Synergy of anti-CD40, CpG and MPL in activation of mouse macrophages

Abstract

Activation of macrophages is a prerequisite for their antitumor effects. Several reagents, including agonistic anti-CD40 monoclonal antibody (anti-CD40), CpG oligodeoxynucleotides (CpG) and monophosphoryl lipid A (MPL), can stimulate activation of macrophages. Our previous studies showed synergy between anti-CD40 and CpG and between anti-CD40 and MPL in macrophage activation and antitumor efficacy in mice. In the present study, we asked whether there was synergy among these three reagents. The activation of adherent peritoneal exudate cells (PEC) obtained from mice injected with anti-CD40 and then treated with CpG and/or MPL in vitro was determined by their ability to suppress proliferation of tumor cells and to produce various cytokines and chemokines in vitro. Cell sorting and histology followed by functional testing showed that macrophages were the main cell population in PEC activated by CD40 ligation in vivo. A combination of anti-CD40, CpG or MPL activated PEC to suppress proliferation of B16 cells and produce nitric oxide far greater than the single reagents or any of the double combinations of these reagents. In addition, the combination of all three reagents activated PEC to secrete IL-12, IFN-γ and MCP-1 to a greater degree than any single reagent or any two combined reagents. These results demonstrate that macrophages can be synergistically activated by anti-CD40, CpG and MPL, suggesting that this novel combined approach might be further investigated as potential cancer therapy.

Keywords: Anti-CD40; CpG; Immunotherapy; Macrophages; Monophosphoryl lipid A.

Figure 1. CD11b⁺ macrophages are activated after anti-CD40 and CpG or MPL treatment

A. PEC from C57BL/6 mice injected IP with 0.5 mg anti-CD40 three days earlier were collected, stained and sorted into 4 subpopulations by a FACSAria cell sorter. The cells were divided as follows: (1) CD11b^high Gr-1^-/int; (2) CD11b^int Gr-1⁺. A separate group of cells showed a CD11b^int/-Gr-1^- phenotype. These were further divided by CD19 and CD11b into populations (3) CD11b^- Gr-1^- CD19⁺ and (4) CD11b^int Gr-1^- CD19⁺. B. Each of the 4 sorted cell populations from Fig. 1A was stained with Wright-Giemsa stain to identify cell types based on morphology (×40 magnification). C. Each sorted cell population was tested in medium, CpG (5 μg/mL) or MPL (5 μg/mL) for the ability to inhibit B16 proliferation ([³H]-thymidine incorporation). D. Each sorted population was tested in medium, CpG (5 μg/mL) or MPL (5 μg/mL) for the ability to produce NO. A one-way ANOVA followed by Tukey test was used to compare [³H]-TdR counts or nitrite concentration between different populations cultured in the same stimulus (medium, CpG or MPL) in comparison to the control cultures of B16 cells in medium, CpG or MPL, but in the absence of any PEC. *P<0.001. ND, not done. Similar results were obtained in two independent experiments performed in triplicate.

Figure 2. Synergistic activation of PEC with anti-CD40 and CpG, anti-CD40 and MPL, and CpG and MPL

A, B: Synergy between anti-CD40 and CpG. PEC were collected from naïve C57BL/6 mice (“naïve”) or mice injected with 0.5 mg anti-CD40 (“anti-CD40”) three days earlier. The adherent PEC were then cocultured with B16 cells in the absence or presence of CpG at the indicated concentrations for 48 hours. The wells were pulsed with [³H]-TdR (1μCi/well) for the last 6 hours of incubation to measure proliferation of B16 cells (A). Results are presented as the mean total number of β-counts over 5 minutes ± SE. Supernatants from the coculture of PEC and B16 cells were tested for nitrite concentration using Griess test (B). Data shown are represented as mean nitrite concentration ± SE. C, D: Synergy between anti-CD40 and MPL. PEC were collected from naïve C57BL/6 mice (“naïve”) or mice injected with 0.5 mg anti-CD40 (“anti-CD40”) three days earlier. The adherent PECs were cocultured with B16 cells in absence or presence of MPL at the indicated concentrations. The tumoristatic (C) and NO-inducing (D) effects of PEC are shown (mean ± SE). E, F: Synergy between CpG and MPL. PEC were collected from naïve C57BL/6 mice. Adherent PEC were cocultured with B16 cells in the absence or presence of CpG, MPL or their combination at the indicated concentrations for 48 hours. The tumoristatic (E) and NO-inducing (F) effects of PEC are shown (mean ± SE). The data were analyzed by two-way ANOVA followed by Bonferroni test. In A, B, C and D: *P <0.00125, **P<0.00025 for the difference between anti-CD40+CpG vs anti-CD40 or CpG alone at the indicated dose of CpG, and between anti-CD40+MPL vs anti-CD40 or MPL alone at the indicated dose of MPL. In E and F: *P<0.0005 for the indicated combination of CpG + MPL vs CpG or MPL alone. The experiment was carried out in triplicate and repeated three times.

Figure 3. Synergy among anti-CD40, CpG and MPL in tumoristasis and NO production of PEC

A, B: Synergy among anti-CD40, CpG and MPL. PEC were collected from C57BL/6 mice injected with anti-CD40 (0.5mg IP) three days earlier. Adherent PEC were cocultured with B16 cells in the absence or presence of CpG, MPL or their combination at the indicated concentrations (ng/ml) for 48 hours. The wells were pulsed with [³H]-TdR (1μCi/well) for the last 6 hours of incubation to measure proliferation of B16 cells (A). Results are presented as the mean total number of β-counts over 5 min ± SE. Supernatants from the coculture of PEC and B16 cells were tested for nitrite concentration using Griess test (B). Data shown are represented as mean nitrite concentration ± SE. A two-way ANOVA followed by Tukey's post-testing method was performed. At the indicated doses of CpG and MPL, the 3-agent combination of anti-CD40, CpG and MPL is compared to the double combination of anti-CD40 and CpG or anti-CD40 and MPL. *P<0.05; **P<0.001. C, D: Synergy among anti-CD40, CpG and MPL at low doses of anti-CD40. PEC were collected from C57BL/6 mice injected with anti-CD40 at the indicated doses three days earlier Adherent PEC were then cocultured in triplicate wells with B16 cells in the absence or presence of CpG (200ng/ml), MPL (20ng/ml) or their combination for 48 hours. The tumoristatic (C) and NO-inducing (D) effects of activated macrophages are shown. The data were analyzed by two-way ANOVA followed by Bonferroni test. At the indicated dose of anti-CD40, the 3-agent combination of anti-CD40, CpG and MPL is compared to anti-CD40 alone, anti-CD40 and CpG, or anti-CD40 and MPL. *P<0.005, **P<0.0005. The experiment was carried out in triplicate and repeated three times.

Figure 4. Synergy among anti-CD40, CpG and MPL in Cytokine and chemokine production of PEC

PEC were collected from naïve C57BL/6 mice or mice injected with anti-CD40 (0.5mg) three days earlier. Adherent cells were then cocultured in triplicate wells with B16 cells in the absence or presence of CpG (200ng/ml), MPL (20ng/ml) or their combination for 48 hours. Supernatants were tested for cytokines and chemokines including IL-12 (A), IFN-γ (B), MCP-1 (C), TNF (D), IL-6 (E) and IL-10 (F) using the CBA method. Data are represented as mean ± SE. A three-way ANOVA followed by Dunnett's post-testing was performed. *P<0.01; **P<0.001. The experiment was carried out in triplicate and repeated two times.