Dendritic cell (DC) therapies are currently being evaluated for the treatment of cancer. The majority of ongoing clinical trials use DCs loaded with defined antigenic peptides or proteins, or tumor-derived products, such as lysates or apoptotic cells, as sources of Ag. Although several theoretical considerations suggest that DCs expressing transgenic protein Ags may be more effective immunogens than protein-loaded cells, methods for efficiently transfecting DCs are only now being developed. In this study we directly compare the immunogenicity of peptide/protein-pulsed DCs with lentiviral vector-transduced DCs, and their comparative efficacy in tumor immunotherapy. Maturing, bone marrow-derived DCs can be efficiently transduced with lentiviral vectors, and transduction does not affect DC maturation, plasticity, or Ag presentation function. Transduced DCs efficiently process and present both MHC class I- and II-restricted epitopes from the expressed transgenic Ag OVA. Compared with peptide- or protein-pulsed DCs, lentiviral vector-transduced DCs elicit stronger and longer-lasting T cell responses in vivo, as measured by both in vivo killing assays and intracellular production of IFN-γ by Ag-specific T cells. In the B16-OVA tumor therapy model, the growth of established tumors was significantly inhibited by a single immunization using lentiviral vector-transduced DCs, resulting in significantly longer survival of immunized animals. These results suggest that compared with Ag-pulsed DCs, vaccination with lentiviral vector-transduced DCs may achieve more potent antitumor immunity. These data support the further development of lentiviral vectors to transduce DCs with genes encoding Ags or immunomodulatory adjuvants to generate and control systemic immune responses.

Dendritic cells (DCs) 3 are the most potent APCs and play a pivotal role in the initiation of immune responses against infections and tumors and in the induction of peripheral tolerance (1, 2). Considerable effort has been made to develop strategies to introduce Ags into DCs to initiate Ag-specific immune responses. Peptide-pulsed DCs have been shown to induce tumor-specific T cell immunity in murine models (3, 4). Delivery of whole proteins using particulate or osmotic-loading approaches has been shown to provide additional advantages of polyepitope delivery and to enable development of synergistic CD4+ and CD8+ T cell responses by simultaneous presentation of epitopes through the MHC class I and class II processing pathways (5, 6, 7). The ability of DCs to actively take-up and present tumor-derived proteins from live or apoptotic tumor cells, or tumor cell lysates has been demonstrated and presents additional theoretical advantages in the efficiency and breadth of the resulting tumor-specific immune responses (8, 9, 10). These results and others provide strong rationale for protein-based DC loading strategies and clearly support the ∼100 clinical trials aimed at inducing antitumor immunity, the majority of which use DCs loaded with peptide or protein tumor Ags (11, 12, 13, 14, 15).

Several studies suggest that surface presentation of MHC class I peptide complexes by peptide- or protein-loaded DCs is relatively short-lived (16, 17, 18, 19), potentially limiting the potency of the resulting immune responses. In addition, recent studies raise the possibility that Ag from Ag-loaded DCs can be cross-presented by resident DCs, contributing to the generation of the immune response (20, 21, 22). Notably, apoptosis of DCs expressing transgenic Ags has been shown to result in enhanced immunity through cross-presentation in vivo (23). Furthermore, the latest studies suggest that cross-presentation favors epitopes derived from stabilized proteins rather than those from rapidly degraded proteins or short peptides (24, 25, 26). Given these observations, it is reasonable to suggest that the use of DCs presenting a sustained source of endogenously synthesized transgenic protein Ag may be advantageous for T cell induction due to both the prolonged presentation by modified DCs and cross-presentation by endogenous DCs.

Viral transduction is an appealing approach for engineering DCs to produce, process, and present specific Ags (27). Adenoviral vectors have been shown to be effective means for transducing DCs (28, 29, 30). However, recent data indicate that adenoviral transduction results in DC maturation and proinflammatory cytokine production through activation of the NF-κB pathway (31). In addition, Ags derived from adenoviral proteins can be presented by transduced DCs, resulting in not only the interference of the desired Ag-specific immune responses, but also the induction of vector-specific immunity that makes repeated immunization problematic (32). Oncoretroviral vectors have been used to deliver genes into DCs with minimal interference of their function, but low transduction efficiencies, presumably due to the fact that oncoretroviral vectors are unable to transduce nondividing cells, limit their applications (33).

To overcome these disadvantages, lentiviral vectors (lvv) have recently been developed to introduce foreign genes into DCs (34, 35, 36, 37, 38). In contrast to oncoretroviral vectors, lvv are able to transduce nondividing cells, such as DCs, at high transduction efficiencies. Importantly, like oncoretroviral vectors, lvv do not encode viral proteins, thereby minimizing the potential for interfering with the function of transduced DCs (39). Recently, third-generation lvv with enhanced safety profiles have been developed and used to efficiently transduce murine DCs and human DCs (36, 37, 40). These improved vectors contain a chimeric Rous sarcoma virus (RSV)/HIV 5′ long terminal repeat (5′LTR) enhancer and promoter to initiate the transcription of genomic viral RNA (41). The stronger chimeric promoter does not require HIV Tat protein, a transactivator in the transcription of HIV genomic RNA, to generate vector transcripts. In addition, the vectors have been made self-inactivating by deleting the majority of U3 region in 3′LTR so that viral RNA cannot be produced in target cells (42). These additional safety modifications further prevent the generation of replication-competent recombinants. In aggregate, these features suggest that this lvv has considerable potential for clinical applications.

Although, in theory, presentation of endogenously expressed Ag by gene-modified DCs should elicit more potent immune responses than peptide- or protein-pulsed DCs, results from comparative investigations are conflicting (32, 43, 44, 45). This may be a critical issue for immunotherapy, particularly in the setting of tumor-bearing hosts or victims of viral infections such as HIV, in whom immunizations will need to overcome pre-existing immunosuppressive mechanisms. We recently demonstrated, by direct comparison, that DCs expressing transgenic Ag after nonviral transfection induced more potent T cell immunity than peptide/protein-pulsed DCs (46). As yet there are no reports comparing the immunogenicity of lvv-transduced DCs with that of peptide/protein-pulsed DCs. In this study, using a third-generation lvv optimized for safety and efficiency, we directly compare these Ag-loading strategies by comparing the kinetics and strength of Ag-specific immune responses induced in vitro and in vivo. We show that DCs transduced with lvv stimulate stronger and long-lasting in vivo CTL activity compared with peptide/protein-pulsed DCs. Furthermore, we demonstrate that DCs modified by lvv can achieve significantly better therapeutic antitumor effects compared with peptide/protein-pulsed DCs. These findings suggest that third-generation lvv have considerable potential for clinical application.

C57BL/6, BALB/c, and OVA TCR transgenic mice (OT-I and OT-II) were purchased from The Jackson Laboratory. The mice were housed under specific pathogen-free conditions in the central animal facility of University of Pittsburgh. Handling of the mice was performed according to institutional guidelines of University of Pittsburgh.

Mouse DCs were generated using standard protocols (47). Briefly, bone marrow cells were depleted of T, B, and NK cells with Ab and complement. Cells were then seeded at 15–20 million/75-cm flask in RPMI 1640 medium containing GM-CSF (1000 U/ml) and IL-4 (1000 U/ml). Half the medium was changed every other day. By day 8, ∼90% of cells were CD11c+.

The third-generation lvv pLenti6/-TOPO plasmid was purchased from Invitrogen-Life Technologies. Genes encoding enhanced GFP (EGFP) or truncated cytoplasmic chicken OVA (first 138 aa were deleted) were cloned into the vector downstream of the CMV promoter using BamHI and XhoI sites. Our previous studies demonstrated that this cytosolically expressed transgenic Ag can be efficiently presented (46). To enhance nuclear import, a DNA fragment containing a central polypurine tract sequence and the central termination sequence, which together form a triple-stranded DNA flap (TRIP), was inserted in the unique site of ClaI in front of CMV promoter as previously described (48). The final plasmid constructs, pLenti-EGFP-TRIP and pLenti-OVA-TRIP, were used to generate lentivirus, as shown diagrammatically in Fig. 1.

FIGURE 1.

Schematic diagram of lvv. pRSV/5′LTR, RSV LTR and HIV LTR chimeric promoter; SD and SA, splicing donor and acceptor sites of HIV mRNA; ψ, RNA packaging signal; RRE, Rev response element sequences; CMVp, CMV promoter used to drive transgene expression; SV40p-Blasticidin, SV40 virus promoter used to drive selection marker blasticidin gene expression; ΔU3/HIV 3′LTR, promoter deleted in U3 region so that the lvv become self-inactivated (SIN); OVA138, truncated cytoplasmic OVA gene. Restriction sites are also indicated.

FIGURE 1.

Schematic diagram of lvv. pRSV/5′LTR, RSV LTR and HIV LTR chimeric promoter; SD and SA, splicing donor and acceptor sites of HIV mRNA; ψ, RNA packaging signal; RRE, Rev response element sequences; CMVp, CMV promoter used to drive transgene expression; SV40p-Blasticidin, SV40 virus promoter used to drive selection marker blasticidin gene expression; ΔU3/HIV 3′LTR, promoter deleted in U3 region so that the lvv become self-inactivated (SIN); OVA138, truncated cytoplasmic OVA gene. Restriction sites are also indicated.

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To produce lvv, human embryonic cells 293T cells were seeded at 5 million/100-mm plate in DMEM and grown for 24 h. Plasmid DNA pLenti-EGFP-TRIP together with packaging plasmids, pLP1, pLP2, and pVSV-G (Invitrogen Life Technologies), were cotransfected into 293T cells using the calcium phosphate precipitation method according to manufacturer’s description (Stratagene). After 48 h, lentivirus was collected and concentrated from medium using ultracentrifugation, followed by ultrafiltration as previously reported (49, 50). Lentiviral titers (transduction units (TU)) were determined using 293T cells. Using this cotransfection approach, the vector titer obtained from the original medium reproducibly ranged from 2–5 × 107 TU/ml. After sequential concentration, the titer determined using 293T cells reached 1.5–3 × 109 TU/ml. Lentiviral vectors produced were designated as EGFP-lvv and OVA-lvv, respectively. Handling of viral vectors was performed according to the guidelines of BSL-2+ laboratories established by the recombinant DNA committee of University of Pittsburgh.

Six million DCs or their progenitors were washed with serum-free RPMI 1640 medium once and resuspended in 200 μl of serum-free medium. Cationic polymer polybrene was added to serum-free medium to a final concentration of 32 μg/ml. One hundred microliters of lvv (1.2–2.4 × 108 TU) in PBS was mixed with 100 μl of polybrene-containing, serum-free medium and incubated at room temperature for 15 min. The cells were mixed with virus and incubate at 37°C for 3 h. The final 400 μl of transduction mixture contained 6 million DCs with 1.2–2.4 × 108 TU of lentivirus. Six milliliters of GM/IL-4-containing medium was added to each tube and divided into two wells (six-well plate). The cells were cultured at 37°C. Half the medium was replaced with fresh GM/IL-4 medium every other day. To load DCs with protein or peptide Ags, DCs were pulsed with OVA protein (200 μg/ml) and or/the class I-restricted peptide SIINFEKL (10 μg/ml) or the class II-restricted peptide ISQAVHAAHAEINEAGR peptide (10 μg/ml), as indicated, in RPMI 1640 medium for 3 h at 37°C. DCs were then washed three times before use.

BALB/c splenic T cells were enriched using nylon wool columns, and B cells, DC, and macrophages were depleted by anti-B220, anti-MHC II (M5114), and anti-macrophage (F4/80) Ab (American Type Culture Collection) and complement (Cedarlane Laboratories). DCs were serially diluted in a round-bottom, 96-well plate started at 2000 cells/well. One hundred microliters of BALB/c cells (0.1 million) were added to each well. Cells were cocultured for 2 days, then 100 μl of medium was collected for measuring cytokine production. [3H]Thymidine was added for an additional 24 h of culture, then cells were harvested, and cell proliferation was measured by [3H]thymidine incorporation.

OT-I and OT-II T cells were isolated from the spleen and lymph nodes (LN) of OVA TCR transgenic mice. The splenocytes and LN cells were enriched on nylon wool columns, then B cells, NK cells, DCs, macrophages, and CD4 or CD8 T cells were deleted using Ab and complement. The purity of OT-I and OT-II T cells was determined to be >95%. DCs (transduced or pulsed) were then serially diluted as in the MLR assay. OT-I or IT-II T cells were added to each well in 100 μl (20,000 cells/well). Cells were cocultured for 2 days, 100 μl of medium was collected for measuring cytokine production. [3H]thymidine was added for an additional 24 h of culture, then cells were harvested, and cell proliferation was measured by [3H]thymidine incorporation.

The quantity of cytokines, IL-12p70 and IFN-γ, were measured using the OPTIA kit from BD Pharmingen according to the manufacturer’s instructions.

Cells were collected and washed with PBS. A half-million cells were added to each staining tube. Anti-CD11c-biotin, anti-B7.2-PE, anti-IFN-γ-allophycocyanin, anti-Vα2-PE, and anti-Vβ5.1–5.2-biotin Abs were purchased from BD Pharmingen. CyChrome- or allophycocyanin-labeled streptavidin was used as secondary reagent at a 1/1,000 dilution. Cells were fixed, and 30,000 events were collected and analyzed.

Mice were inoculated with 0.2 million B16-OVA cells in 50 μl of PBS s.c. Three days later, 500,000 lvv-transduced or SIINFEKL/OVA-pulsed DCs were injected into the footpads of the mice. Tumor growth was monitored by measuring the perpendicular diameter of the tumor, and survival was recorded daily.

In vivo Ag-specific lytic activity was measured by an in vivo killing assay, as previously described (51). Briefly, mouse splenocytes were collected and pulsed with SIINFEKL (250 ng/ml) and were labeled using a high concentration of CFSE (5 μM; CFSEhigh). Mouse splenocytes without SIINFEKL peptide were labeled using a low concentration of CFSE (0.5 μM; CFSElow) as an internal control. Ten million cells of each population were mixed and injected into mice via the tail vein. The relative abundance of CFSEhigh and CFSElow cells in spleen or peripheral blood was determined by flow cytometry at 5 and/or 20 h after injection, as specified in the individual experiments. Specific lysis was calculated according to the following formula: {1 − [(ratio of CFSElow/CFSEhigh of naive mouse)]/[(ratio of CFSElow/CFSEhigh of vaccinated mouse)]} × 100.

Splenocytes were collected and stimulated in vitro for 20 h by adding OVA class I peptide SIINFEKL or class II peptide ISQAVHAAHAEINEAGR. GolgiStop (BD Pharmingen) was added to the culture in the last 4 h of restimulation to block the secretion of IFN-γ. Cells were then collected and stained for CD8 or CD4, and intracellular IFN-γ was detected using an intracellular staining kit according to the manufacturer (BD Pharmingen).

The in vivo Ag presentation assay was performed as previously described (52). Briefly, mice were injected in the footpad with 500,000 DCs that were transduced with lvv expressing OVA or pulsed with SIINFEKL peptide or OVA protein as indicated. At the indicated time points, 5 million OT-I cells labeled with CFSE were administered through the tail vein. Forty-eight hours after injection of OT-I cells, draining popliteal LN and contralateral LN cells were collected and stained with anti-Vα2 and anti-Vβ5.1–5.2 and analyzed for proliferation as described previously (52).

Unpaired t test analysis was used to determine whether the differences between T cell-mediated immune responses induced by peptide/protein-pulsed DCs vs transduced DCs was significant.

Day 2 DCs were transduced in vitro with EGFP-lvv at 40 TU/cell On day 8, DCs were collected and stained for expression of CD11c and B7.2. Approximately 84% of the total cells in culture in both mock- and lvv-transduced cells were CD11c+, and 50% of these expressed EGFP (Fig. 2,A). In addition, when the levels of expression of B7.2 were used as a marker of DC maturation, it was found that approximately equal numbers of mature (CD11c+B7.2high) and immature (CD11c+B7.2low or neg) DCs expressed EGFP (Fig. 2,B). Importantly, DC transduction did not alter the relative proportions of mature vs immature DCs in DC populations (Fig. 2 B), suggesting that lentiviral transduction of DC progenitors or DCs at early stages does not alter DC development or maturation.

FIGURE 2.

Efficiency of lentivirus-mediated transduction of DCs. Day 2 DCs were transduced with EGFP-lvv at 40 TU or mock-transduced with PBS, the buffer used to deliver lvv. Six days later, cells were collected and stained to determine the percentage of CD11c+ (A) or B7.2+ (B) cells expressing transgenic EGFP. Numbers indicate the percentage of events in the indicated quadrant. A typical result from four experiments is presented. C, Bone marrow cells were collected on day 0 and were transduced immediately or cultured in GM/IL-4 medium and transduced on day 2, 4, or 6 as indicated. Cells were collected on day 8 (for cells transduced on days 0 and 2), or day 10 (for cells transduced on days 4 and 6), stained with CD11c, and analyzed by flow cytometry. The percentage of CD11c+EGFP+ cells is shown. For each time point, three transductions were conducted on each six-well plate, and the mean ± SD are shown. The data shown are representative of three experiments.

FIGURE 2.

Efficiency of lentivirus-mediated transduction of DCs. Day 2 DCs were transduced with EGFP-lvv at 40 TU or mock-transduced with PBS, the buffer used to deliver lvv. Six days later, cells were collected and stained to determine the percentage of CD11c+ (A) or B7.2+ (B) cells expressing transgenic EGFP. Numbers indicate the percentage of events in the indicated quadrant. A typical result from four experiments is presented. C, Bone marrow cells were collected on day 0 and were transduced immediately or cultured in GM/IL-4 medium and transduced on day 2, 4, or 6 as indicated. Cells were collected on day 8 (for cells transduced on days 0 and 2), or day 10 (for cells transduced on days 4 and 6), stained with CD11c, and analyzed by flow cytometry. The percentage of CD11c+EGFP+ cells is shown. For each time point, three transductions were conducted on each six-well plate, and the mean ± SD are shown. The data shown are representative of three experiments.

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To evaluate the effect of maturation stage on the efficiency of transduction, we compared the transduction efficiencies of DCs transduced on days 0, 2, 4, and 6 of culture with that of the same batch of viral vector at 20 TU. On day 8, the cells transduced on days 0 and 2 were collected and stained for CD11c to identify DCs. Similarly, on day 10, cells transduced on days 4 and 6 were harvested for staining with anti-CD11c Ab. The expression of EGFP by DCs (CD11c+) was quantified by flow cytometry. As shown in Fig. 3, transduction of day 0 bone marrow cells resulted in 20% EGFP-positive DCs, whereas transduction of day 2 cells resulted in the expression of EGFP in 40% of DCs. Transduction efficiency began to decline at later time points, with 26 and 18% of DCs expressing EGFP resulting from transductions performed on days 4 and 6, respectively. In all samples tested, ∼90% of the cells expressed CD11c (data not shown).

FIGURE 3.

Effect of lentiviral transduction on DC maturation, responses to proinflammatory stimuli, and Ag presentation function. A, DCs transduced with lvv or mock-transduced with PBS on day 2 were harvested on day 7 and treated with 1 μM CpG oligonucleotide or 1 μg/ml LPS overnight or were left untreated. On day 8, the levels of B7.2 expression and IL-12p70 production by CD11c+ cells were determined. Each treatment was performed in triplicate, and the mean ± SD are shown. The data represent a typical result from one of three experiments. B, DCs transduced with lvv OVA-lvv or mock-transduced were cocultured with allogenic splenic T cells for 3 days. T cell proliferation (left) and the level of IFN-γsecreted by activated T cells in the supernatant (right) were determined. Data points are presented as the mean ± SD of triplicate cultures. This result is representative of four experiments.

FIGURE 3.

Effect of lentiviral transduction on DC maturation, responses to proinflammatory stimuli, and Ag presentation function. A, DCs transduced with lvv or mock-transduced with PBS on day 2 were harvested on day 7 and treated with 1 μM CpG oligonucleotide or 1 μg/ml LPS overnight or were left untreated. On day 8, the levels of B7.2 expression and IL-12p70 production by CD11c+ cells were determined. Each treatment was performed in triplicate, and the mean ± SD are shown. The data represent a typical result from one of three experiments. B, DCs transduced with lvv OVA-lvv or mock-transduced were cocultured with allogenic splenic T cells for 3 days. T cell proliferation (left) and the level of IFN-γsecreted by activated T cells in the supernatant (right) were determined. Data points are presented as the mean ± SD of triplicate cultures. This result is representative of four experiments.

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To study the capacity of transduced DCs to retain their ability to respond to proinflammatory stimuli, we investigated whether transduction of DCs with lvv would affect their ability to respond to CpGs or LPS by undergoing terminal maturation and increasing secretion of IL-12. On day 7, 5 days after transduction, CpG oligonucleotides (1 μM) or LPS (1 μg/ml) were added to some DC cultures. After overnight incubation (20 h), DCs were collected and stained for CD11c and B7.2 expression, and the culture medium was assayed for secreted IL-12p70 by ELISA. As shown in Fig. 3 A, both transduced and mock-transduced DCs similarly upregulated B7.2 expression in response to LPS stimulation and to a lesser extent to CpG stimulation. Although neither mock- nor lvv-transduced DCs produced detectable levels of IL-12p70 in the absence of CpGs or LPS proinflammatory stimuli, both transduced and mock-transduced DCs produced significant and comparable levels of IL-12p70 in response to CpG oligonucleotide or LPS. These data indicate that unlike adenovirus, lentiviral transduction alone does not induce DCs to produce Th1-skewing cytokine IL-12, nor does transduction with lvv diminish the capability of DCs to terminally mature and secrete IL-12 in responses to proinflammatory stimuli.

To determine whether lvv transduction affected the Ag presentation function of transduced DCs, we evaluated and compared the capacities of transduced and mock-transduced DCs to stimulate allogenic T cells. Bone marrow cell cultures were transduced on day 2 with OVA-lvv vector encoding a cytoplasmic form of OVA protein or were mock-transduced with PBS. On day 8, DCs were cocultured with BALB/c T cells at varying DC:T cell ratios. The lvv-transduced DCs induced strong allogenic T cell proliferation and IFN-γ secretion (Fig. 3 B), comparable to those induced by mock-transduced DCs.

To investigate the capacity of transduced DCs to present transgenic Ags, DCs transduced with OVA-lvv or PBS mock transduced DCs were used to stimulate class I restricted OT-I or class II-restricted OT-II T cells isolated from OVA TCR transgenic mice. As a positive control, presentations by otherwise identical DC populations pulsed with the corresponding class I-restricted (SIINFEKL) or class II-restricted (ISQAVHAAHAEINEAGR) peptide were compared. As shown in Fig. 4, DCs transduced with OVA-lvv vector presented OVA to both class I- and class II-restricted T cells, as assessed by T cell proliferation and IFN-γ secretion. Remarkably, class I-restricted presentation of transgenic Ag by transduced DCs was comparable to that observed by DCs pulsed with saturating amounts of exogenous peptide (Fig. 4, A and B). Although transduced DCs presented synthesized transgenic Ag to class II-restricted T cells, presentation was less efficient than that observed by peptide-pulsed DCs (Fig. 4 C; using unpaired t test, at 1:10 and 1:20, p < 0.05), presumably due to decreased access of endogenously synthesized peptide to the class II-restricted processing pathway. It is important to note that studies from other laboratories have indicated that inclusion of leader sequences targeting expression to the endoplasmic reticulum or endosome can increase the efficiency of presentation of endogenously expressed class II-restricted epitopes (53).

FIGURE 4.

Presentation of transgenic Ag by transduced DCs. OVA-lvv-transduced, mock-transduced, or SIINFEKL or ISQAVHAAHAEINEAGR peptide-pulsed DCs were cocultured with class I-restricted OT-I (A and B) or class II-restricted OT-II (C and D) T cells at varying DC:T cell ratios. T cell proliferation (A and C) and the level of secreted IFN-γ in the supernatants (only at a ratio of 1:10; B and D) were determined. Data points are presented as the mean ± SD of triplicate cultures. There is no significant difference between pulsed DCs and OVA-lvv-transduced DCs in stimulating OT-I cell proliferation and IFN-γ production. However, there is consistently higher OT-II cell proliferation (p < 0.05) and IFN-γ production (p < 0.01) stimulated by pulsed DCs compared with OVA-lvv-transduced DCs. This result is a representative of three experiments.

FIGURE 4.

Presentation of transgenic Ag by transduced DCs. OVA-lvv-transduced, mock-transduced, or SIINFEKL or ISQAVHAAHAEINEAGR peptide-pulsed DCs were cocultured with class I-restricted OT-I (A and B) or class II-restricted OT-II (C and D) T cells at varying DC:T cell ratios. T cell proliferation (A and C) and the level of secreted IFN-γ in the supernatants (only at a ratio of 1:10; B and D) were determined. Data points are presented as the mean ± SD of triplicate cultures. There is no significant difference between pulsed DCs and OVA-lvv-transduced DCs in stimulating OT-I cell proliferation and IFN-γ production. However, there is consistently higher OT-II cell proliferation (p < 0.05) and IFN-γ production (p < 0.01) stimulated by pulsed DCs compared with OVA-lvv-transduced DCs. This result is a representative of three experiments.

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To compare the strength of T cell responses in vivo, mice were vaccinated with DCs pulsed with SIINFEKL and OVA protein (SIINFEKL/OVA-pulsed DCs) or DCs transduced with OVA-lvv. Mock-transduced DCs were used as a control. Eight days after immunization, we measured in vivo Ag-specific killing and determined intracellular IFN-γ expression by Ag-specific T cells. Determination of Ag-specific lytic activity from measurement of target lysis in peripheral blood 5 h after target injection demonstrated a quantitatively more potent response in OVA-lvv DC-immunized animals, with OVA-lvv-immunized animals demonstrating ∼4 times more Ag-specific in vivo lytic activity than mice immunized with SIINFEKL/OVA-pulsed DCs (82 vs 20% lysis, respectively; Fig. 5, A and B). Not surprisingly, both OVA-lvv-pulsed DC- and SIINFEKL/OVA-pulsed DC-immunized mice demonstrated considerable lytic activity when evaluating target cells localizing in spleen 20 h after target cell injection (99 and 91% lysis, respectively). The CTL-mediated immunity induced by peptide/protein-pulsed DCs we observed under these conditions was consistent with previously reported results of CTL assays using splenic effectors (3).

FIGURE 5.

Comparison of the immune responses elicited by transduced and pulsed DCs. To determine in vivo Ag-specific lytic activity induced by immunization, mice were immunized with 0.5 million OVA-lvv-transduced or SIINFEKL/OVA-pulsed DCs by i.d. injection, and 8 days later a 1/1 mixture of CFSEhigh SIINFEKL-pulsed splenocytes and CFSElow unpulsed splenocytes was injected i.v. Five hours later, blood was collected, and the reduction of CFSEhigh vs CFSElow target cells was determined (A). The same mice were killed 20 h after injection of target cells, and their splenocytes were collected and analyzed for the decrease in CFSEhigh vs CFSElow target cells (A). Numbers indicate the percentage of that target cell population present after 5 or 20 h of in vivo killing. The specific lysis was calculated and is shown as the mean ± SD of three mice (B). C, Splenocytes from immunized or naive mice were restimulated in vitro with SIINFEKL or ISQAVHAAHAEINEAGR peptide for 20 h, as indicated, to define CD4+ and CD8+ T cell pupations and were stained for intracellular expression of IFN-γ. The numbers of IFN-γ+ cells per 100,000 CD4+ or CD8+ T cells were determined as the mean ± SD from three mice (D).

FIGURE 5.

Comparison of the immune responses elicited by transduced and pulsed DCs. To determine in vivo Ag-specific lytic activity induced by immunization, mice were immunized with 0.5 million OVA-lvv-transduced or SIINFEKL/OVA-pulsed DCs by i.d. injection, and 8 days later a 1/1 mixture of CFSEhigh SIINFEKL-pulsed splenocytes and CFSElow unpulsed splenocytes was injected i.v. Five hours later, blood was collected, and the reduction of CFSEhigh vs CFSElow target cells was determined (A). The same mice were killed 20 h after injection of target cells, and their splenocytes were collected and analyzed for the decrease in CFSEhigh vs CFSElow target cells (A). Numbers indicate the percentage of that target cell population present after 5 or 20 h of in vivo killing. The specific lysis was calculated and is shown as the mean ± SD of three mice (B). C, Splenocytes from immunized or naive mice were restimulated in vitro with SIINFEKL or ISQAVHAAHAEINEAGR peptide for 20 h, as indicated, to define CD4+ and CD8+ T cell pupations and were stained for intracellular expression of IFN-γ. The numbers of IFN-γ+ cells per 100,000 CD4+ or CD8+ T cells were determined as the mean ± SD from three mice (D).

Close modal

To evaluate Ag-specific T cell activation by intracellular IFN-γ production, splenocytes from immunized animals were restimulated in vitro with SIINFEKL (class I) or ISQAVHAAHAEINEAGR (class II) peptide for 20 h, then stained for the expression of CD8 or CD4 and cytoplasmic IFN-γ. Consistent with results from the in vivo lysis assay, as shown in Fig. 5, C and D, ∼2% of the spleen CD8+ T cells from mice vaccinated with OVA-lvv DCs produced IFN-γ, whereas only 0.5% of CD8+ T cells from mice vaccinated with SIINFEKL/OVA DCs were IFN-γ positive. Notably, neither immunized group demonstrated significant IFN-γ production from the CD4+ T cell population, with <0.1% of CD4+ T cells producing IFN-γ.

We compared the kinetics of T cells responses induced by OVA-lvv-transduced DCs with responses induced by DCs pulsed with either SIINFEKL peptide or OVA protein. The in vivo CTL activity induced by DCs pulsed with SIINFEKL alone peaked on day 5 and declined to undetectable levels in 2 wk (Fig. 6). Not surprisingly, OVA protein-pulsed DCs induced longer-lasting in vivo CTL activity compared with SIINFEKL-pulsed DCs. However, the in vivo CTL activity induced by OVA protein-pulsed DCs was also dramatically reduced by day 15 (Fig. 6). The magnitude and kinetics of the response induced by DCs pulsed with both peptide and OVA protein were similar to those induced by DCs pulsed with OVA protein alone (data not shown). In contrast, the mice injected with OVA-lvv DCs maintained a high level of CTL activity through day 15 and maintained significant Ag-specific lytic activity through day 41, the latest time point tested (Fig. 6). These data indicate that immunization with OVA-lvv-transduced DCs stimulated considerably longer-lasting T cell immunity than that induced by protein- or peptide-pulsed DCs.

FIGURE 6.

Kinetics of in vivo Ag-specific lytic activity. Kinetics of in vivo Ag-specific lytic activity were compared among mice immunized with OVA-lvv-transduced DCs, OVA protein-pulsed DCs, or SIINFEKL peptide-pulsed DCs. Mice were immunized as described in Fig. 5. At the indicated time points, in vivo killing assays (20 h) were conducted, and specific lysis was determined as described Fig. 5. The 5-h in vivo killing assay was also performed on day 8 when the 20-h assay generated a saturated killing effect (inset). Each time point represents the mean ± SD from three mice. Representative results from three experiments were presented.

FIGURE 6.

Kinetics of in vivo Ag-specific lytic activity. Kinetics of in vivo Ag-specific lytic activity were compared among mice immunized with OVA-lvv-transduced DCs, OVA protein-pulsed DCs, or SIINFEKL peptide-pulsed DCs. Mice were immunized as described in Fig. 5. At the indicated time points, in vivo killing assays (20 h) were conducted, and specific lysis was determined as described Fig. 5. The 5-h in vivo killing assay was also performed on day 8 when the 20-h assay generated a saturated killing effect (inset). Each time point represents the mean ± SD from three mice. Representative results from three experiments were presented.

Close modal

One possible mechanism for the long-lasting CTL activity induced by OVA-lvv-transduced DCs is extended Ag presentation in vivo. To investigate the duration of Ag presentation in vivo, immunized mice were injected with naive OVA-specific OT-I T cells 5, 8, or 12 days after immunization. The CFSE-labeled T cells were collected from the LNs, and in vivo proliferation was determined by flow cytometry as previously described. Interestingly, through day 8 after immunization, all DCs induced similar levels of proliferation (Fig. 7). Furthermore, although OVA-specific T cell proliferation could still be detected 12 days after immunization in all groups, proliferation in mice immunized with OVA-lvv-DCs was considerably lower than that seen in mice immunized with protein- or peptide-pulsed DCs (Fig. 7).

FIGURE 7.

Duration of Ag presentation in vivo. Mice were immunized with OVA-lvv DCs, OVA-DCs, or SIINFEKL-DCs. At different time points, 5 million CFSE-labeled OT-I cells were injected by tail vein. Forty-eight hours later, the cells from the draining and contralateral LNs were collected and stained for Vα2 and Vβ5.1–5.2. Vα2 and Vβ5.1–5.2 double-positive cells were gated and analyzed for CFSE reduction and banding to identify proliferating cells. Cumulative data from two mice are shown and are representative of two separate experiments.

FIGURE 7.

Duration of Ag presentation in vivo. Mice were immunized with OVA-lvv DCs, OVA-DCs, or SIINFEKL-DCs. At different time points, 5 million CFSE-labeled OT-I cells were injected by tail vein. Forty-eight hours later, the cells from the draining and contralateral LNs were collected and stained for Vα2 and Vβ5.1–5.2. Vα2 and Vβ5.1–5.2 double-positive cells were gated and analyzed for CFSE reduction and banding to identify proliferating cells. Cumulative data from two mice are shown and are representative of two separate experiments.

Close modal

To evaluate the potential use of lentiviral-transduced DCs for tumor immunotherapy in vivo, we used a well-defined and very aggressive melanoma tumor model. In this model, the B16 melanoma expresses the cytosolic form of the model Ag OVA. Similar to MO4 (5), the expression of OVA in B16-F10 cells does not increase the immunogenicity of this tumor (data not shown). Initial experiments showed that naive mice vaccinated with OVA-lvv DCs before tumor challenge were completely protected from B16-OVA tumor cell challenge and remained tumor free for at least 2 mo, unlike mice immunized with control mock-transduced DCs or EGFP-lvv DCs, which developed progressively growing tumors comparable to those in unimmunized animals (data not shown). To evaluate the therapeutic potential of this approach, we established s.c. B16-OVA tumors by inoculating 0.2 million cells intradermally into C57/BL6 mice. After 3 days, when the tumor was established with a size of at least 3–4 mm in diameter, mice were immunized by intradermal injection of either 0.5 million SIINFEKL/OVA-pulsed or OVA-lvv-transduced DCs at distant sites. As shown in Fig. 8, animals treated with SIINFEKL/OVA-pulsed DCs showed marginal inhibition of tumor growth and had survival rates no better than those of control mice. In contrast, in mice immunized with transduced DCs, tumor growth was strongly inhibited (p < 0.01 compared with control mice or mice vaccinated with pulsed DCs), and the mice survived significantly longer. In this aggressive tumor model, mice immunized with lvv-transduced DCs demonstrated a significant therapeutic benefit over those immunized with peptide/protein-pulsed DCs.

FIGURE 8.

Immunotherapy of established B16 tumors. Mice were inoculated with 0.2 million B16-OVA cells s.c. Three days later, mice with tumors at least 3–4 mm in diameter were immunized with 0.5 million OVA-lvv-transduced DCs or SIINFEKL/OVA-pulsed DCs. Tumor growth and mouse survival were monitored. Tumor area was calculated by multiplying the width and length of the tumor. The number in the parentheses indicates the number of surviving mice on day 24. Using a t test, tumor size was compared between mice in the transduced group and control mice, between the transduced group and the pulsed group, and between the pulsed group and the control group at each time point. Tumor size in the mice of transduced group was significantly smaller than that in the control and pulsed DC-vaccinated group from days 15–24.

FIGURE 8.

Immunotherapy of established B16 tumors. Mice were inoculated with 0.2 million B16-OVA cells s.c. Three days later, mice with tumors at least 3–4 mm in diameter were immunized with 0.5 million OVA-lvv-transduced DCs or SIINFEKL/OVA-pulsed DCs. Tumor growth and mouse survival were monitored. Tumor area was calculated by multiplying the width and length of the tumor. The number in the parentheses indicates the number of surviving mice on day 24. Using a t test, tumor size was compared between mice in the transduced group and control mice, between the transduced group and the pulsed group, and between the pulsed group and the control group at each time point. Tumor size in the mice of transduced group was significantly smaller than that in the control and pulsed DC-vaccinated group from days 15–24.

Close modal

The remarkable plasticity of DC function enables DCs to specifically stimulate or suppress Ag-specific immune responses depending on signals received from their environment naturally or artificially through strategies designed to manipulate immune function. To initiate immune responses, Ags must be acquired, processed, and presented by DCs, which, because they are professional APCs, are the primary targets for the immunotherapy of a variety of diseases, including infections, cancer, and transplantation. The development of effective strategies to introduce genes encoding Ags or immunoregulatory proteins into DCs has the potential to enable Ag-specific immunoregulation of immune responses. Introducing Ag-encoding genes into DCs could provide a sustained source of Ag for both direct presentation and cross-presentation, enabling presentation of both class I- and class II-restricted antigenic epitopes and theoretically inducing strong and synergistic CD8+ and CD4+ T cell immunity.

Gene delivery into DCs remains problematic. First, the fact that DCs are terminally differentiated poses a considerable obstacle for gene delivery by most vectors. Second, the function and phenotype of DCs are extremely plastic and can be readily influenced by environmental factors in the medium or by gene delivery vectors (54, 55, 56). Thus, the transfection or transduction process can introduce unwanted functions into gene-modified DCs that can limit their subsequent applications. Vectors or gene delivery strategies that achieve high efficiency without perturbing the intrinsic properties and function of DCs will contribute significantly to DC-based immunizations and immunotherapies.

Retroviral vectors, especially HIV-based lvv, may fulfill the requirements for transducing DCs (57). Lentiviral vector has the advantage of transducing nondividing cells and thus should be more effective for transducing DCs (49). Like oncoretroviral vectors, only minimal cis elements are needed for lvv production. The few essential proteins are provided through a trans-complementation mechanism encoded by separate plasmids during vector production. Thus, there are no genes encoding viral proteins in the lvv particles. The low amount of regulatory viral proteins present in the vector particles in the absence of a sustained source from transduced cells is likely to minimize vector effects on the functions of transduced DCs, providing an important advantage to lvv for introducing Ag-encoding genes into DCs (54). In the current study our data confirmed that lvv transduction of DC progenitors or immature DCs did not affect their maturation or intrinsic function (Figs. 2 and 3). Although important for immunization strategies, this may be particularly relevant for efforts to induce Ag-specific regulatory T cells, because the use of lentivectors appears to avoid the vector-mediated immunostimulatory effects of other viral vectors, such as adenovirus (31).

We directly addressed whether maturation would affect the efficiency of lvv transduction of murine DCs. Our study indicated that transduction of day 2 DC progenitors or immature DCs resulted in the best transduction efficiency (Fig. 2 C). Progenitor cells cultured in the presence of GM-CSF and IL-4 appeared to be better targets than those directly isolated from bone marrow, possibly because day 2 bone marrow cells may be in a stage of proliferation secondary to stimulation by GM-CSF and IL-4 cytokines, whereas the day 0 bone marrow cells may be quiescent. When DC populations become more mature with increased time in culture, the transduction efficiency begins to decline, suggesting that mature DCs were less likely to be transduced. These data suggested that although lvv with nuclear localization sequences (TRIP) can transduce quiescent cells, dividing or proliferating cells may offer additional advantages for improving transduction efficiency. It is known that dissolution of the nuclear membrane barrier during cell division facilitates the entry of exogenous DNA into the nucleus and thus increases transduction efficacy. In addition, DNA replication assists the integration of foreign DNA into the host genome. These factors may help explain why cells in the early stage of proliferation are more readily transduced. These results from our analysis of mouse DCs resemble those of previous studies focused on human CD34+-derived DCs (58), indicating that even though lentivirus could be imported into the nucleus through its unique nuclear import protein, cell proliferation still facilitated gene delivery and expression in DCs. It is important to note that others have reported that factors such as the use of 1) higher multiplicity of infection for transduction, 2) increased concentration of vector, 3) cocentrifugation of DCs and viral particles, and 4) repeat transduction can all increase DC transduction efficiency (36, 59, 60). This suggests that the potency of the transduced DCs used in our studies could be even further enhanced by optimizing DC transduction.

Previous studies comparing the immunogenicity of peptide/protein-loaded DCs with transduced DCs have produced mixed results. Using β-galactosidase as a model Ag, it has been shown that retroviral vector-transduced DCs induce stronger CTL activity and greater IFN-γ secretion by responding T cells compared with pulsed DCs (44). However, in the same studies there was no significant difference in antitumor activity between pulsed and transduced DCs under the conditions tested using the colon cancer CT26 lung metastasis tumor model. Using a tumor protection model, it has been shown that immunization with retroviral vector-transduced DCs can better protect mice from B16-OVA tumor challenge than immunization with peptide-pulsed DCs (45). However, tumor therapy was not evaluated. Adenoviral vector-transduced DCs have been compared with peptide/protein-pulsed DCs in two studies. Using a DC cell line, Brossart et al. (43) showed that DCs transfected with adenoviral vector or vaccinia virus expressing the SIINFEKL peptide induce stronger in vitro stimulation of CTL activity than peptide-pulsed DCs. In contrast, Tuettenberg et al. (32) recently showed that adenoviral vector-transduced DCs only induced short-lived immune responses compared with Ag-pulsed DCs in vitro. The evidence suggests that the generation of vector-specific immune responses through repeated stimulation by adenoviral vector-transduced DCs may interfere with the development and potency of the desired Ag-specific T cell responses (32). Nonviral transfection of DCs would obviate this obstacle, and the delivery of Ag-encoding mRNA to DCs has been shown to result in Ag expression and stimulation of Ag-specific T cell responses (61, 62). However, in a direct comparison, mRNA-transfected DCs were less immunogenic in vivo than lvv-transduced DCs (63). Interestingly, although introduction of mRNA into DCs by electroporation resulted in efficient transgene expression, electroporated DCs showed a 10-fold reduction in IL-12p70 secretion compared with unmodified or lentivirally transduced DCs (63). Overcoming this defect by retrovirus-mediated cotransfection of IL-12p70 overcame this limitation, but added an additional level of complexity (63).

We have recently shown that nonviral transfection of DCs can be achieved using naked DNA with very high transfection efficiencies (46). Although the use of plasmid DNA to transfect DCs results in nonintegrating transfection, we demonstrated more sustained expression of H-2Kb-SIINFEKL complexes on the surface of transfected DCs compared with DCs pulsed with SIINFEKL. In direct comparisons among peptide-pulsed DCs, naked DNA transfected DCs, and adenovirus-transduced DCs, DCs transfected with naked DNA induced considerably more potent CD8+ T cell responses in vitro and in vivo than pulsed DCs, resulting in stimulation comparable to that achieved using adenovirus-transduced DCs.

Our current studies demonstrated that in vivo, lentivector-transduced DCs can induce stronger, longer lasting T cell responses than peptide/protein-pulsed DCs. Lentiviral vector-mediated transduction results in integration of transgenes into host DNA. Although this raises concerns of potential mutagenesis, the resulting stable expression of transgenic protein may be an advantage for Ag delivery or the expression of immunomodulatory molecules in DCs to more specifically engineer immune responses.

In the current studies we compared the efficacy of T cell immune responses elicited by lentivector-transduced DCs and Ag-pulsed DCs. We demonstrate that even though in vitro, peptide- or protein-pulsed DCs and transduced DCs stimulated T cell proliferation with similar potencies, in vivo transduced DCs can induce stronger and longer lasting Ag-specific lytic T cell responses. The mechanism for this remains unclear. However, the stronger and longer lasting T cell responses we observed with lentivirus-transduced DCs are unlikely to be due an initially increased ligand density, because the MHC-SIINFEKL complexes detected by 25D1.16 Ab were not higher on OVA-lvv-transduced DCs (data not shown). It is possible that adoptively transferred DCs endogenously expressing transgenic Ag result in more durable presentation of Ag, either by longer lasting direct presentation of MHC-Ag ligand on the cell surface made possible by sustained production of Ag or by providing a more efficient source of Ag for cross-priming. Several studies suggest that diminishing class I peptide ligand density may be a limiting factor for in vivo T cell stimulation (16, 17, 18). Recently published data demonstrate that on mature DCs, five of six peptide class I ligands analyzed had cell surface half-lives of only 8.7–26.6 h when DCs were loaded by peptide pulsing (18). Additional studies suggested that although reduced membrane turnover can increase ligand longevity, newly synthesized MHC molecules do not present pulsed peptides, and it is unlikely that internal pools of pulsed peptide can contribute to ligand density (18). In the case of lvv-transduced DCs, it is likely that the cells stably express the OVA Ag, providing a sustained source of Ag to replace cycling or lost class I peptide complexes, and thus are able to generate stronger and long-lasting T cell responses. Alternatively, the more potent immune responses observed using transduced donor DCs may be a result of more efficient cross-priming by recipient DCs resident in draining lymph nodes or spleen. Several studies now suggest that cross-presentation of Ags delivered by virally infected donor DCs to recipient host lymphoid organ-resident DCs may contribute significantly to the induction of CD8+ T cell responses (20, 21, 23). Furthermore, very recent studies suggest that cross-presentation favors epitopes derived from stabilized proteins, rather than those from rapidly degraded proteins or short peptides (24, 25, 26). In this context, whole transgenic Ags produced by transduced donor DCs may serve as a more efficient source of Ag for cross-presentation than peptide Ags bound to MHC complexes or peptides or proteins internalized through the endosomal pathway of pulsed donor DCs.

Of note, using the OT-1 system to measure Ag-specific T cell proliferation, we were unable to demonstrate longer lasting in vivo Ag presentation. This system is limited however, because it is a measure of activation of naive T cells. It is possible that Ag ligand persistence sufficient to drive or help previously stimulated T cells may be important to support the long-lasting effector responses we observe, but insufficient to drive induction of the naive OT-1 T cells used in the assay. Other recent studies have shown that for maximal CTL induction, a short period of stimulation (24 h), not prolonged Ag presentation, is required (52, 64). These studies suggest that the magnitude and duration of CD8 T cell responses are determined by the initial clonal burst of T cell activation resulting from an initial period of engagement with APCs. Once the T cell immune response is initiated, the T cells are refractory to further stimulation by prolonged Ag presentation (64). Thus, it is possible that lvv-transduced DCs may enable stronger initial activation that results in stronger effector function and greater longevity. It is also possible that stronger lytic activity may eliminate some APCs more quickly (65), so that the in vivo Ag presentation measured by the naive OT-I T cell assay actually appears to be reduced. Additional experiments are needed to address these possibilities.

Importantly, our studies demonstrate that lentivirus-transduced DCs demonstrate some therapeutic efficacy in an aggressive B16 tumor model resistant to immunotherapy. B16 melanoma is a frequently studied tumor model that has been shown to escape or inhibit immune responses through a variety of mechanisms. B16 has well-document Ag escape mechanisms, including down-regulation of class I molecule expression and Ag-processing machinery (66), Growing B16 tumors have been shown to induce CD4+CD25+ regulatory cells that facilitate immune escape (67, 68, 69). In addition, B16 cells produce vesicular endothelial growth factor, which inhibits DC function and T cell immunity, and galectin-1, which is a negative regulator of T cell activation and survival (70). Taken together, these observations suggest that the B16 tumor possesses many of the tumor escape mechanisms used by human tumors and is a formidable model tumor for the evaluation of immunotherapeutic strategies. Interestingly, very recent studies suggest that sustained DC activation may overcome some forms of tumor-induced immunosuppression (71). Together with those results, our studies raise the possibility that sustained Ag expression by lentivirus-transduced DCs in combination with sustained adjuvant signaling may result in more effective immunotherapies. Lentivector-mediated expression of both Ag and costimulatory molecules or cytokines by transduced DCs could enhance or direct T cell immunity. Importantly, because transduction with lentivectors does not induce DC maturation or activation, these vectors may also provide a means to engineer DCs to induce Ag-specific tolerance through the coexpression of Ags and immunoregulatory molecules.

The authors have no financial conflict of interest.

We thank Jeff Plowey, M.S., for genotying the OT-I mice, and Dr. Ronald Germain, National Institutes of Health, for providing the 25D1.16 Ab.

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 National Institutes of Health grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (to Y.H. and L.D.F.) and the National Institute of Allergy and Infectious Diseases (to L.D.F.).

3

Abbreviations used in this paper: DC, dendritic cell; EGFP, enhanced GFP; LN, lymph node; LTR, long terminal repeat; lvv, lentiviral vector; RSV, Rous sarcoma virus; TRIP, triple-stranded DNA flap; TU, transduction unit.

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