Agne Petrosiute

Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity

Cyclin suppresses antitumor immunity Despite the dramatic success of cancer immunotherapy, many types of cancer do not respond. Understanding why could help us to find ways to enhance the overall responsiveness of tumors to immunotherapies. Dorand et al. report that cyclin-dependent kinase 5 (Cdk5), an enzyme that is highly expressed by neurons in many brain cancers, may dampen the ability of T cells to reject tumors. In a mouse model of medulloblastoma, if tumors were Cdk5 deficient, T cells were able to remove them. This heightened antitumor immunity correlated with reduced expression of the inhibitory molecule programmed cell death ligand 1 (PD-L1), a target of current cancer immunotherapies. Science, this issue p. 399 Cyclin-dependent kinase 5 expression by tumors inhibits antitumor immunity in human and mouse medulloblastoma. Cancers often evade immune surveillance by adopting peripheral tissue– tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)–induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4+ T cell–mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.

Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex

Fluorescent imaging coupled with high-resolution femtosecond pulsed infrared lasers allows for interrogation of cellular interactions deeper in living tissues than ever imagined. Intravital imaging of the central nervous system (CNS) has provided insights into neuronal development, synaptic transmission, and even immune interactions. In this review we will discuss the two most common intravital approaches for studying the cerebral cortex in the live mouse brain for pre-clinical studies, the thinned skull and cranial window techniques, and focus on the advantages and drawbacks of each approach. In addition, we will discuss the use of neuronal physiologic parameters as determinants of successful surgical and imaging preparation.

Chemokines as Cancer Vaccine Adjuvants

We are witnessing a new era of immune-mediated cancer therapies and vaccine development. As the field of cancer vaccines advances into clinical trials, overcoming low immunogenicity is a limiting step in achieving full success of this therapeutic approach. Recent discoveries in the many biological roles of chemokines in tumor immunology allow their exploitation in enhancing recruitment of antigen presenting cells (APCs) and effector cells to appropriate anatomical sites. This knowledge, combined with advances in gene therapy and virology, allows researchers to employ chemokines as potential vaccine adjuvants. This review will focus on recent murine and human studies that use chemokines as therapeutic anti-cancer vaccine adjuvants.

Extravascular CX3CR1+ cells extend intravascular dendritic processes into intact central nervous system vessel lumen

Within the central nervous system (CNS), antigen-presenting cells (APCs) play a critical role in orchestrating inflammatory responses where they present CNS-derived antigens to immune cells that are recruited from the circulation to the cerebrospinal fluid, parenchyma, and perivascular space. Available data indicate that APCs do so indirectly from outside of CNS vessels without direct access to luminal contents. Here, we applied high-resolution, dynamic intravital two-photon laser scanning microscopy to directly visualize extravascular CX3CR1+ APC behavior deep within undisrupted CNS tissues in two distinct anatomical sites under three different inflammatory stimuli. Surprisingly, we observed that CNS-resident APCs dynamically extend their cellular processes across an intact vessel wall into the vascular lumen with preservation of vessel integrity. While only a small number of APCs displayed intravascular extensions in intact, noninflamed vessels in the brain and the spinal cord, the frequency of projections increased over days in an experimental autoimmune encephalomyelitis model, whereas the number of projections remained stable compared to baseline days after tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma. Our observation of this unique behavior by parenchyma CX3CR1+ cells in the CNS argues for further exploration into their functional role in antigen sampling and immune cell recruitment.

Triterpenoid inducers of Nrf2 signaling as potential therapeutic agents in sickle cell disease: a review

Sickle cell disease (SCD) is an inherited disorder of hemoglobin in which the abnormal hemoglobin S polymerizes when deoxygenated. This polymerization of hemoglobin S not only results in hemolysis and vasoocclusion but also precipitates inflammation, oxidative stress and chronic organ dysfunction. Oxidative stress is increasingly recognized as an important intermediate in these pathophysiological processes and is therefore an important target for therapeutic intervention. The transcription factor nuclear erythroid derived-2 related factor 2 (Nrf2) controls the expression of anti-oxidant enzymes and is emerging as a protein whose function can be exploited with therapeutic intent. This review article is focused on triterpenoids that activate Nrf2, and their potential for reducing oxidative stress in SCD as an approach to prevent organ dysfunction associated with this disease. A brief overview of oxidative stress in the clinical context of SCD is accompanied by a discussion of several pathophysiological mechanisms contributing to oxidative stress. Finally, these mechanisms are then related to current management strategies in SCD that are either utilized currently or under evaluation. The article concludes with a perspective on the potential of the various therapeutic interventions to reduce oxidative stress and morbidity associated with SCD.

Utilization of Multiphoton Imaging For Real-Time Fate Determination of Mesenchymal Stem Cells in an Immunocompetent Mouse Model.

The clinical application of Mesenchymal Stem Cells (MSCs) for the treatment of a variety of diseases is the focus of intense research. Despite large research efforts many questions regarding MSC biology in vivo remain unanswered. For instance, we do not know for certain whether MSCs exert their therapeutic effects directly within the target tissue or indirectly by influencing the polarization of other cell types, such as macrophages, which can then home to the target tissue microenvironment. To help address this issue, the application of intravital multiphoton microscopy allows for the determination of the dynamic action of intact MSCs versus endogenous host cells at the target tissue site in real time.

Viewing transplantation immunology through today's lens: new models, new imaging, and new insights.

The last several decades have brought significant immunologic advances in the fields of inflammation, infection, and transplantation tolerance. Heretofore, our understanding of how complex immune interactions occur has been limited to static in situ tissue analysis and in vitro dynamic studies using isolated cells devoid of stromal elements typically present in vivo. Recent advances in molecular, flow cytometry, and intravital imaging have provided new insight into the dynamic interactions occurring among a variety of cells within the bone marrow (BM) and immune systems, ranging from undifferentiated hematopoietic progenitors to fully committed effector memory cells, which will likely have direct clinical and translational implications. In this review we highlight how the application of these cutting-edge technologies will sculpt the landscape of the next generation of immunologic advances.

Multifactorial regulators of tumor programmed death-ligand 1 (PD-L1) response

Tumor cells hijack physiological mechanisms to create favorable conditions that allow them to survive and thrive within the hostile tissue and immune microenvironments. The identification and subsequent therapeutic blockade against immune checkpoint molecules including cytotoxic T lymphocyte associated antigen 4 (CTLA-4; CD152), programmed cell death protein 1 (PD-1; CD279) and its ligand programmed death-ligand 1 (PD-L1; CD274; B7-H1) have evoked much excitement in cancer immunotherapy against a variety of chemo-refractory cancers (1-4). Since the initial characterization of the PD-1/PD-L1 axis over 2 decades ago (5-7), over 4,000 articles have been published exploring how this immune checkpoint receptor-ligand pair influence tumor development, survival, and metastasis (2,8). Surprisingly, however, only a handful of studies have described how tumor PD-L1 is regulated at the transcriptional and post-translational levels. Recent studies by Mezzadra et al. (9). and Burr et al. (10) describe a novel post-translational mechanism by which PD-L1 is regulated within primary human dendritic cells and a variety of human tumor cell types, adding to our understanding of how this critical immune regulatory axis is regulated.

Abstract A050: Cdk5 disruption attenuates tumor PD-L1 expression via regulation of IFN-γ signaling components and promotes antitumor immunity

In order to escape normal immune surveillance, tumors mimic peripheral tissue tolerance mechanisms such as the expression of programmed cell death-ligand 1 (PD-L1), the inhibition of which can lead to potent anti-tumor responses. Here we show the contribution of cyclin dependent kinase 5 (Cdk5) to immune evasion. Cdk5 is a proline-directed serine/threonine kinase, which is highly expressed in post-mitotic neurons and directs a variety of homeostatic functions from cytoskeletal rearrangement to neurotransmitter signaling. Studies in xenograft models have linked Cdk5 activity to the generation and metabolic activity of primary tumors within the central nervous system (CNS). Here we show that Cdk5, expressed in both murine and human medulloblastoma (MB) cell lines, plays a major role in the ability of MB to avoid immune detection. First, we observed that decreased Cdk5 expression was associated with an increase in the number of infiltrating CD3+ cells in the tumor mass of clinical MB samples. Additionally, using publicly available datasets, we found that decreased Cdk5 expression is correlated with better overall survival or fewer distant metastasis in melanoma, breast cancer, glioma, and lung adenocarcinoma. Next, using a CRISPR/Cas-9 approach, we silenced Cdk5 in a murine model of Sonic Hedgehog (SHH) pathway MB. Rather than causing intrinsic growth defects in MB, interference of Cdk5 activity sensitizes tumors to killing by the normal host immune system in a CD4+ T cell-dependent manner. This rejection is associated with increased IFN-γ expression in the tumor microenvironment, as well as increased PD-L1 expression by myeloid populations in both subcutaneous and intracranial tumors. Mechanistically, we observed an attenuated response to IFN-γ stimulated expression of programmed death ligand 1 (PD-L1) on MB cells. This blunted response was recapitulated when MB cells were treated with Roscovitine, a non-selective Cdk5 inhibitor. Furthermore, using a quantitative global phosphoproteomics approach, we found that Cdk5 knockdown also increased phosphorylation of S-440 and S-443 on interferon regulatory factor binding protein 2 (IRF2BP2), an upstream co-repressor of interferon regulatory factor 2 (IRF-2). Increased phosphorylation of IRF2BP2 corresponded with stable expression of IRF-2 with a concomitant decrease in surface expression of PD-L1. These observations highlight a critical role for Cdk5 in the immune escape mechanisms of primary CNS tumors and provides new therapeutic targets for PD-1/PD-L1 directed immunotherapy. Citation Format: Rodney D. Dorand, Jr., Joseph Nthale, Jay T. Myers, Deborah S. Barkauskas, Stefanie Avril, Steven M. Chirieleison, Tej K. Pareek, Derek W. Abbott, Duncan S. Stearns, John J. Letterio, Alex Y. Huang, Agne Petrosiute. Cdk5 disruption attenuates tumor PD-L1 expression via regulation of IFN-γ signaling components and promotes antitumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A050.

Establishing a Novel In Vivo Platform to Interrogate the Effects of Pharmacologic and Molecularly Targeted Modulation of CDK5 in the Invasive and Metastatic Potential of Medulloblastoma in Live Experimental Animals

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Originating from embryonal neuroepithelial cells, MB accounts for approximately 20% of all primary pediatric CNS tumors. MB often exhibits an aggressive growth pattern and is one of only a few tumor types with a metastatic potential. It can attain several centimeters in size resulting in mass-occupying lesions most often located in the posterior fossa. Due to invasion into regional subarachnoid and ventricular spaces, widespread seeding of the subarachnoid space may occur. Unfortunately, effective therapy for metastatic MB continues to be a great clinical challenge; therefore, insights into the mechanisms by which MB attains its invasive properties may spark future development of rational new therapeutic strategies.