Th-1 lymphocytes induce dendritic cell tumor killing activity by an IFN-γ-dependent mechanism

Abstract

Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4(+) Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1-activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.

Figures

Figure 1. Activated CD4+ T cells induce DC tumoricidal activity

(A, B) Day 6 CD11c+ DCs were cultured for 48 hours with activated CD4+ T lymphocytes at the indicated ratios. CD11c+ DCs were then separated from T cells, washed and subsequently incubated for an additional 48 hours with (A) 4T1 or (B) B16 tumor cells. Tumor cell survival was determined using a crystal violet assay. LPS-activated (1 µg/ml) DCs ([DC] LPS) were used as positive controls and untreated DCs (Untreated DCs) as negative controls. The data represent the mean +/− SD from triplicate wells. * Significant difference when compared with tumor cells cultured with control untreated DCs (p<0.001). (C) 4T1 or (D) B16 tumor cells were cultured for 48 hours with untreated DCs (Untreated DCs), with LPS-activated (1 µg/ml) KDCs ([DC] LPS), or with DCs that had first been co-cultured for 48 hours separated from activated CD4+ T cells by a micro-porous membrane ([DC] CD4 (TW)) as described in material and methods. Tumor cell viability was determined as described above. Mean +/− SD of triplicate cultures. (*p<0.0001).

Figure 2. Generation and characterization of T-helper lymphocyte lineages

Splenic CD4+CD25−CD62L+ T cells were isolated by magnetic cell sorting and cultured for 3 days in different polarization conditions as described in materials and methods to generate Th-1, Th-2, Th-17 and Treg cells. (A) Phenotypical analysis of the obtained T lymphocytes. Cells were stained with the indicated Ab and were analyzed by flow cytometry. (B) On day 3, cells were washed and re-stimulated with anti-CD3/CD28 antibody coated beads for eight hours and the culture supernatants were collected and analyzed by ELISA. (C) Real time PCR analysis of the cells obtained in (B) for the expression of the indicated cytokine mRNAs. Significant difference when compared to naïve T cell groups (* p<0.05, * p<0.0001).

Figure 3. Th-1 but not Th-2, Th-17 or Treg are capable of inducing DC tumoricidal activity

Day 6 CD11c+ DCs were cultured for 48 hours with the supernatants from Th-1, Th-2, Th-17 and Treg cultures. DCs were then washed extensively and incubated for 48 hours with (A) 4T1 or (B) B16 tumor cells. KDCs activated with LPS (1 µg/ml) ([DC] LPS) were used as positive controls. Tumor cell survival was then determined. Mean +/− SD from triplicate cultures. (*p < 0.001). (C) Phenotype of DCs obtained after 48 hours of culture with the culture supernatant from Th-1, Th-2, Th-17 and Treg cultures. Cells were stained with the indicated Ab and analyzed by flow cytometry.

Figure 4. The induction of DC killing activity by Th-1 lymphocytes depends on IFN-γ

Day 6 DCs were treated with Th-1 supernatant ([DC] Th-1) with or without anti-IFN-γ blocking antibody (+IFN-γ blocking Ab) or isotype control antibody (+Isotype control) or were treated with recombinant IFN-γ (5 ng/ml) ([DC] IFN-γ) for 48 hours. DCs were then washed extensively and plated at 5:1 DC: tumor cell ratio with (A) 4T1 or (B) B16 tumor cells. (C) 4T1 tumor cells were cultured with DC generated from wild-type (WT) or from IFN-γ receptor −/− mice (IFN-γ R−/−) and treated with Th-1 culture supernatant ([DC] Th-1) or IFN-γ ([DC] IFN-γ). (A–C) Tumor cell viability was then evaluated. Mean +/− standard deviation from triplicate cultures. Untreated DCs (Untreated DC) and DCs treated with LPS (1 µg/ml) ([DC] LPS) were used as negative and positive controls respectively. (*p<0.001)

Figure 5. Th-1-induced KDC tumoricidal activity requires a direct cell-cell contact but does not depend on death receptor ligands

(A) 4T1 or B16 tumor cells were cultured separated or not by a transwell insert (TW), with untreated DCs (Untreated DC), or DCs treated with IFN-γ ([DC] IFN-γ) or Th-1 supernatant-treated DCs ([DC] Th-1). * Significant difference compared to the corresponding group without transwell insert (p−/− or (D) perforin−/− mice were treated with IFN-γ or Th-1 supernatant and cultured for 48 hours with 4T1 tumor cells. * Significant difference compared to wild-type DCs (p<0.01). (A,C,D) Tumor cell killing was determined after 48 hours. Mean +/− standard deviation from triplicate cultures. Untreated DCs (Untreated DC) and DCs treated with LPS (1 µg/ml) ([DC] LPS) were used as negative and positive controls respectively.

Figure 6. Th-1 KDC cytotoxic activity depends on Nitric Oxide

(A) Detection of nitrites in the supernatants of DCs treated for 48 hours with the supernatants of Th-1, Th-2, Th-17 and Treg cell cultures, or with LPS or IFN-γ. (B) iNOS expression was determined by Western blot (left panel) and RT-PCR (right panel) in day 8 DCs treated for 24 hours with LPS (1 µg/ml) ([DC] LPS), IFN-γ (5 ng/ml) ([DC] IFN-γ) or Th-1 supernatant ([DC] Th-1). * Significant compared to untreated DCs (p−/− or wild type mice were treated with IFN-γ or Th-1 supernatant and cultured for 48 hours with 4T1 tumor cells. (C,D) Tumor cell killing was determined after 48 hours. Mean +/− standard deviation from triplicate cultures (A,C,D *p<0.001). Untreated DCs and DCs treated with LPS were used as negative and positive controls respectively.

Figure 7. Th-1 KDCs are capable of presenting tumor antigens from the tumor cells they have killed to tumor-specific T cells

CD11c+ DCs were treated with LPS (1 µg/ml) ([DC] LPS), IFN-γ (5 ng/ml) ([DC] IFN-γ) or Th-1 supernatant ([DC] Th-1) for 48 hours. (A) DCs were then washed and cultured for 24 hours with B16 or B16-OVA melanoma cells. DCs were selected from the culture using CD11c microbeads and incubated for an additional 24 hours with B3Z cells (DC:B3Z ratio = 1:10) as outlined in material and methods. The activity of β-galactosidase was measured by evaluating the conversion of its substrate into a chemoluminescent product (RLU, Relative Luminescence Unit). (B) CD11c-GFP-DTR mice were injected with B16-OVA melanoma cells. When tumors become palpable, diphtheria toxin (DT) was administered to deplete host CD11c+ cells. CD11c+ DCs generated in vitro and treated with LPS or IFN-γ or Th-1 supernatant were then injected intratumorally (20×106 cells/tumor). After 36 hours, draining lymph nodes were harvested and CD11c+ cells were isolated using CD11c microbeads. The obtained DCs were then cultured with the OVA-specific T cell line B3Z. Specific recognition of tumor-derived OVA peptide was measured as outlined above. * Significant difference when compared to untreated DC group (p<0.01).