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The Journal of Immunology, 2004, 172: 7832-7840.
Copyright © 2004 by The American Association of Immunologists

Defective Dendritic Cell Function in HIV-Infected Patients Receiving Effective Highly Active Antiretroviral Therapy: Neutralization of IL-10 Production and Depletion of CD4+CD25+ T Cells Restore High Levels of HIV-Specific CD4+ T Cell Responses Induced by Dendritic Cells Generated in the Presence of IFN-{alpha}1

Cédric Carbonneil*, Vladimira Donkova-Petrini*, Albertine Aouba{ddagger} and Laurence Weiss2,*,{dagger},{ddagger}

* Institut National de la Santé et de la Recherche Médicale Unité 430, Institut des Cordeliers, {dagger} Université René Descartes, and {ddagger} Hôpital Européen Georges Pompidou, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated that GM-CSF/IFN-{alpha} combination allowed the differentiation of monocytes from HIV-infected patients into dendritic cells (DCs) exhibiting high CD8+ T cell stimulating abilities. The present study was aimed at characterizing the ability of DCs generated in the presence of GM-CSF and IFN-{alpha} to induce CD4 T cell responses. DCs were generated from monocytes of HIV-infected patients in the presence of GM-CSF with either IFN-{alpha} (IFN-DCs) or IL-4 (IL-4-DCs) for 7 days. Eleven patients receiving highly active antiretroviral therapy and exhibiting CD4 cell counts above 400/mm3 and plasma HIV-RNA <50 copies/ml for at least 1 year were included in the study. Both DC populations were found to be defective in inducing autologous (in response to tuberculin or HIV-p24) or allogeneic CD4 T cell proliferation. Neutralization of IL-10 during the differentiation of IFN-DCs, but not during the DC-T cell coculture, significantly increased their ability to stimulate autologous CD4 T cell proliferation in response to tuberculin and allogeneic CD4 T cell proliferation (4.1-fold and 3.0-fold increases, respectively, at the DC to T cell ratio of 1:10). Moreover, IL-10 neutralization and CD4+CD25+ T cell depletion synergistically act to dramatically increase HIV-p24-specific CD4 T cell responses induced by IFN-DCs (31.7-fold increase) but not responses induced by IL-4-DCs. Taken together, our results indicate that IFN-DCs are more efficient than IL-4-DCs to stimulate CD4+ T cell proliferation, further supporting their use for immune-based therapy in HIV infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs),3 the most potent APCs of the immune system are involved in the induction of primary immune responses, generation of T cell-dependent cellular and humoral immunity, and induction of tolerance. DCs physiologically reside within peripheral tissues, where they act as immune sentinels (1). After contact with danger signals, DCs trigger their maturation and migrate toward secondary lymphoid organs, where they sequentially stimulate CD4+ and CD8+ T lymphocytes (1, 2, 3, 4, 5). It was also reported that DCs, infected by some viruses, might directly stimulate CTLs without CD4+ T cell help (3). Therefore, an impairment of DC functions may induce critical consequences for antiviral cellular immunity.

HIV infection is mainly characterized by a progressive decrease in the number of CD4+ T lymphocytes and a loss of CD4+ and CD8+ T cell responses toward HIV or other pathogens (6, 7). Highly active antiretroviral therapy (HAART) results in reduced HIV-1 replication, increased CD4 cell counts in most treated patients, and progressive but incomplete recovery of CD4+ T cell functions. Thus, treatment of chronic HIV infection does not result in most cases in a full recovery of HIV-specific helper cells and CTL responses leading to clinical trials of HIV-specific immunization in HAART-treated patients (8, 9). In addition, HIV infection has been associated with a depletion of blood and splenic DCs (10). DCs were demonstrated to be trapped in lymph nodes and most DCs were found to exhibit an immature phenotype with low expression of CD80 and CD83 in vivo (11, 12). Although Langerhans cells from HIV-infected patients were reported to be functional (13, 14), whether peripheral DCs exhibit a functional impairment is still debated. Some authors reported on normal functions of blood circulating DCs or monocyte-derived DCs (15, 16). Conversely, Macatonia et al. (17) found that blood DCs isolated from HIV-infected patients did not induce the proliferation of autologous or allogeneic CD4+ T lymphocytes, in contrast with monocyte/macrophages derived from the same patients. Donaghy et al. (18) have further demonstrated that blood DCs were unable to induce T cell proliferation.

Our previous study has demonstrated that DCs, generated from peripheral blood monocytes of HIV-infected patients, in the presence of GM-CSF and IFN-{alpha} (IFN-DCs) in vitro, expressed high levels of MHC and costimulatory molecules and were able to induce IFN-{gamma} production by HIV-specific CD8+ T cells (19). Furthermore, IFN-DCs derived from healthy donors were demonstrated to induce a weak proliferation of purified CD4+ T cells and a strong proliferation of purified CD8+ T cells (55), suggesting that IFN-DCs are able to induce CD8+ T cell-mediated immune responses without CD4+ T cell help. As we believe that in vivo generation of DCs by administration of GM-CSF and IFN-{alpha} before immunization with HIV peptides in HAART-treated patients should represent a promising approach of therapeutic vaccination, it appears critical to investigate whether IFN-DCs derived from HIV-infected patients retain the ability to activate CD4+ T cells.

Thus, in the present study, we characterize IFN-DCs derived from HIV-infected individuals in terms of induction of CD4+ T cell proliferation and IL-10 production. We demonstrate in this study that DCs fail to induce allogeneic, tuberculin purified protein derivate (PPD) and HIV-p24-specific CD4+ T cell proliferation. However, CD4+ T cell proliferation was found to be restored when IFN-DCs were differentiated in the presence of neutralizing anti-IL-10 mAbs. In addition, HIV-p24-specific CD4+ T cell proliferation dramatically increased following depletion of CD4+CD25+ T cells. Thus, IFN-DCs were found to be defective in inducing CD4+ T cell proliferation; however, neutralization of IL-10 during their differentiation and depletion of CD4+CD25+ T cells from responding CD4+ T lymphocytes synergistically act to induce high levels of HIV-p24-specific CD4+ T cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and blood samples

Buffy coats of healthy donors were obtained from the Etablissement Français du Sang (Rungis, France), according to ethical guidelines. Patients included in the study underwent HAART for at least 1 year and exhibited at the time of the study CD4 cell counts above 400 cells/mm3 and a plasma HIV viral load below 50 copies/ml. Heparin-anticoagulated blood samples were collected from 11 HIV-infected patients. Clinical characteristics of the patients are depicted in Table I. Written informed consent was obtained from all the patients, according to human experimentation guidelines from national ethical committees.


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  		Table I. Clinical characteristics of HIV-infected individuals studied

 
In vitro differentiation of DCs

PBMC were isolated from buffy coats or heparin anticoagulated blood samples by Ficoll density gradient centrifugation on Medium for Separation of Lymphocytes (Eurobio, Les Ulis, France). The percentage of monocytes among PBMC was determined by flow cytometry using forward scatter and side scatter properties. Mononuclear cells were resuspended in RPMI 1640 with ultra-glutamine (BioWhittaker, Verviers, France), penicillin-streptomycin (100 IU/ml and 100 µg/ml, respectively; Life Technologies, Paisley, Scotland), and 10% AB serum. Cells were then seeded into 12-well plates (Costar, Cambridge, MA) at the concentration 1 x 106 monocytes/ml. Plates were incubated at 37°C for 1 h. Nonadherent cells were kept for subsequent T lymphocyte isolation. The adherent cell population was rinsed gently and resuspended in RPMI 1640 supplemented with 10% FCS (Dutscher, Brumath, France) and penicillin-streptomycin for 7 days containing GM-CSF (1000 IU/ml) in combination with IFN-{alpha} (500 IU/ml) or IL-4 (500 IU/ml). As previously reported, the recovered cells were not adherent and exhibited a DC morphology (19). The purity of DCs at day 7 of culture was generally 90% with <10% of contaminating T lymphocytes, as assessed by staining with anti-CD3 mAb (BD Biosciences, Le Pont de Claix, France) and flow cytometry. Recombinant human GM-CSF (Leucomax) and recombinant human IFN-{alpha}2b (Introna) were provided by Schering-Plough (Levallois-Perret, France); recombinant human IL-4 was from R&D Systems (Oxon, U.K.). In another set of experiments, control IgG or neutralizing anti-IL-10 mAb (5 µg/ml, clone no. 20116.11 and no. 23738.111, respectively; R&D Systems) were added to the cytokine combinations previously described. Every 2 days, 400 µl of medium were gently removed from each well and replaced by 500 µl of fresh medium containing the appropriate cytokines. Phenotypic and functional characterizations were assessed between days 7 and 9.

Isolation of CD3+, CD4+, and CD4+CD25– T lymphocytes

Nonadherent PBMC were incubated in medium overnight at 37°C. The cellular fraction recovered, referred to as "bulk T cells," contained >85% CD3+ T lymphocytes and <1% monocytes (as assessed by flow cytometry). T lymphocytes were frozen until use. CD4+ T lymphocytes were further purified from bulk T cells using CD4+ isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). This process results in obtaining >90% purity of CD4+ T cells (data not shown). The CD4+CD25+ cells were subsequently removed using magnetic columns (Miltenyi Biotec) according to company guidelines (CD25+ and CD25– fractions were collected separately). This generally resulted in obtaining >90% purity of CD4+CD25– cell population, as assessed by flow cytometry (data not shown).

Analysis of DC phenotype

Surface Ags were analyzed at day 7 of culture by flow cytometry using two- or three-color direct immunofluorescence. The following murine anti-human mAbs conjugated to FITC, PE, or PE-cyanin-5 were used: anti-CD14 FITC, anti-CD16 PE-cyanin-5, anti-DC lysosome-associated membrane protein (LAMP) PE, and anti-CD83 PE-cyanin-5 mAbs, provided by Coulter Immunotech (Marseille, France); anti-HLA-DR FITC mAb (BD Biosciences); anti-CCR5 PE mAb (BD PharMingen, San Diego, CA); anti-CD1a FITC mAb provided by Tébu (Le Perray en Yvelines, France). After saturation with PBS (BioMérieux, Marcy l’étoile, France) containing 10% FCS for 20 min at 4°C, cells were incubated with the appropriate mAbs for 30 min at 4°C, washed twice with PBS containing sodium azide (0.01%) and BSA (0.2%), and then fixed using PBS formaldehyde (1%) (Sigma-Aldrich, St. Louis, MO). Analyses were performed using FACSCalibur and CellQuest software (BD Biosciences) on at least 1000 events. Gating was performed according to light scattering properties.

Induction of cytokine production

At day 7, cells (5 x 104/106 DCs) were stimulated by either L cells transfected with human CD40 ligand (CD40L), L40L, kindly provided by Dr. J. Banchereau (Baylor Institute for Immmunology Research, Dallas, TX), in the presence of recombinant human IFN-{gamma} (1000 IU/ml/106 cells; Roussel-Uclaf, Romainville, France) for 48 h, or by Staphylococcus aureus Cowan (SAC) (100 µg/ml; Calbiochem Novabiochem, San Diego, CA) for 24 h. Cytokine production was assessed at single cell level by flow cytometry after 24 or 48 h of stimulation according to optimal conditions previously defined for the detection of each cytokine.

Intracellular detection of cytokine production by FACS at single cell level

To prevent cytokine secretion, brefeldin A (10 µg/ml/106 cells; Sigma-Aldrich) was added to cell cultures during the last 24 h of stimulation. After stimulation, cells were saturated with RPMI 1640, 10% FCS, incubated with anti-CD83 PE-cyanin-5 mAb (5 µl/106 cells) for 30 min at 4°C and fixed with PBS formaldehyde (4%) (Sigma-Aldrich). Cells were subsequently incubated with anti-IL-10 PE mAb (Diaclone, Besançon, France) or anti-IL-12p40 FITC mAb (10 µl/106 cells; BD PharMingen) in PBS-azide (0.01%), BSA (0.2%), saponin (0.5%) (Sigma-Aldrich) for 25 min at 4°C. Cells were washed twice before flow cytometric analysis.

CD4+ T lymphocyte proliferation assay

This assay was performed as previously described with some modifications (20). Briefly, DCs were pulsed with 5 µg/ml PPD (Statens Seruminstitute, Copenhagen, Denmark) or 2 µg/ml HIV-p24 protein (Protein Science, Meriden, CT) overnight. After extensive washes, DCs were cocultured for 6 days with either 105 CD4+ or CD4+CD25– T lymphocytes at the following DC to T cell ratios, 1:10, 1:20, 1:40, and 1:80. In another set of experiments, control IgG or neutralizing anti-IL-10 mAb (10 µg/ml) were added to the cocultures. At day 5, 1 µCi of [3H]thymidine per well was added during the last 18 h. Cells were then harvested by means of a Mach II 96 cell harvester (Tomtec, Hyamden, CT) and levels of [3H]thymidine incorporation was measured (at day 6) in a Microbeta liquid scintillation counter (Wallac, Turku, Finland). Results were expressed as the mean of stimulation index in cpm = [(cocultures of pulsed-DCs and CD4+ T cells)/(cocultures of unloaded DCs and CD4+ T cells)], obtained in different experiments.

Allogeneic MLR

Purified CD4+ T lymphocytes from either allogeneic HIV-infected or uninfected individuals were cultured at 105 cells per well in 96-well plates in RPMI 1640, 10% FCS. Increasing numbers of DCs, derived from either HIV-infected or uninfected individuals, were added to the culture for 4 days. Cultures were performed in triplicate. [3H]Thymidine (1 µCi per well) was added during the last 18 h. Cells were then harvested by means of a Mach II 96 cell harvester (Tomtec) and levels of [3H]thymidine incorporation were measured in a Microbeta liquid scintillation counter (Wallac). Results were expressed as mean of stimulation index in cpm = [(cocultures of DCs and CD4+ T cells)/(cultures of CD4+ T cells alone)], obtained in different experiments.

Statistical analysis

Data are expressed as mean ± SD. Statistical comparisons were performed using Student’s t test. Significance was considered for p ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Decreased stimulation of allogeneic and autologous CD4+ T lymphocyte proliferation by DCs derived from HIV-infected individuals

We first assessed the ability of DCs, derived from HIV-infected individuals, to induce allogeneic and Ag-specific CD4+ T lymphocyte proliferation. IFN-DCs and DCs generated in the presence of GM-CSF and IL-4 (IL-4-DCs) were cocultured with purified allogeneic CD4+ T cells at different DC: to T cell ratios for 4 days before assessing proliferation. As depicted in Fig. 1A, low proliferation of allogeneic CD4+ T cells from HIV-infected patients was observed when stimulated with DCs whether differentiated in the presence of GM-CSF and IFN-{alpha} or IL-4. In contrast, DCs from HIV-seronegative controls, generated in the same experimental conditions, were able to induce proliferation of allogeneic CD4+ T cells from both healthy and HIV-infected individuals although the proliferation of CD4+ T cells from HIV-infected patients was significantly lower than that of CD4+ T cells from healthy donors (Fig. 1A). Moreover, DCs from HIV-infected individuals were unable to efficiently stimulate the proliferation of allogeneic CD4+ T cells from healthy donors (Fig. 1A). Autologous CD4+ T lymphocytes from HIV-infected patients cocultured at different APC to T cell ratios with DCs pulsed with either PPD or HIV-p24 protein also failed to proliferate (Fig. 1, B and C).



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  		FIGURE 1. IFN-DCs and IL4-DCs only induce weak allogeneic and autologous CD4+ T cell proliferation. DCs were generated from monocytes, as described in Materials and Methods. A, IFN-DCs (filled symbols) and IL4-DCs (open symbols) derived from either HIV-infected patients ({diamond}, {diamondsuit}, {square}, and {blacksquare}) or uninfected healthy donors ({circ}, •, {triangleup}, and {blacktriangleup}) were cocultured at different DC to T cell ratios (1:80 to 1:10) with allogeneic purified CD4+ T lymphocytes, isolated from either HIV-infected or uninfected healthy individuals, for 4 days. Thymidine incorporation was measured after an 18 h pulse with 1 µCi of [3H]thymidine. Results are shown as mean ± SD of stimulation index in cpm = [(cocultures of DCs and CD4+ T cells)/(cultures of CD4+ T cells alone)], obtained in three different experiments. B and C, IFN-DCs (filled symbols) and IL4-DCs (open symbols) derived from either HIV-infected patients ({square} or {blacksquare}) or uninfected healthy donors ({circ} or •) were pulsed with either 5 µg/ml PPD (B) or 2 µg/ml HIV-p24 protein (C). DCs were then cocultured with autologous purified CD4+ T lymphocytes, at different DC/T ratios (1:80 to 1:10), for 6 days. Thymidine incorporation was measured after a 18 h pulse with 1 µCi of [3H]thymidine. Results are shown as mean ± SD of stimulation index in cpm = [(cocultures of pulsed-DCs and CD4+ T cells)/(cocultures of unloaded DCs and CD4+ T cells)], obtained in three different experiments.

 
These results clearly indicate that, in addition to CD4 T cell abnormalities, HIV infection is associated with an intrinsic functional defect of DCs derived from circulating monocytes of patients whether the DCs were differentiated in the presence of IFN-{alpha} or IL-4 in addition to GM-CSF.

IL-10 production by DCs from HIV-infected patients

The functional abilities of DCs derived from HIV-infected patients were further assessed by quantifying IL-10-producing DCs following maturation. Thus, IFN-DCs and IL-4-DCs were stimulated with either SAC or L40L cells plus IFN-{gamma} during 24 or 48 h, respectively. IL-10-producing DCs were then quantified by flow cytometry at the single cell level. Following stimulation with L40L cells plus IFN-{gamma} or SAC, the proportion of IL-10-producing cells did not differ between IFN-DCs and IL-4-DCs (Fig. 2 and Table II). The proportion of IL-10-producing cells following CD40L ligation and IFN-{gamma} treatment of DC from HIV-infected patients was significantly increased compared with the proportion of IL-10-producing DCs derived from healthy donors for both IFN-DCs and IL-4-DCs (p = 0.015 and p = 0.050, respectively) (Table II).4



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  		FIGURE 2. Cytokine production by IFN-DCs and IL-4-DCs from HIV-infected patients following stimulation by SAC or CD40L+IFN-{gamma}. DCs generated from monocytes of HIV-infected individuals were analyzed by flow cytometry for CD83 membrane expression and intracellular production of IL-10 (A and B) and IL-12p40 (C and D). Cells stimulated by either L40L cells in the presence of recombinant human IFN-{gamma} for 48 h (A and C), or by SAC for 24 h (B and D) were stained with the relevant mAb or isotype control. The numbers indicate the percentage of positive cells in each quadrant defined according to the isotypic control. Results are representative of four different patients.

 

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  		Table II. Proportion of IL-10-producing cells following stimulation with SAC or CD40L plus IFN-{gamma}a

 
We have previously reported that the proportion of DCs, from HIV-infected patients, whether differentiated in the presence of IL-4 or IFN-{alpha}, producing IL-12 following stimulation with L40L cells and IFN-{gamma}, was weaker than their counterparts derived from healthy donors (19) and that the proportion of IFN-DCs and IL-4-DCs producing IL-12 was similar following CD40 ligation as illustrated in this study for a representative patient (Fig. 2). Taken together, these results suggest that the IL-12/IL-10 balance is impaired toward an over-production of IL-10 by both DC populations.

Neutralization of IL-10 induced changes in the phenotype of both IFN-DCs and IL-4-DCs

Monocytes from HIV-infected patients were reported to constitutively secrete IL-10 and monocytes from healthy donors were demonstrated to produce high amounts of IL-10 in response to several soluble HIV-derived proteins (21, 22, 23). Previous studies established that autocrine production of IL-10 by DCs exerts modulatory effects on their phenotype (20, 24), on their ability to stimulate CD4+ T cell proliferation (25) (26), and to produce IL-10 following maturation (27).

To gain an insight into the role of IL-10 in the modulation of the differentiation of monocytes toward DCs in HIV-infected patients, the secreted IL-10 was neutralized during the entire differentiation of DCs using anti-IL-10 mAbs.

In the absence of IL-10 neutralization, in contrast to DCs derived from healthy controls, IFN-DCs from HIV-infected individuals retained the expression of CD14 and CD16 molecules, as IL-4-DCs which, however, exhibited a lower expression of these markers. IFN-DCs exhibited a lower expression of CD1a compared with IL-4-DCs. In addition, we observed a weak intracellular expression of DC-LAMP in both IFN-DCs and IL-4-DCs. As previously reported, IFN-DCs expressed higher levels of CCR5, the major HIV coreceptor, as compared with IL-4-DCs, and nearly 100% of cells from both DC populations expressed HLA-DR (19). These phenotypic characteristics were observed in the presence or absence of control IgG (data not shown and Fig. 3). In the presence of neutralizing anti-IL-10 mAbs throughout the differentiation, the expression of CD14, CD16, and CCR5 was significantly decreased on both IL-4-DCs and IFN-DCs. The addition of anti-IL-10 mAbs did not affect the expression of CD1a and CD83 on IL-4-DCs, at the opposite of IFN-DCs, which exhibited a significantly higher expression of both DC markers. Furthermore, the expression of DC-LAMP dramatically increased in IFN-DCs differentiated in the presence of neutralizing anti-IL-10 mAbs whereas DC-LAMP expression only slightly increased in IL-4-DCs (Fig. 3). Finally, expression of HLA-DR was significantly increased on both DC subsets (increase of 1.7-fold (p = 0.050) and 2.1-fold (p = 0.030) of mean fluorescence intensity for IFN-DCs and IL-4-DCs, respectively).



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  		FIGURE 3. Neutralization of IL-10 production influenced the phenotypic characteristics of IFN-DCs and IL-4-DCs. DC were generated from monocytes in the presence of recombinant human GM-CSF and either recombinant human IFN-{alpha} or recombinant human IL-4 and either 5 µg/ml neutralizing anti-IL-10 mAbs or control IgG, for 7 days. Expression of CD16, CD14, CCR5, CD83, CD1a, DC-LAMP, and HLA-DR was analyzed following staining with the relevant mAb or isotype control. Histograms represent the mean ± SD (n = 2) of the percentage of positive cells. *, p ≤ 0.05 between DCs generated in the presence of anti-IL-10 mAb and control IgG.

 
Altogether, these results suggest that the neutralization of autocrine IL-10 induces changes in the phenotype of both DC subsets, more strikingly on IFN-DCs. Phenotypic changes are consistent with a more advanced differentiation stage of anti-IL-10 mAbs treated DCs.

Neutralization of IL-10 during the differentiation increased the ability of IFN-DCs to stimulate allogeneic and PPD-induced proliferation of CD4+ T lymphocytes

To assess the impact of IL-10 on the ability of DCs to stimulate CD4+ T cells, DCs generated in the presence of neutralizing anti-IL-10 mAbs were cocultured with allogeneic or autologous CD4+ T cells. The proliferation of allogeneic CD4+ T cells isolated from HIV-infected individuals, was significantly increased when both IL-4 DCs and IFN-DCs were differentiated in the presence of anti-IL-10 mAbs. However, stimulation indices of allogeneic CD4+ T cell proliferation were higher when IFN-DC were used as APCs, compared with IL-4-DCs (Fig. 4A). The addition of anti-IL-10 mAbs during the differentiation also restored the ability of IFN-DCs to stimulate the proliferation of autologous CD4+ T cells in response to recall Ags such as PPD (Fig. 4B). Nevertheless, the addition of anti-IL-10 mAbs slightly increased the proliferation induced by PPD-loaded IL-4-DCs, the difference being significant only at the DC to T cell ratio of 1:10.



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  		FIGURE 4. Neutralization of IL-10 production increased the ability of IFN-DCs to stimulate CD4+ T lymphocytes. IFN-DCs (closed symbols) or IL4-DCs (open symbols) from HIV-infected patients were differentiated in the presence of either neutralizing anti-IL-10 mAb (5 µg/ml) ({circ} and •) or control IgG mAb (5 µg/ml) ({diamond} and {diamondsuit}) (A), ({square} and {blacksquare}) (B). A, DCs were cocultured with allogeneic purified CD4+ T cells, isolated from HIV-infected individuals at different DC to T cell ratios (1:80 to 1:10), for 4 days. Results are shown as mean ± SD of stimulation index in cpm = [(cocultures of DCs and CD4+ T cells)/(cultures of CD4+ T cells alone)], obtained in two different experiments. B, DCs were pulsed with 5 µg/ml PPD and were further cocultured with autologous purified CD4+ T lymphocytes at different DC to T cell ratios (1:80 to 1:10), for 6 days. Results are shown as mean ± SD of stimulation index in cpm = [(cocultures of pulsed-DCs and CD4+ T cells)/(cocultures of unloaded DCs and CD4+ T cells)], obtained in two different experiments. *, p ≤ 0.05 and **, p ≤ 0.01 between DCs generated in the presence of anti-IL-10 mAb and control IgG.

 
Neutralization of IL-10 and depletion of CD4+CD25+ T lymphocytes dramatically increased HIV-p24-specific T cell proliferation induced by IFN-DCs

We further aimed at characterizing the mechanisms affecting the proliferation of HIV-p24 specific CD4+ T cells induced by DCs. As previously described in Fig. 1C and illustrated in Fig. 5, both IL-4-DCs and IFN-DCs pulsed with HIV-p24 did not induce any proliferation of autologous CD4+ T cells from HIV-infected individuals. Interestingly, as observed for allogeneic and PPD-specific CD4+ T cell proliferation, IFN-DCs differentiated in the presence of anti-IL-10 mAbs recovered their ability to induce the proliferation of autologous HIV-p24-specific CD4+ T cells. The levels of proliferation of CD4+ T cells induced by p24-loaded IL-4-DCs were lower compared with those induced by IFN-DCs (Fig. 5).



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  		FIGURE 5. Neutralization of IL-10 and depletion of CD4+CD25+ T lymphocytes dramatically increased HIV-p24-specific T cell proliferation induced by IFN-DCs. IFN-DCs or IL4-DCs, differentiated in the presence of either neutralizing anti-IL-10 mAb or control IgG mAb (5 µg/ml), were pulsed with 2 µg/ml HIV-p24 protein. DCs were then cocultured with either autologous purified CD4+ T lymphocytes or autologous purified CD4+CD25– T cells, at the DC to T cell ratio 1:10, for 6 days. Neutralizing anti-IL-10 mAb or control IgG mAb (10 µg/ml) were further added throughout the DC-T cell coculture. Results are shown as mean ± SD of stimulation index in cpm = [(cocultures of p24 pulsed-DCs and CD4+ T cells)/(cocultures of unloaded DCs and CD4+ T cells)], obtained in two separate experiments. Values shown for p were calculated according to the Student t test.

 
As Ostrowski et al. (28) previously demonstrated that stimulation of CD4+ T lymphocytes in the presence of HIV-gag peptides induced a high proportion of IL-10-producing CD4+ T cells and considering that IL-10 may impair the proliferation of CD4+ T cells, we also assessed, in the same experiment, the effect of adding anti-IL-10 neutralizing mAbs throughout the proliferation assay. As illustrated in Fig. 5, the neutralization of IL-10 during the coculture of HIV-p24-loaded DCs and CD4+ T cells did not result in any detectable effect. Altogether, these data demonstrate that production of IL-10 throughout the differentiation of IFN-DCs rather than during the DC-T cell coculture is involved in the impairment of the proliferation of HIV-p24-specific CD4+ T cells.

Finally, as we demonstrated using monocytes as APCs, that regulatory CD4+CD25+ T lymphocytes suppressed the proliferation of HIV-p24-specific CD4+CD25– T cells,4 we further assessed the effect of depletion of CD4+CD25+ T lymphocytes on p24-specific CD4+ T cell proliferation induced by DCs. Thus, DCs differentiated in the presence or absence of neutralizing anti-IL-10 mAbs, pulsed with HIV-p24 protein, were cocultured with autologous CD4+CD25– T lymphocytes. As observed with CD4+ T cells, p24-loaded IL-4-DCs did not induce any proliferationof CD4+CD25– T cells. Nevertheless, the proliferation of CD4+CD25– T cells was increased (2.1-fold) when IL-4-DCs differentiated in the presence of anti-IL-10 mAbs were used (Fig. 5). Depletion of CD4+CD25+ T lymphocytes significantly increased (5.4-fold) the levels of p24-specific proliferation of CD4+ T cells induced by IFN-DCs. Interestingly, the use of IFN-DCs differentiated in the presence of anti-IL-10 mAbs as APCs dramatically increased the proliferation of CD4+CD25– T cells (31.7-fold) (Fig. 5). Thus, neutralization of IL-10 during the differentiation of IFN-DCs and depletion of CD4+CD25+ T cells from responding CD4+ T lymphocytes synergistically act to dramatically increase HIV-p24-specific CD4+ T cell proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we characterize IFN-DCs derived from HIV-infected individuals in terms of induction of CD4+ T cell proliferation and IL-10 production. IFN-DCs were found to be defective in inducing allogeneic and autologous CD4+ T cell proliferation in response to PPD and HIV-p24. However, neutralization of IL-10 secreted during the differentiation of IFN-DCs and depletion of CD4+CD25+ T lymphocytes from responding CD4+ T cells synergistically act to induce high levels of HIV-p24-specific CD4+ T cell proliferation.

Both IFN-DCs and IL-4-DCs derived from HIV-infected individuals failed to induce potent allogeneic, PPD and HIV-p24-specific CD4+ T cell proliferation. In contrast, CD4+ T cells from HIV-infected patients retained a relative ability to proliferate in response to allogeneic DCs from healthy controls whereas CD4+ T cells from healthy controls failed to proliferate when stimulated by DCs from patients. Altogether, these observations demonstrate that, in addition to the CD4 T cell defect, an intrinsic defect of DCs from HIV-infected patients is responsible for the impaired proliferation of allogeneic and autologous CD4+ T lymphocytes. These data are in agreement with those of Macatonia et al. (17) and Donaghy et al. (18) demonstrating defective functions of blood circulating DCs. In addition, APC dysfunction has also been supported in a study demonstrating that monocyte-derived DCs infected in vitro with HIV impaired normal T cell functions (29). Conversely, in a study suggesting a normal immune function of monocyte-derived DCs from HIV-infected individuals, the authors restricted the functional analysis to a subpopulation of DCs that expressed CD1a and did not express CD14 and CD16 molecules (15). However, our results indicate that DCs generated from monocytes of HIV-infected individuals retain the expression of CD16 and, at a lesser degree, CD14 (Fig. 3).

Most of the studies have demonstrated that monocytes from HIV-infected patients spontaneously produce high amounts of IL-10 (30, 31) and that monocytes from healthy controls secrete IL-10 when stimulated by soluble HIV proteins (21, 23, 32, 33, 34). Plasma levels of IL-10 were found to be increased in HIV-infected patients and to gradually decrease in patients undergoing HAART without, however, normalization (35). Thus, circulating monocytes from patients, even successfully treated by HAART, can be considered as "pretreated" by IL-10 in vivo prior isolation and differentiation toward DCs.

We first assessed the hypothesis that the DC defect, involved in the impaired activation of allogeneic and autologous CD4+ T lymphocytes, could result from an overproduction of IL-10 throughout the differentiation of DCs and/or an IL-12/IL-10 imbalance following CD40 ligation during the DC/T coculture. Previous studies have demonstrated an impaired production of IL-12 in HIV infection (31, 36, 37). We also have previously demonstrated that both IFN-DCs and IL-4-DCs, derived from HIV-infected patients, produced lower IL-12p40 following CD40 ligation, as compared with DCs from healthy donors (19). IL-12 production by DCs may be inhibited by an over-production of IL-10, as previously suggested (31). Accordingly, we found that, following maturation signals, a higher proportion of both IFN-DCs and IL-4-DCs from HIV-infected patients produced IL-10, compared with DCs from healthy controls. Taken together, these data indicate that DCs from HIV-infected individuals exhibit an IL-10/IL-12 imbalance toward IL-10 production. Furthermore, as the maturation of DCs induced by CD40 ligation mimics the maturation induced by CD4+ T cells, we may consider that an over-production of IL-10 by DCs may also occur during the DC-CD4+ T cell coculture. Therefore, the IL-10/IL-12 imbalance could result in impaired CD4+ T cell proliferation in our system. Thus, we have neutralized IL-10 secretion throughout the coculture of CD4+ T cells and DCs. We found that neutralization of IL-10 during the coculture did not exert any detectable effect suggesting that IL-10, produced by DCs and/or CD4+ T lymphocytes throughout the coculture, was not involved in the defective proliferation of CD4+ T lymphocytes.

We and others have previously demonstrated that an autocrine production of IL-10 by DCs throughout their differentiation can modulate their ability to further stimulate CD4+ T cell proliferation (27). We have thus investigated the phenotypic and functional consequences of neutralizing IL-10 secreted throughout the differentiation of DCs. Neutralization of autocrine IL-10 led to phenotypic changes more striking for IFN-DCs than for IL-4-DCs. Whereas IL-10 neutralization induced an increase in the mean membrane expression of class II molecules and no significant effect on CD80/CD86 expression (data not shown) on both DC populations, the dramatic increase in DC-LAMP expression was restricted to IFN-DCs. As DC-LAMP is known to be up-regulated upon differentiation/maturation of DCs (38, 39), this observation suggests that IFN-DCs generated in the presence of neutralizing anti-IL-10 mAbs reached the last steps of differentiation; this is further supported by the down-regulation of CD14, CD16 and CCR5 molecules (40, 41). In addition, DC-LAMP could be involved in Ag presentation, due to its expression in MHC class II compartments (39). Taken together, these data are consistent with an increased ability of IFN-DCs generated in the presence of neutralizing anti-IL-10 mAbs to present Ag in the context of MHC class II molecules.

We found that neutralization of IL-10 during the differentiation significantly increased the ability of IFN-DCs from patients to stimulate allogeneic and autologous CD4+ T lymphocyte proliferation in response to PPD and HIV-p24 protein. The CD4 T cell proliferation was, in all cases, lower when IL-4-DCs, even generated in the presence of neutralizing anti-IL-10 mAbs, were used as APCs. Altogether, these results clearly indicate that IL-10, produced throughout the differentiation of circulating monocytes toward DCs, plays a major role in inducing the intrinsic functional defects of DCs. Interestingly, the modulatory effect of IL-10 was more striking on IFN-DCs than on IL-4-DCs. This is in agreement with IL-10-induced phenotypic changes on IFN-DCs. It should be noted that IFN DCs from HIV-infected patients did not produce higher amount of IL-10 as compared with IL-4 DCs within their differentiation (data not shown). In addition, it has been demonstrated that IL-10-pretreated monocytes are partially unresponsive to IFN-{alpha} (42). Therefore, monocytes from HIV-infected patients, which may be considered as pretreated in vivo by IL-10, may exhibit an impaired IFN-{alpha} signaling and we can postulate that neutralization of IL-10 throughout the differentiation of IFN-DCs could restore the IFN-{alpha} signaling within these cells. This hypothesis is supported by the observation that IFN-DCs, differentiated in the presence of a higher concentration of IFN-{alpha} (1000 IU/ml) and in the absence of neutralizing anti-IL-10 mAbs, induce a potent proliferation of allogeneic CD4+ T cells (data not shown). Considering that IL-10 was not reported to alter IL-4 signaling, these observations highlight the interplay between IL-10 and IFN-{alpha} with a more potent effect of IL-10 on IFN-DCs than on IL-4-DCs.

Expansion of activated CD4+ T cells is the net result of CD4+ T cell proliferation and activation-induced apoptosis. Low expansion of CD4+ T cells may thus result from defective Ag presentation by APCs, increased apoptosis (reviewed in Ref.43) and/or a CD4 T cell defect including suppression of CD4+ T cell proliferation by regulatory T cells. Several reports suggest that defective Ag presentation, related to alterations in the trafficking or expression of class II molecules, may occur within DCs derived from HIV-infected patients (25, 44, 45). We found however that, following the neutralization of autocrine IL-10, the expression of MHC class II molecules increased on both DC populations. At this point, we cannot exclude that an altered trafficking of MHC class II molecules may be in part responsible for the defective CD4 T cell proliferation induced by DCs from HIV-infected patients.

Activated T cell autonomous death of CD4+ and CD8+ T cells of HIV-infected individuals has been demonstrated to be prevented by IL-2 and by IL-15 (46). IL-15 was reported to activate the proliferation of CD4+CD45RO+ cells and to enhance survival of CD4+CD45RO– cells in HIV-infected individuals (46). IL-15 was previously found to be secreted in significant amounts by immature IFN-DCs but not by IL-4-DCs (47, 48). We may postulate that the ability of IFN-DCs to induce CD4+ T cell proliferation is at least in part related to IL-15 production induced by IFN-{alpha}, and as a result, an enhanced proliferation and survival of expanded CD4+ T cells.

An expansion of a subset of CD4+ T cells coexpressing CD25 has been observed in HIV-infected patients (49, 50). We have demonstrated that peripheral CD4+CD25+ T cells from HIV-infected patients exhibit phenotypic and functional characteristics of regulatory T cells. Thus, depletion of CD4+CD25+ T lymphocytes dramatically increased CD4+ T cell proliferation in response to recall Ags including HIV-p24 protein.4 These observations led us to postulate that suppression by CD4+CD25+ regulatory T cells could be involved in the impairment of CD4+ T cell proliferation induced by DCs. Indeed, our results further demonstrated that regulatory CD4+CD25+ T cells suppressed the proliferation of p24-specific CD4+ T cells, even when p24-pulsed DCs were used as APCs. Interestingly, the depletion of CD4+CD25+ T lymphocytes and the neutralization of IL-10 throughout the differentiation of IFN-DCs act synergistically resulting in a dramatic increase of CD4+ T cell proliferation. Considering that CD4+CD25+ T cells exert their inhibitory functions following TCR ligation (51), we thus may consider that IFN-DCs derived from HIV-infected patients are able to present Ags by means of MHC class II pathway. In contrast to IFN-DCs, p24-pulsed IL-4 DCs, used in the same experimental conditions, only resulted in relatively low levels of proliferation of CD4+CD25– T cells. We cannot exclude that IFN-{alpha}, which has been reported to modulate the generation of regulatory T cells (52), may also favor Ag-specific activation of CD4+CD25+ T cells. Alternatively, immature IL-4-DCs, may exhibit altered trafficking of MHC class II molecules resulting in defective MHC class II pathway presentation.

Taken together, results from the present study indicate that IFN-DCs, in addition to efficiently stimulate CD8+ T cell responses in vitro (19) and in vivo (53), are more efficient than IL-4-DCs to stimulate CD4+ T cell proliferation, further supporting the use of the GM-CSF-IFN-{alpha} combination for immune-based therapy in HIV infection. Whether s.c. administration of GM-CSF and IFN-{alpha} to HIV-infected patients can induce in vivo generation of DCs, as previously suggested for GM-CSF and IL-4 in cancer patients (54) and may represent a DC-based immunotherapeutic approach in HIV infection is currently under investigation. Finally, the present study also emphasizes the relevance of therapeutic strategies aimed at modulating IL-10 production and the CD4+CD25+ T cell subset to enhance specific CD4 T cell responses for immune intervention in HIV infection.


   Acknowledgments
 
We thank Dr. Bayry for helpful discussion on DCs. We also gratefully acknowledge Drs. Piketty and Gonzalez-Canali for including patients in the study and Drs. Tartour and Kaveri for helpful discussions and rereading the manuscript.


   Footnotes
 
1 This work was supported by a grant from Ensemble Contre le SIDA, France. C.C. is a recipient of a fellowship from the Ministère de l’Education Nationale, France. Back

2 Address correspondence and reprint requests to Dr. Laurence Weiss, Hôpital Européen Georges Pompidou, Service d’Immunologie Clinique, 20 rue Leblanc, 75908 Paris Cedex 15 France. E-mail address: laurence.weiss{at}egp.ap-hop-paris.fr Back

3 Abbreviations used in this paper: DC, dendritic cell; HAART, highly active antiretroviral therapy; CD40L, CD40 ligand; SAC, Staphylococcus aureus Cowan I bacteria; PPD, tuberculin purified protein derivate; LAMP, lysosome-associated membrane protein. Back

4 L. Weiss, V. Donkova-Petrini, L. Caccavelli, M. Mallo, C. Carbonneil, and Y. Levy. Expansion of CD4+CD25+ regulatory T cells which suppress HIV-specific CD4 T-cell responses in HIV-infected patients. Submitted for publication. Back

Received for publication September 29, 2003. Accepted for publication March 29, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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