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The Journal of Immunology, 1998, 161: 868-876.
Copyright © 1998 by The American Association of Immunologists

Human Dendritic Cell (DC)-Based Anti-Infective Therapy: Engineering DCs to Secrete Functional IFN-{gamma} and IL-121

Seema S. Ahuja2,*, Srinivas Mummidi*, Harry L. Malech{dagger} and Sunil K. Ahuja*

* University of Texas Health Science Center, San Antonio, TX 78284; and {dagger} Laboratory of Host Defenses, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An imbalance in the Th1- and Th2-type cytokine responses may allow certain microbes to modify the host response to favor their own persistence. We now show that infection/pulsing of human CD34+ peripheral blood hemopoietic progenitor cell-derived dendritic cells (DCs) with Leishmania donovani promastigotes, Histoplasma capsulatum, and Mycobacterium kansasii impairs the constitutive production of IL-12 from these cells. Thus, strategies aimed at modulating a dysregulated Th1/Th2 response to infection would be of great interest. To both augment the host immune response and deliver potent immunomodulatory cytokines such as IL-12 and IFN-{gamma}, our goal is to develop a therapeutic strategy using genetically modified, microbial Ag-pulsed DCs. Toward developing such immunotherapies, we used retrovirus-mediated somatic gene transfer techniques to engineer human DCs to secrete biologically active IL-12 and IFN-{gamma}. DCs pulsed with microbial antigens (e.g., leishmania and histoplasma Ags) were capable of inducing proliferative responses in autologous CD4+ lymphocytes. CD4+ lymphocytes cocultured with IL-12-transduced autologous DCs had enhanced Ag-specific proliferative responses compared with CD4+ lymphocytes cocultured with nontransduced or IFN-{gamma}- transduced DCs. In this cell culture model system we demonstrate that IL-12 has a negative effect on IL-4 secretion that is independent of its ability to induce IFN-{gamma} secretion. Taken together, these results indicate that IL-12-transduced DCs may be specifically suited in inducing or down-modulating Ag-specific Th1 or Th2 responses, respectively, and thus may be useful as adjunctive therapy in those intracellular infections in which a dominant Th1 response is critical for the resolution of infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An imbalance in the Th1- and Th2-type cytokine responses may allow certain pathogenic organisms to replicate within the host to cause disease (reviewed in Refs. 1–3). The signature cytokines of Th1 and Th2 cells are IFN-{gamma} and IL-4, respectively. In this context, IFN-{gamma} and IL-4 are primarily associated with cellular and humoral immunity, respectively. This Th1/Th2 paradigm of acquired immunity has been demonstrated in several animal models of fungal, bacterial, parasitic, and retroviral infections (4, 5, 6, 7, 8, 9). Based on these observations there has been significant interest in restoring and/or modulating the imbalance in the Th1 and Th2 responses by using cytokine/anticytokine therapies for recruiting pathogen-specific protective Th cell responses (10, 11, 12).

The focus of our laboratory is to develop strategies to skew and augment the appropriate Th response to intracellular infections using genetically engineered dendritic cells (DC).3 Over the last few years significant attention has been placed on developing DC-based treatments and vaccine strategies for cancer and HIV-1 infection (12, 13, 14, 15). However, a little explored potential for these strategies is in the development of immunotherapies and vaccines for common intracellular pathogens such as leishmaniasis, histoplasmosis, and the mycobacterial species.

The rationale for using DCs as the focal point for developing anti-infective strategies is based on the unique biologic properties of DCs that make them ideally suited for taking up, processing, and presenting Ag to naive CD4+ T cells, ultimately resulting in Ag-specific immunity (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). Because of their role in initiating a primary T cell response, DCs may also be ideal vehicles to deliver immune-modulating and antimicrobial cytokines such as IL-12 and IFN-{gamma} to appropriate immune sites in vivo (28, 29, 30, 31). The potential advantages of using this mode of delivery is based on the potential of 1) targeted delivery to sites of DC-T cell engagement and 2) minimizing systemic side-effects of these cytokines.

Human and murine studies have documented that the IL-12- and IFN-{gamma}-induced signal transduction cascades play critical roles in the host immune response in a variety of infectious diseases (31, 32, 33, 34, 35). The prevailing paradigm is that IL-12 is a key factor in the initiation of cell-mediated immunity, linking innate immunity to the Ag-specific adaptive immunity. IL-12, a heterodimeric cytokine, is produced primarily by monocytes, macrophages, DCs, and B cells; it stimulates NK and Th1 lymphocytes and rapidly induces the synthesis of IFN-{gamma} from these activated lymphocytes. The IFN-{gamma} produced, in turn, activates macrophages, resulting in increased phagocytosis and superoxide production leading to the clearance of microbes. IFN-{gamma} also leads to an increase in MHC class I and II expression and contributes to efficient Ag presentation to lymphocytes. IL-12 induces Ag-specific Th1 lymphocyte differentiation and further enhances the proliferation of these Ag-specific differentiated lymphocytes, whereas IFN-{gamma} inhibits Th2 cell expansion. Since for most infectious diseases caused by intracellular pathogens a dominant Th2 cytokine response is associated with disease progression while a dominant Th1 response is protective, strategies designed to deliver IL-12 and IFN-{gamma} to immune sites may be ideally suited to enhance a protective Th1 response.

Interestingly, infection of APCs with certain pathogens may impair the production of IL-12. Carrera et al. (36) and Reiner et al. (37) have shown that infection of murine bone-marrow derived macrophages with Leishmania major promastigotes leads to inhibition of IL-12 production. Thus, this pathogen may block the capacity of the host cell to produce potent stimuli for the induction of leishmanicidal activity by IFN-{gamma}. Interestingly, influenza virus-infected human DCs also do not produce IL-12, suggesting that evading the host immune system by inhibiting IL-12 secretion may not be limited to intracellular pathogens (38). In another example, PBMCs from patients infected with HIV-1 are deficient in their production of IL-12 and have suboptimal Th1 immune responses (9, 39, 40, 41). Addition of IL-12 to T cells in vitro restores recall responses to Ags (28). Hilkens et al. have shown recently that contact between DCs and naive Th cells during the initiation of specific immune responses does not result in the efficient induction of IL-12 production (42). Instead, exogenous IL-12-inducing factors such as certain bacterial products (e.g., LPS) or exogenous IFN-{gamma} are required to promote primary Th1-mediated cellular immune responses. Taken together, these studies provide the rationale for developing strategies using both "immune-enhanced" DCs, i.e., DCs engineered to secrete immunomodulatory cytokines such as IL-12, and "educated" DCs, i.e., microbial Ag-pulsed DCs to modulate the host immune response to infections.

In this report we demonstrate that infection/pulsing of human CD34+ peripheral blood hemopoietic progenitor (PBHP) cell-derived DCs infected/pulsed with three intracellular pathogens, namely, Leishmania donovani promastigotes, Histoplasma capsulatum, and Mycobacterium kansasii, impairs the constitutive production of IL-12 from these cells. We show that the IL-12 or IFN-{gamma} secreted by the retrovirally transduced human DCs is biologically active and results in phenotypic and functional changes similar to those observed after exogenous administration of these cytokines. We demonstrate that DCs engineered to secrete IL-12 augment Th1 cytokine responses and inhibit IL-4 secretion from bulk CD4+ T cell clones. These studies highlight the potential benefits of developing DC-based immunotherapeutic strategies for those infections in which a dysregulated Th1/Th2 cytokine response is associated with a poor prognosis or progressive disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of retrovirus-based plasmids

Using overlap PCR strategies we created a bicistronic IL-12-MFGS retroviral expression plasmid in which the 5' long terminal repeat and the internal ribosome entry site element drive the expression of the p40 and p35 subunits of human IL-12, respectively. The fidelity of the PCR amplification product (p40-internal ribosome entry site-p35) was confirmed by full-length sequencing of both DNA strands. The construction of the MFGS-IFN-{gamma} plasmid has been described previously (43). The Moloney murine leukemia virus retroviral vector, MFGS, was a gift from Somatix Therapy (Alameda, CA).

Generation of {Psi} crip producer clones

The calcium phosphate precipitation method was used to transfect 5 x 105 {Psi} crip amphotropic packaging cells with a mixture of the following plasmid DNAs: 38 µg of MFGS-IL-12 and 2 µg of pSV2-neomycin. Single-cell neomycin-resistant {Psi} crip clones were selected and expanded to confluence. The supernatants from these neomycin-resistant {Psi} crip clones contain replication-incompetent viral particles encoding IL-12 that have the potential of transducing heterologous cells. We therefore collected supernatants from these neomycin-resistant {Psi} crip clones and tested their ability to transduce NIH-3T3 cells, i.e., confer NIH-3T3 cells with the ability to secrete human IL-12. The {Psi} crip clone that conferred the maximum IL-12 production from transduced NIH-3T3 cells was designated the high producer clone. The high producer {Psi} crip/MFGS-IFN-{gamma} clone has been described previously (43). As a virus transduction control, a {Psi} crip clone producing the MFGS-lacZ virus was used (the {Psi} crip clone producing MFGS-lacZ is a gift from Somatix Therapy). Transduction was confirmed by staining for ß-galactosidase. The transduction protocol used was described previously (43).

Isolation of CD34+ PBHP cells and PBMCs

Signed informed consent was obtained from all study participants as approved by the institutional review boards at the National Institutes of Health and the University of Texas Health Science Center at San Antonio. Healthy adult volunteers were pretreated with granulocyte CSF (G-CSF; Amgen, Thousand Oaks, CA), and low density cells in the peripheral blood were collected by apheresis on the sixth day. The dose of G-CSF used was 10 µg/kg/day for 5 days. Two donors were apheresed at the Blood Transfusion Department, Clinical Center, National Institutes of Health (Bethesda, MD), and the CD34+ PBHP cells were purified using the Isolex immunomagnetic bead technology (Baxter Healthcare, Mundelein, IL). At the University of Texas Health Science Center at San Antonio the donors were apheresed (Bone Marrow Transplant Apheresis Unit and General Clinical Research Center at Audie Murphy Veterans Hospital), and their CD34+ PBHP cell populations were enriched using the Ceprate SC stem cell concentrating system (Cell-Pro, Bothell, WA). The average yield ranged between 50 and 300 million CD34+ PBHP cells from each donor, and cells were of 30 to 90% purity as analyzed by FACS analysis. The CD34+ PBHP cells were cryopreserved in aliquots of 5 to 10 million cells. PBMCs, monocytes, and lymphocytes from each donor were purified using standard techniques, including gradient density centrifugation and plastic adherence. CD4+ lymphocytes were isolated using the Cell-Pro avidin column and frozen in aliquots of 5 million cells.

Cytokine differentiation of DCs

After thawing, the CD34+ PBHP cells were cultured in Iscove’s Modified Dulbecco’s Medium containing 10% FBS, penicillin/streptomycin, sodium pyruvate, nonessential amino acids, and 4 mM glutamine (complete medium) and supplemented with the following recombinant growth factors (R&D Systems, Minneapolis, MN): 20 ng/ml of stem cell factor (SCF), and 50 ng/ml of granulocyte-macrophage CSF (GM-CSF). IL-4 and TNF-{alpha} at 10 ng/ml (R&D) each were added to the culture on day 6. The cytokine-differentiated CD34+ PBHP cells were kept in culture for a total of 12 to 14 days, and in each experiment the differentiating cells were stained for cell surface markers for DCs, monocytes, and lymphocytes. By day 14 in culture >99% of cells were CD33+, suggesting that the predominant population was of the myeloid series. The proportion of cells that stained for T lymphocytes (CD3) or B lymphocytes (CD19) was consistently <1 to 3%.

FACS

The phenotypic analysis of the hemopoietic cells growing in culture was performed using fluorescent-labeled Abs (PharMingen, San Diego, CA) by standard techniques as described previously (43). The following cell surface Ags were tested: CD34, progenitor cell marker; CD45, leukocyte cell marker; CD33/CD15, myeloid cell marker; CD3/CD2, T lymphocyte marker; CD19, B lymphocyte marker; CD14, monocyte marker; CD1a, DC marker; CD80 and CD86, CD28 ligand (and DC marker); and CD11a/CD11b/CD11c, CD54 adhesion molecules. HLA class I and HLA class II were analyzed by fluorescent-labeled Abs against HLA-DR and MHC class I-specific W6/32 Ab. The stained cells were washed twice in HBSS, fixed in 1% paraformaldehyde, and then analyzed on the FACScan.

Transduction of NIH-3T3 cells

NIH-3T3 cells were transduced for 6 h on 3 consecutive days with viral supernatants diluted 1/1 with complete medium containing 6 µg/ml of protamine as described previously (43). IL-12 and IFN-{gamma} levels in the supernatants of the transduced NIH-3T3 cells were determined 72 h following the third transduction.

Transduction of human CD34+ PBHP cells

The concentration of CD34+ PBHP cells were adjusted to 0.3 x 106/ml before transduction. Transductions were performed in six-well plates for 6 h on 3 consecutive days (days 2, 3, and 4 of cell culture) with viral supernatant diluted 1/1 with complete medium supplemented with 6 µg/ml of protamine and the cytokines, SCF and GM-CSF. To enhance transduction efficiency, the culture plates were centrifuged at 2400 rpm for 1 h at room temperature immediately after addition of the viral supernatant and incubated for an additional 5 h at 37°C and 5% CO2. Subsequently, the cells were pelleted and resuspended in fresh complete medium containing the cytokines SCF and GM-CSF. IL-12 and IFN-{gamma} levels in the supernatants of the transduced PBHP cells were measured 72 h following the third transduction. The transduction efficiency of the retrovirus MFGS-IFN-{gamma} into human PBHP-derived myeloid cells has been described previously (43).

Cytokine ELlSAs

ELISA kits specific for human IFN-{gamma} (Endogen, Cambridge, MA), TNF-{alpha}, IL-1ß, IL-4, and the IL-12 bioactive p70 heterodimer (R&D) were used to measure the cytokine levels in culture supernatants.

Infection/pulsing studies

The source for infectious agents used were as follows. The H. capsulatum G217B strain was a gift from Dr. J. R. Graybill and was maintained in yeast form on Sabouraud’s dextrose agar supplemented with cysteine at 37°C. For in vitro infection the yeast were grown for 48 h in brain heart infusion broth, heat killed at 55°C for 60 min, washed, and then counted. The M. kansasii strain used was a clinical isolate maintained on Löwenstein-Jenson agarose plates containing Middlebrook 7H10 was also a gift from Dr. J. R. Graybill. L. donovani promastigotes 1S strain were grown in Grace’s insect medium and were a gift from Dr. P. C. Melby. All three infectious agents (concentration, 107/ml) were opsonized before infection by resuspending in PBS containing 50% human serum and incubating at 37°C for 30 min. They were then washed once with PBS and incubated with DC at a ratio of 1:1 for 18 to 24 h in complete medium containing growth factors. Following infection, the cells were gently washed several times to remove nonphagocytosed microbes and resuspended in fresh medium containing the appropriate cytokines. Phagocytosis was confirmed by special staining Diff Quick (L. donovani), Gomori methamine silver stain (H. capsulatum), and acid-fast stain (M. kansasii).

T lymphocytes

Purified CD8-negative CD45RA-negative T lymphocytes were cultured under limiting dilution in the presence of PHA (0.5 µg/ml; Burroughs Wellcome, Research Triangle Park, NC) and medium supplemented with 10% human AB serum and 25 U/ml rIL-2 (Life Technologies, Grand Island, NY) for 3 days. These cells were then cocultured with irradiated (3000 rad), Ag-pulsed autologous CD34+ PBHP cell-derived DCs (106 cells/ml). The DCs were cultured overnight in the presence of soluble L. donovani Ag (SLDA; gift from P. C. Melby) (44) or detergent extract from cell wall and cell membrane of H. capsulatum yeast cells (CW/M; gift from G. S. Deepe, Jr.) (45), and then washed before irradiation. Both SLDA and CW/M have been shown to confer protective immunity in mice. The T cells were passaged weekly by coculturing equal number of CD4+ T cells and the pulsed, irradiated DCs for 3 wk in medium containing IL-2. The Ag-reactive CD4+ T cells acquired a memory phenotype CD45RO, and the specificity of Ag reactivity was tested by proliferation in response to DCs pulsed with and without SLDA or CW/M. Before proliferation, the Ag-reactive CD4+ T cells were rested for 3 days in RPMI supplemented with 2% human serum without IL-2, following which they were cocultured with autologous DCs transduced with IFN-{gamma} or IL-12.

Mixed leukocyte reaction

Stimulator cells were CD34+ PBHP-derived DCs nontransduced or transduced with IFN-{gamma} or IL-12, or monocytes from the same donor. Before irradiation the stimulator cells were pulsed with the Ag of interest for 12 h and then irradiated at 3,000 rad and cultured (in triplicate) in 96-well round-bottom plates (1,000–100,000 cells/well) with autologous T lymphocytes or Ag-naive CD4+ or Ag-reactive CD4+ lymphocytes (50,000–100,000 cells/well) for 144 h. To assay for proliferation, 0.5 µCi of [3H]thymidine (New England Nuclear, Boston, MA) was added to each well 8 h before harvesting the cells on glass-fiber filters (Pharmacia, Piscataway, NJ). The filters were counted in a beta scintillation counter.

CFU assays

PBHP cells (n = 100,000) transduced with IFN-{gamma} or IL-12 were plated in triplicate in 1 ml of 0.2% semisolid agarose-containing complete medium with growth factors in 12-well plates and incubated at 37°C with 5% CO2 for 14 days. The total number of CFUs and the cell lineage of the CFUs were identified by standard staining techniques described previously (43).

Assay for superoxide production

On days 7 and 14 of cell culture, a chemiluminescence assay was used to measure superoxide production from transduced CD34+ PBHP-derived cells as described previously (43).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytokine differentiation of CD34+ PBHP cells toward the DC lineage

Previous studies have demonstrated that DCs are heterogeneous and that their ability to stimulate resting T cells depends on the methods of isolation (46, 47, 48, 49, 50). The source of our DCs is cytokine-differentiated CD34+ PBHP cells obtained from the G-CSF-mobilized peripheral blood of normal volunteers. Since our goal is to engineer DCs to secrete cytokines such as IFN-{gamma}, and determine whether the cytokine secreted alters the phenotypic or functional properties of DCs, it was important to characterize the phenotypic and functional characteristics of the nontransduced CD34+ PBHP cell-derived DCs.

Among the five donors analyzed there was significant donor-to-donor variability (~20–30%) in the levels of some cell surface markers (n = 2 for each donor). Nevertheless, in all five donors the cytokine-differentiated PBHP cells were of myeloid origin, as was evident by the high expression levels of CD33 (range, 90–98% cells), and low expression levels of CD3 (range, 0–3%) and CD19 (range, 0–3%). These cells had phenotypic characteristics of DCs such as expression of CD1a (range, 20–81%), CD80 (range, 15–74%), CD86 (range, 18–81%), and HLA-DR (range, 40–99%) and of adhesion molecules such as CD11a (range, 30–85%), CD11b (range, 26–69%), CD11c (range, 33–84%), and CD54 (range, 17–76%). The results of experiments that highlight the ability of these cytokine-differentiated CD34+ PBHP cells to present Ags are shown in Figure 1Go. In these experiments, both unpulsed DCs and tetanus toxoid Ag-pulsed DCs induced autologous CD4+ lymphocytes to proliferate. The ability to induce lymphocytes to proliferate in the absence of stimuli or mitogens is a characteristic of DCs. In parallel with the phenotypic and functional analysis we examined the morphology of the cultured cells. During culture the relatively homogeneous population of small mononuclear CD34+ cells differentiated into a heterogeneous population of adherent and nonadherent cells. It was the loosely adherent clusters of proliferating cells that had a typical DC morphology (data not shown).



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  		FIGURE 1. Proliferation of human CD4+ T lymphocytes cocultured with autologous DCs. The proliferation (mean counts per minute ± SD) of human CD4+ lymphocytes cocultured with autologous monocytes, nonpulsed DCs, or DCs pulsed with tetanus toxoid (TT) were analyzed. Two experiments were performed with cells derived from two donors each (n = 4), and the results shown are from one representative experiment.

 
Infection/pulsing of human DCs impairs IL-12 production

IL-12 production by DCs inducing primary T cell responses could represent a critical point in influencing the Th1/Th2 outcome of the immune response. Hence, we first determined whether IL-12 is constitutively produced by human CD34+ PBHP-derived DCs. We found that the CD34+ PBHP cell-derived DCs constitutively secreted detectable levels of the bioactive IL-12 heterodimer p70 protein into the medium. The range of IL-12 produced was between 5 and 6 pg/ml over a period of 48 h (Fig. 2GoA). In the next series of experiments, we examined whether infection/pulsing of human DCs with L. donovani promastigotes, H. capsulatum, or M. kansasi influences the ability of these cells to secrete IL-12. To performe these experiments we cultured CD34+ PBHP cell-derived DCs with these organisms at a ratio of 1:1 for 24 h. The cells were extensively washed to remove nonphagocytosed organisms, and the supernatants were harvested 4, 8, 24, and 48 h later for cytokine analysis. In these experiments we also measured IL-12 production from DCs pulsed with Ags and following stimulation with LPS or SAC. The levels of IL-12 in DCs pulsed with SLDA or CW/M 24 and 48 h postpulsing were comparable to those in the uninfected, nonpulsed DCs (Fig. 2Go, A and B). DCs stimulated with LPS and SAC produced large quantities of IL-12 (Fig. 2GoD). The peak IL-12 production in response to stimulation with LPS and SAC occurred within the first 24 h poststimulation and then declined precipitously. IL-12 levels could be detected initially in the supernatants of DCs infected/pulsed with the aforementioned organisms. However, as shown in Figure 2GoC, IL-12 could not be detected in these supernatants at the later time points.



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  		FIGURE 2. IL-12 production from DCs. In each experiment the unpulsed/noninfected DCs (A) were washed and resuspended in fresh medium at the same time as the DCs in B, C, and D, and supernatants were harvested at the indicated times thereafter to determine constitutive IL-12 levels. In the pulsing studies (B), DCs were pulsed for 12 h before measuring IL-12 levels at the time points indicated. In the infection studies, cells were infected for 24 h and washed extensively, and then IL-12 was measured at the time points indicated (C). IL-12 levels following stimulation with LPS and SAC are shown in D. To account for the time differences in microbial exposure (24 h), Ag pulsing (12 h), or stimulation with SAC and LPS (6 h), we included at time zero for B, C, and D a control sample from the unpulsed/noninfected DC group (A). Since the values obtained in the control samples were relatively constant, only one representative graph is shown (A). Two experiments were performed with cells derived from two donors each, and the results shown are from one representative experiment. Note differences in scale in A through C and D.

 
Transduction of DCs with retroviral vectors encoding for IFN-{gamma} or IL-12

The lack of IL-12 production by the aforementioned infected human DCs provided additional rationale for genetically engineering DCs to secrete enhanced levels of cytokines such as IL-12. Toward this end we used previously established retroviral gene therapy-based methods (43) to transduce DCs with IL-12 and IFN-{gamma}. We found 10 {Psi} crip clones that could confer NIH-3T3 cells with the ability to secrete IL-12. IL-12 production was estimated as immunoreactive IL-12 per million cells per day. The maximum rate of IL-12 production was about 350 pg/106 cells/day (clone I8). Using a similar strategy we previously reported a high producer IFN-{gamma} {Psi} crip clone, designated G23 in which the maximum rate of IFN-{gamma} production was 7000 pg/106 cells/day (43). We next tested the ability of supernatants derived from clone I8 (IL-12-MFGS) and G23 (IFN-{gamma}-MFGS) to transduce human DCs (Fig. 3Go). IFN-{gamma} levels were undetectable in the supernatants obtained from either sham or MFGS-lacZ-transduced DCs, whereas MFGS-IFN-{gamma}-transduced DCs secreted IFN-{gamma} (Fig. 3GoA). In contrast to sham and MFGS-lacZ transduced-DCs, IL-12-transduced DCs secreted significantly higher amounts of IL-12 (Fig. 3GoB).



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  		FIGURE 3. DC transduction with retroviral vectors encoding for IL-12 and IFN-{gamma}. The amounts of IFN-{gamma} and IL-12 produced from sham-transduced, MFGS-lacZ-transduced, and MFGS-IFN-{gamma}- or MFGS-IL-12-transduced DCs were measured. The data for IFN-{gamma} and IL-12 production are represented as the mean (±SD) rate per 106 cells per day. For IFN-{gamma} determinations, there was a total of six experiments with CD34+ PBHP-derived DCs from four donors, and for IL-12, there was a total of three experiments with CD34+ PBHP-derived DCs from three donors.

 
Cell numbers and phenotypic changes in IFN-{gamma}- and IL-12-transduced DCs

Previous studies have demonstrated that addition of exogenous IFN-{gamma} and IL-12 to progenitor cells derived from bone marrow or cord blood can decrease their cell proliferation rate and the number of CFUs generated (43, 51, 52). For this reason we examined the effect of the IFN-{gamma} and IL-12 produced by the transduced DCs on similar parameters. In these experiments, equal numbers of differentiating CD34+ PBHP cells obtained from a single donor were transduced with supernatants from I8 (MFGS-IL-12 transduced) or G23 (MFGS-IFN-{gamma} transduced) {Psi} crip clones, and changes in cell numbers were determined. On day 14 of culture, there was a mean 21.5-fold increase in cell number in sham-transduced cells, whereas there was only a 15.3-fold increase in cell number in MFGS-IFN-{gamma} transduced DCs (n = 4 experiments; p < 0.2, by paired t test). There was no difference in the number of cells between the sham-transduced and the MFGS-IL-12-transduced PBHP cells (average 19.6-fold increase).

In experiments to determine the effect of the secreted IL-12 and IFN-{gamma} on CFU formation, we first transduced CD34+ progenitor cells with retroviruses for IFN-{gamma} or IL-12, and then aliquots of 100,000 transduced cells were mixed in soft agarose that contained GM-CSF, SCF, and TNF-{alpha}. The CFU assays were performed in triplicate wells. It should be emphasized that the transductions were performed on CD34+ PBHP cells derived from a single donor and that the transductions for both IFN-{gamma} and IL-12 were performed in parallel. Compared with the sham-transduced DCs, there was a 33 ± 7% decrease in the number of CFUs in the IFN-{gamma}- transduced PBHP cells, but no difference in the IL-12-transduced PBHP cells. It should be noted that the levels of IL-12 produced by the transduced PBHP derived DCs were in the picogram range. This is in contrast to previous reports on the effect of exogenously added IL-12 in the nanogram range (51). These higher concentrations of IL-12 have been reported to inhibit CFU formation in soft agarose assays, although the concentration of IL-12 may be supraphysiologic.

In addition to the apparent antiproliferative effects of IFN-{gamma}, there were other phenotypic changes observed in IFN-{gamma}-transduced DCs (Table IGo). 1) IFN-{gamma} transduced DCs had higher numbers of Fc{gamma}RI receptors (CD64). Fc{gamma}RI receptors on monocytes/macrophages and activated neutrophils were up-regulated by IFN-{gamma} and were involved in phagocytosis, immune complex clearance, and Ab-dependent cell-mediated toxicity (30, 43). 2) There was an increase in MHC class I and II expression. As shown in Table IGo, there was a fourfold increase in the mean fluorescence intensity for HLA-DR expression in the IFN-{gamma}-transduced cells. Cells transduced with IFN-{gamma} had a two- to threefold increase in the mean fluorescence intensity for MHC class I expression. 3) We observed a decrease in the expression of adhesion molecules and costimulatory molecules such as CD80 and CD86 in the IFN-{gamma}-transduced DCs (Table IGo). These differences may be accounted for by the finding that the proportion of cells staining for DCs (e.g., CD1a, CD80, and CD86) was lower among IFN-{gamma}-transduced cells, and the proportion of cells positive for CD14, a characteristic marker for monocytes, was higher in this cell population. 4) There was increased PMA-stimulated superoxide production in the IFN-{gamma}-transduced DCs. The levels of superoxide produced (mean ± SD) by the sham-, IFN-{gamma}-, and IL-12-transduced DCs were 449 ± 79, 995 ± 152, and 510 ± 87 luminol units, respectively (n = 3). In contrast, to the cells transduced with IFN-{gamma}, IL-12-transduced PBHP cells gave rise to DCs that exhibited no phenotypic changes compared with nontransduced cells. All comparisons of the phenotypic changes between transduced and nontransduced or sham-transduced cells were made using CD34+ progenitor cells derived from two donors (n = 3).


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  		Table I. Surface markers of DCs transduced with IFN-{gamma} and IL-121

 
IL-12-transduced, PBHP cell-derived DCs can enhance Th1 responses in vitro

To determine whether the IL-12-transduced DCs can skew the T cells toward a Th1 response, we incubated resting T cells with IL-12 transduced or nontransduced DCs. After 36 h of coculture (autologous T cells and DCs) we observed that the T cells formed clusters around the DCs, their cell sizes increased, and their ability to incorporate thymidine increased by 15 to 30% (not statistically significant). This suggested that the T lymphocytes proliferate when they come into contact with the DCs. Furthermore, the engagement of T cells with IL-12 transduced DCs led to the secretion of IFN-{gamma} (Fig. 4GoA) and IL-2 (Fig. 4GoB). By treating the cocultures of T cells and IL-12-transduced DCs with anti-IL-12 mAbs, we could partially inhibit by 70% the increase in levels of both IFN-{gamma} and IL-2. The incomplete inhibition by anti-IL-12 suggests that there may be IL-12-independent factors in DC-T cell cocultures that may be responsible for the increases in IL-2 and IFN-{gamma}. Taken together, these findings suggested that the IL-12 produced by the transduced DCs was biologically active and could induce IL-2 and IFN-{gamma} secretion from the T cells, i.e., enhance further a Th1-like response in vitro.



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  		FIGURE 4. IFN-{gamma} and IL-2 production from T lymphocytes cocultured with IL-12-transduced autologous DCs. The mean (±SD) IFN-{gamma} (A) and IL-2 (B) level in the supernatants of T cells cocultured with either IL-12-transduced or nontransduced autologous DCs is shown. The IFN-{gamma} and IL-2 measurements were performed on cocultures of DCs and T cells derived from a total of four experiments derived from two donors. Note that the cells were derived from two donors, and the phenotypic characteristics of the DCs used in the experiment are shown in Table IGo.

 
Induction of Ag-specific responses to microbial Ags and effects of IL-12 and IFN-{gamma} secreted by transduced DCs on these responses

To perform these experiments we first analyzed the T cell-proliferative responses of normal donors against well-known Ags such as BSA, PPD, and tetanus toxoid and also against SLDA and CW/M (concentrations of 5–50 µg/ml). Of the donors tested, in two donors there was no SLDA, CW/M, or PPD-induced proliferation above the unstimulated controls (Fig. 5GoA), indicating that the likelihood that these two donors had been exposed to mycobacterial organisms, leishmania, or histoplasmosis was low. As a positive control we tested the proliferative responses of PBMCs from these two individuals in response to pulsing with tetanus toxoid (Fig. 5GoA).



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  		FIGURE 5. Proliferative responses in PBMCs and CD4+ T lymphocytes to Ags. A, Thymidine incorporation in PBMCs in response to PPD, SLDA, CW/M, and tetanus toxin (TT) was analyzed. The concentration of the Ags was 5 µg/ml except as indicated. B, Thymidine incorporation in purified naive CD4+ lymphocytes in response to autologous DCs pulsed with the Ags indicated (5 µg/ml). The experiments were conducted in triplicate. Note that the cells were derived from two donors, and the phenotypic characteristics of the DCs used are shown in Table IGo. The data shown are representative of one of four experiments performed on cells derived from two donors.

 
We next harvested CD34+ cells from these two naive donors and cytokine differentiated these cells toward the DC lineage. DCs were first pulsed with the Ags, then irradiated (3000 rad) and subsequently incubated with autologous CD4+ T lymphocytes. As shown in Figure 5GoB, we observed that proliferative responses were induced by low concentrations of tetanus toxoid (5 µg/ml), SLDA, and CW/M (Fig. 5GoB). These findings indicate that CD34+ PBHP-derived DCs could potentially be used to induce microbial Ag-specific proliferative responses in naive individuals. However, the proliferative responses to BSA (lane 3, Fig. 5GoB) indicated that the DCs grown in FBS also present foreign bovine proteins. This implied that the frequency of Ag reactive CD4+ T lymphocytes is likely to be very low in primary DC-T cell interactions. To circumvent this, we generated Ag-specific CD4+ T lymphocytes derived from individual donors and used these Ag-reactive T cells in the subsequent experiments.

We next examined whether transduction of PBHP cell-derived DCs with retroviruses encoding IL-12 or IFN-{gamma} alters Ag-specific proliferative responses. DCs were transduced with IFN-{gamma} or IL-12 and pulsed with either SLDA or CW/M. Following Ag pulsing, the DCs were irradiated and then incubated with CD4+ T lymphocytes. The Ag reactivity of the bulk CD4+ T lymphocytes is indicated in Figure 6Go. As shown in Figure 6Go, DCs transduced with IFN-{gamma} induced lower Ag-specific proliferative responses (compare lanes 6 and 8, and lanes 7 and 9). In contrast, DCs transduced with IL-12 enhanced proliferative responses to both SLDA and CW/M Ags (Fig. 6Go, compare lanes 6 and 10, and lanes 7 and 11).



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  		FIGURE 6. Proliferative responses of naive or Ag-reactive CD4+ lymphocytes in response to autologous DCs transduced with IFN-{gamma} or IL-12. DCs were transduced with MFGS-IL-12 or MFGS-IFN-{gamma} retroviruses as described in Materials and Methods. DCs were pulsed with SLDA or CW/M (Ags for L. donovani and H. capsulatum), irradiated (3000 rad), and cultured with CD4+ lymphocytes for 7 days and then assayed for thymidine incorporation as described. The CD4+ lymphocytes used in these experiments were as follows. Lanes 2 through5, naive CD4+; lanes 6, 8, and 10, CW/M-reactive (autologous) CD4+ lymphocytes; lanes 7, 9, and 11, SLDA-reactive (autologous) CD4+ lymphocytes. The baseline proliferative counts for SLDA-reactive CD4+ T lymphocytes in the presence of CW/M (890 ± 120 cpm) and the CW/M-reactive T lymphocytes in the presence of SLDA (1208 ± 140 cpm) were comparable to background counts, demonstrating the specificity of the Ag-specific responses. Expression of cell surface markers for the DCs used in these experiments is described in Table IGo. The data shown (mean thymidine incorporation ± SD) are representative of one of three experiments performed on cells derived from two donors. Note that the SD arrow bars for lanes 1 through 10 are very small and therefore could not be illustrated in the figure.

 
Since IL-12 is known to prevent the development of IL-4-producing cells, i.e., inhibits the Th2 response, we measured IL-4 levels (Table IIGo) in the supernatants of the cell cultures shown in Figure 6Go. In these experiments, we observed that supernatants from cocultures of CD4+ T cells and DCs transduced with IL-12 had lower IL-4 levels than cocultures of T cells with nontransduced DCs (Table IIGo, compare lane 3 with lanes 5, 10, and 11). The negative effects of IL-12 on IL-4 levels was consistent in both donors. In contrast, the IL-4 levels in supernatants of cocultures that contained IFN-{gamma}-transduced DCs were discordant in the two donors: in donor 1, the IL-4 levels were higher, whereas in donor 2, the levels were lower (Table IIGo, compare lane 3 with lanes 4, 8, and 9).


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  		Table II. IL-4 and IFN-{gamma} production from Ag reactive CD4+ lymphocytes cocultured with autologous DCs transduced with IFN-{gamma} or IL-121

 
It is also important to note that the levels of IFN-{gamma} in the supernatants from the cocultures containing IL-12-transduced DCs were similar to those present in the IFN-{gamma}-transduced DCs (Table IIGo; compare lanes 4, 8, and 9 with lanes 5, 10, and 11). These findings indicate that the lower levels of IL-4 in the supernatants of cocultures containing IL-12-transduced DCs are likely to be independent of IFN-{gamma}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we demonstrate that CD34+ progenitor-derived human DCs infected/pulsed with L. donovani promastigotes, H. capsulatum, and M. kansasii may have an impaired production of IL-12. In part, this observation forms the basis for developing a two-pronged immunotherapeutic strategy that is based on gene delivery of immunomodulatory cytokines via DCs and the potent Ag-presenting properties of DCs. To this end, we have used retroviral techniques to engineer DCs with the ability to secrete biologically active IFN-{gamma} and IL-12. The IFN-{gamma} produced by the transduced DCs acted in an autocrine manner and had antiproliferative effects on the DCs. The secreted IFN-{gamma} also altered the phenotypic markers on DCs. In contrast, we provide evidence that the IL-12 produced by the transduced DCs did not alter the phenotypic characteristics of DCs and that these engineered DCs not only enhance Th1 responses but also inhibited a Th2 response, i.e., IL-4 production. Furthermore, the inhibition of IL-4 by IL-12 appeared to be independent of IFN-{gamma}, suggesting a direct effect of IL-12 on Th cells.

In this study we used G-CSF-mobilized CD34+ cells from normal donors for the ex vivo generation of DCs. Markers specific for DCs are currently unavailable so DCs are phenotypically defined with multiple marker panels. We studied the phenotypic markers on DCs obtained by the cytokine-induced differentiation of CD34+ PBHP cells from five normal donors and observed a significant variation in the level of surface markers thought to be characteristic of DCs. However, morphologically and functionally the DCs obtained from different donors were similar. This degree of heterogeneity in phenotypic markers has been described previously in DCs derived from both peripheral blood DC and G-CSF-mobilized CD34+ cells from cancer patients as well as other sources of DCs, such as isolated Langerhans cells (46, 47, 48, 49, 50). Nevertheless, the ability to enrich for rare CD34+ PBHP cells and the ability to expand them in vitro toward the DC lineage point to the feasibility of the proposed immunotherapeutic strategy. It is conceivable that G-CSF mobilization may not be feasible in all clinical situations, but techniques to generate DCs from peripheral blood monocytes are also in place (13, 14, 15).

The cytokine profile of DCs is gradually being elucidated. It should be emphasized that it is conceivable that this profile may differ depending on the species of the DCs tested, the method of their isolation and culture conditions, and the methods used for detection. To date, most studies addressing this issues have focused on bone marrow-derived murine DCs, and in most human studies the source of DCs have been cytokine-differentiated monocytes. A limitation of nonmobilized peripheral blood DCs or monocyte-derived DCs is their low yield. In contrast, cytokine differentiation of G-CSF-mobilized human CD34+ PBHP cells provides a relatively abundant source of DCs. After a single apheresis we typically harvest between 50 and 300 million CD34+ cells. In a typical experiment we use between 1 and 2 million CD34+ cells. After differentiation toward the DC lineage, i.e., by days 10 to 14, there is a 20- to 50-fold expansion of the cell number, yielding approximately 20 to 100 million cells. Thus, multiple experiments can be performed on DCs derived from a single donor, thereby decreasing inter- and intraexperiment variabilities.

Because IL-12 production during the initiation of the primary immune responses that DCs initiate would be expected to augment a Th1 response, it was critical to determine whether the human CD34+ PBHP-derived DCs were capable of producing IL-12. Our data show that these DCs secrete bioactive IL-12. Our findings are in agreement with those of Verhasselt et al. (53) and Kang et al. (54), who demonstrated basal production of IL-12 by DCs derived from human PBMCs and Langerhans cells (skin DCs), respectively. Kinetic analysis of the production of IL-12 in response to stimulation with SAC and LPS revealed that maximal production occurred during a small time frame (optimally within 24 h).

Studies specifically examining the effect of infection/pulsing of human DCs with L. donovani promastigotes, H. capsulatum, and the mycobacterial species studied in this report have not been described. Considering the central role of APCs such as DCs and macrophages in the initiation as well as the effector functions of the immune response, infection of these cells might impair their ability to provide the appropriate regulatory cytokines for enhanced production of IFN-{gamma}. We now show that human CD34+ PBHP cell-derived DCs infected/pulsed with L. donovani promastigotes, H. capsulatum, and M. kanasii have impaired IL-12 production. It should be emphasized that the specific nature of the organism infecting DCs is also likely to have a profound effect on the nature of the cytokine response. For example, in vitro experiments with other intracellular pathogens, such as Mycobaterium tuberculosis, have revealed that exposure of human DCs to this organism results in strong induction of IL-12 (55).

The precise mechanisms that led to this impaired IL-12 production in DCs are not yet known. Nevertheless, these findings in conjunction with findings by several other investigators (36, 37, 38, 39, 40, 41) lend credence to the idea of developing immunotherapies using IL-12 in conjunction with DCs to overcome this block and/or to enhance microbe Ag-specific responses. DCs are an ideal target cell for introducing genes, since trafficking studies in chimpanzees suggest that in vitro differentiated DCs migrate to the appropriate immune sites necessary for DC-T cell interactions and in many respects behave similarly to the endogenous Langerhans cells (26). This property combined with their Ag-presenting properties suggest that DCs may be an ideal vehicle for delivering anti-infective immunomodulatory cytokines.

To date, six groups have described their experience with ex vivo gene transfer into human DCs by a variety of methods (56, 57, 58, 59, 60, 61). The source of the DCs was either bone marrow or cord blood CD34+ precursors or nondividing PBMC. In these reports the gene transfered was a tumor-associated Ag or a reporter/marker gene. For example, Szabolcs et al. reported recently that human DCs retrovirally transduced with murine CD2 express a normal phenotype and retain potent T cell stimulatory capacity (56). The findings reported here significantly extend these findings and to our knowledge comprise the first report describing the engineering of human G-CSF-mobilized, CD34+ PBHP cell-derived DCs to secrete IL-12 and IFN-{gamma}.

We observed no difference in the phenotypic markers of DCs engineered to secrete human IL-12. In contrast, transduction of DCs with IFN-{gamma} induced phenotypic and anti-proliferative changes. These changes were similar to those that we have observed previously when we engineered CD34+ PBHP cell-derived monocytes to secrete IFN-{gamma} and to changes observed in monocytes/macrophages that have been stimulated in vitro by IFN-{gamma} (43). As noted previously, the lack of any effect of IL-12 on the phenotypic markers and proliferation of DCs and CFU of progenitor cells may be related to the fact that the amount of IL-12 secreted by these DCs is significantly less than that usually added exogenously to cell cultures. The anti-proliferative and phenotypic changes induced by the autocrine effects of the secreted IFN-{gamma} and the ability of IL-12 to potentiate a Th1 response point to the expression of biologically active cytokines. In agreement with Szabolcs (56), these findings document that transduced DCs retain potent T cell stimulatory capacity, and we now show that the differences in T cell stimulation that occur (see Fig. 6Go) are related to the autocrine effects of the transgene.

In addition to demonstrating that the IL-12-transduced DCs enhance a Th1 response, we show that these cells may also lead to the inhibition of the development of IL-4-producing T cells. There has been some debate in the literature about whether IL-12 acts on Th cells through the induction of IFN-{gamma} (62, 63, 64). However, in agreement with Macatonia et al. (63) and Heinzel et al. (64), our data demonstrate that the IL-12 inhibition of IL-4 secretion by lymphocytes is independent of the effects of IFN-{gamma}. Among other actions, IL-4 is thought to be primarily responsible for expanding the Th2 response and suppressing the secretion and macrophage-activating effects of Th1-associated cytokines including IFN-{gamma} (reviewed in Refs. 1–3). Taken together, the present study highlights the potential utility of using IL-12-engineered DCs in skewing the Th responses toward a dominant type 1 response.

Previous studies have documented that IL-12 inhibits IL-4 secretion by T cells. In our system, the effect of IFN-{gamma} on IL-4 secretion differed in the two individuals we studied; in one instance there was a decrease in IL-4 secretion, and in the other there was an increase in IL-4 secretion. We cannot readily explain these discordant results. Nevertheless, this observation may be consistent with both human and murine studies showing that the genetic background of an individual is likely to be an important determinant of IL-12 and IFN-{gamma} responsiveness. Thus, genetic differences in cytokine-mediated responses may influence disease progression following infection. For example, Guler et al. have shown that the genetic background of T lymphocytes influences the development of the Th phenotype, resulting in either resistance or susceptibility of certain mouse strains to pathogens such as L. major (65, 66). Holland et al. have described rare patients who have refractory disseminated nontuberculous mycobacterial infections without HIV infection and have abnormal IL-12 regulation. IFN-{gamma} has been used successfully with antimycobacterials for treatment of these patients (31).

In on-going experiments we are currently testing the in vivo anti-infective efficacy of Th1 cytokines delivered by genetically modified and microbial Ag-pulsed murine DCs in animal models of tuberculosis and leishmaniasis. Potential targets for anti-infective gene therapy include chronic infectious diseases such as those analyzed in this study as well HIV-1. Another target would be patients who have a defined defect in the IL-12 and/or IFN-{gamma} signal transduction cascades, putting them at risk for mycobacterial (8, 9, 11, 29, 33, 39) and perhaps other infections. It should be emphasized that the studies outlined here represent the early stages of our efforts toward developing DC-based anti-infective immunotherapies, and future studies will be needed to rigorously define the optimal conditions and safety profiles for such therapies.


   Acknowledgments
 
We thank R. A. Clark, P. C. Melby, and A. Infante for helpful discussions and critical reading of the manuscript. We thank P. C. Melby, G. Deepe, Jr., J. R. Graybill for reagents. We thank the staff of the National Institutes of Health Blood Bank Center (National Institutes of Health, Bethesda, MD) and the Bone Marrow Transplant Unit and the General Clinical Research Center (Audie Murphy VA Medical Center, San Antonio, TX) for their help in apheresis of the normal donors.


   Footnotes
 
1 This work was supported by a Kleberg Foundation grant (to S.S.A. and S.K.A.), a Howard Hughes Medical Research Institute New Faculty Award (to S.S.A.), NIH Grant P50 CA58183 National Institutes of Health Grant P50 CA58183, and American Cancer Society Grant IRG-116R (to S.S.A.). Back

2 Address correspondence and reprint requests to Dr. Seema S. Ahuja, Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284. E-mail address: Back

3 Abbreviations used in this paper: DC, dendritic cell; PBHP, peripheral blood hemopoietic progenitor; G-CSF, granulocyte CSF; SCF, stem cell factor; GM-CSF, granulocyte-macrophage CSF; SLDA, soluble Leishmania donovani Ag; CW/M, detergent extract from cell wall and cell membrane of Histoplasma capsulatum yeast cells; SAC, Staphylococcus Aureus cowan strain; PPD, purified protein derivative. Back

Received for publication December 10, 1997. Accepted for publication March 10, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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