HIV-1-Specific CTL Responses Primed In Vitro by Blood-Derived Dendritic Cells and Th1-Biasing Cytokines

DC generation

PBMC were isolated from heparinized peripheral blood obtained by venipuncture from normal donors using density centrifugation. After four or five washes with HBSS (Life Technologies, Grand Island, NY), 10⁷ cells/ml serum-free AIM-V medium (Life Technologies) were plated in flasks and incubated for 1–1.5 h at 37°C. Nonadherent cells were removed with gentle washes, and plastic-adherent cells were cultured for 5–10 days in AIM-V medium supplemented with 1000 U/ml rIL-4 and rGM-CSF. Following this culture period, nonadherent cells (DC) were harvested and further purified as necessary by discontinuous density centrifugation on a layer of Nycoprep 1.064 (Nycomed, Oslo, Sweden):LSM (Organon-Teknika, Durham, NC), 9:1, 1000 × g for 10 min. Cells generated in this fashion were determined to be >90% DC based on morphology and the expression of a CD3/CD14/CD16/CD20-negative, MHC class II⁺, CD40⁺, CD80⁺, CD86⁺ phenotype assessed by direct immunofluorescence assays monitored by flow cytometry. Day 7 yields were approximately 5–15% of starting normal donor PBMC numbers.

Plasmid DNA

The plasmid pCMV-A-hIFNα2b was constructed by ligating a NotI-EcoRI fragment containing the hIFNα2b cDNA (kindly provided by Dr. Paul Zavodny, Schering-Plough Research Institute, Kenilworth, NJ) into CMV-A. pCMV-A-hIL-12 (p40-IRES-p35) was constructed by ligating a BamHI fragment containing the IRES sequence from EMCV followed by the human p35 cDNA 22 into pCMV-A-p40 (kindly provided by Dr. Will Swain, Auragen/Geniva, Madison, WI). The plasmid pCI-Nef was constructed by subcloning the ORF of HIV1-Nef (LAI strain) into the expression plasmid pCI (Promega, Middleton, WI). Using PCR techniques, a SalI-NotI-fragment was generated from a proviral construct containing HIV-Nef (kindly provided by S.-Y. Kim, Seoul, Korea) and ligated into pCI. The insert was sequenced in both directions to exclude mutations introduced by PCR. Plasmids were grown in Escherichia coli strain DH5α and purified using Qiagen Endofree Plasmid Maxi Kits (Qiagen, Chatsworth, CA).

Particle-mediated gene transfer to DC

Plasmid DNA was precipitated onto 2.6-μm gold particles at a density of 2 μg of DNA per mg of particles. Briefly, gold particles and DNA were resuspended in 100 μl of 0.05 M spermidine (Sigma Chemical, St. Louis, MO), and DNA was precipitated by the addition of 100 μl of 1 M CaCl₂. Particles were washed in dry ethanol to remove H₂O, resuspended in dry ethanol containing 0.075 mg/ml PVP (Sigma), and coated onto the inner surface of Tefzel tubing using a tube loader. The tubing was cut into 0.5-inch segments, resulting in the delivery of 0.5 mg of gold coated with 1 μg of plasmid DNA per transfection with the Accell helium pulse gun. Gold particles, tubing, tube loader, and the Accell helium pulse gun were kindly provided by Auragen/Geniva. DC were transfected in suspension in six-well plates. DC were harvested and pelleted by centrifugation, and 2 × 10⁶ cells were resuspended in 20 μl of fresh medium and spread evenly in the center of each well. Cells were bombarded at a pressure of 300 psi of helium, and fresh culture medium was added immediately.

Induction of primary T cell responses

For protein pulsing experiments, day 5–10 DC were pulsed overnight with recombinant HIV-1 Nef (LAI strain) (AGMED, Bedford, MA) at a concentration of 20 μg/ml, washed, irradiated with 3000 rad, and cocultured with autologous nonadherent PBMC at a ratio of 1 DC:20–50 PBMC in AIM-V medium supplemented with 5% human AB serum. In some experiments, recombinant cytokine hIL-12 (final concentration, 500 pg/ml) or hIFNα-2b (final concentration, 100 U/ml) (Schering-Plough) was added to the DC-PBMC coculture on day 1 of induction. Proliferating responder T cells were restimulated weekly with irradiated, autologous DC pulsed with 5 μg/ml Nef overnight and grown in AIM-V 5% human AB serum with 10 U/ml rIL-2. For gene delivery experiments, DC cultured for 5–10 days were irradiated with 3000 rad and transfected with the pCI-Nef plasmid by particle-mediated transfer as described above. For coexpression of cytokines, DC were transfected first with pCI-Nef, then in a subsequent “shot” with pCMV-A-hIFNa2b or pCMV-A-hIL-12 plasmids, and cocultured with autologous responder cells as described above. Responder T cells were restimulated weekly with autologous DC transfected with pCI-Nef only. At the end of each restimulation period (7–10 days), T cells were tested for Nef-specific proliferation and lytic activity.

Cytotoxicity assays

Autologous B-lymphoblastoid cell line (B-LCL) or MHC class I-matched allogeneic B-LCL were incubated with 10–20 μg/ml of Nef peptide and 100–200 μCi of ⁵¹Cr in a total volume of 200 μl for 1–2 h at 37°C before use as targets. Targets were washed and added to plates at 5 × 10³ cells/well in 100 μl. Responder T cells were plated at varying (2 or 3) E:T ratios in triplicate and assayed for cytotoxicity in a standard chromium release assay. The percentage of specific ⁵¹Cr release was calculated as 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release). Lytic activity was expressed as the percentage specific lysis, or percentage lysis of HIV Ag-expressing targets minus percentage lysis of non-HIV Ag-expressing targets.

Peptides

Peptides spanning the entire Nef protein (LAI strain of HIV-1) were kindly supplied by Dr. Bruce Walker (Massachusetts General Hospital, Boston, MA) and consisted of sequential 20-amino acid (aa) peptides overlapping by 15 aa. P1–P6 represent pools of these overlapping peptides, with 4 peptides per pool (except P6, which contains the terminal 3 peptides) with P1 containing the N-terminus and P6 containing the C-terminus of Nef. The minimal epitope peptides were synthesized at the University of Pittsburgh Cancer Institute Peptide Synthesis facility using standard F-moc chemistry and purified by reverse-phase HPLC, with purity exceeding 90% based on mass spectrometry for m.w. All minimal epitope peptides are based on the LAI strain amino acid sequence of HIV-1 Nef 21 .

HLA typing

HLA typing was performed by Dr. P. Morel (University of Pittsburgh) or Children’s Hospital HLA Laboratory (University of Pittsburgh Medical Center) using standard serotyping assays.

MHC class I peptide binding assay

Binding of peptides to HLA-A2 and HLA-B7 were assessed using a class I reconstitution assay, as previously described 23 . Briefly, the class I-reduced B cell line transfectant expressing HLA-A2.1 (C1R.A2) or HLA-B7 (C1R.B7) was treated with a citrate-phosphate solution (pH 3.3) to denature preexisting class I complexes, as determined by loss of binding to conformation-dependent mAb (W6/32). Acid-treated cells were incubated with varying concentrations of peptides overnight in the presence of β2-microglobulin (Sigma, 5 μg/ml). Cells were washed, fixed, stained with FITC-conjugated W6/32 mAb or anti-A2 mAb (BB7.2), and evaluated by flow cytometric analysis.

Flow cytometry

For immunophenotyping, DC or T cell responders were washed in HBSS supplemented with 1% BSA and 0.1% NaN₃ and incubated (30 min at 4°C) with one of the following mAb: phycoerythrin (PE)-conjugated anti-HLA-DR (Becton Dickinson, Mountain View, CA), FITC-conjugated anti-CD80 (Ancell, Bayport, MN), FITC-conjugated anti-CD86 (PharMingen, San Diego, CA), FITC-conjugated anti-CD40 (PharMingen), PE-conjugated anti-CD3 (Becton Dickinson), FITC-conjugated anti-CD4 (Becton Dickinson), PE-conjugated anti-CD8 (Becton Dickinson), FITC-conjugated anti-CD14 (Becton Dickinson), PE-conjugated anti-CD16 (Becton Dickinson), and FITC-conjugated anti-CD20 (Becton Dickinson). DC were also stained with corresponding isotype-matched control mAb (PharMingen). Surface expression was analyzed using a FACScan flow cytometer (Becton Dickinson) and Lysis II software, with data being collected on 5000 to 10000 viable cells.

MHC class I-restricted, Nef-specific T cell responses can be primed in vitro by autologous DC from seronegative donors, engineered to coexpress HIV-1 Nef and Th1-biasing cytokines IL-12 or IFN-α

We have previously shown that DC engineered to express tumor Ags in vitro are potent stimulators of MHC class I-restricted, tumor-specific CTL 16 . In the present study, we sought to determine whether DC engineered to express an HIV Ag could induce primary HIV-specific T cell responses in vitro and whether this in vitro vaccine model could serve to identify immunogenic epitopes as potential vaccine targets. Cultured DC were transfected with HIV-1 Nef plasmid cDNA by particle-mediated gene transfer as described above. Expression of HIV-1 Nef was confirmed in 1–5% of DC by immunohistochemical staining with Nef-specific mAb (data not shown). Additionally, in previous studies, transfection of cultured DC with cDNA encoding hIL-12 and/or IFN-α cDNA using a gene gun was observed to result in modest levels of cytokine production (50–150 pg/million DC/48 h), increased expression of DC-associated costimulatory molecules, and enhanced induction of tumor-reactive CTL in vitro 16 . We investigated whether coinsertion of the plasmids encoding hIL-12 and hIFN-α with the Nef-encoding plasmid could enhance the efficiency of induction of Nef-specific CTL or alter the pattern of CTL epitopes recognized.

HIV-uninfected donors expressing the common HLA class I molecules HLA-A2, HLA-A3, or HLA-B7 were initially evaluated as responders. Using bioballistic gene transfer, cultured DC were transfected with pCI-Nef alone or with pCI-Nef plus IL-12 or IFN-α cDNA, irradiated, and used to stimulate autologous responder T cells. Responder T cells were restimulated with autologous DC expressing Nef, without coexpression of IL-12 or IFN-α, and tested for the ability to lyse HLA-matched target cells pulsed with defined Nef epitope peptides or overlapping, pooled Nef peptides. CTL responses restricted by several different HLA class I molecules and targeting to defined epitope peptides, as well as larger, overlapping peptides, were noted in multiple individuals. Fig. 1⇓ depicts the HLA-B7-restricted Nef peptide-specific CTL responses induced by autologous DC engineered to express Nef alone or to coexpress Nef and hIL-12 or hIFN-α during priming. A response against only the P6 pool of peptides was noted following stimulation with DC-Nef alone, whereas IL-12-primed T cells responded to the P6 pool and weakly to the P2 peptide pool. IFN-α-primed T cells did not respond strongly to the P6 peptides, but instead displayed low level lysis of several other peptide pools. These results suggest that in certain cases the addition of cytokines to primary DC-T cell cocultures may result in differences in the epitope specificity of the Ag-specific T cell responses generated. These data may reflect differences in processing or presentation of peptides under differing cytokine conditions or reflect skewing of the T cell repertoire resulting from error in sampling of low frequency responses.

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FIGURE 1.

Lysis of HLA-B7⁺ targets pulsed with pooled Nef peptides by T cells primed in vitro with Nef-transfected DC. Nonadherent PBMC from HIV-seronegative donor LP1.31 (HLA-B7⁺) were stimulated with autologous DC transfected with HIV-1 nef cDNA with or without hIL-12 or IFN-α cDNA as described in Materials and Methods and tested after two restimulations for their ability to lyse B7⁺ target cells (C1R.B7) pulsed with pooled, overlapping Nef peptides, P1–P6, or no peptide at an E:T ratio of 20:1. Lytic activity was expressed as the percentage peptide-specific lysis, or percentage lysis of peptide-pulsed targets minus percentage lysis of targets without peptide.

No HLA B7-restricted CTL epitopes had previously been identified in the C-terminal region of Nef spanned by the P6 peptides, suggesting that CTL with a novel epitope specificity were generated using this approach. A single B7-restricted epitope within the P2 pool, Nef 68–76 (FPVTPQVPL), was previously identified in HIV-infected individuals by Haas et al. 24 . This peptide was synthesized, was determined to be a strong HLA-B7 binder (Fig. 2⇓A) in an HLA-B7 reconstitution assay 23 , and was used to pulse B7-expressing targets in a chromium release assay. Specific killing of this peptide was mediated by responder cells from the donor depicted in Fig. 1⇑, which were stimulated with DC-Nef + IL-12 and to a lesser extent DC-Nef + IFN-α, but not DC-Nef alone (Fig. 2⇓B). Interestingly, no cytotoxic response to the P3 pool of peptides, which contains another previously reported immunodominant B7-restricted epitope (Nef 128–137, TPGPGVRYPL) 24 , was identified in this donor following any of the evaluated priming conditions.

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FIGURE 2.

Recognition of HLA-B7-restricted Nef epitope by T cells primed with DC transfected with nef and cytokine cDNA. A, Defined epitope peptides Nef 68–76 (FPVTPQVPL, amino acid sequence located within the P2 peptide pool) and Nef 71–79 (TPQVPLRPM, located within the P3 peptide pool) were tested for HLA-B7 binding as described in Materials and Methods. HIV gp41 843–851 (IPRRIRQGL) and Mart-1 27–35 (AAGIGILTV, A2-restricted epitope) peptides were run as positive and negative controls, respectively. B, Donor LP1.31 bulk T cells stimulated with autologous DC transfected with HIV-1 nef cDNA with or without hIL-12 or IFN-α cDNA and restimulated twice with Nef-expressing DC as described (Fig. 1⇑) were tested for recognition of HLA-B7⁺ targets pulsed with defined B7-restricted Nef epitope peptide, Nef 68–76. Donor LP1.31 bulk T cells stimulated with MAGE1-transfected DC were also evaluated as a negative control. The E:T ratio was 10:1.

In addition to evaluating CTL reactivity against pooled, overlapping peptides, DC-stimulated responder T cells were also tested directly against targets pulsed with defined peptide epitopes, in some cases. Three well-characterized HLA-A2-restricted CTL epitopes in Nef have been previously identified in HIV-infected individuals: Nef 136–145 (PLTFGWCYKL), Nef 180–189 (VLEWRFDSRL), and Nef 190–198 (AFHHVAREL) 21, 24, 25 . Using the described MHC class I reconstitution assay 23 , we determined the relative binding affinities of each of these peptides for HLA-A2.1, with relative A2 binding of Nef 136 > Nef 180 ≫ Nef 190 reproducibly detected in multiple assays (Fig. 3⇓A). Nef 190–198 bound weakly to HLA-A2.1 in this sensitive assay, but we and others have been unable to detect binding of this peptide to HLA-A2.1 using the T2 binding assay 26 . PBMC from nine HIV-1-seronegative donors expressing HLA-A2 were stimulated with DC engineered to express HIV-1 Nef with or without IL-12 or IFN-α. Following three stimulations with DC transfected with nef only, responder cells were tested for their ability to kill targets expressing only HLA-A2.1 (C1R.A2) when pulsed with the above epitope peptides. Nef peptide-specific CTL responses (>10% specific lysis) were identified in five of the nine donors, with two donors responding to two different peptides (Table I⇓). Equal numbers of responses against peptides 136–145 and 190–198 were identified, whereas only a single donor responded weakly to Nef peptide 180–189. This pattern of CTL responses would not necessarily have been predicted based only on binding affinities to HLA-A2.1 (Fig. 3⇓A), as Nef 180–189 consistently bound to HLA-A2 better than did Nef 190–198, yet was poorly recognized by responder T cells in this system.

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FIGURE 3.

Nef peptide-specific, HLA-A2-restricted CTL responses augmented by priming with IL-12 and IFN-α. A, The following peptides, identified as HLA-A2-restricted CTL epitopes in HIV-infected individuals, were tested for A2.1 binding as described: Nef 136–145 (PLTFGWCYKL), Nef 180–189 (VLEWRFDSRL), and Nef 190–198 (AFHHVAREL). Peptides derived from influenza matrix (Flu 58–66, GILGFVFTL) and Mage-1 (aa 161–169, EADPTGHSY, HLA-A1-restricted epitope) were run as positive and negative controls, respectively. B, Nonadherent PBMC from donor 1 (Table I⇓) stimulated with autologous DC transfected with HIV-1 nef cDNA with or without hIL-12 or IFN-α cDNA and restimulated twice with Nef-expressing DC as described were tested for recognition of HLA-A2⁺ targets (C1R.A2) pulsed with defined A2-restricted Nef epitope peptides, Nef 136–145, Nef 180–189, and Nef 190–198, or no peptide. Lytic activity was expressed as the percentage peptide-specific lysis or percentage lysis of peptide-pulsed targets minus percentage lysis of targets without peptide.

With only one exception, the addition of Th1-biasing cytokines during priming enhanced the induction of CTL responses (Table I⇑ and Figs. 3⇑B and 4). Overall, IL-12 was slightly more effective than IFN-α in enhancing the induction of such responses. Figs. 3⇑B and 4, A and B, depict the Nef peptide-specific responses of the two donors in whom responses against more than one peptide were identified (donors 1 and 5; Table I⇑). The pattern of CTL responses in donor 1 responders (Fig. 3⇑B) shows that the addition of either IL-12 or IFN-α during the primary stimulation led to detection of stronger CTL responses and that the pattern of epitope specificity generated under all conditions was similar. As shown in Fig. 4⇓, Nef peptide-specific lysis was only detected in those T cell cultures coprimed with IL-12, with specific lysis of Nef 190 > N180 > N136-pulsed targets mediated by CTL from donor 5.

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FIGURE 4.

Nef peptide-specific, HLA-A2-restricted CTL responses augmented by priming with IL-12. Nonadherent PBMC from donor 5 (Table I⇑) stimulated with autologous DC transfected with HIV-1 nef cDNA with or without hIL-12 or IFN-α cDNA and restimulated twice with Nef-expressing DC as described were tested for recognition of HLA-A2⁺ targets (C1R.A2) pulsed with defined A2-restricted Nef epitope peptides, Nef 136–145 (PLTFGWCYKL), Nef 180–189 (VLEWRFDSRL), and Nef 190–198 (AFHHVAREL), or no peptide. A, Lysis of peptide-pulsed targets by the three bulk responder populations at a single E:T ratio. B, Lysis of peptide-pulsed targets by bulk responder T cells from the same donor primed with DC transfected with nef and IL-12 cDNA at three E:T ratios.

MHC class I-restricted Nef-specific T cell responses can be primed in vitro by autologous DC from seronegative donors, pulsed with recombinant Nef protein and recombinant IL-12 or IFN-α

DCs have been reported to have the ability to process exogenous proteins for presentation by MHC class I Ags 27, 28 . We therefore sought to determine whether primary class I-restricted responses to the HIV-1 Nef protein could be induced by DC pulsed with recombinant protein and whether induction in the presence of recombinant Th1-biasing cytokines would enhance or alter this process. DC cultured from HIV-seronegative donors were pulsed with recombinant HIV-1 Nef protein, irradiated, and used as APC to stimulate autologous nonadherent T cells as described in Materials and Methods. Responder cells were restimulated weekly with irradiated, Nef protein-pulsed DC and maintained in media with low dose rhIL-2. HIV-specific cytotoxicity was evaluated using HLA-matched or partially matched B-LCL pulsed with defined minimal Nef epitope peptides or with pools of larger peptides spanning the entire Nef protein sequence (P1–P6). MHC class I or class II restriction of responses was determined either by using targets matched only at certain class I alleles or by blocking with anti-MHC I monomorphic (W6/32) or MHC II monomorphic (L243) mAbs. Strong MHC class II-restricted responses to HIV Nef were noted in the majority of donors tested (data not shown; C. C. Wilson, manuscript in preparation), and Nef-specific CTL responses were detected in three of six donors tested. Significant CTL responses were generally first noted after two or three rounds of stimulation, with marked enhancement of responses noted after four or five stimulations. The majority of responder T cells stimulated in this fashion were CD3⁺CD4⁺ (70–90% after three stimulations), but one donor generated primarily a CD3⁺CD8⁺ T cell response in response to stimulation with DC pulsed with Nef protein (Fig. 5⇓A). Fig. 5⇓A illustrates HLA-A3-restricted killing of target cells pulsed with a previously identified Nef peptide (Nef 73–84, QVPLRPMTYK) by T cells from an HLA-A3⁺, B7⁺ HIV-seronegative donor, after five stimulations with DC pulsed with rNef protein. The B7-restricted peptide (Nef 128–137, TPGPGVRYPL) determined to be an immunodominant epitope in HIV-infected individuals 24 was not recognized by this bulk T cell population.

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FIGURE 5.

MHC class I-restricted recognition of Nef epitope peptides after in vitro priming with DC pulsed with Nef protein and enhancement with addition of rIL-12 or IFN-α during T cell priming. A, Nonadherent PBMC from donor I.D. (HLA-A3⁺, B7⁺) were stimulated with autologous DC pulsed overnight with recombinant HIV-1 Nef protein and restimulated with Nef protein-pulsed DC weekly for 4 wk. Bulk responder T cells were tested for their ability to lyse HLA-A3⁺ or HLA-B7⁺ targets pulsed with or without defined Nef epitope peptides, Nef 73–82 (A3 restricted) and Nef 128–137 (B7 restricted), in a 4-h standard chromium release assay. Responder cells were phenotypically 91% CD3⁺CD8⁺ and 9% CD3⁺CD4⁺. B, Nonadherent PBMC from donor D.M. (HLA-A11⁺, A24⁺, B54⁺) were stimulated with autologous DC pulsed overnight with recombinant HIV-1 Nef protein and either rIL-12 or rIFN-α as described in Materials and Methods. After two subsequent stimulations with DC pulsed with rNef protein (without cytokines), responder T cells were tested for the ability to lyse autologous B-LCL pulsed with pooled, overlapping Nef peptides, P1–P6, or no peptide at an E:T ratio of 20:1. Lytic activity was expressed as the percentage peptide-specific lysis, or percentage lysis of peptide-pulsed targets minus percentage lysis of targets without peptide. Specific lysis could be blocked by the addition of anti-MHC mAb (W6/32) (data not shown).

The addition of either rIL-12 or IFN-α to the cultures, in concentrations similar to those expressed by gene gun-transfected DC, only during the initial stimulation, led to the subsequent enhancement of HIV peptide pool-specific CTL responses, as depicted in a representative assay in Fig. 5⇑B. T cells from this donor (D.M., HLA-A11⁺, A24⁺, B54⁺) specifically recognized autologous targets pulsed with the P3 and P6 pooled Nef peptides. These responses were enhanced following priming with IL-12 or IFN-α. Although minimal epitope specificities were not mapped in this case, it is interesting to note that both previously identified A11-restricted Nef epitopes, Nef 75–82 (PLRPMTYK) and Nef 84–92 (AVDLSHFLK) 17, 29 , are located within the P3 pool of Nef epitopes tested.

Mechanism of cytokine-mediated enhancement of DC-dependent T cell induction

To determine the mechanism by which IL-12 and IFN-α enhanced the induction of HIV-specific T cell responses in this system, we evaluated the effect of each cytokine on T cell and DC phenotype. T cells stimulated with DC transfected with the nef gene or pulsed with Nef protein were predominantly of CD3⁺CD4⁺ phenotype (80 ± 11%, 74 ± 16% respectively). As previously described, T cell cultures induced by DC cotransfected with IL-12 or IFN-α cDNA and tumor Ag cDNA displayed cytokine-dependent phenotypic changes 16 . In these experiments, the coexpression or addition of IL-12 to DC at the time of T cell priming led to a significant (95% confidence, Student’s t test) expansion of CD8⁺ T cells, markedly reducing the calculated CD4:CD8 ratio after three stimulations (Table II⇓). Despite the addition of IL-12 during T cell priming, the majority of DC-stimulated cultures still contained a predominance of CD4⁺ T cells (absolute CD4:CD8 ratio > 1) (Table II⇓ legend). IFN-α, expressed or added during priming of T cells, had a minimal effect on subsequent T cell phenotype, only slightly increasing the CD4:CD8 ratio in most cases (Table II⇓).

We next evaluated the impact of each cytokine on DC phenotype. We previously reported that the coexpression of IL-12 or IFN-α with tumor Ag in DC by bioballistic gene transfer led to phenotypic changes in the DC, with up-regulation of MHC and costimulatory molecules most markedly following expression of IFN-α. In these experiments, we observed that the addition of rIFN-α to DC cultures also up-regulated expression of CD40, CD80, CD86, and MHC I and II on GM-CSF/IL-4-cultured DC, whereas rIL-12 had little or no impact on DC phenotype, even at concentrations of up to 10 ng/ml cytokine (Fig. 6⇓).

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FIGURE 6.

Effect of recombinant cytokines IL-12 and IFN-α on DC phenotype. DC were grown for 7 days in hIL-4 and GM-CSF and then treated with the addition of the indicated cytokine at 10 ng/ml for 48 h, at which time FACS analysis was performed. Fold control mean fluorescence channel (MFC) represents the ratio of cytokine-treated DC MFC to untreated DC MFC.