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, 18 (9), 663-73

Therapeutic Effectiveness of Intratumorally Delivered Dendritic Cells Engineered to Express the Pro-Inflammatory Cytokine, Interleukin (IL)-32

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Therapeutic Effectiveness of Intratumorally Delivered Dendritic Cells Engineered to Express the Pro-Inflammatory Cytokine, Interleukin (IL)-32

Y Qu et al. Cancer Gene Ther.

Abstract

Interleukin-32 (IL-32) is a pro-inflammatory cytokine conditionally produced by T cells, natural killer (NK) cells, monocytes, epithelial cells and keratinocytes, which has an important role in host resistance against infectious disease. Interestingly, elevated levels of IL-32 transcripts in fine needle aspirates of tumor tissue have also been correlated with objective clinical responses in cancer patients receiving immunotherapy. To evaluate the antitumor impact of IL-32 gene therapy, we treated BALB/c mice bearing established subcutaneous CMS4 sarcomas with intratumoral (i.t.) injections of syngenic dendritic cells (DCs) engineered to express human IL-32β complementary DNA (that is, DC.IL32). Although ectopic expression of IL-32β by DC resulted in only modest phenotypic changes in these antigen-presenting cells, DC.IL32 produced higher levels of IL-12p70 than control DC. DC.IL32 were more potent activators of type-1 T-cell responses in vitro and in vivo, with i.t. administration of DC.IL32 leading to the CD8(+) T-cell-dependent (but CD4(+) T-cell- and NK cell-independent) suppression of tumor growth. Effective DC.IL32-based therapy promoted infiltration of tumors by type-1 (that is, CXCR3(+)VLA-4(+)GrB(+)) CD8(+) T cells and CD11b(+)CD11c(+) host myeloid DC, but led to reductions in the prevalence of CD11b(+)Gr1(+) myeloid-derived suppressor cells and CD31(+) blood vessels.

Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Expression and bioactivity of hIL-32β in mouse CD11c+ DCs
When compared to control DCs (DC.null and DC.ψ5), DC.IL-32 expressed higher intracellular expression levels of IL-32β protein as determined by flow cytometry (panel A; MOI = 250) and ELISA (panel B; MOI ranging from 50–500) as outlined in Materials and Methods. For the ELISA assays (B), both DC culture supernatants and DC lysates were analyzed for IL-32β concentration. In C, cell-free supernatants were harvested 48h after DC infection with no adenovirus, Ad.ψ5 (MOI = 500) or Ad.IL32β (at MOI ranging from 50–500), and then used to supplement cultures of the Raw 264.7 murine macrophage cell line. After 18h, supernatants were recovered and analyzed for TNF-α content by ELISA. Recombinant IL-32α (400 pg/ml) was used as positive control for induction of TNF-α from Raw 264.7 cells. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 2
Figure 2. Ectopic expression of IL-32β enhances the immunostimulatory phenotype of DC
DC.IL32 (MOI = 250) or control DC (DC.null or DC.ψ5) were prepared as described in Materials and Methods. Forty-eight hours after adenoviral infection, DC were harvested and analyzed by flow cytometry for cell surface expression of MHC and costimulatory molecules, as well as, CCR7 required for DC migration to secondary lymphoid tissues (A). In B., cell-free supernatants were recovered from the individual DC cohorts 24h after stimulation with CD40L+J558 cells (as described in Materials and Methods) and analyzed for the indicated cytokines by specific ELISA. Results are reported as the mean ± SD of triplicate determinations. NS = not significant; **p < 0.01.
Figure 3
Figure 3. DC.IL32 promote enhanced Type-1 polarization, but not proliferation, of alloreactive CD8+ T cells in vitro
In A., CFSE-labeled (H-2d) CD8+ splenocytes were stimulated with H-2b control DC (DC.null or DC.ψ5) or DC.IL32for 3 days as outlined in Materials and Methods. Cells were then harvested and CD8+ gated cells analyzed for CSFE fluorescence dilution by flow cytometry. In B., the percentage of proliferating CD8+ T cells in each of the indicated cultures is reported as mean ± SD. Cell-free supernatants were also harvested from these DC-T cell cultures and analyzed for IFN-γ and IL-10 concentrations using commercial ELISA (panel C). Data are reported as mean ± SD from triplicate determinations. All experiments are representative of data obtained in 3 independent experiments. NS = Not significant; *p < 0.05; **p < 0.01.
Figure 4
Figure 4. Intratumoral delivery of DC.IL-32β is effective in suppressing CMS4 tumor growth in vivo via a CD8+ T cell-dependent mechanism
In A., BALB/c mice bearing day 7 established CMS4 tumors (approximately 40 mm2 in size) received i.t. injections of 106 DC.IL32 or control DC, with a second identical injection of DC provided on day 14 post-tumor inoculation. To determine the relevance of CD4+ T cells, CD8+ T cells or asGM1+ NK cells in protection associated with i.t. delivery of DC.IL32, replicate cohorts of treated animals were also injected with specific depleting antibodies as described in Materials and Methods. Tumor growth was monitored every 4 days. Data are reported as the mean ± SD for 5 animals/group. **p < 0.01 versus DC.null or DC.ψ5. In B., Kaplan-Meier plots are provided for treatment with 1 × 106 (n = 15 mice) or 2.5 × 106 (n = 10 mice) DC.IL32 versus DC.null or DC.ψ5 treatment, respectively. p < 0.05 for DC.IL32 (106) versus DC.ψ5 (106) or DC.null (106); p < 0.001 for DC.IL32 (2.5 × 106) versus DC.ψ5 (2.5 × 106) or DC.null (2.5 × 106), and p < 0.01 for DC.IL32 (2.5 × 106) versus DC.IL32 (106). In C., to evaluate therapy-induced activation of anti-tumor Tc1 cells, on day 21 post-tumor inoculation, CD8+ T cells were isolated from the spleens and TDLN of CMS4-bearing animals treated with DC.null, DC.ψ5 or DC.IL32. These effector cells were then analyzed for their ability to recognize relevant CMS4 tumor cells versus unrelated H-2d MethA sarcoma cells based on IFN-γ secretion analyzed by ELISA as described in Materials and Methods. Data are reported as the mean ± SD of triplicate determinations. **p < 0.01 versus MethA.
Figure 5
Figure 5. Intratumoral delivery of DC.IL32 promotes increased tumor infiltration by T cells and host DC, reductions in tumor vascularity and MDSC content, and the increased apoptotic death of tumor/stromal cells in the TME
CMS4 tumor-bearing mice were treated as described in Fig. 4. On day 21 post-tumor inoculation, tumors were harvested and tissue sections analyzed for expression of CD4+ T cells, CD8+ T cells, CD11b+Gr1+ (MDSC) cells, and NG2+CD31+ vascular structures by immunofluorescence microscopy as outlined in the Materials and Methods. Representative images (A) and quantified numbers of events per high power field (HPF; reported as mean ± SD for 10 HPF per slide; panel B) are depicted. *p < 0.05; **p < 0.01 versus DC.null or DC.ψ5. CD31 vessel area is reported in microns2. For C, BALB/c mice bearing established day 7 CMS4 tumors were treated with i.t. delivery of DC.IL32 or control DC pre-labeled with CFSE (as described in Materials and Methods). On day 21 post-tumor inoculation, tumors were harvested and tissue sections analyzed for: the presence of injected DC (CD11c+CFSE+) versus host DC (CD11c+CFSEneg) and the presence of apoptotic DC (TUNEL+CFSE+) versus tumor/tumor stromal cells (TUNEL+CFSEneg) by immunofluorescence microscopy as outlined in Materials and Methods. **p < 0.01 versus DC.null or DC.ψ5. Quantified numbers of events per high power field (HPF; reported as mean ± SD for 10 HPF per slide) are reported in panel D. Similar data were obtained in 3 independent experiments performed.
Figure 6
Figure 6. Intratumoral delivery of DC.IL32 results in increased numbers of host-derived CD11c+ DC in the tumor-draining lymph nodes (TDLN)
BALB/c mice bearing established day 7 CMS4 tumors were treated with i.t. delivered DC.IL32 or control DC (pre-labeled with CFSE) on day 7. A, on day 8 post-tumor inoculation, TDLN were harvested and single-cell suspensions analyzed by flow cytometry for CD11c+ cells of host (CFSEneg/dim) versus therapy (CFSEbright+) origin. B, absolute numbers of each cell population were calculated as the product of total cell yield and the frequency of the indicated cell subset as deduced in panel A. Similar data were obtained in 3 independent experiments performed. *p < 0.05 versus DC.null or DC.ψ5.

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