Keynote Speaker: Stefan Bauer, Universität Marburg, Institut für Immunologie, Marburg, Germany
Immunology and cytometry could be considered the old married couple in the field of cytometry: in the first days of cytometric analysis, the investigation of immune cell populations resulted in paving the way for their phenotyping and characterization.
Most higher organisms have two immune responses: the adaptive or acquired immune response and the innate immune response. In this session, we will focus on the innate immune system, which recognizes pathogens by germ line encoded pattern recognition receptors and defends invading pathogens either directly or by activating the adaptive immune response. With new discoveries and applications in the cytometry field, even minute changes in physiology within a single cell can now be tracked and monitored. Immune cells are now able to be categorized according to their secretory capacities or even transient changes in intracellular protein levels and modifications of signaling molecules.
Toll-like receptors and the recognition of nucleic acids
Philipps-Universität Marburg, Germany;
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are indispensable components in living organisms. DNA stores genetic information while RNA serves as genome for some viruses is a prerequisite for protein translation and has important regulatory functions.
Evidence accumulated over the past few decades that nucleic acid when released from pathogens or host cells can activate the innate immune system and lead to enhanced anti-tumour activity and type I interferon production. Details on nucleic acid recognizing receptors such as Toll-like receptors will be discussed.
Exploring innate immune signaling via genome-wide shRNA screens
University of Bonn, Germany;
The innate immune system is the first line of defense against invading pathogens. Members of the Toll-like receptor (TLR) family act as primary sensors that detect a wide range of microbial components and elicit innate immune responses. All TLR signaling pathways culminate in the activation of the transcription factor nuclear factor-κB (NF-κB), which controls the expression of an array of inflammatory genes. In addition, each TLR elicits specific antimicrobial defenses owing to differential usage of intracellular adaptor proteins or due to activation of cell-specific signaling cascades. Given the extraordinary complexity of immune regulation via TLRs, this can be best investigated with systematic, genome-wide loss of function approaches. Here we describe a comprehensive strategy for large-scale pooled short hairpin RNA (shRNA) screening in macrophages that combines FACS sorting for genome-wide negative selection and next-generation sequencing for deconvolution of candidate genes. We applied this methodology to explore the TLR4 signalling cascade and were able to identify known regulators as well as unknown candidates. We are convinced that a more detailed understanding of the molecular networks underlying TLR signal transduction will reveal new drug targets and strategies to fine tune TLR-mediated immune responses.
NK Cells in Hodgkin Lymphoma are impaired but can be activated
University Hospital Cologne, Germany, Department of Internal Medicine I, Laboratory for Immunotherapy;
Introduction: NK cells represent the key component of the innate immune system to recognize and eliminate cancer cells. Defects in NK cell function including impaired cytotoxicity/cytokine secretion, aberrant receptor expression profile, NK cell number and NK cell anergy are reported in non Hodgkin lymphoma and correlate with a bad prognosis. So far, nothing is known about the phenotype of peripheral NK cells and serum levels of ligands for NK cell receptors in Hodgkin Lymphoma (HL) patients. Here, cytotoxicity, expression pattern of activating NK cell receptors and the serum levels of several ligands for the key cytotoxic receptors NKG2D and NKp30 are determined.
Methods: The cytotoxicity of NK cells isolated from HL patients was analysed by europium release assay using the HL cell line L428 as target cells. The serum level of the NKp30-ligand BAT3 and ligands for NKG2D (MICA, MICB and ULBP1,2,3) was estimated in sera of 117 HL patients and 40 healthy donors by ELISA. The expression pattern of NKp30, NKp44, NKp46, CD16 and the activation markers CD25, CD69 and CD71 was determined by 4-colour FACS analysis of peripheral blood lymphocytes.
Results: The cytotoxicity assays reveal a significantly reduced killing efficacy of NK cells from HL patients against the Hodgkin cell line L428 in comparison to NK cells from healthy donors. Correlating with the impaired NK cell function, we observed that the serum level for BAT3 and MICA was significantly elevated in HL patients, whereas other ligands (MICB and ULBP1,2,3) remained unchanged. NKG2D showed a significantly decreased expression on NK cells of HL patients. No significant difference was observed for all other receptors and activation markers tested.
Conclusion: Our results suggest that soluble BAT3 and MICA, ligands for NKp30 and NKG2D, contribute to the NK cell inhibition in HL patients. Since soluble ligands for NK cell receptors are known to inhibit NK cell-cytotoxicity, the release of these ligands might represent an immune escape mechanism of HL tumors to avoid detection and killing by the innate immune system. To overcome NK cell inhibition in HL patients we design, express and purify bispecific proteins (immunoligands) that target NKG2D and a HL-specific tumorantigen. Work to activate HL-derived NK cells ex vivo will be discussed.