Listeria monocytogenes-derived listeriolysin O has pathogen-associated molecular pattern-like properties independent of its hemolytic ability

Abstract

There is a constant need for improved adjuvants to augment the induction of immune responses against tumor-associated antigens (TAA) during immunotherapy. Previous studies have established that listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, exhibits multifaceted effects to boost the stimulation of immune responses to a variety of antigens. However, the direct ability of LLO as an adjuvant and whether it acts as a pathogen-associated molecular pattern (PAMP) have not been demonstrated. In this paper, we show that a detoxified, nonhemolytic form of LLO (dtLLO) is an effective adjuvant in tumor immunotherapy and may activate innate and cellular immune responses by acting as a PAMP. Our investigation of the adjuvant activity demonstrates that dtLLO, either fused to or administered as a mixture with a human papillomavirus type 16 (HPV-16) E7 recombinant protein, can augment antitumor immune responses and facilitate tumor eradication. Further mechanistic studies using bone marrow-derived dendritic cells suggest that dtLLO acts as a PAMP by stimulating production of proinflammatory cytokines and inducing maturation of antigen-presenting cells (APC). We propose that dtLLO is an effective adjuvant for tumor immunotherapy, and likely for other therapeutic settings.

Fig 1

Construction, purification, and characterization of a detoxified form of listeriolysin O (dtLLO). (A) dtLLO protein, HPV-16 E7 protein, and a genetic fusion protein of dtLLO and E7 were extracted from BL21(DE3) transformants after IPTG (isopropyl-β-d-thiogalactopyranoside) induction and then purified using a Ni²⁺ column. Each purified protein was then subjected to 4 to 12% SDS-PAGE analysis, and the gel was stained with Coomassie blue. Each protein is visualized in its respective labeled lane. (B) Western blot analysis to detect LPS contamination after endotoxin removal in protein preparations from BL21(DE3) transformants. Briefly, endotoxin was removed from each purified protein preparation by use of Norgen Proteospin endotoxin removal columns, and subsequently, proteins or LPS (20 ng; positive control) was subjected to 4 to 12% SDS-PAGE analysis. After transfer to a polyvinylidene difluoride (PVDF) membrane, Western blot analysis with anti-LPS antibody was performed to demonstrate removal of endotoxin from purified protein preparations. (C) Detoxification of LLO by mutation of the cholesterol binding domain results in reduced hemolytic activity. Hemolytic activity of dtLLO was determined using an SRBC lysis assay. Briefly, LLO WT and dtLLO protein preparations were initially activated by incubation at an acidic pH (1× PBS-cysteine buffer, pH 5.4) and subsequently were incubated with sheep red blood cells. The number of hemolytic units was determined by measuring released hemoglobin (optical density at 570 nm) with each treatment.

Fig 2

dtLLO augments antitumor efficacy of E7 protein vaccination. (A) The ability of dtLLO alone to act as an adjuvant in tumor immunotherapy was determined using the TC-1 tumor model. Mice (8/group) were implanted subcutaneously with 10⁵ TC-1 tumor cells and subsequently vaccinated with protein vaccines (dtLLO, E7, dtLLO-E7, and dtLLO plus E7). Tumor loads are depicted as the tumor volume for each treatment group throughout the course of the experiment (P = 0.002 for the dtLLO and dtLLO-plus-E7 groups compared to the E7 group, using the nonparametric Wilcoxon signed-rank test). (B) Representative tumor load study depicting the percentage of TC-1 tumor-free mice in each treatment group throughout the course of the experiment. (C) Mean percentage of tumor-free mice around day 54 for each treatment group at the end of three separate tumor load studies.

Fig 3

dtLLO augments tumor-specific adaptive immune responses. (A) Augmentation of E7-specific T cell responses was measured in the spleen by intracellular IFN-γ staining. Splenocytes were harvested from mice in each treatment group from the tumor load study depicted in Fig. 2 and stimulated with IL-2 alone or IL-2 along with an E7-specific CTL epitope peptide (RAHYNIVTF). Splenocytes were then processed for flow cytometric analysis of intracellular production of IFN-γ. Each dot plot depicts activated E7-specific CD8⁺ T cells from a different treatment group as the percentage of total activated CD8⁺ T cells in the spleen. (B) Increased infiltration of E7-specific CD8⁺ TILs into TC-1 tumors after dtLLO protein vaccination. Tumor-infiltrating lymphocytes were isolated from TC-1 tumors at the end of the experiment, pooled within the treatment group, and stained with a tetramer that recognizes E7-specific CTLs. (Bottom row) Tumor-infiltrating E7-specific CTLs are depicted as the percentage of total tumor-infiltrating activated CD8⁺ CD62L^low CD11b⁻ E7 tetramer⁺ cells for each treatment group. (Top row) E7-specific CTLs in the spleens of mice from each treatment group.

Fig 4

dtLLO treatment of BMDCs stimulates induction of mRNAs for proinflammatory cytokines. (A) dtLLO treatment stimulates production of TNF-α mRNA. dtLLO was purified from BL21(DE3) IpxM, followed by endotoxin removal. BMDCs were stimulated for 16 h with LPS, LPS pretreated with a specific inhibitor of LPS signaling (polymyxin B [PMXB]), dtLLO, dtLLO pretreated with PMXB, or PMXB alone. After stimulation, cells were processed for RNA isolation, cDNA conversion, and qPCR analysis with TNF-α primers. TNF-α mRNA production after stimulation is depicted in relation to TNF-α mRNA levels in the untreated BMDC controls. (B and C) TNF-α expression by BMDCs with dtLLO stimulation is dependent on native, intact dtLLO. To determine if the dtLLO protein was responsible for the signaling observed in panel A, the dtLLO protein was either heat denatured by boiling for 10 min (dtLLO-Heat) or treated with proteinase K for 60 min (dtLLO-PK). BMDCs were subsequently stimulated with untreated dtLLO, dtLLO-Heat, dtLLO-PK, LPS alone, LPS-Heat, or LPS-PK, followed by TNF-α (B) and IL-12 (C) qPCR analyses. (D and E) Signaling by dtLLO is independent of TLR4. BMDCs were isolated from tlr4^−/− mice and stimulated with dtLLO, dtLLO-Heat, or dtLLO-PK, in addition to poly(I-C) as a positive control and LPS as a negative control. After stimulation for 2 h, cells were processed for qPCR analysis of TNF-α (D) and IL-12 (E) mRNA expression. **, P < 0.01; ***, P < 0.001.

Fig 5

dtLLO treatment facilitates expression of mRNAs for markers associated with BMDC maturation. (A and B) dtLLO treatment induces expression of costimulatory molecules. BMDCs were stimulated with dtLLO, dtLLO-Heat, dtLLO-PK, LPS, LPS-Heat, or LPS-PK, followed by CD40 (A) and MHC II (B) qPCR analyses. (C and D) Costimulatory molecule expression by dtLLO is independent of TLR4. BMDCs were isolated from tlr4^−/− mice and stimulated with dtLLO, dtLLO-Heat, or dtLLO-PK, in addition to poly(I-C) as a positive control and LPS as a negative control. After stimulation for 2 h, cells were processed for qPCR analysis of CD40 (C) and MHC II (D) mRNA expression. **, P < 0.01; ***, P < 0.001.