo studies in the SCID-hu chimera model have shown that immature thymocytes are infected by HIV-1 and implicate HIV as the direct (3, 4, 5) and indirect (6, 7) cause of thymocyte death. Although information on infection of thymocytes in vivo is limited, a report analyzing thymic tissue obtained from an infant with perinatal HIV-1 infection demonstrated infection in immature thymocytes (1). Pathologic abnormalities in the thymus, such as thymic involution and thymocyte depletion, are common findings at autopsy (8, 9), although the significance of these observations is complicated by the fact that thymic involution and atrophy may occur as a sequel to the debilitating opportunistic infections that accompany AIDS. In addition to HIV infection of thymocytes, data from SCID-hu mice (4) demonstrated the presence of HIV RNA in thymic epithelial cells and degeneration of thymic epithelium. Taken together, these findings suggest that HIV infection of the thymus is likely to thwart efforts to reconstitute immune function in HIV-infected individuals. This is further complicated by the fact that the predominant population of HIV-infected individuals are adults, who, as a consequence of their age, have less residual thymic function, which impacts on the ultimate regenerative capacity of their immune system. This has been further illustrated in studies that have evaluated thymic function in HIV-infected individuals (10) and SIV-infected macaques (11) by measuring TCR excionsal circles. These studies documented a reduced number of TCR exscionsal circles correlated with poor CD4 recovery after antiretroviral therapy and with reduced thymic function as a consequence of HIV or SIV infection.
The exact mechanisms of HIV/SIV-induced thymocyte depletion have not been well characterized, and the relative contributions of direct vs indirect mechanisms of thymocyte depletion remain controversial. Infection of thymocytes by SIV and HIV support direct mechanisms of apoptosis. However, during thymocyte maturation both bcl-2 and Fas-Fas ligand pathways contribute to the regulation of apoptosis during both positive and negative selection (12, 13, 14), and disruption of these pathways as a consequence of retroviral infection may contribute to increased apoptosis. In these studies, we evaluated the role of bcl-2 and Fas in SIV-induced thymocyte depletion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten newborn rhesus macaques (Macaca mulatta) were housed in accordance with standards of the Association for Assessment and Accreditation of Laboratory Animal Care. The investigators adhered to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The animals were born to dams that were negative for Abs to HIV-1, SIV, type D retrovirus, and simian T cell leukemia virus type 1.
Animals were i.v. inoculated with equal doses (10 ng of SIVp27) of a
pathogenic (SIVmac239) molecular clone of SIV. This is 
20 ng/kg p27,
which is equivalent to the dose used in adult animals. In addition,
four healthy, age- and sex-matched rhesus monkeys were sacrificed as
normal controls. SIVmac239 is a pathogenic molecular clone of SIV
(15).
Quantitation of viral load
Peripheral blood was collected for viral isolation before inoculation and at days 3, 7, 14, 21, 35, and 50 after inoculation. Quantitative viral cultures were performed as described previously (16).
Briefly, serial 3-fold dilutions were performed in duplicate beginning with 106 PBMC. PBMC dilutions were cocultured with 105 CEMX174 cells in a volume of 1 ml. Cultures were split 1:2 twice weekly until day 21, when the cultures were assayed for virus production by enzyme immunoassay for SIV p27 (Coulter, Miami, FL). Results are expressed as the number of SIV+ cells/106 PBMC.
Virion-associated SIV RNA in plasma was quantified by using a real-time RT-PCR assay on an Applied Biosystems Prism 7700 sequence detection system (Foster City, CA) as described previously (17, 18). Results shown are averages of duplicate determinations. Analyses of viral RNA levels were performed by Drs. Jeffrey Lifson and Michael Piatak (Science Applications International Corp.-Frederick, Frederick, MD).
Tissue collection and processing
Two animals from each group were sacrificed at 3, 7, 14, 21, and 50 days postinfection by i.v. injection of sodium pentobarbital. Thymus and other tissues were collected in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin by routine histologic techniques. Adjacent sections were subjected to in situ hybridization and immunohistochemistry. Adjacent blocks of fresh tissue were collected for flow cytometry and snap-frozen in optimum cutting temperature compound (OCT, Miles, Elkhart, IN) by immersion in 2-methylbutane cooled in dry ice for immunohistochemistry.
Flow cytometric analysis of thymocyte progenitors
Thymic tissue was obtained at the time of euthanasia, minced into small fragments, and then digested into a single-cell suspension by incubation in PBS with 0.5 mg/ml of collagenase (Sigma, St. Louis, MO) and 2 U/ml DNase1 (Sigma) at 37°C for 60 min with frequent agitation. The cell suspension was then washed once in PBS with 2% normal mouse serum and filtered through a 70-µm nylon mesh. Abs used for immunophenotyping of rhesus thymocytes included anti-CD3 (6G12) (kindly provided by J. Wong, Massachusetts General Hospital) (19), anti-CD4 (OKT4) (Ortho Diagnostics, Raritan, NJ), anti-CD8 (Leu-2a) (Becton Dickinson, San Diego, CA), anti-MHC class I (w632) (Dako, Carpinteria, CA), anti-HLA-A, B, C (PharMingen, San Diego, CA), anti-Fas (Immunotech, Miami, FL), anti-Fas ligand (PharMingen), and anti-CD34 (Qbend-10) (Immunotech). Cells were stained in the presence of staining media (PBS with 2% mouse serum). After Ab staining, the cells were fixed with fresh 2% paraformaldehyde. Three- and four-color flow cytometry analysis of the cells was performed using a FACScan with CellQuest software (Becton Dickinson).
For intracellular cytokine staining, previously cryopreserved thymus
cells were quick thawed and washed three times in PBS (Life
Technologies, Grand Island, NY). Cells were surface stained with
anti-CD4 FITC (custom conjugate), anti-CD4 PE (custom
conjugate), and anti-CD8 peridinin chlorophyll protein (Becton
Dickinson) for 30 min at 4°C in the dark. Cells were washed once with
stain media (2% normal mouse serum (Sigma) in PBS). One hundred
microliters of reagent A from the Fix and Perm cell permeabilization
kit (Caltag, South San Francisco, CA) was added to each tube. Cells
were incubated for 15 min at room temperature in the dark. Cells were
washed once. One hundred microliters of reagent B from the Fix and Perm
cell permeabilization kit (Caltag) was added to each tube. The
appropriate intracellular cytokine Ab conjugates were also added (rat
IgG2a PE, IL-10 PE, IL-2 PE, IL-4 PE, IL-7 PE, IL-12 PE, IFN-
PE,
and TNF-
PE, all from PharMingen). Cells were incubated for 15 min
at room temperature in the dark. Samples were washed once in stain
media as before. Cells were resuspended in 250 µl of 2%
paraformaldehyde and then analyzed using a FACScalibur (Becton
Dickinson).
For the detection of apoptosis using TdT, isolated thymocytes were
stained (1 x 106 cells/tube) for the
presence of surface CD4 and CD8 before their permeabilization/fixation
with 0.1% Tween 20 in 2% paraformaldehyde (37°C for 30 min,
followed by 30 min at room temperature) for the subsequent assessment
of apoptosis employing Oncor’s ApopTag kit (Oncor, Gaithersburg, MD).
Each tube was given 76 µl equilibration buffer and gently vortexed,
and then 3 ml of PBS was added. The tubes were spun at 1500 rpm for
5 min at 25°C as is the case for all centrifugations in the
protocol.
The liquid was aspirated and each tube’s content resuspended in 38 µl reaction buffer and 16 µl TdT enzyme. The tubes were capped, vortexed, and placed in a 37°C heat block (covered with foil) for 30 min. Then 3 ml/tube of the stop/wash buffer were added upon removal from the heat block. The tubes were centrifuged, the liquid aspirated off, and the cells were resuspended in 56 µl blocking solution and 49 µl antidigoxigen FITC Ab and then incubated at room temperature for 30 min. The excess Ab was removed by two 0.1% Triton X-100/PBS washes, and the cells were resuspended in 300 µl/tube of 2% paraformaldehyde for flow analysis using a FACScalibur (Becton Dickinson) the next day. Analysis was performed through a "live" gate established on forward scatter (FSC)3/side scatter (SSC) plots to exclude debris and monocytic macrophages.
For detection of apoptosis using Phi Phi Lux (OncoImmunne, Gaithersburg, MD), 2 x 106 thymocytes/tube were aliquoted into 1.5-ml conical microcentrifuge tubes. Microfuge tubes were spun at 1200 rpm for 7 min at 4°C to allow for removal of all media by vacuum aspiration. Then 150 µl of the 10 µM Phi Phi Lux G1D2 caspase substrate and 10 µl FBS were then added to each tube. The cells were gently resuspended. The open tubes were incubated in a 5% CO2 incubator at 37°C for 60 min in the dark.
Cells were washed with 500 µl/tube of OncoImmune and resuspended in 500 µl/106 cells of fresh flow cytometry media and placed on ice for FACS analysis. For additional surface staining, the cells were resuspended in 50 µl flow cytometry media with the appropriate amount of Ab. The cells were incubated on ice for 15 min, washed, spun, and resuspended in 500 µl flow cytometry media for FACS analysis using a FACScalibur (Becton Dickinson). Analysis was performed through a live gate established on FSC/SSC plots to exclude debris and larger monocytic macrophages.
Localization of SIV-infected cells
Localization of infected cells was performed by immunohistochemistry for viral Ags and in situ hybridization for viral DNA and RNA. In situ hybridization was performed on formalin-fixed paraffin-embedded sections. The DNA probe used was labeled with digoxigenin-11-dUTP by random priming (Boehringer Mannheim, Indianapolis, IN) as previously described (2, 16). Sections were examined microscopically and scored semiquantitatively on a scale as follows. The absence of positive cells was given a score of negative; 1–5 positive cells per section were given a score of 1+; 5–15 positive cells per 10x field were given a score of 2+; 15–30 positive cells per 10x field were given a score of 3+; and >30 positive cells per 10x field were given a score of 4+.
Adjacent snap-frozen blocks of tissue were used in immunohistochemical procedures to localize virus as previously described (2, 20, 21). Briefly, tissue sections cut at 6 µm were fixed in 2% paraformaldehyde and immunostained using an avidin-biotin-HRP complex technique with diaminobenzidine as the chromogen. The primary Ab used was Senv71.1 (a gift from C. Colignon and C. Thiriart, SmithKline Beacham, Belgium), which recognizes SIVgp120.
Immunophenotype of infected cells
To examine the expression of bcl-2 in relation to the presence of SIV-infected cells, we performed double labels for SIVgp120 (Senv71.1) and bCL-2 (Becton Dickinson). Briefly, immunohistochemistry for SIVgp120 was performed as described previously (2) using Vector red (Vector Laboratories, Burlingame, CA) followed by monoclonal anti-bCL-2 directly conjugated to FITC. The slides were then examined by immunofluorescence and confocal microscopy taking advantage of the intense fluorescence of Vector red. Controls consisted of thymus from uninfected age-matched controls and substitution of isotype-matched Ig for primary Ab.
Confocal microscopy was performed using a Leica TCS SP laser scanning microscope (Leica Microsystems, Exton, PA) fitted with a x100 Leica objective (PL APO, 1.4NA) and using the Leica image software. Images were collected at 512 x 512 pixel resolution. The stained cells were optically sectioned in the z-axis, and the images in the different channels (photomultiplier tubes) were collected simultaneously. The step size in the z-axis was varied from 0.2 to 0.5 µm to obtain 30–50 optical sections per imaged field.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the early effects of SIV on the neonatal thymus, 10 newborn macaques were injected i.v. with 10 ng of SIV p27. This is approximately equivalent to 20 ng/kg of SIV p27, which is an equivalent dose to what we have previously used in juvenile and adult animals (2). Two animals from each group were sacrificed at 3, 7, 14, 21, and 50 postinfection, and thymus was collected for flow cytometry, histopathology, immunohistochemistry, and in situ hybridization. Peripheral blood was collected at these same time points and used to evaluate cell-associated viral load and lymphocyte subset analysis in all animals alive at that time. Four additional, healthy age-matched animals were sacrificed as normal controls.
SIV isolation and quantitation
Virus was recovered from all animals by 3 days postinfection.
Viral loads rose quickly and peaked at 7–14 days postinfection, (Fig. 1
). Cell-associated viral loads in
neonates were as high as 4 x 104
virus-positive cells per 106 PBMC, which is as
high or higher than what is typically observed in juvenile or adult
animals infected with the same viral stocks, but the difference did not
reach statistical significance (data not shown).
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Coincident with peak viral loads at 7 days postinfection, virus
was detected in the thymic medulla by immunohistochemistry for SIVgp120
and in situ hybridization for SIV nucleic acid (Table I and Fig. 2). Most of the infected cells were
present in the thymic medulla with rare positive cells in the cortex
consistent with previous observations (2, 22, 23).
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) and 50 days (data not shown)
postinfection. The apparent thinning of the cortex and subsequent
rebound in cortical thickness occurred coincident with alterations in
thymocyte apoptosis (see below).
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Alterations in thymocyte subpopulations was evident at 3 days
postinfection, where we observed a decrease in the relative frequency
of CD4+CD8+ double-positive
cells. This subset continued to decline with a nadir at 21 days
postinfection followed by a return to baseline levels by 50 days
postinfection (Fig. 4A). This
relative decrease in double-positive cells was accompanied by a
relative increase in
CD4-CD8+ single-positive
cells (from 2% at baseline to 7% by day 21), and a relative increase
in CD4-CD8- cells (from
1% at baseline to 15% by day 21) (Fig. 4B). Using three-
and four-color flow cytometry, it was evident that a significant number
of the CD4- CD8- cells
were in fact CD34+ progenitors (data not shown).
Consistent with our previous observations in juvenile macaques infected
with SIV (2), we observed a rebound phenomenon in thymic T
progenitors during the course of acute SIV infection. The most profound
alterations observed both by histology and flow cytometry were evident
by 14–21 days postinfection. Thymic atrophy and involution was
followed by profound increases in CD34+
progenitors at 14 and 21 days postinfection and restoration of normal
thymic architecture as well as thymocyte phenotype. By day 50
postinfection, the frequency of CD34+ T
progenitors in the thymus had also decreased to levels observed before
SIV infection (Fig. 4A). The phenotypic changes described
above were not detected in normal age-matched controls (data not
shown), demonstrating that these fluctuations were a consequence of SIV
infection.
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We analyzed the expression of MHC-class I on double-positive and
single-positive thymocytes in normal and SIV-infected neonates using
two Abs that cross-react with rhesus epitopes. As previously described
by others (7), we detected a lower level of MHC class I
expression on CD4+CD8+
thymocytes compared with CD4+ and
CD8+ thymocytes in normal thymic tissue (Fig. 5). In tissue derived from age-matched
SIV-infected (day 21 postinfection) neonates, the level of MHC class I
molecules on double-positive cells was similar to uninfected controls.
In contrast, in SIV-infected animals the difference in surface MHC
class I expression was most evident in CD4+
single-positive cells where thymocytes from SIV-infected animals
expressed levels of class I MHC in a range comparable to
CD4+CD8+ double-positive
thymocytes. To control for differences that may result from instrument
settings, samples from naive and SIV-infected neonates were stained and
analyzed on the same day using the same instrument settings. As
alterations in the microenvironment have been incriminated in inducing
change in MHC class I expression (7), we also examined
cytokine levels in thymus from SIV-infected neonates. Additional
unstimulated samples were stained to detect intracellular levels of
IL-2, IL-4, IL-7, IL-10, IL-12, IFN-, and TNF-. No significant
differences were detected in thymus from SIV-infected neonates as
compared with uninfected animals (data not shown). As we and others
have previously published (2, 22, 23), the number of
thymocytes infected with SIV is <5% of cells, whereas MHC class I
down-regulation affects >70% of all thymocytes. Thus, down-regulation
of MHC-class I in the thymus is likely an indirect effect of SIV
infection, in contrast to the direct effects reported due to infection
of cells with either HIV or SIV (24).
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SIV infection contributes to an increase in apoptosis during acute
SIV infection (2). As shown in Figs. 6A and
7, we were able to detect an increase in
apoptotic cells 3 days postinfection using TdT staining and detection
by flow cytometry. The frequency of TdT+ cells
peaked at 14 days postinfection, coincident with the peak viremia and
thymic involution (Figs. 1
and 7). In addition, the
TdT+ cells were more frequent in
CD4+ and
CD4+CD8+ cells than
CD8+ cells (data not shown). This population bias
suggests an association with SIV replication.
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B, thymus from
SIV-infected macaques demonstrates a marked increase in the activity of
mitochondrial caspases, indicative of apoptosis. This increase in
activity is biased toward CD4+ cells, although it
is increased in all subsets. This confirms the observations made using
TdT to detect apoptosis. In addition, this observation is supportive of
indirect mechanisms associated with the induction of apoptosis as the
frequency of apoptotic cells exceeds the relative number of
SIV-infected cells. Furthermore, as previously described
(2), most infected cells are in the thymic medulla,
whereas the majority of changes we observed are in immature thymocytes
that would be located in the thymic cortex. Alterations in Fas, Fas ligand, and bcl-2 in the thymus of SIV-infected neonates
Thymocyte depletion during acute SIV infection is accompanied by
an increase in apoptosis as determined by TdT incorporation of labeled
nucleotides as well as increased mitochondrial caspases activity. The
frequency of apoptotic cells increased dramatically shortly after peak
viral loads were observed in both blood and tissues including the
thymus. We examined the two major pathways associated with apoptosis,
namely Fas and bcl-2. The surface levels of Fas on thymocytes from
normal neonates is generally low (1–3% of all cells) and does not
demonstrate any specific phenotypic distribution (data not shown).
After SIV infection, the level of surface Fas changes modestly,
increasing to a maximum of 8% of thymocytes on day 14 postinfection
(Fig. 8A). Although the
overall alterations in surface Fas expression were modulated in all
thymocytes, when gating on single-positive cells more significant
fluctuations were detected. The level of Fas on
CD4+ single-positive thymocytes increased four to
five times when compared with either
CD4+CD8+ thymocytes or
CD8+ single-positive cells (data not shown). In
normal age-matched neonates, the level of Fas expression was generally
equivalent in CD4+ and CD8+
single-positive thymocytes. This data demonstrates that up-regulation
of Fas on CD4+ thymocytes may contribute to an
increase in apoptosis of this subset. This was accompanied by increases
in surface expression of Fas ligand predominantly on
CD4+CD8+ and
CD8+ single-positive thymocytes, particularly at
14 and 21 days postinfection (Fig. 8A), demonstrating modest
fluctuations in both Fas and Fas ligand in acute SIV infection.
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).
However, it was difficult to quantitate bcl-2 levels in various
thymocyte subsets using this technique. Therefore, we analyzed bcl-2
expression using flow cytometry where levels could be quantitated in
various subsets using mean channel fluorescence.
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B). The lowest levels of bcl-2 occur coincident with
the largest amount of apoptosis, detected by TdT and Phi Phi Lux
staining. By day 21, the levels of virus have reached a set point, and
this is associated with the resumption of normal thymic function.
Specifically, by day 21, the number of apoptotic cells is drastically
reduced, and the number of bcl-2 cells begins to approach baseline
levels. The level of bcl-2 on a per cell basis recovers only by day 50
postinfection. Based on these observations, it would appear that
dysregulation of the bcl-2 pathway is the predominant mechanism
associated with the apoptosis that accompanies retroviral infection of
the thymus. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As programmed cell death is an integral feature of normal thymocyte development, we examined the frequency of apoptotic cells in normal and SIV-infected neonatal thymus. To further determine the potential pathways associated with alterations in the number of apoptotic thymocytes, we determined the levels of Fas, Fas ligand, and bcl-2 in both normal and SIV-infected neonatal tissue.
Destruction of the thymus has been demonstrated to contribute to a failure to regenerate new T cells during HIV or SIV infection and thus impact directly on the course of disease. Understanding how thymopoiesis is disrupted is the first step to design interventive strategies that may aid in sparing the thymus during retroviral infection. Consistent with previous observations (2), we detected a rapid increase in the frequency of apoptotic thymocytes during neonatal SIV infection. In general, this occurred shortly after peak viral loads were detected. A greater number of apoptotic cells were detected using flow cytometry, and this was coincident with thymic atrophy detected by histology. The two major mechanisms of apoptosis are associated with either up-regulation of Fas expression, which allows Fas ligand to induce a signal for apoptosis, or bcl-2, which protects cells from apoptosis-inducing signals. We examined the levels of both Fas and bcl-2 expression in the thymus of SIV-infected neonates and were able to demonstrate that the levels of bcl-2 are substantially decreased just before and during the period of peak apoptosis. Both the frequency of bcl-2-positive cells and the level of bcl-2 on a per cell basis were found to be significantly decreased. This mechanism is observed in normal T cell ontogeny, where cells that are not positively selected in the thymus down-regulate bcl-2 expression (12). In concert with this, bcl-2 is up-regulated or induced to re-express in cells that are destined to undergo positive selection (12, 13). However, these fluctuations are much more pronounced in acute SIV infection and are observed in the majority of thymocytes. Again, consistent with the fact that productively SIV-infected cells are detectable at a low frequency using in situ hybridization and immunohistochemistry (1, 2), an indirect mechanism must be implicated as being associated with this down-regulation of bcl-2 expression. This would imply that the presence of SIV in only a small number of cells within the thymus is adequate to disrupt the microenvironment, and this in turn results in decreased bcl-2 expression and thus increased apoptosis. The accompanying increase in cellular proliferation associated with thymocyte apoptosis may contribute to some of the alterations observed in bcl-2 expression, although the decrease in bcl-2 observed was evident to some extent in all thymocyte subsets, including double-positive and single-positive cells. Of potential relevance is our previous observation of infection of cells of monocyte-macrophage lineage in the thymus of SIV-infected macaques (2). These cells contribute to establishing the thymic microenvironment, and so retroviral infection of these cells may further disrupt this environment. It should be further noted that bcl-2 is down-regulated in both CD4+CD8+ double-positive and CD4+ or CD8+ single-positive thymocytes. However, it is predicted that the consequence of bcl-2 down-regulation will be the most overt in CD4+CD8+ double-positive cells, as bcl-2 participates to a lesser degree in the apoptosis mechanisms in single-positive thymocytes. Furthermore, the bulk of negative selection occurs during the double-positive stage. Double-positive thymocytes also comprise the major population of cells in the thymus, so disruption of this subset will most likely result in substantial effects at the immature stage, but depletion of these cells will have obvious consequences on the generation of single-positive T cells.
It is well documented that Fas contributes to the induction of apoptosis in single-positive thymocytes and peripheral T cells (14, 26, 27, 28). In general, Fas is expressed at low levels in normal thymocytes. When we examined bulk thymocytes, modest differences in Fas expression were evident. However, when we examined the level of Fas in single-positive thymocytes, we detected a striking difference, with much greater increases in surface Fas on CD4+ single-positive thymocytes. This is in fact consistent with what might be expected based on the fact that Fas contributes to cell death of single-positive and peripheral T cells and not double-positive thymocytes, as well as the fact that CD4+ thymocytes are the primary target of HIV and SIV infection (26, 27, 28). It would be predicted that this is again a consequence of an indirect mechanism, as the frequency of cells with increased surface Fas expression appears to exceed the number of SIV-infected cells. The contribution of proliferating cells to alterations in Fas expression cannot be excluded.
It has recently been reported that Nef is capable of inducing Fas expression in T lymphoid cells (14). Although it is unlikely that all CD4+ single-positive thymocytes with increased Fas expression are directly infected, it is possible that this may be one of the mechanisms responsible for increased Fas expression in this subset of cells, as both intracellular and extracellular Nef have been shown to induce Fas expression. As predicted by studies in normal animals, SIV infection results in disruption of the antiapoptotic pathway governed by bcl-2 in double-positive thymocytes and the Fas apoptosis-inducing pathway in single-positive CD4+ thymocytes.
It has been demonstrated that apoptosis may be a favorable response in
HIV infection where the virus is induced to replicate in dying cells
(29). Alternatively, it has been postulated that the
induction of Fas expression may result in death of Fas
ligand-positive CTL to either SIV or HIV infection (14).
This may then facilitate immune evasion of CD4+
SIV or HIV-infected single-positive thymocytes, and these cells could
enter the peripheral circulation, contributing to the dissemination
of virally infected cells to distant sites. An additional mechanism of
immune evasion that has recently been reported in PBLs is the
down-regulation of MHC class I molecules (30, 31). This
has been shown to be a direct consequence of HIV or SIV infection,
predominately as a consequence of Nef. The down-regulation of MHC class
I prevents complete recognition of the cell by Ag-specific CTL,
allowing them to escape immunosurveillance. This is similar to our
observation in thymocytes of SIV-infected neonates where we detected a
decrease in MHC class I expression, although in this in vivo model it
is unlikely to only be a direct effect as the frequency of SIV-infected
cells is too low to account for the global down-regulation of MHC class
I. In addition, this was not detected in animals infected with
SIVmac239
Nef, a less pathogenic deletion mutant of SIV (data not
shown). It is feasible that an indirect mechanism contributes to this
phenomenon in SIV-infected monkeys and that this in turn contributes to
evasion of the immune response. This is in contrast to a recent report
that demonstrated an increase in MHC class I expression in the thymus
during HIV-1 infection (7). It should be noted that this
observation was in the SCID-hu model in the context of HIV and the
absence of an immune response. The situation in vivo in an animal with
a relatively intact immune system (albeit immature) in the context of
acute SIV infection may in fact be different due to differences in the
microenvironment of the thymus in the two animal models. The
consequences of SIV infection of the thymus result in thymocyte
depletion due to increased apoptosis, and this may contribute to the
ultimate failure of the regenerative response of the immune system.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Rosenzweig, Division of Immunology, New England Regional Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772.
3 Abbreviations used in this paper: FSC, forward scatter; SSC, side scattter.
Received for publication April 5, 2000. Accepted for publication July 5, 2000.
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References |
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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.
chain. J. Exp. Med. 189:1489.This article has been cited by other articles:
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  E. G. Meissner, L. Zhang, S. Jiang, and L. Su Fusion-Induced Apoptosis Contributes to Thymocyte Depletion by a Pathogenic Human Immunodeficiency Virus Type 1 Envelope in the Human Thymus J. Virol., November 15, 2006; 80(22): 11019 - 11030. [Abstract] [Full Text] [PDF]
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 K. B. Gurney and C. H. Uittenbogaart Human Immunodeficiency Virus Persistence and Production in T-Cell Development Clin. Vaccine Immunol., November 1, 2006; 13(11): 1237 - 1245. [Abstract] [Full Text] [PDF]
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 A. Miyake, K. Ibuki, Y. Enose, H. Suzuki, R. Horiuchi, M. Motohara, N. Saito, T. Nakasone, M. Honda, T. Watanabe, et al. Rapid dissemination of a pathogenic simian/human immunodeficiency virus to systemic organs and active replication in lymphoid tissues following intrarectal infection. J. Gen. Virol., May 1, 2006; 87(Pt 5): 1311 - 1320. [Abstract] [Full Text] [PDF]
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 A. Muthukumar, D. Zhou, M. Paiardini, A. P. Barry, K. S. Cole, H. M. McClure, S. I. Staprans, G. Silvestri, and D. L. Sodora Timely triggering of homeostatic mechanisms involved in the regulation of T-cell levels in SIVsm-infected sooty mangabeys Blood, December 1, 2005; 106(12): 3839 - 3845. [Abstract] [Full Text] [PDF]
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E. Priceputu, I. Rodrigue, P. Chrobak, J. Poudrier, T. W. Mak, Z. Hanna, C. Hu, D. G. Kay, and P. Jolicoeur The Nef-Mediated AIDS-Like Disease of CD4C/Human Immunodeficiency Virus Transgenic Mice Is Associated with Increased Fas/FasL Expression on T Cells and T-Cell Death but Is Not Prevented in Fas-, FasL-, Tumor Necrosis Factor Receptor 1-, or Interleukin-1{beta}-Converting Enzyme-Deficient or Bcl2-Expressing Transgenic Mice J. Virol., May 15, 2005; 79(10): 6377 - 6391. [Abstract] [Full Text] [PDF]
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R. A. Reyes, D. R. Canfield, U. Esser, L. A. Adamson, C. R. Brown, C. Cheng-Mayer, M. B. Gardner, J. M. Harouse, and P. A. Luciw Induction of Simian AIDS in Infant Rhesus Macaques Infected with CCR5- or CXCR4-Utilizing Simian-Human Immunodeficiency Viruses Is Associated with Distinct Lesions of the Thymus J. Virol., February 15, 2004; 78(4): 2121 - 2130. [Abstract] [Full Text] [PDF]
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 M.-T. Nugeyre, V. Monceaux, S. Beq, M.-C. Cumont, R. H. T. Fang, L. Chene, M. Morre, F. Barre-Sinoussi, B. Hurtrel, and N. Israel IL-7 Stimulates T Cell Renewal Without Increasing Viral Replication in Simian Immunodeficiency Virus-Infected Macaques J. Immunol., October 15, 2003; 171(8): 4447 - 4453. [Abstract] [Full Text] [PDF]
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N. Schmitt, L. Chene, D. Boutolleau, M.-T. Nugeyre, E. Guillemard, P. Versmisse, C. Jacquemot, F. Barre-Sinoussi, and N. Israel Positive Regulation of CXCR4 Expression and Signaling by Interleukin-7 in CD4+ Mature Thymocytes Correlates with Their Capacity To Favor Human Immunodeficiency X4 Virus Replication J. Virol., May 15, 2003; 77(10): 5784 - 5793. [Abstract] [Full Text] [PDF]
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A. Shen, M. C. Zink, J. L. Mankowski, K. Chadwick, J. B. Margolick, L. M. Carruth, M. Li, J. E. Clements, and R. F. Siliciano Resting CD4+ T Lymphocytes but Not Thymocytes Provide a Latent Viral Reservoir in a Simian Immunodeficiency Virus-Macaca nemestrina Model of Human Immunodeficiency Virus Type 1-Infected Patients on Highly Active Antiretroviral Therapy J. Virol., April 15, 2003; 77(8): 4938 - 4949. [Abstract] [Full Text] [PDF]
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D. L. Sodora, J. M. Milush, F. Ware, A. Wozniakowski, L. Montgomery, H. M. McClure, A. A. Lackner, M. Marthas, V. Hirsch, R. P. Johnson, et al. Decreased Levels of Recent Thymic Emigrants in Peripheral Blood of Simian Immunodeficiency Virus-Infected Macaques Correlate with Alterations within the Thymus J. Virol., August 28, 2002; 76(19): 9981 - 9990. [Abstract] [Full Text] [PDF]
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T. Igarashi, C. R. Brown, R. A. Byrum, Y. Nishimura, Y. Endo, R. J. Plishka, C. Buckler, A. Buckler-White, G. Miller, V. M. Hirsch, et al. Rapid and Irreversible CD4+ T-Cell Depletion Induced by the Highly Pathogenic Simian/Human Immunodeficiency Virus SHIVDH12R Is Systemic and Synchronous J. Virol., January 1, 2002; 76(1): 379 - 391. [Abstract] [Full Text] [PDF]
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 S. C. De Rosa, D. K. Mitra, N. Watanabe, L. A. Herzenberg, L. A. Herzenberg, and M. Roederer V{delta}1 and V{delta}2 {gamma}{delta} T cells express distinct surface markers and might be developmentally distinct lineages J. Leukoc. Biol., October 1, 2001; 70(4): 518 - 526. [Abstract] [Full Text] [PDF]
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E. Guillemard, M.-T. Nugeyre, L. Chene, N. Schmitt, C. Jacquemot, F. Barre-Sinoussi, and N. Israel Interleukin-7 and infection itself by human immunodeficiency virus 1 favor virus persistence in mature CD4+CD8{-}CD3+ thymocytes through sustained induction of Bcl-2 Blood, October 1, 2001; 98(7): 2166 - 2174. [Abstract] [Full Text] [PDF]
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