Indirect recruitment of a CD40 signaling pathway in dendritic cells by B7-DC cross-linking antibody modulates T cell functions

Abstract

The human IgM B7-DC XAb protects mice from tumors in both therapeutic and prophylactic settings. Its mechanism of action is mediated by its binding to B7-DC/PD-L2 molecules on the surface of dendritic cells (DCs) to induce a multimolecular cap and subsequent activation of signaling cascades that determine a unique combination of DC phenotypes. One such phenotype, the B7-DC XAb-induced antigen accumulation in mTLR-matured DCs, has been linked to signaling through TREM-2, but the signals required for other DC phenotypes critical for the therapeutic effects in animal models remain unclear. Here, FRET and co-immunoprecipitation studies show that CD40 is recruited to the multi-molecular complex by B7-DC XAb. Signals emanating from CD40 are important, as CD40(-/-) DCs treated with B7-DC XAb (DC(XAb)) activated DAP12, but failed to activate NFkappaB, and were not protected from cell death upon cytokine withdrawal or treatment with Vitamin D(3). CD40(-/-) DC(XAb) also failed to secrete IL-6 and were unable to support the conversion of T regulatory cells into IL-17+ effector T cells in vitro. Importantly, the expression of CD40 was required for the overall ability of B7-DC XAb to induce anti-tumor CTL, to provide protection from a number of tumor types, and for DC(XAb) to be effective anti-tumor vaccines in vivo. These results indicate that B7-DC XAb modulation of DC phenotypes is through its ability to indirectly recruit common signaling molecules and elements of their endogenous signaling pathways through targeted binding to a cell-specific surface determinant.

Figure 1. CD40 is rapidly recruited into complexes containing MHC MHC class II on dendritic cells treated with B7-DC XAb.

(A) DCs were pre-stained with APC-labeled antibody against MHC class II and PE-labeled antibody against CD40 for 15 min. An aliquot of cells was analyzed by flow cytometry (0 min time point). The remaining cells were then treated with 10 µg/ml IgM control antibody (filled histograms) or B7-DC XAb (open histograms). Cells were sampled at the indicated time points and analyzed for a FRET (FL3 channel). (B) Lysates were prepared from untreated DC (0′) or DC treated with control antibody or B7-DC XAb for the indicated times and subjected to immunoprecipitation using an antibody against MHC class II. The resultant complexes were resolved by SDS-PAGE, transferred to PVDF membrane, and probed for CD40. IgH serves as a loading control. The results shown are representative of 2 experiments.

Figure 2. DCs require CD40 for B7-DC XAb mediated NFκB activation and protection from cell death.

(A) NFκB activation in wild type (WT), CD40^−/− or TREM2^−/− (KO) DC was determined after 15 min stimulation with control antibody, B7-DC XAb, anti-CD40, or LPS (TLR4 ligand). Cells were fixed, stained and imaged using a confocal microscope. Upper left panels show FITC-anti-p65 staining, upper right panels show DAPI staining of nuclei, lower panels show merged images of the upper panels. (B) Wild-type or CD40^−/− (KO) DC were treated with control antibody or B7-DC XAb for 5 min. DAP12- (left) or Syk- (middle) specific immunoprecipitates or whole cell lysate for ERK (right) were resolved and blotted using a phosphotyrosine-specific antibody. IgH or total ERK serves as a loading control. (C) Cell death was induced in wild type (filled bars) or CD40^−/− (open bars) DC by treatment with vitamin D₃. All cultures were subsequently incubated with Alamar Blue Dye. Cell viability was measured using a fluorescence plate reader after 24 hours and is expressed as relative fluorescence units (RFU). (D) Similar to (C) except cell death was induced by cytokine withdrawal. Wild type (filled bars) or CD40^−/− (open bars) DC were cultured in the presence or absence of GM-CSF and IL-4 (+ or − Cytokine) for 24 h in the presence of 10 µg/ml control antibody, B7-DC XAb, anti-MHC class II IgM antibody 25-9-3, or the combination of 1 µg/ml B7-DC XAb and 0.1 µg/ml RANKL as indicated. The results shown are representative of 2 experiments.

Figure 3. DCs require CD40 in order to secrete IL-6 and convert Tregs to Th17 cells in vitro.

(A) IL-6 was measured in culture supernatants of wild type or CD40^−/− (KO) DC stimulated for 48 h with control antibody, B7-DC XAb, anti-CD40 or TLR agonists as indicated. Statistical analysis was performed using one-way ANOVA. IL-6 secreted by WT DCs stimulated with B7-DC XAb was significantly higher than IL-6 secreted by WT DCs stimulated with control antibody; IL-6 secreted by wild type or CD40^−/− DC in response to TLR ligands was significantly higher than IL-6 induced in WT DC by B7-DC XAb (p<0.01). Error bars represent the standard deviation from the mean of triplicate cultures. (B) Enriched populations of DO11.10 non-Tregs or Tregs (>95% pure) were cultured with OVA-pulsed and control- or B7-DC XAb-treated DC isolated from wildtype, (C and E) CD40^−/− mice and (D) IL-6−/− mice. After 48 h, expression profiles for FoxP3 and IL-17A were determined by intracellular staining and flow cytometry. All experiments were conducted at least twice.

Figure 4. CD40 is required for the DC^XAb-induced generation of anti-tumor CTL responses in vivo.

Wild type or CD40^−/− mice were engrafted with B16 melanoma (A) or WEHI-3 leukemia (B) and treated intravenously with 30 µg B7-DC XAb or control antibody. On day 7, cells from draining lymph nodes were used as effectors in CTL assays. (A) CTL against ⁵¹Cr-labeled B16 tumor targets (Left) or unrelated EL-4 controls (Right). Filled squares show CTL for wild-type mice receiving B7-DC XAb. (B) CTL against ⁵¹Cr-labeled WEHI-3 tumor targets (Left) or unrelated, MHC-matched P815 controls (Right). Filled triangles show the CTL response from wild type mice receiving B7-DC XAb. Symbols and lines for all other treatment groups are nearly superimposable. The results shown are representative of 2 experiments.