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(Journal of Leukocyte Biology. 2002;71:652-658.)
© 2002 by Society for Leukocyte Biology

Erythrocytes deliver Tat to interferon-{gamma}-treated human dendritic cells for efficient initiation of specific type 1 immune responses in vitro

Silvia Corinti*, Laura Chiarantini{dagger}, Sabrina Dominici{dagger}, Maria Elena Laguardia{dagger}, Mauro Magnani{dagger} and Giampiero Girolomoni*

* Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome, Italy; and
{dagger} Institute of Biochemistry Giorgio Fornaini, University of Urbino, Italy

Correspondence: Dr. Silvia Corinti, Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Via Monti di Creta 104, 00167 Roma, Italy. E-mail: s.corinti{at}idi.it


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ABSTRACT
 
Dendritic cells (DC) can represent an important target for vaccine development against viral infections. Here, we studied whether interferon-{gamma} (IFN-{gamma}) could improve the functions of DC and analyzed human red blood cells (RBC) as a delivery system for Tat protein. Monocyte-derived DC were cultured in human serum and matured with monocyte-conditioned medium (MCM) in the presence or not of IFN-{gamma}. Tat was conjugated to RBC (RBC-Tat) through avidin-biotin bridges. Stimulation of DC with IFN-{gamma} increased the release of interleukin (IL)-12 and tumor necrosis factor-{alpha} and inhibited the production of IL-10. Moreover, IFN-{gamma}-treated DC up-regulated the release of CXCL10 (IP-10) markedly and reduced the secretion of CCL17 TARC significantly, attracting preferentially T-helper (Th)1 and Th2 cells, respectively. DC internalized RBC-Tat efficiently. Compared with DC pulsed with soluble Tat, DC incubated with RBC-Tat elicited specific CD4+ and CD8+ T-cell responses at a much lower antigen dose. DC matured in the presence of MCM were more effective than immature DC in inducing T-cell proliferation and IFN-{gamma} release. Finally, immature and mature DC exposed to IFN-{gamma} were better stimulators of allogeneic T cells and induced a higher IFN-{gamma} production from Tat-specific CD4+ and CD8+ T lymphocytes. In conclusion, erythrocytes appear an effective tool for antigen delivery into DC, and IFN-{gamma} could be used advantageously for augmenting the ability of DC to induce type 1 immune responses.

Key Words: T lymphocytes • AIDS • cytokines • chemokines • vaccination


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INTRODUCTION
 
Dendritic cells (DC) are essential in the initiation of immune responses. DC reside in unperturbed tissues in an immature form, where they are adapted for capturing and accumulating antigens. A variety of danger signals, including microorganisms, dying cells, or proinflammatory cytokines, induce the terminal differentiation, also known as maturation, of DC [1 2 3 4 ]. Mature DC migrate to secondary lymphoid organs and acquire a potent T-cell-stimulating capacity. DC maturation is strengthened by T-cell-derived signals, such as the CD40 ligand (CD40L) [1 2 3 ]. Interferon-{gamma} (IFN-{gamma}) has also been shown to promote the terminal maturation of mouse DC [5 ] and to increase interleukin (IL)-12 release from mouse and human DC [6 , 7 ]. Mature DC express higher levels of presenting and costimulatory molecules and release large amounts of IL-12, thereby preferentially stimulating T helper (Th)1 responses. Given their central role in the immune system, DC can represent an important target for vaccine development against viral infections. In addition, the possibility of generating large numbers of autologous DC from precursors has made the use of DC feasible and very attractive as a means for inducing or boosting protective immune responses [8 9 10 11 ]. An important issue in using DC in immunotherapy is to optimize antigen delivery to stimulate CD4+ and CD8+ antigen-specific T cells. Antigens administered to DC in a soluble form are preferentially routed to the major histocompatibility complex (MHC) class II pathway and elicit CD4+ T-cell responses primarily. Various approaches have been developed to assure an efficient access of exogenous antigens to the MHC class I processing pathway and thus induce CD8+ T-cell responses [12 13 14 ]. The use of particulate antigens in the form of apoptotic cells or antigen coupled to latex beads, encapsulated in liposomes, or expressed on recombinant bacteria has been demonstrated to elicit CD4+ and CD8+ T-cell responses [12 , 15 16 17 18 ]. Red blood cells (RBC) are an interesting tool for antigen delivery because they can be obtained and conjugated easily to protein antigens via biotin-avidin bridges [19 ]. Moreover, inoculation of RBC coated with viral antigens was shown to induce protective immune responses higher then those obtained using classical adjuvant in vivo [20 , 21 ].

In this study, we evaluated whether RBC could deliver the HIV regulatory protein Tat into DC and the capacity of such DC to induce specific immune responses in vitro. Because in HIV infection there is a progressive loss of CD4+ T-cell help to DC maturation that may limit the efficacy of antiviral CD8+ responses [22 ], we also examined whether IFN-{gamma} could be of value in improving the functions of DC.


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MATERIALS AND METHODS
 
DC preparation and stimulation
DC were generated from peripheral blood mononuclear cells (PBMC) of healthy individuals, as described previously [16 ]. Briefly, Percoll-purified (Pharmacia, Uppsala, Sweden) monocytes (>90% CD14+) were cultured at 1 x 106 cells/ml in RPMI 1640 containing 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen Italia, San Giuliano Milanese, Italy), 0.05 mM 2-mercaptoethanol (Merck, Darmstadt, Germany), and 2% human serum (complete RPMI), and were supplemented with 100 ng/ml human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; Mielogen®, Schering-Plough, Milan, Italy) and 200 U/ml human recombinant IL-4 (R&D Systems, Abingdon, UK) at 37°C with 5% CO2. Medium was changed after 3 days, and at day 6 of culture, cells were recovered and depleted of CD2+ and CD19+ cells by means of immunomagnetic beads coated with specific monoclonal antibodies (mAb; Dynal, Oslo, Norway). Monocyte-conditioned medium (MCM) was prepared as described [23 ]. In brief, PBMC from buffy coats were plated on bacterial Petri dishes coated with 0.5 µg/ml human immunoglobulin (Ig; Istituto Sieroterapico Berna, Como, Italy). After 2 h, nonadherent cells were removed, and adherent cells were cultured overnight. The supernatant was then filtered and stored at -20°C until use. MCM was added (1:1 v/v) to DC cultures for induction of maturation at day 6. Where indicated, DC were treated with 10 µg/ml lipopolysaccharide (LPS; from Salmonella typhimurium, Sigma-Aldrich, Milan, Italy), 1 µg/ml soluble CD40L (sCD40L; Alexis Corp., San Diego, CA), and/or 50 U/ml human recombinant IFN-{gamma} (R&D Systems).

Flow cytometry analysis of DC
The mAb fluorescein isothiocyanate (FITC)-conjugated, anti-HLA-DR, anti-CD14, anti-CD1a, anti-CD86, anti-CD40, and control mouse Ig were purchased from BD PharMingen (San Diego, CA). FITC-conjugated anti-CD54 and anti-CD80 and pure anti-CD83 came from Immunotech (Marseille, France). Pure anti-MHC class I was from Dako (Glostrup, Denmark). DC, untreated or stimulated for 18 h with MCM, LPS, or sCD40L in the presence or not of IFN-{gamma}, were washed and then incubated with FITC-conjugated mAb diluted in phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS) and 0.01% NaN3 for 40 min at 4°C. When pure mAb were used, a second incubation with a FITC-coupled goat (Fab')2 anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) was performed.

Cytokine and chemokine release from DC
Supernatants were collected after 18 h of DC cultures and were stored at -80°C. Enzyme-linked immunosorbent assay (ELISA) for IL-12 (p70) and tumor necrosis factor {alpha} (TNF-{alpha}) were performed using OptEIATM kits from BD PharMingen, per the manufacturer’s protocol. IL-10 was measured using a matched pair of mAb from BD PharMingen. Regulated on activation, normal T expressed and secreted (RANTES; CCL5) and IFN-{gamma}-inducible protein of 10 kDa (IP-10, CXCL10) were detected with Ab pairs from R&D Systems. Thymus and activation-regulated chemokine (TARC, CCL17) and macrophage-derived chemokine (MDC, CCL22) were measured with ELISA kits from R&D Systems.

Preparation of RBC-conjugated Tat (RBC-Tat)
Recombinant Tat protein was expressed in Escherichia coli and purified to homogeneity by heparin-affinity chromatography. Tat or bovine ubiquitin (Sigma-Aldrich) was biotinylated by incubation with N-hydroxysuccinimmidobiotin (Pierce, Rockford, IL) in N,N-dimethylformamide (Sigma-Aldrich), as described previously [19 ]. Excess biotin was removed by dialysis overnight at 4°C against PBS. Protein concentration was determined by the Bradford method (Bio-Rad, Richmond, CA). Tat protein had undetectable endotoxin levels (<10 pg/mg) by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). Human RBC (10% hematocrit) were coupled to biotinylated Tat or ubiquitin by means of a biotin-avidin-biotin bridge, as described previously [19 , 24 ]. The percentage of RBC conjugated to Tat was evaluated by flow cytometry using a rabbit FITC-conjugated anti-Tat Ab (16 µg/ml). Based on the relative number of biotin molecules bound to human RBC (up to approximately 3000) and the capacity of RBC-bound avidin to bind one to three biotinylated proteins, we have estimated that every RBC carried between 3000 and 9000 molecules of biotinylated Tat, corresponding to 4.8–14.4 x 10-5 pg Tat protein per RBC [24 ].

Phagocytosis assay
RBC were stained with PKH-26 fluorescent dye (Sigma-Aldrich), per the manufacturer’s protocol. DC (106 cells/ml) were incubated with PKH-26-stained RBC (DC:RBC ratio, 1:4) in complete RPMI at 37°C or 4°C, and at selected time points, uptake was stopped by adding cold PBS containing 2% FBS and 0.01% NaN3. Cells were then washed and analyzed by flow cytometry. Surface-binding values obtained by incubating cells at 4°C were subtracted from values measured at 37°C.

Mixed leukocyte reaction (MLR) assay
T lymphocytes were purified (>95% CD3+) from the heavy density fraction (50–60%) of Percoll gradients followed by immunomagnetic depletion using a mixture of anti-MHC class II and anti-CD19 mAb-conjugated beads (Dynal). Immature or mature DC treated or not with IFN-{gamma} were washed and then cultured in 96-well microculture plates in serial dilutions (5x103–40 cells/well) together with purified, allogeneic CD3+ T lymphocytes (1.5x105 cells/well) in complete RPMI supplemented with 5% human serum. Cocultures were pulsed after 3–5 days with 1 µCi/well 3H-thymidine (Amersham, Little Chalfont, UK) for about 16 h at 37°C and were then harvested onto fiber-coated 96-well plates (Packard Instruments, Groningen, The Netherlands). Radioactivity was measured in a ß-counter (TopcountTM, Packard Instruments).

Induction of Tat-specific T-cell responses
CD4+ and CD8+ T cells were purified (>92%) by negative selection using immunomagnetic beads coated with anti-CD8 and anti-CD4 mAb, respectively. Autologous DC (106 cells/ml) were pulsed with soluble, recombinant Tat (0–5 µg/ml) or RBC-Tat (DC:RBC ratio, 1:4). DC pulsed with RBC-conjugated ubiquitin served as control. Where indicated, MCM (±IFN-{gamma}) was added to DC culture 3 h later. After 18 h, DC were washed and cultured at 5 x 103 cells/well with 105 purified CD4+ or CD8+ T cells in 96-well flat-bottom plates. T-cell proliferation was determined after 6 days by 3H-thymidine incorporation, and results are given as mean cpm ± SD of triplicate cultures. IFN-{gamma} and IL-4 were measured in the T-cell supernatants, 48–72 h after activation by ELISA, using matched pairs of mAb from BD PharMingen. T cells proliferating in response to RBC-Tat were expanded by adding 20 U/ml recombinant human IL-2 at day 6 and were then restimulated with DC pulsed with RBC-Tat at days 10–15, as described previously [25 ]. Antigen specificity of RBC-Tat-responsive T cells was assessed after 2–3 cycles of stimulation using mature DC (5x103 cells/well) pulsed with soluble Tat (5 µg/ml), RBC-Tat, or RBC-ubiquitin (RBC:DC ratio, 4:1). 3H-thymidine uptake was measured after 72 h.

Statistical analysis
The unpaired two-tailed Student’s t-test was used to compare differences in DC membrane marker expression, cytokine and chemokine release, T-cell proliferation, and IFN-{gamma} release. P values <=0.05 were considered significant.


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RESULTS
 
IFN-{gamma} regulates DC production of cytokines and chemokines and increases the allostimulatory capacity of DC
Monocyte-derived DC were propagated in medium containing 2% human serum and then matured with MCM, a culture system that allows the use of DC in humans [23 ]. Immature DC were CD1a-negative uniformly and expressed lower levels of CD40, CD80, and HLA-DR but higher amounts of CD86, MHC class I, and CD83 compared with DC cultured in bovine serum, as described previously [26 , 27 ]. Treatment with MCM, LPS, or sCD40L increased the expression of MHC class I, CD80, and CD83, whereas CD86 did not change significantly (Table 1) . In the first series of experiments, we tested the effects of IFN-{gamma} on various DC functions. Table 1 shows that IFN-{gamma} did not change the membrane phenotype of immature DC or DC maturated with MCM. Similarly, IFN-{gamma} did not affect the immunophenotype of DC coactivated with LPS or sCD40L. Conversely, IFN-{gamma} enhanced TNF-{alpha} release and increased IL-12 secretion from mature DC markedly, whereas production of IL-10 was reduced significantly (Fig. 1 ). IFN-{gamma} also had interesting effects on chemokine production by DC. Immature DC produced high levels of TARC and MDC, low amount of RANTES, and no IP-10. As expected, IFN-{gamma} up-regulated the release of IP-10, a chemokine with a prominent role in the attraction of type 1 T lymphocytes [28 ]. In contrast, IFN-{gamma} reduced the production of TARC significantly, which attracts type 2 cells preferentially via the CCR4 receptor [29 ], whereas it did not affect secretion of MDC, another CCR4 agonist, and RANTES (Fig. 2 ). To test the effects of IFN-{gamma} on the antigen-presenting functions of DC, the primary MLR assay was used. Mature DC were better stimulators of allogeneic T cells compared with immature DC, and IFN-{gamma} augmented the T-cell-activating properties of immature and mature DC (Fig. 3 ). Moreover, IFN-{gamma}-treated DC stimulated T cells to a higher IFN-{gamma} production (immature DC: 0.1±0.05 vs. 0.6±0.8 ng/ml; P<0.05; mature DC: 4.3±0.7 vs. 7.2±1.1 ng/ml; P<0.03).


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  		Table 1. IFN-{gamma} Does Not Affect the Membrane Phenotype of DC Cultured in Human Serum



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  		Figure 1. IFN-{gamma} enhances the release of IL-12 and TNF-{alpha} and reduces IL-10 secretion from DC, which were generated from purified peripheral blood CD14+ cells cultured for 6 days with GM-CSF and IL-4. Thereafter, CD2+ and CD19+ cells were removed by immunomagnetic beads, and DC stimulated with LPS (10 µg/ml), sCD40L (1 µg/ml), or MCM (1:1 v/v) in the presence (solid bars) or not (open bars) of IFN-{gamma} (50 U/ml). After 18 h, supernatants were collected, and IL-12 (A), TNF-{alpha} (B), and IL-10 (C) were measured by ELISA. Results are expressed as mean ± SD of triplicate cultures. *, P < 0.05 versus IFN-{gamma}-untreated DC. One of four experiments from three different donors is shown.



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  		Figure 2. IFN-{gamma} augments and decreases DC production of IP-10 and TARC, respectively. DC were stimulated with LPS, sCD40L, or MCM in the absence (open bars) or presence (solid bars) of IFN-{gamma}. After 18 h, IP-10 (A), TARC (B), RANTES (C), and MDC (D) were measured in the supernatants by ELISA. Results are expressed as mean ± SD of triplicate cultures. *, P < 0.05 versus IFN-{gamma}-untreated DC. Shown is one of three or four experiments performed.



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  		Figure 3. IFN-{gamma} increases the allostimulatory capacity of DC. Immature DC (A) or DC stimulated with LPS (B), sCD40L (C), or MCM (D) were treated concomitantly (solid symbols) or not (open symbols) with IFN-{gamma}. After 18 h, DC were washed extensively and then cocultured in graded numbers with purified, allogeneic CD3+ T cells. 3H-thymidine uptake was measured after 3 days. Results are the mean cpm ± SD of triplicate cultures. *, P < 0.05 versus IFN-{gamma}-untreated DC. One experiment out of four performed is shown.

RBC-Tat are internalized effectively by DC and are efficient at inducing Tat-specific T-cell responses in vitro
Next, we evaluated whether RBC-Tat were delivered into DC and could induce specific T-cell responses. RBC were coated with Tat by means of avidin-biotin bridges. This method allowed the preparation of about 80% RBC-Tat (Fig. 4A ), and each RBC carried 4.8–14.4 x 10-5 pg Tat protein, corresponding to 48–144 pg Tat/106 RBC [24 ]. DC internalized RBC effectively and rapidly, with a half-maximum uptake after 8 h (Fig. 4B) . Phagocytosis of RBC was confirmed by transmission electron microscopy and was inhibited markedly by pretreatment of DC with cytochalasin D (unpublished results). The ability of DC to internalize RBC was not affected by the maturation stage of DC or by treatment with IFN-{gamma}. In subsequent experiments, we compared DC pulsed with RBC-Tat or soluble Tat in their capacity to induce primary T-cell responses. To this end, DC were prepared from healthy and HIV-seronegative donors (and thus naive for HIV antigens), exposed to soluble Tat or RBC-Tat, and then incubated with autologous, purified CD4+ or CD8+ T lymphocytes. As shown in Figure 5A and 5B , DC could induce a strong CD4+ and CD8+ T-cell proliferation to a concentration of soluble Tat >0.5 µg/ml. However, DC incubated with RBC-Tat were much more efficient at inducing specific CD4+ and CD8+ T-cell responses compared with DC pulsed with soluble Tat. Indeed, incubation of DC with RBC-Tat at a 4:1 ratio, corresponding to an antigen dose of about 0.4 ng/ml, was sufficient to elicit an effective CD4+ and CD8+ activation in terms of cell proliferation and IFN-{gamma} production (Fig. 5C 5D 5E 5F ). Thus, using RBC-Tat, 1250-fold less amount of antigen was required to obtain a T-cell response similar to that induced by soluble antigen. DC pulsed with RBC-conjugated ubiquitin did not elicit any T-cell response. Moreover, DC matured with MCM were more potent than immature DC at presenting RBC-Tat and soluble Tat, with significantly stronger T-cell proliferation (P<0.03) and IFN-{gamma} release (P<0.05). Treatment of DC with IFN-{gamma} did not change their capacity to induce T-cell proliferation. However, DC exposed to IFN-{gamma} stimulated T cells to a significantly higher IFN-{gamma} production (P<0.05; Fig. 5E and 5F ). Very limited amounts of IL-4 were released by T cells or could be visualized by intracellular staining in all conditions tested (unpublished results). To assess the antigen specificity of T cells responding to RBC-Tat, T-cell lines were generated by multiple cycles of stimulation and then tested for their reactivity to Tat and an irrelevant antigen. As shown in Figure 6 , CD4+ and CD8+ T-cell lines proliferated in response to soluble Tat and RBC-Tat, whereas no response was detected with RBC-conjugated ubiquitin. This finding excluded the possibility that the T-cell response to RBC-Tat was directed against avidin-biotin-modified RBC.



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  		Figure 4. RBC-Tat are internalized by DC. (A) Flow cytometry analysis of RBC-Tat stained with a rabbit anti-Tat Ab (solid curve). As control, RBC-conjugated ubiquitin were stained with the same Ab (open curve). (B) DC were cultured in medium alone ({circ}) or in the presence of MCM ({square}) for 18 h at 37°C. Thereafter, DC were washed and incubated with PKH-26-stained RBC (DC:RBC ratio, 1:4) at 37°C. At various time points, cells were washed with cold PBS, and RBC accumulation was monitored by flow cytometry. Dashed line represents the fluorescence of DC pulsed with PKH-26-stained RBC and maintained at 4°C. Fluorescence of cells incubated at 4°C was subtracted from the fluorescence of cells incubated at 37°C.



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  		Figure 5. RBC-Tat is more efficient than soluble Tat at inducing specific T-cell responses. (A and B) DC were incubated for 18 h with increasing doses of soluble Tat and were then cocultured with autologous, purified CD4+ or CD8+ T-cell lines. (C–F) DC (106 cells/ml) were pulsed with two doses of soluble Tat or with RBC-Tat at a DC-to-RBC ratio of 1:4, corresponding to about 0.4 ng/ml soluble Tat. As control, DC were also pulsed with RBC-conjugated ubiquitin. After 3 h, DC cultures were left untreated or added with MCM in the presence or not of IFN-{gamma} for an additional 18 h. DC (5000 cells/well) were then used to activate CD4+ (C and E) and CD8+ (D and F) T cells. 3H-thymidine uptake (A–D) and IFN-{gamma} secretion (E and F) were measured. *, P < 0.05 between immature and mature DC; {circ}, P < 0.03 between DC treated or not with IFN-{gamma}. Similar results were obtained in two (A and B) or seven (C–F) independent experiments from three different donors.



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  		Figure 6. Antigen specificity of T-cell lines generated with RBC-Tat. CD4+ (A) and CD8+ (B) T cells proliferating in response to DC pulsed with RBC-Tat, treated with MCM, IFN-{gamma}, or MCM plus IFN-{gamma}, or left untreated (x-axes) were expanded by adding 20 U/ml IL-2 at day 6 and were then restimulated with DC pulsed with RBC-Tat at days 10–15. Antigen specificity of these T cells was assessed after 2–3 cycles of stimulation using mature DC (5x103 cells/well) pulsed with soluble Tat (5 µg/ml), RBC-Tat or RBC-ubiquitin (RBC:DC ratio, 4:1), and CD4+ or CD8+ T cells (5x104 cells/well). 3H-thymidine uptake was measured after 72 h.


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DISCUSSION
 
High-level, HIV-specific immune responses are associated with a delay in disease progression and with protection from infection in high-risk, exposed individuals. CD4+ and CD8+ T-cell responses are necessary to guarantee effective immunity [22 , 30 ]. CD8+ T cells can lyse HIV-infected cells efficiently and thus prevent virus spreading. CD4+ T cells are important for inducing maturation of DC that in turn are essential for CD8+ T-cell priming and the maintenance of CD8+ T-cell memory. Moreover, CD4+ and CD8+ T cells can secrete IFN-{gamma}, which inhibits HIV replication [31 , 32 ], or chemokines that block virus entry into cells. The early and specific impairment of the CD4+ T-cell response following HIV infection limits the efficacy of CD8+ immune responses [22 ]. Therefore, anti-HIV vaccines capable of stimulating CD4+ and CD8+ T cells and administered before the appearance of major immune damage may have improved, protective efficacy. Tat has been indicated as a relevant target for vaccination against HIV infection because it is produced early after infection, is released extracellularly by infected cells, and is essential for virus replication. Moreover, Tat is involved in the pathogenesis of AIDS-associated Kaposi’s sarcoma and can exert multiple immune-suppressive activities [33 , 34 ]. Finally, studies in nonhuman primates have suggested that Tat-specific CD8+ T lymphocytes are potent inhibitors of initial simian immunodeficiency virus replication [35 ], and vaccination with Tat protein was effective in controlling virus replication [36 , 37 ].

In this study, we compared the capacity of DC pulsed with soluble Tat and RBC-Tat to induce specific CD4+ and CD8+ T-cell responses in vitro. DC prepared in medium devoid of bovine serum internalized RBC-Tat efficiently and rapidly, a process that was not affected by the maturation stage of DC or by treatment with IFN-{gamma}. Compared with DC treated with soluble Tat, DC pulsed with RBC-Tat were much more efficient at eliciting specific CD4+ and CD8+ T-cell responses, requiring about 1250-fold less antigen to induce a similar T-cell response. This antigen delivery system uses a limited amount of Tat and can thus be advantageous compared with others that use transduction of DC with Tat DNA or RNA, for instance. In fact, Tat has well-recognized, immunosuppressive effects such as induction of T-cell apoptosis [38 ], and an unrestricted production of the protein by DC may impair the generation of an optimal T-cell response. The use of RBC as an antigen-delivery system can also offer another advantage. In fact, RBC can enhance T-cell expansion and survival by counteracting intracellular, oxidative stress [39 ].

In keeping with previous observations [26 , 27 ], DC generated from monocytes under serum-free conditions expressed lower amounts of CD40, CD80, and HLA-DR but higher levels of CD86, MHC class I, and CD83 compared with DC cultured in bovine serum. Treatment with MCM, LPS, or sCD40L increased the expression of MHC class I, CD80, and CD83 but not of CD86. Moreover, DC matured with MCM were shown to be more effective than immature DC at inducing CD4+ and CD8+ T-cell proliferation and IFN-{gamma} release [40 ]. In addition, we investigated whether IFN-{gamma} could improve the functions of DC and substitute for CD4+ T-cell help, because IFN-{gamma} is an inducer of IL-12 production in mature DC [6 , 7 ]. Indeed, DC maturated in the presence of IFN-{gamma} showed a significantly augmented IL-12 and TNF-{alpha} secretion. In contrast, IFN-{gamma} down-regulated the production of IL-10, a cytokine with relevant immunosuppressive activities on DC functions [41 ], demonstrating that IL-12 and IL-10 are regulated by IFN-{gamma} reciprocally. Moreover, IFN-{gamma}-treated DC up-regulated the release of IP-10 markedly and reduced the secretion of TARC significantly, chemokines attracting mainly Th1 and Th2 cells, respectively. The preferential release of IP-10 as well as of other Th1-active chemokines [42 ], together with the high secretion of IL-12, can be very important for an effective Th1 polarization of the immune response. Other than recruiting Th1 cells, IP-10 can in fact mediate an antigen-independent activation signal directly on T cells and favor Th1-dominated responses [28 ]. Finally, DC exposed to IFN-{gamma} induced a higher IFN-{gamma} production from Tat-specific CD4+ and CD8+ T lymphocytes, although their proliferative capacity was not affected.

In aggregate, the results show that erythrocytes can be used effectively to deliver Tat antigen into DC and that IFN-{gamma} enhances the capacity of DC to induce type 1 immune responses, which are involved primarily in the protection against viral infections. This approach might be applied for the design of vaccination strategies against HIV infection, including in vivo active immunization or ex vivo priming of T cells for adoptive T-cell therapy.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Istituto Superiore di Sanità (AIDS projects), the Ministero della Sanità, and the Associazione Italiana per la Ricerca sul Cancro.

Received August 24, 2001; revised October 26, 2001; accepted December 5, 2001.


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