Katherine H. Parker

Myeloid-Derived Suppressor Cells

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.

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-6–dependent, MDSC-driven immune escape, establishing IDO as an overarching factor directing the establishment of a protumorigenic environment.

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.

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.

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.

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.

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.

Abstract 3665: IDO1 is an integrative determinant of tumor-promoting, pathogenic inflammation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The tryptophan-catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) has been implicated as a mediator of immune tolerance in the reproductively essential process of shielding the ‘foreign’ fetus from maternal immunity. By extrapolation, we and others hypothesized that tumors might elevate IDO1 under selective pressure by the immune system. Our finding that loss of the Bin1 tumor suppressor gene potentiates the superinduction of IDO1 provided the first discreet example of a molecular pathway through which this immune escape process can occur. However, the involvement of IDO1 in tumorigenesis has turned out to be far more complex. Tumors that inherently lack IDO1 expression have been demonstrated to induce IDO1 expression in antigen presenting cells of the host, providing an alternative mechanism for immune escape. We have also found from studies in the classical DMBA/TPA skin carcinogenesis model that IDO1 can be induced by the inflammatory tumor-promoting process itself independent of the presence of an initiated tumor. Thus, IDO1 can be a factor in tumor promotion throughout the entire immunoediting process. With the rapid pace of development of IDO inhibitors, which are currently being evaluated in clinical trials, we are interested in determining whether mouse tumor models might provide additional insight into the optimal therapeutic application of these agents based on the underlying biology. In current studies, we have found that IDO1-nullizygous mice are resistant to both KRAS-induced lung adenocarcinomas and pulmonary breast carcinoma metastases. Micro-computed tomographic imaging confirmed that lung tumor burden was correspondingly lower in IDO1-nullizygous mice. Surprisingly, this analysis also revealed a significantly reduced pulmonary blood vessel density in IDO1-nullizygous mice. Elevation of the inflammatory cytokine IL6 (interleukin 6) was greatly attenuated in conjunction with the loss of IDO1, consistent with in vitro evidence that IDO1 potentiates IL6 production. MDSCs (myeloid derived suppressor cells) from IDO1-nullizygous animals exhibited reduced T cell suppressive activity that could be rescued by IL6. IL6 could likewise reverse the pulmonary metastasis resistance exhibited by IDO1-nullizygous mice. Together, our findings provide support for the emerging concept of IDO1 as a prototypical, integrative immune modifier that bridges inflammation, vascularization and immune escape to foster the establishment of a pathogenic, tumor-promoting environment. Citation Format: Alexander J. Muller, Courtney Smith, Mee Young Chang, James DuHadaway, Arpita Mondal, Hollie Flick, Katherine Parker, Daniel Beury, Suzanne Ostrand-Rosenberg, George C. Prendergast. IDO1 is an integrative determinant of tumor-promoting, pathogenic inflammation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2014-3665

Abstract 461: Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cancer immunotherapy aims to harness the power of the immune system to fight advanced stages of cancer. A main hindrance to cancer immunotherapy is tumor-induced immune suppression. Within the tumor microenvironment are a plethora of immune suppressive cells including Myeloid-Derived Suppressor Cells (MDSC). MDSC are present in nearly all types of cancer blocking T cell activation and shifting immunity towards a type 2 tumor-promoting phenotype through cross-talk with macrophages that enhances the secretion of pro-tumor molecules such as IL-10, and simultaneously drops IL-12 production by macrophages. MDSC accumulation is induced by pro-inflammatory cytokines including IL-1β, IL-6 and VEGF as well as alarmins such as S100A8/9. Because the alarmin HMGB1 is increased in many cancers, we are determining if HMGB1 drives MDSC. Inhibition of HMGB1 in vitro by chemical inhibitors Glycyrrhizin and Ethyl Pyruvate decreased cross-talk-induced IL-10 production by MDSC and IL-6 production by macrophages. Ethyl Pyruvate also reduced MDSC suppressive capacity in transgenic T cell suppression assays, suggesting that HMGB1 regulates multiple aspects of MDSC function. Treatment of BALB/c mice with 4T1 mammary carcinoma with HMGB1 inhibitors Glycyrrhizin and Ethyl Pyruvate reduced lung metastases and did not impact primary tumor progression. Treatment of C57BL/6 mice with MC38 colon carcinoma with an HMGB1 neutralizing antibody (2G7) reduced MDSC levels in the blood, spleen, and tumor. HMGB1 consists of two domains, the pro-inflammatory B box domain and an anti-inflammatory A box domain. Treatment of C57BL/6 mice with MC38 tumor with A box significantly reduced primary tumor growth and decreased MDSC levels in the blood. These findings indicate that full-length HMGB1 drives inflammation and tumor progression, while the anti-inflammatory A box domain counteracts full-length HMGB1 and reduces tumor growth and MDSC. These results suggest that HMGB1 drives MDSC accumulation and tumor progression and that A box can be used to target MDSC-mediated tumor-induced immune suppression. (Research supported by NIH RO1CA115880 and RO1CA84232) Citation Format: Katherine Parker, Suzanne Rosenberg, Pratima Sinha, Huan Yang, Kevin Tracey, Jianhua Li. Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2013-461

Abstract 2876: Inflammation of the tumor microenvironment is regulated by myeloid derived suppressor cell and macrophage crosstalk.

Myeloid-Derived Suppressor Cells (MDSC) polarize macrophages to a Type II tumor-promoting phenotype via MDSC-macrophage crosstalk. We previously demonstrated that MDSC produce IL-10 which inhibits macrophage production of IL-12, and MDSC production of IL-10 is dependent on TLR4 and enhanced by cell-cell contact with macrophages. However, the role of inflammation in MDSC-macrophage crosstalk is not well defined. To determine the role of inflammation, we utilized wild type, IL-6-/-, and IL-10-/- mice bearing syngeneic 4T1 mammary carcinoma. IL-10 is classically an anti-inflammatory cytokine. In contrast, IL-6 is a pro-inflammatory cytokine that is secreted by activated Type 1 macrophages, is found at higher levels in tumor-bearing patients, and is an inducer of MDSC. 4T1-induced MDSC and macrophages express both IL-6 and IL-10 receptors, suggesting that they have the potential to respond to both cytokines. To determine if IL-6 and IL-10 contribute to tumor progression by modulating MDSC-macrophage crosstalk, MDSC from wild type or IL-10-/- mice bearing 4T1 tumors were cultured with macrophages from wild type or IL-6-/- BALB/c mice. Although MDSC are typically pro-inflammatory cells, they significantly decreased macrophage production of IL-6, suggesting that MDSC can also function as anti-inflammatory cells. IL-6 levels are important in vivo since primary 4T1 tumors grow more slowly in IL-6-deficient mice, and these mice have significantly extended survival compared to wild type BALB/c mice. Anti-inflammatory effects are further exacerbated by macrophages themselves, since their production of IL-6 increases MDSC production of IL-10. However, macrophage-produced IL-6 affects MDSC indirectly, as incubation of MDSC with exogenous IL-6 in the absence of macrophages does not increase IL-10 production. Although IL-10 is classically considered as an anti-inflammatory cytokine, it contributes to tumor progression because 4T1-bearing IL-10-/- mice have delayed primary tumor progression and extended survival vs. wild type mice. These data demonstrate that MDSC have both pro-inflammatory and anti-inflammatory effects and that MDSC-macrophage cross-talk contributes to the overall milieu of IL-10 and IL-6 within the tumor microenvironment. Since IL-6 is a signature cytokine of anti-tumor M1-like macrophages, these data also suggest that although MDSC may decrease inflammation through their production of IL-10, this effect neither promotes the development of tumoricidal macrophages nor decreases tumor progression. Supported by NIH R01 CA115880, RO1CA84232, and DOD BCRP W81XWH-11-1-0115 Citation Format: Daniel W. Beury, Katherine H. Parker, Suzanne Ostrand-Rosenberg. Inflammation of the tumor microenvironment is regulated by myeloid derived suppressor cell and macrophage crosstalk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2876. doi:10.1158/1538-7445.AM2013-2876