Steven M. Leonardo

Conserved gammaherpesvirus kinase and histone variant H2AX facilitate gammaherpesvirus latency in vivo

Many herpesvirus-encoded protein kinases facilitate viral lytic replication. Importantly, the role of viral kinases in herpesvirus latency is less clear. Mouse gammaherpesvirus-68 (MHV68)-encoded protein kinase orf36 facilitates lytic replication in part through activation of the host DNA damage response (DDR). Here we show that MHV68 latency was attenuated in the absence of orf36 expression. Unexpectedly, our study uncovered enzymatic activity-independent role of orf36 in the establishment of MHV68 latency following intraperitoneal route of infection. H2AX, an important DDR protein, facilitates MHV68 lytic replication and may be directly phosphorylated by orf36 during lytic infection. In this study, H2AX deficiency, whether systemic or limited to infected cells, attenuated the establishment of MHV68 latency in vivo. Thus, our work reveals viral kinase-dependent regulation of gammaherpesvirus latency and illuminates a novel link between H2AX, a component of a tumor suppressor DDR network, and in vivo latency of a cancer-associated gammaherpesvirus.

Altered B Cell Development and Anergy in the Absence of Foxp3

The importance of regulatory T cells in immune tolerance is illustrated by the human immune dysregulatory disorder IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), caused by a lack of regulatory T cells due to decreased or absent expression of Foxp3. Although the majority of work on regulatory T cells has focused on their ability to suppress T cell responses, the development of significant autoantibody titers in patients with IPEX suggests that regulatory T cells also contribute to the suppression of autoreactive B cells. Using a murine model, deficient in the expression of Foxp3, we show that B cell development is significantly altered in the absence of regulatory T cells. Furthermore, we identify a loss of B cell anergy as a likely mechanism to explain the production of autoantibodies that occurs in the absence of regulatory T cells. Our results suggest that regulatory T cells, by either direct or indirect mechanisms, modulate B cell development and anergy.

Ataxia telangiectasia mutated kinase controls chronic gammaherpesvirus infection.

ABSTRACT Gammaherpesviruses, such as Epstein-Barr virus (EBV), are ubiquitous cancer-associated pathogens that interact with DNA damage response, a tumor suppressor network. Chronic gammaherpesvirus infection and pathogenesis in a DNA damage response-insufficient host are poorly understood. Ataxia-telangiectasia (A-T) is associated with insufficiency of ataxia-telangiectasia mutated (ATM), a critical DNA damage response kinase. A-T patients display a pattern of anti-EBV antibodies suggestive of poorly controlled EBV replication; however, parameters of chronic EBV infection and pathogenesis in the A-T population remain unclear. Here we demonstrate that chronic gammaherpesvirus infection is poorly controlled in an animal model of A-T. Intriguingly, in spite of a global increase in T cell activation and numbers in wild-type (wt) and ATM-deficient mice in response to mouse gammaherpesvirus 68 (MHV68) infection, the generation of an MHV68-specific immune response was altered in the absence of ATM. Our finding that ATM expression is necessary for an optimal adaptive immune response against gammaherpesvirus unveils an important connection between DNA damage response and immune control of chronic gammaherpesvirus infection, a connection that is likely to impact viral pathogenesis in an ATM-insufficient host.

Expansion of follicular helper T cells in the absence of Treg cells: implications for loss of B-cell anergy.

The maintenance of B‐cell anergy is essential to prevent the production of autoantibodies and autoimmunity. However, B‐cell extrinsic mechanisms that regulate B‐cell anergy remain poorly understood. We previously demonstrated that regulatory T (Treg) cells are necessary for the maintenance of B‐cell anergy. We now show that in Treg‐cell‐deficient mice, helper T cells are necessary and sufficient for loss of B‐cell tolerance/anergy. In addition, we show that the absence of Treg cells is associated with an increase in the proportion of CD4+ cells that express GL7 and correlated with an increase in germinal center follicular helper T (GC‐TFH) cells. These GC‐TFH cells, but not those from Treg‐cell‐sufficient hosts, were sufficient to drive antibody production by anergic B cells. We propose that a function of Treg cells is to prevent the expansion of TFH cells, especially GC‐TFH cells, which support autoantibody production.

Cutting Edge: In the Absence of Regulatory T Cells, a Unique Th Cell Population Expands and Leads to a Loss of B Cell Anergy

The absence of regulatory T cells (Tregs) results in significant immune dysregulation that includes autoimmunity. The mechanism(s) by which Tregs suppress autoimmunity remains unclear. We have shown that B cell anergy, a major mechanism of B cell tolerance, is broken in the absence of Tregs. In this study, we identify a unique subpopulation of CD4+ Th cells that are highly supportive of Ab production and promote loss of B cell anergy. Notably, this novel T cell subset was shown to express the germinal center Ag GL7 and message for the B cell survival factor BAFF, yet failed to express markers of the follicular Th cell lineage. We propose that the absence of Tregs results in the expansion of a unique nonfollicular Th subset of helper CD4+ T cells that plays a pathogenic role in autoantibody production.

Imprime PGG-Mediated Anti-Cancer Immune Activation Requires Immune Complex Formation

Imprime PGG (Imprime), an intravenously-administered, soluble β-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-β glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy.

Abstract 5034: Imprime PGG decreases regulatory T cell suppression and enhances T cell proliferation and differentiation revealing additional mechanisms for its anti-tumor activity

Imprime PGG is a soluble yeast-derived β-glucan immunomodulator being developed for treatment of cancer in combination with anti-tumor antibodies (Abs). Imprime PGG binds to complement receptor (CR)3 on innate immune cells (neutrophils and monocytes), and primes them to exert anti-tumor activity against Ab-targeted, complement opsonized tumor cells. Numerous studies in syngeneic and human xenogeneic tumor models in mice have demonstrated that administration of Imprime PGG in combination with anti-tumor Ab treatment reduces tumor growth and prolongs survival beyond that observed with either agent alone. In clinical trials, Imprime PGG in combination with tumor-targeting Abs has also been associated with improvements in objective tumor response and survival. Imprime PGG9s mechanism of action has been extensively studied in the context of the innate immune system. Whether Imprime PGG may also play a role in the modulation of adaptive immune responses and the cross-talk that exists between the innate immune system and T-cell responses was unclear. Recent work has revealed that Imprime PGG is capable of activating not only the innate immune system but also has the potential of inhibiting the polarization of macrophages into the immunosuppressive M2 phenotype. Since considerable cross-talk exists between the innate and adaptive immune cells in the immunosuppressive tumor microenvironment, we investigated whether Imprime PGG may also play a role in direct or indirect modulation of one of the adaptive immunosuppressive cell types, regulatory T cells (Tregs). We demonstrated that plasma from Imprime PGG treated whole blood (WB) was able to decrease the suppressive capability of Tregs on conventional CD4+ T cells. We also determined that plasma from Imprime PGG treated WB was able to enhance the proliferation of both CD4+ and CD8+ T cells in response to CD3/28 beads and to peptide stimulation in vitro. This enhanced proliferation was accompanied by upregulation of the transcription factor Tbet and increased IFN-γ production suggesting Imprime PGG may drive polarization of T cells to a Th1, anti-tumor phenotype. Interestingly, these effects were greatest when Imprime PGG was added to WB and not to isolated plasma, indicating that the effects of Imprime PGG on the T cell compartment are indirect and may involve cross-talk from the innate immune system. These data are the first to show that Imprime PGG may exert anti-tumor activity not only by re-directing the innate immune system against opsonized tumor cells, but also by re-orienting the adaptive immune compartment toward a Th1, anti-tumor phenotype. Citation Format: Steven M. Leonardo, Keith Gorden, Ross Fulton, Lindsay Wurst. Imprime PGG decreases regulatory T cell suppression and enhances T cell proliferation and differentiation revealing additional mechanisms for its anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5034. doi:10.1158/1538-7445.AM2015-5034

Abstract B019: Imprime PGG, a soluble β-glucan, binds to and activates dendritic cells resulting in enhanced T cell priming, expansion, and cytokine production

Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in both objective tumor response and survival. Mechanistic work has revealed that Imprime acts to stimulate a coordinated, anti-cancer immune response in conjunction with antibody therapy. Imprime represents a pathogen associated molecular pattern (PAMP) and, as such, can be efficiently and effectively recognized by cells of the innate immune system, triggering macrophage repolarization, neutrophil activation, monocyte-derived dendritic cell maturation, NK cell activation and, via cross-talk with the adaptive immune system, expansion of CD4 and CD8 T cells. As PAMPs have been shown to stimulate dendritic cells (DCs), which are critical for generating robust and durable anti-tumor responses, we sought to better understand the effects of Imprime on DCs. Here we demonstrate that Imprime binds to various DC subsets, stimulating the critical antigen presenting functional activity of these DC subsets. Following i.v. administration in mice, Imprime bound both classical splenic DCs, including CD8α+ cross-presenting DCs, and migratory DC subsets within peripheral lymph nodes. In vitro treatment of human whole blood showed binding in the inflammatory DC subset (Lin-HLA-DR+CD11c+CD16+) as well as the classical (Lin-HLA-DR+CD11c+CD1c+) DC subset. Binding of Imprime to both mouse and human DCs resulted in the upregulation of MHC class II and the co-stimulatory molecules CD80/86 that are critical for antigen presentation and T cell activation. Furthermore, in vivo treatment of mice with Imprime in combination with H-2Kb-restricted OVA257-264 peptide resulted in enhanced expansion of adoptively transferred OVA-specific OT-I CD8 T cells. In contrast to mice immunized with OVA peptide alone, which did not generate a functional CD8 T cell response, Imprime co-administration resulted in functional OT-I capable of ex vivo degranulation and production of the cytokines IFN-γ and IL-2. These data suggest that, in addition to previous studies showing Imprime primes monocytes, macrophages, and neutrophils, Imprime also enhances T cell activation and expansion by directly stimulating dendritic cell maturation and efficient antigen presentation. These data demonstrate that Imprime PGG treatment may enhance the adaptive immune response necessary for durable tumor control and, together with previously published data, indicate that Imprime PGG treatment triggers an orchestrated anti-cancer immune response involving both the innate and adaptive immune systems. Citation Format: Ross B. Fulton, Steven M. Leonardo, Kyle S. Michel, Michael E. Danielson, Keith B. Gorden, Jeremy R. Graff. Imprime PGG, a soluble β-glucan, binds to and activates dendritic cells resulting in enhanced T cell priming, expansion, and cytokine production. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B019.

Abstract A160: Imprime PGG binds to neutrophils through complement, Fc, and dectin-1 receptors, priming these cells for enhanced ROS production and tumor cell cytotoxicity

Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. In clinical studies, including randomized Phase 2 clinical trials in the 1st-line treatment of stage IV non-small cell lung cancer with bevacizumab, Imprime treatment has shown promising efficacy in both objective tumor response rates and survival. In numerous syngeneic and xenogeneic mouse tumor models, Imprime treatment in combination with an anti-tumor antibody reduced tumor growth and prolonged survival beyond that observed with antibody alone. Mechanistic studies have now demonstrated that, with endogenous anti-β-glucan antibodies (ABA), Imprime forms an immune complex. This Imprime-ABA complex then activates the classical complement pathway and is subsequently opsonized by iC3b. This immune complex then binds to and primes innate immune cells, including macrophages, monocytes and neutrophils, triggering a coordinated immune attack against antibody-targeted cancer cells. In this study, we sought to explore more fully the functional consequences of Imprime-ABA immune complex binding to, and priming of, neutrophils. Neutrophils are the first line of defense against fungal infections. Neutrophils detect fungal infections through recognition of β-glucans on the surface of yeast and fungi. These β-glucans serve as fungal pathogen associated molecular patterns (PAMPs) that are efficiently and effectively recognized by receptors on innate immune cells, initiating a coordinated immune response to infection. Here we demonstrate that Imprime, in individuals with high ABA levels, is recognized as a PAMP, directly interacting with neutrophils via multiple receptors, priming these cells and ultimately leading to enhanced effector responses including reactive oxygen species (ROS) generation and tumor cell killing. Our data now show that Imprime elicits enhanced neutrophil survival as measured by cell viability dye exclusion using flow cytometry. Imprime treatment also enhances the responsiveness of neutrophils to Fc receptor activation. As measured by luminol-based read-outs, ROS production in response to anti-Fc receptor antibody-coated beads is profoundly enhanced by Imprime treatment. Moreover, Imprime treated neutrophils show a remarkable surge in ROS production compared to vehicle–treated neutrophils when exposed to B cell lymphoma cells decorated with anti-CD20 monoclonal antibodies (i.e. rituximab). These Imprime-treated neutrophils also exhibit a robust increase in cytotoxicity against these B cell lymphoma cells coated with rituximab using in vitro Calcein AM dye release assays. We next sought to delineate which receptors might be most critical for Imprime binding and priming of neutrophils. As demonstrated by antibody blocking studies, the Imprime-ABA complex can bind to both complement receptors (notably CR3) and Fc receptors. Imprime can also block binding of a dectin-1 agonizing antibody to dectin-1 and can bind directly to HL-60 and HEK cells lines engineered to overexpress dectin-1 by retroviral transduction. Collectively, these data show for the first time that Imprime directly primes neutrophils to recognize and kill antibody-decorated tumor cells and interacts with the Fc, complement and dectin-1 receptors on the surface of neutrophils. Citation Format: Steven M. Leonardo, Ross B. Fulton, Lindsay R. Wurst, Keith B. Gorden, Adria Bykowski Jonas, Xiaohong Qui, Anissa SH Chan, Jeremy R. Graff. Imprime PGG binds to neutrophils through complement, Fc, and dectin-1 receptors, priming these cells for enhanced ROS production and tumor cell cytotoxicity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A160.

Modulation of B-cell tolerance by murine gammaherpesvirus 68 infection: requirement for Orf73 viral gene expression and follicular helper T cells.

Viruses such as Epstein–Barr virus (EBV) have been linked to mechanisms that support autoantibody production in diseases such as systemic lupus erythematosus. However, the mechanisms by which viruses contribute to autoantibody production remain poorly defined. This stems in part, from the high level of seropositivity for EBV (> 95%) and the exquisite species specificity of EBV. In this study we overcame these problems by using murine gammaherpesvirus 68 (MHV68), a virus genetically and biologically related to EBV. We first showed that MHV68 drives autoantibody production by promoting a loss of B‐cell anergy. We next showed that MHV68 infection resulted in the expansion of follicular helper T (Tfh) cells in vivo, and that these Tfh cells supported autoantibody production and a loss of B‐cell anergy. Finally, we showed that the expansion of Tfh cells and autoantibody production was dependent on the establishment of viral latency and expression of a functional viral gene called Orf73. Collectively, our studies highlighted an unexpected role for viral latency in the development of autoantibodies following MHV68 infection and suggest that virus‐induced expansion of Tfh cells probably plays a key role in the loss of B‐cell anergy.