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CD4+ recent thymic emigrants are infected by HIV in vivo, implication for pathogenesis

Fabre-Mersseman, Véroniquea,b,c,d; Dutrieux, Jacquesa,b,c,d; Louise, Annee; Rozlan, Sandraf; Lamine, Auréliag; Parker, Raphaëllea,b,c,d; Rancez, Magalih; Nunes-Cabaço, Helenai; Sousa, Ana Ei; Lambotte, Olivierg; Cheynier, Rémia,b,c,d,j

Author Information

aInstitut Cochin, Département Immunologie-Hematologie, France

bInserm, U1016, France

cCNRS, UMR 8104, France

dUniversité Paris Descartes, Faculté de Médecine René Descartes, UMR-S 8104, Paris, France

ePlatforme de cytomètrie en flux (Imagopôle), Institut Pasteur, Paris, France

fCytheris S.A., Technopolis, France

gINSERM U802, Université Paris-Sud, Bicêtre, France

hLaboratoire de Transmission et Dissémination Virales, Université Paris Diderot, Paris Cedex, France

iUnidade de Imunologia Clínica, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal

jDépartement de Virologie, Institut Pasteur, Paris Cedex, France.

Received 6 November, 2010

Revised 7 March, 2011

Accepted 24 March, 2011

Correspondence to Rémi Cheynier, Institut Cochin, Bâtiment Gustave Roussy, 27 rue du Faubourg Saint Jacques, Paris 75014, France. Tel: +33 1 40 51 65 41; e-mail: remi.cheynier@inserm.fr

doi: 10.1097/QAD.0b013e3283471e89

Abstract

Introduction

Despite the efficacy of long-term HAART in HIV-infected patients, HIV eradication remains impossible to achieve. Indeed, in the vast majority of patients, treatment interruption leads to rebound in viremia and decrease in the CD4+ T-cell counts, suggesting that local viral replication still proceeds in the secondary lymphoid organs of HAART-treated HIV-infected patients [1]. Resting CD4+ T cells have been described as a major contributor to the pool of HIV-infected cells in chronically HIV-infected patients [2–4]. Infected in periphery during antigen-induced proliferation, memory T cells have a long lifespan and may return to a resting state after achievement of the immune response. Similarly, naive T cells are long-lived cells; however, they are characterized by limited proliferation history [5]. Considering the importance of cell activation/proliferation for the achievement of the HIV-infection process, the mechanisms leading to naive T-cell infection remain poorly understood.

Infection of naive T cells may take place either in periphery or in the thymus. Infection of thymocyte has been demonstrated in thymic organ cultures [6] and in humanized mice [7] as well as in vivo[8]. Moreover, thymic function is rapidly affected during primary HIV infection, suggesting active viral replication in this organ [9]. On the contrary, mature naive T cells might also be infected, in particular in the context of lymphoid tissues [10] and during interkeukin (IL)-7-dependent proliferation [11]. However, infection of naive T cells after stimulation by IL-7 remains controversial. Although productive HIV infection requires at least partial cell activation and transition to the G1b stage of the cell cycle [12,13], the cells proliferating in response to IL-7 stimulation in naive T-cell subset are mostly recent thymic emigrants (RTEs) [14–16].

RTE can be identified from mature CD4+ naive T cells (non-RTEs) according to their overexpression of CD31 and/or PTK7 [17,18]. Moreover, as a consequence of their small proliferation history, RTEs have a higher signal joint T-cell receptor excision circle (sjTREC) content than non-RTEs [19,20]. Indeed, sjTRECs, by-products of the excision of the T-cell receptor (TCR)δ locus that precedes TCRα chain rearrangement during T-cell differentiation in the thymus, are nonreplicating molecules, therefore, lost by dilution during mitosis.

The capacity of the thymus to produce RTEs is mostly dependent upon thymocyte proliferation [21]. Indeed, between rearrangements at the T-cell receptor beta (TCRB) and T-cell receptor alpha (TRCA) loci that, respectively, occur at the late double negative and early double positive stages of differentiation, thymocytes go through extensive cell cycling. This proliferation directly correlates with subsequent thymic export and participates in the generation of T-cell repertoire diversity [21–23]. The extent of cell proliferation between late double negative and early double positive in the thymus can be estimated in patients through measuring the ratio between sjTREC and DβJβ deletion circles [Dβ-Jβ T-cell receptor excision circle(DJβTREC)] frequencies in peripheral blood cells [24]. Because of the fact that these by-products of TCR rearrangement processes do not replicate during mitosis, increased precursor T-cell proliferation leads to the dilution of DJβTRECs but not of the sjTREC that is produced after this proliferation step, thus to an increase of the sj/βTREC ratio [9].

Using a highly sensitive real-time PCR assay, we precisely quantified HIV DNA in both RTE and non-RTE circulating naive T cells from viremic and HAART-treated aviremic HIV-infected patients. Analysis of intrathymic precursor T-cell and circulating T-cell proliferation allowed a better understanding of HIV infection in these naive T-cell subsets.

Patients and methods

Study individuals

Twenty-six HIV-1-infected patients were enrolled in this study (Table 1): 11 viremic individuals with viral load varying from 3.6 × 103 to 3.2 × 105 copies/ml and CD4 counts: 162–797 cells/μl and 15 aviremic individuals with no detectable plasma viremia, CD4 counts: 356–1118 cells/μl. Samples were obtained with the individuals' informed consent. All viremic individuals (except individual #6) were untreated at sampling. Patients #2 and #3 were off therapy for more than 2 years at sampling but previously received HAART. Aviremic individuals were all under HAART. Peripheral blood mononuclear cells (PBMCs) were purified on Ficoll density gradient (Eurobio, Courtaboeuf, France). Samples from a group of healthy individuals (n = 13), gathered from the French blood bank, were included as control.

Table 1
Table 1:
Profiles of viremic and aviremic individuals enrolled in the study.

Flow cytometric cell sorting and phenotypic analysis

Fluorescence activated cell sorting (FACS) of naive and memory T-cell subsets was performed on PBMCs from HIV-infected individual. PBMCs were labeled 45 min with anti-CD3 conjugated to allophycocyanin (SP34-2; BD Biosciences, Le Pont de Claix, France), anti-CD4 conjugated to fluorescein isothiocyanate (FITC) (M-T310; Dako, Trappes, France), anti-CD45RA conjugated to phycoerythrin (HI100; BD Biosciences), anti-CCR7 conjugated to phycoerythrin-Cy7 (3D12; BD Biosciences), anti-CD31 conjugated to biotin (WM59; AbD Serotec, Düsseldorf, Germany) monoclonal antibodies (mAbs) and streptavidin conjugated to phycoerythrin-Cy5 (BD Biosciences). Cells were washed and fixed overnight in phosphate-buffered saline (PBS) containing 2% paraformaldehyde. T-cell subsets were sorted by MoFlo (Beckman Coulter, Villepinte, France) as indicated in Fig. 1 a. Postsorting purity was assessed and ranged from 84.0 to 98.0% (Supplemental Fig. 2, http://links.lww.com/QAD/A136).

Fig. 1
Fig. 1:
Infection of CD4+ naive T-cell subsets. (a) T-cell sorting strategy. Freshly isolated peripheral blood mononuclear cells from an HIV-1-infected patient were stained with monoclonal antibodies directed against CD3, CD4, CCR7, CD45RA and CD31 molecules. CD4+ recent thymic emigrants [recent thymic emigrant (RTE): CD3+ CD4+ CD45RA+ CCR7+ CD31high], naive CD4+ T cells (non-RTE: CD3+ CD4+ CD45RA+ CCR7+ CD31low), memory CD4+ T cells (CD3+ CD4+ CD45RA–), naive CD8+ CD31high T cells (CD3+ CD4– CD45RA+ CCR7+ CD31high) and naive CD8+ CD31low T cells (CD3+ CD4– CD45RA+ CCR7+ CD31low) were FACS sorted. signal joint T-cell receptor excision circle (b) and HIV DNA (c) concentrations were measured by real-time quantitative PCR in purified T-cell subsets from viremic (gray circles) and aviremic (white diamonds) patients. Median values for each T-cell subset are indicated by horizontal bars. Statistical differences as compared with RTE CD4+ (a) or memory CD4+ (b) are shown (*: P ≤ 0.05; **: P ≤ 0.01). (d) Quantification of HIV proviral DNA in RTE and non-RTE CD4+ T cells from viremic (left) and aviremic (right) patients. Samples with a too low number of recovered non-RTE naive T cells are indicated by “*”. PBL,; RTE, recent thymic emigrant; sjTREC, signal joint T-cell receptor excision circle.

Phenotypic analyses were performed on cryopreserved total blood samples conserved in 10% dimethyl sulfoxide. White blood cells were washed three times with PBS supplemented with 10% fetal calf serum. Cells were then incubated for 15 min with conjugated mAbs. For intracellular labeling, cells were permeabilized with the Cytofix/Cytoperm Kit (BD Biosciences) before incubation with specific mAbs according to the manufacturer's instructions. Monoclonal antibodies used for phenotypic analyses were anti-CD4 conjugated to Alexa Fluor 700 (OKT-4; eBioscience, Frankfurt, Germany), anti-CD8 conjugated to pacific blue (RPA-T8; BD Biosciences), anti-CD45RA conjugated to phycoerythrin (HI100; BD Biosciences), anti-CCR7 conjugated to phycoerythrin-Cy7 (3D12; BD Biosciences), anti-CD31 conjugated to biotin (WM59; AbD Serotec), strepatvidin conjugated to phycoerythrin-Texas-Red (BD Biosciences) and anti-Ki-67 conjugated to FITC (MIB-1; Dako). Living cells were identified using the LIVE/DEAD Fixable Dead Cell Stains kit (Invitrogen, Cergy Pontoise, France), according to the manufacturer's instructions. Analyses were performed on a cyan cytofluorometer (Beckman Coulter) flow cytometer interfaced to summit 4.3 and analyzed with FlowJo 8.7 software.

Signal joint T-cell receptor excision circle, DβJβ T-cell receptor excision circle, total and integrated HIV DNA quantifications

sjTRECs and DJβTRECs were quantified in FACS-sorted cells using real-time quantitative PCR, with a technique adapted and simplified from Dion et al.[24] as described [25]. Briefly, sorted cells (106 cells) were lysed in Tween-20 (0.05%), Nonidep P-40 (0.05%) and proteinase K (100 μg/ml) for 30 min at 56 °C, and then 15 min at 98 °C. Multiplex PCR amplification was performed for sjTREC together with the CD3γ chain, in a final volume of 100 μl (10 min initial denaturation at 95 °C, then 22 cycles of 30 s at 95 °C, 30 s at 60 °C, 2 min at 72 °C) using outer 3′/5′ primer pairs [26]. PCR conditions in the LightCycler (Roche Applied Science, Meylan, France) experiments performed on 1/100th of the initial PCR products in JumpStart mix (Sigma-Aldrich, Lyon, France), were 1 min initial denaturation at 95 °C, then 40 cycles of 1 s at 95 °C, 10 s at 60 °C, 15 s at 72 °C. Measurements of the fluorescent signals were performed at the end of annealing steps. TREC and CD3γ LightCycler quantifications were performed in independent experiments, using the same first-round serial dilution standard curve. Similarly, DJβ1TRECs (DJβ1.1–1.6) and DJβ2TRECs (DJβ2.1–2.7) were quantified in multiplex quantitative PCR assays in LightCycler Probes Master (Roche Applied Science, Meylan, France). This highly sensitive nested quantitative PCR assay made it possible to detect one TREC molecule in 105 cells for any excision circle. The sj/βTREC ratio was calculated as previously described (sj/βTREC = sjTREC/105 cells/(DJβ1TRECs/105cells + DJβ2TRECs/105cells) [9].

A similar method was used to quantify total HIV DNA. Gag and Env sequences were amplified together with the CD3γ chain in triplicate using the ‘outer’ 3′/5′ primer pairs (Supplemental Table 1, http://links.lww.com/QAD/A136), using the same conditions as described above. PCR products were diluted 10-fold and quantified by real-time PCR in JumpStart mix (Sigma) with 1 pmole of probes and 24 pmoles of the ‘inner’ 3′/5′ primer pairs indicated in supplemental Table 1, http://links.lww.com/QAD/A136. As standard curves, we used plasmids containing one copy of both the CD3γ and either Gag or Env amplicon. The results were expressed as absolute number of HIV copies per 105 cells.

Integrated HIV DNA was quantified by Alu-PCR as previously described [27,28].

Interleukin-7 plasma concentration

Plasma IL-7 concentrations were quantified by the ultrasensitive Quantikine HS IL-7 immunoassay Kit (R&D Systems, Lille, France), according to the manufacturer's instructions.

Statistical analyses

The correlations between variables were analyzed by Spearman's rank test (http://www.u707.jussieu.fr/biostatgv). Results were compared using the Mann–Whitney U test. P values of less than 0.05 were considered statistically significant.

Results

Both recent thymic emigrants and non-recent thymic emigrant naive T cells are HIV infected in vivo

To determine the contribution of RTE and non-RTE circulating naive T cells to the pool of HIV-infected cells, HIV DNA was quantified in five T-cell subsets (CD31high naive CD4+ T cells, CD31low naive CD4+ T cells, memory CD4+ T cells, CD31high naive CD8+ T cells and CD31low naive CD8+ T cells) purified from PBMCs sampled from untreated viremic (n = 11) and HAART-treated aviremic (n = 15) HIV-infected patients. RTEs were purified according to their high expression of CD31 (Fig. 1a) [18]. To confirm the enrichment of RTE in the CD31high sorted T cells, the sjTREC frequency was quantified by real-time PCR in each T-cell subset purified from 15 patients (Fig. 1b). In the CD4+ compartment, RTEs contained two-fold to 15-fold more sjTREC/105 cells than non-RTEs naive CD4+ T cells (median 1802 and 531 sjTREC/105 cells in RTE and non-RTE, respectively, P = 0.002) and seven-fold to 22-fold more than memory CD4 T cells (median 69 sjTREC/105 cells, P = 0.016). As expected, CD31 expression on naive CD8+ T cells did not correlate with sjTREC frequency [18].

We then quantified HIV DNA in the different T-cell subsets using a highly sensitive real-time PCR assay that allows detecting one HIV copy in 105 cells (Supplemental Fig. 1, http://links.lww.com/QAD/A136). Two viral genes (Gag and Env) were quantified in parallel in each sample in order to prevent the lack of detection of viral DNA due to point mutations in the primer sequences. Gag and/or Env sequences could be amplified for all the studied patients. Gag sequences were undetectable in patients #2 and #5, Env sequences were undetectable in patients #3, #10, #12, #16, #28 and #29. In all the patients with amplifiable Gag and Env sequences, both frequencies were similar (data not shown). Quantification of integrated proviral DNA in RTE CD4+ T cells isolated from 10 HAART-treated patients, performed using a semiquantitative method, [27,28] showed that total viral DNA mostly reflects integrated proviral DNA confirming that the vast majority of the quantified HIV DNA was integrated (data not shown).

For all T-cell subsets, similar magnitude of HIV-infection was observed in viremic and aviremic patients (Fig. 1c). As expected, HIV DNA concentrations were lower in any naive CD4+ T-cell subset than in the memory CD4+ compartment, in both groups (P ≤ 0.005; Fig. 1c). The amount of CD31high cells isolated from patients #2, #11 and #12 was insufficient to accurately quantify proviral DNA; these patients were, thus, excluded from further analysis. In both groups, most patients showed only slightly higher HIV DNA frequencies in non-RTE CD4+ T cells than in RTE CD4+ T cells (median fold difference = 1.4, P = 0.015; Fig. 1d). Interestingly, in the aviremic patients group, HIV DNA frequency in RTEs correlated to that in non-RTE CD4+ T cells (r = 0.847, P < 0.001). That was not the case for HIV DNA frequency in memory CD4+ T cells. In viremic patients, such a correlation was not observed.

Recent thymic emigrant and non-recent thymic emigrant naive CD4+ T cells are similarly HIV infected in vivo

We then assessed, by FACS, the percentage of each T-cell subset among PBMCs in all the patients and evaluated the relative contribution of the diverse T-cell subsets to the pool of HIV DNA-containing cells. In viremic and aviremic patients, both RTE and non-RTE CD4+ T cells represented about 5% of PBMCs, memory CD4+ T cells (central, effector and transitional memory) representing a larger subset (14%; Fig. 2a). In the two groups of patients, the distribution of CD4+ T cells in the different subsets was similar, except for memory T cells that represented 10% in viremic and 16% in aviremic treated patients (P < 0.001, Fig. 2a). Calculation of the relative contribution of each subset (Fig. 2b) showed that memory CD4+ T cells represent the major HIV-infected subset (85%). In contrast, infected RTE and non-RTE CD4+ T cells, respectively, represented 4.5% (1–56%) and 5.2% (1–30%) of the circulating infected cells (Fig. 2b). However, although the range of contribution of both RTE and non-RTE subsets was huge, RTE and non-RTE CD4+ T cells similarly contribute to the pool of HIV DNA-containing cells in most individual patient (Fig. 2b). Finally, naive CD8+ T cells barely contributed to HIV infection.

Fig. 2
Fig. 2:
Contribution of naive CD4+ RTE and non-RTE T cells to the HIV+ T-cell pool. (a) Distribution of T-cell subsets among peripheral blood mononuclear cells. Data from viremic patient are represented with gray circles, from aviremic patient with white diamonds and from healthy controls with black triangles. Statistical differences are shown (*: P≤ 0.05; **: P≤ 0.01). (b) Contribution of the different T-cell subsets to the pool of HIV-infected cells. Data from viremic and aviremic patients are represented with gray circles and white diamonds, respectively. Median values for each T-cell subsets are indicated by horizontal bars. Statistical differences as compared with memory CD4+ are shown (**: P ≤ 0.01). (c) Correlations between viral DNA content in RTE and non-RTE CD4+ T cells (top panel) and between viral DNA content in RTE and plasma HIV RNA (bottom panel) for low-RTE proviral load (<100 HIV copies/106 RTEs – gray diamonds) and high-RTE proviral load (>100 HIV copies/106 RTEs – black squares) patients. Spearman's correlation coefficient and the associated probability are shown when significant. PBMCs, peripheral blood mononuclear cells; RTE, recent thymic emigrant.

These results demonstrated that despite the fact that a vast majority of HIV DNA is contained in memory CD4+ T cells, both RTE and non-RTE CD4+ T cells also contribute to the HIV-infected T-cell pool in most patients. Altogether, infected naive CD4+ T cells accounted for 12% (0.1%–58%) of the HIV-infected T cells.

Considering that RTE infection rates did not globally differ between viremic and aviremic patients included in this study but spanned over three logs in both patient groups, we classified patients according to HIV proviral load in RTEs. Accordingly, patients #3, #4, #6, #8, #9, #13, #17, #19, #24 and #33 who demonstrated an RTE proviral load higher than 100 copies/106 cells were defined as the ‘high-RTE proviral load’ group, whereas patients #1, #5, #7, #10, #14, #15, #16, #20, #23, #28 and #29 (<100 copies/106 cells) were considered as ‘low-RTE proviral load’ patients (Table 2). Interestingly, a strong correlation between HIV proviral load in RTE and non-RTE subsets was observed in the high-RTE proviral load group (r = 0.984, P < 0.0002; black squares, Fig. 2c top panel) as well as in the low-RTE proviral load patients (r = 0.787, P < 0.005; gray diamonds, Fig. 2c top panel). Similarly, infection rate in the memory compartment correlated with both RTE and non-RTE proviral load in high-RTE proviral load group (r = 0.898 and r = 0.839, respectively, P≤ 0.002; data not shown). In contrast, RTE proviral DNA did not correlate with plasma viral loads (Fig. 2c bottom panel). These data demonstrate that RTEs can be infected in both untreated and HAART-treated patients, independently from plasma viral load, suggesting that the extent of ongoing viral replication is not a key factor for neo-infection in this subset.

Table 2
Table 2:
HIV DNA-load, interleukin-7 plasma levels and proliferation data in patients.

Recent thymic emigrant infection rate correlates with interleukin-7 plasma levels

We then compared, in high-RTE and low-RTE proviral load patients, various parameters that might be implicated in the infection of naive T-cell subsets. Considering that cell cycling may play a role in HIV infection and integration into the host genome [13,29–31], we evaluated circulating cell cycling, as defined by Ki-67 expression, in both RTE and non-RTE naive T-cell subsets as well as intrathymic precursor T-cell proliferation through calculation of the sj/βTREC ratio, which estimates the extent of thymocyte proliferation between TCRB rearrangement and the excision of the T-cell receptor delta (TCRB) locus [9], in the two groups of patients (Table 2). Interestingly, the frequency of cycling cells in the RTE compartment was significantly higher than in any other T-cell subset (median %Ki-67+ cells = 17.5% in RTE as compared with 1.5% in non-RTE and 7.4% in memory CD4+ T cells; P < 0.0001 with any other T-cell subset; Fig. 3a). However, RTE cycling rates were similar in low-RTE and high-RTE proviral load patients (Fig. 3b). Similarly, the sj/βTREC ratio was comparable in both groups of patients (Fig. 3c). In contrast, quantification of plasma IL-7 concentration showed that this essential cytokine for the regulation of both central and peripheral naive T-cell homeostasis was significantly higher in patients presenting high-RTE infection rate (median IL-7 plasma levels = 0.6 pg/ml and 2.1 pg/ml in low-RTE and high-RTE proviral load patients, respectively, P = 0.012; Fig. 3d). This data translated into a statistically significant correlation between IL-7 plasma levels and RTE infection rate (r = 0.607, P = 0.0035; Fig. 3 g), whereas neither peripheral nor intrathymic proliferation correlated with RTE infection rate (Fig. 3e and f). Similarly, but to a lesser extent, non-RTE infection rate also correlated with IL-7 plasma concentrations (r = 0.493, P = 0.032; data not shown). Such correlation did not exist for memory CD4+ T cells.

Fig. 3
Fig. 3:
Infection of naive CD4+ T cells correlates with plasma interleukin-7 levels. Ki-67 expression in various T-cell subsets purified from peripheral blood of HIV-infected patients (a). Statistical differences as compared with recent thymic emigrant (RTE) CD4+ are shown (**: P ≤ 0.01). Ki-67 expression in CD4+ RTE and non-RTE T cells (b), the signal joint T-cell receptor excision circle/DβJβ T-cell receptor excision circle (sj/βTREC) ratio; (c) and plasma interleukin (IL)-7 concentration (d) were measured for patients with low-RTE proviral load (<100 HIV copies/106 RTEs, gray diamonds) and high-RTE proviral load (>100 HIV copies/106 RTEs, black squares). Statistical difference is shown (*: P ≤ 0.05). Relationship between HIV infection frequency in RTEs and Ki-67 expression (e), sj/βTREC ratio (f) and plasma IL-7 concentration (g) as measured for patients with low-RTE proviral load (gray diamonds) and high-RTE proviral load (black squares). IL, interleukin; RTE, recent thymic emigrant; sj/β TREC, single joint β T-cell receptor excision circle.

These results suggest that infection of RTE CD4+ T cells was neither a consequence of their intrathymic proliferative history nor linked to their peripheral proliferation. However, the relationship between IL-7 plasma levels and RTE infection suggests a role for this cytokine in the RTE infection process.

Discussion

Through quantification of the in-vivo HIV-1 infection rate in various T-cell subsets, we here evidenced that, while memory CD4+ T cells effectively represent the most infected T-cell subset in circulating blood [4,32,33], naive CD4+ T cells also significantly contribute to the HIV-infected T-cell population. Among these, RTEs as defined as CD31high naive CD4+ T cells, represent about half. Indeed, although CD4+ naive T-cell subsets contain more than 20% of the HIV-infected cells in one-third of the studied patients and up to 50% in a few, in most patients with high-HIV infection in the naive T-cell subset, RTEs represent more than 40% of the HIV+ cells in this subset. Similarly, in the low-RTE proviral group, six out of 12 patients showed more than 40% of the HIV+ naive CD4+ T cells in the RTE compartment. Such data confirmed previous observations showing that non-RTEs are slightly more frequently infected than RTEs [2,34]. HIV infection of these naive T cells could have occurred either in periphery, during homeostatic regulation of T-cell subsets, or in the thymus, both phenomena depending upon IL-7 stimulation [16,35–38].

In periphery, as part of the homeostatic regulation of circulating T-cell numbers occurring in secondary lymphoid organs, naive T cells are subjected to IL-7-induced stimulation. Cell cycling was for long time considered as necessary for HIV infection and integration in the host genome. Thus, the fact that RTEs are more responsive to IL-7 than non-RTEs [16], leading to higher Ki-67 expression in this subset (Fig. 3a), suggests that RTEs might be more sensitive to HIV infection. Thus, the slight difference we observed between RTE and non-RTE infection rates fits with HIV infection occurring mostly in the RTE subset due to their higher IL-7-induced proliferation capacity. However, several publications showed that proliferation per se is indeed not required for HIV-1 infection [12,39,40], initiation of cell activation being sufficient to put the cells in an infectable state. As RTEs and non-RTEs are equally sensitive to IL-7 in terms of induction of STAT-5 phosphorylation and BCl-2 expression [16], if infection was occurring in periphery, then non-RTE should be much more infected than RTEs as a consequence of their longer lifespan. Thus, our data argues for the majority of infection occurring earlier in T-cell development, probably within the thymus.

Various thymocyte subsets (Immature single positive, double positive and SP-CD4) are susceptible to HIV infection [6,41–46]. It could, thus, be argued that infected RTEs acquired HIV during their thymic maturation. However, we and others [2,47] barely observed infected naive CD8+ T cells in vivo (both CD31high and CD31low subsets; Fig. 1). Together with the lack of correlation between RTE infection and intrathymic precursor T-cell proliferation (sj/βTREC ratio), this strongly suggests that infection at the double positive stage or earlier does not significantly contribute to the extent of circulating HIV+ T-cell pool. These data argue for infection occurring in the SP-CD4 subset that expresses the IL-7 receptor and demonstrate a higher proliferation and survival capacity in the presence of IL-7 and/or HIV [48–51]. Moreover, these cells express HIV coreceptors [52,53] and were shown to express high levels of P24 antigen following HIV infection [52], in particular in the presence of IL-7 [51,54]. Altogether, our data suggest that, RTEs acquired HIV infection, in an IL-7-dependent manner, during the late stages of thymopoiesis or early-on during their extrathymic life.

Whether therapeutic use of recombinant IL-7 could lead to similar effect remains to be evaluated. Indeed, IL-7 administration leads to a burst of intrathymic and peripheral T-cell stimulation [26,55,56] that allows reconstituting circulating naive T-cell compartments and, subsequently, to reduced endogenous IL-7 production. Thus, on the long term, IL-7 therapy could lead to less RTE neo-infection.

Surprisingly, in none of the T-cell subsets was the infection rate reflecting plasma viral load. Indeed, patients harboring high-RTE or low-RTE proviral load were equally distributed in untreated viremic patients and aviremic patients under HAART. Moreover, even within the viremic patients group, plasma viral loads did not correlate with HIV DNA load, in any T-cell subset. Although DNA viral load was shown to correlate with plasma viral load during primary HIV infection [57], HAART-induced decrease in HIV DNA load in patients initiating antiviral therapy is usually of small amplitude (less than a log) as compared with changes in plasma viral loads [57–59]. Considering the large range of HIV DNA load in the patients we studied, such a small difference in a cross-sectional study could not reach statistical significance. Moreover, untreated patients' characteristics could also explain their similarity with HAART-treated patients in terms of DNA viral load. Indeed, viremic patients studied here (except for patient #6) were untreated and progressing to disease slowly enough to delay treatment initiation. In viremic patients, the infection rate of both RTEs and non-RTEs correlates with that of memory CD4+ T cells, suggesting persistent viremia as a common source of infection. In contrast, in aviremic individuals, although the infection rates in RTE and non-RTE subsets strongly correlates (r = 0.847, P < 0.001), that is not the case for memory T cells. Thus, a common infection mechanism probably exists for RTEs and non-RTEs in aviremic patients, different from that of memory T cells.

In conclusion, we herein demonstrated that HIV-infected CD4+ RTEs represent a substantial part of the HIV+ T-cell pool in both untreated viremic and HAART-treated patients. The analysis of various parameters implicated in the homeostatic regulation of this subset strongly suggested that infection of RTEs occurred at the end of or immediately after their maturation in the thymus, through a mechanism dependent upon IL-7 plasma levels. The physiopathological consequences of RTE infection remain to be established as well as the impact of IL-7 therapy, which is presently under clinical evaluation for the reconstruction of the CD4+ T-cell subsets in HAART-treated HIV-infected patients.

Acknowledgements

The authors thank M. T. Rannou, nurses, and patients for their cooperation.V.F-M. was the recipient of an Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) postdoctoral fellowship. This work was financed by the ANRS and the Institut Pasteur.

References

1. Bailey JR, Sedaghat AR, Kieffer T, Brennan T, Lee PK, Wind-Rotolo M, et al. Residual human immunodeficiency virus type 1 viremia in some patients on antiretroviral therapy is dominated by a small number of invariant clones rarely found in circulating CD4+ T cells. J Virol 2006; 80:6441–6457.
2. Brenchley JM, Hill BJ, Ambrozak DR, Price DA, Guenaga FJ, Casazza JP, et al. T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis. J Virol 2004; 78:1160–1168.
3. Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA, Yassine-Diab B, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med 2009; 15:893–900.
4. Ostrowski MA, Chun TW, Justement SJ, Motola I, Spinelli MA, Adelsberger J, et al. Both memory and CD45RA+/CD62L+ naive CD4(+) T cells are infected in human immunodeficiency virus type 1-infected individuals. J Virol 1999; 73:6430–6435.
5. Macallan DC, Wallace D, Zhang Y, De Lara C, Worth AT, Ghattas H, et al. Rapid turnover of effector-memory CD4(+) T cells in healthy humans. J Exp Med 2004; 200:255–260.
6. Bonyhadi ML, Su L, Auten J, McCune JM, Kaneshima H. Development of a human thymic organ culture model for the study of HIV pathogenesis. AIDS Res Hum Retroviruses 1995; 11:1073–1080.
7. Gobbi A, Stoddart CA, Locatelli G, Santoro F, Bare C, Linquist-Stepps V, et al. Coinfection of SCID-hu Thy/Liv mice with human herpesvirus 6 and human immunodeficiency virus type 1. J Virol 2000; 74:8726–8731.
8. Salemi M, Burkhardt BR, Gray RR, Ghaffari G, Sleasman JW, Goodenow MM. Phylodynamics of HIV-1 in lymphoid and nonlymphoid tissues reveals a central role for the thymus in emergence of CXCR4-using quasispecies. PLoS ONE 2007; 2:e950.
9. Dion ML, Poulin JF, Bordi R, Sylvestre M, Corsini R, Kettaf N, et al. HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation. Immunity 2004; 21:757–768.
10. Eckstein DA, Penn ML, Korin YD, Scripture-Adams DD, Zack JA, Kreisberg JF, et al. HIV-1 actively replicates in naive CD4(+) T cells residing within human lymphoid tissues. Immunity 2001; 15:671–682.
11. Steffens CM, Managlia EZ, Landay A, Al-Harthi L. Interleukin-7-treated naive T cells can be productively infected by T-cell-adapted and primary isolates of human immunodeficiency virus 1. Blood 2002; 99:3310–3318.
12. Yamashita M, Emerman M. The cell cycle independence of HIV infections is not determined by known karyophilic viral elements. PLoS Pathog 2005; 1:e18.
13. Korin YD, Zack JA. Progression to the G1b phase of the cell cycle is required for completion of human immunodeficiency virus type 1 reverse transcription in T cells. J Virol 1998; 72:3161–3168.
14. Jaleco S, Kinet S, Hassan J, Dardalhon V, Swainson L, Reen D, Taylor N. IL-7 and CD4+ T-cell proliferation. Blood 2002; 100:4676–4677, author reply 4677–4678.
15. Dardalhon V, Jaleco S, Kinet S, Herpers B, Steinberg M, Ferrand C, et al. IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+ T cells. Proc Natl Acad Sci U S A 2001; 98:9277–9282.
16. Azevedo RI, Soares MV, Barata JT, Tendeiro R, Serra-Caetano A, Victorino RM, Sousa AE. IL-7 sustains CD31 expression in human naive CD4+ T cells and preferentially expands the CD31+ subset in a PI3K-dependent manner. Blood 2009; 113:2999–3007.
17. Haines CJ, Giffon TD, Lu LS, Lu X, Tessier-Lavigne M, Ross DT, Lewis DB. Human CD4+ T cell recent thymic emigrants are identified by protein tyrosine kinase 7 and have reduced immune function. J Exp Med 2009; 206:275–285.
18. Kimmig S, Przybylski GK, Schmidt CA, Laurisch K, Mowes B, Radbruch A, Thiel A. Two subsets of naive T helper cells with distinct T cell receptor excision circle content in human adult peripheral blood. J Exp Med 2002; 195:789–794.
19. Kohler S, Wagner U, Pierer M, Kimmig S, Oppmann B, Mowes B, et al. Postthymic in vivo proliferation of naive CD4+ T cells constrains the TCR repertoire in healthy human adults. Eur J Immunol 2005; 35:1987–1994.
20. McFarland RD, Douek DC, Koup RA, Picker LJ. Identification of a human recent thymic emigrant phenotype. Proc Natl Acad Sci U S A 2000; 97:4215–4220.
21. Almeida AR, Borghans JA, Freitas AA. T cell homeostasis: thymus regeneration and peripheral T cell restoration in mice with a reduced fraction of competent precursors. J Exp Med 2001; 194:591–599.
22. Arstila TP, Casrouge A, Baron V, Even J, Kanellopoulos J, Kourilsky P. A direct estimate of the human alphabeta T cell receptor diversity. Science 1999; 286:958–961.
23. Dulude G, Cheynier R, Gauchat D, Abdallah A, Kettaf N, Sekaly RP, Gratton S. The magnitude of thymic output is genetically determined through controlled intrathymic precursor T cell proliferation. J Immunol 2008; 181:7818–7824.
24. Dion ML, Sekaly RP, Cheynier R. Estimating thymic function through quantification of T-cell receptor excision circles. Methods Mol Biol 2007; 380:197–213.
25. Parker R, Dutrieux J, Beq S, Lemercier B, Rozlan S, Fabre-Mersseman V, et al.Interleukin-7 treatment counteracts IFN{alpha} therapy-induced lymphopenia and stimulates SIV-specific CTL responses in SIV-infected rhesus macaques.Blood 2010; 116:5589–5599.
26. Beq S, Nugeyre MT, Fang RH, Gautier D, Legrand R, Schmitt N, et al. IL-7 induces immunological improvement in SIV-infected rhesus macaques under antiviral therapy. J Immunol 2006; 176:914–922.
27. Brussel A, Sonigo P. Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus. J Virol 2003; 77:10119–10124.
28. Sagot-Lerolle N, Lamine A, Chaix ML, Boufassa F, Aboulker JP, Costagliola D, et al. Prolonged valproic acid treatment does not reduce the size of latent HIV reservoir. AIDS 2008; 22:1125–1129.
29. Pierson TC, Zhou Y, Kieffer TL, Ruff CT, Buck C, Siliciano RF. Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection. J Virol 2002; 76:8518–8531.
30. Swiggard WJ, Baytop C, Yu JJ, Dai J, Li C, Schretzenmair R, et al. Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli. J Virol 2005; 79:14179–14188.
31. Vatakis DN, Kim S, Kim N, Chow SA, Zack JA. Human immunodeficiency virus integration efficiency and site selection in quiescent CD4+ T cells. J Virol 2009; 83:6222–6233.
32. Schnittman SM, Denning SM, Greenhouse JJ, Justement JS, Baseler M, Kurtzberg J, et al. Evidence for susceptibility of intrathymic T-cell precursors and their progeny carrying T-cell antigen receptor phenotypes TCR alpha beta + and TCR gamma delta + to human immunodeficiency virus infection: a mechanism for CD4+ (T4) lymphocyte depletion. Proc Natl Acad Sci U S A 1990; 87:7727–7731.
33. Sleasman JW, Aleixo LF, Morton A, Skoda-Smith S, Goodenow MM. CD4+ memory T cells are the predominant population of HIV-1-infected lymphocytes in neonates and children. AIDS 1996; 10:1477–1484.
34. Wightman F, Solomon A, Khoury G, Green JA, Gray L, Gorry PR, et al. Both CD31(+) and CD31 naive CD4(+) T cells are persistent HIV type 1-infected reservoirs in individuals receiving antiretroviral therapy. J Infect Dis 2010; 202:1738–1748.
35. Fry TJ, Connick E, Falloon J, Lederman MM, Liewehr DJ, Spritzler J, et al. A potential role for interleukin-7 in T-cell homeostasis. Blood 2001; 97:2983–2990.
36. Chu YW, Memon SA, Sharrow SO, Hakim FT, Eckhaus M, Lucas PJ, Gress RE. Exogenous IL-7 increases recent thymic emigrants in peripheral lymphoid tissue without enhanced thymic function. Blood 2004; 104:1110–1119.
37. Swainson L, Kinet S, Mongellaz C, Sourisseau M, Henriques T, Taylor N. IL-7-induced proliferation of recent thymic emigrants requires activation of the PI3K pathway. Blood 2007; 109:1034–1042.
38. Guimond M, Veenstra RG, Grindler DJ, Zhang H, Cui Y, Murphy RD, et al. Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4+ T cells. Nat Immunol 2009; 10:149–157.
39. Lewis P, Hensel M, Emerman M. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J 1992; 11:3053–3058.
40. Lewis PF, Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 1994; 68:510–516.
41. Chene L, Nugeyre MT, Barre-Sinoussi F, Israel N. High-level replication of human immunodeficiency virus in thymocytes requires NF-kappaB activation through interaction with thymic epithelial cells. J Virol 1999; 73:2064–2073.
42. Chene L, Nugeyre MT, Guillemard E, Moulian N, Barre-Sinoussi F, Israel N. Thymocyte-thymic epithelial cell interaction leads to high-level replication of human immunodeficiency virus exclusively in mature CD4(+) CD8(-) CD3(+) thymocytes: a critical role for tumor necrosis factor and interleukin-7. J Virol 1999; 73:7533–7542.
43. Stanley SK, McCune JM, Kaneshima H, Justement JS, Sullivan M, Boone E, et al. Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse. J Exp Med 1993; 178:1151–1163.
44. Su L, Kaneshima H, Bonyhadi M, Salimi S, Kraft D, Rabin L, McCune JM. HIV-1-induced thymocyte depletion is associated with indirect cytopathogenicity and infection of progenitor cells in vivo. Immunity 1995; 2:25–36.
45. Bonyhadi ML, Rabin L, Salimi S, Brown DA, Kosek J, McCune JM, Kaneshima H. HIV induces thymus depletion in vivo. Nature 1993; 363:728–732.
46. De Rossi A, Calabro ML, Panozzo M, Bernardi D, Caruso B, Tridente G, Chieco-Bianchi L. In vitro studies of HIV-1 infection in thymic lymphocytes: a putative role of the thymus in AIDS pathogenesis. AIDS Res Hum Retroviruses 1990; 6:287–298.
47. McBreen S, Imlach S, Shirafuji T, Scott GR, Leen C, Bell JE, Simmonds P. Infection of the CD45RA+ (naive) subset of peripheral CD8+ lymphocytes by human immunodeficiency virus type 1 in vivo. J Virol 2001; 75:4091–4102.
48. Guillemard E, Nugeyre MT, Chene L, Schmitt N, Jacquemot C, Barre-Sinoussi F, Israel N. Interleukin-7 and infection itself by human immunodeficiency virus 1 favor virus persistence in mature CD4(+)CD8(-)CD3(+) thymocytes through sustained induction of Bcl-2. Blood 2001; 98:2166–2174.
49. Fry TJ, Mackall CL. Interleukin-7: from bench to clinic. Blood 2002; 99:3892–3904.
50. Munitic I, Williams JA, Yang Y, Dong B, Lucas PJ, El Kassar N, et al. Dynamic regulation of IL-7 receptor expression is required for normal thymopoiesis. Blood 2004; 104:4165–4172.
51. Napolitano LA, Stoddart CA, Hanley MB, Wieder E, McCune JM. Effects of IL-7 on early human thymocyte progenitor cells in vitro and in SCID-hu Thy/Liv mice. J Immunol 2003; 171:645–654.
52. Zaitseva MB, Lee S, Rabin RL, Tiffany HL, Farber JM, Peden KW, et al. CXCR4 and CCR5 on human thymocytes: biological function and role in HIV-1 infection. J Immunol 1998; 161:3103–3113.
53. Taylor JR Jr, Kimbrell KC, Scoggins R, Delaney M, Wu L, Camerini D. Expression and function of chemokine receptors on human thymocytes: implications for infection by human immunodeficiency virus type 1. J Virol 2001; 75:8752–8760.
54. Pedroza-Martins L, Boscardin WJ, Anisman-Posner DJ, Schols D, Bryson YJ, Uittenbogaart CH. Impact of cytokines on replication in the thymus of primary human immunodeficiency virus type 1 isolates from infants. J Virol 2002; 76:6929–6943.
55. Beq S, Rozlan S, Gautier D, Parker R, Mersseman V, Schilte C, et al.Injection of glycosylated recombinant simian IL-7 provokes rapid and massive T-cell homing in rhesus macaques.Blood 2009; 114:816–825.
56. Levy Y, Lacabaratz C, Weiss L, Viard JP, Goujard C, Lelievre JD, et al. Enhanced T cell recovery in HIV-1-infected adults through IL-7 treatment. J Clin Invest 2009; 119:997–1007.
57. Ngo-Giang-Huong N, Deveau C, Da Silva I, Pellegrin I, Venet A, Harzic M, et al. Proviral HIV-1 DNA in subjects followed since primary HIV-1 infection who suppress plasma viral load after one year of highly active antiretroviral therapy. AIDS 2001; 15:665–673.
58. Ibanez A, Puig T, Elias J, Clotet B, Ruiz L, Martinez MA. Quantification of integrated and total HIV-1 DNA after long-term highly active antiretroviral therapy in HIV-1-infected patients. AIDS 1999; 13:1045–1049.
59. Viard JP, Burgard M, Hubert JB, Aaron L, Rabian C, Pertuiset N, et al. Impact of 5 years of maximally successful highly active antiretroviral therapy on CD4 cell count and HIV-1 DNA level. AIDS 2004; 18:45–49.
Keywords:

HIV; HIV pathogenesis; HIV reservoir; interleukin 7; naive CD4+ T cells; recent thymic emigrant; T-cell activation; T-cell homeostasis; T-cell proliferation; thymic function; thymus

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