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The Journal of Immunology, 2005, 174: 758-766.
Copyright © 2005 by The American Association of Immunologists

Langerhans Cells Derived from Genetically Modified Human CD34+ Hemopoietic Progenitors Are More Potent Than Peptide-Pulsed Langerhans Cells for Inducing Antigen-Specific CD8+ Cytolytic T Lymphocyte Responses1

Jianda Yuan*,{ddagger},||, Jean-Baptiste Latouche2,{ddagger},¶,||, John L. Reagan*,{ddagger},||, Glenn Heller§,||,#, Isabelle Riviere{ddagger},¶,||,#, Michel Sadelain{ddagger},¶,||,# and James W. Young3,*,{dagger},{ddagger},||,#

* Laboratory of Cellular Immunobiology; {dagger} Allogeneic Bone Marrow Transplantation and Clinical Immunology Services; {ddagger} Division of Hematologic Oncology, Department of Medicine; § Biostatistics Service, Department of Biostatistics and Epidemiology; and Laboratory of Gene Transfer and Gene Expression, Gene Transfer and Somatic Cell Engineering Facility, Immunology Program, || Memorial Sloan-Kettering Cancer Center; and # Weill Medical College of Cornell University; New York, NY 10021


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34+ hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34+ hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20–30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100–150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-{gamma} secretion (100–123 spot-forming cells/105 CD8+ T cells) than did fluMP-pulsed LCs (10–91 spot-forming cells/105 CD8+ T cells). CD8+ T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8+ T cell responses than are peptide-pulsed LCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs)4 have emerged as effective immunogens in vivo for pairing tumor or viral Ags with the requisite secondary and tertiary signals needed to initiate cellular immune responses (1, 2, 3). Investigators are exploring a variety of tactics, however, to achieve more robust and sustained presentation of Ag, even by potent DCs.

One approach is to modify DCs genetically by introducingvectors that express a particular Ag of interest. Investigators have used lentivirus (4, 5, 6), oncoretrovirus (7, 8), adenovirus (9, 10), or plasmid DNA (11, 12, 13, 14, 15) Oncoretroviral vectors are the only ones that require proliferating target cells for transduction.

Many investigators are also exploiting the phagocytic capacity of monocyte-derived DCs (moDCs) by loading these cells with dying (16, 17, 18, 19, 20, 21, 22, 23) or Ab-opsonized cells (24) for cross-presentation to autologous T cells. Neither gene transduction nor cross-presentation of cellular Ag requires prior knowledge of a specific antigenic peptide or its restricting MHC allele. Unlike passive loading of single class I MHC-restricted peptides, these methods can also potentially provide helper epitopes to stimulate CD4+ T cells that support more robust primary CTL responses as well as development of immunologic memory (25, 26, 27, 28).

Human DC subsets are increasingly well defined, with attendant divisions of labor for stimulating innate and adaptive lymphocyte responses (23, 29, 30, 31). For example, CD34+ hemopoietic progenitor cell (HPC)-derived Langerhans cells (LCs) are more potent stimulators of CTL responding either to passively loaded peptide or to cross-presented tumor Ag than are moDCs, even though LCs are less phagocytic (23). moDCs are more potent stimulators of resting NK cells (30), but LCs help sustain NK cell viability (31).

We have explored the use of retrovirus for the introduction of Ag(s) into dividing CD34+ HPCs that subsequently differentiate into LCs. These Ags can be presented over time by potent LCs to stimulate Ag-specific CTL. Challenges for clinical application include the need to avoid culture in FCS, optimization of cytokines for adequate expansion of CD34+ HPCs in the absence of FCS without compromising retroviral transduction, and the presentation of Ag(s) that can elicit an actual immune response.

We have therefore undertaken these studies with these challenges in mind. To establish the parameters for optimal transduction, we have first characterized the immune response to a recall viral Ag such as the influenza matrix protein. To that end we designed a retroviral vector encoding the specific gene for the HLA-A*0201-restricted influenza matrix peptide (fluMP; GILGFVFTL). We have compared gene-transduced and peptide-loaded LCs with respect to typical DC phenotype and immunogenicity as well as the stimulation of MHC-restricted, fluMP-specific CD8+ CTL responses. Our long term goal is to transduce CD34+ HPCs with genes expressing full-length Ag(s), which would allow the DC progeny to synthesize, process, and present endogenous Ag tailored to self-MHC alleles. This would broaden the range of MHC molecules that could present immunogenic, but undefined, peptide Ags.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cytokines

Sterile recombinant, LPS-, pyrogen-, and mycoplasma-free human cytokines included GM-CSF (sargramostim, Leukine; previously Immunex, now Berlex); FLT-3 ligand (FL), IL-4, TNF-{alpha}, TGF-{beta}1, thrombopoietin (TPO), c-kit ligand (KL) or stem cell factor, IL-1{beta}, IL-6 (all from R&D Systems), and PGE2 (Calbiochem). All cytokines were supplied carrier-free by the manufacturer. but were reconstituted using human serum albumin (1% final concentration; 25% human serum albumin, NDC 63546-251-05, pharmaceutical grade, manufactured by Swiss Red Cross, distributed by Alpine Biologics) in PBS. Cytokine doses are specified below for the generation and maturation of CD34+ HPC-derived human DCs (23).

Oncoretroviral vectors, packaging cell lines, and titration of retroviral stocks

The SFG retroviral vector (32) encoding the Ag(s) of interest was placed under the transcriptional control of the Moloney murine leukemia virus long terminal repeat. Bicistronic retroviral constructs were made that carried the gene encoding either enhanced GFP (eGFP) or fluMP. This gene was joined by an internal ribosomal entry site (IRES) to a gene that encoded puromycin-N-acetyltransferase resistance (puroR; Fig. 1; retroviral vectors GIP and (fluMP-IRES-puroR) (FIP)) (33). The fluorescent marker served to monitor gene expression, and the flu peptide was the HLA-A*0201-restricted 9-mer, GILGFVFTL, which was used to stimulate and monitor MHC-restricted, Ag-specific CTL responses. High titer PG13 packaging cell lines were generated and selected for resistance to 0.5 µg/ml puromycin (Sigma-Aldrich) (34). Viral stocks were collected from these packaging cell lines and were used to transduce CD34+ HPCs before differentiation into LCs.



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  		FIGURE 1. Schematic representation of the SFG-GIP and SFG-FIP vectors. Bicistronic vectors were generated to express either eGFP or the HLA-A*0201-restricted, fluMP, GILGFVTL, linked in either case by an IRES to a puromycin resistance gene (puroR). The splice donor site (SD), splice acceptor site (SA), and extended packaging signal ({psi}+) are indicated.

 
Packaging cell lines were routinely passed and cultured in DMEM (Invitrogen Life Technologies) with 1% penicillin/streptomycin (Invitrogen Life Technologies), L-glutamine (Invitrogen Life Technologies), and 50 µM 2-ME (Invitrogen Life Technologies), supplemented with 10% FCS (Gemini BioProducts). For retroviral stocks generated under FCS-free conditions, these cells were thoroughly washed free of the FCS-containing medium and then recultured in serum- and plasma-free X-VIVO 15 (BioWhittaker). Supernatants for retroviral stocks were then collected daily over 3 days.

Retroviral vector titers were determined by infection of indicator HeLa cells (CCL-2; American Type Culture Collection) using serial dilutions of the retroviral vector stock. HeLa cells were transduced with cell-free (0.45 µm pore size filtration; Acrodisc; Pall) retroviral supernatants over 16 h in the presence of 8 µg/ml polybrene (Sigma-Aldrich). One replicate well was used at the time of infection to measure the exact viable target cell number. Seventy-two to 96 h from the start of the transduction, HeLa cells were harvested. The eGFP expression was assessed by flow cytometry, or puromycin-resistant colonies were enumerated. The fraction of HeLa cells that yielded between 10 and 50% eGFP-positive cells or viable (puromycin-resistant) colonies was used to calculate the titer as follows: (percentage of eGFP-positive or percentage of puromycin-resistant, viable colonies) x (HeLa cell number at time of infection) x (dilution factor) = infectious particles/ml.

FluMP synthesis

FluMP was synthesized using the known HLA-A*0201-restricted 9-mer sequence, GILGFVFTL (ResGen; Invitrogen Life Technologies). Peptide sequence and m.w. were confirmed on receipt, resuspended in 50% (v/v) RPMI 1640-DMSO (Sigma-Aldrich), and stored frozen at –20°C. Peptides were thawed once for dilution and use; peptides were never refrozen and rethawed.

Retroviral transduction and differentiation of CD34+ HPC-derived DCs

All human specimen collections adhered to protocols approved by the Memorial Hospital, Memorial Sloan-Kettering Cancer Center, Institutional Review and Privacy Board. CD34+ HPCs were isolated using positive immunomagnetic selection (CD34+ isolation and LS separation columns; Miltenyi Biotec) as previously described (23).

CD34+ HPCs were initially cultured at 2 x 105 cells/3 ml/well in a six-well tissue culture plate (Costar; Corning) in the presence of TPO (50 ng/ml; R&D Systems), c-kit ligand (KL; 20 ng/ml; R&D Systems), and FLT-3 ligand (FL; 50 ng/ml; R&D Systems) in X-VIVO15. After 24 h, the cycling CD34+ HPCs were transferred to a new non-tissue culture-treated, six-well plate (Costar) that had been coated with RetroNectin (Takara Bio) at 15 µg/ml. CD34+ cells in X-VIVO15 and cytokines were infected with viral stock at a 1:1 ratio for 24 h, which was 24–48 h after the start of culture. The infection was repeated on the same dividing CD34+ cells 24 h later, which was then 48–72 h after the start of culture. Thereafter, the expanding CD34+ HPCs were washed free of retroviral stock and existing cytokines and medium, then replated to new tissue culture-treated, six-well plates (Costar). These cells were then cultured in X-VIVO15 medium and cytokines with terminal maturation to generate gene-transduced LCs, exactly as previously described (23).

Cell cycle analysis

Cells were fixed in 0.4% paraformaldehyde (Sigma-Aldrich) and permeabilized in 50% (v/v) PBS (MSKCC Media Lab) and 50% (v/v) 0.2% Triton X-100 (Sigma-Aldrich). Cells were stained first by anti-Ki-67-FITC (IM 0606; Immunotech), and the DNA was subsequently stained with 7-aminoactinomycin D (no. 9400; Sigma-Aldrich). Cells were analyzed by flow cytometry using a FACScan (BD Biosciences Immunocytometry Systems) for cell cycle entry into S/G2/M phase (35).

T lymphocytes

T cells were obtained from tissue culture plastic (Falcon brand tissue culture plates, no. 35-3003; BD Biosciences)-nonadherent PBMCs. T cells were further purified by nonadherence and elution from nylon wool columns (Polysciences). The CD3+ purity was at least 95%.

Phenotypic analyses by flow cytometry

FITC-, PE-, and CyChrome-conjugated mouse anti-human mAbs included anti-CD11b, anti-CD14, anti-CD25, anti-CD83, anti-HLA-DR, and anti-CD86 (BD Biosciences). Isotype controls included IgG1-FITC, IgG1-PE, and IgG2a-FITC (DakoCytomation). Flow cytometric assessment used a FACScan (BD Biosciences Immunocytometry Systems), gating for collection and analysis of live events. Cells transduced by eGFP could be measured directly in the FITC channel, with counterstaining of other epitopes using fluorochrome conjugates detected in one of the other flow cytometer channels. For analysis of specific epitope expression by DCs, candidate cells were gated for viable, large forward scatter, HLA-DRbright cells, and 10,000 events were collected.

MLRs

DCs were cocultured with 105 purified allogeneic T cells (alloMLRs) in triplicate, round-bottom microwells at serial ratios from 30:1 to 1000:1 (T:DC), in complete RPMI 1640 (containing 10 mM HEPES (Life Technologies), 1% penicillin/streptomycin (MSKCC Media Lab Core Facility), 50 µM 2-ME (Invitrogen Life Technologies), and 1% L-glutamine (Invitrogen Life Technologies)), with 10% heat-inactivated (56°C, 30 min), autologous or single donor human plasma or serum. DCs were extensively washed to remove cytokines and irradiated (1500 rad) with 137Cs before adding to T cells. The resulting proliferation of responder T cells was based on the incorporation of [3H]TdR (1 µCi/well; NEN) during the last 12 h of a 4- to 5-day culture, as measured in a beta scintillation counter (Betaplate; Wallac).

Induction and measurement of influenza virus-specific CTL

Mature LCs were generated from HLA-A*0201 healthy donors. These LCs had been transduced as cycling CD34+ HPCs using a retroviral vector expressing the genes encoding the HLA-A*0201-restricted influenza matrix peptide and puromycin resistance (FIP-transduced-LCs). Alternatively, a separate cohort of LCs were loaded with the HLA-A*0201-restricted 9-mer, GILGFVFTL, at 10 µM for 1 h at room temperature (fluMP-pulsed-LCs). Mature fluMP-pulsed LCs or FIP-transduced LCs were cocultured with 106 purified autologous HLA A*0201 donor T cells per well in a 24-well plate at 30:1 T cell:LC ratio. These cultures used complete RPMI 1640 (see above) supplemented with 10% heat-inactivated, autologous plasma. T cell responses were assessed by ELISPOT and tetramer reactivity after one and/or two 5- to 7-day rounds of stimulation.

HLA-A*0201-restricted, fluMP-specific tetramer production and measurement of tetramer-positive T cells

HLA-A*0201-restricted fluMP streptavidin-PE-labeled tetramers (produced by Tetramer Core Facility, Sloan-Kettering Institute) were used to detect fluMP-specific T cells. Four-color staining and flow cytometric analyses (FACSCalibur, BD Biosciences Immunocytometry Systems) were performed with anti-CD62L-FITC, anti-CD3-CyChrome, anti-CD8-allophycocyanin (BD Biosciences), and tetramer-PE. T cells sensitized by fluMP-pulsed-LCs or FIP-transduced LCs were assayed for tetramer staining after each stimulation. Cells were considered positive for tetramer staining when they formed a clear population with a mean fluorescent intensity that was at least 1 log above the negative control staining. Up to 100,000 events were collected progressively after live gating on lymphocytes by forward and side scatter.

IFN-{gamma} ELISPOT assay

Multiscreen IP plates (Millipore) were coated with 10 µg/ml anti-human IFN-{gamma} (1-D1K; DiaPharma Group) in PBS overnight at 4°C. Unbound Ab was removed by three washings with PBS. After blocking the plates with 10% heat-inactivated autologous plasma/RPMI 1640 medium (1 h, 37°C), 105 cells/well were seeded in triplicate. FluMP-pulsed K562-A*0201-transfected cells (gift from Dr. T. Wolfel, University of Mainz) (36) or mature FIP-transduced LCs were used as APCs in the ELISPOT assays, both at 1 x 104 cells/well. CD8+ T cell responders were purified by positive immunomagnetic selection (Miltenyi Biotec). Replicate control wells contained CD8+ T cells with unloaded APCs, medium alone, or PHA (2 µg/ml; Sigma-Aldrich). Medium was complete RPMI 1640 with 10% heat-inactivated autologous plasma, all in a final volume of 200 µl/well. Cells were incubated at 37°C in 5% CO2 in a water-saturated incubator for 20 h. IFN-{gamma} production was then detected by the addition of a 1/500 dilution biotinylated mouse-anti-human IFN-{gamma} (clone 7-B6-1; Mabtech). After washing the wells five or six times with PBS/0.05% Tween 20, peroxidase-labeled streptavidin was added. The spots were developed with 3-amino-9-ethyl-carbazole substrate Vector ABC kit (Vector Laboratories). Spot development was stopped after 4 min by washing with running distilled water. The plates were examined under a stereomicroscope, and spots were evaluated with an Automated ELISPOT Reader System with KS 4.3 software (Carl Zeiss).

Statistics

The test of equality of means between groups was based on the two-sample t statistic. Pearson’s correlation coefficient was used as the measure of association between factors.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pre-expansion of CD34+ HPCs with TPO, KL, and FL maintains cell cycle entry and supports subsequent cytokine-driven differentiation into typical DC progeny

Removal of FCS from cultures of CD34+ HPCs compromises the expansion of DC progenitors (8, 23, 37, 38). Certain cytokines, e.g., TGF-{beta}1 or TNF-{alpha}, may also decrease susceptibility to retroviral gene transduction. We therefore asked whether initial expansion of CD34+ HPCs by cytokines without a differentiating effect toward DCs would achieve cell cycle entry similar to that supported by our standard cytokine combination for expansion and differentiation into LCs. Secondly, we determined whether pre-expansion altered subsequent susceptibility to cytokine-supported LC differentiation.

We tested the combination of TPO, KL, and FL in supporting CD34+ HPC expansion, because these cytokines optimize CD34+ cell division and prevent apoptosis (39, 40, 41). This triple cytokine combination was assessed against other combinations that specifically support expansion and differentiation of LCs (FL, KL, GM-CSF, TNF-{alpha}, and TGF-{beta}1) or dermal-interstitial DCs (DDC-IDCs) (FL, KL, GM-CSF, TNF-{alpha}, and IL-4) (23). Using a flow cytometry-based method, we determined the percentage of cells in S/G2/M phase (35) (Fig. 2). The rate of cell cycle entry was comparable across the different cytokine combinations and was highest between 24 and 48 h, which should correspond to the greatest susceptibility to retroviral gene transduction and DNA integration. To ensure that as many cycling cells as possible would be transduced, however, we elected to expose the target cells to retrovirus twice, initially from 24–48 h and again from 48–72 h



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  		FIGURE 2. Different cytokine combinations support comparable cell cycle entry by CD34+ hemopoietic progenitor cells. CD34+ HPCs cells were cultured as described in Materials and Methods using cytokine and medium conditions that supported the distinct generation of LCs (FL, KL, GM-CSF, TNF, and TGF-{beta}1) or DDC/IDCs (FL, KL, GM-CSF, TNF, TGF-{beta}1, and IL-4) and compared with TPO, KL, and FL. Cells were stained with anti-Ki-67-FITC and 7-aminoactinomycin D at the indicated time points along the x-axis. The double-positive cells by cytofluorography represent those cells in the S/G2/M phase of the cell cycle.

 
We next determined whether initial expansion in TPO, FL, and KL affected the phenotype and function of differentiated LC or DDC-IDC progeny (23). Flow cytometry data confirmed that all DC progeny achieved comparable phenotypic profiles after expansion, differentiation, and maturation, regardless of initial exposure to TPO, FL, and KL (pre-expansion vs standard exposure to DC expanding and differentiating cytokines (23) (Fig. 3A). Note the absence of the monocyte marker CD14 by all DCs, the expression of CD11b only by DDC-IDCs, but not by LCs (23), and the comparable expression of the activation and maturation epitopes CD83, CD25, and CD86, especially among the LCs. Comparable maturation phenotype between LCs or DDC-IDCs, pre-expanded or not with TPO, FL, and KL (only pre-expanded DCs shown; Fig. 3B), corresponded to comparably potent function in a standard assay of DC immunogenicity, the allogeneic MLR (alloMLR; Fig. 3C). There was even a trend toward higher allogeneic T cell proliferation when stimulated by pre-expanded LCs or DDC-IDCs, compared with their counterparts in standard cultures. We concluded that pre-expansion of CD34+ HPCs in TPO, FL, KL with subsequent cytokine-driven DC differentiation and maturation supports the phenotype and potent immunostimulatory function expected of standard DCs.



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  		FIGURE 3. Pre-expansion of CD34+ HPCs with TPO, KL, and FL does not alter normal DC maturation type or function. LCs and DDC/IDCs were generated from CD34+ HPCs with or without pre-expansion in TPO, KL, and FL as described in Materials and Methods. These were analyzed by flow cytometry on days 13–14 after the change of culture to DC-specific cytokines, which was 3 days after the original start of culture for those cells that underwent pre-expansion. Terminal maturation was accomplished by ~48-h exposure to an inflammatory cytokine combination (23 42 ). A, LCs or DDC/IDCs were gated on viable, large forward scatter, HLA-DRbright cells, and the percentage of total candidate DCs that expressed a given epitope was determined (n = 2–4 independent experiments for each of the epitopes, with mean percentage ± SD shown). The bars from left to right for each condition correspond to CD11b, CD14, CD25, CD83, and CD86. B, The fluorescent intensity of each marker is shown in a representative set of dot plots from pre-expanded LCs and DDC-IDCs. Because we subsequently transduced all DC progenitors during pre-expansion with TPO, KL, and FL, DC epitope expression without pre-expansion is not shown, but was similar. C, The allogeneic MLR was used as a standard assay of DC immunostimulatory function (x-axis, T:DC ratios) based on the extent of [3H]TdR incorporation by proliferating responder T cells (y-axis, log2 scale) in the last 12 h of a 4- to 5-day coculture. One representative alloMLR of three independent assays is shown with the triplicate means of [3H]TdR incorporation ± SD. D, The process of retroviral transduction did not alter the normal expression of a characteristic activation/maturation epitope like CD83. Some of the LC progeny already expressed CD83 because of physical cell manipulation in culture, even before cytokine-driven maturation. CD83 increased appropriately with maturation of either transduced (eGFP or FIP) or mock-transduced LCs. E, LC progeny from the same CD34+ HPC culture exposed to the same retroviral supernatant were not all transduced. Hence, only a proportion of the LC progeny expressed the transgene, yet all LCs regardless of transduction expressed the same maturation epitopes at comparable log10 fluorescent intensities. Shown in this study are two representative maturation and activation epitopes, CD83 and CD86.

 
We next investigated whether retroviral transduction itself would in any way alter the terminal activation and maturation phenotype of DCs under conditions of cytokine-driven CD34+ HPC pre-expansion and subsequent differentiation in the absence of FCS. Some autoactivation occurred due to physical manipulation of the cells in culture, even before addition of the maturation cytokines (42). The important point, however, was that transduced LCs matured comparably to mock-transduced LCs. Shown are FIP- or eGFP-transduced vs mock-transduced LCs expressing CD83, which is a standard DC surface epitope that characterizes not only successful differentiation along the DC pathway, but also stable maturation (23, 43, 44, 45) (Fig. 3D). Moreover, within the same culture exposed to retroviral vector encoding the gene for eGFP, both transduced and nontransduced LCs expressed comparable levels of CD83 as well as the costimulatory ligand CD86 (Fig. 3E).

RetroNectin supports efficient retroviral gene transduction in target CD34+ HPCs undergoing expansion and differentiation into LCs.

Fibronectin provides a substrate for target cells that can enhance retroviral gene transduction (46). A recombinant form of fibronectin, i.e., RetroNectin, allegedly exerts the same or greater effect (40, 47). We therefore compared gene expression by LCs after retroviral transduction of CD34+ HPCs cultured on non-tissue culture-treated plates precoated with RetroNectin, polybrene, 10% plasma as a natural source of fibronectin, or medium only. RetroNectin supported the most efficient gene transduction by ~2-fold, after two serial exposures to retrovirus from 24–48 h and again from 48–72 h. Histograms from a representative experiment (Fig. 4A) as well as the average percent transduced LCs from four independent experiments are shown (Fig. 4B). The LC progeny sustained this high GFP expression over 2 wk of subsequent culture (not shown) and achieved the expected mature phenotype and functional activity, as depicted in Fig. 3.



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  		FIGURE 4. RetroNectin (RN) facilities retroviral gene transduction of CD34+-derived DCs. Cell culture plates were coated as described in Materials and Methods with RetroNectin (15 µg/ml), polybrene (8 µg/ml), 10% plasma, or X-VIVO15 medium (negative control). CD34+ cells were added to these plates in the presence of TPO, KL, and FL in X-VIVO15 and transduced with eGFP retroviral supernatants at a 1:1 ratio of supernatant to medium. A, Histograms show the cell frequency and log eGFP gene expression 72 h after completion of transduction and transfer to DC-supportive cytokine conditions, with the percentage of positive cells indicated on each histogram. B, The percentage of cells that were eGFP positive after pre-expansion and transduction on each of the indicated subsets is shown as the mean of three independent experiments ± SD. *, p = 0.048; **, p = 0.005; ***, p = 0.008 (n = 3; two sample t statistic).

 
Differences in retroviral titer and donor susceptibility contribute to the efficiency of transduction and expression of the transgene

CD34+ HPCs were exposed twice to retroviral supernatant from 24–48 and 48–72 h during initial expansion in TPO, FL, and KL. Thereafter, expansion and differentiation continued in LC-specific cytokines. Preliminary experiments demonstrated that transduction and gene expression were compromised in the absence of pre-expansion and RetroNectin, compared with those in cells cultured in the presence of FCS (8). Having already converted to FCS-free methods for generating DCs from CD34+ HPCs, albeit at the expense of overall DC progenitor expansion (23, 37, 48), we tested transduction efficiency using retroviral stocks generated from packaging cells cultured in the presence or absence of FCS. Sixteen eGFP transductions (10 retroviral supernatants with and six without FCS) were evaluated for expression of the marker gene in the LC progeny (Fig. 5A). The absence of FCS, although essential for any future clinical application, did, in fact, somewhat compromise gene transduction efficiency, as manifested by lower eGFP expression (mean, 41.8 vs 26.6%; day 12). There was also a trend toward reduced expression after maturation from days 12–14 (mean, 41.8%, decreasing to 35.3% in the presence of FCS; mean, 26.6%, decreasing to 21.4% in the absence of FCS). Transgene expression in the absence of FCS was considerably higher, however, when CD34+ HPCs were pre-expanded in TPO, FL, and KL than when they were not (not shown).



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  		FIGURE 5. Efficiency of retroviral gene transfer and expression by CD34+ HPC-derived LCs exhibits viral titer-, donor-, and time-dependent variability. A, CD34+ HPCs were pre-expanded from 0–72 h with TPO, KL, and FL on RetroNectin-coated plates. Cells were exposed twice to FCS-containing or FCS-free retroviral supernatants carrying the eGFP vector from 24–48 and 48–72 h (supernatant:medium ratio, 1:1), followed by thorough washing and reculture in LC-supportive cytokines as described. Candidate HLA-DRbright LC progeny were gated on day 12 (precytokine maturation) and day 14 (postcytokine maturation) from the start of LC-specific culture, and the percentage of these cells that expressed eGFP was determined by flow cytometry. A scatter plot depicting the percentage from each independent experiment (n = 10 with FCS-containing supernatants; n = 6 with FCS-free supernatants) and the calculated mean (bar) is shown. Transduction was somewhat less efficient by the FCS-free supernatants with some decline upon maturation, but was comparable or superior to previous results in FCS-containing medium without pre-expansion or RetroNectin (8 ). B, The percentage of mature, day 14 LCs expressing the eGFP transgene from A was plotted against the titer of each retroviral supernatant used. There was a positive correlation, but only r = 0.51 (Pearson’s correlation coefficient), suggesting that other factors in addition to viral titer contributed to transduction efficiency and gene expression. C, Three different FCS-free retroviral supernatants with moderate range titers (2.34, 3.22, and 3.89 x 105 U/ml) were tested on CD34+ HPCs from each of four different healthy donors, and the mean percentage of eGFP-positive LCs ± SD is shown (top). The mean percentage of eGFP-positive LCs ± SD is also shown for each of these three supernatants tested on the four different healthy donors. D, Depicted is the percentage of total LCs from three different donor CD34+ HPCs expressing the eGFP transgene after exposure to one of three different retroviral supernatants. Regardless of peak expression, there was only a 25–30% decline in eGFP-positive LCs over 4 wk.

 
We next evaluated the correlation between retroviral titer and eGFP expression by transduced LCs (Fig. 5B). Although higher titers usually supported greater transduction and, hence, eGFP expression, the r value was only 0.51. This suggested that other factors influenced the final outcome, especially in the case of FCS-free retroviral stocks. Three different FCS-free retroviral supernatants with intermediate range titers (2.34, 3.22, and 3.89 x 105 U/ml) were therefore tested on CD34+ HPCs from each of four different healthy donors, and the data were evaluated in two different ways. The mean percentage of LC progeny that expressed eGFP after separate transductions by all three retroviral stocks was determined for each donor and indicated donor-dependent variability in retroviral transduction susceptibility (Fig. 5C, top). Alternatively, the mean percentage of LC progeny from all four donors that expressed eGFP was calculated for each of the retroviral stocks. This demonstrated more modest differences in transduction efficiency and gene expression, comparable to the similar, intermediate range titers among the three different retroviral stocks (Fig. 5C, bottom). Finally, regardless of the peak transduction and gene expression by a given donor’s DCs, there was sustained expression after maturation, with a decline of only 25–30% over 28 days (Fig. 5D). We concluded that differences in both donor susceptibility and retroviral titer were important contributors to the final transgene expression in LCs. This was especially true for expression by mature LCs after transduction by FCS-free retroviral supernatants. Expression was nevertheless sustained by a sizeable proportion of cells over 4 wk.

Genetically modified CD34+ HPC-derived Langerhans cells express and present an immunogenic viral Ag that stimulates a more robust CD8+ cytotoxic T cell response than do peptide-pulsed LCs

Given the parameters established using the marker gene eGFP, we undertook critical experiments to prove that retrovirally transduced LCs could, in fact, present vector-encoded Ag in an immunogenic form that would stimulate CD8+ CTL responses. To focus specifically on the effect of retroviral transduction on gene expression and Ag presentation, we evaluated the response to influenza and thereby obviated issues related to baseline cellular immunity.

CD34+ HPCs were pre-expanded in TPO, KL, and FL and serially exposed twice to retroviral supernatant with the vector encoding the HLA-A*0201-restricted fluMP (see also Fig. 1). The vector also encoded the gene for puromycin resistance, and the titer for each retroviral stock was determined on transduced HeLa cells cultured at the same time as the CD34+ HPCs. After transduction and 2-wk culture and maturation of LC progeny, these FIP-transduced-LCs were used to stimulate autologous T cells. Comparison conditions included fluMP-pulsed LCs (23) and eGFP-transduced LCs, all at a DC:T cell ratio of 1:30 for 6–7 days. Binding of the T cell responders to flu-MP/HLA-A*0201 tetramers confirmed expansion of fluMP-specific CTLs. The expansion of fluMP-specific CTLs was more robust after a second 7-day stimulation by FIP-transduced LCs compared with that stimulated by fluMP-pulsed LCs (Fig. 6A). Stimulation by eGFP-transduced LCs did not increase tetramer-positive cells above baseline (not shown). Within either population of T cells responding to LC stimulation against fluMP, the tetramer-positive cells were the only T cells to expand (Fig. 6B; the stimulation index of flu-MP/HLA*A0201 tetramer-positive cells was calculated as the absolute number of tetramer-positive cells at the end of each stimulation over the number of tetramer-positive cells in the initial culture).



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  		FIGURE 6. Stimulation of HLA-A*0201/fluMP-tetramer specific CD8+ T cells by FIP-transduced or fluMP-pulsed LCs. Autologous HLA-A*0201-positive T cells were stimulated by FIP-transduced LCs or fluMP-pulsed LCs at a T cell:LC ratio of 30:1 without addition of any exogenous cytokines. A, Dot plots of CD8+ HLA-A*0201/fluMP-reactive T cells are shown after one and two rounds of stimulation by the respective LCs, with a truly discrete tetramer-positive population consistently arising only after a second round of stimulation (representative of three independent experiments). The overall increase over 0.2% baseline tetramer-positive cells was ~130-fold for T cells stimulated by FIP-transduced LCs and ~30-fold after stimulation by fluMP-pulsed LCs. B, The stimulation index for CD8+ HLA-A*0201/fluMP reactive, tetramer-positive cells was calculated as the absolute number of tetramer-positive cells at the end of each stimulation over the number of tetramer-positive cells at the initial culture. The mean of three independent experiments ± SD is shown. The difference between the transduced and peptide-pulsed LCs after the second round of stimulation was significant (p = 0.028, two sample t statistic).

 
In addition to tetramer reactivity, we confirmed that transduced LCs stimulated higher levels of functionally active CTL compared with peptide-pulsed LCs. Autologous T cells were stimulated twice by mature FIP-transduced LCs or autologous FluMP-pulsed LCs at a T:DC ratio of 30:1. After the second stimulation, CD8+ T cells were positively selected and replated in the presence of either fluMP-pulsed K562-A*0201 transfectants or FIP-transduced LCs. These served as targets for stimulation of IFN-{gamma} secretion by the responder CD8+ T cells, which was measured in an ELISPOT assay (Fig. 7A). FIP-transduced LCs stimulated significantly greater IFN-{gamma} responses by CD8+ T cell responders than did fluMP-pulsed LCs (p < 0.01, two sample t statistic; n = 3). For a given method of Ag loading, however, LCs stimulated comparable IFN-{gamma}-producing CD8+ T cells against each of the target cells. This confirmed that the CD8+ CTL reactivity was directed against fluMP and not against copresented retroviral Ags, because the response against the transduced targets was the same as that against the peptide-pulsed targets. These data do not exclude a role, however, for nonspecific helper epitopes provided by retroviral expression in the gene-modified LCs during primary stimulation, because only class I MHC-restricted responses were measured in this study. We also confirmed a strong concordance between the number of HLA-A*0201-fluMP tetramer-positive cells after two rounds of stimulation and the amount of IFN-{gamma} secretion, as measured in secondary ELISPOT assays (r = 0.828; Fig. 7B).



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  		FIGURE 7. Stimulation of IFN-{gamma}-secreting, CD8+ fluMP-specific CTL by FIP-transduced LCs or fluMP-pulsed LCs. Autologous T lymphocytes were sensitized by FIP-transduced LCs or fluMP-pulsed LCs at a 1:30 ratio (DC:T cells). After two 7-day rounds of stimulation, ELISPOT assays measuring IFN-{gamma} secretion by positively selected CD8+ T cell responders were performed with one of two target cells: HLA-A*0201-transfected K562 cells pulsed with fluMP or FIP-transduced LCs. A, FluMP-specific CD8+ CTLs induced by FIP-transduced LCs targeted either the fluMP-pulsed K562-A*0201 transfectants (range 100–123, mean ± SD, 107 ± 15) or the fluMP-transduced LCs (range 84–121, mean ± SD, 105 ± 19). The fluMP-pulsed LCs also stimulated substantial fluMP-specific CD8+ CTLs, but at a lower frequency with a broader range against either the fluMP-pulsed K562-A*0201 transfectants (range 16–77, mean ± SD, 47 ± 31) or the fluMP-transduced LCs (range 10–91, mean ± SD, 50 ± 40). Data are from three independent experiments. The negative controls were unpulsed K562-A*0201 transfectants. *, p = 0.009; **, p = 0.004 (n = 3; two sample t statistic). B, The frequency of IFN-{gamma} spot-forming cells also correlates positively with HLA-A*0201/fluMP tetramer-reactive CD8+ T cells (r = 0.828, Pearson’s correlation coefficient; n = 11 independent experiments).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that gene-modified human DCs derived from CD34+ HPCs can generate robust Ag-specific CTL. Oncoretroviral transduction and gene expression by DC progeny occur under FCS-free culture conditions in the presence of TPO, FL, and KL (39, 40, 41) using a recombinant fibronectin (RetroNectin) substrate. Subsequent expansion and differentiation yield gene-modified, but otherwise normal, immunostimulatory DC progeny. Neither pre-expansion, retroviral transduction, nor expression of the transgene alters the expected phenotype and function of the mature, activated DCs. Approximately 20–30% of the LC progeny exhibit sustained expression of the transgene over at least 28 days. This efficiency of retroviral transduction is at least comparable and often better than what was previously achieved in the presence of FCS but without pre-expansion and/or RetroNectin (8). Both variations in donor susceptibility and retroviral titer contribute to the efficiency of transduction and eventual gene expression.

The specific human cell surface receptors for retroviral entry can depend on culture medium and its components, including FCS, without which there is lower gene expression after transduction (49, 50) (Fig. 5A). Another limiting factor has been that the packaging cell lines yield lower titers of retrovirus in the absence of FCS. Cytokines that expand and differentiate CD34+ HPCs into DCs in the absence of FCS (23) also never reproducibly support sufficient levels of transduction. Whether cytokines such as TGF-{beta}1 or TNF-{alpha} in the standard LC cultures might also interfere with transduction during exposure to a retroviral vector remains speculative. We have found that the combination of TPO, KL, and FL in the presence of recombinant fibronectin (RetroNectin) avoids this potential interference. It also optimizes CD34+ cell cycle entry and confers resistance to apoptosis (39, 40, 41, 46, 47), while avoiding the obstacle of FCS to any clinical applications of transduced LCs.

We have concentrated on retroviral transduction and expression of the immunogenic fluMP compared with passive peptide loading onto the DC surface. By using influenza, we avoided many unknowns about potential baseline reactivity against a self/tumor Ag. We also focused on LCs derived from CD34+ HPCs, because these are more potent stimulators of Ag-specific CTL than either CD34+ HPC-derived DDC-IDCs or moDCs (23). The moDCs differentiate, but do not undergo cell division, from their blood monocyte precursors and hence are not in cycle for retroviral transduction.

Gene-modified LCs expressing fluMP stimulate a much higher frequency of HLA-A*0201-restricted, fluMP tetramer-reactive T cells, than do fluMP-pulsed LCs, even though both are very active at T:LC (total) ratios of 30:1 and higher. For the 20–30% transduced LCs, this corresponds to ratios of T:LCs (transduced) of 100–150:1 and higher, and it is consistent with the expected potent immunogenicity of mature, activated LCs. Tetramer-positive T cells also account for all of the expansion in these cultures, because only class I MHC-restricted Ag is presented. CTLs stimulated by the retrovirally transduced LCs react with the specific fluMP Ag and do not show increased reactivity against targets expressing retroviral Ags from the vector. This contrasts with the immunogenicity of other vectors like adenovirus or poxvirus, which can in turn limit their longer term efficacy if used in cellular vaccines. Our data do not exclude a role, however, for nonspecific helper epitopes provided by retroviral expression in the gene-modified LCs during primary stimulation.

We have used a Moloney murine leukemia virus-based vector in these studies. Transcriptional silencing of transgene expression has been observed over time when using this vector (51), but we found expression out to 28 days despite some decline from peak levels. Although this would compromise gene modification of cells used for replacement therapies, this is not a problem for DC-based vaccines bearing an immunogenic transgene. The expected duration of gene expression is no less than the anticipated life span of an injected DC and is sufficient for T cell stimulation. Long term immunity may require some form of chronic exposure to Ag-loaded dendritic cells, but this is an issue for all forms of DC vaccines.

We conclude that even though only 20–30% of the LC progeny are transduced, expression of the transgene encoding fluMP is both adequate, even at low stimulator doses, and sufficiently sustained to recruit additional reactive T cells with repeated rounds of stimulation. This underscores an important advantage of transduced gene expression in CD34+ HPC-derived DCs. Unlike DCs bearing exogenously pulsed peptide, the Ag is not lost at an unknown rate from surface MHC molecules without being replenished. Moreover, vectors encoding longer polypeptides or even full-length protein Ags should enable the DCs themselves to process and present antigenic peptides on a variety of MHC molecules. This would obviate a major obstacle to the broader use of DC-based vaccines by not requiring prior knowledge of specific immunogenic peptides and their restricting MHC alleles.


   Acknowledgments
 
We gratefully acknowledge the discussions and critical review of the manuscript by Drs. Alan Houghton and Christian Munz. We thank the nurses and physicians of the Allogeneic Bone Marrow Transplantation Service as well as the Allogeneic Transplant and Cytotherapy Laboratory staffs at Memorial Sloan-Kettering Cancer Center for assistance with sample procurement and processing. We also greatly appreciate the formative technical assistance of Humilidad Gallardo in the early stages of this work.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants P01CA59350 (to all authors) and R01CA83070 (to J.W.Y.) from the National Cancer Institute, National Institutes of Health; and by William H. Goodwin and Alice Goodwin of the Commonwealth Cancer Foundation for Research and the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center. Back

2 Current address: Laboratoire de Génétique Moléculaire, Institut National de la Santé et de la Recherche Médicale, Unité 614-IFRMP, Centre Hospitalier Universitaire, Faculté de Médecine et de Pharmacie, 22 boulevard Gambetta, 76183 Rouen, France. Back

3 Address correspondence and reprint requests to Dr. James W. Young, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail address: youngjw{at}mskcc.org Back

4 Abbreviations used in this paper: DC, dendritic cell; alloMLR, allogeneic MLR; eGFP, enhanced GFP; FIP, fluMP-IRES-puroR; FL, FLT-3 ligand; fluMP, influenza matrix peptide; HPC, hemopoietic progenitor cell; IRES, internal ribosomal entry site; KL, c-Kit ligand; LC, Langerhans cell; DDC-IDC, dermal-interstitial DC; moDC, monocyte-derived DC; puroR, puromycin-N-acetyltransferase resistance; TPO, thrombopoietin. Back

Received for publication September 10, 2004. Accepted for publication November 2, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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