IL-12 triggers a programmatic change in dysfunctional myeloid-derived cells within mouse tumors

Abstract

Solid tumors are complex masses with a local microenvironment, or stroma, that supports tumor growth and progression. Among the diverse tumor-supporting stromal cells is a heterogeneous population of myeloid-derived cells. These cells are alternatively activated and contribute to the immunosuppressive environment of the tumor; overcoming their immunosuppressive effects may improve the efficacy of cancer immunotherapies. We recently found that engineering tumor-specific CD8(+) T cells to secrete the inflammatory cytokine IL-12 improved their therapeutic efficacy in the B16 mouse model of established melanoma. Here, we report the mechanism underlying this finding. Surprisingly, direct binding of IL-12 to receptors on lymphocytes or NK cells was not required. Instead, IL-12 sensitized bone marrow-derived tumor stromal cells, including CD11b(+)F4/80(hi) macrophages, CD11b(+)MHCII(hi)CD11c(hi) dendritic cells, and CD11b(+)Gr-1(hi) myeloid-derived suppressor cells, causing them to enhance the effects of adoptively transferred CD8(+) T cells. This reprogramming of myeloid-derived cells occurred partly through IFN-γ. Surprisingly, direct presentation of antigen to the transferred CD8(+) T cells by tumor was not necessary; however, MHCI expression on host cells was essential for IL-12-mediated antitumor enhancements. These results are consistent with a model in which IL-12 enhances the ability of CD8(+) T cells to collapse large vascularized tumors by triggering programmatic changes in otherwise suppressive antigen-presenting cells within tumors and support the use of IL-12 as part of immunotherapy for the treatment of solid tumors.

Trial registration: ClinicalTrials.gov NCT01236573.

Figures

Figure 1. Antitumor immunity of IL-12–engineered pmel-1 CD8+ T cells (IL-12 cells) is not dependent on type I self polarization but does require an endogenous response to secreted IL-12.

(A) Representative intracellular flow cytometry plot for IL-12 expression in WT or Il12rb2–/– CD8+ cells. (B) Representative histogram for CD62L, IL-2Rα, IL-7Rα, and Sca-1 expression in WT or Il12rb2–/– IL-12 cells. (C) Intracellular staining for IFN-γ and TNF-α in WT or Il12rb2–/– IL-12 cells stimulated with PMA/ionomycin. All flow cytometry data in A–C are representative of at least 3 independent experiments. Numbers represent percentage of cells in each quadrant. (D) Tumor treatment with 105 WT or Il12rb2–/– IL-12 cells transferred into sublethally irradiated (5-Gy TBI) C57BL/6 mice bearing 10-day established subcutaneous B16 tumors (n = 5). All data are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment (NT) control. (E) Antitumor immunity of 105 WT IL-12 cells transferred into sublethally irradiated WT or Il12rb2–/– C57BL/6 mice bearing subcutaneous B16 tumors established for 10 days. All data are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment control; ΨP < 0.05, compared with WT IL-12 cells in Il12rb2–/– host.

Figure 2. IL-12–dependent sensitization of bone marrow–derived cells is critical for tumor regression.

(A) WT C57BL/6 mice were lethally irradiated (6-Gy + 6-Gy TBI) and reconstituted with either WT (WT→WT) or Il12rb2–/– (Il12rb2–/–→WT) bone marrow. Six weeks after the formation of chimeras, mice (n = 10/group) were implanted with subcutaneous B16 melanomas, sublethally irradiated (5-Gy TBI), and given 105 IL-12 cells after tumors were established for 10 days. Data are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment control; **P < 0.05, compared with IL-12 cells into Il12rb2–/–→WT mice. (B) Il12rb2–/– C57BL/6 mice were lethally irradiated (6-Gy + 6-Gy TBI) and reconstituted with either WT (WT→Il12rb2–/–) or Il12rb2–/– (Il12rb2–/–→Il12rb2–/– ) bone marrow. Six weeks after the formation of chimeras, mice (n = 10/group) were implanted with subcutaneous B16 melanomas, sublethally irradiated, and given 105 IL-12 cells after tumors were established for 10 days. Data are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment control; **P < 0.05, compared with IL-12 cells in Il12rb2–/–→Il12rb2–/– mice.

Figure 3. IL-12–induced IFN-γ production and sensitization by host cells are partially required for antitumor immunity.

(A) Antitumor immunity of 2.5 × 105 WT or Ifng–/– CD8+ T cells expressing the P-TCR and IL-12 transferred into sublethally irradiated (5-Gy TBI) C57BL/6 mice (n = 5/group) bearing subcutaneous B16 tumors established for 10 days. In the same experiment, 2.5 × 105 WT CD8+ T cells expressing the P-TCR and IL-12 were adoptively transferred into sublethally irradiated WT or Ifng–/– C57BL/6 mice (Ifng–/–h; n = 5) bearing subcutaneous B16 tumors established for 10 days. All data are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment control; **P < 0.05, compared with P-TCR + IL-12 (WT > Ifng–/–h). (B) Antitumor immunity of 105 IL-12 cells following transfer into sublethally irradiated WT or Ifngr–/– C57BL/6 recipient mice (Ifngr–/–h) bearing subcutaneous B16 tumors established for 10 days. All data are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment control; **P < 0.05, compared with IL-12 cells in Ifngr–/–h mice.

Figure 4. IL-12–induced sensitization of endogenous immunity is independent of host T, B, and NK cells, but increases tumor infiltration of adoptively transferred T cells.

(A) Antitumor immunity following the transfer of 105 IL-12 cells into sublethally irradiated WT or Rag1–/– C57BL/6 mice (n = 5) bearing subcutaneous B16 tumors established for 10 days. (B) Tumor treatment of 105 IL-12 cells adoptively transferred into sublethally irradiated tumor-bearing WT or Rag1–/– C57BL/6 mice depleted of NK cells. All data in A and B are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment control. (C) Representative flow cytometry plots (left panel) and enumeration (right panel) of adoptively transferred T cells (CD8+ thy1.1+) from single-cell tumor suspensions 3 and 7 days following treatment with 105 IL-12 or mock cells in NK-depleted Rag1–/– mice. Data are expressed as mean ± SEM, *P < 0.05, Student t test. All flow cytometry plots gated on live PI– cells and numbers represent percentage of CD8+ Thy1.1+ cells. (D) Tumor sections from sublethally irradiated WT mice 7 days following the treatment of 105 IL-12 or mock cells were stained for thy1.1 on transferred T cells (green), CD31 expressed on endothelial cells (red), and DAPI (blue) to stain the nucleus. Stained sections were analyzed using fluorescence confocal microscopy. Original magnification, ×40.

Figure 5. Tumors from mice receiving IL-12 compared with mock cells display an increase in the intrinsic capabilities for antigen processing and presentation.

(A) Dendrogram of whole transcriptome analysis displaying the 407 gene transcripts common to both day 3 and day 7 that were differentially expressed in tumors following the adoptive transfer of 105 IL-12 cells compared with mock cells into sublethally irradiated mice bearing subcutaneous B16 tumors. Data are representative of 4 independent experimental samples. (B) Unbiased gene-ontology enrichment analysis for pathways under immune system processes from tumors of mice sublethally irradiated and treated with 105 IL-12 compared with mock cells. Pathway analysis formulated using Ingenuity software. (C) Expression pattern (FC) from tumors of mice treated with IL-12 over mock cells for different gene transcripts involved in the antigen-processing and -presentation pathway. Data were generated using Ingenuity software analysis. (D) The top 8 upregulated and downregulated gene transcripts from tumors of mice treated with 105 IL-12 over mock cells from A. Data represent mean ± SEM of the FC from 4 independent samples. For A–D, differentially expressed genes were identified by 1-way repeated measures ANOVA (P < 0.01) corrected by Benjamini-Hochberg FDR method (P < 0.05), and this gene list was further filtered for between-group α levels of P < 0.01 and an FC criterion of more than 2.0 for genes differentially expressed from tumors treated with IL-12 over mock cells.

Figure 6. Enhanced antitumor immunity triggered by IL-12 is dependent on in vivo cross presentation of tumor antigens.

(A) The percentage of PI–, NK1.1–, CD3–, CD11b+, and IL-12Rβ2+ cells from single-cell suspensions of well-established B16 tumors. (B) Flow cytometric analysis with backgating for PI–CD3–B220–NK1.1–CD11b+ cells from tumors 7 days following treatment with 105 IL-12 or mock cells. (C) CD11b+ cells from B were examined for expression of H-2Db with quantification of mean fluorescence intensity for 4 independent mice (right panel). *P < 0.05, t tests compared with no treatment. All plots are representative of at least 3 independent experiments. (D) Antitumor immunity in sublethally irradiated WT, Iab–/–, or B2m–/– C57BL/6 mice bearing established B16 tumors and treated with 105 IL-12 cells. All data are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment control; ΨP < 0.05, compared with IL-12 cells into B2m–/– host. (E) Representative flow cytometry plots for transferred T cells in tumors and spleens harvested from sublethally irradiated WT or B2m–/– C57BL/6 mice 7 days following the transfer of 105 IL-12–expressing pmel-1 Ly5.1+CD8+ T cells (IL-12P-Ly5.1) and 105 IL-12–expressing open-repertoire thy1.1+CD8+ T cells (IL-12OR-Thy1.1) into the same mouse. Data are representative of 3 independent samples. Numbers represent percentage of Thy1.1+Ly5.1– or Thy1.1–Ly5.1+ cells. (F) Confocal microscopy for tumor sections from mice treated in D (Thy1.1, green; CD31, red; DAPI, blue). Original magnification, ×40.

Figure 7. MDSCs, macrophages, and dendritic cells residing within B16 tumors of mice treated with IL-12 cells potently stimulate the proliferation of pmel CD8+ T cells.

(A) B16 tumors established on C57BL/6 mice for 10 days were excised 8 hours following 5-Gy TBI and examined by multicolor flow cytometry for CD45, NK1.1, TCRb, B220, CD11b, CD11c, I-Ab, F4/80, Ly6C, and Ly6G cells. Flow cytometry plot is representative of at least 3 independent samples and gated on live, NK1.1–, TCRb–, and B220– cells. Numbers represent percentage of cells within each quadrant. (B) Single-cell tumor suspensions were created 5 days following treatment of established B16 tumors with IL-12 or mock cells, and CD11b+GR1hi MDSCs, CD11b+F4/80hi macrophages, and CD11b+CD11chi dendritic cells were sorted by flow cytometry and cocultured for 72 hours with CFSE-labeled untouched pmel CD8+ T cells at a 10:1 T cells/APC ratio. Flow cytometry plots represent 3 independent samples.

Figure 8. IL-12 cells (H-2b) are capable of causing complete regression of established Cloudman S91 melanomas (H-2d) on C57BL/6 × DBA F1 mice (H-2b/d).

(A) H-2Db and H-2Dd expression of B16 and Cloudman S91 melanoma cell lines exposed to 675 ng/ml of IFN-γ for 48 hours. (B) Antitumor immunity of 5 × 105 IL-12 or mock cells adoptively transferred into sublethally irradiated C57BL/6 × DBA F1 H-2b/d mice bearing subcutaneous Cloudman S91 tumors established for 21 days. All data are expressed as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, Wilcoxon’s rank-sum test compared with no treatment and mock. (C) Flow cytometry plots for macrophages (CD11b+ F4/80hi), dendritic cells (CD11b+CD11chi), MDSC-M (CD11b+Ly6ChiLyGlo), and MDSC-G (CD11b+Ly6CMid-hiLy6Ghi) within established Cloudman S91 melanomas on C57BL/6 × DBA F1 H-2b/d mice 10 days following treatment with IL-12 or mock cells. All plots gated on PI– live cells; numbers represent percentage of cells in each quadrant. (D) Data from C were quantified through a flow cytometry count and normalized to 1 gram of tumor. Data are expressed as mean ± SEM. *P < 0.05, 1-way ANOVA compared with NT and mock controls.

Figure 9. Schematic diagram summarizing the proposed mechanism for tumor destruction induced by IL-12.

Myeloid-derived cells within the tumor microenvironment are sensitized by IL-12 to create an acute inflammatory environment and improve the ability of antigen-specific CD8+ T cells to collapse large established tumors.