The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ranjbar, S.
Right arrow Articles by Goldfeld, A. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ranjbar, S.
Right arrow Articles by Goldfeld, A. E.
The Journal of Immunology, 2004, 172: 1953-1959.
Copyright © 2004 by The American Association of Immunologists

Mycobacterium tuberculosis Recall Antigens Suppress HIV-1 Replication in Anergic Donor Cells via CD8+ T Cell Expansion and Increased IL-10 Levels1

Shahin Ranjbar*, Nary Ly{dagger}, Sok Thim{ddagger}, Jean-Marc Reynes{dagger} and Anne E. Goldfeld2,*

* Center for Blood Research and Department of Medicine, Harvard Medical School, Boston, MA 02115; {dagger} Unité de Virologie, Institut Pasteur du Cambodge, Phnom Penh, Cambodia; and {ddagger} Cambodian Health Committee, Phnom Penh, Cambodia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mycobacterium tuberculosis (MTb) is the leading cause of death in the setting of AIDS. MTb enhances the pathogenicity and accelerates the course of HIV disease and, furthermore, infection with HIV-1 increases the risk of reactivation or reinfection with MTb. In this study, we show that host-specific recall responses to one pathogen, MTb, has a direct effect upon the regulation of a second pathogen, HIV-1. Using cells from immunocompetent former tuberculosis (TB) patients who displayed either a persistently positive (responsive) or negative (anergic), delayed-type hypersensitivity (DTH) reaction to intradermal injection of purified protein derivative (PPD), we investigated the effect of recall Ags to MTb upon the replication of HIV-1 primary isolates in vitro. We show that HIV-1 replication of a T cell-tropic isolate was significantly impaired in MTb-stimulated PBMC from PPD-anergic donors. Furthermore, these donors displayed a significant increase in CD8+ T cells and IL-10 levels and lower levels of IL-2 and TNF-{alpha} relative to PPD-responsive donors in response to PPD stimulation. Strikingly, CD8+ T cell depletion and blocking of IL-10 significantly increased HIV-1 replication in these PPD-anergic donors, indicating that an immunosuppressive response to MTb recall Ags inhibits HIV-1 replication in PPD-anergic individuals. Therefore, immunotherapeutic approaches aimed at recapitulating Ag-specific MTb anergy in vivo could result in novel and effective approaches to inhibit HIV-1 disease progression in MTb/HIV-1 coinfection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tuberculosis (TB)3 and AIDS are the largest causes of infectious disease death globally (1). In countries with a high incidence of TB and AIDS, reactivation of latent TB disease associated with HIV-1-induced immunosuppression is the major cause of TB disease in coinfected individuals (2). In addition, a proportion of TB cases are also due to infection with a second MTb strain (3) in patients who previously achieved a chemotherapeutic TB cure. Notably, TB disease accelerates the course of HIV-1 disease progression (4, 5) and is the largest single cause of death in the setting of AIDS (6). Since HIV-1 plasma viral load strongly predicts progression to AIDS and death (7, 8), host-specific immune responses to TB reactivation or reinfection that influence HIV-1 replication could thus be expected to impact survival of coinfected patients.

To investigate the impact of host-specific recall responses to Mycobacterium tuberculosis (MTb) Ags upon HIV-1 replication, we established an ex vivo HIV-1 infection model using cells from a unique cohort of immunocompetent HIV-negative former Cambodian pulmonary TB patients. These individuals, all of whom achieved a chemotherapeutic and microbiological cure of TB, do not manifest a delayed-type hypersensitivity (DTH) skin response to intradermal injection of purified protein derivative (PPD), but do manifest a response to the injection of other Ags such as mumps or Candida (9, 10). Concordant with these in vivo results, T cells from PPD-anergic donors display impaired responses to PPD but not to other Ags in vitro. By contrast, T cells from former TB patients with a positive skin reaction to PPD (PPD-responsive donors) in vivo respond equally to PPD and other Ags in vitro (9, 10). HIV-1 infection of cells from these PPD-anergic and PPD-responsive donors thus afforded us the unique opportunity of investigating Ag-specific recall responses to MTb upon replication of HIV-1 primary isolates.

Strikingly, we demonstrate that MTb Ag stimulation of PPD-anergic donor cells results in an immunosuppressive state resulting in a significant impairment of T-tropic HIV-1 replication. We show that there is a disproportionate expansion of CD8+ T cells and increased IL-10 levels in anergic donor PBMC upon MTb Ag challenge and that CD8+ T cell depletion and blocking IL-10 in vitro results in a significant restoration of HIV-1 replication. Thus, an immunosuppressive state induced by a host-specific recall response to MTb Ags can control the level of HIV-1 replication in certain individuals. These results provide new insight into immune responses to MTb infection and suggest that host-specific responses to MTb have a significant impact upon HIV-1 disease pathogenesis in the coinfected host.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Donors

PBMC were obtained from 12 healthy individuals with a history of pulmonary TB with either a positive intradermal skin reaction (>10 mm) to PPD (5TU; Adventis Pasteur, Swiftwater, PA) (PPD+; six individuals) or negative (no skin reaction, anergic) to PPD (six individuals) during and following successful TB chemotherapy. The patients were recruited from a cohort of consenting volunteers identified by the Cambodia Health Committee in the Svay Rieng Province, Cambodia, as previously described (9, 10). The diagnosis of clinical TB was made on the basis of medical history, physical examination, and the detection of acid-fast bacilli in sputum by light microscopy. All 12 had completed TB therapy according to the protocol of the Cambodian National TB program: isoniazid/rifampin/pyrazinamide/ethambutol for 2 mo followed by ethambutol and isoniazid for 6 additional months. All patients were tested for clearance of acid-fast bacilli from their sputum at 2 and 6 mo after beginning anti-TB therapy and at the end of therapy. All PPD-anergic as well as PPD-responsive donors displayed a positive skin reaction to another intradermal Ag such as Candida or mumps and all 12 were HIV-1 and HIV-2 negative as determined by the ELISA assay as previously described (10). Whole heparinized blood was collected and kept at room temperature and processed <24 h after collection. The experiments were repeated on sets of 12 individuals (6 anergic and 6 PPD-responsive) three different times over a 2-year period except for the CD8+ T cell depletion experiment, which was performed twice.

Viruses

The viruses used in this study were HIV-1KR25 (T-tropic) and HIV-198IN22 (M-tropic). HIV-1 KR25 was isolated from a 28-year-old Cambodian male with advanced AIDS (S.R. and A.E.G., unpublished data) according to standard techniques (11). HIV-198IN22 was obtained from the Centralized Facility for AIDS Reagents, National Institute for Biological Standard and Control, United Kingdom and was isolated from a 27-year old male from India without evidence of immunocompromise (12). We determined the secondary receptor usage of HIV-1KR25 and HIV-198IN22 by incubating them with a set of U87.CD4 cell lines expressing CCR1-CCR3, CCR5, or CXCR4 receptor (13) and found that HIV-1KR25 utilizes only CXCR4 and is thus T-tropic (X4), whereas HIV-198IN22 utilizes only CCR5 as its secondary receptor and is thus M-tropic (R5; data not shown).

Cell culture and PPD stimulation

PBMC were isolated by centrifugation on Ficoll-Hypaque gradients as previously described (10), 2 x 106 PBMC were cultured in 24-well plates in 1.5 ml of RPMI 1640 medium supplemented with 10% FCS containing 20 U/ml penicillin and 20 µg/ml streptomycin and were mock stimulated or stimulated with 10 µg/ml PPD as indicated. Separate cultures were set up for each time point and the cells were not washed and the medium was not changed for the duration of the experiment. At days 3, 8, and 14 poststimulation, culture supernatants were collected and IL-10, IL-2, IFN-{gamma}, and TNF-{alpha} levels of cultures were measured using OptEIA ELISA kits (BD PharMingen, San Diego, CA).

HIV-1 infection

The cells were set up as above and were mock stimulated or stimulated with PPD (10 µg/ml) or PHA (3.5 µg/ml) and IL-2 (20 IU/ml) and were incubated at 37°C for 2 days before being infected with 10 tissue culture-effective dose (TCID) 50%/ml HIV-1KR25 or 50 TCID50/ml HIV-198IN22. The cultures were further incubated at 37°C in 5% CO2 and 95% humidity for 1, 6, 12, and 17 days after HIV-1 infection, after which supernatants were collected and IL-10, IL-2, IFN-{gamma}, and TNF-{alpha} levels of cultures were measured as described above. The HIV-1 p24 levels were determined in infected cultures by measuring p24 Ag (picograms per milliliter) in the supernatants using a p24 ELISA kit (NEN, Boston, MA). The medium of the cultures that were terminated at days 1, 6, and 12 postinfection were not changed for the duration of the experiment; however, cultures terminated at day 17 were fed with fresh patient PBMC on day 11 postinfection to maintain viability of the cultures.

CD8+ T cell depletion

CD8+ T cells were depleted from PBMC by positive selection using magnetic beads coated with an anti-CD8 mAb (Dynal Biotech, Great Neck, NY). The purity of the cells was ~96% as judged by flow cytometry (data not shown). CD8+ T cells depleted or bulk PBMC were stimulated with 10 µg/ml PPD for 2 days before infection with 10 TCID50 HIV-1KR25. Supernatants were collected at day 3 postinfection and virus replication was determined by measuring HIV-1 p24 Ag in the culture supernatants.

Neutralization of IL-10 and TNF-{alpha}

mAbs to IL-10 (22 µg/ml; R&D Systems, Minneapolis, MN) or TNF-{alpha} (2.5 µg/ml; R&D Systems) were added at the time of PPD stimulation of PBMC cultures (time zero) and cells were infected with HIV-1KR25 or HIV-198IN22 2 days later as described above.

Cell proliferation

The proliferation of total PBMC from PPD-anergic and PPD-responsive donors was assessed by culturing 2 x 105 cells/well in a final volume of 200 µl of medium containing 10 µg/ml PPD or 3.5 µg/ml PHA and 20 IU/ml IL-2. Cultures were incubated for 3 and 5 days at 37°C and pulsed with 1 µCi of [3H]thymidine for the final 20 h. [3H]Thymidine incorporation was expressed in cpm.

Flow cytometry

The percentage of CD3+CD4+, CD3+CD8+ T cells and CD4+CXCR4+ cells was determined by flow cytometry using conjugated mAbs CD3-FITC, CD4-Cy5, CD8-PE, and CXCR4-PE (R&D Systems). Triple staining was performed by incubation of 2 x 105 cells with the conjugated Abs in HBSS with 2% FCS for 20 min at 4°C. Cells were then washed in HBSS and resuspended in FACS buffer containing 2.7% formaldehyde for flow cytofluorometric analysis. Nonspecific staining was controlled for by incubation of cells with appropriately labeled mouse IgG1 Abs (R&D Systems). Data were acquired and analyzed with CellQuest software (BD Biosciences, Mountain View, CA).

Statistical analysis

Results are expressed as a mean ± SEM. The Mann-Whitney U test was used to analyze statistical differences between two groups. A value of p < 0.05 was taken as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytokine profiles of PPD-anergic and PPD-responsive donors are distinct and are not influenced by HIV-1 infection

We isolated PBMC from six HIV-negative immunocompetent persistently PPD-responsive and six PPD-anergic former pulmonary TB patients who had achieved chemotherapeutic cure of TB. Long-term follow-up of the anergic individuals after successful treatment revealed that lack of DTH to PPD was not a transient phenomenon associated with active pulmonary TB, but was an Ag-specific and persistent finding in the anergic individuals (9, 10).

Cells were mock stimulated or stimulated with PPD and were either uninfected or infected with HIV-1 two days after PPD stimulation and the levels of IL-10, IL-2, IFN-{gamma}, and TNF-{alpha} were measured (Fig. 1). By 3 days after PPD stimulation, there was a significant increase in the production of all four cytokines in both PPD-anergic and -responsive cultures. However, although after PPD stimulation, IL-2, IFN-{gamma}, and TNF-{alpha} levels were higher in PPD-responsive cultures, by contrast, IL-10 levels were higher in PPD-anergic cultures at all time points (Fig. 1A).



View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 1. Cytokine profiles of PPD-anergic and PPD-responsive donors are distinct. PBMC from PPD-anergic ({square}) and PPD- responsive ({blacksquare}) donors were cultured at day 0 and either mock stimulated (UN) or PPD-stimulated (PPD stim) for the times indicated. Levels of IL-10, IL-2, IFN-{gamma}, and TNF-{alpha} were determined in HIV-1-uninfected (A), T-tropic HIV-1KR25-infected (B), or M-tropic HIV-198IN22-infected cultures (C). The cells were infected with virus at day 2 after PPD stimulation and the cytokines were measured at days 3, 8, and 14 poststimulation. Separate cultures were set up for each time point and the cells were not washed and the medium was not changed for the duration of the experiment. Results are expressed as a mean ± SEM and are representative of three separate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

 
We next sought to determine whether the different cytokine milieu elicited by PPD stimulation in anergic vs PPD-responsive PBMC was influenced by HIV-1 infection or viral tropism. A subset of PPD-anergic or PPD-responsive donor PBMC were thus infected with a T lymphocyte (T)-tropic HIV-1KR25 (Fig. 1B) or a macrophage (M)-tropic HIV-198IN22 (Fig. 1C) primary isolate 2 days after PPD or mock stimulation, and cytokine levels were measured 1, 6, and 12 days after infection and thus 3, 8, and 14 days after PPD stimulation.

Without PPD prestimulation, there were no detectable changes in the cytokine profiles of HIV-1-infected vs uninfected cells (Fig. 1). Whereas HIV-1 infection of PPD-stimulated cells further increased the levels of all four cytokines as compared with HIV-1-uninfected PPD-stimulated cultures, the pattern of cytokine induction in PPD-anergic and PPD-responsive cells remained unchanged (Fig. 1). Thus, the cytokine profile of PPD-stimulated cells is distinct in PPD-anergic and PPD-responsive PBMC and is not changed by HIV-1 infection.

T-tropic HIV-1 replication is significantly lower in the PPD-stimulated PBMC from PPD-anergic compared with PPD-responsive donors

We next measured p24 levels in the infected culture supernatants to determine whether the different cytokine milieus of PPD-anergic and -responsive PBMC affected HIV-1 replication. Strikingly, infection of PPD-stimulated cells with T-tropic HIV-1KR25 resulted in dramatically lower virus replication in PPD-anergic compared with PPD-responsive cultures (Fig. 2A). In PPD-stimulated and HIV-1KR25-infected anergic cultures, p24 levels were ~5-fold lower (p < 0.001) at day 6 and 3-fold lower (p < 0.005) at day 12 postinfection as compared with PPD-responsive cultures, whereas no difference was observed in p24 levels of both subgroups stimulated with the positive control PHA/IL-2 (Fig. 2A). By contrast, the p24 levels in the PPD-responsive cultures were similar whether stimulated with PPD or PHA/IL-2 at all time points. Notably, infection with the M-tropic HIV-198IN22 after stimulation with PPD or with PHA/IL-2 resulted in very low levels of virus replication and there was no difference in the p24 levels between PPD-anergic and -responsive donors up to 17 days postinfection (Fig. 2B).



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 2. T-tropic HIV-1, but not M-tropic HIV-1, replication is significantly impaired in PPD-anergic as compared with PPD-responsive donor cells. PBMC from PPD-anergic ({square} and PPD-responsive ({blacksquare}) donors were unstimulated (UN) or stimulated with PPD or PHA/IL-2 for 2 days before infection with T-tropic HIV-1KR25 (A) or M-tropic HIV-198IN22 (B). p24 levels were determined at days 6, 12, and 17 postinfection. The medium of the cultures that were terminated at days 6 and 12 was not changed for the duration of the experiment. However, cultures terminated at day 17 were fed with fresh patient PBMC on day 11 postinfection to maintain viability of the cultures. Results are expressed as a mean ± SEM and are representative of three separate experiments.*, p < 0.05; **, p < 0.01; ***, p < 0.005. Please note the different scales used for measuring p24 levels in cell infected with T-tropic HIV-1KR25 (A) or M-tropic HIV-198IN22 (B).

 
Blocking of IL-10 significantly increases HIV-1 replication in anergic donor cells and blocking of TNF-{alpha} significantly decreases HIV-1 replication in PPD-responsive donor cells

Given the higher levels of IL-10 in anergic PBMC compared with IL-10 levels in PPD-responsive PBMC and given the ability of IL-10 to inhibit Ag-specific responses in general (14, 15, 16), we next tested the effect of neutralizing IL-10 upon HIV-1KR25 p24 levels. Using an anti-IL-10 mAb at the time of PPD stimulation, p24 levels were significantly increased in PPD-anergic HIV-1KR25-infected cultures at day 6 (p < 0.05) and day 12 (p < 0.01) postinfection. By contrast, blocking IL-10 only minimally increased p24 levels in PPD-responsive cultures at all time points. Thus, blocking IL-10 relieved the inhibition of HIV-1KR25 replication in PPD-anergic cells.

Since TNF-{alpha} levels were higher in PPD-responsive cultures, and given that TNF-{alpha} enhances HIV-1 replication (17), we next tested the effect of blocking TNF-{alpha} upon p24 levels in both groups of donor cells. When PPD-responsive cultures were treated with an anti-TNF-{alpha} mAb at the time of PPD stimulation, p24 levels of HIV-1KR25-infected cultures were significantly decreased at day 6 (p < 0.005) and day 17 (p < 0.01) postinfection, whereas p24 levels were minimally reduced in the PPD-anergic cultures at all time points after infection (Fig. 3A). By contrast, there was no observable effect of blocking TNF-{alpha} or IL-10 on p24 levels in cultures infected with the M-tropic HIV-198IN22 (Fig. 3B). This could be due to very low levels of HIV-198IN22 replication within the time points studied.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 3. Blocking IL-10 and TNF-{alpha} have opposite effects upon T-tropic HIV-1 replication. PBMC from PPD-anergic ({square}) and PPD-responsive ({blacksquare}) donors were stimulated with PPD in the presence or absence of anti-IL-10 or anti-TNF-{alpha} mAbs as indicated for 2 days before being infected with HIV-1KR25 (A) or HIV-198IN22 (B). The p24 levels were measured at days 6, 12, and 17 after virus infection. Results are expressed as a mean ± SEM and are representative of three separate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

 
Thus, the immunosuppressive cytokine milieu (with relatively higher IL-10 levels) elicited by MTb recall Ags in PPD-anergic cultures compared with the immunostimulatory milieu elicited in PPD-responsive cultures (with relatively higher TNF-{alpha} and IL-2 levels) had a significant impact upon the replication of a HIV-1 T-tropic primary isolate. Furthermore, blocking TNF-{alpha} or IL-10 did not influence the replication of the M-tropic HIV-1 primary isolate in the period of time studied.

CXCR4 receptor expression and cellular proliferation in PPD-stimulated PPD-anergic and PPD-responsive PBMC are equivalent

Several reports have demonstrated an important role for CD4+ T cell activation and CXCR4 receptor expression in the ability of T- tropic HIV-1 to establish a productive infection (18, 19, 20). Since cytokines can influence the expression of CXCR4, we next examined the percentage of CD4+ cells expressing CXCR4 and the intensity (mean fluorescence intensity (MFI)) of this receptor in PPD-anergic and -responsive PBMC stimulated with PPD. While following PPD stimulation for 3 days, the percentage of CD4+ cells expressing CXCR4 was slightly higher in PPD-responsive cells and the MFI of CXCR4 was marginally higher in anergic cells, PPD stimulation significantly increased the CXCR4 intensity in both groups of donor CD4+ cells (Fig. 4). Therefore, differences in CXCR4 receptor expression and intensity could not be responsible for the significant difference in HIV-1KR25 p24 levels observed between PPD-anergic and -responsive PBMC stimulated with PPD.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 4. CXCR4 expression is similar in PPD-anergic and -responsive PBMC. PPD-anergic and -responsive PBMC were freshly isolated ({square}) or were stimulated with PPD ({blacksquare}) for 3 days and the percentage of CD4+CXCR4+ cells was determined. Data are represented as the difference between specifically stained cells and nonspecifically stained cells obtained by setting a quadrant marker for nonspecific staining in dot plots. Data on MFI are represented as the difference between MFI of specifically stained cells and nonspecifically stained cells obtained by setting a quadrant marker for nonspecific staining in dot plots. Results are expressed as a mean ± SEM and are representative of three separate experiments.

 
We next stimulated bulk PBMC from anergic and PPD-responsive PBMC with PPD or PHA/IL-2 for 3 and 5 days and assessed cellular proliferation by (21) thymidine incorporation. In response to PPD stimulation, proliferation was slightly higher in the PPD-responsive cells as compared with anergic cells, whereas there was no difference in the two donor subgroups stimulated with PHA/IL-2 (Fig. 5). Intriguingly, PHA/IL-2 stimulation resulted in equivalent T-tropic virus p24 levels in PPD-anergic and -responsive cultures (Figs. 2 and 5). However, although proliferation was equivalent in PPD-stimulated anergic and PPD-responsive cultures, p24 levels were significantly different in the two donor groups (Figs. 2 and 5). Thus, proliferation of bulk PBMC stimulated with MTb recall Ag is not concordant with T-tropic HIV-1 replication in anergic and PPD-responsive PBMC.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 5. Cellular proliferation of PPD-anergic and -responsive bulk PBMC is equivalent. The proliferation of total PBMC from PPD-anergic ({square}) and PPD-responsive ({blacksquare}) donors was measured by [3H]thymidine incorporation (expressed in cpm) in response to stimulation with PPD or PHA/IL-2 for 3 and 5 days as indicated. Results are expressed as a mean ± SEM and are representative of three separate experiments.

 
PPD-anergic CD8+ T lymphocytes are significantly increased in response to PPD stimulation

Previous studies have shown that both CD4+ and CD8+ T cells play an active role in the immune response to MTb Ags (22, 23). Although PPD presentation by autologous APC to CD4+ T cells results in significantly lower CD4+ T cell proliferation in PPD-anergic cells as compared with PPD-responsive cells (9), the effect of PPD stimulation upon CD8+ T cell expansion in this system was unknown. As shown in Fig. 6, no difference in the CD4:CD8 ratio was observed in resting PPD-anergic and -responsive donor cells. Strikingly however, after 3 days of PPD stimulation, there was a significant increase in the percentage of CD8+ T cells (from ~20 to 33%, p < 0.01) and there was a decrease in the CD4+ T cells (from ~39 to 25%), resulting in a dramatic decrease in the CD4:CD8 ratio of PPD-anergic PBMC (from 1.92 to 0.77; Fig. 6). By contrast, PPD stimulation of PPD-responsive PBMC resulted in nonsignificant increases in both CD8+ T cells (from 18 to 26%) and CD4+ T cells (from 30 to 33%) and the CD4:CD8 ratio remained above one (1.23; Fig. 6). The percentage of CD4CD8 double-positive cells was also increased in both subgroups to a similar level. Thus, following MTb recall Ag stimulation, there is a disproportional expansion of CD8+ T cells and a lack of CD4+ T cell proliferation in PPD-anergic compared with PPD-responsive PBMC from hosts previously exposed to MTb.



View larger version (57K):
[in this window]
[in a new window]
  		FIGURE 6. CD8+ T lymphocytes differentially expand in anergic PBMC in response to PPD stimulation. The percentage of CD3+CD4+, CD3+CD8+ T cells was determined by flow cytometry. Data are represented as the difference between specifically stained cells and nonspecifically stained cells obtained by setting a quadrant marker for nonspecific staining in dot plots. The dot plot is representative of PBMC freshly isolated or PPD stimulated for 3 days from six PPD-anergic and six PPD-responsive donors. Results are expressed as a mean ± SEM and are representative of three separate experiments. **, p < 0.01.

 
MTb-activated CD8+ T cells significantly suppress HIV-1KR25 levels in PPD-anergic PBMC

Given that activated CD8+ T cells are known to suppress HIV (24, 25, 26), we next investigated the effect of depleting CD8+ T cells from PPD-anergic and PPD-responsive bulk PBMC upon HIV-1KR25 replication. Bulk PBMC and CD8+ T cell-depleted PBMC from both donor subgroups were PPD stimulated for 2 days before being infected with HIV-1KR25 and p24 levels were then measured 3 days following infection. Depletion of CD8+ T cells significantly increased p24 levels in PPD-anergic (~2.5-fold, p < 0.01) cells, whereas there was only a nonsignificant increase in p24 levels in PPD-responsive cultures (~1.7-fold, p > 0.1; Fig. 7). Taken together, these results demonstrate that MTb Ag-stimulated CD8+ T cells differentially suppress HIV-1 expression in PPD-anergic donor PBMC.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 7. CD8+ T cells differentially suppress HIV-1 replication in PPD-anergic PBMC. CD8+ T cells were depleted from PBMC by positive selection using magnetic beads coated with an anti-CD8 mAb (Dynal Biotech). The purity of the cells was ~96% as judged by flow cytometry (data not shown). p24 levels were measured 3 days after HIV-1KR25 infection of PPD-prestimulated, PPD-anergic, and PPD-responsive bulk PBMC ({square} or CD8+-depleted PBMC ({blacksquare}). Results are expressed as a mean ± SEM and are representative of two separate experiments. **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that an immunosuppressive and reversible host response to MTb recall Ags resulting in increased IL-10 levels and CD8+ T cell expansion significantly suppresses HIV-1KR25 replication in cells from PPD-anergic donors in vitro. These results thus demonstrate that PPD-anergic individuals have an advantage in controlling HIV-1 viral load if re-challenged with MTb Ags.

Even using an optimal anti-TB chemotherapeutic regimen, it is estimated that ~5–10% of patients with apparent cure, relapse with TB disease (27). Thus, host-specific responses to TB reactivation or reinfection that result in higher viral loads can be expected to promote HIV-1 disease progression, which in turn causes further depletion of CD4+ cells, resulting in MTb disease progression (2). Consistent with these observations, isoniazid prophylaxis in HIV-infected individuals who are latently infected with TB has been reported to delay the onset of HIV-1-related illnesses and to prolong survival in the coinfected host (5).

Intriguingly, our data also indicate that MTb recall Ags differentially induce viral replication of a T-tropic, but not an M-tropic, HIV-1 primary isolate up to 17 days postinfection. Consistent with other studies (28, 29) using primary M-tropic HIV-1 isolates from patients in an early stage of HIV-1 infection, we have observed that replication of the M-tropic HIV-198IN22 (50 TCID50) was significantly less (more than a 1000-fold less) than what was observed using the T-tropic primary isolate HIV-1KR25 (10 TCID50). We note that both of the isolates used in this study had only three short passages within normal PBMC cultures. Our data demonstrate PPD stimulation of PBMC from PPD-responsive donors does not differentially activate M-tropic replication as it does when the T-tropic virus is used within the early time points studied. Given the low levels of HIV-198IN22 replication we observed, it is not surprising that we were unable to detect any effect of blocking IL-10 or TNF-{alpha} in these cultures even stimulated with PPD or PHA and IL-2.

In the case of the T cell-tropic HIV-1 infection, TNF-{alpha} is significantly increased by PPD stimulation of PBMC from PPD-responsive donors and its neutralization results in a significant decrease of HIV-1 replication. By contrast neutralization of IL-10 has a nonsignificant impact upon HIV-1 replication in the PPD-responsive PBMC. Thus, PPD-stimulated TNF-{alpha} is directly linked to an increase in HIV-1 replication in PBMC from PPD-responsive donors. Furthermore, IL-2 and IFN-{gamma} are differentially increased in PPD-responsive donors and may also contribute to the observed increase in viral replication of the T-tropic isolate. Given that HIV-1 viral load is directly linked to HIV-1 and AIDS disease progression (30), these data provide a direct demonstration that the state of Ag-specific activation of the immune system by MTb and potentially the stage of HIV-1 infection as reflected by the tropism of the infecting viral strain has a direct impact upon HIV-1 progression in the setting of rechallenge with MTb Ags.

Notably, CXCR4-using T-tropic viruses, which infect CD4+ T cells, predominate comparatively late in HIV-1 infection (31, 32). Thus, MTb Ag-specific CD4+ T cell activation that occurs later in HIV-1 infection, when T cell-tropic viruses predominate, may be particularly destructive. Furthermore, our data support the concept that MTb infection, reinfection, or reactivation may in fact promote HIV-1 disease through the creation of a cytokine and cellular milieu that favors the transition of M-tropic to T-tropic viral strains and the rapid expansion of T-tropic viruses in the PPD-responsive host.

Since more than one-third of the global population is infected with MTb, mostly in the regions with high a incidence of HIV-1, the majority of individuals infected with HIV-1 are PPD responsive before the occurrence of HIV-1-associated immunosuppression (33). TNF-{alpha} has a clear role in increasing HIV-1 replication via its engagement of the signaling pathway, resulting in activation of the transcription factor NF-{kappa}B, which binds to the HIV-1 long terminal repeat and thus drives HIV-1 transcription (34, 35). Thus, our demonstration of the differential increase of TNF-{alpha} and HIV-1 replication in PPD-stimulated PPD-responsive cultures underscores the importance of chemotherapy for TB as early as possible in the HIV-1/MTb-coinfected host.

CD8+ T cells control HIV-1 levels by inhibiting viral infection and replication via the production of soluble antiviral factors, including CD8+ antiviral factor, which is capable of inhibiting both M- and T- tropic HIV-1 replication (24, 26, 36) and by their ability to directly kill infected cells (37, 38). Previously we have shown that MTb Ag-specific anergic patients have a constitutive increase in CD4+ IL-10-producing T cells and increased levels of soluble IL-10 in PPD-stimulated PBMC supernatants (9, 10). Given that IL-10 is a chemotactic factor for CD8+ T lymphocytes and enhances both the precursor frequency and activity of CD8+ T lymphocytes (39), the increased levels of IL-10 in PPD-anergic individuals may in fact predispose them to the observed differential increase in CD8+ T cell expansion and subsequent suppression of HIV-1 replication in response to MTb recall Ags.

In the case of individuals with persistent MTb Ag-specific anergy after successful Tb treatment, MTb recall challenge causes an expansion of CD8+ T cells and increased IL-10 production. Thus a host-specific environment that is unfavorable to T-tropic HIV-1 replication is created. Finally, the identification of a subset of individuals whose cells have an innate advantage in controlling HIV-1 viral load in response to MTb recall challenge suggests that immunotherapeutic approaches aimed at recapitulating Ag-specific MTb anergy in vivo could result in novel treatment strategies of the MTb/HIV-1-coinfected host.


   Acknowledgments
 
We are deeply indebted to the generosity of the patients who participated in this study and to the Cambodian Health Committee staff, particularly Sun Sath, Riel Sarom, and Sok Sophat whose assistance was invaluable. We thank H. Holmes and the Centralized Facility for AIDS Reagents (National Institute for Biological Standard and Control, U.K.) and S. Osmanov of the World Health Organization-Joint United Nations Programme on HIV/AIDS (Geneva, Switzerland) for the gift of HIV-198IN22. We are grateful to Prema Shankar, Judy Lieberman, Philippe Glaziou, Jean-Louis Sarthou, Julio Delgado, Keith Crawford, Marie Nguyen, Gianfranco Pancino, and Francoise Barre-Sinoussi for helpful discussions.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (to A.E.G.) (HL59838) and the Campbell Foundation (to S.R.). Back

2 Address correspondence and reprint requests to Dr. Anne.E. Goldfeld, Center for Blood Research, Harvard Medical School, 800 Huntington Avenue, Boston, MA 02115. E-mail address:goldfeld{at}cbr.med.harvard.edu Back

3 Abbreviations used in this paper: TB, tuberculosis; MTb, Mycobacterium tuberculosis; PPD, purified protein derivative; DTH, delayed-type hypersensitivity; TCID50, 50% tissue culture-effective dose; MFI, mean fluorescence intensity. Back

Received for publication August 14, 2003. Accepted for publication November 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. World Health Organization. 2001. The World Health Report 2001 144. World Health Organization, Geneva.
  2. Bloom, B.. 1994. Tuberculosis: Pathogenesis, Protection, and Control ASM Press, Washington, DC.
  3. van Rie, A., R. Warren, M. Richardson, T. C. Victor, R. P. Gie, D. A. Enarson, N. Beyers, P. D. van Helden. 1999. Exogenous reinfection as a cause of recurrent tuberculosis after curative treatment. N. Engl. J. Med. 341:1174.[Abstract/Free Full Text]
  4. Whalen, C., C. R. Horsburgh, D. Hom, C. Lahart, M. Simberkoff, J. Ellner. 1995. Accelerated course of human immunodeficiency virus infection after tuberculosis. Am. J. Respir. Crit. Care Med. 151:129.[Abstract]
  5. Pape, J. W., S. S. Jean, J. L. Ho, A. Hafner, W. D. J. Johnson. 1993. Effect of isoniazid prophylaxis on incidence of active tuberculosis and progression of HIV infection. Lancet 342:268.[Medline]
  6. Corbett, E. L., C. J. Watt, N. Walker, D. Maher, B. G. Williams, M. C. Raviglione, C. Dye. 2003. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 163:1009.[Abstract/Free Full Text]
  7. Mellors, J. W., A. Munoz, J. V. Giorgi, J. B. Margolick, C. J. Tassoni, P. Gupta, L. A. Kingsley, J. A. Todd, A. J. Saah, R. Detels, et al 1997. Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann. Intern. Med. 126:946.[Abstract/Free Full Text]
  8. Mellors, J. W., C. R. Rinaldo, Jr, P. Gupta, R. M. White, J. A. Todd, L. A. Kingsley. 1996. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 272:1167. [Published erratum appears in 1997 Science 275:14.]. [Abstract]
  9. Boussiotis, V. A., E. Y. Tsai, E. J. Yunis, S. Thim, J. C. Delgado, C. C. Dascher, A. Berezovskaya, D. Rousset, J. M. Reynes, A. E. Goldfeld. 2000. IL-10-producing T cells suppress immune responses in anergic tuberculosis patients. J. Clin. Invest. 105:1317.[Medline]
  10. Delgado, J. C., E. Y. Tsai, S. Thim, A. Baena, V. A. Boussiotis, J. M. Reynes, S. Sath, P. Grosjean, E. J. Yunis, A. E. Goldfeld. 2002. Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia. Proc. Natl. Acad. Sci. USA 99:7576.[Abstract/Free Full Text]
  11. Rubsamen-Waigmann, H., H. von Briesen, H. Holmes, A. Bjorndal, B. Korber, R. Esser, S. Ranjbar, P. Tomlinson, B. Galvao-Castro, E. Karita, et al 1994. Standard conditions of virus isolation reveal biological variability of HIV type 1 in different regions of the world: WHO Network for HIV Isolation and Characterization. AIDS Res. Hum. Retroviruses 10:1401.[Medline]
  12. Rodenburg, C. M., Y. Li, S. A. Trask, Y. Chen, J. Decker, D. L. Robertson, M. L. Kalish, G. M. Shaw, S. Allen, B. H. Hahn, et al 2001. Near full-length clones and reference sequences for subtype C isolates of HIV type 1 from three different continents. AIDS Res. Hum. Retroviruses 17:161.[Medline]
  13. Bjorndal, A., H. Deng, M. Jansson, J. R. Fiore, C. Colognesi, A. Karlsson, J. Albert, G. Scarlatti, D. R. Littman, F. E. M. . 1997. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J. Virol. 71:7478.[Abstract]
  14. Schwartz, R. H.. 1996. Models of T cell anergy: is there a common molecular mechanism?. J. Exp. Med. 184:1.[Free Full Text]
  15. Groux, H., M. Bigler, J. E. de Vries, M. G. Roncarolo. 1996. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J. Exp. Med. 184:19.[Abstract/Free Full Text]
  16. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
  17. Fauci, A.. 1996. Host factors and the pathogenesis of HIV-induced disease. Nature 384:529.[Medline]
  18. Bukrinsky, M. I., T. L. Stanwick, M. P. Dempsey, M. Stevenson. 1991. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science 254:423.[Abstract/Free Full Text]
  19. Ramilo, O., K. D. Bell, J. W. Uhr, E. S. Vitetta. 1993. Role of CD25+ and CD25- T cells in acute HIV infection in vitro. J. Immunol. 150:5202.[Abstract]
  20. Kinter, A., J. Arthos, C. Cicala, A. S. Fauci. 2000. Chemokines, cytokines and HIV: a complex network of interactions that influence HIV pathogenesis. Immunol. Rev. 177:88.[Medline]
  21. Barthel, R., A. V. Tsytsykova, A. K. Barczak, E. Y. Tsai, C. C. Dascher, M. B. Brenner, A. E. Goldfeld. 2003. Regulation of tumor necrosis factor {alpha} gene expression by mycobacteria involves the assembly of a unique enhanceosome dependent on the coactivator proteins CBP/p300. Mol. Cell. Biol. 23:526.[Abstract/Free Full Text]
  22. Kaufmann, S. H.. 2001. How can immunology contribute to the control of tuberculosis?. Nat. Rev. Immunol. 1:20.[Medline]
  23. Serbina, N. V., V. Lazarevic, J. L. Flynn. 2001. CD4+ T cells are required for the development of cytotoxic CD8+ T cells during Mycobacterium tuberculosis infection. J. Immunol. 167:6991.[Abstract/Free Full Text]
  24. Zhang, L., W. Yu, T. He, J. Yu, R. E. Caffrey, E. A. Dalmasso, S. Fu, T. Pham, J. Mei, J. J. Ho, W. Zhang, P. Lopez, D. D. Ho. 2002. Contribution of human {alpha}-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor. Science 298:995.[Abstract/Free Full Text]
  25. Walker, C. M., D. J. Moody, D. P. Stites, J. A. Levy. 1986. CD8+ lymphocytes can control HIV infection in vitro by suppressing virus replication. Science 234:1563.[Abstract/Free Full Text]
  26. Migueles, S. A., A. C. Laborico, W. L. Shupert, M. S. Sabbaghian, R. Rabin, C. W. Hallahan, D. Van Baarle, S. Kostense, F. Miedema, M. McLaughlin, et al 2002. HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors. Nat. Immunol. 3:1061.[Medline]
  27. Weis, S. E., P. C. Slocum, F. X. Blais, B. King, M. Nunn, G. B. Matney, E. Gomez, B. H. Foresman. 1994. The effect of directly observed therapy on the rates of drug resistance and relapse in tuberculosis. N. Engl. J. Med. 330:1179.[Abstract/Free Full Text]
  28. Asjo, B., L. Morfeldt-Manson, J. Albert, G. Biberfeld, A. Karlsson, K. Lidman, E. M. Fenyo. 1986. Replicative capacity of human immunodeficiency virus from patients with varying severity of HIV infection. Lancet 2:660.[Medline]
  29. Schwartz, S., B. K. Felber, E. M. Fenyo, G. N. Pavlakis. 1989. Rapidly and slowly replicating human immunodeficiency virus type 1 isolates can be distinguished according to target-cell tropism in T-cell and monocyte cell lines. Proc. Natl. Acad. Sci. USA 86:7200.[Abstract/Free Full Text]
  30. Novitsky, V., P. Gilbert, T. Peter, M. F. McLane, S. Gaolekwe, N. Rybak, I. Thior, T. Ndung’u, R. Marlink, T. H. Lee, M. Essex. 2003. Association between virus-specific T-cell responses and plasma viral load in human immunodeficiency virus type 1 subtype C infection. J. Virol. 77:882.
  31. Schuitemaker, H., M. Koot, N. A. Kootstra, M. W. Dercksen, R. E. de Goede, R. P. van Steenwijk, J. M. Lange, J. K. Schattenkerk, F. Miedema, M. Tersmette. 1992. Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population. J. Virol. 66:1354.[Abstract/Free Full Text]
  32. Koot, M., I. P. Keet, A. H. Vos, R. E. de Goede, M. T. Roos, R. A. Coutinho, F. Miedema, P. T. Schellekens, M. Tersmette. 1993. Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS. Ann. Intern. Med. 118:681.[Abstract/Free Full Text]
  33. American Thoracic Society and the Centers for Disease Control and Prevention 2000. Targeted tuberculin testing and treatment of latent tuberculosis infection. Am. J. Respir. Crit. Care Med. 161:S221.[Free Full Text]
  34. Nabel, G., D. Baltimore. 1987. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 326:711.[Medline]
  35. Israel, N., U. Hazan, J. Alcami, A. Munier, F. Arenzana-Seisdedos, F. Bachelerie, A. Israel, J. L. Virelizier. 1989. Tumor necrosis factor stimulates transcription of HIV-1 in human T lymphocytes, independently and synergistically with mitogens. J. Immunol. 143:3956.[Abstract]
  36. Barker, E., K. N. Bossart, J. A. Levy. 1998. Primary CD8+ cells from HIV-infected individuals can suppress productive infection of macrophages independent of {beta}-chemokines. Proc. Natl. Acad. Sci. USA 95:1725.[Abstract/Free Full Text]
  37. Walker, B. D., E. S. Rosenberg, C. M. Hay, N. Basgoz, O. O. Yang. 1998. Immune control of HIV-1 replication. Adv. Exp. Med. Biol. 452:159.[Medline]
  38. Lieberman, J., P. Shankar, N. Manjunath, J. Andersson. 2001. Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection. Blood 98:1667.[Abstract/Free Full Text]
  39. Groux, H., M. Bigler, J. E. de Vries, M. G. Roncarolo. 1998. Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells. J. Immunol. 160:3188.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CVIHome page
B. L. Anderson, R. J. Welch, and C. M. Litwin
Assessment of Three Commercially Available Serologic Assays for Detection of Antibodies to Mycobacterium tuberculosis and Identification of Active Tuberculosis
Clin. Vaccine Immunol., November 1, 2008; 15(11): 1644 - 1649.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
N. Tanaka, Y. Hoshino, J. Gold, S. Hoshino, F. Martiniuk, T. Kurata, R. Pine, D. Levy, W. N. Rom, and M. Weiden
Interleukin-10 Induces Inhibitory C/EBP{beta} through STAT-3 and Represses HIV-1 Transcription in Macrophages
Am. J. Respir. Cell Mol. Biol., October 1, 2005; 33(4): 406 - 411.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
C. Renoux, S. Wain-Hobson, B. Hurtrel, and R. Cheynier
Antigenic Stimulation Specifically Reactivates the Replication of Archived Simian Immunodeficiency Virus Genomes in Chronically Infected Macaques
J. Virol., September 1, 2005; 79(17): 11231 - 11238.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ranjbar, S.
Right arrow Articles by Goldfeld, A. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ranjbar, S.
Right arrow Articles by Goldfeld, A. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS