Dendritic cell-activating vaccine adjuvants differ in the ability to elicit antitumor immunity due to an adjuvant-specific induction of immunosuppressive cells

Abstract

Purpose: We questioned whether the vaccine adjuvant combination of TLR-7 ligand agonist, imiquimod, with granulocyte macrophage colony-stimulating factor (GM-CSF) would result in enhanced dendritic cell recruitment and activation with increased antigen-specific immunity as compared with either adjuvant used alone.

Experimental design: The adjuvant effects of GM-CSF and imiquimod were studied in ovalbumin (OVA) and MMTVneu transgenic mice using peptide-based vaccines. Type I immunity, serum cytokines, myeloid-derived suppressive cells (MDSC), and regulatory T cells (Treg) levels were examined.

Results: Both GM-CSF and imiquimod equally induced local accumulation and activation of dendritic cells. Both adjuvants effectively enhanced OVA-specific T-cell responses. We further evaluated the antitumor efficacy of adjuvant GM-CSF and imiquimod immunizing against murine insulin-like growth factor-binding protein-2 (IGFBP-2), a nonmutated oncoprotein overexpressed in the tumors of MMTVneu transgenic mice. Tumor growth was significantly inhibited in the mice receiving IGFBP-2 peptides with GM-CSF (P = 0.000), but not in imiquimod vaccine-treated groups (P = 0.141). Moreover, the addition of imiquimod to GM-CSF negated the antitumor activity of the vaccine when GM-CSF was used as the sole adjuvant. While GM-CSF stimulated significant levels of antigen-specific T-helper cell (T(H))1, imiquimod induced elevated serum interleukin (IL)-10. Both MDSC and Tregs were increased in the imiquimod-treated but not GM-CSF-treated groups (P = 0.000 and 0.006, respectively). Depleting MDSC and Treg in animals immunized with imiquimod and IGFBP-2 peptides restored antitumor activity to the levels observed with vaccination using GM-CSF as the sole adjuvant.

Conclusion: Adjuvants may induce regulatory responses in the context of a self-antigen vaccine. Adjuvant triggered immunosuppression may limit vaccine efficacy and should be evaluated in preclinical models especially when contemplating combination approaches.

Figures

Figure 1. GM-CSF and imiquimod, as vaccine adjuvants, equally induce the mobilization and maturation of dendritic cells

(A) Total number of CD11c+ DC per vaccine DLN (y-axis) per adjuvant group (x-axis). Bars represent the mean (± SE), n=5/group. (B) Total number of CD11c+ CD86+ mature DC per vaccine DLN (y-axis) per adjuvant group (x-axis). n=5/group. * = p<0.05 and ** = p<0.005 compared to control. ns=not significant. (C) Representative dot plots of CD11c+ (X-axis) CD86+ (y-axis) DC in DLN of mice treated with PBS, GM-CSF, and imiquimod as adjuvants respectively.

Figure 2. Both GM-CSF and imiquimod are effective adjuvants in stimulating antigen specific T cell immunity after OVA peptide-based immunization

(A) Number (×105) of OVA p323 specific (DO11.10 TCR+) CD4+ T cells in DLN (y-axis) per experimental group (x-axis). Bars represent the mean (± SE), n=9/group; (B) Percentage of OVA p323 specific DO11.10 CD4+ T cells of all splenic T cells (y axis) per experimental group (x-axis), n=9/group; (C) Number (×105) of OVA p257 specific (OVA p257 H2-Kb tetramer+) CD8+ T cells in DLN (y-axis) per experimental group (x-axis). Bars represent the mean (± SE), n=7/group. (D) Percentage of OVA p257 specific OT-1 CD8+ T cells of all splenic T cells (y axis) per experimental group (x-axis), n=7. Representative Flow histograms of CFSE labeled CD4+ DO11.10 cells (E) and CD8+ OT-1 cells (F); PBS, GM-CSF, and imiquimod used with OVA p323 (E) or OVA p257 vaccination (F). * indicates p<0.05 and ** indicates p<0.005 vs. PBS control. Results were reproduced in two independent experiments.

Figure 3. GM-CSF and imiquimod differ in the ability to stimulate anti-tumor immunity when administered with an IGFBP-2 peptide vaccine

(A) Growth of MMC tumor (mm3) (y-axis) in mice immunized with PBS control (●), GM-CSF (▲), and IGFBP-2 peptide vaccine with PBS (V) (▼) or GM-CSF (■), (x-axis). Data is shown as the mean ± SE, n=5/group. (B) Growth of MMC tumor (mm3) (y-axis) in mice immunized with PBS control (●), imiquimod (▲), and IGFBP-2 peptide vaccine with PBS (▼) or imiquimod (■), (x-axis). Data represent mean ± SE, n=5/group. The experiments were repeated on two independent occasions with similar results. (C) IGFBP-2 specific IFN-gamma secretion (y-axis); bars represent PBS alone or IGFBP-2 peptides in PBS, GM-CSF and imiquimod (x-axis). Shown are the mean (± SE) of spots/2×105 cells, n=5/group. (D) Columns represent the mean (± SE) of IL-10 (pg/ml) (y-axis) from PBS alone or IGFBP-2 peptides in PBS, GM-CSF and imiquimod (x-axis), n=12/group. In all above graphs, * indicates p<0.05 and ** indicates p<0.005 in comparison with IGFBP-2 peptides with PBS group. (E) Growth of MMC tumor (mm3) (y-axis) in mice immunized with PBS control (●), and IGFBP-2 peptide vaccine with PBS (▼) GM-CSF (■), imiquimod (▲), and GM-CSF and Imiquimod (◆) (x-axis). Data represent mean ± SE, n=3/group.

Figure 4. Imiquimod, as a vaccine adjuvant, increases systemic levels of MDSC and Treg cells in IGFBP-2 immunized mice

(A) % of CD11b+Gr-1+ MDSC on spleen (y-axis) and (B) % of Foxp3+ cells in splenic CD4+ T cells (y-axis). Experimental groups on x-axis; Control: PBS alone; PBS+peptides, GM-CSF+peptides, imiquimod+peptides and GM-CSF+imiquimod+peptides, n=5/group. Bars represent the mean. * indicate p<0.05 and ** indicates p<0.005 vs. controls. Representative dot plots of MDSC (C) and Treg cells (D) from the different experimental groups.

Figure 5. Selective depletion of Treg or MDSC during vaccination with imiquimod as an adjuvant restored the anti-tumor effect of IGFBP-2 immunization

(A) Schema of IGFBP2 immunization and the administration of anti-CD25 and anti-Gr-1 mAb. (B) Growth of MMC tumor (mm3) (y-axis) in mice immunized with imiquimod (○), imiquimod+CD25 Ab (▽), and IGFBP-2 peptide vaccine with PBS (V) (■), imiquimod (▲), imiquimod+ CD25 Ab (▼), and GM-CSF (◆) (x-axis). (C) Growth of MMC tumor (mm3) (y-axis) in mice immunized with imiquimod (○), imiquimod+ Gr-1 Ab (▽), and IGFBP-2 peptide vaccine with PBS (■), imiquimod (▲), imiquimod+Gr-1 Ab (▼), and GM-CSF (◆) (x-axis). (D) The % of tumor growth (y-axis): bars represent controls and the experimental groups. Data are shown as the mean ± SE, n=5/group. * indicate p<0.05 and ** indicates p<0.005 vs. controls.