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Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215; and
New England Regional Primate Research Center, Southboro, MA 01772
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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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It is hypothesized that tuberculosis may accelerate HIV disease through activation of the immune system, which may result in increased viral replication in the HIV/M. tuberculosis-coinfected individuals. In vitro studies have shown that M. tuberculosis can up-regulate HIV-1 replication in chronically or acutely infected T or macrophage cell lines 11, 12 . M. tuberculosis or purified protein derivative (PPD)3 can stimulate activation of the CD4+ lymphocytes from PPD-positive individuals, which results in an increase in HIV-1 replication 13 . Pleural fluids and lymphocytes from patients with tuberculous pleuritis can enhance HIV-1 replication in the in vitro system 14 . Furthermore, a transient increase in plasma HIV RNA was noted in a small group of HIV-1-infected patients with active tuberculosis 13 , although another study was unable to demonstrate such a change in virus loads in HIV-1-infected tuberculosis patients before and after antituberculosis treatment 15 . However, it is not clear how a M. tuberculosis coinfection can compromise the immune system in HIV-infected individuals and whether M. tuberculosis-driven immune activation can accelerate HIV-1 disease. Additional in vivo studies are clearly needed to characterize the natural history of HIV-1/M. tuberculosis coinfection and the immunopathogenesis in HIV/M. tuberculosis-coinfected individuals.
SIV-infected nonhuman primates have proved to be powerful models for the study of AIDS 16, 17, 18 . Using the SIV/macaque animal model, we have explored T cell responses to AIDS viruses and studied the AIDS immunopathogenesis 19, 20, 21, 22, 23, 24, 25, 26 . More recently, we reported that an SIVmac-infected monkey developed a tuberculosis-like disease and an accelerated SIVmac disease following Mycobacterium bovis BCG coinfection 25 . This observation suggests that SIVmac/BCG-coinfected monkeys may be a valuable animal model for studying HIV-Mycobacterium interactions and the reciprocal impact on Mycobacterium- and HIV-induced diseases. In the present study, we utilized the SIVmac/BCG coinfection model to study the impact of mycobacterial coinfection on the AIDS virus-mediated disease. We found that BCG coinfection in SIVmac-infected monkeys resulted in a prolonged stimulation of T cell subpopulations, which correlated with marked changes in virus loads and accelerated SIVmac disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rhesus monkeys (Macaca mulatta) were used in these studies. The monkeys used in the chronic SIVmac/BCG coinfection were 3 to 5 years old; those used in acute BCG infection were 1-year-old naive animals. These animals were maintained in accordance with the guidelines of the Committee on Animals for Harvard Medical School and the Guide for the Care and Use of Laboratory Animals46 . All monkeys were inoculated i.v. with SIVmac 251 strain, as described previously 19 .
M. bovis BCG coinfection
M. bovis BCG (Pasteur strain) was generously provided by Dr. Scott Koenig, MedImmune, Gaithersburg, MD. BCG was stored in liquid nitrogen and thawed immediately before inoculation. Four groups of monkeys were included for BCG infection studies: 1) 8 monkeys were infected with SIVmac for 3 to 18 mo and then coinfected with BCG; 2) 2 naive monkeys were inoculated simultaneously with SIVmac and BCG; 3) as controls, 4 normal monkeys not infected with SIVmac were infected with BCG alone; 4) as another controls, 10 monkeys were infected only with SIVmac in this study. Monkeys were inoculated i.v. with 106 CFU of BCG. After BCG inoculation, the monkeys were followed for the signs of clinical illness. Monkeys that died from SIVmac/BCG coinfection were subjected to a necropsy study.
Isolation and fractionation of lymphocyte populations in blood and lymph nodes
PBMC were isolated from EDTA blood of the monkeys using Ficoll/diatrizoate gradient centrifugation. Peripheral lymph nodes were obtained by standard biopsy procedures before and after BCG inoculation, and were carefully teased to generate single-cell suspensions. CD4+ or CD8+ lymphocytes were purified using anti-CD4 or anti-CD8 Ab-conjugated Dynabeads (Dynal, Great Neck, NY), as described previously 19 . PBMC or lymph node cells were incubated with these immunomagnetic beads for 30 min at room temperature and then selected in two cycles with a magnetic particle concentrator. Monocytes/macrophages in blood or tissues were purified by adherence to culture flasks through 1 h of incubation, as described previously 21 . Monocytes/macrophages purified by this method contained less than 2% of CD4+ lymphocytes.
mAbs and flow cytometric analysis
The following anti-human CD mAbs that cross-reacted with corresponding macaque Ags were used: phycoerythrin (PE)-conjugated anti-monkey CD3 (FN18, Biosource, Camarillo, CA), PE-conjugated anti-human CD4 (Ortho Diagnostic Systems, Raritan, NJ), PE-conjugated anti-human CD8 (Dako, Carpinteria, CA). Whole blood staining was used following the instructions of the immunolysing kit, ImmunoPrep, from Coulter (Hialeah, FL). Single-cell suspensions from lymph nodes were stained using standard methods. Two-color flow cytometric analyses were performed on an XL flow cytometer (Coulter). Lymphocytes were gated by forward- and side-scatter characteristics, and up to 10,000 gated cells were analyzed.
mRNA extraction and cDNA synthesis
mRNA was extracted from unfractionated or fractionated
lymphocytes using guanidinium thiocyanate and oligo(dT) spun columns
(mRNA extraction kit, Pharmacia, Piscataway, NJ). The first-strand cDNA
was synthesized in a 20-µl final volume at 42°C for 1 h using
0.2–1 µg of mRNA, 1 µg of random hexanucleotides, and 5 U of
reverse transcriptase (Promega, Madison, WI). The samples were heated
for 5 min at 95°C to terminate the reaction.
TCR-ß CDR3 length analyses
CDR3 length analyses were done by two methods: autoradiogram display after 32P labeling as described previously 22, 26 ; and spectratyping through fluorescence labeling in the Genescan analysis 28 .
Autoradiogram analysis of CDR3 lengths. cDNAs were amplified by PCR for expression of 24 Vß families using individual Vß-specific primers and a Cß-specific primer as described previously 22, 26 . The second round of PCR was performed using nested Vß primers and a Cß primer, designed as described 26 . The internal Cß primer was labeled at its 5' end with 32P. The TCR-ß DNA from the first round PCR was amplified for 15 cycles under the following conditions: 95°C for 30 s; 55°C for 30 s; and 72°C for 30 s. The amplified TCR ß-chains bearing various CDR3 lengths were visualized as a series of radiolabeled bands, 3 bases apart, on a 6% polyacrylamide sequencing gel. The selected Vß families that exhibited a change in CDR3 lengths were repeated at least once by the same method, followed by quantitation using the Genescan method.
Spectratyping analysis of CDR3 lengths. The PCR was done under the conditions as described above. The derived PCR products were then amplified in the second-round PCR using individual nested Vß primers and the internal Cß primer coupled with the Fam fluorophore (Applied Biosystems, Foster City, CA). The 15-cycle PCR was performed in a 15-µl volume with 0.2 µM concentrations of each primer under the same conditions as described above for autoradiogram analysis. One microliter of each reaction product was mixed with deionized formamide and a TMRA-500 size standard and then electrophoresed on a 5% acrylamide gel. Data were analyzed for size and fluorescence intensity using the Genescan software. Verifying experiments using the samples derived at three time points from four normal monkeys indicated that these CDR3 length analyses were highly reproducible. Further cloning and sequencing in conjunction with the CDR3 length display allowed for the prediction of CDR3 lengths, which were expressed as the predicted number of amino acids.
Molecular cloning and sequencing of TCR-ß CDR3 length-bearing cDNA
This was done using PCR-based cloning technique 21, 23, 26 . Briefly, selected Vß-bearing cDNA exhibiting dominant CDR3 lengths was cut from the gel using the autoradiogram as a guidance. The cDNA in the gel piece was recovered by incubating the gel piece at 100°C for 3 min before the standard ethanol precipitation. The selected cDNA was then amplified by PCR for 30 cycles using nested Vß-specific primers containing an EcoRI restriction site and the Cß primer containing an XbaI restriction site. For a longitudinal clonotypic analysis, the same CDR3 length-bearing cDNAs from the CD4+ or CD8+ PBL sampled at different time points from the monkeys were similarly isolated for cloning and sequencing. PCR was performed for 30 cycles as previously described 26 . To minimize PCR-generated misincorporation, plaque-forming unit DNA polymerase was used in the PCR reactions. The PCR products were digested with EcoRI and XbaI and ligated into the plasmid pSP65 (Promega) for cloning and sequencing. At least 25 clones were analyzed for each cDNA sample. The frequency of the clonotypic sequences was determined based on the percentage of the CDR3-restricted clones in the total clones bearing the same CDR3 lengths.
Quantitative competitive PCR (QC-PCR)
A QC-PCR assay for measuring plasma HIV RNA was adapted to measure SIV RNA in plasma 29 . In these experiments, a pSP72 plasmid containing the SIVmac gag fragment DNA 20 was used to create the mutant competitor by engineering a 75-bp internal deletion. Subsequently, the wild-type and mutant competitor RNA were generated from the plasmid DNA using an in vitro transcription system, as described previously 22 . For viral RNA extraction, 200 µl of plasma from each time point were added to a tube containing 1800 µl of 20 mM Tris buffer and then ultracentrifuged at 40,000 rpm for 70 min in a Sorval rotor. The pellets of viral particles were digested with proteinase K and DNase, and treated with phenol/chloroform for RNA extraction. The viral RNA was precipitated in ethanol and spun down for 15 min at 14,000 rpm. The extracted RNA was aliquoted into 6 different tubes, which contained individually 0, 10, 50, 100, 500, or 1000 copies of SIVmac gag competitor RNA. The RNA mixtures were reverse-transcribed to cDNA and competitively amplified by a 35-cycle PCR using a pair of SIVmac gag-specific primers. The sequences for these oligonucleotides were as follows: sense primer, 5'-TAA ATG CCT GGG TAA AAT-3' (gag position 461–478 of SIVmac 251); antisense primer 5'-TGG TAT GGG GTT CTG TTG TCT GT-3' (gag position 752–774 of SIVmac 251). The amplified PCR products containing wild-type (314 bp) and competitor (239 bp) were separated on 2% agarose gels and measured for their densities in a GS 700 Imaging Densitometer (Bio-Rad, Richmond, CA). Quantitation was achieved by data analysis using Molecular Analyst system software (Bio-Rad). The coefficient variation of intra- and interassays using this protocol was <20%. The sensitivity of the QC-PCR was 1 x 103 RNA copies in 1 ml of plasma. As a complementary study, plasma SIV RNA was also quantitated by the branched DNA (bDNA) assay (Chiron, Emeryville, CA). This assay allows for detecting a minimum of 104 RNA copies/ml.
PCR-based semiquantitation for intracellular SIVmac RNA
To analyze intracellular SIVmac RNA expression, purified
CD4+ lymphocytes and CD14+ cells, as well as
adherent monocytes, were subjected to mRNA extraction and cDNA
synthesis. The 356-bp fragment was amplified from cellular cDNA by a
30-cycle PCR using a pair of SIVmac gag-specific primers as described
above and previously 30 . The PCR products were separated on a 2%
agarose gel, transferred onto a nylon membrane, and then hybridized to
a 32P-labeled internal oligonucleotide. The sequence of the
hybridizing primer was 5'-GCC AGG ATT TCA GGC ACT GTC AGA AGG CTG-3'
(gag position 513–542 of SIVmac 251). The defined copies of
SIV gag cDNA were always included as standards for the
semiquantitation. Radioactivity was quantitated using AMBIS 100 22, 24, 26 . To normalize SIVmac RNA expression levels, the housekeeping
gene ß-actin was similarly quantitated using the same amount of
cellular cDNA. The sequences of actin oligonucleotides were as follows:
sense primer, 5'-CCC CCA TGC CAT CCT GCG TCT G-3'; antisense primer,
5'-CAT GAT GGA GTT GAA GGT AGT TT-3'; hybridizing primer, 5'-GAC CTG
ACT GAC TAC CTC ATG AAG ATC CTC AC-3'. The semiquantitation was
achieved by calculating the copy number of SIV gag RNA in 1.66 x
10-16 M actin-containing cellular cDNA (106
cells).
Quantitation of SIVmac Ags
SIV virus loads in the acute infection was assessed by measuring SIV Gag p27 proteins in plasma using an Ag capture kit (Coulter).
Statistical analysis
Two methods of statistics were undertaken to determine whether BCG infection significantly enhanced a decline of CD4+ PBL and accelerated the progression to clinical AIDS in SIVmac-infected monkeys. The paired Student t test, as described previously 19, 31 , was used to examine the difference in decline rates of CD4+ PBL before and after BCG coinfection in the monkeys chronically infected with SIVmac. The CD4+ PBL decline rates in a SIVmac infection (before BCG coinfection) was calculated and expressed as the declining number of cells/µl/6 mo using the data obtained from 2–6 mo of postSIVmac infection through the time of pre-BCG inoculation. The decline rates in the SIVmac/BCG coinfection were derived from the CD4+ PBL counts obtained from the different time points of entire course of BCG coinfection. In addition, the unpaired Student t test was utilized to analyze different decline rates of CD4+ PBL between the SIVmac/BCG-coinfected monkeys and the monkeys infected with SIVmac alone. The CD4+ PBL decline rates in the controls of SIVmac-infected monkeys were calculated using the CD4+ PBL counts from 2–20 mo after SIVmac infection through the last time points of follow-up. Furthermore, the difference in the surviving time between the SIVmac/BCG-coinfected monkeys and the controls of the SIVmac-infected monkeys was also evaluated by the Student t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To optimally characterize BCG-driven T cell responses, normal
monkeys not infected with SIVmac were inoculated with BCG and assessed
for evidence of in vivo stimulation of selected Vß-expressing
CD4+ and CD8+ T cell subpopulations. Purified
CD4+ and CD8+ cells obtained from different
time points after BCG inoculation were subjected to a molecular
analysis of CDR3 lengths in each of 24 Vß gene families.
Interestingly, BCG infection resulted in a striking change in CDR3
lengths in selected Vß-expressing CD4+ and
CD8+ PBL subpopulations of the monkeys. CDR3 lengths used
by selected Vß family-expressing CD4+ and
CD8+ PBL subpopulations underwent a change from gaussian
distributions to one or two predominant lengths after the BCG
inoculation (Fig. 1
, A and
B). This BCG-induced change in CDR3 lengths was most evident
in PBL of the infected monkeys 1 to 3 wk after BCG inoculation.
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C). These clonotypic sequences were undetectable in 30 cDNA
clones derived from PBL of the monkey before and 2 mo after BCG
inoculation. These results, therefore, provide molecular evidence
suggesting that BCG Ags stimulate a dominant clonal expansion of
CDR3-restricted Vß+CD4+ and CD8+
cell subpopulations during acute BCG infection of monkeys. BCG coinfection induced a prolonged clonal expansion of selected CDR3-bearing CD4+ and CD8+ T cell subpopulations in SIVmac-infected monkeys
We then sought to determine whether BCG infection could result in
similar activation and expansion of T cell subpopulations in
SIVmac-infected monkeys. Eight monkeys previously infected with SIVmac
for 3–18 mo were inoculated i.v. with BCG and examined for an
alteration in CDR3 lengths. Like normal monkeys not infected with
SIVmac, SIVmac-infected monkeys exhibited one or two dominant CDR3
lengths in selected Vß-expressing CD4+ and
CD8+ PBL subpopulations following BCG inoculation (Fig. 2, A and
B). Furthermore, molecular analysis of the dominant CDR3
length-bearing ß cDNA revealed that the clonal expansion, as seen in
normal monkeys, was also identified in these selected Vß-expressing
cell subpopulations during the acute BCG coinfection of the
SIVmac-infected animals (Fig. 2C). Surprisingly, while the
clonal expansion was transiently identified in normal monkeys not
infected with SIVmac, the clonotypic sequences in the selected CDR3
length-bearing Vß+ cells were persistent during the
chronic BCG infection and became dominant at the end stage of
SIVmac/BCG coinfection (Fig. 2C). Nevertheless, these
clonotypic sequences were not readily identified in the periods of
pre-BCG inoculation (Fig. 2C), suggesting that the expansion
of the CDR3-restricted cell subpopulations was driven by BCG Ags. These
findings, therefore, suggested that BCG Ags stimulated a prolonged
clonal expansion of the selected Vß-expressing CD4+ and
CD8+ PBL subpopulations in SIVmac-infected monkeys
following the BCG coinfection.
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BCG-induced T cell activation may increase the ability of AIDS
virus to replicate in CD4+ lymphocytes and macrophages. To
address this issue, we examined whether CDR3-dependent activation of
selected Vß-expressing cell subpopulations resulted in a change in
SIV loads in the SIVmac/BCG-coinfected animals. The change in
intracellular and plasma SIV RNA was investigated using PCR-based
quantitation and branch DNA assay, respectively (Fig. 3). As expected, the SIVmac-infected
monkeys showed an apparent increase in viral loads during the acute
phase of BCG coinfection (Fig. 4).
Increases of 3–100-fold in SIV cDNA in CD4+ PBL or
CD4+ L.N. cells were detected in the SIVmac-infected
monkeys 1–3 wk after BCG inoculations (Fig. 4A). Up to
6-fold increases in SIV RNA were detected in lymph node macrophages of
the coinfected monkeys after BCG inoculation despite the lack of
significant change in their blood monocytes (Fig. 4B).
Consistent with the up-regulation of SIV RNA expression in
CD4+ T cells and macrophages was a marked increase in
plasma SIV RNA in the SIVmac-infected monkeys (Fig. 4C). A
maximum increase of 40- to 1000-fold in plasma RNA was identified by
bDNA assay and QC-PCR in 6 of 8 SIVmac-infected monkeys following
BCG inoculation (Fig. 4C). Furthermore, a sustained increase
in virus loads in four of the infected monkeys coincided with the
prolonged dominance of clonotypic sequences in CD4+
lymphocytes, suggesting a correlation between the BCG-driven clonal
activation and increased virus loads in the SIV/BCG-coinfected monkeys
(Fig. 4D). Thus, these results suggest that BCG-induced T
cell activation may be the driving force for the increased virus
replication in the SIVmac-infected monkeys.
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Clinical studies have demonstrated that an enhanced decline of
CD4+ PBL counts during the chronic HIV infection is
indicative of the development of clinical AIDS 45 . We therefore
sought to determine whether BCG-driven T cell activation and associated
increase in virus loads in SIVmac-infected monkeys resulted in an
enhanced decline of CD4+ PBL and acceleration of SIVmac
infection. Interestingly, the SIVmac/BCG-coinfected monkeys
demonstrated an increased decline of CD4+ PBL counts (Fig. 5A), with the decline rates
increasing from <200/µl/6 mo up to 1560/µl/6 mo a few months after
BCG inoculation (Table I). The lymphocyte
count of monkey 266, whose CD4+ in the blood was
980/mm3 before BCG inoculation, dropped to
600/mm3 2 mo after BCG inoculation, and 1 mo later declined
further to 200/mm3 (Fig. 5A). The other
coinfected monkeys also displayed such a rapid decline of
CD4+ lymphocyte counts in the blood after the BCG
inoculation (Fig. 5A, Table I). In contrast, the control
SIVmac-infected monkeys had a slower decline of their CD4+
lymphocyte counts in the blood during the chronic SIVmac infection
(Fig. 5B, Table I). The control monkeys infected with BCG
alone did not show a decline of CD4+ PBL after BCG
inoculation (Fig. 5C).
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A).
The profound depletion of CD4+ PBL coincided with the
persistent SIV p27 antigenemia in the SIVmac/BCG-coinfected but not the
control SIVmac-infected monkeys (Fig. 6B). Moreover,
the enhanced SIVmac infection appeared to be associated with
BCG-induced immune activation. The monkey 347 developed T cell
activation-related toxic shock syndrome characterized by severe
dehydration and collapse in circulation on day 18 after the
simultaneous SIVmac/BCG inoculation. The monkey had to be
treated intensively with fluid infusion for a recovery from the shock
syndrome. The other animal also developed a transient change in mental
status. In contrast, the control SIVmac-infected or BCG-infected
monkeys did not show any noticeable signs of clinical illness or such a
marked depletion of CD4+ PBL during the acute infection
(Fig. 6A). Thus, the results in the naive monkeys
coinfected with SIVmac/BCG were consistent with those seen in
the chronically coinfected monkeys, indicating that BCG coinfection
induced the rapid decline of CD4+ PBL in SIVmac-infected
monkeys.
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The enhanced decline of CD4+ PBL counts in SIVmac/BCG-coinfected monkeys resulted in the rapid progression to clinical AIDS
Since BCG coinfection accelerated SIVmac disease
characterized by the enhanced decline of CD4+ PBL, we
expected to see that these coinfected monkeys died rapidly from
clinical AIDS-like syndrome. The clinical course of AIDS is temporally
compressed in the SIVmac-infected monkeys as compared with the
HIV-1-infected humans, with animals usually dying 2–5 yr after
infection. As expected, the enhanced decline of CD4+ PBL
inevitably led to a rapid death in the SIVmac/BCG-coinfected monkeys.
All SIVmac-infected monkeys died 2 to 7 mo after BCG inoculation (Table I). At the end stage of SIVmac/BCG coinfection, all these monkeys
developed a disseminated BCG infection, as detected by acid-fast
staining of tissue sections and PCR-based quantitation of BCG rRNA
isolated from the tissues 25 . Surprisingly, four coinfected monkeys
developed disseminated tuberculosis-like disease. The BCG-induced
tuberculosis was characterized clinically by diarrhea,
colitis/peritonitis, and weight loss and pathologically by
nonnecrotizing granulomas in multiple tissues, which has been described
in some patients coinfected with HIV-1/M. tuberculosis 32 .
On the other hand, the SIVmac-induced disease was also apparent in the
monkeys that died after SIVmac/BCG coinfection (Table I). Profound
depletion of lymphocytes in lymphoid tissues, opportunistic infections,
lymphoma, and the fatal thrombosis induced by SIVmac 16 were seen in
these coinfected animals. In contrast, the control SIVmac-infected
monkeys exhibited a natural course of SIVmac infection and survived for
up to 5 years after the viral infection (Table I). The normal monkeys
inoculated with BCG alone survived BCG infection without any noticeable
signs of clinical illness (Table I). A statistical analysis of
difference in surviving time between the coinfected monkeys and control
SIVmac-infected animals indicated that BCG coinfection significantly
shortened the course of SIV infection in the coinfected monkeys
(p < 0.001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, dominant responses of CDR3-restricted T cell
subpopulations were transiently detected in the acute BCG infection of
normal SIV-negative and SIVmac-infected monkeys. The transient
expansion of CDR3-restricted T cell subpopulations was followed by an
undetectability of clonotypic sequences after acute BCG infection in
the normal monkeys not infected with SIVmac. In SIVmac-infected
monkeys, however, the dominant responses of the CDR3-restricted T cell
subpopulations preceded persistence of clonotypic sequences in the
selected CDR3 length-bearing T cells. The dominant responses of
CDR3-restricted T cell subpopulations may be driven by the high level
of BCG Ags produced in the acute infection, since expansion of these
cell subpopulations was associated with a resolution of BCG infection
in normal monkeys. In the SIVmac-infected monkeys, the transition from
the acute to chronic stage of BCG infection may help to explain the
waning of the dominant expansion of CDR3-restricted T cells. BCG loads
during the chronic BCG coinfection may be lower than those during the
acute phase in the SIVmac-infected monkeys. The decreased level of BCG
loads during the chronic BCG infection may result in the reduced
magnitude of clonal expansion in the coinfected monkeys. This notion is
also supported by the finding that the clonal dominance in the selected
CDR3 length-bearing T cells reoccurs at the time the animals die from
BCG dissemination (Fig. 2C). Nevertheless, we cannot exclude
the additional possibility that the decreased magnitude of the dominant
responses during the chronic BCG coinfection may also result from some
degree of the clonal exhaustion driven by the SIVmac-BCG interaction.
Our study demonstrated that BCG-driven activation and expansion of selected Vß-expressing cell subpopulations were associated with the increase in intracellular SIV RNA expression as well as in plasma viremia in SIVmac-infected monkeys. The up-regulation of SIVmac replication in these coinfected animals was comparable with the transient increase in viremia observed in HIV-1-infected individuals following influenza vaccination or recall Ag immunization 35, 36, 37, 38 . Our results were also paralleled to the recent observation demonstrating that T cell activation can be a driving force for the selected variants of AIDS viruses 39, 40 . The increase in virus loads in the SIV/BCG-coinfected monkeys may result from the BCG-driven activation and expansion of CD4+ T cells in that this increase was most evident at the time dominant expansion of CDR3-restricted T cell subpopulations was seen during the acute BCG infection. In addition, this increase in SIV loads was correlated with the prolonged clonal expansion driven by BCG in some of the coinfected monkeys. Furthermore, the increase in intracellular SIV RNA expression was more striking in CD4+ T cells than in macrophages during the acute BCG infection. Several possibilities may be considered as the explanation for the T cell activation-associated increase in the virus loads. The BCG Ags-activated CD4+ cells may become more infectable as a result of induction of an expression of chemokine receptors required for virus entry 41, 42 . Moreover, activation signals in CD4+ T cells may trans-activate the latent virus and facilitate the virus replication 43 . Finally, the proinflammatory cytokines produced by BCG Ags-activated CD4+ T cells may directly or indirectly up-regulate viral replication in SIVmac-infected CD4+ T cells 43 . This cytokine-mediated up-regulation of SIV replication may also help to explain an increase in SIV RNA expression in tissue macrophages following BCG coinfection in the SIVmac-infected monkeys. BCG-infected macrophages may more readily be infected by SIVmac in the coinfected animals. In fact, increased viral production in macrophages has recently been reported in HIV-1-infected patients with Mycobacterium coinfection 44 .
In contrast to those reports studying a role of immune stimulation in
AIDS virus pathogenesis 35, 36, 37, 38, 39, 40 , our studies in the SIV/BCG
coinfection model provide important information concerning the clinical
and pathological consequence of prolonged T cell activation in the
coinfected individuals. The prolonged clonal expansion of T cells
during the chronic BCG coinfection coincided with an enhanced decline
of CD4+ PBL counts and accelerated progression to a
clinical AIDS-like syndrome in SIVmac-infected monkeys. These findings
suggest that BCG-driven T cell activation may be an underlying
mechanism for the acceleration of SIVmac disease. BCG-induced T cell
activation may increase the ability of SIV to deplete CD4+
T cells through up-regulating SIVmac replication. This correlation
between the increased virus load and enhanced decline of
CD4+ PBL counts was indeed observed in the naive monkeys
coinfected with SIV/BCG (Fig. 6) as well as in some of the chronically
coinfected animals (Figs. 4 and 5). On the other hand, the prolonged T
cell activation in persistent BCG infection can certainly compromise
and exhaust the immune system to a greater extent, which may inevitably
result in an enhanced decline of CD4+ T cells and
accelerated progression to a AIDS-like syndrome in the
SIVmac/BCG-coinfected monkeys. This BCG-induced immune exhaustion may
help to explain an accelerated decline of CD4+ PBL counts
without sustained increases in viral loads in some coinfected monkeys.
The BCG-induced acceleration of the SIVmac disease was characterized by an enhanced decline of CD4+ PBL as well as rapid progression to a clinical AIDS-like syndrome in the coinfected monkeys. Like HIV-1-infected humans 45 , chronically SIVmac-infected monkeys exhibited a slow decline of CD4+ PBL counts before BCG coinfection. Following BCG coinfection, the decline rates of CD4+ PBL can increase greatly to a level as high as 1560 cells/µl/6 mo. In fact, the accelerated decline of CD4+ PBL is observed in the HIV-1-infected patients at the time when they develop clinical AIDS 46 . The fast decline of CD4+ PBL counts is also seen in the rapidly progressing persons infected with HIV-1 46 . However, M. tuberculosis-induced acceleration of CD4+ PBL declines has not formally been demonstrated in HIV-1/M. tuberculosis-coinfected persons. The natural history of HIV/M. tuberculosis coinfection may not well be unveiled in the clinical setting, since anti-tuberculosis intervention must be initiated once tuberculosis is diagnosed in HIV-1-infected patients. Moreover, diagnosis of M. tuberculosis coinfection still remains a challenge in HIV-infected persons. These factors may underestimate the effect of tuberculosis on the immune system of the coinfected patients.
BCG-induced acceleration of SIV disease was more striking in the naive monkeys than in the chronically SIVmac-infected monkeys. A marked depletion of CD4+ PBL was noted in the naive monkeys 2 wk after the simultaneous SIV/BCG inoculation, whereas in the SIVmac-infected monkeys the increased decline of CD4+ PBL counts became apparent 2 to 4 mo after BCG coinfection. The difference in the pace of CD4+ PBL decline between these two groups of monkeys may be related to the status of SIVmac infection and the magnitude of BCG-driven T cell activation. In the naive monkeys, we have observed that SIVmac/BCG coinfection can lead to a toxic shock syndrome, a T cell activation-related disorder that we have seen in the monkeys challenged with a superantigen 26 . Such a profound T cell activation can certainly render CD4+ T cells more infectable and destroyable during SIVmac/BCG coinfection. Moreover, the burst of viral replication may be more striking after the SIVmac/BCG coinfection in the monkeys naive for anti-SIVmac immune responses. This notion is indeed supported by the finding demonstrating the correlation between the CD4+ T cell depletion and prolonged SIV p27 antigenemia in the naive monkeys. In the chronically SIVmac-infected animals, however, the superimposed BCG infection may not be able to induce such a marked increase in virus burden in the presence of established immune responses to SIV. In addition, the magnitude of BCG-driven T cell activation may be lower in the SIVmac-infected monkeys, since the repertoire of T cells responding to BCG Ags is usually shrunk or depressed due to the global depletion of CD4+ T cells in the chronic infection.
Coincident with the accelerated decline of CD4+PBL counts was a rapid progression to clinical AIDS-like syndrome in the SIVmac/BCG-coinfected monkeys. The SIVmac/BCG-coinfected monkeys died within 2 to 7 mo after BCG inoculation. The impact of mycobacterial coinfection is indeed twofold in these SIVmac/BCG-coinfected animals. The SIVmac-infected but not normal monkeys can develop disseminated BCG infection or tuberculosis-like disease following BCG coinfection. On the other hand, AIDS-like syndromes such as lymphocytic depletion in lymphoid tissues, opportunistic infections, lymphoma and SIVmac-induced arteriopathy, and thrombosis are observed in these monkeys that died from the SIVmac/BCG coinfection 16 . Therefore, the coinfected monkeys can die from clinical AIDS that is linked to either Mycobacterium-related sequelae or SIVmac disease.
Thus, the results in SIVmac/BCG-coinfected monkeys indicate that BCG infection can accelerate a progression of SIVmac disease, strongly supporting the current hypothesis that mycobacterial coinfection can enhance viral pathogenicity as well as accelerate the progression to clinical AIDS in HIV-infected individuals. The findings in the present studies, therefore, suggest that the prolonged T cell activation driven by Mycobacterium may be a mechanism underlying the acceleration of AIDS virus-induced disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Zheng W. Chen, Harvard Medical School, Beth Israel Deaconess Medical Center, 330 Brookline Avenue RE113, East Campus, Boston, MA 02215. E-mail address:
3 Abbreviations used in this paper: PPD, purified protein derivative; BCG, bacille Calmette-Guérin; CDR3, complementarity-determining region 3; SIVmac, simian immunodeficiency virus of macaques; PE, phycoerythrin; QC-PCR, quantitative competitive polymerase chain reaction; bDNA, branched DNA.
Received for publication July 24, 1998. Accepted for publication October 14, 1998.
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