A Bacterial and Ganglioside-based Nanoparticle Initiates Reprogramming of Macrophages and Promotes Antitumor Phenotypes.

Macrophages represent the most abundant immune component of the tumor microenvironment and often exhibit protumorigenic (M2-like) phenotypes that contribute to disease progression. Despite their generally accepted protumorigenic role, macrophages can also display tumoricidal (or M1-like) behavior, revealing that macrophages can be functionally reprogrammed, depending on the cues received within the tumor microenvironment. Moreover, such plasticity may be achieved by pharmacologic or biologic interventions. To that end, we previously demonstrated that a novel immunomodulator termed the "very small size particle" (VSSP) facilitates maturation of dendritic cells and differentiation of myeloid-derived suppressor cells to APCs with reduced suppressive activity in cancer models. VSSP was further shown to act in the bone marrow to drive the differentiation of progenitors toward monocytes, macrophages, and dendritic cells during emergency myelopoiesis. However, the underlying mechanisms for VSSP-driven alterations in myeloid differentiation and function remained unclear. In this study, in mouse models, we focused on macrophages and tested the hypothesis that VSSP drives macrophages toward M1-like functional states via IRF8- and PU.1-dependent mechanisms. We further hypothesized that such VSSP-mediated actions would be accompanied by enhanced antitumor responses. Overall, we showed that (1) VSSP drives naive or M2-derived macrophages to M1-like states, (2) the M1-like state induced by VSSP occurs via IRF8- and PU.1-dependent mechanisms, and (3) single-agent VSSP induces an antitumor response that is accompanied by alterations in the intratumoral myeloid compartment. These results provide a deeper mechanistic underpinning of VSSP and strengthen its use to drive M1-like responses in host defense, including cancer.

RIPK3 Activation Leads to Cytokine Synthesis that Continues after Loss of Cell Membrane Integrity.

Necroptosis is a form of programmed cell death that is defined by activation of the kinase RIPK3 and subsequent cell membrane permeabilization by the effector MLKL. RIPK3 activation can also promote immune responses via production of cytokines and chemokines. How active cytokine production is coordinated with the terminal process of necroptosis is unclear. Here, we report that cytokine production continues within necroptotic cells even after they have lost cell membrane integrity and irreversibly committed to death. This continued cytokine production is dependent on mRNA translation and requires maintenance of endoplasmic reticulum integrity that remains after plasma membrane integrity is lost. The continued translation of cytokines by cellular corpses contributes to necroptotic cell uptake by innate immune cells and priming of adaptive immune responses to antigens associated with necroptotic corpses. These findings imply that cell death and production of inflammatory mediators are coordinated to optimize the immunogenicity of necroptotic cells.

Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity.

Although the signaling events that induce different forms of programmed cell death are well defined, the subsequent immune responses to dying cells in the context of cancer remain relatively unexplored. Necroptosis occurs downstream of the receptor-interacting protein kinases RIPK1 and RIPK3, whose activation leads to lytic cell death accompanied by de novo production of proinflammatory mediators. Here, we show that ectopic introduction of necroptotic cells to the tumor microenvironment promotes BATF3+ cDC1- and CD8+ leukocyte-dependent antitumor immunity accompanied by increased tumor antigen loading by tumor-associated antigen-presenting cells. Furthermore, we report the development of constitutively active forms of the necroptosis-inducing enzyme RIPK3 and show that delivery of a gene encoding this enzyme to tumor cells using adeno-associated viruses induces tumor cell necroptosis, which synergizes with immune checkpoint blockade to promote durable tumor clearance. These findings support a role for RIPK1/RIPK3 activation as a beneficial proximal target in the initiation of tumor immunity. Considering that successful tumor immunotherapy regimens will require the rational application of multiple treatment modalities, we propose that maximizing the immunogenicity of dying cells within the tumor microenvironment through specific activation of the necroptotic pathway represents a beneficial treatment approach that may warrant further clinical development.

Comparing the effects of different cell death programs in tumor progression and immunotherapy.

Our conception of programmed cell death has expanded beyond apoptosis to encompass additional forms of cell suicide, including necroptosis and pyroptosis; these cell death modalities are notable for their diverse and emerging roles in engaging the immune system. Concurrently, treatments that activate the immune system to combat cancer have achieved remarkable success in the clinic. These two scientific narratives converge to provide new perspectives on the role of programmed cell death in cancer therapy. This review focuses on our current understanding of the relationship between apoptosis and antitumor immune responses and the emerging evidence that induction of alternate death pathways such as necroptosis could improve therapeutic outcomes.

ß-Adrenergic Signaling in Mice Housed at Standard Temperatures Suppresses an Effector Phenotype in CD8+ T Cells and Undermines Checkpoint Inhibitor Therapy.

The immune context of tumors has significant prognostic value and is predictive of responsiveness to several forms of therapy, including immunotherapy. We report here that CD8+ T-cell frequency and functional orientation within the tumor microenvironment is regulated by ß2-adrenergic receptor (ß-AR) signaling in host immune cells. We used three strategies-physiologic (manipulation of ambient thermal environment), pharmacologic (ß-blockers), and genetic (ß2-AR knockout mice) to reduce adrenergic stress signaling in two widely studied preclinical mouse tumor models. Reducing ß-AR signaling facilitated conversion of tumors to an immunologically active tumor microenvironment with increased intratumoral frequency of CD8+ T cells with an effector phenotype and decreased expression of programmed death receptor-1 (PD-1), in addition to an elevated effector CD8+ T-cell to CD4+ regulatory T-cell ratio (IFNγ+CD8+:Treg). Moreover, this conversion significantly increased the efficacy of anti-PD-1 checkpoint blockade. These data highlight the potential of adrenergic stress and norepinephrine-driven ß-AR signaling to regulate the immune status of the tumor microenvironment and support the strategic use of clinically available ß-blockers in patients to improve responses to immunotherapy. Cancer Res; 77(20); 5639-51. ©2017 AACR.

HSPs drive dichotomous T-cell immune responses via DNA methylome remodelling in antigen presenting cells.

Immune responses primed by endogenous heat shock proteins, specifically gp96, can be varied, and mechanisms controlling these responses have not been defined. Immunization with low doses of gp96 primes T helper type 1 (Th1) immune responses, whereas high-dose immunization primes responses characterized by regulatory T (Treg) cells and immunosuppression. Here we show gp96 preferentially engages conventional and plasmacytoid dendritic cells (pDCs) under low and high doses, respectively, through CD91. Global DNMT-dependent epigenetic modifications lead to changes in protein expression within these antigen-presenting cells. Specifically, pDCs upregulate neuropilin-1 to enable the long term interactions of pDCs with Treg cells, thereby enhancing suppression of Th1 anti-tumour immunity. Our study defines a CD91-dependent mechanism through which gp96 controls dichotomous immune responses relevant to the therapy of cancer and autoimmunity.

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.

Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes.

Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive network that drives cancer escape by disabling T cell adaptive immunity. The prevailing view is that MDSC-mediated immunosuppression is restricted to tissues where MDSC co-mingle with T cells. Here we show that splenic or, unexpectedly, blood-borne MDSC execute far-reaching immune suppression by reducing expression of the L-selectin lymph node (LN) homing receptor on naïve T and B cells. MDSC-induced L-selectin loss occurs through a contact-dependent, post-transcriptional mechanism that is independent of the major L-selectin sheddase, ADAM17, but results in significant elevation of circulating L-selectin in tumor-bearing mice. Even moderate deficits in L-selectin expression disrupt T cell trafficking to distant LN. Furthermore, T cells preconditioned by MDSC have diminished responses to subsequent antigen exposure, which in conjunction with reduced trafficking, severely restricts antigen-driven expansion in widely-dispersed LN. These results establish novel mechanisms for MDSC-mediated immunosuppression that have unanticipated implications for systemic cancer immunity.

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.

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.

Mild cold-stress depresses immune responses: Implications for cancer models involving laboratory mice.

Physiologically accurate mouse models of cancer are critical in the pre-clinical development of novel cancer therapies. However, current standardized animal-housing temperatures elicit chronic cold-associated stress in mice, which is further increased in the presence of tumor. This cold-stress significantly impacts experimental outcomes. Data from our lab and others suggest standard housing fundamentally alters murine physiology, and this can produce altered immune baselines in tumor and other disease models. Researchers may thus underestimate the efficacy of therapies that are benefitted by immune responses. A potential mediator, norepinephrine, also underlies stress pathways common in mice and humans. Therefore, research into mechanisms connecting cold-stress and norepinephrine signaling with immune depression in mice could highlight new combination therapies for humans to simultaneously target stress while stimulating anti-tumor immunity.

Establishment of tumor-associated immunity requires interaction of heat shock proteins with CD91.

Host antitumor adaptive immune responses are generated as a result of the body's immunosurveillance mechanisms. How the antitumor immune response is initially primed remains unclear, given that soluble tumor antigens generally are quantitatively insufficient for cross-priming and tumors generally lack the classical pathogen-associated molecular patterns to activate costimulation and initiate cross-priming. We explored the interaction of the tumor-derived heat shock proteins (HSP) with their common receptor (CD91) on antigen-presenting cells (APC) as a mechanism for host-priming of T-cell-mediated antitumor immunity. Using targeted genetic disruption of the interaction between HSPs and CD91, we demonstrated that specific ablation of CD91 in APCs prevented the establishment of antitumor immunity. The antitumor immunity was also inhibited when the transfer of tumor-derived HSPs to APCs was prevented using an endogenous inhibitor of CD91. Inhibition was manifested in a reduction of cross-presentation of tumor-derived antigenic peptides in the lymph nodes, providing a molecular basis for the observed immunity associated with tumor development. Our findings demonstrate that early in tumor development, the HSP-CD91 pathway is critical for the establishment of antitumor immunity.

Identification of the cellular sentinels for native immunogenic heat shock proteins in vivo.

Select members of the heat shock proteins (HSPs) family, such as gp96, elicit immune responses specific to their chaperoned peptides. Although immunologic effects of HSPs on APCs described to date have largely been demonstrated with cell lines or primary cells in culture, their collective responses in vitro have been consistent with priming immune responses. In this study, we examine the physiologically relevant APCs in mice that are targeted after vaccination with native, murine HSPs, and we characterize those cells. Gp96 accesses the subcapsular region of the draining lymph node, and it is internalized predominantly by CD11b(+) cells in this locale. Cells acquiring gp96 can transfer protective antitumor immunity to naive mice by actively cross-presenting gp96-chaperoned peptides and providing costimulation. Our studies illustrate how HSPs act to alert the immune system of cellular damage and will be of paramount importance in immunotherapy of patients with cancer and infectious disease.

CD91-Dependent Modulation of Immune Responses by Heat Shock Proteins: A Role in Autoimmunity.

Heat shock proteins (HSPs) have been known for decades for their ability to protect cells under stressful conditions. In the 1980s a new role was ascribed for several HSPs given their ability to elicit specific immune responses in the setting of cancer and infectious disease. These immune responses have primarily been harnessed for the immunotherapy of cancer in the clinical setting. However, because of the ability of HSPs to prime diverse immune responses, they have also been used for modulation of immune responses during autoimmunity. The apparent dichotomy of immune responses elicited by HSPs is discussed here on a molecular and cellular level. The potential clinical application of HSP-mediated immune responses for therapy of autoimmune diseases is reviewed.

Human dendritic cells adenovirally-engineered to express three defined tumor antigens promote broad adaptive and innate immunity.

Dendritic cell (DC) immunotherapy has shown a promising ability to promote anti-tumor immunity in vitro and in vivo. Many trials have tested single epitopes and single antigens to activate single T cell specificities, and often CD8(+) T cells only. We previously found that determinant spreading and breadth of antitumor immunity correlates with improved clinical response. Therefore, to promote activation and expansion of polyclonal, multiple antigen-specific CD8(+) T cells, as well as provide cognate help from antigen-specific CD4(+) T cells, we have created an adenovirus encoding three full length melanoma tumor antigens (tyrosinase, MART-1 and MAGE-A6, "AdVTMM"). We previously showed that adenovirus (AdV)-mediated antigen engineering of human DC is superior to peptide pulsing for T cell activation, and has positive biological effects on the DC, allowing for efficient activation of not only antigen-specific CD8(+) and CD4(+) T cells, but also NK cells. Here we describe the cloning and testing of "AdVTMM2," an E1/E3-deleted AdV encoding the three melanoma antigens. This novel three-antigen virus expresses mRNA and protein for all antigens, and AdVTMM-transduced DC activate both CD8(+) and CD4(+) T cells which recognize melanoma tumor cells more efficiently than single antigen AdV. Addition of physiological levels of interferon-α (IFNα) further amplifies melanoma antigen-specific T cell activation. NK cells are also activated, and show cytotoxic activity. Vaccination with multi-antigen engineered DC may provide for superior adaptive and innate immunity and ultimately, improved antitumor responses.

A role for the heat shock protein-CD91 axis in the initiation of immune responses to tumors.

For over 100 years, it has been established that tumor-specific immune responses can frequently be detected in the tumor-bearing host. Whether or not these immune responses are capable of controlling the growth of the tumor is influenced by many factors. However, the mechanism by which the immune responses are initiated in the first place has remained a dilemma. In this chapter, we present evidence that heat shock protein-peptide complexes released by tumor cells are the entity responsible for initiating the immune responses. Interaction of the extracellular HSP with its receptor CD91 is necessary for priming the immune response. We propose that the disruption of the HSP-CD91 interaction may be an active mechanism by which tumors prevent the generation of immune responses against it.

Francisella tularensis LVS-induced Interleukin-12 p40 cytokine production mediates dendritic cell migration through IL-12 Receptor ß1.

Three cytokines use the IL-12p40 cytokine subunit namely: IL-12p70 (IL-12-comprised of IL-12p40 and IL-12p35), IL-23 (comprised of the IL-12p40 and IL-23p19 subunits) and homodimeric IL-12p40 (IL-12(p40)(2)). Following activation, immature dendritic cells (DCs) upregulate the chemokine receptor Chemokine-C-Receptor 7 (CCR7), and migrate in response to homeostatic chemokines such as chemokine (C-C motif) ligand 19 (CCL19). Induction of the cytokine IL-12p40 in response to pathogen-exposure, likely in its homodimeric form, is one of the primary events that mediates migration of DCs in response to CCL19. Here we show that following exposure to Francisella tularensis Live Vaccine Strain (LVS), DCs produce IL-12p40 and promote the migration of DCs to the chemokine CCL19 in an IL-12Rß1- and IL-12p(40)(2)-dependent manner. Induction of IL-12p40 and resulting chemokine responsiveness in DCs is TLR2-dependent and coincides with the uptake of F. tularensis LVS and activation of DCs. Importantly, we show that IL-12Rß1 signaling is required for DC migration from the lung to the draining lymph node following F. tularensis LVS exposure and coincides with accumulation of IL-12p40 expressing DCs in the draining lymph nodes. Together, these findings illustrate that IL-12p40 is induced rapidly in response to F. tularensis LVS and is required for DC migration through an IL-12Rß1-IL-12(p40)(2) dependent mechanism.

Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.

The importance of T helper type 1 (Th1) cell immunity in host resistance to the intracellular bacterium Francisella tularensis is well established. However, the relative roles of interleukin (IL)-12-Th1 and IL-23-Th17 cell responses in immunity to F. tularensis have not been studied. The IL-23-Th17 cell pathway is critical for protective immunity against extracellular bacterial infections. In contrast, the IL-23-Th17 cell pathway is dispensable for protection against intracellular pathogens such as Mycobacteria. Here we show that the IL-23-Th17 pathway regulates the IL-12-Th1 cell pathway and was required for protective immunity against F.tularensis live vaccine strain. We show that IL-17A, but not IL-17F or IL-22, induced IL-12 production in dendritic cells and mediated Th1 responses. Furthermore, we show that IL-17A also induced IL-12 and interferon-gamma production in macrophages and mediated bacterial killing. Together, these findings illustrate a biological function for IL-17A in regulating IL-12-Th1 cell immunity and host responses to an intracellular pathogen.