Lucas A. Horn

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.

The Programmed Death-1 Immune-Suppressive Pathway: Barrier to Antitumor Immunity

Programmed death ligand 1 (PD-L1, also known as B7 homolog 1 or CD274) is a major obstacle to antitumor immunity because it tolerizes/anergizes tumor-reactive T cells by binding to its receptor programmed death-1 (CD279), renders tumor cells resistant to CD8+ T cell– and FasL-mediated lysis, and tolerizes T cells by reverse signaling through T cell–expressed CD80. PD-L1 is abundant in the tumor microenvironment, where it is expressed by many malignant cells, as well as by immune cells and vascular endothelial cells. The critical role of PD-L1 in obstructing antitumor immunity has been demonstrated in multiple animal models and in recent clinical trials. This article reviews the mechanisms by which PD-L1 impairs antitumor immunity and discusses established and experimental strategies for maintaining T cell activation in the presence of PD-L1–expressing cells in the tumor microenvironment.

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.

Phagocytic ability declines with age in adult Drosophila hemocytes

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age‐related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1‐week‐ and 5‐week‐old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.

Combined MYC activation and Pten loss are sufficient to create genomic instability and lethal metastatic prostate cancer

Genetic instability, a hallmark feature of human cancers including prostatic adenocarcinomas, is considered a driver of metastasis. Somatic copy number alterations (CNA) are found in most aggressive primary human prostate cancers, and the overall number of such changes is increased in metastases. Chromosome 10q23 deletions, encompassing PTEN, and amplification of 8q24, harboring MYC, are frequently observed, and the presence of both together portends a high risk of prostate cancer-specific mortality. In extant genetically engineered mouse prostate cancer models (GEMM), isolated MYC overexpression or targeted Pten loss can each produce early prostate adenocarcinomas, but are not sufficient to induce genetic instability or metastases with high penetrance. Although a previous study showed that combining Pten loss with focal MYC overexpression in a small fraction of prostatic epithelial cells exhibits cooperativity in GEMMs, additional targeted Tp53 disruption was required for formation of metastases. We hypothesized that driving combined MYC overexpression and Pten loss using recently characterized Hoxb13 transcriptional control elements that are active in prostate luminal epithelial cells would induce the development of genomic instability and aggressive disease with metastatic potential. Neoplastic lesions that developed with either MYC activation alone (Hoxb13-MYC) or Pten loss alone (Hoxb13-Cre∣Pten(Fl/Fl)) failed to progress beyond prostatic intraepithelial neoplasia and did not harbor genomic CNAs. By contrast, mice with both alterations (Hoxb13-MYC∣Hoxb13-Cre∣Pten(Fl/Fl), hereafter, BMPC mice) developed lethal adenocarcinoma with distant metastases and widespread genome CNAs that were independent of forced disruption of Tp53 and telomere shortening. BMPC cancers lacked neuroendocrine or sarcomatoid differentiation, features uncommon in human disease but common in other models of prostate cancer that metastasize. These data show that combined MYC activation and Pten loss driven by the Hoxb13 regulatory locus synergize to induce genomic instability and aggressive prostate cancer that phenocopies the human disease at the histologic and genomic levels.

A Soluble Form of CD80 Enhances Antitumor Immunity by Neutralizing Programmed Death Ligand-1 and Simultaneously Providing Costimulation

Haile and colleagues report that CD80-Fc effectively maintained PD-1+ T-cell activation and IFNγ production by blocking PD-L1-mediated immune suppression and costimulating CD28, and suggest the development of CD80-Fc as a therapeutic agent. Tumor cells use various methods of immunosuppression to overcome antitumor immunity. One such method is that of programmed death ligand-1 (PD-L1 or B7-H1), which upon binding its receptor PD-1 on T cells triggers apoptotic death of the activated T cells. Overexpression of the costimulatory molecule CD80 on PD-L1+ tumor cells, or inclusion of a soluble form of CD80 (CD80-Fc), maintains the activation of PD-1+–activated T cells. Using T cells from CD28-deficient mice and antibodies to block CD28 on human T cells, we now report that a soluble form of CD80 mediates this effect by simultaneously neutralizing PD-1–PD-L1-mediated immunosuppression and by providing CD80–CD28 costimulation, and is more effective than antibodies to PD-L1 or PD-1 in maintaining IFNγ production by PD-1+ activated T cells. Therefore, soluble CD80 may be a more effective therapeutic than these checkpoint antibodies for facilitating the development and maintenance of antitumor immunity because it has the dual functions of preventing PD-L1–mediated immunosuppression and simultaneously delivering the second signal for T-cell activation. Cancer Immunol Res; 2(7); 610–5. ©2014 AACR.

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.

Age- and diet-specific effects of variation at S6 kinase on life history, metabolic, and immune response traits in Drosophila melanogaster.

Life history theory hypothesizes that genetically based variation in life history traits results from alleles that alter age-specific patterns of energy allocation among the competing demands of reproduction, storage, and maintenance. Despite the important role that alleles with age-specific effects must play in life history evolution, few naturally occurring alleles with age-specific effects on life history traits have been identified. A recent mapping study identified S6 kinase (S6k) as a candidate gene affecting lipid storage in Drosophila. S6k is in the target of rapamycin pathway, which regulates cell growth in response to nutrient availability and has also been implicated to influence many life history traits from fecundity to life span. In this article, we used quantitative complementation tests to examine the effect of allelic variation at S6k on a range of phenotypes associated with metabolism and fitness in an age-, diet-, and sex-specific manner. We found that alleles of S6k have pleiotropic effects on total protein levels, glycogen storage, life span, and the immune response and demonstrate that these allelic effects are age, diet, and sex specific. As many of the genes in the target of rapamycin pathway are evolutionarily conserved, our data suggest that genes in this pathway could play a pivotal role in life history evolution in a wide range of taxa.

Novel strategies for inhibiting PD-1 pathway-mediated immune suppression while simultaneously delivering activating signals to tumor-reactive T cells

We previously developed cell-based vaccines as therapeutics for metastatic cancers. The vaccines were aimed at activating type I CD4+T cells and consisted of tumor cells transfected with genes encoding syngeneic MHC class II and CD80 costimulatory molecules, and lacking the MHC II-associated invariant chain. The vaccines showed some efficacy in mice with sarcoma, melanoma, and breast cancer and activated MHC class II syngeneic T cells from breast, lung, and melanoma patients. During the course of the vaccine studies, we observed that CD80 not only costimulated naïve T cells, but also bound to PD-L1 and prevented tumor cell-expressed PD-L1 from binding to its receptor PD-1 on activated T cells. A soluble form of CD80 (CD80-Fc) had the same effect and sustained IFNγ production by both human and murine PD-1+ activated T cells in the presence of PD-L1+ human or mouse tumor cells, respectively. In vitro studies with human tumor cells indicated that CD80-Fc was more effective than antibodies to either PD-1 or PD-L1 in sustaining T cell production of IFNγ. Additionally, in vivo studies with a murine tumor demonstrated that CD80-Fc was more effective than antibodies to PD-L1 in extending survival time. Studies with human T cells blocked for CD28 and with T cells from CD28 knockout mice demonstrated that CD80-Fc simultaneously inhibited PD-L1/PD-1-mediated immune suppression and delivered costimulatory signals to activated T cells, thereby amplifying T cell activation. These results suggest that CD80-Fc may be a useful monotherapy that minimizes PD-1 pathway immune suppression while simultaneously activating tumor-reactive T cells.

Soluble CD80 protein delays tumor growth and promotes tumor infiltrating lymphocytes

Virginia Clements, and Suzanne Ostrand-Rosenberg Therapeutic strategies that simultaneously neutralize PD-1 suppressive mechanisms while activating tumor-reactive T cells are needed. The use of soluble CD80 in mice achieved both of these goals, which supports its potential usefulness as an immunotherapeutic for cancer patients. Tumor cells use various immune-suppressive strategies to overcome antitumor immunity. One such method is tumor expression of programmed death ligand-1 (PD-L1), which triggers apoptotic death or anergy upon binding programmed death-1 (PD-1) on T cells. Our previous in vitro cellular studies with human and mouse PD-L1+ tumor cells demonstrated that a soluble form of the costimulatory molecule CD80 prevented PD-L1–mediated immune suppression and restored T-cell activation by binding PD-L1 and blocking interaction with PD-1. We now report that in vivo treatment of established syngeneic PD-L1+ CT26 colon carcinoma and B16F10 melanoma tumors with CD80-Fc delays tumor growth and promotes tumor-infiltrating T cells. Studies with PD-1−/− and CD28−/− mice demonstrate that soluble CD80 acts in vivo by simultaneously neutralizing PD-1 suppression and activating through CD28. We also report that soluble CD80 mediates its effects by activating transcription factors EGR1-4, NF-κB, and MAPK, downstream signaling components of the CD28 and T-cell receptor pathways. Soluble CD80 binds to CTLA-4 on activated human peripheral blood mononuclear cells. However, increasing quantities of CTLA-4 antagonist antibodies do not increase T-cell activation. These results indicate that soluble CD80 does not suppress T-cell function through CTLA-4 and suggest that CTLA-4 acts as a decoy receptor for CD80, rather than functioning as a suppressive signaling receptor. Collectively, these studies demonstrate that soluble CD80 has therapeutic efficacy in vivo in mouse tumor systems and that its effects are due to its ability to inhibit PD-1–mediated suppression while concurrently activating T cells through CD28. Cancer Immunol Res; 6(1); 59–68. ©2017 AACR.