Pseudomonas aeruginosa–mannose-sensitive hemagglutinin (PA-MSHA) as a drug may kill tumor cells and has been used clinically. However, the antitumor immune response of PA-MSHA is not completely understood. In this study, we found that treating Lewis lung carcinoma (3LL)-bearing mice with PA-MSHA plus 3LL antigen led to slower tumor progression and longer survival. After PA-MSHA treatment, T-cell number and dendritic cell maturation were both increased significantly at the tumor site. In addition, PA-MSHA in vitro stimulation resulted in the maturation of bone marrow–derived dendritic cells (BMDCs) from naive mice, showing higher costimulatory molecule expression, more cytokine secretion, lower endocytic activity, and stronger capacity to enhance T-cell activation. Toll-like receptor (TLR)4 but not TLR2 was required in the maturation process. More importantly, PA-MSHA–induced DCs were essential for PA-MSHA to enhance activation, expansion, and interferon (IFN)-γ secretion of TLR4-mediated T cells, which play a role in the antitumor effect of PA-MSHA. Thus, this study reveals PA-MSHA as a novel TLR4 agonist that elicits antitumor immune response to slow tumor progression.
Heat-killed Pseudomonas aeruginosa (PA) is a Gram-negative bacterium that has been combined with mannose-sensitive hemagglutination (MSHA) fimbriae (PA-MSHA) to chemically inactivate P. aeruginosa and lower its toxicity by lining it with straight, fragile MSHA fimbriae (Liu et al., 2009). PA-MSHA has been approved by the US Food and Drug Administration and used as a drug in anti-infection, antivirus, and anticancer therapies, including for lung cancer, colon cancer, breast cancer, and so forth. However, the antitumor effect of PA-MSHA is not yet fully understood. A previous study demonstrated that PA-MSHA exhibits antitumor effect mainly attributed to direct tumoricidal activity (Liu et al., 2010). However, several studies have suggested that PA-MSHA stimulates innate immune cells, such as macrophages, natural killer cells, and dendritic cells, to induce Th1-type immune responses or to modulate the immune response (Mu, 1986; Hou et al., 2012). A recent study also showed that PA-MSHA enhances antigen-presenting function by activating dendritic cells (DCs) via Toll-like receptor (TLR) signaling (Hou et al., 2012).
Toll-like receptors are initially identified for their ability to recognize many exogenous pathogens, including bacterial lipopolysaccharide (LPS), bacterial flagellum, and bacterial and viral single- and double-stranded RNA (Akira et al., 2006; Adams, 2009; Galluzzi et al., 2012). Many studies have indicated that the signaling that occurs upon recognition of pathogen-associated molecular patterns (PAMPs) by TLRs is attributable to the induction of DC maturation. DCs express a variety of TLRs through which they recognize a number of microbial compounds (Jarrossay et al., 2001; Kawasaki et al., 2001; Iwasaki and Medzhitov, 2004; Napolitani et al., 2005; Auray et al., 2010). Upon challenge with microbial stimulation, immature DCs undergo a complex process of maturation, resulting in the upregulation of costimulatory molecules and the major histocompatibility complex (MHC), which is required for T-cell priming and the induction of antigen-specific immunity (Banchereau and Steinman, 1998). Thus, TLR-mediated activation of DCs is important for the establishment of an effective anticancer immune response (Vacchelli et al., 2012). Therefore, the use of TLR agonists to mature DCs either ex vivo or in situ is an attractive method for initiating or promoting antitumor responses (Baxevanis et al., 2013).
Lung cancer is the most common cause of death from cancer, as it is responsible for approximately 13.5% of all cancer deaths (Ferlay et al., 2007; Prado-Garcia et al., 2012), and the 5-year survival rate in lung cancer is less than 15% (Jemal et al., 2009). The limited success of chemotherapy has exposed the need to adopt new innovative therapeutic strategies for the treatment of lung cancer. Treatment approaches using pathogenic bacteria or their products as systemic adjuvants, boosting the efficacy of antitumor immunotherapy, are promising.
Therefore, our study was designed to investigate whether and how PA-MSHA could induce antitumor immune response in Lewis lung carcinoma (3LL)–bearing mice. In this study, subcutaneous injection of PA-MSHA plus 3LL antigen (Ag) slowed lung cancer progression and prolonged survival against 3LL-tumor challenge. In addition, we found more T cells and mature DCs in tumors after PA-MSHA treatment. The in vitro experiments further confirmed that PA-MSHA could directly induce the maturation of DCs in a TLR4-dependent way. Moreover, with the presence of PA-MSHA–induced DCs, PA-MSHA promoted the activation, expansion, and interferon (IFN)-γ secretion of TLR4-mediated T cells, which play a role in the inhibition of tumor growth by PA-MSHA. Our results support the finding that PA-MSHA enhances host antitumor immune response to resist tumor progression.
Materials and Methods
Animals and Cell Lines.
Six to eight-week-old female C57BL/6 wild-type and nude mice were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). Six- to eight-week-old female C57BL/6 TLR4−/− and TLR2−/− mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). The mice were acclimatized in our housing facility and maintained under specific pathogen-free barrier conditions. All animal experiments were approved by the Animal Care and Use Committee of Fudan University and followed its regulations.
The 3LL cell line was kindly provided by Dr. Hong-Ming Hu (Earle A. Chiles Research Institute, Portland, OR). The 3LL cells were maintained in complete RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS; Gibco/Life Technologies, Grand Island, NY), 100 μg/ml streptomycin, and 100 U/ml penicillin at 37°C and 5% CO2. The 3LL protein Ag used in this study was 3LL cell line cultured at 37°C for 48 hours, heat-inactivated, purified by centrifugation, protein concentration examined by enzyme-linked immunosorbent assay (ELISA), and resuspended in phosphate-buffered saline (PBS; Gibco/Life Technologies).
The P. aeruginosa injection, the generic name of PA-MSHA, was provided by Beijing Wanter Bio-Pharmaceutical Co., Ltd (Beijing, China). The recombinant murine proteins granulocyte-macrophage colony–stimulating factor (GM-CSF), interleukin-4 (IL-4), and interleukin-2 (IL-2) were purchased from R&D Systems (Minneapolis, MN). Escherichia coli LPS was obtained from Sigma-Aldrich (St. Louis, MO). The following mouse antibodies were purchased from eBioscience (San Diego, CA): phycoerythrin (PE)-αCD8 (clone: 53-6.7), PE-Cy7-αCD4 (clone: BM8), APC-Cy7-αCD11c (clone: N418), APC-αCD86 (clone: GL1), PE-αCD80 (clone: 16-10A1), FITC-αTLR1 (clone: 602), FITC-αTLR2 (clone: 602), PE-αTLR4 (clone: UT41), PE-αTLR6 (clone: 14502011), FITC-αI-Ab (clone: 14502011), and PE-Cy7-αCD40L (clone: MR1).
Treatment of Tumors with PA-MSHA.
For treatment experiments, we used a skin tumor model that facilitated tumor measurement (Luo et al., 2011). Mice were injected in the right flank with 1 × 105 3LL cells per mouse on day 0. The mice were then randomly assigned to six groups (five to six mice per group) and treated subcutaneously with or without 106, 107, or 108 PA-MSHA, combined subcutaneously with or without 10 μg of 3LL protein Ag on days 3, 10, and 17 or as indicated. The size of the tumors was measured twice per week using a caliper, and the product of the perpendicular diameters was determined. The volume of the tumors was calculated (largest diameter × smallest diameter2)/2 (Cerchietti et al., 2009). The mice were sacrificed when the tumors reached 12 mm in diameter or when the tumor began to ulcerate, or when the mice became ill because of metastatic disease.
Generation of Bone Marrow-Derived Dendritic Cells.
Mouse bone marrow–derived dendritic cells (BMDCs) were obtained from wild-type TLR4−/− or TLR2−/− mice according to the following protocol. In brief, bone marrow cells were flushed from the femurs and tibias. After lysis of erythrocytes, these cells were plated in a 60-mm tissue culture dish and cultured for 90 minutes. After that, nonadherent cells were removed and discarded by gently washing with RPMI 1640 medium. These adherent cells were adjusted at a concentration of 1 × 106 cells/ml in complete RPMI 1640 medium with 20 ng/ml GM-CSF at 37°C and 5% CO2 and plated at 5 ml per well in six-well plates (Corning Life Sciences (Lowell, MA). Every other day, the medium was replaced with fresh complete RPMI 1640 medium containing 20 ng/ml GM-CSF. After 7 days, these cells were purified using anti-CD11c microBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) (Sanecka et al., 2011). These purified cells were stimulated with 10 μl/ml PBS, 107/ml PA-MSHA, or 200 ng/ml LPS for 48 hours.
BMDC Endocytic Activity.
BMDC endocytic activity was determined by the uptake of fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich) (Hou et al., 2012). After stimulation with 10 μl/ml PBS, 107/ml PA-MSHA, or 200 ng/ml LPS for 48 hours, DCs were incubated with staining buffer (PBS with 2% FBS) containing 200 μg/ml FITC-dextran at 4°C in the dark for 1 hour. After that, cells were washed with PBS with 2% FBS and analyzed by flow cytometry. The proportion of dextran + CD11c+ cells in the CD11c+ population was measured as the index of DC phagocytic function
Isolation of Cells in Tumor.
The tumors were weighed, minced into small fragments, and digested in 0.1 mg/ml DNase (Sigma-Aldrich) and 1 mg/ml collagenase IV (Sigma-Aldrich) at 37°C for 1 hour (Lai et al., 2011). The dissociated cells were then prepared for analysis of T cells by flow cytometry.
T-Cell Proliferation Assay.
T cells were isolated from the draining lymph nodes of wild-type or TLR4−/− 3LL-bearing mice by magnetic-activated cell sorting (MACS) system (Miltenyi Biotec) according to the manufacturer’s protocol. Proliferation assays consisted of the following protocol (Sanecka et al., 2011; Brennan et al., 2012). In brief, fresh T cells were labeled with 3 μM carboxyfluorescein N-succinimidyl ester (CFSE) (Invitrogen, Carlsbad, CA) for 3.5 minutes at room temperature and stopped by PBS containing 5% FBS. In some experiments, T cells were prestimulated with 10 μl/ml PBS or 107/ml PA-MSHA for 2 days before CFSE staining. CFSE-labeled T cells were cocultured with or without PBS-, PA-MSHA-, or LPS-induced DCs at the ratio of 8:1. Three days later, the proliferation was assessed by fluorescence-activated cell sorter (FACS; Beckman Coulter, Brea, CA) analysis of the CFSE dilution.
To analyze the expression of various surface markers, 1.0 × 106 cells were suspended in PBS containing 2% FBS and 0.5% sodium azide and then incubated with anti-mouse CD16/CD32 antibody to block nonspecific binding to the Fcγ receptors (Kim et al., 2003). Next, the cells were incubated with PE-αCD8, PE-Cy7-αCD4, APC-Cy7-αCD11c, APC-αCD86, PE-αCD80, FITC-αI-Ab, FITC-αTLR1, FITC-αTLR2, PE-αTLR4, PE-αTLR6, or PE-Cy7-αCD40L for 30 minutes at 4°C in the dark. After three washes, these cells were resuspended in 300 μl of PBS containing 2% FBS and analyzed by FACS. The CyAn ADP Analyzer (Beckman Coulter) was used for the data analysis.
Cell culture supernatants were collected and analyzed for IL-12p70, IL-10, tumor necrosis factor (TNF)-α, IL-6, IL-1β, or IFN-γ using a standard sandwich ELISA (eBioscience), according to user instruction manual.
All data are expressed as the mean ± S.E.M. of at least three different experiments. The statistical analysis was executed using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA). Analysis of variance (ANOVA) followed by Student t test was adopted in studies. Survival curve datasets were analyzed by the Mann–Whitney U test. A value of P < 0.05 was considered to be statistically significant.
PA-MSHA Slows Tumor Progression and Prolongs 3LL-Bearing Mice Survival in a Dose- and Frequency-Dependent Manner.
Previous studies have reported that PA-MSHA exhibits antitumor effect by the direct tumoricidal activity (Liu et al., 2009, 2010) and also induces innate immune response against HIV in an infectious disease model (Hou et al., 2012). Here, to explore whether antitumor effect of PA-MSHA is also related to the induction of immune response, its therapeutic effect on lung cancer was investigated first. C57BL/6 mice were administered 1 × 105 3LL cells subcutaneously on day 0 and then randomly divided into six groups, with five to six mice per group. On days 3, 10, and 17, these mice were treated with or without 10 μg s.c. of inactivated 3LL antigen, combined with or without different subcutaneous doses of PA-MSHA. The curative effect was determined by the tumor size, which was measured twice per week, and the survival time (Lo et al., 2003). As shown in Fig. 1, A and B, tumor grew slowly with the 108 PA-MSHA + Ag group. On day 25, the tumor size of 108 PA-MSHA + Ag group was the smallest one, and the survival time of these mice was significantly extended compared with PBS group. Lower doses of PA-MSHA + Ag were less effective, and neither Ag nor PA-MSHA alone exhibited any curative effect. The best curative effect required three injections of 108 PA-MSHA + Ag on days 3, 10, and 17. Single or double injections were all ineffective (Fig. 1, C and D). The above data indicate that the effects of PA-MSHA on delaying tumor progression and extending survival time in 3LL-bearing mice are dependent on the dose and frequency of injection.
T Cell Number and Dendritic Cell Maturation in Tumor Increased after PA-MSHA Treatment.
To investigate whether PA-MSHA influences host immune cells, especially tumor-infiltrating lymphocytes, 3LL-bearing mice were treated next with or without 10 μg s.c. of inactivated 3LL antigen, combined with PBS or different subcutaneous doses of PA-MSHA on days 3, 10, and 17 separately. Tumor tissues were removed from the treated tumor-bearing mice on day 20. To normalize for differences in tumor size at the time sacrificed, we assessed the numbers of tumor-infiltrating lymphocytes (TILs) per milligram of tumor tissue (Lai et al., 2011). Single-cell suspensions were prepared from the tumor tissues with equal amounts and analyzed for cell types by flow cytometry. As shown in Fig. 2, A and B, the cell number of CD8+ and CD4+ T cells were increased greatly in 108 PA-MSHA plus Ag group compared with other groups (Fig. 2, A and B). Furthermore, we also tested the expression level of costimulatory molecules on CD11c+ tumor-infiltrating DCs. It is noteworthy that, along with increasing doses of PA-MSHA, the expression of CD86, CD80, and I-Ab was elevated gradually (Fig. 2, C–E), showing a mature phenotype on the tumor-infiltrating DCs. In addition, similar results were also observed in the draining lymph nodes (data not shown).
PA-MSHA Induces the Maturation of Dendritic Cells.
To determine whether PA-MSHA has a direct effect on dendritic cells, we isolated bone marrow cells from naive mice and cultured them under standard conditions for 7 days to induce BMDC. After that, the cells were stimulated with PBS, PA-MSHA, or LPS for an additional 2 days to induce the maturation. Two days later, FACS results showed that, compared with PBS stimulation group, the expression levels of the costimulatory molecules CD86, CD80, and I-Ab were increased significantly in PA-MSHA stimulation group; these levels were similar to those in LPS stimulation positive control group (Fig. 3A). In addition, the culture supernatant was collected to measure the release of cytokines by BMDC under different stimulation. Compared with PBS group, PA-MSHA significantly upregulated the secretion of IL-12, TNF-α, IL-6, and IL-1β, and downregulated the IL-10 secretion slightly (Fig. 3, B and C). There also was no statistically significant difference between PA-MSHA group and LPS group (Fig. 3, B and C). The DC phagocytosis was detected next by using FITC-dextran. Similar to the effect of LPS, compared with PBS group, PA-MSHA markedly decreased the proportion of dextran + CD11c+ cells, showing that upon PA-MSHA stimulation, the ability of DCs to phagocytize dextran was decreased greatly (Fig. 3D). Moreover, T cells derived from the draining lymph nodes of 3LL tumor-bearing mice were stained with 3 μM CFSE and then cocultured with PBS-stimulated or PA-MSHA–stimulated or LPS-stimulated DCs for 72 hours, combined with mitomycin-inactivated 3LL Ag and 100 U/ml rIL-2. The proliferation and CD40L expression of T cells were detected by FACS, and IFN-γ secretion in the culture supernatant was measured by ELISA. Markedly, PA-MSHA–stimulated DCs promoted T-cell proliferation, upregulated CD40L expression of T cells, and increased IFN-γ secretion compared with PBS-stimulated DCs (Fig. 3, E–G). The similar results were also observed in the presence of LPS-stimulated DCs (Fig. 3, E–G). Our results suggest that the direct stimulation of BMDC by PA-MSHA leads to the maturation of DC not only in phenotype but also in function.
TLR4, but Not TLR2, Is Required for PA-MSHA–Induced DC Maturation.
PA-MSHA is a kind of Gram-negative bacterium preparation that belongs to PAMPs, and there are many types of pattern recognition receptor (PRR) expressed on dendritic cells, for example, TLRs (Hemmi and Akira, 2005). Thus, we examined whether PA-MSHA promotes DC maturation through PAMP-PRR interaction. 3LL tumor–bearing mice were administered 10 μg s.c. of inactivated 3LL antigen combined with three subcutaneous injections of 100 μl of PBS or 108 PA-MSHA on days 3, 10, and 17. On day 20, lymph nodes were collected, and TLR expression on DCs was analyzed by FACS. As shown in Fig. 4A, compared with the PBS + Ag group, only TLR4 expression on DCs in the PA-MSHA + Ag group was increased significantly, implying that PA-MSHA might induce DC maturation through TLR4. To further confirm the above result, BMDCs derived from wild-type TLR4−/− and TLR2−/− mice were cultured with GM-CSF for 7 days and stimulated with PBS, PA-MSHA, or LPS for an additional 2 days. The expression level of costimulatory molecules was analyzed by FACS, and the secretion of cytokines was measured by ELISA. Under PBS stimulation, there was no difference in costimulatory molecule expression and cytokine secretion among all three types of BMDCs (Fig. 4, B and C). However, under PA-MSHA or LPS stimulation, wild-type BMDCs significantly increased CD86, CD80, and I-Ab expression and IL-12p70, TNF-α, IL-6, and IL-1β secretion (Fig. 4, B and C). Similar results were also observed in TLR2−/− BMDCs, whereas no remarkable increase in costimulatory molecule expression and cytokine secretion was detected in TLR4−/− BMDCs after PA-MSHA or LPS stimulation (Fig. 4, B and C). The dramatically decreased phagocytic function was found in both wild-type BMDCs and TLR2−/− BMDCs after PA-MSHA stimulation, whereas the reduction in phagocytic function disappeared in TLR4−/− BMDCs (Fig. 4D). Likewise, the lack of TLR4 on DCs impaired the ability of PA-MSHA–induced DCs to promote the proliferation, CD40L expression, and IFN-γ secretion of T cells, although no visible impairments were detected in the lack of TLR2 group compared with wild-type group (Fig. 4, E–G). These data further demonstrate that the TLR4 pathway plays a vital role in PA-MSHA–induced DC maturation.
PA-MSHA–Induced DCs Are Required in PA-MSHA–Promoted T-Cell Response in a TLR4-Dependent Way.
Our previous study and other reports have identified that TLRs are expressed not only on innate immune cells but also on adoptive immune cells (Medzhitov et al., 1997; Gelman et al., 2004; Zhang et al., 2011). Also, the frequency and cell number of CD4+ and CD8+ T cells were upregulated significantly after PA-MSHA treatment (Fig. 2, A and B). Hence, we further evaluated whether PA-MSHA directly activates T cells to induce antitumor immune response. First of all, T cells were isolated from the draining lymph nodes of 3LL tumor–bearing mice and stained with CFSE. After that, the T cells were stimulated with PBS or PA-MSHA for 48 hours and then cocultured with or without PA-MSHA–induced DCs for another 72 hours, combined with mitomycin-inactivated 3LL Ag and 100 U/ml rIL-2. The proliferation of T cells was measured by FACS. It is noteworthy that, without PA-MSHA–induced DCs, there was no difference in T-cell proliferation, no matter whether or not T cells were prestimulated with PA-MSHA. However, with PA-MSHA–induced DCs, the proliferation of PA-MSHA–stimulated T cells was much more enhanced than that of PBS-stimulated T cells (Fig. 5A), indicating that the presence of PA-MSHA–induced DCs is required for the direct effect of PA-MSHA on T cells. Next, T cells were isolated from not only wild-type but also TLR4−/− tumor-bearing mice. After staining with CFSE, T cells were stimulated with PA-MSHA for 48 hours and then cocultured with PA-MSHA–induced DCs for another 72 hours, combined with mitomycin-inactivated 3LL Ag and 100 U/ml rIL-2. As expected, even in the presence of PA-MSHA–induced DCs, TLR4 deficiency on T cells blocked the proliferation, CD40L expression, and IFN-γ secretion of T cells (Fig. 5, B–D). To further confirm the involvement of T cells in PA-MSHA–mediated tumor inhibition, nude mice, which lack T cells, were also inoculated with 3LL tumor cells subcutaneously. 3LL-bearing nude mice or wild-type mice were treated with 10 μg s.c. of inactivated 3LL antigen, combined with subcutaneous PBS or PA-MSHA on days 3, 10, 17. The curative effect of PA-MSHA was partially impaired in nude mice (Fig. 5E), which indicates T cells indeed play a role in PA-MSHA–mediated tumor inhibition. We then treated 3LL tumor-bearing TLR4−/− or wild-type mice with 10 μg s.c. of inactivated 3LL antigen, combined with subcutaneous PBS or PA-MSHA on days 3, 10, 17. As shown in Fig. 5F, PA-MSHA–mediated tumor inhibition was impaired in TLR4−/− mice compared with that in wild-type mice, indicating that TLR4 is critical in PA-MSHA–mediating antitumor immunity. Taken together, antitumor effect of PA-MSHA is partially attributable to T cell–mediated antitumor immunity, which might result from the effect of PA-MSHA on T cells in a TLR4-dependent way.
In the current study, we report that PA-MSHA not only has direct tumoricidal activity but also elicits an antitumor immune response, resulting in slower tumor growth and longer survival of the tumor-bearing host. To our knowledge, this is the first time that PA-MSHA has been shown to induce DC maturation in a TLR4-dependent manner to promote the activation and expansion of T cells and, further, to modulate the proliferation of T cells via TLR4 directly, which might lead to the effective inhibition of tumor growth. Previous studies mainly focused on the antitumor activities of PA-MSHA, such as the inhibition of the tumor cell cycle, the suppression of tumor cell proliferation, and the activation of apoptosis-associated pathways (Liu et al., 2010). Although several articles have described PA-MSHA as an adjuvant that could activate monocytes, macrophages, natural killer cells, and antigen-presenting cells to trigger innate immune response and even Th1-type immune response (Hou et al., 2012), none has ascribed PA-MSHA–mediated tumor inhibition to the immune cells or the immune response.
We note that 108 PA-MSHA therapy significantly slowed tumor growth and prolonged the survival of 3LL tumor–bearing mice (Fig. 1, A–B), and lower doses (107 or 106) show less or no efficacy. In addition, 108 PA-MSHA induces the elevated T-cell count and the maturation of DCs in tumor (Fig. 2). Previously, another study showed that more than 108 PA-MSHA could directly inhibit the proliferation, as well as induce apoptosis and G0–G1 cell-cycle arrest of tumor cells (Liu et al., 2010). These data indicate that 108 PA-MSHA is probably an ideal dose for the successful antitumor therapy using PA-MSHA, which elicits not only an induction of tumor cell apoptosis but also an elevation of antitumor immunity. Contradictorily, a recent study found that in an HIV-infected model, 108 PA-MSHA induced immunosuppressive responses, whereas only 102–104 PA-MSHA induced beneficial immune response (Hou et al., 2012). These contradictory results might be attributable to different models, which suggest that it is essential to carefully choose a proper dose for the application of PA-MSHA in different diseases.
PA-MSHA characterized by the expression of MSHA fimbriae is a Gram-negative bacterium preparation, which is the main source of TLR4 agonists (Ashkar et al., 2008; Mossman et al., 2008; Mian et al., 2010; Abdul-Careem et al., 2011). In addition, only TLR4 expression on DCs was increased after PA-MSHA treatment (Fig. 4A). Accordingly, TLR4 deficiency but not TLR2 deficiency impaired the effect of PA-MSHA on DCs (Fig. 4, B–G). These results all strongly suggest that PA-MSHA is a novel TLR4 agonist. More interestingly, recent studies, including ours, have reported that TLRs express not only on the innate immune cells but also on the adoptive immune cells, such as T cells (Zarember and Godowski, 2002; Gelman et al., 2004; Komai-Koma et al., 2004; Caron et al., 2005; Cottalorda et al., 2006; Zhang et al., 2011). Although the expression levels of TLRs on T cells are much lower than those on DC, macrophages, and B cells, the direct TLR signaling in T cells plays a key role in T-cell activation, proliferation, survival, and even cytotoxicity (Bendigs et al., 1999; Caron et al., 2005; Cottalorda et al., 2006; Gelman et al., 2006; Quigley et al., 2009). Therefore, we also investigated the direct effect of PA-MSHA on T cells. Unexpectedly, no prominent enhancement of T-cell proliferation was detected after PA-MSHA stimulation directly (Fig. 5A). However, when PA-MSHA–induced DCs were cocultured, the direct stimulation of PA-MSHA greatly enhanced T-cell activation and expansion and IFN-γ secretion in a TLR4-dependent manner (Fig. 5, A–D), implying that priming of T cells by DCs is required and PA-MSHA stimulation might provide the costimulatory signaling in T-cell activation.
Although the curative effect of PA-MSHA was impaired without T cells in tumor-bearing hosts, the antitumor effect of PA-MSHA still could be observed, with statistical significance, when T cells were deficient (Fig. 5E). Therefore, besides the direct tumoricidal activity, our results could not yet exclude other immune cells, such as NK cells or B cells, both of which have been reported to play a role in antitumor immunity (Li et al., 2011; Stojanovic and Cerwenka, 2011). Considering the high expression of TLR4 on NK cells and B cells, PA-MSHA is probably involved in the modulation of them, but further investigation is needed.
In summary, our results demonstrate that PA-MSHA, as a novel TLR4 agonist, can induce dendritic cell maturation to give T-cell priming for the activation, expansion, and IFN-γ secretion of T cells, and to boost T-cell proliferation directly. As a result, tumor growth is inhibited and mice survival is prolonged. Our findings offer a new perspective on the antitumor effect of PA-MSHA that could lead to a novel therapeutic strategy utilizing PA-MSHA.
Participated in research design: M. Zhang, Luo, Chu.
Conducted experiments: M. Zhang, Luo, Y. Zhang, Wang, Lin, Yang.
Contributed new reagents or analytic tools: Hu, Wu.
Performed data analysis: M. Zhang, Luo, Chu.
Wrote or contributed to the writing of the manuscript: M. Zhang, Luo, Chu.
- Received December 11, 2013.
- Accepted March 10, 2014.
M.Z. and F.L. contributed equally to this work and share authorship as first co-authors.
This work was supported by National Science Foundation of China [Grant 81072408, 91229110]; and the Science and Technology Commission of Shanghai Municipality [13JC1407700] in China.
- bone marrow–derived dendritic cell
- carboxyfluorescein N-succinimidyl ester
- dendritic cell
- enzyme-linked immunosorbent assay
- fetal bovine serum
- granulocyte-macrophage colony–stimulating factor
- bacterial lipopolysaccharide
- Pseudomonas aeruginosa mannose-sensitive hemagglutinin
- pathogen-associated molecular pattern
- phosphate-buffered saline
- Toll-like receptor
- tumor necrosis factor-α
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics