A novel function of API5 (apoptosis inhibitor 5), TLR4-dependent activation of antigen presenting cells

Abstract

Dendritic cell (DC)-based vaccines are recognized as a promising immunotherapeutic strategy against cancer. Various adjuvants are often incorporated to enhance the modest immunogenicity of DC vaccines. More specifically, many of the commonly used adjuvants are derived from bacteria. In the current study, we evaluate the use of apoptosis inhibitor 5 (API5), a damage-associated molecular pattern expressed by many human cancer cells, as a novel DC vaccine adjuvant. We showed that API5 can prompt activation and maturation of DCs and activate NFkB by stimulating the Toll-like receptor signaling pathway. We also demonstrated that vaccination with API5-treated DCs pulsed with OVA, E7, or AH1-A5 peptides led to the generation of OVA, E7, or AH1-A5-specific CD8 + T cells and memory T cells, which is associated with long term tumor protection and antitumor effects in mice, against EG.7, TC-1, and CT26 tumors. Additionally, we determined that API5-mediated DC activation and immune stimulation are dependent on TLR4. Lastly, we showed that the API5 protein sequence fragment that is proximal to its leucine zipper motif is responsible for the adjuvant effects exerted by API5. Our data provide evidence that support the use of API5 as a promising adjuvant for DC-based therapies, which can be applied in combination with other cancer therapies. Most notably, our results further support the continued investigation of human-based adjuvants.

Keywords: API5; TLR4; adjuvants; cancer vaccines; dendritic cells.

Figure 1.

API5 as a DAMP that interacts with TLR4. (A) API5 expression level of various cancer cells were detected using western blot analysis. (B) To confirm that API5 protein is a DAMP, expression of API5 by cancer cells with or without doxorubicin treatment was measured using western blot analysis. (C) The production of recombinant human API5 protein via bacterial protein purification system was confirmed using CBB (coomassie brilliant blue) and western blot analysis. (D) Binding between API5 and TLR2 or TLR4 was assessed using immunoprecipitation. (E) TLR4 expressing HEK293 cells were transfected with plasmid encoding luciferase under NF-kB promoter, and treated with API5 (0.5μg/ml), GFP (0.5μg/ml) or LPS (100ng/ml), and assessed for luciferase signal. Data are presented as mean ± SD (** = p < 0.01).

Figure 2.

Recombinant human API5 protein matures and activates mouse and human APCs. (A) Wild type murine dendritic cells were treated with recombinant API5 (1μg/ml or 5μg/ml), GFP (5μg/ml) or LPS (100ng/ml). Supernatants were collected and levels of various cytokines were assessed using ELISA. (B) Murine DCs were treated with API5 (1μg/ml or 5μg/ml) protein, GFP (5μg/ml) or LPS (100ng/ml) and assessed for the expression of various maturation markers (CD40, CD80, CD86 and MHCI) using flow cytometry. (C) Murine DCs were treated with API5 (5μg/ml) protein for 0, 10, 20, 30, 40, 50 or 60 minutes, lysed, and assessed for the expression of various MAPk and IkB-α using western blot analysis. (D) Expression of migration marker CCR7 by murine DCs with or without API5 or LPS treatment were measured using flow cytometry. (E-F) Macrophage-like human THP-1 cells were treated with API5 (5μg/ml), GFP (5μg/ml) or LPS (100ng/ml). Cells and supernatants were collected and assessed for the expression of various macrophage maturation markers and secretion of various cytokines using flow cytometry and ELISA, respectively. Data are presented as mean ± SD (** = p < 0.01, *** = p < 0.001, N.S. = not significant).

Figure 3.

API5-mediated DC vaccination produces antigen-specific CD8 ± T cells and prevents tumor formation. Wild type C57BL/6 mice (5 mice per group) were immunized with 1) PBS, 2) immature DC, 3) immature DC loaded with antigen peptide, 4) API5 treated mature DC, 5) API5 treated mature DC loaded with antigen peptide, and 6) LPS treated mature DC loaded with antigen peptide, twice at one week intervals. One week following respective vaccination, the abundance of OVA (A), E7 (C) or AH1-A5 (E) specific CD8+ and IFN-γ+ T cells in splenocytes of mice were measured using flow cytometry. Kaplan-Meier survival analysis of mice(5 mice per group) challenged with TC-1 (B), EG.7 (D) or CT26 (F) tumor cells, at one week after the second DC vaccination. Data are presented as mean ± SD (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Figure 4.

Long-term antigen-specific CD8 ± T cell memory and tumor protective effect produced by API5 treated DC vaccination. Mice (5 mice per group) were vaccinated with PBS or API5 treated mature DC loaded OVA (A), E7 (C) or AH1-A5 twice at one week intervals. (E). Seven weeks after second vaccination, mice were challenged with or without EG.7, TC-1 or CT26 tumor cells, respectively. One week after tumor challenge, splenocytes of mice were collected and assessed for the abundance of antigen specific CD8+ T cells by flow cytometry. The tumor free survival of EG.7 (B), TC-1 (D) or CT26 (F) tumor challenged mice cells were followed for up to 30 days after tumor challenge. Data are presented as mean ± SD (* = p < 0.05, ** = p < 0.01).

Figure 5.

API5-treated DC vaccination reduces tumor growth and prolongs survival. Mice (5 mice per group) were challenged with EG.7 (A), TC-1 (C) or CT26 (E) tumor cells. Three and ten days after tumor challenge, mice were treated with 1) PBS, 2) immature DC, 3) immature DC loaded with antigen peptide, 4) API5 treated mature DC, 5) API5 treated mature DC loaded with antigen peptide, and 6) LPS treated mature DC loaded with antigen peptide, in the footpad twice at one week intervals. Tumor volume was measured using digital caliper. (B, D, F) Mice survival were assessed using Kaplan-Meier analysis. Data are presented as mean ± SD (* = p ≤ 0.05, ** = p ≤ 0.01).

Figure 6.

DC activating effect of API5 is TLR4 dependent. Wild type, TLR2 KO or TLR4 KO murine DCs were treated with API5 (5μg/ml) protein or LPS (100ng/ml). (A) Supernatants were collected and levels of various cytokines were assessed using ELISA. (B) Expression of various maturation markers (CD40, CD80, CD86 and MHCI) by treated cells was analyzed using flow cytometry. (C) Wild type, TLR2 KO or TLR4 KO murine DCs were treated with API5 (5μg/ml) protein for 0, 10, 30, or 60 minutes, lysed, and assessed for the expression of various MAPk and IkB-α using western blot analysis. (D) Mice (5 mice per group) were vaccinated with wild type or TLR4 KO DCs treated with API5 and pulsed with E7 peptide twice at one week intervals. One week after second vaccination, splenocytes of mice were collected and assessed for E7-specific CD8+ T cells. (E-F) Mice (5 mice per group) were challenged with TC-1 cells. Three days after tumor challenge, mice were vaccinated with wild type or TLR4 KO DCs treated with API5 and pulsed with E7 peptide twice at one week intervals. (E) Tumor growth was measured using digital caliper. (F) Mice survival were assessed using Kaplan-Meier analysis. Data are presented as mean ± SD (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).

Figure 7.

The effect of various API5 protein fragments on inducing DC activation and maturation. (A) Fragments of API5 protein were constructed (API5 full [aa1-504], API5 forward [aa1-230], API5 middle [aa108-391] and API5 end [aa320-504]). (B) Various API5 protein fragments were purified using E.coli system and confirmed using western blot analysis. (C) DCs were treated with various API5 protein fragments and assessed for secretion of TNF-α and IL-6 cytokine levels using ELISA. (D) Expression of maturation marker (CD40 and CD80) by various API5 fragment treated DCs were measured using flow cytometry. Data are presented as mean ± SD (** = p ≤ 0.01, *** = p ≤ 0.001).