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The Journal of Immunology, 2001, 166: 3210-3217.
Copyright © 2001 by The American Association of Immunologists

In Vivo CD40-CD154 (CD40 Ligand) Interaction Induces Integrated HIV Expression by APC in an HIV-1-Transgenic Mouse Model1

Claire Chougnet*, Corona Freitag{dagger},§, Marco Schito{dagger}, Elaine K. Thomas{ddagger}, Alan Sher2,{dagger} and Gene M. Shearer2,3,*

* Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; {dagger} Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892; {ddagger} Immunex, Seattle, WA 98101; and § Howard Hughes Medical Institute, National Institutes of Health Research Scholars Program, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because of their relative resistance to viral cytopathic effects, APC can provide an alternative reservoir for latently integrated HIV. We used an HIV-transgenic mouse model in which APC serve as the major source of inducible HIV expression to study mechanisms by which integrated virus can be activated in these cells. When admixed with transgenic APC, activated T lymphocytes provided a major contact-dependent stimulus for viral protein expression in vitro. Using blocking anti-CD154 mAb as well as CD154-deficient T cells, the HIV response induced by activated T lymphocytes was demonstrated to require CD40-CD154 interaction. The role of this pathway in the induction of HIV expression from APC in vivo was further studied in an experimental model involving infection of the HIV-transgenic mice with Plasmodium chabaudi parasites. Enhanced viral production by dendritic cells and macrophages in infected mice was associated with up-regulated CD40 expression. More importantly, in vivo treatment with blocking anti-CD154 mAb markedly reduced viral expression in P. chabaudi-infected animals. Together, these findings indicate that immune activation of integrated HIV can be driven by the costimulatory interaction of activated T cells with APC. Because chronic T cell activation driven by coinfections as well as HIV-1 itself is a characteristic of HIV disease, this pathway may be important in sustaining viral expression from APC reservoirs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The persistence of HIV-1 in latent cellular reservoirs that survive antiviral therapy represents a major obstacle to the eradication of this important pathogen (1, 2, 3, 4, 5). Although T lymphocytes are considered to be the major source of latent virus (6), there is a growing body of evidence suggesting that APC can also serve as reservoirs (1, 7, 8, 9, 10). Indeed, because these cells are relatively resistant to cytopathic effects of HIV, they may constitute a major source of persistent infection, particularly in the preterminal stages of AIDS (11, 12). Although immune activation is thought to represent a driving force for activation of HIV-1 from latent reservoirs, little is known about the mechanisms responsible for this process, which is probably differently regulated than activation from the resting T cell reservoir.

We previously characterized an in vivo experimental model for immune activation of HIV-1 in which the production of infectious virus is induced in HIV-1-transgenic (Tg)4 mice by infection with intracellular pathogens (13, 14). The line 166 mice that we use carry complete DNA copies of the HIV-1 genome, including an unaltered LTR and provide a tool for identifying in vivo mechanisms of immune activation analogous to the murine Tg model, which has been effectively used for studying the immunopathogenesis of hepatitis B (15). After in vivo or in vitro microbial stimulation, splenocytes from HIV-Tg animals produce low levels of infectious virus recoverable by coculture with a human T cell line. Interestingly, previous results in this model demonstrated that expression of HIV could not be induced in activated T cells. Instead, distinct APC populations were implicated as sources of HIV Ags and infectious virus (14). Nevertheless, although T cells are not the source of virus, their activation could be a driving factor in the activation-induced expression of viral mRNA and proteins, because activated T cells are known to be a potent stimulator of all lineages of APC. Interactions between CD40, present on APC, and CD154 (CD40 ligand), the expression of which is up-regulated on activated T cells, have been described as a major intercellular pathway involved in APC stimulation (16, 17). Thus, antigenic T cell activation and cellular contact with APC could provide signals that induce expression of integrated HIV. Because chronic T cell activation is a prominent feature of human HIV disease (18, 19, 20), this pathway may provide a major stimulus for the activation of latent virus from APC reservoirs.

In this study we demonstrate in HIV-Tg mice that activated T cells induce the expression of integrated virus from APC in vitro, and that this response is highly dependent on CD40-CD154 interaction. More importantly, we show that the same pathway plays a major role in the induction of viral expression in vivo as a consequence of parasitic infection. The model chosen for the latter experiments involves infection with blood stages of the murine malaria parasite Plasmodium chabaudi. Because these forms do not invade APC or other cells that would harbor the Tg, they cannot directly stimulate HIV expression. Together our in vitro and in vivo findings support the concept that CD40-CD154 interaction can provide a potent stimulus for viral induction from APC reservoirs, particularly in the context of the strong T cell activation resulting from concomitant infections.


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

The HIV-Tg mouse line 166 was derived as previously described (13) by pro-nuclear injection of FVB/N mouse embryos with proviral DNA encoding the entire genome of the NL4–3 molecular clone, a T cell tropic strain of HIV-1. The resulting animals contained ~20–60 copies of the proviral transgenes present at single integration sites and transmitted them in a stable Mendelian fashion. Homozygous 166 male were bred to female C57BL/6 and (166 x C57BL/6)F1, and the offspring were used. The presence of HIV-Tg in the F1 was assessed by screening for HIV p24 antigenemia in the blood of these animals. Mice were maintained in an escape-proof facility within the animal care facilities of the National Institute of Allergy and Infectious Diseases (Bethesda, MD). Non-Tg (FVB/N x C57BL/6)F1 mice were bred in the same facility. C57BL/6 CD154 knockout (CD154-/-) mice or C57BL/6 wild-type (wt) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals between 8 and 20 wk of age were used in all experiments. All protocols were approved by the National Institute of Allergy and Infectious Diseases institutional review board.

Infection with malaria parasites

Blood stage infection with Plasmodium chabaudi chabaudi (AS) was maintained by weekly passages in naive mice as previously described (21). Experimental infections were initiated with i.p. inoculation of 105 P. chabaudi-parasitized erythrocytes. Parasitemia was monitored by examination of Diff-Quick (Dade Behring, Dudingen, Switzerland)-stained thin blood smears made from tail blood.

Media, Abs, and Ag preparation

Complete medium (CM), consisting of RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM {beta}-ME, 5 mM HEPES, and 2 mM glutamine, was used in all cultures. Recombinant murine CD154 trimer (rCD154) and rat anti-murine CD154 mAb (M158) were provided by Immunex (Seattle, WA). A rat anti-{beta}-galactosidase Ab (GL113), prepared from ascites and partially purified by ammonium sulfate precipitation, was used as control Ab. Both Ab were used in vivo. Anti-CD3, anti-CD154, anti-IL-12, and anti-IFN-{gamma} mAb and matched isotype control mAb (PharMingen, San Diego, CA) were used in vitro (10 µg/ml). LPS was used at 1 µg/ml (Sigma, St. Louis, MO). Trophozoite stage P. chabaudi-parasitized RBC (pRBC) were prepared from EDTA-treated blood from infected mice just before peak parasitemia. PBMC were removed using Lympholyte-M separation medium (Cedarlane Laboratories, Hornby, Ontario, Canada), and the RBC pellet was washed in CM. Parasitemia was assessed on the pellet, and pRBC were used at a concentration of 106/ml. Equivalent numbers of RBC obtained from uninfected mice were used as a control (cRBC).

Cell populations

To obtain enriched populations of dendritic cells (DC), spleens were digested with collagenase H (1 mg/ml; Roche, Indianapolis, IN) for 30 min at 37°C, followed by incubation with Iscove’s medium (Life Technologies) containing 5 mM EDTA for 5 min at 37°C. A single-cell suspension was then prepared, and RBC were lysed by ammonium chloride treatment. Cells were overlaid on a 14.5% metrizamide-CM gradient (Sigma) and spun for 15 min at 2000 rpm at room temperature. The resulting band was harvested and washed twice in CM. Cells were incubated with anti-mouse CD16/CD32 (PharMingen; 10 µg/ml) for 10 min at 4°C, to block Fc receptors, then with anti-mouse CD11c-coated magnetic beads (Miltenyi Biotech, Auburn, CA) and separated on MACS separation columns. The cells were >60% CD11c+; the remaining cells were B220+, as previously described (22). CD4+ and CD8+ T cells were not detectable in the preparations. Macrophages (M{phi}) were prepared by positive selection, using CD11b-coated beads (Miltenyi Biotech) and were >60% CD11b+. To improve the purity of the M{phi} population, the cells were further adhered for 2 h at 37°C in culture plates. Splenic T cells were prepared by negative selection, using T cell enrichment columns (R&D Systems, Minneapolis, MN), according to the manufacturer’s instruction. Purified T cells were >80% CD3+ (results not shown). In some experiments, unseparated splenocytes, prepared after collagenase digestion of the spleen, were used.

Measurement of cytokine production and of virus activation

Enriched DC and T cells were cocultured at a 1:5 ratio with DC at a final concentration of 106/ml. T cells were either unstimulated or stimulated with immobilized anti-CD3 (10 µg/ml). In some experiments T cells and DC were cultured in the separate chambers of Transwell plates (Costar, Cambridge, MA). Unseparated splenocytes were cultured at the final concentration of 5 x 106/ml, enriched DC and M{phi} were cultured at 106/ml. Supernatants were harvested at 24, 48, and 72 h for measurement of IL-12 p40, HIV p24, and IFN-{gamma}, respectively. IL-12 p40 and IFN- {gamma} were measured using OptEIA sets (detection limit, 30 pg/ml; PharMingen). Levels of HIV p24 was determined by ELISA (detection limit, 2 pg/ml; Coulter, Miami, FL). Plasma HIV p24 levels were assayed on EDTA-treated plasma obtained from individual mice. To control for individual variability in baseline p24 levels, antigenemia was calculated as the fold increase in p24 level post- vs pre-P. chabaudi infection for each individual mouse.

In vivo treatment with anti-CD154 Ab

HIV-Tg female mice were treated with anti-CD154 or control Ab (200 µg/mouse i.p.), starting on day 0 and every 2 days thereafter and were infected with pRBC on day 0, as described above. Uninfected controls were treated with anti-CD154 Ab or control Ab, respectively. All animals were euthanized on day 9, and spleens were harvested for determination of HIV p24 production and in situ hybridization. Animals were also individually bled on day -1 before infection and on day 9 to determine changes in plasma p24 antigenemia.

lp;&6qLocalization of virus expression by in situ hybridization

Portions of spleen from HIV-Tg mice were fixed in 1.3 M aqueous formaldehyde for 24 h. In situ hybridization was performed as previously described (14) (Molecular Histology, Gaithersburg, MD). Briefly, two sets of mounted 6-µm paraffin sections were dewaxed and treated with protease to expose viral nucleic acid. They were hybridized with a 33P-labeled antisense probe (HIV-1, IIIB) that represents 9 kb of the HIV-1 genome. Sense probe hybridization was also performed as a control. Slides were stained with hematoxylin and eosin for morphological assessment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activated T cells induce virus activation in splenic DC from HIV-Tg mice

DC enriched from spleens of HIV-Tg mice presented with a profile of immature DC, characterized by low to moderate expression of CD80 and CD86, and high expression of class II molecules (data not shown). Without stimulation, low to undetectable amounts of HIV p24 and IL-12 p40 were detected in these DC cultures (<50 pg/ml). After LPS stimulation, high levels of HIV p24 and IL-12 p40 were detected (mean, 2, 146.7 ± 142.9 and 478.7 ± 84 pg/ml for p24 and p40, respectively).

Because activated T cells are known to be a potent stimulator of DC function, we studied whether this signal could induce the expression of the HIV-Tg in DC. Purified T cells from syngeneic non-Tg mice were cocultured with splenic DC enriched from HIV-Tg mice in the presence or the absence of anti-CD3. Interactions between DC and activated T cells, but not with unactivated T cells, induced an ~10-fold increase in the production of both HIV p24 and IL-12 p40, a cytokine known to be up-regulated in DC as a consequence of T cell interaction. In contrast, anti-CD3 did not stimulate directly DC in the absence of T cells (Fig. 1Go).



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  		FIGURE 1. Stimulation of virus expression from enriched splenic HIV-Tg DC by activated T cells. HIV p24 and IL-12 p40 production were measured in supernatants of 48- or 24-h cultures, respectively, from enriched DC populations from HIV-Tg mice (pool of four to eight mice). DC were cultured in the presence or the absence of T cells (TC) from syngeneic non-HIV-Tg mice (pool of two to four mice). Cells were either unstimulated (medium) or stimulated with immobilized anti-CD3. The results shown are the mean ± SD of duplicate or triplicate cultures.

 
In vitro virus activation is partially dependent on CD40-CD154 interaction

Because activated T cells provide a signal to DC that results in HIV induction, we investigated whether this signal was contact dependent. Cultures were performed in Transwell plates, with anti-CD3-activated T cells added to one chamber and DC to the other. The prevention of contact between the two cellular populations almost completely abrogated the activation of HIV and IL-12 in DC (Table IGo). We next studied whether CD40-CD154-dependent signaling events are involved in the activation of HIV-Tg. Addition of a blocking anti-CD154 reduced the production of HIV p24 induced by activated T cells (p < 0.05, by t test), whereas an isotype control Ab had no effect on p24 production (p > 0.4; Fig. 2GoA). As expected, anti-CD154 Ab was efficient at reducing IL-12 p40 production in the same cultures (Fig. 2GoA). Stimulation of HIV-Tg DC with rCD154 induced production of both HIV p24 and IL-12 p40 (Fig. 2GoB), confirming the role of CD154 in activation of HIV-Tg.


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  		Table I. Induction of HIV expression in DC by activated T cells is contact dependent

 


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  		FIGURE 2. Production of HIV p24 by isolated splenic DC is partially CD40 dependent. HIV p24 and IL-12 p40 production were measured in supernatants of 48- or 24-h cultures, respectively, from enriched DC populations from HIV-Tg mice (pool of four to eight mice). Cells were cultured in the presence or the absence of T cells (TC) from syngeneic non HIV-Tg mice (A). Cells were either unstimulated (medium) or stimulated with immobilized anti-CD3 in the presence or the absence of anti-CD154 or control Ab (10 µg/ml). *, A significant difference between anti-CD154- and isotype-treated cultures (p < 0.05, by paired t test). Cells were cultured with rCD154 (5 µg/ml; B) or in the presence or the absence of TC from wt or CD154-/- C57BL/6 mice (C). The results shown are the mean ± SD of duplicate or triplicate cultures.

 
To further assess the importance of CD40-CD154 interaction, T cells from CD154-/- mice and wt C57BL/6 mice were compared for the induction of HIV from DC. Because DC were generated from line (166 x C57BL/6)F1 mice, wt T cells induced some activation of DC (~5-fold increase in p24 and ~2-fold increase in p40 production) that was probably due to the recognition of H-2q Ags on the surface of DC by the T cells. As expected, anti-CD3-stimulated wt T cells induced a large increase in p24 (~40-fold) and p40 (~10-fold) production. In contrast, activated CD154-/- T cells did not induce IL-12 production and only minimally induced HIV p24 production (Fig. 2GoC). Anti-CD3-stimulated T cells from both wt and CD154-/- mice produced high levels of IFN-{gamma} (>40,000 pg/ml), ruling out a lack of activation of CD154-/- T cells as the mechanism for the lack of stimulation of the HIV-Tg.

Because HIV expression induced by activated T cells or rCD154 was in most cases accompanied by the production of IL-12 p40, we investigated whether the induction of this proinflammatory cytokine or its end product IFN-{gamma} is indirectly responsible for the viral up-regulation observed in vitro. As shown in Table IIGo, simultaneous neutralization of IL-12 and IFN-{gamma} failed to significantly alter DC production of p24 triggered by either T cell-related stimulus (all p > 0.1, by paired t test), arguing that HIV-Tg is not induced as a consequence of IL-12 up-regulation.


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  		Table II. In vitro neutralization of IL-12 and IFN-{gamma} production does not alter HIV expression by DC

 
Activated T cells from P. chabaudi-infected mice stimulate viral expression by HIV-Tg APC

To evaluate whether CD40-CD154 interaction also plays a role in HIV-Tg induction in vivo, we examined viral expression in HIV-Tg mice experimentally infected with P. chabaudi blood stage parasites. First, we studied the effect of stimulation with plasmodial Ag of splenocytes obtained from 4-wk P. chabaudi-infected animals, at the time when malaria-specific T cells are present in spleen (21, 23). Addition of pRBC induced an ~3-fold increase in p24 production by splenocytes from malaria-infected HIV-Tg mice, compared with that induced by addition of cRBC (Fig. 3Go). In contrast, no increase in p24 production was observed after stimulation of splenocytes of uninfected mice with pRBC, ruling out a direct activation of HIV-Tg by plasmodial Ag. Addition of blocking anti-CD154 mAb to the culture significantly decreased the induction of p24 after stimulation of splenocytes from P. chabaudi-infected mice with pRBC (p = 0.05, by paired t test), whereas it had no effect on p24 production after addition of cRBC or in cultures of splenocytes from uninfected mice (all p > 0.6, by paired t test; Fig. 3Go).



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  		FIGURE 3. Ag-dependent activation of HIV p24 production is partially CD40-CD154 dependent. Splenocytes from P. chabaudi-infected (4 wk postinfection) or uninfected HIV-Tg mice were stimulated with pRBC (106/ml) or cRBC in the presence or the absence of anti-CD154 or control mAb (10 µg/ml). Each bar represents the mean (±SD) p24 production of three or four mice assayed individually. *, A significant difference between P. chabaudi-infected and uninfected mice (p < 0.05, by unpaired t test); #, a significant difference between anti-CD154- and control Ab-treated cultures (p < 0.05, by paired t test).

 
At the time of acute parasitemia (days 8–9 postinfection), spontaneous production of p24 was dramatically increased in APC populations isolated from P. chabaudi-infected animals compared with that in uninfected mice, with increases of >10-fold in unseparated splenocytes and M{phi} and >4-fold in DC (Fig. 4Go). Interestingly, after in vitro stimulation by rCD154, cells isolated from infected mice produced significantly more p24 than cells isolated from uninfected mice (>20-fold increase for splenocytes or M{phi} and >3-fold increase for DC; Fig. 4Go). In contrast, production of p24 after LPS stimulation was identical in the two groups of mice (data not shown). Furthermore, CD40 expression was up-regulated on DC and M{phi} from P. chabaudi-infected animals (Fig. 5Go).



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  		FIGURE 4. HIV p24 production by isolated splenic APC populations during acute malaria. Mean p24 ± SD in supernatants of 48-h cultures of unseparated splenocytes, DC, or M{phi} populations isolated from spleens from HIV-Tg animals (days 8–10 postinfection) or from uninfected littermates (pool of two to four animals). Cells were either unstimulated (medium) or stimulated with rCD154 (5 µg/ml).

 


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  		FIGURE 5. Increased expression of CD40 on splenic APC during the acute phase of P. chabaudi infection. DC and M{phi} populations were isolated from spleens from HIV-Tg animals on days 8–10 post-P. chabaudi infection or from uninfected littermates (pool of two to four animals). Cells were stained for cell type-specific surface markers in conjunction with anti-CD40 Ab or isotype control. CD40 expression was analyzed on cells gated on the expression of CD11c for DC and CD11b for M{phi}. A minimum of 5000 events were analyzed.

 
In vivo blockade of CD40-CD154 interaction markedly inhibits viral activation

To further establish the involvement of CD40-CD154 interaction in the activation of integrated HIV, we treated HIV-Tg mice with a blocking anti-CD154 mAb during the acute phase of malaria (days 0–9). Anti-CD154-treated mice as well as control Ab-treated mice developed parasitemia that peaked on day 8. The two groups of mice did not significantly differ in terms of either the kinetics of the infection or the peak parasitemia (Fig. 6GoA). Importantly, while the P. chabaudi-infected animals treated with the control Ab presented with an ~2.5-fold increase in plasma p24 antigenemia, in vivo treatment with the anti-CD154 Ab completely prevented this increase (Fig. 6GoB; p < 0.05, by unpaired t test). In addition, production of p24 by splenocytes obtained from infected anti-CD154-treated mice was reduced by about 60% compared with that by splenocytes from infected control Ab-treated mice (p < 0.001; Fig. 6GoC). In contrast, in the absence of parasitic infection, treatment with the anti-CD154 Ab did not affect the basal levels of p24 in plasma or after splenocyte culture (both p > 0.15; Fig. 6Go, B and C).



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  		FIGURE 6. Effect of anti-CD154 Ab treatment on malaria-induced virus expression in vivo. HIV-Tg mice (six animals per group) were treated with anti-CD154 Ab or control Ab (200 µg/mouse i.p., starting on day 0 and every 2 days thereafter), and were infected i.p. with P. chabaudi parasites on day 0. Control HIV-Tg mice (three or four animals per group) were treated with anti-CD154 Ab or control Ab using the regimen described above, but did not receive parasites. A, Mean parasitemia (±SD) of anti-CD154 or control Ab-treated mice. B, Plasma p24 levels, calculated as the fold increase in the p24 postinfection (day 9) vs the preinfection level for each individual mouse. C, Mean spontaneous production of p24 (±SD) in supernatants of 48-h splenocyte cultures. Each bar represents the mean (±SD) p24 production of three to six mice assayed individually. The p values represent a significant difference between anti-CD154 or control Ab-treated mice (p < 0.05, by unpaired t test).

 
Viral expression in tissues was also assessed by in situ hybridization with a probe derived from the HIV genome, as represented on Fig. 7Go. The level of hybridization clearly increased in spleens of P. chabaudi-infected mice treated with the control Ab (day 9 postinfection), compared with that in uninfected HIV-Tg animals, either with or without anti-CD154 Ab treatment. Increased HIV-1 RNA expression in P. chabaudi-infected animals was concentrated in the marginal zone of the spleen rather than in the T cell areas, suggesting that the cells expressing the viral mRNA are likely to be DC and/or M{phi}. Importantly, the number as well as the intensity of hybridization signals were decreased in the anti-CD154 Ab-treated P. chabaudi-infected animals, in parallel with the in vitro p24 production levels. Interestingly, in spleen sections from these animals much of the residual hybridization appeared to be localized within the periarterial lymphatic sheath as opposed to the marginal zone, suggesting a possible switch in expression to a different cell type as a consequence of anti-CD154 treatment. Hybridization with sense control riboprobe was performed in the same tissues and was uniformly negative (data not shown).



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  		FIGURE 7. Localization of HIV-1 mRNA expression by in situ hybridization in spleens of P. chabaudi-infected vs uninfected mice after in vivo treatment with anti-CD154 or control Ab. All sections were hybridized with HIV antisense riboprobe. Results are representative fields observed in sections from spleens of two mice per group (day 9 postinfection). The different magnifications used (x50, x100, or x400) are indicated. Images obtained with two different magnifications are represented for P. chabaudi-infected mice. They were digitally captured using a SPOT camera (Diagnostic Instruments, Sterling Heights, MI).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study used HIV-Tg mice as a model of integrated HIV to demonstrate that interaction between APC and activated T cells constitutes a major mechanism in the activation of latent HIV present in the APC. In particular, the CD40-CD154 pathway is clearly involved, because the in vitro and in vivo blocking of these interactions significantly reduced the expression of the integrated virus following activation. Many microbes and microbial products have been described to enhance HIV-1 replication in acutely, chronically, and latently infected cells through the induction of proinflammatory cytokines (24, 25, 26). In this study we describe another pathway by which microbes could induce the expression of a latent integrated virus via T cell activation and signals subsequently given to APC.

Using an in vitro coculture model, we demonstrated that activated T cells up-regulate p24 production from DC in a contact-dependent manner. A major T cell-dependent signaling mechanism for APC is CD40-CD154 ligation (16, 17). By means of several different approaches, i.e., blocking interaction with an anti-CD154 mAb, comparing the effects of T cells from CD154-/- vs wt mice, or stimulating DC with rCD154, we demonstrated that CD40-CD154 interaction plays a major role in HIV-Tg activation. Consistent with the involvement of the CD40-CD154 system in the induction of IL-12 production (27, 28), IL-12 p40 was coinduced with HIV-Tg in most of our experiments (Figs. 1Go and 2Go and Table IGo). Nevertheless, because neutralization of IL-12 (as well as IFN-{gamma}) failed to alter p24 production (Table IIGo), it is clear that the T cell-dependent induction of HIV-Tg is not an indirect consequence of IL-12 stimulation.

As shown previously by us and other investigators (29, 30, 31, 32, 33), the induction of HIV from human APC through CD40 is a complex response that depends on the type and maturity of the cells as well as the viral strain used. CD40-stimulated immature DC and M{phi} decreased the replication of CCR5-using HIV (29, 30, 31), whereas the opposite was observed in the case of CXC chemokine receptor 4 coreceptor-using viruses (29, 30). In addition, CD40-mediated stimulation of human B cells induced CXC chemokine receptor 4 coreceptor and CD4 surface expression, priming them for HIV infection and allowing them to serve as a potential viral reservoir (32). Mature DC were shown to provide a drug-resistant reservoir for integrated HIV-1 and interaction with activated T cells or soluble rCD154 stimulated viral replication in these cells (33). Our in vitro data investigating HIV expression by enriched DC from Tg mice strongly support the conclusions of the latter study.

Although the above in vitro evidence argues for a role for T cell-APC interaction in induction of latent virus, it has previously been difficult to confirm the importance of this pathway in vivo in HIV-infected humans due to the paucity of cells with integrated virus and their likely sequestration in secondary lymphoid organs. The line 166 HIV-Tg mice used by us offer a unique tool for testing the relevance of this pathway in an in vivo model. The experimental system we used in this study involves infection of HIV-Tg mice with the malaria parasite P. chabaudi. Previous experiments demonstrated that this protozoan rapidly induces increased expression of HIV proteins and mRNA in spleen and increased p24 antigenemia in parallel with the acute phase of parasitemia (34). In our model, production of HIV p24 was found to be dramatically elevated in DC and M{phi} isolated from P. chabaudi-infected animals compared with that in uninfected mice and, importantly, was shown in this study to be associated with up-regulated CD40 expression on the same cells. Proinflammatory cytokines are induced during early P. chabaudi infection, with plasma TNF-{alpha} and IFN-{gamma} levels peaking just prior to the peak parasitemia (35). This increased IFN-{gamma} production could be the driving factor in the up-regulated expression of CD40 (36). Alternatively and nonexclusively, some microbial products, such as Toxoplasma gondii soluble tachyzoite Ag, directly increase CD40 expression independently of IFN-{gamma} (37). Moreover, these APC populations purified from infected mice were more susceptible to stimulation with rCD154 ex vivo. This increase in CD40 expression is probably a critical step in viral induction and probably explains why T cell activation in itself is an insufficient stimulus for p24 production in vitro, as demonstrated by our previous results with anti-CD3-stimulated splenocytes from naive animals (14).

Although proinflammatory cytokines are probably implicated in the up-regulated virus expression following P. chabaudi infection through the induction of CD40 on APC, their neutralization did not decrease microbial-induced viral expression (34), ruling out a direct role in the activation of APC. Interestingly, in the case of hepatitis B virus-Tg mice, malaria-induced proinflammatory cytokines have the opposite role, causing the suppression of hepatitis B virus replication and gene expression, emphasizing the distinct mechanisms regulating transgene expression in these two viral models (38).

The key evidence supporting a role for CD40-CD154 interaction in viral induction in vivo was the markedly reduced up-regulation of HIV p24 and mRNA expression following treatment of P. chabaudi-infected HIV-Tg mice with a blocking anti-CD154 mAb. Nevertheless, production of p24 by splenocytes from anti-CD154-treated infected animals and hybridization levels in their spleen remained higher than those in uninfected animals. Similarly, in our in vitro experiments, p24 production was only partially inhibited by anti-CD154 mAb treatment. One explanation of these findings is that the anti-CD154 mAb failed to completely block CD40-CD154 interaction. However, the same Ab induced a more pronounced inhibition of IL-12 p40 production in our in vitro culture system. These results suggest that additional mechanisms may participate in the up-regulation of virus expression. For example, T cell costimulation of APC function can also be mediated by other members of the TNF/TNF receptor superfamily, such as TRANCE/TRANCE receptor or OX40 ligand/OX40 (39, 40). Moreover, T lymphocyte engagement of surface adhesion molecules has been reported to enhance HIV replication in chronically infected monocytic cell lines (41). Alternatively, the activation pathways of the HIV and IL-12 p40 genes require different thresholds of activation and are, therefore, not equally sensitive to the same levels of inhibition.

Taken together, our findings in the HIV-Tg model support the concept that APC provide a significant reservoir for viral activation by coinfecting microbial agents. We have now demonstrated that such microbial activation can be triggered through distinct pathways. Thus, in the case of two pathogens, Toxoplasma gondii and Mycobacterium avium, that invade APC, activation of the pro-virus in these cells can occur independently of T lymphocytes (13, 14). In contrast, as revealed in this study, blood stage malaria parasites that do not infect APC require T cells and CD40-CD154 interaction for viral induction. Although involving distinct extracellular triggers, these two mechanisms may share the same downstream signaling pathway. Thus, we have recently shown that IL-1 and microbial products that signal through Toll-like receptors are the most potent activators of the HIV-Tg in resting APC populations (M. Schito, manuscript in preparation). Toll-like receptors and CD40 are thought to trigger NF-{kappa}B through a common TNF receptor-associated factor-6-dependent mechanism (17, 42) and, therefore, may induce HIV-1 through a related signaling cascade.

Because chronic T cell activation accompanies many infections, including HIV infection itself (18, 19, 20), CD40-CD154 interaction should play a prominent role in many situations of immune activation that HIV-infected individuals confront. A further implication is that strategies designed to flush out resting T cell reservoirs through activation may indirectly trigger latent virus from APC. If so, it will be important to determine to what extent the latter cells are susceptible to cytopathic and/or antiviral drug effects and thus can be eliminated without viral spread. Although some aspects of this model restrict its relevance to human HIV disease, the line 166 HIV-Tg mice used in our studies may nevertheless offer a unique tool not only for identifying in vivo mechanisms of immune activation, but also for testing pharmacological and immunological interventions that target viral reservoirs at the APC level.


   Acknowledgments
 
We thank Drs. Brian Kelsall, Genevieve Milon, and Dinah Singer for helpful discussion and review of the manuscript. We also thank Dr. Cecil Fox for performing the in situ hybridizations, and Dr. Malcolm Martin for his help in providing and maintaining the HIV-Tg mouse colony.


   Footnotes
 
1 This work was supported in part by funding from the National Institutes of Health Intramural AIDS Targeted Anti-Viral Program. Back

2 A.S. and G.M.S. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Gene M. Shearer, Experimental Immunology Branch, National Cancer Institute, Building 10, Room 4B36, National Institutes of Health, 4 Center Drive, MSC 0425, Bethesda, MD 20892-0425. Back

4 Abbreviations used in this paper: Tg, transgenic; DC, dendritic cells; cRBC, control RBC; M{phi}, macrophages; pRBC, parasitized RBC; wt, wild type; CM, complete medium; rCD154, recombinant murine CD154 trimer. Back

Received for publication November 13, 2000. Accepted for publication December 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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