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(Journal of Leukocyte Biology. 2002;72:735-742.)
© 2002 by Society for Leukocyte Biology

IFN-{gamma} and IL-12 differentially regulate CC-chemokine secretion and CCR5 expression in human T lymphocytes

Giuliana Losana*,{dagger}, Chiara Bovolenta{ddagger},, Laura Rigamonti*, Igor Borghi*,{dagger}, Frederic Altare§,, Emmanuelle Jouanguy§, Guido Forni*,{dagger}, Jean-Laurent Casanova§, Barbara Sherry||, Manuela Mengozzi||,, Giorgio Trinchieri#,, Guido Poli{ddagger}, Franca Gerosa** and Francesco Novelli*,{dagger}

* Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy;
{dagger} Experimental Medicine Research Center, San Giovanni Battista Hospital, Turin, Italy;
{ddagger} AIDS Immunopathogenesis Unit, Dibit, San Raffaele Scientific Institute, Milan, Italy;
§ Laboratory of Human Genetics of Infectious Diseases, Neckér Medical School, Paris, France;
|| The Picower Institute for Medical Research, Manhasset, New York;
# Wistar Institute, Philadelphia, Pennsylvania; and
** Department of Pathology, University of Verona, Italy

Correspondence: Dr. Francesco Novelli, Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale San Luigi Gonzaga, 10043 Orbassano, Italy; E-mail: franco.novelli{at}unito.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-12, especially in the presence of neutralizing anti-IL-4 monoclonal antibodies, primed CD45RO- T clones for high CCL3/macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}) and CCL4/MIP-1ß levels. In CD4+ and CD8+ clones from two patients deficient for IL-12Rß1 (IL-12Rß1-/-), production of CCL3/MIP-1{alpha} and CCL4/MIP-1ß was defective. CD4+ clones from two patients deficient for interferon-{gamma} (IFN-{gamma}) R1 (IFN-{gamma}R1-/-) produced somewhat decreased CCL4/MIP-1ß levels. IL-12 failed to prime CD4+ or CD8+ healthy clones for high CCL5/regulated on activation, normal T expressed and secreted (RANTES) production, although its secretion was impaired in CD4+ clones from IL-12Rß1-/- and IFN-{gamma}R1-/- patients. CCR5 surface expression was up-regulated in resting peripheral blood mononuclear cells and CD4+ clones from both kinds of patients, rendering them more susceptible to CCR5-dependent (R5) HIV-1 infection. Neutralization of IFN-{gamma} increased CCR5 expression and decreased CC-chemokine secretion by CD4+ clones from healthy and IL-12Rß1-/- individuals, suggesting an IFN-{gamma}-dependent control of CCR5 expression. These data provide the first documented analysis of chemokine secretion and chemokine receptor expression on T cells from IL-12 and IFN-{gamma} receptor-deficient patients and dissect the role of IL-12 and IFN-{gamma} on inducing inflammatory chemokine secretion and down-regulating CCR5 expression in human T cells.

Key Words: chemokine receptor • cytokine receptors • AIDS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-12, a phagocyte- and dendritic cell-derived cytokine, is a major inducer of interferon-{gamma} (IFN-{gamma}) by T and natural killer (NK) cells [1 , 2 ]. It is composed of two subunits, p40 and p35, which form the p70 heterodimer that binds a heterodimeric receptor composed of the IL-12Rß1 and IL-12Rß2 chains [2 , 3 ].

T helper cell type 1 (Th1) cells play a pivotal role in eradicating intracellular bacterial and viral infections, and IL-12 induces stable priming for IFN-{gamma} in naive T lymphocytes [4 ]. In vivo, the efficient recruitment of effector Th1 cells into inflammatory tissues requires the expression of receptors for inflammatory (CC) chemokines that promote selective migration of effector cells [5 ]. Th1 lymphocytes preferentially express the CCR5 receptor for CCL3/macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), CCL4/MIP-1ß, and CCL5/regulated on activation, normal T expressed and secreted (RANTES) [6 7 8 ]. Thus, the Th1 phenotype appears to be characterized by a specific profile of chemokine production and related receptor expression in addition to IFN-{gamma} production. However, although IL-12 plays a role in inducing CC-chemokine production by peripheral T cells [8 9 10 ], it has remained unclear whether it provides any direct signal to promote CC-chemokine production by developing Th cells.

The biological effects of IL-12 are often interconnected with those of IFN-{gamma}, a cytokine that has pleiotropic and immunological functions. IFN-{gamma} is secreted by Th1 and NK cells [11 ] and binds a heterodimeric receptor formed by the IFN-{gamma}-binding R1 chain (IFN-{gamma}R1) and the transducing R2 chain (IFN-{gamma}R2) [11 ]. Although the role of IFN-{gamma} in controlling CC-chemokine production in T cells has never been investigated, there is evidence for its involvement in such production in other cell types. For example, CC-chemokine secretion and CC-receptor up-regulation are induced by IFN-{gamma} in human monocytes [12 , 13 ], and CCL5/RANTES is induced by IFN-{gamma} and tumor necrosis factor {alpha} in human endothelial cells [14 ]. Moreover, CCL5/RANTES production is impaired in IFN-{gamma}R1- and IFN-{gamma}-deficient mice [15 , 16 ], further implicating IFN-{gamma} in the regulation of CC-chemokine production.

In the present study, we examined the ability of IL-12 to prime CD45RO- T cell clones from healthy donors for CC-chemokine production and evaluated such production and CCR5 expression in CD4+ and CD8+ T cell clones from patients genetically deficient for IL-12Rß1 (IL-12Rß1-/-) and IFN-{gamma}R1 (IFN-{gamma}R1-/-). The CCR5 (R5)-dependent HIV-1 infection of resting peripheral blood mononuclear cells (PBMC) and CD4+ clones from healthy and receptor-deficient individuals was also evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
RPMI 1640, fetal calf serum (FCS), L-glutamine, penicillin, streptomycin, gentamycin, and trypan blue dye were from Life Technologies (Grand Island, NY). Phytohemagglutinin (PHA), phorbol 12-myristate 13-acetate (PMA), paraformaldehyde, 2-mercaptoethanol, and Tween 20 were from Sigma Chemical Co. (St. Louis, MO). Dimethyl sulfoxide, phosphate-buffered saline (PBS), bovine serum albumin (BSA), MgCl2, and sodium azide were from Merck Chemicals (Milan, Italy). KCl, NaCl, and HCl were from Fluka (Milan, Italy). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD4, FITC-conjugated mouse anti-human CD8, phycoerythrin (PE)-conjugated mouse anti-human CD3 monoclonal antibody (mAb), and isotype-control PE-conjugated mouse immunoglobulin G (IgG)2a, FITC-conjugated mouse IgG2a, PE-conjugated mouse IgG1, and FITC-conjugated mouse IgG1 were from Dako (Milan, Italy). FITC-conjugated mouse anti-human CCR5 and PE-conjugated mouse anti-human CXCR4 mAb were from PharMingen (San Diego, CA). Anti-CD45RO mAb Leu45RO and FITC goat anti-mouse Ig were from Becton Dickinson (Milan, Italy). Mouse {gamma}123 mAb (IgG1) neutralizes the antiviral activity of IFN-{gamma} [17 ], and mouse 5A4 mAb (IgG1) neutralizes the activity of IL-4 [18 ]. Recombinant human (r-h)IL-2 was from EuroCetus (Milan, Italy), and r-hIFN-{gamma} was from Genzyme (Milan, Italy). Plastic was from Falcon (Becton Dickinson).

Generation of CD4+ and CD8+ T cell clones from CD45RO- T cells
T cell clones from CD45RO- T cells were generated as described [19 ]. Briefly, PBMC from three healthy donors were stained by indirect immunofluorescence using the anti-CD45RO mAb Leu45RO and FITC-goat anti-mouse Ig. Cells were sorted using a Coulter Elite cell sorter (Coulter, Hialeah, FL). Before sorting, 37% of lymphocytes were CD45RO+; after sorting, purified CD45RO- cells were contaminated with 1.2% CD45RO+. Immediately after sorting, T cells were cloned by limiting dilution. In brief, 0.5 PBMC/well were seeded in 96-well round-bottom tissue-culture plates in 200 µl RPMI-1640 medium with 10% FCS in the presence of PHA (5 µg/ml), {gamma}-irradiated (50 Gy) PBMC (2.5x104 cells/well), and RPMI-8866 B lymphoblastoid cells (104 cells/well) in the presence of IL-12 (2.5 ng/ml), IL-4 (50 U/ml), neutralizing anti-IL-12 mAb C8.6 (ascites 1:400), or neutralizing anti-IL-4 mAb 5A4. After 1 week, irradiated PBMC and RPMI-8866 were added as feeder cells; clones were maintained in culture with IL-2, fed and split as required. After 4 weeks, clones were washed, incubated for 24 h in medium containing IL-2 (50 U/ml) only, washed again, and stimulated for 18 h with soluble anti-CD3 mAb and PMA; IFN-{gamma} and chemokines were measured in the cell-free supernatant fluid by RIA and enzyme-linked immunosorbent assay (ELISA), respectively.

T cell cultures and clone generation from healthy IFN-{gamma}R1-/- and IL-12Rß1-/- individuals
PHA-derived T cell clones and T lymphoblasts from three healthy donors, two IFN-{gamma}R1-/- [20 , 21 ], and two IL-12Rß1-/- [22 , 23 ] patients were generated as previously described [24 , 25 ]. After 4 weeks, clones were analyzed for expression of CD4, CD8, CCR5, and CXCR4 molecules and for CC-chemokine production.

Evaluation of CC-chemokine production in T cell clones
To evaluate CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES production, T cell clones (106 cells) were cultured for 72 h in complete medium containing IL-2 (60 U/ml); cell-free supernatants were collected and stored at -70°C until analyzed by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). The minimum detectable dose is 10 pg/ml for CCL3/MIP-1{alpha}; 4 pg/ml for CCL4/MIP-1ß; and 8 pg/ml for CCL5/RANTES.

Flow cytometry
For analysis of membrane expression of CCR5 and CXCR4 in clones from healthy and deficient individuals, cells (106) were incubated with CCR5-FITC or CXCR4-PE mAb for 30 min at 4°C, followed by two washes with cold PBS supplemented with 0.2% BSA and 0.05% sodium azide (PBS-azide). For intracytoplasmic detection of CCR5, cells (106) were fixed with 100 µl cold 4% paraformaldehyde in PBS, incubated for 1 h at 4°C, washed, and incubated for 20 min with PBS supplemented with 0.2% Tween 20. After two washes with PBS-azide, cells were labeled with CCR5-FITC as described above. Expression of membrane and intracellular protein was analyzed with a FACScan flow cytometer (Becton Dickinson).

HIV-1 strains and coreceptor use
The HIV-1BaL strain was used as prototype of CCR5 (R5) macrophage-tropic virus and the HIV-1LAI/IIIB strain as prototype of CXCR4 (X4) T tropic virus. Two primary HIV-1 isolates (HIV-1SR5 and HIV-1SR-3) were obtained by cocultivation of PBMC from HIV-1-infected individuals with allogeneic PHA-blasts from seronegative donors without further passage. The macrophage-tropic infectious molecular clone HIV-1NL(Ad8) (kindly donated by Eric Freed, National Institutes of Health, Bethesda, MD) was generated by replacing 1.7 kb of the env gene of the HIV-1LAI/IIIB strain with the homologous portion of the R5 HIV-1ADA strain. All viruses were characterized for coreceptor use by analyzing their ability to use selected chemokine receptors, including CXCR4, CCR2b, CCR3, and CCR5, as described [26 ]. As expected, HIV-1BaL, HIV-1NL(Ad8), and HIV-1SR5 used only CCR5, and HIV-1LAI/IIIB used only CXCR4, whereas HIV-1SR-3 used CCR5 and CXCR4.

HIV-1 infections
T cell clones or lines were maintained in culture by periodical restimulation (every 9–12 days) with PHA, IL-2 in the presence of irradiated PBMC as feeder cells. IL-2 was replaced every 3 days to allow growth in a state of ongoing activation. This represents the standard condition of our studies. Approximately 10 days after the last restimulation, T cells were infected with different HIV-1 strains at the multiplicity of infection of 0.1 in 48-well plates in the presence of complete medium containing IL-2 (100 U/ml) to avoid interference with the presence of feeder cells. Fifty percent of the medium was collected every 3 days and stored at -80°C until tested for reverse transcriptase (RT) activity as described [26 , 27 ]; fresh medium was then added.

Statistical analysis
Differences between control and deficient clones in CC-chemokine production and CCR5 expression were evaluated with Pearson’s t test (GraphPad Prism 3, GraphPad Software, Inc., San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-12 induction and IL-4 inhibition of priming for CC-chemokine secretion by T cell clones derived from CD45RO- T lymphocytes
Increasing evidence points to the critical role of type I cytokines and in particular IL-12 in regulating CC-chemokine production by human T cells [9 , 28 , 29 ]. However, a requirement for IL-12 in the differentiation of CC-chemokine-producing T cells has not been demonstrated. As selection phenomena as a result of induction of apoptosis might occur in a bulk polyclonal culture, previous in vitro analyses of CC-chemokine regulation in bulk polyclonal T cell cultures were unable to distinguish between direct effects on the differentiation of Th clones versus indirect effects favoring expansion of Th1 cells or inhibition of Th2 cell growth [9 , 10 , 30 ]. To distinguish between direct and indirect effects of IL-12, we analyzed the effect of IL-12 or anti-IL-12-neutralizing mAb on CC-chemokine production of human CD4+ and CD8+ T cells by using a limiting dilution cloning system that allows the clonal expansion of almost every single T cell from peripheral blood [19 ]. Through this approach, the effects on CC-chemokine production can be evaluated on any single cell progeny.

CD4+ and CD8+ T cells with a naive phenotype (CD45RO-) were cloned by limiting dilution in the presence of feeder cells, IL-2, PHA, and one of the following cytokine/mAb combinations: IL-12; IL-12 + anti-IL-4 mAb; IL-12 + IL-4; anti-IL-12 mAb; and IL-4 + anti-IL-12 mAb. After 4 weeks of culture, clones were washed, stimulated overnight with soluble anti-CD3 mAb and PMA, and evaluated by RIA and ELISA for IFN-{gamma} and CC-chemokine secretion (Fig. 1 ). Production of IFN-{gamma}, IL-10, and IL-4 from these clones has been previously reported [19 ]. CD4+ and CD8+ clones generated in the presence of anti-IL-12-neutralizing mAb, with or without IL-4, produced negligible amounts of IFN-{gamma}, CCL3/MIP-1{alpha}, and CCL4/MIP-1ß, whereas they produced very high levels of these three proteins when generated in the presence of IL-12. Addition of rIL-4 minimally affected this high production of CCL3/MIP-1{alpha} and CCL4/MIP-1ß, whereas neutralization of endogenous IL-4 enhanced their production two- to fivefold but only slightly increased production of IFN-{gamma}. CCL5/RANTES was produced by all the clones, particularly the CD8+ clones, and only a slight enhancement of CCL5/RANTES production was observed in CD4+ clones but not in CD8+ clones generated in the presence of IL-12 with or without anti-IL-4 mAb (Fig. 1) .



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  		Figure 1. IFN-{gamma} and CC-chemokine secretion by CD4+ and CD8+ clones generated from CD45RO- T cells. CD45RO- clones were generated by limiting dilution in the presence of one of the following cytokine/mAb combinations: IL-12 ({blacksquare}); IL-12 + anti-IL-4 mAb (•); IL-12 + IL-4 (gray {triangleup}); anti-IL-12 mAb ({square}); and IL-4 + anti-IL-12 mAb ({circ}), as described in Materials and Methods. After 4 weeks of culture, clones were washed, stimulated overnight with soluble anti-CD3 mAb and PMA, and evaluated by RIA and ELISA for IFN-{gamma} and CC-chemokine secretion. Data are given as mean ± SEM of CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES production (ng/ml) as a function of IFN-{gamma} production (U/ml) by all clones analyzed in each cloning condition. The number of clones analyzed was: CD4+: IL-12 n = 22, IL-12 + anti-IL-4 mAb n = 26, IL-12 + IL-4 n = 43, anti-IL-12 mAb n = 29, anti-IL-12 mAb + IL-4 n = 38; CD8+: IL-12 n = 17, IL-12 + anti-IL-4 mAb n = 12, IL-12 + IL-4 n = 12, anti-IL-12 mAb n = 8, anti-IL-12 mAb + IL-4 n = 19.

 
These data indicate that IL-12, especially when IL-4 is neutralized, primes CD4+ and CD8+ CD45RO- cells for high CCL3/MIP-1{alpha} and CCL4/MIP-1ß production. By contrast, IL-12 fails to prime CD45RO- cells for high CCL5/RANTES production.

The effect of IL-12 in priming for MIP-1{alpha}/CCL3 and MIP-1ß/CCL4 was much less pronounced in CD4+ clones derived from T cells with a memory phenotype (CD45RO+) than in those derived from CD45RO- cells; even CCL5/RANTES was fourfold higher in CD45RO+ clones (data not shown). This probably reflects in vivo priming and commitment to a pattern of cytokine production and indicates that the plasticity of CD45RO- in acquiring a given cytokine profile is less stochastic than that of clones from already in vivo-primed CD45RO+ cells.

CC-chemokine secretion by T cells from IFN-{gamma}R1-/- or IL-12Rß1-/- patients
To better analyze the impact of the IL-12/IFN-{gamma} axis on CC-chemokine production by human T cells, we evaluated the constitutive secretion of CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES by CD4+ and CD8+ T cell clones from IFN-{gamma}R1-/- and IL-12Rß1-/- patients.

CD4+ and CD8+ T cell clones were generated in the absence of polarizing conditions from PHA-activated T lymphoblasts of three healthy individuals and the two IFN-{gamma}R1-/- and two IL-12Rß1-/- patients. CC-chemokine production was measured as described in Materials and Methods. As shown in Figure 2 , clones from healthy individuals constitutively secreted CCL5/RANTES at levels comparable with those of clones generated from peripheral blood lymphocytes in the presence of IL-12 or anti-IL-12 mAb following maximal stimulation with anti-CD3 mAb plus PMA (Fig. 1) . By contrast, constitutive production of CCL3/MIP-1{alpha} and CCL4/MIP-1ß by the cultured clones was 10- to 50-fold lower than that induced by anti-CD3 mAb plus PMA, as shown in Figure 1 .



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  		Figure 2. CC-chemokine secretion by T cell clones from healthy and the IFN-{gamma}R1-/- and IL-12Rß1-/- individuals. Production of CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES in CD4+ (upper panels) and CD8+ (lower panels) T clones from three healthy, two IFN-{gamma}R1-/-, and two IL-12Rß1-/- individuals was evaluated by ELISA in the supernatants of clones cultured for 72 h after the last passage in complete medium containing IL-2 (60 U/ml). Data are given as mean ± SEM chemokine production of all clones analyzed. n indicates number of clones analyzed.

 
CD4+ clones generated from the IFN-{gamma}R1-/- patients produced similar amounts of CCL3/MIP-1{alpha} but significantly lower amounts of CCL4/MIP-1ß (P<0.05) and CCL5/RANTES (P<0.001) as compared with CD4+ clones from healthy donors (Fig. 2) . CD8+ clones from the IFN-{gamma}R1-/- patients produced all three chemokines at levels similar to those of CD8+ clones from healthy donors. CD4+ clones from the IL-12Rß1-/- patients produced significantly lower levels of CCL3/MIP-1{alpha} (P<0.001), CCL4/MIP-1ß (P<0.001), and CCL5/RANTES (P<0.001) than the clones from healthy donors, whereas their CD8+ clones produced lower levels of CCL3/MIP-1{alpha} (P<0.001) and CCL4/MIP-1ß (P<0.001) but not of CCL5/RANTES.

These data confirm the role of IL-12 in controlling CCL3/MIP-1{alpha} and CCL4/MIP-1ß secretion by CD4+ and CD8+ and suggest a restricted role for IFN-{gamma} in controlling the production of CCL5/RANTES by CD4+ T cells only.

CCR5 surface expression on T cells from healthy and deficient individuals
Expression of CCR5 was evaluated in fresh PBMC and in CD4+ T cell clones from the IFN-{gamma}R1-/- or IL-12Rß1-/- patients after 10 days of culture in the presence of IL-2 (Fig. 3 ). Its surface expression was almost undetectable in PBMC from healthy individuals but was higher with an increase of over 15-fold in percentage and of threefold in mean fluorescence intensity (MFI) in IFN-{gamma}R1-/- and over tenfold in percentage and threefold in MFI in IL-12Rß1-/- PBMC (Fig. 3A) . In CD4+ clones from healthy individuals, CCR5 was nearly absent, whereas its expression was high in those from the deficient patients (Fig. 3B) . No difference in CXCR4 surface expression was observed between healthy and deficient PBMC and CD4+ clones (Fig. 3A and 3B) . Flow cytometry on permeabilized cells revealed similarly high levels of intracytoplasmic CCR5 expressed by CD4+ clones from controls and deficient patients, indicating that synthesis is unaffected (Fig. 3B) . The increased CCR5 expression in CD4+ clones from the deficient patients did not affect their chemotactic response to CCL4/MIP-1ß and CCL5/RANTES, which was similar to that of healthy CD4+ clones (data not shown).



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  		Figure 3. Surface and cytoplasmic expression of CCR5 and CXCR4 in PBMC and T clones. (A) Results of flow cytometry analysis of surface expression of CCR5 and CXCR4 in PBMC from healthy IFN-{gamma}R1-/- and IL-12Rß1-/- individuals. (B) Similar analysis of CXCR4 (left panels) and CCR5 surface (middle panels) and CCR5 cytoplasmic (right panels) expression of three representative CD4+ clones, one from healthy, one from IFN-{gamma}R1--, and one from IL-12Rß1-/- individuals. CCR5 and CXCR4 expression was detected by FITC-conjugated anti-CCR5 and PE-conjugated anti-CXCR4 mAb, respectively (shaded). For cytoplasmic expression, T cells were permeabilized and stained as described in Materials and Methods. Thin lines indicate background fluorescence on the surface and in the cytoplasm as detected by FITC-conjugated isotype-matched control IgG (for CCR5) and by PE-conjugated isotype-matched control IgG (for CXCR4). In the box are indicated the percentage of positive cells (%), the MFI, and the number of tested sample (n°).

 
IFN-{gamma}-dependent down-regulation of CCR5 surface expression in CD4+ clones
To test whether CCR5 modulation was dependent on IFN-{gamma}, we assessed CCR5 expression and CC-chemokine production in CD4+ clones cultured for 24 h in the presence of 50 µg/ml anti-IFN-{gamma}-neutralizing {gamma}123 mAb or 100 U/ml IFN-{gamma} (Fig. 4 ). Neutralization of IFN-{gamma} significantly increased the expression of CCR5 and decreased the production of all three CC-chemokines in healthy and IL-12Rß1-/- CD4+ clones (Fig. 4A and 4B) . In IL-12Rß1-/- CD4+ clones, IFN-{gamma} significantly increased the production of CCL5/RANTES and down-regulated CCR5 surface expression but caused no significant increase in CCL3/MIP-1{alpha} and CCL4/MIP-1ß production (Fig. 4C) . Addition of CCL5/RANTES prevented the up-modulation of CCR5 induced by the neutralization of IFN-{gamma} in CD4+ clones from healthy or IL-12Rß1-/- individuals (data not shown). As expected, neither exogenous IFN-{gamma} nor anti-IFN-{gamma} mAb affected CCR5 expression and CC-chemokine production in CD4+ IFN-{gamma}R1-/- clones (data not shown). These data strongly suggest that IFN-{gamma} down-regulates CCR5 surface expression by enhancing CC-chemokine secretion.



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  		Figure 4. Effects of IFN-{gamma} on CC-chemokine production and CCR5 surface expression. Healthy (A) and IL-12Rß1-/- (B) CD4+ clones were cultured in complete medium containing IL-2 in the presence of 50 µg/ml control isotype-matched mouse IgG1a mAb or anti-IFN-{gamma}-neutralizing mAb ({gamma}123). IL-12Rß1-/- CD4+ clones (C) were cultured in the presence or absence of IFN-{gamma} (100 U/ml). After 24 h, CCR5 surface expression and CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES secretion in the supernatant were evaluated. The results are expressed as means ± SEM of percentage of CCR5+ cells and pg/ml chemokine production. Significances are indicated as * = 0.01 < P < 0.05; ** = P < 0.01; NS = not significant. The number of CD4+ clones analyzed was: controls with or without anti-IFN-{gamma} mAb for CCR5 expression, 12; for CC-chemokine production, 9; IL-12Rß1-/- with or without anti-IFN-{gamma} mAb for CCR5 expression, 5; for CC-chemokine production, 5; IL-12Rß1-/- with or without IFN-{gamma} for CCR5 expression, 13; for CC-chemokine production, 6.

 
HIV-1 replication in CD4+ T cells from the IFN-{gamma}R1-/- and IL-12Rß1-/- patients
As PBMC and CD4+ clones from the deficient patients displayed enhanced surface expression of CCR5 and reduced CC-chemokine production, we investigated whether these cells were more susceptible to HIV-1 infection (Fig. 5 ). PHA-activated T lymphoblasts or CD4+ clones from healthy and IFN-{gamma}R1-/- individuals were infected with the laboratory-adapted macrophage-tropic R5 HIV-1BaL strain and were cultured for 32 days in complete medium containing IL-2. HIV-1 replicated in T lymphoblasts from healthy donors with a peak of RT activity at day 14 post-infection, whereas virus production peaked at day 11 and was threefold greater in T lymphoblasts from one IFN-{gamma}R1-/- patient (Fig. 5A) . The kinetics of R5 HIV-1BaL replication differed in CD4+ clones from healthy and IL-12Rß1-/- patients, with a peak of RT activity at day 25 post-infection in the CD4+ clone from a healthy donor and an accelerated peak of virus replication at day 14 in a CD4+ clone obtained from one IL-12Rß1-/- patient (Fig. 5B) . RT activity decrement after 32 days post-infection correlates with a T cell viability decrement of approximately 75% observed in healthy and deficient individuals.



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  		Figure 5. HIV-1 replication in CD4+ T cells from healthy and deficient individuals. (A) Kinetics of R5-HIV-1 replication in PHA-lymphoblasts derived from healthy and IFN-{gamma}R1-/- individuals and cultured in complete medium containing IL-2 (100 U/ml). (B) Kinetics of R5-HIV-1 replication in CD4+ T cell clones derived from healthy and IL-12Rß1-/- individuals. (C) Kinetics of X4-HIV-1 replication in PHA-lymphoblasts derived from healthy and IFN-{gamma}R1-/- individuals. (D) Kinetics of X4-HIV-1 replication in CD4+ T cell clones derived from healthy and IL-12Rß1-/- individuals. (E) A single CD4+ clone from a control and three CD4+ clones and one CD8+ clone from an IFN-{gamma}R1-/- patient infected with laboratory-adapted R5-HIV-1BaL and HIV-1NL(Ad8) or with the primary isolates HIV-1SR-5 and HIV-1SR-3. The Mg++-dependent RT activity was evaluated after 14 days of culture.

 
Unlike from HIV-1BaL, the replication X4-dependent HIV-1IIIB became evident only after 20 days in IL-2-stimulated, healthy T cells. This is not surprising, as differently from R5-dependent, laboratory-adapted and primary-isolate X4-dependent virus replication in T cells is strictly dependent on their progression through the cell cycle, as can be achieved following a recent T cell receptor (TCR) stimulation [31 ]. However, the replication of HIV-1IIIB was slightly increased in IL-2-stimulated, deficient T cells (Fig. 5C and 5D) . The increased rate of replication correlated with a different rate of death following X4-dependent infection; the viability of healthy T cells being approximately 50% and 70% decreased compared with IFN-{gamma}R1 and IL-12Rß1-deficient cells, respectively.

The augmented susceptibility of deficient T cells to R5-dependent HIV replication was further confirmed by infection of three CD4+ clones from the IFN-{gamma}R1-/- patients with different R5-HIV-1 strains (BaL, Ad8) and with the primary isolates HIV-1SR-5 and HIV-1SR-3; the latter were R5X4-dualtropic for coreceptor use (Fig. 5E) . No RT activity was detected after 14 days in a control CD8+ clone from an IFN-{gamma}R1-/- patient. The majority of HIV-1 strains tested did not replicate in a CD4+ clone from a healthy individual after 14 days of cultivation, consistent with the delayed kinetics of virus multiplication observed in these clones as compared with healthy PHA lymphoblasts (Fig. 5A) . By contrast, efficient replication of all virus strains was observed in the CD4+ clones from the IFN-{gamma}R1-/- patients (Fig. 5E) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this paper, we dissect the role of IL-12 and IFN-{gamma} on CC-chemokine production and CCR5 expression by human T cells by generating PHA-derived clones obtained from peripheral lymphocytes of healthy IL-12Rß1-/- and IFN-{gamma}R1-/- individuals. We found that IL-12 very efficiently primes CD4+ and CD8+ clones for high CCL3/MIP-1{alpha} and CCL4/MIP-1ß production. Neutralization of endogenous IL-4 further enhanced this priming effect, but the presence of high concentrations of IL-4 did not inhibit it. The present results also demonstrate that priming by IL-12 is an absolute requirement for CCL3/MIP-1{alpha} and CCL4/MIP-1ß production, as T cell clones generated in the presence of anti-IL-12-neutralizing mAb produce negligible amounts of these two CC-chemokines, irrespective of the neutralization of IL-4, and their severely reduced production in IL-12Rß1-/- but not in IFN-{gamma}R1-/- T cell clones supports this conclusion.

IL-12 only moderately primed the CD4+ clones for CCL5/RANTES production. However, this production and to a lesser extent that of CCL4/MIP-1ß were dramatically reduced in CD4+ clones and PHA-activated PBMC from IL-12Rß1-/- and IFN-{gamma}R1-/- patients. These data suggest that the production of CCL5/RANTES, which unlike CCL3/MIP-1{alpha} and CCL4/MIP-1ß is a constitutively released rather than TCR-induced chemokine [32 ], probably requires a signal mediated by endogenous IFN-{gamma}. As IL-12 does not prime for CCL5/RANTES production, its reduced secretion by CD4+ clones from IL-12Rß1-/- patients can be mainly attributed to a reduced ability to produce IFN-{gamma}. Indeed, severely reduced IFN-{gamma} production has been reported in patients deficient in the genes encoding IL-12Rß1 [22 , 23 , 33 ] or IL-12p40 [34 ], and CD4+ and CD8+ clones from those patients display decreased but not absent production of IFN-{gamma} [25 ]. Moreover, the clinical phenotype of IL-12Rß1-/- patients is probably also attributable to IL-23, a novel, IFN-{gamma}-inducing cytokine that contains the p40 subunit of IL-12, heterodimerizes with the p19 subunit, and binds the IL-12Rß1 chain [35 ]. However, the control of CCL5/RANTES secretion by IFN-{gamma} and IL-12 appears to be restricted to CD4+ T cells, as CD8+ clones from both types of patients were indistinguishable from those derived from healthy individuals with respect to CCL5/RANTES levels.

Analysis of the direct effects of IFN-{gamma} on CC-chemokine production in T cells is difficult, as activated T cells cultured in the presence of IL-2 constitutively produce endogenous IFN-{gamma} [17 ]. As activated human T cells respond to IFN-{gamma} [17 , 20 , 36 ], this cytokine may control CC-chemokine secretion through an autocrine mechanism. Consistent with this hypothesis, the constitutive CC-chemokine production by CD4+ clones derived from healthy and IL-12Rß1-/- individuals was decreased in the presence of anti-IFN-{gamma}-neutralizing mAb.

CCR5 surface expression was enhanced in PBMC and CD4+ T cell clones from IFN-{gamma}R1-/- and IL-12Rß1-/- patients, whereas intracytoplasmic CCR5 levels were comparable in deficient and healthy CD4+ T cells. These findings suggest a constitutive dysregulation of CCR5 surface expression and/or internalization in the absence of IFN-{gamma} and IL-12 and rule out the hypothesis of an increase of CCR5 gene transcription by IL-12 and IFN-{gamma}. IFN-{gamma} neutralization increased CCR5 expression in healthy and IL-12Rß1-/- CD4+ clones, and this was correlated with a simultaneous decrease in secretion of all three CC-chemokines. Conversely, the addition of IFN-{gamma} down-regulated CCR5 and up-regulated CC-chemokine secretion by IL-12Rß1-/- CD4+ clones. Thus, it appears that IFN-{gamma}, independently of IL-12, down-regulates the surface expression of CCR5, mostly by enhancing CC-chemokine secretion, consistent with the failure of anti-IFN-{gamma} mAb to block the down-regulation of cell surface CCR5 expression by CCL5/RANTES. As CD8+ T cells from deficient patients and healthy individuals display a similarly low CCR5 surface expression and as IFN-{gamma} induces CCR5 expression and CC-chemokine production in human monocytic cells [12 , 13 ], our results point to the existence of different cell-lineage-specific, IFN-{gamma}-dependent pathways of CCR5 down-regulation. This pathway seems restricted to IL-2-stimulated CD4+ T cells, as in TCR-stimulated CD4+ and CD8+ cells, the IL-12/signal transducer and activator of transcription (STAT)-4 and IFN-{gamma}/STAT-1 pathways converge in up-regulating CCR5 expression [37 38 39 ]. Thus, it seems that IFN-{gamma}- and IL-12-dependent regulation of CCR5 expression plays a role in different phases of T cell activation, with IL-12 favoring CCR5 up-regulation on recently activated T cells and IFN-{gamma} favoring its down-regulation on long-term, stimulated T cells.

It is of interest that although CCR5 levels differed markedly in healthy versus deficient CD4+ T cells, their chemotactic response to CCL4/MIP1ß and CCL5/RANTES was the same. Thus, dysregulation of CCR5 expression, probably as a result of recycling failure, does not appear to affect the chemotactic response of deficient T cells. Consistent with their increased CCR5 surface expression, CD4+ T cells from deficient patients show an enhanced susceptibility to HIV-1 infection. Thus, our findings provide direct evidence for a general protection of CD4+ T cells from HIV-1 infection by IFN-{gamma} and IL-12. IFN-{gamma} has been reported to exert different effects during HIV-1 infection of T cells [40 , 41 ] ranging from enhancement to inhibition of the HIV life cycle or no effect [42 43 44 ]. Our data and those from other studies point to the important role of IFN-{gamma} in protecting CD4+ T cells from HIV infection, as we have previously shown in monocytic cells [27 ]. CD4+ T lymphoblasts from IFN-{gamma}R1-/- patients showed a faster kinetics of R5-HIV-1 replication compared with that from healthy individuals. Moreover, CD4+ clones from IFN-{gamma}R1-/- patients were much more susceptible to infection by laboratory-adapted or primary R5 and R5X4 isolates. Compared with healthy T cells, both types of deficient T cells displayed an earlier onset of R5-HIV-1 infection. However, R5-HIV-1 replicated more efficiently in IFN-{gamma}R1-/- T cells, and this enhanced replication may be attributable to their complete impairment of IFN-{gamma}-induced, antiviral activity [27 ]. By contrast, IFN-{gamma} antiviral activity may be induced by the residual IFN-{gamma} production observed in IL-12Rß1-/- patients [23 , 25 , 45 ].

Despite comparable CXCR4 levels displayed by healthy and deficient T cells, X4-HIV-1 replication was only slightly increased in IFN-{gamma}R1-/- and IL-12Rß1-/- cells. This difference seems to be a result of a different rate of death, suggesting a role of IFN-{gamma} and IL-12 in regulating the death of X4-HIV-1-infected T cells. Our data show that replication of X4-HIV in T cells is slower than that of R5-HIV. This may explain the fact that the dual tropic virus HIV-1SR-3 does not productively infect the healthy CD4+ clone after 14 days.

These data provide the first documented analysis of the patterns of CC-chemokine secretion, CCR5 expression, and HIV infection on T cells from patients with genetic defects, preventing a response to IL-12 or IFN-{gamma}. They clearly demonstrate that IL-12 primes CD4+ and CD8+ T cells for high CCL3/MIP-1{alpha} and CCL4/MIP-1ß production. Although IFN-{gamma} does not prime T cells directly for high CC-chemokine production, it is essential for the efficient secretion of CCL5/RANTES and to a lesser extent, for CCL4/MIP-1ß secretion by CD4+ cells, resulting in down-regulation of CCR5 and protection from R5-HIV-1 infection.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Istituto Superiore di Sanità (AIDS National Programm on AIDS, project: "Pathogenesis, Immunity and Vaccine") to F. N. and G. P.; Associazione Italiana Ricerca sul Cancro (AIRC) to F. N. and G. F.; Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST), Consiglio Nazionale delle Ricerche [(CNR) Biotechnology Program L.95/95] to G. F.; MURST ex 40% to F. N., G. F., and F. G.; Programme National de Reserche Medicale, Institut Universitarie de France, Programme National de Reserche Fundamentale en Microbiologie, Legs Poix, Maladies Infectionses et Parasitaires, Lise-Meitner Programme to J-L. C. We thank Dr J. Iliffe for critically reading the manuscript. We also thank Daniela Boselli for technical support.


   FOOTNOTES
 
Current address of Chiara Bovolenta: Genera S.p.A., Milan, 20132, Italy. Back

Current address of Manuela Mengozzi: Mario Negri Institute of Pharmacological Research, 20157, Milan, Italy. Back

Current address of Giorgio Trinchieri: Schering-Plough Laboratory of Immunological Research, Dardilly, 69571, France. Back

Current address of Frederic Altare: CNRS UMR 5089, Toulouse, 31077, France. Back

Received March 30, 2002; accepted June 18, 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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