Summit 2014: Scientific Presentations

Scientific Presentations

SoCal Flow Program Committee is honored to host these distinguished faculty for the scientific program presentations.


Zbigniew Darzynkiewicz, MD, PhD (Director of the Brander Cancer Research Institute at the New York Medical College and the Professor of Pathology, Medicine and Microbiology/Immunology at the same College): “Cytometric Assessment of DNA Damage – and mTOR – signaling, the Factors Contributing to Aging and Senescence”

Two mechanisms are being proposed as a cause of aging and senescence. Persistent DNA damage by reactive oxygen species (ROS), by-products of oxidative phosphorylation, is one of them (ROS mechanism). Activation of the mTOR/S6K1 signaling pathway by nutrients and growth factors is considered to be an alternative (mTOR) mechanism. The flow- and laser scanning- cytometric methods were developed to measure the level of the constitutive DNA damage/ROS- as well as of mTOR/S6K1- signaling. Specifically, activation of ATM and expression of γH2AX in untreated cells detected with phospho-specific Abs reports constitutive DNA damage induced by endogenous ROS. The phosphorylation of Ser235/236-of S6 ribosomal protein (RP), of Ser2448 of mTOR and of Ser65 of 4EBP1 informs on constitutive signaling along the mTOR/S6K1 pathway. The reported anti-aging agents: rapamycin, metformin, 2-deoxyglucose, berberine, resveratrol, vitamin D3 and aspirin, all decreased the level of constitutive DNA damage signaling. They also decreased intracellular level of ROS and mitochondrial trans-membrane potential ΔΨm, the marker of mitochondrial energizing. All these agents also reduced phosphorylation of mTOR, RP-S6 and 4EBP1 in A549, TK6, WI-38 cells and in mitogenically stimulated human lymphocytes. The most effective was rapamycin. Although the primary target of each on these agents may be different the data are consistent with the downstream mechanism in which the reduction of translation rate through mTOR/S6K1 signaling is coupled with a decrease in energy production through oxidative phosphorylation and leads to a decline in the level of ROS, mitochondrial potential and oxidative DNA damage. The decreased rate of translation induced by these agents may slow down cells hypertrophy and alleviate other features of cell aging/senescence. The reduced oxidative DNA damage is expected to lower predisposition to neoplastic transformation which may result from defective DNA repair at the sites coding for oncogenes or tumor suppressor genes. The data suggest that combined assessment of constitutive γH2AX expression, mitochondrial activity (ROS, ΔΨm) and mTOR signaling provides an adequate gamut of cell responses to evaluate effectiveness of suspected gero-suppressive agents by cytometry.

Marcin Kortylewski, PhD (Assistant Professor, Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute, City of Hope Medical Center): “Dissecting mechanisms of STAT3-mediated immunosuppression in mouse and human tumor models using flow Cytometry”.

Signal Transducer and Activator of Transcription-3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. We previously developed an immunostimulatory strategy based on targeted Stat3 and not gene silencing in Toll-like Receptor 9 (TLR9)-positive hematopoietic cells using siRNA-based strategy (CpG-siRNA conjugates). In our recent studies, we evaluated therapeutic effect of systemic STAT3 blocking/TLR9 triggering on disseminated acute myeloid leukemia (AML) in vivo utilizing multiple flow cytometry-based assays. We used a model of transplantable mouse Cbfb/MYH11/Mpl-induced leukemia, which mimics human inv(16)AML. Our results demonstrated that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential in majority of mice. These antitumor effects were mediated by effector CD8+ T cells but did not require host’s TLR9+ antigen-presenting cells. Instead, CpG-Stat3 siRNA had direct immunogenic effect on AML cells in vivo leading to upregulation of the surface expression of MHC class II, costimulatory molecules and proinflammatory mediators, such as IL-12, while downregulating expression of coinhibitory PD-L1 molecule. Systemic administration of CpG-Stat3 siRNA generated potent tumor antigen-specific immune responses, increased the ratio of tumor-infiltrating CD8+ for T cells to Tregs in various organs and resulted in CD8+ T cell-dependent regression of leukemia. TLR9 is also commonly expressed in human AML, including leukemic progenitors (CD34+CD38+) and stem cells (CD34+CD38). Our preliminary results indicate that STAT3 blocking/TLR9 triggering can alleviate immunosuppressive potential of primary patients’ AML cells while promoting autologous T cell activation. These findings underscore the potential of using CpG-Stat3 siRNA strategy to break tumor tolerance and induce potent immunity against AML and potentially other TLR9-positive blood cancers.

Craig Walsh, PhD (Associate Director, Institute for Immunology, Member, Multiple Sclerosis Research Center, Associate Professor, Department of Molecular Biology & Biochemistry, UC Irvine, California): “Using flow cytometry to investigate T cell death mechanisms and immune tolerance”.

Our group is interested in the mechanisms that control T cell homeostasis, the process that dynamically regulates the expansion and contraction of antigen specific T cells. In particular, we are focused on the means by which cell death modulates this important balance. A major project in the lab is to understand the biology of a protein known as FADD, and how this protein contributes to T cell function. Although FADD is known to activate a form of cell death known as apoptosis, we have discovered that FADD also contributes to the activation and survival of antigenically-stimulated T cells. These intriguing results raise an important question: how can a single molecule both induce apoptosis under some circumstances and promote survival and expansion of T cells under other conditions. Understanding this paradox will contribute to our knowledge of how T cell homeostasis is maintained and will also help us understand how such a balance is disturbed in autoimmunity and lymphoproliferative disease. A second protein the lab is investigating is DRAK2, an apoptosis regulating kinase. Mice with an engineered deficiency in this death-inducing kinase are known to be hyper-sensitive to antigenic stimulation under specific conditions. We are dissecting the signaling pathways that lead to such hyper-responsiveness. We are also conducting studies to determine how the structure of the kinase regulates its function. Finally, we are determining if this kinase plays a role in the prevention of autoimmune diseases such as rheumatoid arthritis. We believe that a more complete understanding of the physiological roles of such apoptotic regulatory pathways will contribute greatly to the amelioration of diseases of the immune system. Flow cytometry is a major tool utilized by our group to investigate signal transduction and immune responsiveness in T cells.

Scott G Kitchen, PhD (Assistant Professor of Medicine, Director, UCLA Humanized Mouse Core Laboratory, Division of Hematology/Oncology. UCLA, California): “A Stem Cell based approach for engineering Anti-HIV Immunity”.

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling HIV replication.  We are interested in the development of ways to genetically enhance the HIV-specific CTL response to allow long-term viral suppression or viral clearance.  We have previously demonstrated that human hematopoietic stem cells (HSCs) can be modified with a molecularly cloned HIV-specific T cell receptor (TCR) to develop into fully functional CTL that can suppress HIV replication in a humanized mouse model.  A fundamental issue with this approach is the notion that these TCRs are human leukocyte antigen (HLA)-restricted, and therefore their use therapeutically is highly restricted to individuals with specific HLA genotypes.

We are currently investigating the use of non-HLA restricted chimeric antigen receptors (CARs) that allow the recognition of HIV when expressed by a CTL.  Here we report that the use of a CD4-ζ chain CAR that contains the extracellular portion of the CD4 molecule fused to the intracellular TCR-zeta signaling domain.  The lentiviral vector containing the CD4-ζ CAR also expresses small hairpin (sh)RNAs specific for CCR5 and the HIV LTR to protect the developing cells from infection.  We determined that CD4-ζ CAR transduced HSCs can differentiate into functional CD4 and CD8 T cells as well as NK cells in vivo in humanized mice.  Importantly, we found that CD4-ζ containing cells can functionally respond to HIV over long periods of time and significantly suppress HIV replication following infection.  Thus, this system allows the close examination of the engineering of antiviral immunity and non-HLA restricted HIV-specific CTL responses in vivo.  Our results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.

Maurice O’Gorman, PhD, MSc, D(AMBLI), MBA (Chief, Division of Laboratory Medicine; Director, Clinical Laboratories; Director, Clinical Immunology and Flow Cytometry Laboratory; Professor, Clinical Pathology and Pediatrics, Children’s Hospital Los Angeles, California): “The Use of Flow Cytometry for the Assessment of Primary Immunodeficiency Disease”.

Over the past 3 decades, flow cytometry has emerged as an invaluable technology in clinical laboratories and has contributed significantly to the biological understanding and the diagnostic evaluation of patients with suspected immune system abnormalities. The unparalleled ability to simultaneously identify at a single cell level, characteristic physical properties (light scatter), functions, and specific gene products at rates of thousands of cells per second has resulted in the development of a large repertoire of diagnostic, prognostic, and monitoring assays. Primary immunodeficiency represents a unique opportunity for the application of flow cytometry based testing with over 120 classified single genetic disorders for which the majority have known genetic associations. Many genetic abnormalities result in the expression of cellular abnormalities which are readily detected by flow cytometry. Cellular abnormalities detected by flow cytometry can be broadly grouped as follows: (1) abnormalities in the relative representation of a specific leukocyte subset(s), e.g. absence of B cells in X-linked a-gammaglobulinemia; (2) loss or abnormal expression of a specific cell-associated marker(s), e.g. absence of CD40-ligand on activated T cells in X-linked hyper IgM syndrome; (3) loss or abnormal expression of a cellular function, e.g. absence of an oxidative burst in the granulocytes of patients with chronic granulomatous disease. The presentation will involve an overview of the primary immunodeficiency diseases in the context of the current IUIS classification tables, a review of the associated abnormalities, and a discussion of the flow cytometry assays that have been developed to measure these abnormalities.

Keith Kelley, MS (Senior Scientist, Department of Medical Sciences, Amgen Inc., Thousand Oaks, California): “Applications of Flow Cytometry to the Characterization of Aggregates in Therapeutic Proteins”.

The formation of particulates and aggregates of therapeutic proteins during their production, fill into syringes or vials and storage is a major concern because the degree of aggregate formation varies as a function of (even) minor processing and storage differences. Both sub visible and visible particulates and aggregates can induce immunogenic responses directed against the therapeutic. The effects of such interactions can alter their pharmacokinetic properties, can neutralize the activity of the therapeutic, can induce hypersensitivity reactions and in some cases can react with endogenous host proteins to mediate life-threatening events. Detection and characterization of particulates and aggregates can be challenging due to their potentially large size range (which transverses into the sub visible space) and varied physical properties. No single analytical platform or method serves to meet the needs of this workstream. In this study we therefore compared flow and imaging cytometry with currently accepted standard methods (MFI™ Micro-Flow Imaging, HIAC) to determine particle count, relative size and morphological characteristics. In addition particles were physically separated on a cell sorter based on scatter profiles and subjected to subsequent microscopic observation, imaging cytometry and component analysis. Overall, we find that flow and imaging cytometry technologies hold value as orthogonal techniques in particulate and aggregate studies, with the size range of detected particles greater than MFI or HIAC. Moreover, the cytometry platforms offer a means to characterize particles in much the same way that they are commonly leveraged to provide detailed characterization of cells.