Reversal of tumor-induced dendritic cell paralysis by CpG immunostimulatory oligonucleotide and anti-interleukin 10 receptor antibody

Abstract

Progressing tumors in man and mouse are often infiltrated by dendritic cells (DCs). Deficient antitumor immunity could be related to a lack of tumor-associated antigen (TAA) presentation by tumor-infiltrating DCs (TIDCs) or to a functional defect of TIDCs. Here we investigated the phenotype and function of TIDCs in transplantable and transgenic mouse tumor models. Although TIDCs could encompass various known DC subsets, most had an immature phenotype. We observed that TIDCs were able to present TAA in the context of major histocompatibility complex class I but that they were refractory to stimulation with the combination of lipopolysaccharide, interferon gamma, and anti-CD40 antibody. We could revert TIDC paralysis, however, by in vitro or in vivo stimulation with the combination of a CpG immunostimulatory sequence and an anti-interleukin 10 receptor (IL-10R) antibody. CpG or anti-IL-10R alone were inactive in TIDCs, whereas CpG triggered activation in normal DCs. In particular, CpG plus anti-IL-10R enhanced the TAA-specific immune response and triggered de novo IL-12 production. Subsequently, CpG plus anti-IL-10R treatment showed robust antitumor therapeutic activity exceeding by far that of CpG alone, and elicited antitumor immune memory.

Figure 1.

TIDCs may express diverse phenotypes but are immature in their majority. (A) DCs were enriched from the indicated solid tumors and analyzed for the expression of CD11c, CD11b, CD8α, and B220 by flow cytometry (log scale). (B) DCs were enriched from peripheral lymph node or from the indicated solid tumors and the expression of MHC class II, CD40, and CD86 molecules was analyzed among gated CD11c⁺ cells (solid line) compared with isotype control (gray histograms) by flow cytometry (log scale). Data are representative of two to five experiments.

Figure 2.

TIDCs are not activated by LPS plus IFN-γ plus anti-CD40 stimulation. (A) Control BM-DCs or enriched C26–6CK TIDCs were cultured overnight with medium alone or with the combination of LPS, IFN-γ plus anti-CD40 antagonist antibody. Flow cytometry histograms (log scale) show the expression of MHC class II, CD40, and CD86 among gated CD11c⁺ cells (solid lines) in unstimulated (top panels) or stimulated (bottom panels) DC populations. The percentage of positive cells was determined by comparison with isotype control (gray histogram) and is indicated in the top right corner of each histogram. Results are representative of more than five experiments. (B) The ability to respond to LPS plus IFN-γ and anti-CD40 activation was analyzed by measuring IL-12p70 levels in 18 h culture supernatants in various DC populations: DCs isolated from C26–6CK tumors or axillary draining lymph nodes from the same tumor-bearing animals; DCs enriched from hepatocarcinoma developing in x/myc transgenic mice or from normal liver; BM-DCs cultured in the presence or absence of supernatant from C26 tumors. ELISA results are expressed as the mean concentration ± SEM of triplicate cultures. Similar results were obtained in two to five experiments, depending upon the conditions tested.

Figure 3.

Combination of CpG 1668 and anti–IL-10R antibody overcome TIDC paralysis in vitro. (A and B) BM-DCs and TIDCs enriched from C26–6CK tumors were activated overnight with either LPS, IFN-γ, and anti-CD40 (white bars), or CpG 1668 (black bars) in the presence (anti–IL-10R) or absence (none) of anti–IL-10R antibody. Culture supernatants were assayed for IL-12p70 and TNFα content. Results are expressed as the mean concentration ± SEM of triplicate cultures and are representative of more than three experiments. (C) Mixed leukocyte reaction. Irradiated populations of enriched TIDCs, previously activated overnight with none (□), CpG 1668 (○), CpG plus anti–IL-10R (▪), LPS plus IFN-γ plus anti-CD40 (•), or anti–IL-10R plus LPS plus IFN-γ plus anti-CD40 (♦), were cultured with allogeneic purified T cells. Proliferation is expressed as the mean cpm incorporation ± SEM for triplicates.

Figure 4.

CpG 1668 plus anti–IL-10R activates tumor-infiltrating DCs in vivo. Mice bearing C26–6CK tumors were injected intratumorally with 5 μg CpG 1668 or control GpG sequence and/or intraperitoneally with 250 μg anti–IL-10R or isotype control antibody. 2 h after injection, mice were killed, and TIDCs enriched and stained for intracellular IL-12 p40/p70 and surface CD11c. The percentage of IL-12–positive cells among CD11c-enriched TIDCs was determined by comparison with isotype control and is indicated in the top right corner.

Figure 5.

Induction of tumor antigen–specific immune responses by TIDCs and modulation by CpG plus anti–IL-10R. (A) TIDC cross-present tumor antigen–derived peptides to T cells. The ability of TIDCs from C26–6CK tumors grown in H-2^d × H-2^b F1 mice to present tumor-derived antigenic peptides was assessed by measuring the secretion of IFN-γ by the CTL clone E/88 (H-2^d CTL, white bars) and CTL cell line TG905 (H-2^b CTL, black bars). Positive controls consisted of DCs enriched from H-2^d × H-2^b spleens (APC) and pulsed with the relevant peptide(s) as well as MCA38 H-2^b and C26 H-2^d cell lines which both express the antigen. (B and C) TIDCs stimulated with CpG 1668 plus anti–IL-10R induce tumor-associated antigen MHC class I–restricted responses in vivo. TIDCs enriched from C26–6CK tumors were cultured overnight with medium alone (TIDC) or CpG 1668 plus anti–IL-10R antibody (TIDC + CpG + a-IL10R), then injected intracutaneously into naive mice. Controls consisted of uninjected mice (negative control) or mice injected with irradiated C26 cells (irrad. C26). Mice were killed 5 d after injection. Experiments were performed with organs pooled from three naive mice and similar results obtained in two separate experiments. (B) Spleens from injected animals were cultured with irradiated C26 cells and IL-2 and cytotoxicity measured against P815 H-2^d target cells, alone (white bars) or loaded with the AH-1 antigenic peptide of C26 (black bars). Results are expressed as the mean cytotoxicity ± SEM per triplicate wells at an effector/target ratio of 1:100. (C) Total cell suspensions from pooled draining popliteal lymph nodes were analyzed for IFN-γ–producing cells after overnight culture without (white bar) or with (black bar) AH-1 peptide. Results are expressed as the mean number of spots ± SD for six different wells.

Figure 6.

DCs from various transplantable or transgene-induced tumors are refractory to activation with LPS, IFN-γ plus anti-CD40 but not with CpG 1668 plus anti–IL-10R. The ability of TIDC populations enriched from the indicated tumors to produce IL-12 in response to LPS plus IFN-γ and anti-CD40 or CpG 1668 plus anti–IL-10R was analyzed: (A) by intracellular staining for IL-12p40/p70 together with surface CD11c staining (log scale), percentages of CD11c⁺ cells expressing intracellular IL-12 are indicated in the top right quadrant; (B) by measuring IL-12 p70 levels in TIDC culture supernatant by ELISA. Results (log scale) are expressed as the mean concentration ± SEM from triplicate cultures.

Figure 7.

Treatment with CpG 1668 plus anti–IL-10R induces tumor rejection. Groups of seven mice were injected subcutaneously at day 0 with 5 × 10⁴ C26 (A) or B16F0 (B) tumor cells. Mice were treated at day 7, 14, and 21 (arrows) with control antibody, 5 μg CpG 1668 injected intratumorally, 250 μg anti–IL-10R antibody intraperitoneally, or CpG plus anti–IL-10R. Tumor incidence and survival were monitored in all groups for the indicated times.

Figure 8.

Treatment with CpG 1668 plus anti–IL-10R induces T cell and NK cell–mediated tumor rejection as well as antitumor immune memory. Groups of seven mice were injected subcutaneously at day 0 with 5 × 10⁴ C26 tumor cells. (A) Mice were treated at day 7, 14, and 21 (arrows) with control or 5 μg CpG 1668 injected intratumorally plus 250 μg anti–IL-10R antibody intraperitoneally. Indicated groups of mice receiving anti–IL-10R plus CpG were further treated with anti-CD4, anti-CD8, or Asialo-GM1 depleting antibodies as described in Materials and Methods. (B) Immunodeficient SCID mice were treated at day 7, 14, and 21 (arrows) with control, 5 μg CpG 1668 injected intratumorally, 250 μg anti–IL-10R antibody intraperitoneally or CpG plus anti–IL-10R. (C) Mice were injected intratumorally at day 7, 14, and 21 (arrows) with 5 × 10⁴ enriched tumor-infiltrating DCs activated for 2 h in vitro with none, LPS plus anti-CD40 plus anti–IL-10R, or CpG plus anti–IL-10R, as well as intraperitoneally with 250 μg anti–IL-10R antibody. (D) Mice treated with CpG plus anti–IL-10R that had rejected a C26 tumor inoculated at day 0 (n = 12) were rechallenged at day 45 with 5 × 10⁴ C26 cells in the contralateral flank and compared naive mice challenged with the same inoculum. Tumor incidence and survival were monitored in all groups for the indicated times.