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. 2012 Jan 3;20(3):644–651. doi: 10.1038/mt.2011.283

Combined Tbet and IL12 Gene Therapy Elicits and Recruits Superior Antitumor Immunity In Vivo

Yanyan Qu 1, Lu Chen 2, Devin B Lowe 1, Walter J Storkus 1,2,3, Jennifer L Taylor 1,*
PMCID: PMC3293621  PMID: 22215017

Abstract

We have recently shown that intratumor (i.t.) injection of syngenic dendritic cells (DC) engineered to express the transcription factor Tbet (TBX21) promotes protective type-1 T cell-mediated immunity via a mechanism that is largely interleukin (IL)-12p70-independent. Since IL-12 is a classical promoter of type-1 immunity, the current study was undertaken to determine whether gene therapy using combined Tbet and IL-12 complementary DNA (cDNA) would yield improved antitumor efficacy based on the complementary/synergistic action of these biologic modifiers. Mice bearing established subcutaneous (s.c.) tumors injected with DC concomitantly expressing ectopic Tbet and IL12 (i.e., DC.Tbet/IL12) displayed superior (i) rates of tumor rejection and extended overall survival, (ii) cross-priming of Tc1 reactive against antigens expressed within the tumor microenvironment, and (iii) infiltration of CD8+ T cells into treated tumors in association with elevated locoregional production of CXCR3 ligand chemokines. In established bilateral tumor models, i.t. delivery of DC.Tbet/IL12 into a single lesion led to slowed growth or regression at both tumor sites. Furthermore, DC.Tbet/IL12 pulsed with tumor antigen-derived peptides and injected as a therapy distal to the tumor site prevented tumor growth and activated robust antigen-specific Tc1 responses. These data support the translation use of combined Tbet and IL-12p70 gene therapy in the cancer setting.

Introduction

The use of dendritic cells (DC) as therapeutic agents and/or vaccine components has received profound attention over the past 15 years, with numerous clinical trials validating the immunogenicity of these cells.1,2,3 Although objective responses have been observed in a minority of cancer patients treated with DC-based immunotherapy,4,5 such responders provide hope that the application of ex vivo generated DC or development of means through which to activate the increasingly complex range of DC subsets in vivo may lead to improved clinical efficacy. Since type-1 T cell responses (particularly CD8+ T cells; i.e., Tc1) have been associated with most beneficial outcomes as a consequence of immunotherapeutic intervention,6,7,8 many including ourselves, have focused on ways to condition patient DC to exert a predictable type-1 functional polarization potential on responder T cells.9,10,11,12

In addition to employing culture conditions that incorporate pro-inflammatory cytokines or toll-like receptor agonists (in order to largely bolster DC production of IL-12p70 and/or diminished IL10 production), one may genetically engineer DC to attain so-called “DC1” functional status.10,11,12,13,14,15,16 In particular, DC1 engineered to express ectopic IL-12p70 or the T cell transactivator protein TBX21 (aka Tbet) have been shown to serve as effective antigen presenting cells (APC) for the promotion of potent Tc1 responses in vitro and in vivo that are competent to mediate tumor rejection.13,14,15 Interestingly, DC1 engineered to express Tbet (i.e., DC.Tbet) appear capable of preferentially activating type-1 T cell responses from naive T cell precursors via a mechanism that depends minimally on the action of IL-12p70.14,15

Given the lack of major operational overlap between DC1-associated IL-12p70 and Tbet in supporting type-1 immunity from naive responder T cells, we hypothesized that the engineering of DC to express high levels of both proteins might yield an “uber-DC1” capable of cross-priming a superior level of protective Tc1-mediated immunity in the cancer setting. We directly investigated this possibility using CMS4 (H-2d) sarcoma or B16 (H-2b) melanoma in syngenic mice applying genetically altered DC1 either as a therapeutic agent directly injected into established tumor lesions or as a vaccine adjuvant injected distal to tumor sites. In both formats, DC.Tbet/IL12 were determined to activate greater systemic levels of antitumor Tc1 in association with improved treatment outcome when compared with all formats of control DC evaluated. These data suggest that DC.Tbet/IL12 or alternate conditional means to promote higher levels of Tbet and IL-12p70 in therapeutic DC1 may lead to enhanced rates of objective clinical response in patients with cancer.

Results

Intratumoral administration of DC engineered to coexpress ectopic Tbet and IL-12p70 cDNA provides superior antitumor therapeutic efficacy

One million control or genetically engineered DC were injected directly into subcutaneous (s.c.) CMS4 sarcoma tumors established for 7 days in syngenic BALB/c mice. An identical treatment was provided to each cohort of animals 1 week later (i.e., day 14 post-tumor inoculation). As shown in Figure 1a, while mice treated with DC infected with Ad.ψ5 (i.e., DC.ψ5) displayed progressive tumor growth that was indistinguishable from untreated mice, animals treated with DC.Tbet, DC.IL12 or DC.Tbet/IL12 exhibited either slowed tumor progression (for DC.Tbet and DC.IL12) or regression (for DC.Tbet/IL12). Mice treated with DC.Tbet/IL12 exhibited a statistically meaningful benefit over DC.Tbet or DC.IL12 monotherapy (i.e., P < 0.05) after day 25 (Figure 1a), which was also reflected in extended overall survival (Figure 1b; DC.Tbet/IL12 versus DC.Tbet (P = 0.006), DC.IL12 (P = 0.029) or DC.ψ5 (P < 0.00007), with P = 0.009 for DC.Tbet versus DC.ψ5 and P = 0.003 for DC.IL12 versus DC.ψ5).

Figure 1.

Figure 1

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CMS4 tumor-bearing mice treated with intratumoral administration of DC.Tbet/IL12 display superior antitumor benefit and extended overall survival. BALB/c mice bearing established day 7 subcutaneous CMS4 sarcomas were left untreated or they were treated with an intratumoral injection of 106 control DC (DC.ψ5) or DC.Tbet, DC.IL12 or DCTbet/IL12. Mice were managed in an identical manner 1 week later (i.e., day 14 post-tumor inoculation). (a) Tumor size (reported as the mean ± SD for five mice/group in a representative experiment of three performed) of and (b) animal survival (for a total of 10 mice/cohort analyzed in two independent experiments) were monitored every 3–4 days until study completion. *P < 0.05 at the indicated time points for DC.Tbet/IL12 versus all other cohorts analyzed. DC, dendritic cell; IL, interleukin; Tbet, T-box expressed in T cells.

DC.Tbet/IL12 gene therapy leads to a stronger and more diverse repertoire of type-1 CD8+ T cells recognizing antigens within the CMS4 tumor microenvironment

Splenic CD8+ T cells were harvested from all cohorts on day 21 post-tumor inoculation and analyzed for reactivity against syngenic DC alone or DC pulsed with peptides extracted by mild acid elution17 from enzymatically digested CMS4 tumors by interferon-γ (IFN-γ) enzyme-linked immunosorbent assay. Data provided in Figure 2 suggests that the profile of type-1 CD8+ T cell reactivity against peptides contained in individual high-performance liquid chromatography (HPLC) fractions differs dramatically between the various treatment cohorts, with untreated or DC.ψ5-treated animals exhibiting a general lack of response to in vivo presented peptides, while mice treated with DC.Tbet, DC.IL12 or DC.Tbet/IL12 demonstrate response to a range of peptide-containing fractions. In particular, Tc1 cells isolated from mice treated with DC.Tbet/IL12 displayed the strongest and most complex pattern of peptide recognition among all cohorts, with such recognition proving to be H-2d class I-restricted (Figure 2).

Figure 2.

Figure 2

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Spleen CD8+ T cells isolated from mice treated with intratumoral DC.Tbet/IL12 display the strongest and most dynamic type-1 response against antigens/peptides presented in the CMS4 tumor microenvironment. CMS4 tumor-bearing mice were treated as outlined in Figure 1. On day 21 post-tumor inoculation, splenocytes were isolated from two mice/cohort and CD8 MACS performed. Day 21 CMS4 tumors from untreated BALB/c mice were enzymatically disrupted, with the resulting single-cell suspension serving as a source of natural peptides that were isolated and then RP-HPLC separated as described in Materials and Methods. Spleen CD8+ T cells were cocultured with syngenic DC alone, DC pulsed with aliquots from each RP-HPLC fraction (#10–50) or (positive control) ConA mitogen for 24 hours. To investigate the class I-dependence of T cell recognition in the DC.Tbet/IL12 treatment cohort, control IgG or anti-H-2d class I Abs were added during the culture period as described in Materials and Methods. Cell-free supernatants were harvested from triplicate determinations and analyzed for IFN-γ by ELISA. Data are representative of two independent experiments performed. Ab, antibody; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; IL, interleukin; MACS, magnetic-assisted cell sorting; RP-HPLC, reverse-phase high-performance liquid chromatography; Tbet, T-box expressed in T cells.

Effective gene therapy results in the cross-priming of Tc1 reactive against tumor cells and tumor-associated pericytes and vascular endothelial cells, with improved recruitment of CD8+ TIL

Although the data provided in Figure 2 suggest that the systemic CD8+ T cell repertoire becomes educated to react against antigens expressed within the tumor microenvironment, particularly after treatment with DC.Tbet/IL12, the identity of specific peptide sequences and the tumor/stromal cells presenting such peptides remained unknown. To determine relevant cellular targets, we analyzed immune CD8+ T cell reactivity against flow cytometry-sorted CD31neg platelet-derived growth factor receptor-β (PDGFRβ)+ pericytes and CD31+PDGFRβneg vascular endothelial cells (VEC) (isolated from the tumors or tumor-uninvolved kidneys of day 21 untreated animals; Figure 3a), in addition to CMS4 tumor cells themselves. The absence of tumor cells in the sorted pericyte and VEC populations was confirmed by reverse transcription-PCR analysis for the tumor-associated MuLV gp70 env gene product (Figure 3b). As depicted in Figure 3c, Tc1 cells isolated from mice treated with DC.Tbet, DC.IL12 or DC.Tbet/IL12 recognized tumor cells and pericytes/VEC purified from the tumors, but not the kidneys of tumor-bearing animals to a comparable degree. The superior antitumor impact of such T cells in the DC.Tbet/IL12 treatment cohort may instead reflect the dramatically increased recruitment of CD8+ T cells into the CMS4 tumor microenvironment (Figure 4a; P < 0.05 versus all other cohorts).

Figure 3.

Figure 3

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Spleen CD8+ T cells isolated from mice treated with intratumoral DC.Tbet/IL12 display the strongest type-1 response against CMS4 tumor cells and pericytes and VEC isolated from the tumor microenvironment. (a) MACS CD8+ T cells isolated from day 21 CMS4 tumors as outlined in Figure 2 were analyzed for reactivity by 24 hours coculture with flow-sorted PDGFRβ+CD31neg pericytes and PDGFRβnegCD31+ VEC isolated from untreated day 19 CMS4 tumors or the kidneys of untreated day 19 CMS4-bearing mice (as described in Materials and Methods), as well as, CMS4 (relevant) and MethA (irrelevant H-2d) tumor cells. (b) Sorted populations of pericytes and VEC were devoid of detectable CMS4 cell contamination based on lack of expression of the tumor-associated MuLV gp70 env gene product based on RT-PCR analysis. (c) After harvesting the CD8+ T cell/target cell cocultures, cell-free supernatants were analyzed for IFN-γ levels by ELISA. *P < 0.05 versus untreated or Dc.ψ5; **P < 0.05 versus all other cohorts. Data are from one representative experiment of two performed. DC, dendritic cell; FITC, fluorescein isothiocyanate; IFN, interferon; IL, interleukin; MACS, magnetic-assisted cell sorting; PDGFRβ, platelet-derived growth factor receptor-β RT-PCR, reverse transcription-PCR; Tbet, T-box expressed in T cells; VEC, vascular endothelial cell.

Figure 4.

Figure 4

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DC.Tbet/IL12 gene therapy leads to superior infiltration of CD8+ T cells and production of CXCR3 ligand chemokines in the tumor microenvironment. Day 21 tumor and kidney (from the same tumor-bearing animal) sections harvested from mice treated as outlined in Figure 1 were analyzed for expression of (a) CD8 or (b) CD31 and the chemokines CXCL9/Mig and CXCL10/IP-10 (for tumor) by fluorescence microscopy as described in Materials and Methods. For each section (right hand subpanels), 10 high-power fields (HPF; ×40) were quantified for the number of chemokine+ cells and results reported as the mean ± SD per HPF. *P < 0.05 versus untreated or Dc.ψ5; **P < 0.05 versus all other cohorts. Data are from one representative experiment of three performed. DAPI, 4′,6-diamidino-2-phenylindole; DC, dendritic cell; IL, interleukin; Tbet, T-box expressed in T cells.

Intratumor DC.Tbet/IL12 gene therapy results in enhanced production of type-1 T cell recruiting chemokines in the CMS4 tumor microenvironment

Since protective Tc1 are recruited into tumors by CXCR3 ligand chemokines,6 we next analyzed day 21 tumor sections for perivascular expression of CXCL9/Mig and CXCL10/IP-10 by immunofluorescence microscopy (Figure 4b). We observed that the frequency of cells expressing/producing these chemokines within the tumor was increased (versus untreated or DC.ψ5-treated conditions) after treatment with DC.Tbet, DC.IL12 or DC.Tbet/IL12, with the greatest increase associated with DC.Tbet/IL12-based therapy (P < 0.05 versus all cohorts for both chemokines).

DC.Tbet/IL12-based vaccines elicit robust antigen-specific Tc1 responses in association with antitumor efficacy in vivo

To determine whether DC.Tbet/IL12 engineered cells would also represent a preferred “adjuvant” in therapeutic vaccines, we treated CMS4 bearing BALB/c mice or B16-bearing C57BL/6 mice with syngenic DC/peptide (hemoglobin-β (HBB)33–42 for CMS4; (ref. 17) or tyrosinase-related protein 2 (TRP2)180–188 for B16 (ref. 18))-based vaccines on days 7 and 14 post-tumor inoculation. Although DC.ψ5/peptide vaccines failed to alter the progressive growth and demise of tumor-bearing animals in either model system, vaccines based on peptide-loaded DC.Tbet, DC.IL12 or DC.Tbet/IL12 either dramatically slowed tumor growth or they promoted disease regression (Figure 5a,c). Such antitumor impact was directly associated with the degree of antigen-specific Tc1 reactivity detected in the spleens of these treated animals (Figure 5b,d). Although we were unable to discern a treatment benefit advantage for the DC.Tbet/IL12/peptide vaccine versus the DC.IL12/peptide vaccine in the CMS4 model (Figure 5a; presumably due to the very strong antitumor action of the DC.IL12-based treatment), we noted a statistical advantage for the combined gene vaccine (versus all other treatment cohorts) for tumor growth suppression of B16 melanoma (Figure 5c) and for specific Tc1 activation in both models (Figure 5b,d).

Figure 5.

Figure 5

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DC.Tbet/IL12 + peptide-based vaccines elicit potent therapeutic immunity in vivo. Mice bearing established syngenic subcutaneous tumors (CMS4 sarcoma in BALB/c mice or B16 melanoma in C57BL/6 mice) in their right flanks were left untreated or they were treated with the indicated DC-based vaccines on days 7 and 14 on the left flank. (a,c) Tumor size (mean ± SD for 10 animals/group) was then monitored every 3–4 days.(b,d) On day 21, two mice were sacrificed and MACS CD8 splenocytes analyzed were cocultured with relevant tumor (either CMS4 or B16), irrelevant tumor (MethA or MCA205, respectively) or irrelevant tumor loaded with the vaccine-targeted peptide (either HBB33–42 or TRP2180–188, respectively) for 24 hours. Cell-free supernatants were assessed for IFN-γ levels by ELISA. *P < 0.05 versus all other cohorts. Data are from one representative experiment of two performed for each tumor model. DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; HBB, hemoglobin-β IFN, interferon; IL, interleukin; Tbet, T-box expressed in T cells; TRP2, tyrosinase-related protein 2.

Discussion

The major finding in the current report is that ectopic (over)expression of both Tbet and IL-12p70 complementary DNA (cDNA) in DC conveys a superior level of cross-priming potential for protective type-1 CD8+ T cells in the tumor-bearing host. This enhanced potential was displayed in therapeutic models in which genetically engineered DC were injected directly into established s.c. tumors or at a site distal to tumor lesions after being pulsed ex vivo with a relevant tumor-associated antigenic peptide (i.e., a vaccine formulation). Provision of DC.Tbet/IL12 into the tumor site allowed these APC to acquire not only tumor, but also stromal (i.e., pericytes and VEC) antigens in situ, leading to systemic activation of Tc1 reactive against tumor cells as well as tumor-associated pericytes and VEC. Indeed, in bilateral established tumor models, i.t. delivery of DC.Tbet/IL12 into one tumor site resulted in dramatically suppressed tumor growth of both the treated and untreated lesions (Supplementary Figure S1).

Although DC.Tbet and DC.IL12 also exhibit the biologic potential to activate a protective CD8+ T cell repertoire, the magnitude and diversity of specificity (based on the pattern of recognition of HPLC fractionated peptides isolated from resected tumor/stromal cell populations) displayed by the DC.Tbet/IL12-primed Tc1 repertoire was greater than that associated with the DC.Tbet- or DC.IL12-based treatments. Importantly, from a safety perspective, the H-2d class I-restricted CD8+ T effector cells promoted by i.t. administration of DC.Tbet/IL12 failed to recognize pericytes or VEC flow-sorted from the tumor-uninvolved kidneys of treated mice.

Interestingly, the superior efficacy of i.t. DC.Tbet/IL12 therapy was also associated with higher levels of CD8+ T cell recruitment into tumor lesions, likely the result of locoregional production of CXCR3 chemokines such as CXCL9/Mig and/or CXCL10/IP-10 which were profoundly enhanced in their expression in DC.Tbet/IL12-treated lesions. Since the majority of DC injected into tumors fail to leave, and may die within, the tumor microenvironment,13,19 these results are consistent with the ability of i.t.-delivered DC.Tbet/IL12 to condition both the tumor and secondary lymphoid microenvironments in a manner conducive to therapeutic Tc1 differentiation in the periphery and the subsequent delivery of circulating T effector cells into tumor sites. Based on our previous report that IL12 gene insertion into DC extends their lifespan within the tumor microenvironment,13 we would expect that DC.Tbet/IL12 would also live longer and sustain their protective functionality within this hostile niche.

Importantly, even when not asked to take up environmental antigens or to condition the tumor microenvironment, DC.Tbet/IL12 consistently served as a superior “biologic adjuvant” when used in therapeutic peptide-based (synthetic and/or natural acid-eluted; Figure 5 and Supplementary Figure S2) vaccine formulations injected distal to tumors. Interestingly, therapeutic vaccines based on a mixture of synthetic TRP2180–188 peptide and natural tumor-derived peptides appeared to provide somewhat stronger anti-B16 melanoma benefit to treated animals when compared to vaccines formulated as either synthetic or natural peptides (Supplementary Figure S2). This could result from: (i) complementation in the largely nonoverlapping responder Tc1 repertoires directed against each set of peptides, and/or (ii) the influence of type-1 helper (Tc1 or Th1) responses that bolster Tc1 reactivity against both sets of target epitopes. These possibilities will be further investigated in prospective studies.

The direct mechanism of action associated with the DC.Tbet/IL12 treatment benefit remains unknown. While intrinsic Tbet expression appears crucial to the ability of DC to drive protective or pathologic type-1 T cell responses,20,21,22,23,24 it remains unclear how ectopic overexpression of Tbet confers improved type-1 polarizing potential on these APC. In our previous reports,14,15 we observed that DC.Tbet were efficient activators of type-1 T cell responses from naive T cell precursors and that such activation required close DC–T cell proximity, but not the “usual suspect” pro-inflammatory cytokines, including DC-elaborated IL-12p70, IL-23, IL-27 or IFN-γ itself. Indeed, our pilot data suggest that DC.Tbet developed from IL-12p35−/− mice display comparable antitumor efficacy to wild-type DC.Tbet when injected directly into s.c. tumor lesions (L. Chen, unpublished results). In this context, IL12-independent pathways for type-1 T cell induction have been reported, in which enhanced (IFN-α/β or IL-18; (refs. 25,26,27) or decreased (IL-10, (ref. 28)) cytokine production by APC have been suggested as dominant drivers for Th1/Tc1 response bias. Interestingly, CpG ODN adjuvants that require APC expression of Tbet24 preferentially promote type-1–mediated immunity in an IL12-independent manner via a mechanism involving downregulation of IL-10 production and co-inhibitory molecule expression by DC.24,28

Alternatively or additionally, LPS-activated murine CD8neg DC have been reported to induce Th1 responses via an IL12-independent mechanism that involves Delta-4 (DLL4)-NOTCH signaling29 and DEC-205+ DC foster Th1 responses via a CD70-CD27-dependent pathway that does not require IL-12p70.30 Although our previous in vitro results obtained appear to refute the dominant use of the NOTCH- or CD70-dependent signaling pathways by human DC.Tbet in activating Th1/Tc1 cells from naive responders,14 preliminary gene array analyses suggest that murine DC.Tbet may be coordinately enriched in NOTCH and depleted of IL-10/TGF-β signaling pathway competency (Supplementary Figure S3 and L. Chen, unpublished results). Interestingly, these endpoints may be operationally inter-related since NOTCH-mediated signaling has been reported to suppress p38 mitogen-activated protein kinase (MAPK) activation,31 and enhanced p38 MAPK activation has been linked with tumor-induced DC dysfunction,32 as well as, the propensity of DC to produce IL-10 in support of Treg responses.33,34,35 Pharmacologic antagonists of p38 MAPK have recently been demonstrated to normalize the functionality of DC isolated from cancer patients32,36 and to dramatically enhance the immunogenicity of DC-based vaccines in supporting type-1 immune responses.34 Importantly, our preliminary analyses (Figure 6) indeed support the preferential inactivation of p38 MAPK and the coordinate activation of extracellular signal-regulated kinase (ERK) in DC.Tbet/IL12. Prospective studies will determine whether blockade of ERK activation attenuates the potent therapeutic antitumor efficacy of DC.Tbet/IL12 and identify upstream elements responsible for differential ERK over p38 MAPK activation in these type-1 polarized APC.

Figure 6.

Figure 6

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DC.Tbet/IL12 express lower levels of activated p38 MAPK and higher levels of activated ERK than control DC. DC were generated from BALB/c mice and and left uninfected or they were infected for 48 hours with the indicated recombinant adenoviruses, before cell lysates were generated and western blot analyses performed using specific antibody probes as described in the Materials and Methods. Data are from one representative experiment of two performed. DC, dendritic cell; ERK, extracellular signal-regulated kinase; IL, interleukin; MAPK, mitogen-activated protein kinase; Tbet, T-box expressed in T cells.

Overall, our data suggest that co-delivery of IL-12p70 cDNA (in DC.Tbet/IL12 cells) provides a non-redundant “boost” to the therapeutic benefits associated with ectopic Tbet cDNA engineering of DC, making autologous DC.Tbet/IL12 an intriguing vaccine/treatment agent for translation into clinical trials designed for patients with cancer. When considering reported regression rates and the overall survival benefit of DC-based therapies in extensively studied B16 melanoma models, single modality DC.Tbet/IL12 appeared equitable or superior in its therapeutic efficacy versus: (i) i.t. delivery of DC engineered to express mCD40L37 or IFN-α38; or (ii) DC plus cotherapies such as anti-PD-L1 antibody + adoptive T cell transfer,39 nonmyeloablative chemotherapy +/− adoptive T cell transfer,40 CpG adjuvant,41 IL-2,42 photodynamic therapy,43 sunitinib6 or anti-MARCO antibody,44 among others. Future work will determine whether the benefits of DC.Tbet/IL12-based therapy can be improved by combination with agents that (i) enhance type-1 T cell activation, delivery into tumors and effector cell sustainability (i.e., such as PD-1 or TIM-3 antagonists; refs. 39,45), and/or (ii) mitigate regulatory immunity (such as anti-CTLA4 Ab or sunitinib; refs. 6,46).

Materials and Methods

Mice. Female 6–8 week old C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All animals were handled under aseptic conditions per an Institutional Animal Care and Use Committee-approved protocol and in accordance with recommendations for the proper care and use of laboratory animals.

Cell lines and culture. The MethA and CMS4 sarcomas syngenic to BALB/c (H-2d) mice and the B16 melanoma syngenic to C57BL/6 (H-2b) mice have been described previously.15,47 Tumor cell lines were free of mycoplasma contamination and were maintained in complete medium (RPMI-1640 media supplemented with 10% heat-inactivated fetal bovine serum, 100 µg/ml streptomycin, 100 U/ml penicillin, and 10 mmol/l -glutamine, all reagents were purchased from Invitrogen, Carlsbad, CA) at 5% CO2 tension in a 37 °C humidified incubator.

Adenoviral vectors. E1/E3-substituted, replication-defective (Ad5-derived) recombinant adenoviruses encoding mTbet and mIL-12p70, as well as the empty control Ad.ψ5 vector, have been described previously.15,47 All vectors were produced, qualified and supplied by the University of Pittsburgh's Vector Core Facility (shared resource).

Generation of bone marrow-derived DC and transduction with adenoviral vectors in vitro. DC were generated from the tibias/femurs of BALB/c mice, as previously described.15 Briefly, bone marrow precursors were cultured for 7 days in complete medium supplemented with 1,000 units/ml recombinant murine granulocyte/macrophage colony-stimulating factor and 1,000 units/ml recombinant murine IL-4 (both from Peprotech, Rocky Hill, NJ). CD11c+ DC were then purified using specific MACS beads (Miltenyi Biotec, Auburn, CA) and infected with recombinant adenovirus at multiplicity of infections of 50–250 as indicated in text for 48 hours. Intracellular staining using specific monoclonal antibodies (mAbs) and flow cytometry was used to document transgene expression in Ad.mTbet-infected DC as previously described.15 In addition, cell-free supernatants were evaluated for levels of mIL-12p70, tumor necrosis factor-α and IL-10 secretion using cytokine-specific commercial enzyme-linked immunosorbent assays per the manufacturer's protocol (BD Biosciences, Franklin Lakes, NJ). Triplicate determinations were used in all instances, with data reported as the mean ± SD. Consistent with our previous published data,13,15 we observed that DC.ψ5 produced ≤30 pg IL-12p70/ml/106 DC/48 hours and expressed <2.5% Tbet+ cells, while DC.IL12 and DC.Tbet/IL12 produced ≥8,000 pg IL-12p70/ml/106 DC/48 hours, and DC.Tbet and DC.Tbet/IL12 expressed >45% Tbet+ cells (Supplementary Figure S3). All DC-evaluated failed to express detectable levels (i.e., >7.8 pg/ml/ml/106 DC/48 hours) of IL-10 (data not shown), unless they were stimulated via CD40 ligation using J558.CD40 cells as previously described (ref. 15; Supplementary Figure S3).

Tumor therapy models. Syngenic mice received s.c. injection with tumor cells (5 × 105 CMS4 or 1 × 105 B16) in the right flank on day 0. On day 7, mice were randomized into treatment cohorts (containing five animals each) exhibiting comparable mean tumor sizes (i.e., ~40–60 mm2). For intralesional therapy, one million DC (control DC.null or DC.ψ5, DC.mTbet, DC.IL12 or DC.Tbet/IL12) were then injected directly into tumors in a total volume of 50 µl (in phosphate-buffered saline) on days 7 and 14 post-tumor inoculation. For DC-based vaccine models, each type of DC was left untreated or pulsed for 4 hours at 37 °C with 10 µmol/l HBB33–42 (H-2Ld-presented in the CMS4 model; ref. 17) or TRP2180–188 (H-2Kb-presented in the B16 model; ref. 18) peptide, then washed with phosphate-buffered saline before being injected s.c. in the left flank (i.e., contralateral to the tumor) on days 7 and 14 post-tumor inoculation. Tumor size was assessed every 3 or 4 days and recorded in mm2 by determining the product of the largest perpendicular diameters measured by vernier calipers.

Evaluation of specific CD8+ T cell responses. CD8+ splenocytes were harvested (and pooled from two mice/group) 7 days after the second round of DC-based therapy (i.e., day 21 after tumor inoculation) and analyzed for reactivity against tumor (relevant or negative control syngenic) cells, peptide-pulsed (negative control, syngenic) tumor cells, or day 19 (flow-sorted) B16-derived PDGFRβ+CD31neg pericytes or PDGFRβnegCD31+ VEC isolated as previously described.47 The lack of tumor cell contamination in these sorted populations was confirmed by reverse transcription-PCR analysis using primers specific to the tumor cell-associated MuLV gp70 env gene product as previously reported.48 In some experiments, T cell responses were also evaluated against syngenic DC pulsed with natural major histocompatibility complex -presented peptides isolated CMS4 tumors. To generate these peptides, day 21 tumors were harvested from untreated mice, aseptically minced and digested with DNase, collagenase, and hyaluronidase (all reagents from Sigma-Aldrich, St Louis, MO), as previously described.17 After filtration through a 70-µm mesh (BD Biosciences), viable cells were washed five times with phosphate-buffered saline by centrifugation. Peptides were acid-eluted using citrate-phosphate buffer (pH 3.3) from this viable cell mixture, desalted, and consequently separated on reverse-phase HPLC, as described previously.17 Individual HPLC fractions were then lyophilized to remove organic solvents and then reconstituted in 100 µl of phosphate-buffered saline and stored at −80 °C until use for T-cell assays. Where indicated, the class I-restricted nature of CD8+ T cell recognition was assessed by inclusion of 30 µg/well of anti-H-2d class I mAbs (i.e., 10 µg/well each of the anti-H-2Kd (31-3-4s; American Type Culture Collection, Manassas, VA), anti-H-2Dd (34-4-21s; American Type Culture Collection) and anti-H-2Ld (28-14-8; Santa Cruz Biotechnology, Santa Cruz, CA) mAbs or 30 µg/well of control IgG (Sigma-Aldrich).

Imaging of tumor tissues. Tumor samples were prepared and sectioned as previously reported.15,17 Briefly, tumor tissues were harvested and fixed in 2% paraformaldehyde (Sigma-Aldrich) at 4 °C for 1 hour, then cryoprotected in 30% sucrose for 24 hours. Tumor tissues were then frozen in liquid nitrogen and 6 mm cryosections prepared. For analysis of T cells, sections were stained with purified rat anti-mouse CD8α mAb (BD Pharmingen, San Diego, CA) for 1 hour. After washing, sections were stained with Alexa Fluor 488-conjugated goat anti-rat secondary antibody (Jackson ImmunoResearch, West Grove, PA). Cell nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI) as previously described.15 For analysis of CD31 and chemokines, the tissue sections were first incubated with rat anti-mouse CD31 and goat-anti-mCXCL9 or goat anti-mCXCL10 (both from R&D Systems, Minneapolis, MN) for 1 hour at room temperature, then washed with 0.5% bovine serum albumin and stained with Alexa Fluor 488-conjugated goat anti-rat antibody (Invitrogen) and Cy3-conjugated donkey anti-goat pAb (Jackson ImmunoResearch). After washing, sections were then covered in Gelvatol (Monsanto, St Louis, MO) and a coverslip applied. Slide images were acquired using an Olympus 500 scanning confocal microscope (Olympus America, Center Valley, PA). The fluorescent cell numbers per high power field were analyzed using Metamorph Imaging software (Molecular Devices, Sunnyvale, CA).

Western blot analyses. DC were generated from BALB/c mice and infected with recombinant adenovirus for 48 hours and lysed for 30 minutes at 4 °C in protein lysis buffer (1 × 107 cells/ml) containing 10 mmol/l Tris-HCL, pH 8.0, 150 mmol/l NaCl, 1% Triton X-100, 0.5% NP-40, 0.2 mmol/l sodium orthovanadate, 1 mmol/l EDTA, and 1× protease inhibitors (all from Sigma-Aldrich). Cell debris was removed by centrifugation at 13,500 rpm for 30 minutes, with the supernatant subsequently boiled in sample buffer containing 2-mercaptoethanol for 5 minutes. Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% Ready Gels (Bio-Rad, Hercules, CA) before electro-blotting onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membrane was then blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (Sigma-Aldrich) for 1 hour at room temperature, before being probed overnight at 4 °C with primary rabbit antibodies (all from Cell Signaling Technology, Danvers, MA) reactive against p38 MAPK, phospho-p38 MAPK, p44/42 MAPK (ERK1/2), phospho-p44/42 (pERK1/2), or anti-mouse β-actin (Abcam, Cambridge, MA). After washing, membranes were incubated with secondary goat anti-rabbit antibody coupled to HRP (Cell Signaling Technology) or goat anti-mouse (Bio-Rad) for 1 hour at room temperature. Antibody–antigen complexes were then detected using the Western Lightning Plus-ECL chemiluminescent detection system according to the manufacturer's instructions (PerkinElmer, Waltham, MA).

Statistical analysis. A two-tailed Student's t-test was used for data analysis with differences between groups considered significant at a value of P < 0.05.

SUPPLEMENTARY MATERIAL Figure S1. DC.Tbet/IL12 therapy applied to a “primary” tumor lesion provides systemic therapeutic benefit. Figure S2. Therapeutic vaccination using DC.Tbet/IL12 pulsed with TRP and/or acid-eluted whole tumor peptides inhibits B16 melanoma progression. Figure S3. Expression of secreted cytokines and intracellular Tbet protein by control and gene-modified DC.

Acknowledgments

The authors wish to thank Adriana Larregina and Binfeng Lu for their helpful comments and discussion during the preparation of this manuscript. This work was supported by NIH grants P01 CA100327 (W.J.S.) and R01 CA140375 (W.J.S., J.L.T.). D.B.L. was supported by a Postdoctoral Fellowship (PF-11-151-01-LIB) from the American Cancer Society. This project used the UPCI Vector Core Facility and was supported in part by University of Pittsburgh Cancer Institute CCSG, NIH P30 CA047904. The other authors declared no conflict of interest.

Supplementary Material

Figure S1.

DC.Tbet/IL12 therapy applied to a “primary” tumor lesion provides systemic therapeutic benefit.

Click here for additional data file. (10.3KB, tiff)
Figure S2.

Therapeutic vaccination using DC.Tbet/IL12 pulsed with TRP and/or acid-eluted whole tumor peptides inhibits B16 melanoma progression.

Click here for additional data file. (9.9KB, tiff)
Figure S3.

Expression of secreted cytokines and intracellular Tbet protein by control and gene-modified DC.

Click here for additional data file. (13.3KB, tiff)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

DC.Tbet/IL12 therapy applied to a “primary” tumor lesion provides systemic therapeutic benefit.

Click here for additional data file. (10.3KB, tiff)
Figure S2.

Therapeutic vaccination using DC.Tbet/IL12 pulsed with TRP and/or acid-eluted whole tumor peptides inhibits B16 melanoma progression.

Click here for additional data file. (9.9KB, tiff)
Figure S3.

Expression of secreted cytokines and intracellular Tbet protein by control and gene-modified DC.

Click here for additional data file. (13.3KB, tiff)

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