Survival of the fittest: how myeloid-derived suppressor cells survive in the inhospitable tumor microenvironment.

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they are significant contributors to the immune suppressive tumor microenvironment (TME). The TME is a hostile locale due to deficiencies in oxygen (hypoxia) and nutrients, and the presence of reactive oxygen species (ROS). The survival of tumor cells within the TME is partially governed by two mechanisms: (1) Activation of the transcription factor Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) which turns on genes that attenuate oxidative stress; and (2) The presence of High Mobility Group Box Protein-1 (HMGB1), a damage-associated molecular pattern molecule (DAMP) that induces autophagy and protects against apoptosis. Because Nrf2 and HMGB1 promote tumor cell survival, we speculated that Nrf2 and HMGB1 may facilitate MDSC survival. We tested this hypothesis using Nrf2+/+ and Nrf2-/- BALB/c and C57BL/6 mice and pharmacological inhibitors of HMGB1. In vitro and in vivo studies demonstrated that Nrf2 increased the suppressive potency and quantity of tumor-infiltrating MDSC by up-regulating MDSC production of H2O2 and decreasing MDSC apoptosis. Decreased apoptosis was accompanied by a decrease in the production of MDSC, demonstrating that MDSC levels are homeostatically regulated. Pharmacological inhibition of autophagy increased MDSC apoptosis, indicating that autophagy increases MDSC half-life. Inhibition of HMGB1 also increased MDSC apoptosis and reduced MDSC autophagy. These results combined with our previous findings that HMGB1 drives the accumulation of MDSC demonstrate that HMGB1 maintains MDSC viability by inducing autophagy. Collectively, these findings identify Nrf2 and HMGB1 as important factors that enable MDSC to survive in the TME.

HPV-specific antibodies at the oral cavity up to 30 months after the start of vaccination with the quadrivalent HPV vaccine among mid-adult aged men.

BACKGROUND: HPV-16 and HPV-18 cause most oropharyngeal cancers, which are increasing in incidence among males. Although HPV vaccines are highly effective against a number of HPV-associated cancers, efficacy for oropharyngeal cancers has not yet been demonstrated. In addition, the level of antibodies required for protection against oral HPV infection is unknown. METHODS: 150 men ages 27-45 years from Tampa, FL, USA, and Cuernavaca, Mexico, received Gardasil at Day 1, Months 2, and 6. Then, sera and oral gargles were collected one month, 12 months, and 24 months after completion of the three doses (Month 7, 18 and 30 of the study) and tested for anti-HPV-16 and HPV-18 IgG antibody levels by a L1 VLP ELISA. RESULTS: All participants developed detectable serum anti-HPV-16 and anti-HPV-18 antibodies and most had detectable antibodies in oral gargles at Month 7 (HPV-16: 93.2%; HPV-18: 72.1%). By months 18 and 30, oral antibodies were detectable in a lower number of participants (HPV-16, 39.8% and 29.6%; HPV-18, 10.7% and 4.6% of individuals, respectively). Overall, oral HPV-16- and 18-specific antibody levels, normalized to total IgG at months 7, 18, and 30, correlated with serum levels (HPV-16, R2 = 0.93; HPV-18, R2 = 0.91). CONCLUSIONS: Reduced detectability of oral and serum HPV-16 and HPV-18 antibodies was observed at months 18 and 30 after initiation of the quadrivalent vaccination. However, when detectable, serum and oral HPV-16 and HPV-18 antibody levels were strongly correlated.

Evaluation of HPV-16 and HPV-18 specific antibody measurements in saliva collected in oral rinses and merocel® sponges.

BACKGROUND: Current Human papillomavirus (HPV) L1 VLP vaccines protect against HPV-16 and HPV-18-associated cancers, in females and males. Although correlates of protection have not been identified, HPV-specific antibodies at sites of infection are thought to be the main mechanism of protection afforded by vaccination. Oral sampling has gained increased attention as a potential alternative to serum in monitoring immunity to vaccination and understanding local immunity in oral cancers. METHODS: Serum was collected via venipuncture, and saliva was collected via oral rinses and Merocel® sponges from healthy volunteers: 16 unvaccinated females, 6 females (ages 24-41) and 6 mid-adult aged males (ages 27-45) recipients of three doses of the HPV-16/18/6/11 vaccine (Gardasil®). Mid-adult male vaccine trial participants were compared to female participants. Samples were tested for anti-HPV-16 and anti-HPV-18 immunoglobulin G levels by an L1 virus-like particle-based enzyme-linked immunosorbent assay (ELISA). RESULTS: All vaccinated participants had detectable serum anti-HPV-16 and anti-HPV-18 antibodies. Optimal standard concentration range and sample serial dilutions for oral rinses were determined. The standard curve was not affected by the type of solution examined. Reproducibility of HPV-16 and HPV-18 antibody titers in mouthwash (overall CV < 10%) or in Merocel® extraction buffer was robust (CV < 13%). Excellent assay linearity (R2 > 0.9) was observed for sera spiked controls in both solutions. HPV-16 and HPV-18 specific antibodies were detectable in saliva from vaccine recipients, both in mouthwash and in Merocel® sponges but levels were several logs lower than those in serum. CONCLUSIONS: This study confirms the application of HPV-16 and HPV-18 ELISAs currently used in sero-epidemiological studies of immunogenicity of HPV vaccines for use with oral samples. Oral samples may be a useful resource for the detection of HPV-16 and HPV-18-specific antibodies in saliva following vaccination.

High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy.

Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by driving them into a proautophagic state.

Myeloid-Derived Suppressor Cells: Critical Cells Driving Immune Suppression in the Tumor Microenvironment.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress innate and adaptive immunity. MDSCs are present in many disease settings; however, in cancer, they are a major obstacle for both natural antitumor immunity and immunotherapy. Tumor and host cells in the tumor microenvironment (TME) produce a myriad of pro-inflammatory mediators that activate MDSCs and drive their accumulation and suppressive activity. MDSCs utilize a variety of mechanisms to suppress T cell activation, induce other immune-suppressive cell populations, regulate inflammation in the TME, and promote the switching of the immune system to one that tolerates and enhances tumor growth. Because MDSCs are present in most cancer patients and are potent immune-suppressive cells, MDSCs have been the focus of intense research in recent years. This review describes the history and identification of MDSCs, the role of inflammation and intracellular signaling events governing MDSC accumulation and suppressive activity, immune-suppressive mechanisms utilized by MDSCs, and recent therapeutics that target MDSCs to enhance antitumor immunity.

Cross-talk among myeloid-derived suppressor cells, macrophages, and tumor cells impacts the inflammatory milieu of solid tumors.

MDSC and macrophages are present in most solid tumors and are important drivers of immune suppression and inflammation. It is established that cross-talk between MDSC and macrophages impacts anti-tumor immunity; however, interactions between tumor cells and MDSC or macrophages are less well studied. To examine potential interactions between these cells, we studied the impact of MDSC, macrophages, and four murine tumor cell lines on each other, both in vitro and in vivo. We focused on IL-6, IL-10, IL-12, TNF-α, and NO, as these molecules are produced by macrophages, MDSC, and many tumor cells; are present in most solid tumors; and regulate inflammation. In vitro studies demonstrated that MDSC-produced IL-10 decreased macrophage IL-6 and TNF-α and increased NO. IL-6 indirectly regulated MDSC IL-10. Tumor cells increased MDSC IL-6 and vice versa. Tumor cells also increased macrophage IL-6 and NO and decreased macrophage TNF-α. Tumor cell-driven macrophage IL-6 was reduced by MDSC, and tumor cells and MDSC enhanced macrophage NO. In vivo analysis of solid tumors identified IL-6 and IL-10 as the dominant cytokines and demonstrated that these molecules were produced predominantly by stromal cells. These results suggest that inflammation within solid tumors is regulated by the ratio of tumor cells to MDSC and macrophages and that interactions of these cells have the potential to alter significantly the inflammatory milieu within the tumor microenvironment.

HMGB1 enhances immune suppression by facilitating the differentiation and suppressive activity of myeloid-derived suppressor cells.

Chronic inflammation often precedes malignant transformation and later drives tumor progression. Likewise, subversion of the immune system plays a role in tumor progression, with tumoral immune escape now well recognized as a crucial hallmark of cancer. Myeloid-derived suppressor cells (MDSC) are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. Thus, MDSCs may define an element of the pathogenic inflammatory processes that drives immune escape. The secreted alarmin HMGB1 is a proinflammatory partner, inducer, and chaperone for many proinflammatory molecules that MDSCs develop. Therefore, in this study, we examined HMGB1 as a potential regulator of MDSCs. In murine tumor systems, HMGB1 was ubiquitous in the tumor microenvironment, activating the NF-κB signal transduction pathway in MDSCs and regulating their quantity and quality. We found that HMGB1 promotes the development of MDSCs from bone marrow progenitor cells, contributing to their ability to suppress antigen-driven activation of CD4(+) and CD8(+) T cells. Furthermore, HMGB1 increased MDSC-mediated production of IL-10, enhanced crosstalk between MDSCs and macrophages, and facilitated the ability of MDSCs to downregulate expression of the T-cell homing receptor L-selectin. Overall, our results revealed a pivotal role for HMGB1 in the development and cancerous contributions of MDSCs.

IDO is a nodal pathogenic driver of lung cancer and metastasis development.

UNLABELLED: Indoleamine 2,3-dioxygenase (IDO) enzyme inhibitors have entered clinical trials for cancer treatment based on preclinical studies, indicating that they can defeat immune escape and broadly enhance other therapeutic modalities. However, clear genetic evidence of the impact of IDO on tumorigenesis in physiologic models of primary or metastatic disease is lacking. Investigating the impact of Ido1 gene disruption in mouse models of oncogenic KRAS-induced lung carcinoma and breast carcinoma-derived pulmonary metastasis, we have found that IDO deficiency resulted in reduced lung tumor burden and improved survival in both models. Micro-computed tomographic (CT) imaging further revealed that the density of the underlying pulmonary blood vessels was significantly reduced in Ido1-nullizygous mice. During lung tumor and metastasis outgrowth, interleukin (IL)-6 induction was greatly attenuated in conjunction with the loss of IDO. Biologically, this resulted in a consequential impairment of protumorigenic myeloid-derived suppressor cells (MDSC), as restoration of IL-6 recovered both MDSC suppressor function and metastasis susceptibility in Ido1-nullizygous mice. Together, our findings define IDO as a prototypical integrative modifier that bridges inflammation, vascularization, and immune escape to license primary and metastatic tumor outgrowth. SIGNIFICANCE: This study provides preclinical, genetic proof-of-concept that the immunoregulatory enzyme IDO contributes to autochthonous carcinoma progression and to the creation of a metastatic niche. IDO deficiency in vivo negatively impacted both vascularization and IL-6­dependent, MDSC-driven immune escape, establishing IDO as an overarching factor directing the establishment of a protumorigenic environment.

Tumor-induced myeloid-derived suppressor cell function is independent of IFN-γ and IL-4Rα.

Myeloid-derived suppressor cells (MDSCs) are present in most cancer patients and experimental animals where they exert a profound immune suppression and are a significant obstacle to immunotherapy. IFN-γ and IL-4 receptor alpha (IL-4Rα) have been implicated as essential molecules for MDSC development and immunosuppressive function. If IFN-γ and IL-4Rα are critical regulators of MDSCs, then they are potential targets for preventing MDSC accumulation or inhibiting MDSC function. Because data supporting a role for IFN-γ and IL-4Rα are not definitive, we have examined MDSCs induced in IFN-γ-deficient, IFN-γR-deficient, and IL-4Rα-deficient mice carrying three C57BL/6-derived (B16 melanoma, MC38 colon carcinoma, and 3LL lung adenocarcinoma), and three BALB/c-derived (4T1 and TS/A mammary carcinomas, and CT26 colon carcinoma) tumors. We report that although MDSCs express functional IFN-γR and IL-4Rα, and have the potential to signal through the STAT1 and STAT6 pathways, respectively, neither IFN-γ nor IL-4Rα impacts the phenotype, accumulation, or T-cell suppressive potency of MDSCs, although IFN-γ and IL-4Rα modestly alter MDSC-macrophage IL-10 crosstalk. Therefore, neither IFN-γ nor IL-4Rα is a key regulator of MDSCs and targeting these molecules is unlikely to significantly alter MDSC accumulation or function.