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AIDS:
doi: 10.1097/01.aids.0000210607.63138.bc
Basic Science

Differential susceptibility of human thymic dendritic cell subsets to X4 and R5 HIV-1 infection

Schmitt, Nathaliea; Nugeyre, Marie-Thérèsea; Scott-Algara, Daniela; Cumont, Marie-Christineb; Barré-Sinoussi, Françoisea; Pancino, Gianfrancoa; Israël, Nicolea,†

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From the aUnité de Régulation des Infections Rétrovirales, Institut Pasteur, Paris, France

bUnité de Recherche et d'Expertise Physiopathologie des Infections Lentivirales, Institut Pasteur, Paris, France.

Nicole Israel passed away after the submission of this article. We dedicate this publication to her memory.

Received 23 August, 2005

Revised 2 November, 2005

Accepted 6 December, 2005

Correspondence to N. Schmitt/G. Pancino, Unité de Régulation des Infections Rétrovirales, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. Tel: +33 1 4568 8738; fax: +33 1 4568 8957 e-mail: nathalis@bhcs.com/gpancino@pasteur.fr

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Abstract

Objectives: Human thymus can be infected by HIV-1 with potential consequences on immune regeneration and homeostasis. We previously showed that CD4 thymocytes preferentially replicate CXCR4 tropic (X4) HIV-1 dependently on interleukin (IL)-7. Here we addressed the susceptibility of thymic dendritic cells (DC) to HIV-1 infection.

Methods: We investigated the replication ability of CXCR4 or CCR5 (R5) tropic HIV-1 in thymic micro-explants as well as in isolated thymic CD11clowCD14 DC, CD11chighCD14+ DC and plasmacytoid DC subsets.

Results: Thymic tissue was productively infected by both X4 and R5 viruses. However, X4 but not R5 HIV-1 replication was enhanced by IL-7 in thymic micro-explants, suggesting that R5 virus replication occurred in cells other than thymocytes. Indeed, we found that R5 HIV-1 replicated efficiently in DC isolated from thymic tissue. The replicative capacity of X4 and R5 viruses differed according to the different DC subsets. R5 but not X4 HIV-1 efficiently replicated in CD11chighCD14+ DC. In contrast, no HIV-1 replication was detected in CD11clowCD14 DC. Both X4 and R5 viruses efficiently replicated in plasmacytoid DC, which secreted interferon-α upon HIV-1 exposure. Productive HIV-1 infection also caused DC loss, consistent with different permissivity of each DC subset.

Conclusions: Thymic DC sustain high levels of HIV-1 replication. DC might thus be the first target for R5 HIV-1 infection of thymus, acting as a Trojan horse for HIV-1 spread to thymocytes. Furthermore, DC death induced by HIV-1 infection may affect thymopoiesis.

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Introduction

The thymus is thought to be a critical compartment for HIV-1 infection in humans [1–3] and in animal models of AIDS [4–6]. Indeed, thymuses from paediatric patients with rapid disease progression or from adults with AIDS show severe thymocyte depletion associated with a profound disorganization of the thymic epithelial network [7–12].

HIV-1 uses the chemokine receptors CXCR4 or CCR5 for entry into the cells. Viruses using CCR5 (R5 viruses) are prevalent during the early stages of infection [13] and the emergence of viruses using CXCR4 (X4 viruses) is a marker of poor prognosis [14,15]. In vitro, X4 viruses replicate more efficiently than R5 viruses in thymocytes [16] and caused thymocyte depletion in SCID-hu Thy/Liv mice [17]. In contrast, R5 viruses replicate, at least during early HIV-1 infection, in stromal cells in the thymic medulla. These stromal cells include cells coexpressing CD11c and high levels of HLA-DR, suggesting a dendritic cell (DC) phenotype. Indeed, thymic CD11c+ cells have been characterized as DC due to phenotypic and functional properties including their capacity to stimulate naive CD4 T-cell proliferation [18,19]. Thymic CD11c+ DC can be divided into two subsets on the basis of CD14 expression [19]. A thymic cell subset exhibiting a phenotype close to that of blood plasmacytoid DC (pDC) has also been characterized [18].

HIV-1-infected DC have been detected in skin, mucosa and blood [20–23]. Blood DC from HIV-1-infected patients contain proviral DNA and exhibit impaired allostimulatory functions [22]. Moreover, low blood DC counts correlate with high HIV-1 RNA viral loads [24]. Blood DC are able to replicate X4 and R5 viruses in vitro although at low levels [25–27]. In these compartments, immature DC may also capture incoming virions at sites of infection through surface lectins, for example DC-SIGN, and transport them to lymphoid tissues [28].

In the thymus, although DC have been shown to be susceptible to HIV-1 infection [29–31], relatively few reports have analysed HIV-1 replication and its impact in each thymic DC subset. Whereas it has recently been reported that thymic pDC are susceptible to both X4 and R5 viruses [30,31], little information is available on the ability of thymic CD11c+ DC to replicate HIV-1 [29]. Nevertheless, thymic DC play a key role in thymopoiesis, notably they present self antigen and induce negative selection of auto-reactive T-cell clones [32,33]. Consequently, infection of thymic DC might affect thymocyte maturation.

We investigated the replication ability of X4 and R5 HIV-1 in thymic micro-explants and in isolated thymic DC subsets. X4 and R5 viruses replicated at comparable levels in thymic micro-explants despite the preferential replication of X4 viruses by thymocytes. CD11chighCD14+ DC only replicate R5 viruses whereas pDC were permissive to both X4 and R5 viruses. Thymic DC appeared to be susceptible to HIV-1 cytopathogenic effects since productive infection was followed by cell death.

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Methods

Viruses

NL4-3 and NLAD8 HIV-1 (refered to as NL4-3 and NLAD8) were produced by transfecting COS-7 cells with molecular clones pNL4-3 [34] or pNLAD8 [35]. Cell supernatants were harvested 2 and 3 days after transfection. Viral titres were determined by infecting peripheral blood mononuclear cells (PBMC) with serial dilutions of virus [36]. HIV-1 p24gag antigen concentrations were determined in culture supernatants using an HIV-1 p24 immunoassay (Beckman-Coulter, Villepinte, France).

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Thymuses

Fresh thymus fragments were obtained during elective cardiac surgery (Hôpital Necker, Paris, France and Marie Lannelongue, Le Plessis Robinsson, France) on HIV-1-seronegative children (age range, 6 days to 24 months).

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Thymocyte isolation

Single-positive CD4+CD8CD3+ (SP CD4), and the immature CD4−/+CD8CD3 (consisting of a pool of triple-negative CD4CD8CD3 and intermediate CD4+CD8CD3) thymocytes were obtained by negative selection as previously described [37]. Briefly, SP CD4 thymocytes were obtained by depletion cycles using monoclonal antibodies (mAb) directed against CD8, CD10, CD14, CD34 and CD83 and anti-mouse IgG-coated magnetic beads. Immature CD4+/−CD8CD3 thymocytes were obtained by depletion cycles using mAb directed against CD3, CD8, CD14 and CD83 and anti-mouse IgG-coated magnetic beads. All mAb were from Beckman-Coulter.

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DC isolation

Tissues were dissociated by gentle teasing, and incubated with collagenase IV (5 mg/ml, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and DNase (150 U/ml, Sigma-Aldrich) for 1 h at 37°C, and 5 mM EDTA was added for the last 10 min. Dissociated cells were subjected to Percoll gradient centrifugation (52%) and the low density fraction, containing DC, was collected. The Fc receptor was saturated with human IgG (Tegeline, LFB, Courtaboeuf, France). The samples were immunodepleted of contaminating cells using CD3 (UCHT1 and X35), CD34 (QBEnd10), CD19 (J4.119) and CD56 (C218) mouse mAb (all from Beckman-Coulter) and anti-mouse IgG-coated magnetic beads (Dynal-Biotech, Oslo, Norway). CD11c+ DC were isolated by positive selection of CD11c-expressing cells using biotinylated CD11c mAb (BU15, Beckman-Coulter) and anti-biotin-coated magnetic microbeads (Miltenyi-Biotec, Bergisch-Gladbach, Germany). Possible residual CD14+ cells were then removed from the negative fraction with CD14 microbeads (Miltenyi-Biotec). pDC were isolated by positive selection for CD123 expression on CD11cCD14 cells using a biotinylated CD123 mAb (9F5, BD-Pharmingen, Mountain View, CA, USA) and anti-biotin-coated magnetic microbeads. The purity of DC populations obtained was about 80%.

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Phenotypic analysis of thymic DC subsets

Cells were incubated for 30 min at 4°C with a mixture of IgG1 (679.1Mc7, Beckman-Coulter), IgG2a (U7.27, Beckman-Coulter) and hIgG. Cells were then washed and immunostained with labelled mouse mAb for 30 min at 4°C. mAb specific for CD4 (13B8.2), CD11c (BU15) and CD14 (RMO52) were from Beckman-Coulter; those for CD123 (9F5), HLA-DR (L243), CCR5 (2D7) and CXCR4 (12G5) from BD-Pharmingen. For intracellular labelling, cells were permeabilized with Cytofix/Cytoperm (BD-Pharmingen) before incubation with mAb specific for HIV-1 core antigen (KC57, Beckman-Coulter). Cell counts were calculated using Flow-Count Fluorospheres (Beckman-Coulter). At least 10 000 events per sample were analysed in all experiments (except when DC cell mortality was too high, in this last case, at least 5000 events were analysed). Acquisitions were performed with an XL-4C cytofluorometer using XL-2 software (Beckman-Coulter). Analysis were performed using EXPO v.2 Analysis software (Beckman-Coulter).

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Infection and culture of thymocytes

Mature SP CD4 or immature CD4−/+CD8CD3 thymocytes were infected with NL4-3 at a multiplicity of infection (m.o.i.) of 1.6 × 10−4 or NLAD8 at a m.o.i of 2.8 × 10−3. The m.o.i for NLAD8 was higher to ensure substantial levels of replication in thymocytes despite the low susceptibility of these cells to R5 viruses. After 1 h at 37°C, the cells were washed three times, and cultured in complete medium (RPMI 1640 containing 10% FCS, 1 mM L-glutamine, 10 mM HEPES and antibiotics) with 10 ng/ml tumour necrosis factor (TNF)-α and 10 ng/ml interleukin (IL)-7 (both from PeproTech, Rocky Hill, NJ, USA).

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Infection and culture of thymic DC

DC were inoculated with NL4-3 or NLAD8 at a m.o.i of 2 × 10−2 for 2 h at 37°C. The cells were then washed three times, and cultured in 96-well plates (250 000 cells per well) in 200 μl of complete medium with 20 ng/ml IL-3 (R&D Systems, Minneapolis, MN, USA). Half of the supernatants were collected each for 2 or 3 days and wells were replenished with fresh medium.

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Infection and culture of thymic micro-explants

Thymus fragments were sliced into approximate 2-mm blocks and incubated with NL4-3 or NLAD8 [103 50% tissue culture infectious dose (TCID50)/block] for 3 h at 37°C. After thorough washing, tissue blocks were placed on top of collagen sponge gels (Pfizer, Paris, France) in culture medium (RPMI 1640, 10% foetal calf serum, 1 mM L-glutamine, antibiotics, fungizone, gentamicin, non-essential amino acids and sodium pyruvate) at the air–liquid interface and cultured as described previously [38]. At the times indicated, 5 ng/ml IL-7 and 1 ng/ml TNF-α were added to culture medium.

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Interferon-α ELISA

Interferon (IFN)-α concentrations in cell culture supernatants were determined using the Hu-IFN-α ELISA kit (RDI, Flanders, NJ, USA) which detects human IFN-αA, α2, αA/D, αB2, αC, αD, αG, αH, αI, αJ and αK (threshold value, 10 pg/ml).

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Results

R5 HIV-1 replicates at high level in human thymic tissue

HIV-1 replicates in two thymocyte subsets in vitro, mature single-positive CD4 (SP4) and intermediate CD4+CD8CD3 [37], and X4 viruses replicate more efficiently than R5 viruses in these cells [16]. We investigated whether replication of X4 viruses was also favoured in the intact thymic tissue by comparing the permissivity of thymic micro-explants and thymocytes to X4 and R5 virus infection. We used the molecular clones NL4-3 and NLAD8 which differ from each other only in the env gene: it confers CXCR4 tropism on NL4-3 and CCR5 tropism on NLAD8. Thymocytes (mature SP4 and immature CD4+/−CD8CD3) were purified and infected with NL4-3 or NLAD8 and then cultured with IL-7 and TNF-α, which are both required for HIV-1 replication in these cells [37]. NL4-3 or NLAD8 infected micro-explants were also cultured in the presence of IL-7 and TNF-α, or in their absence, to evaluate the contribution of thymocytes and cells other than thymocytes in X4 and R5 virus replication. NL4-3 and NLAD8 replication patterns in micro-explants (Fig. 1a) and in thymocytes (Fig. 1b) were markedly different. Consistent with previous findings, NL4-3 replicated at higher level than NLAD8 in purified thymocytes in the presence of IL-7 and TNF-α. In contrast, NL4-3 and NLAD8 replicated at similar levels in thymic micro-explant cultures in the presence of IL-7 and TNF-α. NL4-3 replication in thymic tissue was strongly increased by the addition of exogenous IL-7 and TNF-α, consistent with the preferential replication of X4 viruses in thymocytes, whereas replication of NLAD8 was not affected. NL4-3 replication in micro-explants in the presence of IL-7 with or without exogenous TNF-α was similar (data not shown) indicating that this effect was mostly due to IL-7. It was presumably due to thymocyte activation by IL-7 [37] and not to IL-7 anti-apoptotic effect, since the survival of thymocytes in micro-explant cultures was similar in presence and absence of exogenous IL-7 as assessed by cell counting and flow-cytometry analysis of thymocyte subsets on days 5, 7 and 12 of culture (data not shown). The R5 virus replicated more efficiently in thymic micro-explants than in purified thymocytes suggesting that it replicates in cells other than thymocytes.

Fig. 1
Fig. 1
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Thymic DC express HIV-1 coreceptors

We tested whether thymic DC cells can be productively infected by HIV-1 and thus contribute to the R5 virus replication observed in thymic micro-explants. We identified three DC subsets in thymic tissue on the basis of the expression of the thymic DC markers CD11c, CD14 and CD123 [18,19]: pDC, CD11chighCD14+ DC and CD11clowCD14 DC (Fig. 2a). To obtain sufficient DC to study HIV-1 infection, we developed a new enrichment strategy. We first isolated CD11c+ DC by positive selection of cells expressing CD11c. CD11c+ DC were distinguished on the basis of CD14 expression (Fig. 2a) into CD11chighCD14+ DC (CD11chigh CD14+ CD123low/− HLA-DRint) and CD11clowCD14 DC (CD11clow CD14 CD123low/− HLA-DRhigh) (Fig. 2b). Importantly, all CD14+ thymic cells expressed CD11c (Fig. 2a) and were thus obtained in the CD11c-positive fraction. Thymic pDC (CD123high CD11c CD14 HLA-DRint) were isolated in a second step by selection of cells expressing CD123.

Fig. 2
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We analysed the expression of molecules required for HIV-1 entry on each freshly isolated DC subset. CD4, CXCR4 and CCR5 were detected in all three thymic DC subsets (Fig. 2b). Conversely, DC-SIGN was not detected on thymic DC by flow cytometry. Furthermore, cells expressing DC-SIGN are found in the cortex of thymic tissue sections by immunohistochemistry, whereas DC are mainly in the medulla (data not shown).

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Thymic DC can be productively infected by HIV-1

Freshly isolated thymic pDC and CD11c+ DC (comprising CD11clowCD14 and CD11chighCD14+ DC) were infected with NL4-3 or NLAD8 and then cultured in the presence of IL-3. As previously described [18,39,40], IL-3 allowed pDC to be maintained in culture protecting a substantial proportion (approximately 30%) of the cells from spontaneous cell death. Culture in the presence of IL-3 also allowed the survival of CD11c+ DC, probably because of endogenous production of other cytokines needed for cell survival (N. Schmitt, M-C Cumont, M-T Nugeyre, B Hutrel, F Barré-Sinoussi, D Scott Algara and N Israël, unpublished data).

After NLAD8 infection of CD11c+ DC, viral production increased throughout the monitoring period to very high levels [1170 ng/ml p24 at day 11 post infection (p.i.)] (Fig. 3a, left). In contrast, the levels of p24 remained at background levels after NL4-3-infection.

Fig. 3
Fig. 3
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We then evaluated the respective contribution of CD11clowCD14 and CD11chighCD14+ DC to R5 HIV-1 replication in CD11c+ DC cultures by performing double staining of CD11c+ DC for CD14 and intracellular p24 (Fig. 3b, left). On day 12, CD14+ cells represented 73% of uninfected CD11c+ DC cultures. Similar proportions of CD14+ cells were found in NL4-3-infected cultures, but CD14+ cells decreased to 50% in NLAD8-infected cultures. Intracellular p24 detection showed that NLAD8 mainly replicated in CD11chighCD14+ DC: 42% ± 2.5% (Fig. 3c) of these cells i.e., 21% of total cells (Fig. 3b, left) were positive for p24 whereas p24 could not be detected in a significant proportion of CD11clowCD14 DC (2.2 ± 2.3%) (Fig. 3c). Again, there was no detectable replication of NL4-3 in either CD11c+ DC subset.

In pDC cultures, the replication of NL4-3 was similar to that of NLAD8 during the first days of culture (130 and 96 ng/ml p24 respectively on day 6 p.i.) (Fig. 3a, right). Thus pDC replicated both X4 and R5 viruses whereas CD11chighCD14+ DC only replicated R5 viruses.

However, NL4-3 and NLAD8 replication kinetics diverged 6 days p.i. in pDC cultures: NL4-3 production decreased (58 ng/ml at day 11 p.i.), whereas NLAD8 production increased throughout the monitoring period to high levels (410 ng/ml at day 11 p.i.).

In pDC culture, a progressive modification of cell surface phenotype was detected. This change involved a progressive decrease of CD123 (from day 1), a shift from CD45RA to CD45RO (from day 4), and appearance of markers including CD11b, CD11c (both from day 2) and also CD14 (from day 4) (N. Schmitt et al. unpublished data). We thus tried to determine whether these modifications could explain differences in X4 and R5 virus replication occurring after day 6. We analysed CD14 and CD14+ subsets separately by CD14-p24 costaining (Fig. 3b, right). On day 12, CD14+ cells made up approximately 55–68% of cells in both uninfected and NL4-3-infected cultures, but only 30% of cells in NLAD8-infected cultures. p24 was detected in 37 ± 8% (Fig. 3c) of CD14+ cells, i.e., 10% of total cells (Fig. 3b, right) in pDC cultures infected with NLAD8. In contrast, no significant p24 expression could be detected in CD14+ cells in NL4-3 infected pDC culture (1.4 ± 2.2%, n = 3) (Fig. 3c). These results indicate that NLAD8 but not NL4-3 efficiently replicates in the CD14+ cell subpopulation of pDC cultures and may thus account for differences in X4 and R5 virus replication patterns found 6 days p.i. in pDC cultures.

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HIV-1 infection induces IFN-α secretion by pDC

We then looked for the effects of HIV-1 on thymic DC. We first measured IFN-α levels in culture supernatants in infected and uninfected pDC and CD11c+ DC cultures. No IFN-α was detectable in supernatants from either uninfected or infected CD11c+ DC (data not shown). In contrast, IFN-α was detected in pDC supernatants after both NL4-3 and NLAD8 infections but not in uninfected cultures (Fig. 4). IFN-α secretion was detected from day 4 p.i., peaked on days 6 or 8 and then decreased. The decrease in IFN-α production in NL4-3-infected pDC cultures was associated with a decrease in viral production, but IFN-α secretion also decreased in NLAD8-infected pDC cultures despite increasing viral replication. This decrease of IFN-α production paralleled the increase of the CD14+ phenotype in pDC cultures, suggesting that these CD14+ cells were not able to synthesize IFN-α in response to HIV-1.

Fig. 4
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HIV-1 infection induces thymic DC death

To evaluate the impact of HIV-1 infection on thymic DC viability and further clarify the kinetics of viral replication, we determined the number of viable cells for each subset of thymic DC, each infected or not infected with NL4-3 or NLAD8, on days 5 and 8 of culture (Fig. 5). Viable cells, identified on the basis of their morphological characteristics (forward and side scatter) and Propidium iodide-annexin V staining (Fig. 5a) were counted by flow cytometry. Viable cell counts were not lower for either NL4-3- or NLAD8-infected CD11clowCD14 DC than uninfected cells consistent with the inefficient replication of HIV-1 in this thymic DC subset. In contrast, the number of viable cells in CD11chighCD14+ DC cultures infected by NLAD8 declined progressively as compared to uninfected controls, consistent with these cells being highly susceptible to R5 virus infection. The viability of CD11chighCD14+ DC was not affected by NL4-3 infection as would be expected from the failure of NL4-3 to replicate in this subset. In contrast, both NLAD8 and NL4-3 infections decreased the viable cell count in pDC cultures in agreement with the permissivity of pDC to both X4 and R5 viruses.

Fig. 5
Fig. 5
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Discussion

X4 HIV-1 replicates more efficiently than R5 in thymocytes and IL-7 favours viral replication in these cells [16,37]. Here, we show that R5 HIV-1 can efficiently infect thymic micro-explants, independently of exogenous IL-7 (Fig. 1). Thymic DC expressed high levels of CCR5 (Fig. 2) whereas thymocytes did not [16,41]. Accordingly, we show that R5 viruses display a high replicative capacity in thymic DC. The efficient replication of R5 HIV-1 in thymic tissue may therefore result, at least partially, from the productive infection of thymic DC. This extends previous data indicating a preferential entry of R5 viruses into thymic DC [29] and is consistent with findings in the SCID-hu Thy/Liv mouse [17] and rhesus macaque [6] models showing HIV-1 or SIV replication in dendritic/macrophage-like cells in thymus. Previous studies reported that thymic epithelial cells do not efficiently replicate HIV-1 [42] and that CD34 progenitors appear to be poorly infectable [43,44]. However, we cannot exclude the possibility that other cell types not studied here contribute to R5 virus replication in human thymus.

The high susceptibility of thymic DC to R5 HIV-1 might have important physiopathological consequences in HIV-1 infection. Infection of thymic DC by R5 viruses might be the first step in thymus infection especially during the early stages of HIV-1 infection when R5 viruses are prevalent. The close localization of DC and thymocytes may also favour the infection of thymocytes by R5 viruses. Moreover, if X4 variants are generated during active viral replication of R5 viruses in thymic DC, they might be transmitted to thymocytes which preferentially replicate X4 HIV-1 [16].

By studying the susceptibility of thymic DC subsets to HIV-1 separately, we evidenced variations in the replicative ability of R5 or X4 viruses. Among thymic CD11c+ DC, CD14+ cells are highly permissive to R5 infection whereas CD14 cells are not (Fig. 3) despite both subsets expressing CD4 and HIV-1 coreceptors CXCR4 and CCR5. Whether CD11chighCD14+ DC and CD11lowCD14 DC are two distinct subsets or the second subset is the mature counterpart of the first one, as suggested by previous reports [19] was not addressed in this study. Should CD11clowCD14 DC represent mature thymic DC, their decreased permissivity to HIV-1 replication might be related to a post-integration block as described for mature monocyte-derived DC [45]. Further studies are needed to elucidate these issues. pDC unlike CD11c+ DC replicated both R5 and X4 HIV-1. Until day 6 p.i., NL4-3 and NLAD8 replicated similarly in thymic pDC (Fig. 3). These data are consistent with the detection of similar amounts of p24-positive pDC in foetal thymic organ cultures [31] and thymus implants in SCID-hu mice [30] infected with X4 and R5 viruses, and also with viral replication in blood pDC [25,27]. However, after day 6, NL4-3 replication decreased and NLAD8 replication progressively increased. These differences in viral replication kinetics coincide with the appearance in pDC culture of CD14+ cells with high replicative capacity for R5 but not for X4 viruses.

The better replication of NLAD8 than of NL4-3 in thymic CD11chighCD14+ DC is most likely related to coreceptor tropism, as the only difference between NLAD8 and NL4-3 is the env gene. However, CXCR4 expression on thymic CD11chighCD14+ DC is consistent and comparable to that of CCR5 (Fig. 2) and thus differences in coreceptor expression do not explain the differential replication of the two viruses. This phenomenon is reminiscent of the reduced permissivity to X4 viruses also observed with macrophages [46]. Differences in the biochemical properties of CXCR4 in macrophages and in T cells have been suggested to affect the association of CXCR4 with CD4 [47,48]. However, some primary X4 isolates replicate in macrophages [49] and it remains to be determined whether the mechanisms underlying the restriction of X4 viruses in thymic CD11chighCD14+ DC are similar to those reported for macrophages.

We also show that HIV-1 induces functional changes in thymic microenvironment. Indeed, HIV-1 induces IFN-α secretion by thymic pDC according to previous studies on SCID-hu Thy/Liv mouse and blood pDC [27,31]. The decrease of IFN-α after day 6–8 is in agreement with a previous report showing that the ability of pDC to produce IFN-α was decreased after maturation into DC by culture with IL-3 [50].

We found that HIV-1 infection kills thymic DC and that cell death correlates with the level of viral replication. Indeed, R5 viruses induce cell death from both pDC and CD11chighCD14+ DC whereas X4 viruses only induce a pDC death. Viability of CD11clowCD14 DC is not altered by either X4 or R5 viruses in agreement with the absence of viral replication in these cells. Thymic DC death caused by HIV-1 infection may alter thymic homeostasis and impair thymocyte differentiation in vivo in particular thymocyte negative selection.

In conclusion, we demonstrate that thymic pDC can be productively infected by both X4 and R5 HIV-1 and that a high level of viral replication is reached by R5 viruses in CD11chighCD14+ DC. It has recently been shown that blood myeloid DC are more susceptible to R5 than to X4 viruses whereas blood pDC were equally susceptible to both isolates [51]. However, the ability of thymic DC to replicate HIV-1 efficiently distinguish them from blood DC which replicate HIV-1 at low levels [25–27]. Also, only a small proportion of immature DC from tissue compartments such as epithelial tissue are productively infected by HIV-1 [52–54]. The high susceptibility of thymic DC to R5 HIV-1 may confer on them the role of a Trojan horse for R5 virus infection of the thymus during the early phases of infection. Furthermore, HIV-1 infection kills thymic DC, and the consequence could be altered thymopoiesis.

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Acknowledgments

We thank Dr Sonia Berrih-Aknin (Hôpital Marie Lannelongue, Le Plessis-Robinson, France) and Professor Leca (Hôpital Necker, Paris, France) for providing us with thymuses. We thank Anne Hosmalin for helpful discussions and Francine Brière for useful information.

Supported by Sidaction. N. Schmitt was successively the recipient of fellowships from the French Ministry of Education and Research (MENESR) and from Sidaction.

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Keywords:

thymus; HIV-1; dendritic cells; viral tropism; cell death

© 2006 Lippincott Williams & Wilkins, Inc.

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