CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection.

Tissue-resident memory CD8 T (TRM) cells defend against microbial reinfections at mucosal barriers; determinants driving durable TRM cell responses in non-mucosal tissues, which often harbor opportunistic persistent pathogens, are unknown. JC polyomavirus (JCPyV) is a ubiquitous constituent of the human virome. With altered immunological status, JCPyV can cause the oft-fatal brain demyelinating disease progressive multifocal leukoencephalopathy (PML). JCPyV is a human-only pathogen. Using the mouse polyomavirus (MuPyV) encephalitis model, we demonstrate that CD4 T cells regulate development of functional antiviral brain-resident CD8 T cells (bTRM) and renders their maintenance refractory to systemic CD8 T cell depletion. Acquired CD4 T cell deficiency, modeled by delaying systemic CD4 T cell depletion until MuPyV-specific CD8 T cells have infiltrated the brain, impacted the stability of CD8 bTRM, impaired their effector response to reinfection, and rendered their maintenance dependent on circulating CD8 T cells. This dependence of CD8 bTRM differentiation on CD4 T cells was found to extend to encephalitis caused by vesicular stomatitis virus. Together, these findings reveal an intimate association between CD4 T cells and homeostasis of functional bTRM to CNS viral infection.

The Granulocyte Progenitor Stage Is a Key Target of IRF8-Mediated Regulation of Myeloid-Derived Suppressor Cell Production.

Alterations in myelopoiesis are common across various tumor types, resulting in immature populations termed myeloid-derived suppressor cells (MDSCs). MDSC burden correlates with poorer clinical outcomes, credited to their ability to suppress antitumor immunity. MDSCs consist of two major subsets, monocytic and polymorphonuclear (PMN). Intriguingly, the latter subset predominates in many patients and tumor models, although the mechanisms favoring PMN-MDSC responses remain poorly understood. Ordinarily, lineage-restricted transcription factors regulate myelopoiesis that collectively dictate cell fate. One integral player is IFN regulatory factor (IRF)-8, which promotes monocyte/dendritic cell differentiation while limiting granulocyte development. We recently showed that IRF8 inversely controls MDSC burden in tumor models, particularly the PMN-MDSC subset. However, where IRF8 acts in the pathway of myeloid differentiation to influence PMN-MDSC production has remained unknown. In this study, we showed that: 1) tumor growth was associated with a selective expansion of newly defined IRF8lo granulocyte progenitors (GPs); 2) tumor-derived GPs had an increased ability to form PMN-MDSCs; 3) tumor-derived GPs shared gene expression patterns with IRF8-/- GPs, suggesting that IRF8 loss underlies GP expansion; and 4) enforced IRF8 overexpression in vivo selectively constrained tumor-induced GP expansion. These findings support the hypothesis that PMN-MDSCs result from selective expansion of IRF8lo GPs, and that strategies targeting IRF8 expression may limit their load to improve immunotherapy efficacy.

Mechanisms overseeing myeloid-derived suppressor cell production in neoplastic disease.

Perturbations in myeloid cell differentiation are common in neoplasia, culminating in immature populations known as myeloid-derived suppressor cells (MDSCs). MDSCs favor tumor progression due to their ability to suppress host immunity or promote invasion and metastasis. They are thought to originate from the bone marrow as a result of exposure to stromal- or circulating tumor-derived factors (TDFs). Although great interest has been placed on understanding how MDSCs function, less is known regarding how MDSCs develop at a transcriptional level. Our work explores the premise that MDSCs arise because cancer cells, through the production of certain TDFs, inhibit the expression of interferon regulatory factor-8 (IRF8) that is ordinarily essential for controlling fundamental properties of myeloid cell differentiation. Our interest in IRF8 has been based on the following rationale. First, it is well-recognized that IRF8 is a 'master regulator' of normal myelopoiesis, critical not only for producing monocytes, dendritic cells (DCs), and neutrophils, but also for controlling the balance of all three major myeloid cell types. This became quite evident in IRF8-/- mice, whereby the loss of IRF8 leads to a disproportionate accumulation of neutrophils at the expense of monocytes and DCs. Second, we showed that such myeloid populations from IRF8-/- mice exhibit similar characteristics to MDSCs from tumor-bearing mice. Third, in a reciprocal fashion, we showed that enforced expression of IRF8 in the myeloid system significantly mitigates tumor-induced MDSC accumulation and improves immunotherapy efficacy. Altogether, these observations support the hypothesis that IRF8 is an integral negative regulator of MDSC biology.

Generation of a C57BL/6 MYC-Driven Mouse Model and Cell Line of Prostate Cancer.

INTRODUCTION: Transgenic mouse modeling is a favorable tool to reflect human prostate tumorigenesis and interactions between prostate cancer and the microenvironment. The use of GEMMs and derived cell lines represent powerful tools to study prostate cancer initiation and progression with an associated tumor microenvironment. Notably, such models provide the capacity for rapid preclinical therapy studies including immune therapies for prostate cancer treatment. METHODS: Backcrossing FVB Hi-MYC mice with C57BL/6N mice, we established a Hi-MYC transgenic mouse model on a C57BL/6 background (B6MYC). In addition, using a conditional reprogramming method, a novel C57BL/6 MYC driven prostate adenocarcinoma cell line was generated. RESULTS: Our results demonstrate that disease progression is significantly delayed in B6MYC when compared to their FVB counterparts. Current data also indicates infiltrating immune cells are present in pre-cancer lesions, prostate intraepithelial neoplasia (PIN). Further, immunophenotyping of this immune infiltrate demonstrates the predominant population as myeloid-derived suppressor cells (MDSC). Also, we successfully generated a B6MYC-CaP cell line, and determined that this new PCa cell line express markers of luminal epithelial lineage. DISCUSSION: This novel model of PCa provides a new platform to understand the cross talk between MYC driven prostate cancer and the microenvironment. Importantly, these models will be an ideal tool to support the clinical development of immunotherapy as well as other novel therapeutic strategies for prostate cancer treatment. Prostate 76:1192-1202, 2016. © 2016 Wiley Periodicals, Inc.

Tumor-induced myeloid dysfunction and its implications for cancer immunotherapy.

Immune function relies on an appropriate balance of the lymphoid and myeloid responses. In the case of neoplasia, this balance is readily perturbed by the dramatic expansion of immature or dysfunctional myeloid cells accompanied by a reciprocal decline in the quantity/quality of the lymphoid response. In this review, we seek to: (1) define the nature of the atypical myelopoiesis observed in cancer patients and the impact of this perturbation on clinical outcomes; (2) examine the potential mechanisms underlying these clinical manifestations; and (3) explore potential strategies to restore normal myeloid cell differentiation to improve activation of the host antitumor immune response. We posit that fundamental alterations in myeloid homeostasis triggered by the neoplastic process represent critical checkpoints that govern therapeutic efficacy, as well as offer novel cellular-based biomarkers for tracking changes in disease status or relapse.

Relevance of Interferon Regulatory Factor-8 Expression in Myeloid-Tumor Interactions.

Perturbations in myelopoiesis are a common feature in solid tumor biology, reflecting the central premise that cancer is not only a localized affliction but also a systemic disease. Because the myeloid compartment is essential for the induction of adaptive immunity, these alterations in myeloid development contribute to the failure of the host to effectively manage tumor progression. These "dysfunctional" myeloid cells have been coined myeloid-derived suppressor cells (MDSCs). Interestingly, such cells not only arise in neoplasia but also are associated with many other inflammatory or pathologic conditions. MDSCs affect disease outcome through multiple mechanisms, including their ability to mediate generalized or antigen-specific immune suppression. Consequently, MDSCs pose a significant barrier to effective immunotherapy in multiple disease settings. Although much interest has been devoted to unraveling mechanisms by which MDSCs mediate immune suppression, a large gap has remained in our understanding of the mechanisms that drive their development in the first place. Investigations into this question have identified an unrecognized role of interferon regulatory factor-8 (IRF-8), a member of the IRF family of transcription factors, in tumor-induced myeloid dysfunction. Ordinarily, IRF-8 is involved in diverse stages of myelopoiesis, namely differentiation and lineage commitment toward monocytes, dendritic cells, and granulocytes. Several recent studies now support the hypothesis that IRF-8 functions as a "master" negative regulator of MDSC formation in vivo. This review focuses on IRF-8 as a potential target suppressed by tumors to cripple normal myelopoiesis, redirecting myeloid differentiation toward the emergence of MDSCs. Understanding the bases by which neoplasia drives MDSC accumulation has the potential to improve the efficacy of therapies that require a competent myeloid compartment.

MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

Interferon regulatory factor-8 (IRF8), originally identified as a leukemic tumor suppressor, can also exert anti-neoplastic activities in solid tumors. We previously showed that IRF8-loss enhanced tumor growth, which was accompanied by reduced tumor-cell susceptibility to apoptosis. However, the impact of IRF8 expression on tumor growth could not be explained solely by its effects on regulating apoptotic response. Exploratory gene expression profiling further revealed an inverse relationship between IRF8 and MMP3 expression, implying additional intrinsic mechanisms by which IRF8 modulated neoplastic behavior. Although MMP3 expression was originally linked to tumor initiation, the role of MMP3 beyond this stage has remained unclear. Therefore, we hypothesized that MMP3 governed later stages of disease, including progression to metastasis, and did so through a novel IRF8-MMP3 axis. Altogether, we showed an inverse mechanistic relationship between IRF8 and MMP3 expression in tumor progression. Importantly, the growth advantage due to IRF8-loss was significantly compromised after silencing MMP3 expression. Moreover, MMP3-loss reduced spontaneous lung metastasis in an orthotopic mouse model of mammary carcinoma. MMP3 acted, in part, in a cell-intrinsic manner and served as a direct transcriptional target of IRF8. Thus, we identified a novel role of an IRF8-MMP3 axis in tumor progression, which unveils new therapeutic opportunities.

Tumor-induced STAT3 signaling in myeloid cells impairs dendritic cell generation by decreasing PKCßII abundance.

A major mechanism by which cancers escape control by the immune system is by blocking the differentiation of myeloid cells into dendritic cells (DCs), immunostimulatory cells that activate antitumor T cells. Tumor-dependent activation of signal transducer and activator of transcription 3 (STAT3) signaling in myeloid progenitor cells is thought to cause this block in their differentiation. In addition, a signaling pathway through protein kinase C ßII (PKCßII) is essential for the differentiation of myeloid cells into DCs. We found in humans and mice that breast cancer cells substantially decreased the abundance of PKCßII in myeloid progenitor cells through a mechanism involving the enhanced activation of STAT3 signaling by soluble, tumor-derived factors (TDFs). STAT3 bound to previously undescribed negative regulatory elements within the promoter of PRKCB, which encodes PKCßII. We also found a previously undescribed counter-regulatory mechanism through which the activity of PKCßII inhibited tumor-dependent STAT3 signaling by decreasing the abundance of cell surface receptors, such as cytokine and growth factor receptors, that are activated by TDFs. Together, these data suggest that a previously unrecognized cross-talk mechanism between the STAT3 and PKCßII signaling pathways provides the molecular basis for the tumor-induced blockade in the differentiation of myeloid cells, and suggest that enhancing PKCßII activity may be a therapeutic strategy to alleviate cancer-mediated suppression of the immune system.