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(Journal of Leukocyte Biology. 2002;72:913-920.)
© 2002 by Society for Leukocyte Biology

Restricted replication of primary HIV-1 isolates using both CCR5 and CXCR4 in Th2 but not in Th1 CD4+ T cells

Elisa Vicenzi*, Paola Panina-Bodignon{dagger}, Giuliana Vallanti*, Pietro Di Lucia{dagger} and Guido Poli*

* AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milano, Italy; and
{dagger} BioXell S.p.A., Milano, Italy

Correspondence: Dr. Guido Poli, P2-P3 Laboratories, DIBIT, Via Olgettina n. 58, 20132, Milano, Italy. E-mail: poli.guido{at}hsr.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously reported that CCR5-dependent human immunodeficiency virus type-1 (HIV-1; R5), but not CXCR4-restricted (X4) virus, efficiently replicates in T helper cell type 1 (Th1), Th2, or Th0 polyclonal T cells obtained from human umbilical cord blood (CB lines). The X4 virus restriction was env-dependent but did not occur at the level of viral entry. Here, we describe that in contrast to these monotropic HIVs, primary HIV-1 isolates capable of using CCR5 or CXCR4 indifferently for entry (i.e., R5X4 viruses) efficiently replicated in Th2 but not in Th1 CB lines. Although Th1 cells secreted significantly higher amounts of the three CCR5-binding chemokines in comparison with Th2 cells, this restriction was not explained by a defective infection of Th1 cells. Interferon-{gamma} (IFN-{gamma}) down-regulated CCR5 in Th1 cells and inhibited, whereas interleukin-4 (IL-4) up-regulated CXCR4 and enhanced the spreading of R5 and R5X4 viruses in polarized CB lines. However, both cytokines did not rescue the replication of X4 and dualtropic viruses in both types of CB lines or in Th1 cells, respectively, whereas addition of anti-IL-4- or anti-IFN-{gamma}-neutralizing antibodies did not activate virus expression. These findings together suggest the existence of post-entry restriction pathways influenced by gp120 Env/chemokine coreceptor interaction that may significantly contribute to the superior capacity of R5 and R5X4 HIV-1 strains to spread in vivo in comparison to X4 monotropic viruses.

Key Words: IFN-{gamma} • IL-4 • CCL3 • CCL4 • CCL5


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human immunodeficiency virus type-1 (HIV-1) infects human T lymphocytes and macrophages via interaction with the CD4 primary receptor and a chemokine receptor, usually CCR5 or CXC chemokine receptor (CXCR)4. Other chemokine receptors, including CCR2b and CCR3, are occasionally used, whereas the chemokines binding to these entry coreceptors exert competitive, inhibitory effects on viral infection and replication [1 ]. The definition of the modality of HIV-1 infection via interaction with CD4 and a chemokine receptor has allowed a better understanding of the relative importance of these host determinants in the spreading epidemic. In this regard, it was previously observed that macrophage-tropic viruses were often characterized by their lack of ability of infecting T cell lines in vitro and were therefore defined as nonsyncytia-inducing (NSI) strains in contrast to T cell tropic viruses [syncytia-inducing (SI) viruses]. T cell lines, including the MT-2 cell line used as the reference model for discrimination of SI versus NSI viruses, express CD4 and CXCR4, but not CCR5, thus allowing the replication of only a fraction of HIV isolates [2 ]. Thus, it became evident that the propagation of HIV-1 is mostly sustained by CCR5-dependent (R5) NSI viruses, whereas the emergence of CXCR4-using strains only occurs in approximately 50% of individuals infected with clade B viruses (dominant in Western Europe and the United States) in their advanced stage of infection. Worthy of note, the absence of CCR5 expression, as a result of a homozygous configuration of the allelic variant CCR5{Delta}32, confers a virtually universal protection from infection [3 , 4 ], whereas the emergence of CXCR4-using strains in countries prevalently infected by nonclade B HIVs is rare [5 , 6 ]. These observations indicate that CCR5 use is evolutionarily advantageous for HIV at the individual and at the population level, whereas CXCR4 use is only adopted in some individuals in an advanced state of immunodeficiency.

Several correlates supporting the superior capacity of R5 HIVs of multiplicating in the host have been reported. First, CCR5 was found to be more available at the genital mucosa level than CXCR4 [7 ], an observation later explained by the abundant, constitutive secretion of the CXCR4 ligand CXCL12 (previously known as stromal-derived factor-1{alpha}) [8 ]. Secondly, although R5 and X4 viruses can infect human macrophages, only the former usually gives raise to a productive infection [9 , 10 ]. Finally, the extracellular release of the HIV protein Tat can exert CXCL12-like inhibitory activity on HIV-1 infection without affecting or even enhancing the replication of R5 viruses [11 , 12 ]. In this scenario, we have described that R5 viruses, primary isolates or tissue culture-laboratory adapted (TCLA)-derived, could efficiently propagate in T helper cell type 1 (Th1), Th2, or Th0 T cell clones and cord blood-derived CD4+ T cell lines (CB lines). In contrast, CXCR4-monotropic (X4) viruses did not replicate or propagated very poorly in these cells. This pattern was determined by the interaction between gp120 Env and CCR5 or CXCR4 but, surprisingly enough, was not caused by a defective ability of X4 HIVs to infect these cells but rather by an inefficient spreading thereafter [13 ].

In the present study, we have extended our analysis to a panel of primary HIV-1 isolates that was previously characterized in terms of its simultaneous capacity of interacting with CCR5 and CXCR4 [14 ]. These viruses replicated efficiently in Th2 (or Th0) CB lines but did not propagate in Th1 cells, implying that the simultaneous use of both chemokine receptors determines an intermediate level of restriction of the viral-replicative capacity linked to the state of polarization of the host T cells. As most infected individuals are characterized by a progressive skewing of their cytokine profile toward a Th0/Th2 pattern, particularly in the advanced phase of infection [15 ], this observation provides additional evidence that this immunological perturbation may foster the maintenance of an R5 phenotype or the emergence of dualtropic R5X4 variants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Primary HIV-1 isolates
Primary HIV isolates from individuals with progressing HIV-1 disease were generated by cocultivation of 4 x 106 peripheral blood mononuclear cells (PBMC) with a mixture of 3 x 106 allogeneic phytohemagglutinin (PHA-P, Sigma Chemical Co., St. Louis, MO)-stimulated PBMC (PHA blasts) from two uninfected individuals in 10 ml complete RPMI-1640 medium (BioWhittaker, Verviers, Belgium) containing 10% fetal calf serum (FCS; Sigma Chemical Co.) and 10 U/ml recombinant interleukin-2 (rIL-2; Boehringer Mannheim, Mannheim, Germany). At the peak of virus replication, monitored in the culture supernatants by a standard Mg++-dependent reverse transcriptase (RT) assay [13 ], supernatants were pooled, aliquoted, and stored at -80°C. The viral infectious titers were determined by the Reed-Muench formula [16 ] after infection of PHA blasts by serial dilutions of the viral stocks in sextuplicates over 4 weeks of infection. The viral phenotype (i.e., SI or NSI) was determined by infection of MT-2 cells [2 ]. In addition, 105 U87 astrocytic cells stably expressing CD4 or CD4 plus CCR2b, CCR3, CCR5, or CXCR4 [17 ] were infected with 100 µl undiluted primary isolates in 1 ml Dulbecco’s modified Eagle’s medium plus 15% FCS. As described in detail elsewhere [14 ], these viruses remained dualtropic after repeated passages on U87 cells expressing individual coreceptors (CCR5 vs. CXCR4) together with CD4 and were sensitive to the inhibitory effect of CCL5/regulated on activation, normal T cell expressed and secreted (RANTES) or the CXCR4 antagonist AMD3100.

Generation and infection of polarized and unpolarized CD4+ CB lines
Human neonatal leukocytes were isolated from umbilical CB by Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) density gradient. Approximately 50% of these cells were CD4+ cells with a naive (CD45 RA+) phenotype, as reported [18 ], and were further enriched in this cell subset by negative selection with a single round of anti-CD8 monoclonal antibody (mAb)-conjugated immunomagnetic beads (Mini-MACS, Miltenyi Biotec GmBH, Bergisch-Gladbach, Germany). Th1 and Th2 differentiation was obtained by a stimulation of these cells by PHA (Wellcome, Beckenham, UK) and a combination of anti-IL-4 Ab (PharMingen, San Diego, CA) plus IL-12 (Hoffman-LaRoche Inc., Nutley, NJ) or anti-IL-12 Ab (gift of M. Gately, Hoffman-La Roche) plus IL-4 (PharMingen) for 72 h, respectively, as described [18 ]. Cell lines were washed and maintained at approximately 1 x 106 cells/ml in RPMI 1640, 5% FCS, antibiotics, and 100 U/ml rIL-2 (Hoffman-La Roche) for 15–21 days before HIV infection. The cells were cultured in IL-2-enriched medium before and after infection. Analysis of the expression of CCR5 and CXCR4 in terms of mean fluorescence intensity (MFI) was performed at day 12 before infection. Secretion of CCL3/macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), CCL4/MIP-1ß, and CCL5/RANTES was determined at the day before and throughout the infections by commercial enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN).

Single-cell analysis of interferon-{gamma} (IFN-{gamma}) and IL-4 production was performed as previously described [19 ]. Briefly, T cell lines were collected 7–10 days after priming, washed, and restimulated with phorbol myristate acetate (PMA; 50 ng/ml; Sigma Chemical Co.) and ionomycin (1 µg/ml; Sigma Chemical Co.) for 2 h at 37°C in complete medium. Brefeldin A (10 µg/ml; Sigma Chemical Co.) was added to the cultures, which were incubated for an additional 2 h. The cells were fixed with 2% paraformaldehyde and permeabilized with saponin. Fixed cells were stained with anti-human (h)IFN-{gamma}-fluorescein isothiocyanate (PharMingen), anti-hIL-4-phycoeryethrin (PharMingen), and anti-hCD4-Quantum Red (Sigma Chemical Co.) or anti-hCD8-Quantum Red (Sigma Chemical Co.) mAb following a protocol provided by the manufacturer. Samples were analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ).

Infection of CB lines with TCLA and primary viruses was performed 15–20 days after their initial stimulation, as reported [13 ]. Cells (1x106) were incubated with approximately 20,000 cpm RT activity equivalents, corresponding in the case of TCLA viruses to multiplicities of infection (MOI) between 0.8 and 1, in 96-well plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ). The kinetics of HIV-1 replication were measured by RT activity on the supernatants collected every 3–4 days and stored at -80°C.

Real-time polymerase chain reaction (PCR) quantification of HIV-1 DNA
The kinetics and levels of HIV-DNA accumulation in Th1 and Th2 cells were determined by the TaqMan assay with an ABI 7700 Prism instrument (Applied Biosystems, Foster City, CA) as recently described [13 ]. Briefly, HIV-1LAI/IIIB, HIV-1BaL, and primary HIV stocks were treated with 70 U/ml DNAse (Roche Diagnostic Corp., Indianapolis, IN) for 30 min at room temperature. Viral stocks containing equal amount of RT activity equivalents (20,000 cpm) were incubated with 1 x 106 Th1 or Th2 cells for 30 min at 37°C. Cells were then washed extensively and seeded in 24-well flat-bottomed tissue-culture plastic plates (Falcon, Becton Dickinson Labware). Cell aliquots corresponding to 1 x 106 cells were harvested at different time points and washed twice with phosphate buffer, and cell pellets were obtained by centrifugation at 12,000 rpm for 5 min. Pellets were then lysed in a proteinase K-containing buffer and DNA was extracted by phenol-chloroform and was ethanol-precipitated. DNA preparations (50 ng) were amplified with primers derived from the p24 gag region. Their sequence and position in the HIV-1HXB2 clone [20 ], indicated in parentheses, are: forward, 5'-ACATCAAGCAGCCATGCAAAT-3' (1368–1388); reverse, 5'-ATCTGGCCTGGTGCAATAGG-3' (1472–1453); and probe (FAM), 5'-CATCAATGAGGAAGCTGCAGAATGGGATAGA-3' (TAMRA; 1401–1431). An external standard curve was obtained by serial dilutions of pNL 4-3 DNA with a linear distribution (r=0.99) between 10 and 106 copies. Intersample variation was monitored by parallel Sybr green PCR (Applied Biosystems) with an ABI 7700 Prism instrument measuring total cellular DNA with primers specific for human glyceraldehydes 3-phosphate dehydrogenase, forward primer (5'-ACCACAGTCCATGCCATCACT-3') and reverse primer [5'-GGCCATCACGCCACAG(Inosine)TT-3']. The thermal cycling conditions were 50°C for 2 min, 95°C for 12 min, and 40 cycles of 95°C for 15 s and 65°C for 1 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
R5X4 primary HIV-1 isolates replicate in Th2 but not in Th1 CB lines
CB lines were generated as described in Materials and Methods and were tested for their polarization toward Th1 or Th2 according to their pattern of cytokine expression. Th1 CB lines were highly positive for IFN-{gamma} expression but negative or only weakly positive for IL-4 synthesis (Fig. 1A , b ), whereas Th2 showed the opposite pattern (Fig. 1A , d ). We have previously reported that Th1 and Th2 CB lines efficiently supported R5 but not X4 virus replication [13 ], as shown here in Figure 2A . As previously observed [21 22 23 ], Th1 cells spontaneously secreted substantially higher levels of the three CCR5 binding than Th2 CB lines (Fig. 1B) .



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  		Figure 1. Polarization of CB lines along Th1 or Th2 pathways. (A) CD4+ cell-enriched CB mononuclear cells were stimulated in the presence of polarizing cytokines, as described in Materials and Methods. Seven to 10 days after the initial stimulation, CB lines were then restimulated with PMA and ionomycin in the presence of Brefeldin A and were then stained for expression of IFN-{gamma} or IL-4 by specific mAb. Most cells originally stimulated in the presence of IL-12 and anti-IL-4 mAb have acquired and maintained a Th1 profile (Fig. 1A , b ), whereas those conditioned by IL-4 and anti-IL-12 mAb are essentially Th2 cells ((Fig. 1 d) . No significant expression of IFN-{gamma} or IL-4 was observed in the absence of restimulation (Fig. 1a and 1c ). (B) Th1 CB lines maintained in IL-2-enriched medium and tested before infection secrete substantially higher levels of CCR5-binding chemokines than parallel, autologous Th2 CB lines (n=4).

 


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  		Figure 2. Differential replicative ability of HIV-1 strains in Th1 and Th2 CB lines. (A) TCLA R5 HIV-1BaL efficiently replicate, whereas X4 HIV-1IIIB/LAI does not propagate in Th1 or Th2 CB lines. (B) R5X4 dualtropic virus spreading in Th2 but not Th1 CB lines. These results were obtained from an individual experiment out of 10 independently performed with CB lines established from 10 donors infected with the different dualtropic viruses.

 
A panel of well-characterized, primary dualtropic viruses [14 ] was tested in the same CB lines in identical culture conditions. Although with variable kinetics, these viruses efficiently replicated in Th2 CB lines of several independent donors, they were very inefficient or did not replicate at all in the parallel Th1 CB lines (Fig. 2B) . This pattern was not a reflection of an increased cytopathicity of these viruses in Th1 cells, as determined by trypan blue dye exclusion criteria and [3H]-thymidine incorporation (data not shown). When dualtropic viruses were tested in unpolarized Th0 CB lines, efficient replication was also observed (not shown).

Kinetics of HIV-DNA accumulation in Th1 and Th2 CB lines
As for X4 viruses, we have analyzed the capacity of dualtropic HIVs of infecting Th1 and Th2 CB lines by quantifying the levels of HIV-DNA accumulation early after infection. In this regard, it has been estimated that a single round of HIV replication occurs in 20–24 h [24 ]. Therefore, the quantification of the levels of HIV-DNA synthesis within the first 1–3 days can be assumed as a faithful indication of the efficiency of viral entry in CD4+ cells. Consistently with the permissive pattern of replication observed in Th1 and Th2 CB lines, similar kinetics of HIV-DNA accumulation of the R5 HIV-1BaL was observed in these cells (Fig. 3 , upper), implying that the constitutive low levels of secretion of CCR5-binding chemokines by Th1 cells did not play a substantial role in limiting the infection and spreading of a CCR5-dependent virus. When dualtropic viruses were tested, a progressive accumulation of HIV DNA occurred in Th1 and Th2 CB lines although with a substantially greater efficiency in the latter (Fig. 3 , lower). However, some level of viral replication, although below the threshold of detection of Mg++-dependent RT activity, likely occurred, as a modest increase of HIV-DNA accumulation could be observed at the latest time point tested (i.e., 200 h post-infection; Fig. 3 , lower) also in Th1 cells. Thus, the replicative restriction of dualtropic viruses in Th1 cells resembles that previously observed with X4 viruses in both cell types in that both occurred, at least in part, at a post-entry level [13 ].



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  		Figure 3. Limited accumulation of HIV-1 DNA from primary dualtropic HIV-1 in Th2 but not Th1 CB lines. A similar kinetic profile of R5 HIV-1-DNA synthesis in Th1 and Th2 cells is shown in the upper panel. Comparable levels of HIV DNA were achieved after 24 h from infection in Th1 and Th2 CB lines, but efficient accumulation of HIV DNA was observed only in Th2 cells at further times.

 
Opposite modulatory effects of IFN-{gamma} and IL-4 on HIV-1 replication in Th1 and Th2 CB lines
CD4+ T cell polarization from naive to Th1 or Th2 cells implies profound gene rearrangements and the selective or preferential expression of cell surface markers and functions [25 , 26 ]. Among other cytokines, Th1 cells express and secrete IFN-{gamma}, a crucial, proinflammatory cytokine that potently activates mononuclear phagocytes. Conversely, IL-4 is a major differentiation factor for Th2 cells that further secrete this cytokine [25 , 26 ]. In addition, a number of studies have previously described that IFN-{gamma} can down-modulate CCR5 (directly or via enhanced secretion of its ligands), whereas IL-4 up-regulates the levels of surface expression of CXCR4 [27 28 29 30 31 ]. Therefore, we tested whether addition of IFN-{gamma} or IL-4 affected the observed patterns of permissive or restricted replication of R5, X4, or dualtropic R5X4 viruses.

IFN-{gamma} showed modest inhibitory effects on the multiplication of R5 HIV-1 in Th1 and Th2 CB lines (Fig. 4 ), and this effect was more consistent when tested on the replication of R5X4 viruses in Th2 CB lines (Fig. 5 ). This effect was associated with decreased expression of CCR5 on Th1 cells (with a MFI of 29±10 and 43±12 in IFN-{gamma} treated vs. controls, respectively), whereas expression of CCR5 on Th2 CB lines was barely evident in the presence or absence of IFN-{gamma} (data not shown). Conversely, IL-4 enhanced R5 HIV-1 replication in Th1 and Th2 cells and dualtropic virus spreading in Th2 CB lines (Figs. 4 and 5 , respectively) and also enhanced CXCR4 expression on Th1 and Th2 cells, as previously described [27 28 29 30 31 ] (data not shown). However, neither IFN-{gamma} nor IL-4 stimulation of Th1 or Th2 cells rescued the replication of X4 virus or that of R5X4 HIV in Th1 CB lines (Figs. 4 and 5 , respectively).



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  		Figure 4. Regulatory effect of IL-4 and IFN-{gamma} on the replication of R5 or X4 monotropic HIV-1 in Th1 and Th2 CB lines. IL-4 up-regulated, whereas IFN-{gamma} moderately inhibited, the replication of R5 HIV-1BaL in Th1 and Th2 CB lines (upper panels). No significant effects on virus multiplication were observed when CB lines were infected with the X4 HIV-1IIIB/LAI strains and stimulated with either cytokine (lower panels).

 


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  		Figure 5. Regulatory effect of IL-4 and IFN-{gamma} on the replication of dualtropic HIV-1 in Th1 and Th2 CB lines. A functional pattern similar to that observed for monotropic HIV strains was observed in Th2 CB lines in that IL-4 induced and IFN-{gamma} inhibited virus replication. No induction of virus multiplication was observed in Th1 CB lines infected by these viruses.

 
We further investigated the possibility that endogenous expression of IFN-{gamma} or IL-4 could determine the restricted pattern observed with dualtropic viruses. Neutralizing concentrations of anti-cytokine mAb or of an isotype control mAb were added at the moment of infection to Th1 and Th2 CB lines. No significant effects were observed by the Ab on the replication of R5 or R5X4 HIVs in Th2 cells, and no induction of virus expression in Th1 cells or of X4 HIV in Th1 and Th2 cells was observed (data not shown). This finding was not surprising, as CB lines are maintained in a semi-quiescent state in IL-2-enriched medium for several days after the original mitogenic stimulation, as determined by the expression of activation markers including CD69, CD25, and human leukocyte antigen-DR (data not shown). Therefore, these cells were not expected to express substantial levels of IL-4 or IFN-{gamma} in the absence of restimulation. Thus, neither endogenous IL-4 nor IFN-{gamma} seems to play a crucial role in the restriction patterns observed after HIV-1 infection of polarized CB lines.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have observed a dichotomous pattern of replication of R5X4 dualtropic viruses that efficiently replicated in Th2 (and Th0) CB lines but failed to propagate into Th1 cells. This restriction resembled that previously described for X4 viruses in polarized and unpolarized CB lines in that it occurred at a post-entry level of the virus life cycle following synthesis of HIV DNA. IFN-{gamma} (Th1) and IL-4 (Th2) exerted opposite, modulatory effects on HIV-1 replication in CB lines but did not rescue R5X4 multiplication in Th1 cells or X4 virus spreading in Th1 or Th2 CB lines.

The emergence of viral variants expanding or shifting their dependency from CCR5 to CXCR4 has been estimated to occur in approximately 50% of individuals [2 ]. Several studies have indicated that this event would translate into a more accelerated disease progression [32 , 33 ]. In most cases, an expansion of chemokine coreceptor use to CXCR4, giving raise to dualtropic R5X4 viruses, occurs, whereas the generation of monotropic X4 viruses is quite rare [34 35 36 ]. In some cases, dualtropic viruses appear to be a mixture of monotropic R5 and X4 quasispecies, whereas in other cases, the dominant HIV seems to possess a gp120 Env molecule capable of interacting with both coreceptors [37 , 38 ], as in the case of the viruses investigated in the present study. In this regard, we have previously characterized the molecular features and the replicative properties of these "truly dualtropic" viruses comparably sensitive to the inhibitory effects of CCL5/RANTES or AMD3100 [14 ], as previously described by others [39 ].

The factors influencing the expansion or the shift in chemokine coreceptor use in vivo remain largely speculative. The presence of a CCR2-64I allelic variant has been reported [40 , 41 ] to determine a higher prevalence of CXCR4-using primary isolates, although the consequences of this bias on disease progression remain unclear. As this mutation is on the one hand silent at the CCR2 protein level but is in linkage dysequilibrium with a mutation in the CCR5 promoter [42 ], it is commonly believed that the protective effects of the mutation on disease progression are exerted via a diminished expression of the primary HIV coreceptor [43 ]. Furthermore, a homozygous configuration of the IL-4 promoter allelic variant IL-4-589T has been reported linked to elevated immunoglobulin E serum levels and a higher prevalence of CXCR4-using viruses than in control individuals [44 ]. In this regard, higher levels of CCR5 expression and secretion of CCR5-binding chemokines characterize Th1 cells [21 22 23 ,45 , 46 ]. Previous studies have indeed observed that freshly derived or mitogen-restimulated Th1 cells allow a more efficent entry of R5 HIVs in comparison to Th2 cells, although the subsequent raise of CCL3, CCL4, and CCL5 secretion curtailed the overall efficiency of virus replication in these cells [22 , 23 ]. Conversely, CXCR4, although expressed on polarized and unpolarized cells, is under the control of IL-4, and therefore, it is expressed at higher levels on the surface of Th0 and Th2 cells [27 , 28 , 30 ].

Most early studies on T cell infection were performed using TCLA X4 viruses, such as the LAI/IIIB strain, infecting mitogen-activated PBMC (T cell blasts) as target cells. Several studies have demonstrated that T cell blasts are exquisitely sensitive to these virus replications, whereas stimulation of PBMC with IL-2, a cytokine known to up-regulate CCR5 expression in PBMC [47 ], in the absence of mitogenic prestimulation resulted in the equal or even superior ability of NSI/macrophage-tropic/R5 viruses to propagate in culture [48 , 49 ]. The observed superiority of R5 viruses to multiplicate in polarized or unpolarized CB lines was not attributable to an intrinsic bias of neonatal cells to be productively infected by these viruses [13 ], although R5 HIV efficiently propagated in CD69+ thymocytes despite the low frequency of CCR5+ thymocytes (<1%) [50 ]. In addition, our results suggest that engagement of CCR5 (alone or together with CD4) by gp120 Env of R5 viruses triggers an activation signal for HIV replication missing when an X4-enveloped virus infects the same T cell. In support of this hypothesis, previous observations have identified important differences in the signaling capacity of R5 and X4 viruses [51 , 52 ] and that trimeric gp120 Env from R5 but not from X4, HIV, or simian immunodeficiency virus could induce Ca++ fluxes in T cells [53 ]. Although subsequent work has indicated a quantitative rather than a qualitative difference between the two types of gp120 Env [54 ], these observations may contribute to explain the dichotomous pattern that we have observed.

The capacity of CD4+ Th cells to undergo a differential program of differentiation into Th1 or Th2 cells has profound implications for the host-defensive capacity against invading pathogens [25 , 26 ]. Concerning HIV, it has been proposed early that infection by the virus was consequent to a substantial failure of the immune system to cope with the virus by mounting a nonprotective, Th2-dependent, humoral response. Indeed, highly exposed but seronegative individuals showed evidence of a Th1-biased, specific immune response to the virus [55 , 56 ]. Individuals in the advanced stages of disease usually have a Th0/Th2-biased immune response [15 ]. In this latter study, it was reported that polarized and unpolarized CD4+ T cell clones from infected individuals were susceptible to HIV infection, as demonstrated by the presence of HIV DNA but that only Th0 or Th2 clones and not Th1 cells could give raise to a productive infection. Unfortunately, no information on the viral phenotype (NSI/SI) was associated with this early study. However, the observation that the viral isolates were obtained from patients in their AIDS stage allows the speculation that at least some of these viruses were indeed dualtropic R5X4 HIVs, thus displaying a restricted pattern of replication superimposable to that illustrated in the present study. We can therefore speculate that a "Th0/Th2 bias" of most CD4+ T cells may favor the replication of R5 or R5X4 dualtropic viruses, which together represent the most frequent species in terms of coreceptor use identified in the advanced clade B infection. In addition, the almost absolute dominance of R5 viruses in India [57 ] and sub-Saharan Africa across the different viral clades has been recently correlated to an increased "inflammatory" state of the host as a result of concurrent infection and malnutrition, resulting among other effects in an increased expression of CCR5 at the cell surface [58 , 59 ].

IFN-{gamma} and IL-4 exerted modulatory effects on productive HIV-1 replication in CB lines, although they failed to rescue X4 replication in Th1 or Th2 cells or dualtropic virus spreading in Th1 CB lines. Of interest, IFN-{gamma} inhibited, whereas IL-4 enhanced virus multiplication in the permissive cells, thus enforcing the dualistic paradigm of a divergent role of Th1 versus Th2 polarization in HIV disease discussed above. On the one hand, several studies have linked the expression of IFN-{gamma} with the up-regulation of CCR5 on T cells and/or of CCR5-binding chemokines, both in vitro [22 , 23 , 60 , 61 ] and in vivo [62 63 64 ]. In apparent contrast, we have observed a diminished expression of CCR5 in CB lines maintained in IL-2-enriched medium and stimulated with IFN-{gamma}. In this regard, stimulation of purified T lymphocytes with another Th1-related cytokine, IL-12, in combination with IL-2 caused down-regulation of CCR5 and reduced HIV replication by increased secretion of CCL3 and CCL4 [65 ]. Conversely, IL-4 has been clearly shown to up-regulate CXCR4 expression, whereas IFN-{gamma} has been reported to exert an opposite effect on adult T cells [27 28 29 30 31 ] and thymocytes [50 ]. In addition, signaling-dependent regulation of post-entry HIV replication has been implicated for R5 and X4 viruses in mature and immature T cells [50 ], as reviewed in detail elsewhere [66 , 67 ].

In conclusion, we have described a model system of primary CD4+ T cells derived from neonatal CB, providing insightful information about the differential capacity of primary viruses displaying gp120 Env molecules interacting with different chemokine coreceptors of establishing productive versus latent infection of T cells. This system can be further exploited to identify the signaling correlates of these different modalities of HIV-1 infection.


   ACKNOWLEDGEMENTS
 
This study was supported in part by grants of the III° National Program of Research Against AIDS of the Istituto Superiore di Sanità, Roma, Italy. We thank Barbara Panzeri and Silvia Ghezzi for their technical support and Francesco Sinigaglia for insightful and critical discussions.

Received January 11, 2002; revised May 27, 2002; accepted July 15, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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