Practical Course HP-F2: Bioinformatics, Applied Tumor Immunity, and Stress Response in Stem Cells

Type: Practical Course with Student Seminars

part I: Analysis of differential expression using RNA-seq data

Date: 5.-11. November 2020

Location: TP Conference Center, INF 582; conference room: Palo Alto

Hosts/Supervisors: Cihan Erkut (cihan.erkut@nct-heidelberg.de), Michelle Michelhans

Topics:

High-throughput technologies have become essential tools in almost every biomedical research field. These techniques allow to perform a large number of measurements simultaneously, and thus enable the profiling of biological systems at unprecedented speed and resolution.
One example is gene expression profiling, where the expression levels of thousands of genes are determined in parallel to create a global picture of the cellular state. The comparison of expression profiles to determine differential expression between healthy and diseased cells can reveal detailed insights into the biology of the disease.

Content:

  • Experimental design
  • Processing of RNA-seq data
  • Statistical considerations
  • Diagnostic plots and result visualization
  • Annotation and downstream analysis

 

part II: CARTs & AAVLPs: immunologic weapons against cancer

Date: 16.-20. November 2020

Location: DKFZ Teaching Lab

Hosts/Supervisors: Silke Uhrig-Schmidt, Patrick Schmidt (silke.uhrig-schmidt@nct-heidelberg.de, patrick.schmidt@nct-heidelberg.de)

Topics:

In recent years, immunotherapy has become of great interest to researchers, clinicians and pharmaceutical companies, particularly in its promise to treat various forms of cancer. By activating a patient’s own immune system, especially CAR T cell therapy achieved success in specifically eradicating tumor cells of hematologic malignancies. However, CAR T cells are sharp weapons that may overshoot or miss their target which often leads to life-threatening consequences. Thus, in order to offer a safe and efficient immunotherapy, CAR T cells need to be precisely controlled. One way to do so is the development of AAV-based virus-like particles (AAVLPs) that structurally mimic the cellular target of a CAR. The specific CAR-AAVLP interaction leads to temporal unavailability of the CAR on the T cell surface which is directly linked to reduced cytotoxic activity of the T cell. Likewise, a control of undesired cross-reactivity of CAR T cells by designer AAVLPs could be anticipated for future use in cancer immunotherapy.

Content:

• Isolation, sorting and activation of T cells from whole blood by Ficoll gradient
• Lentiviral transduction of T cells with CARs, expression analysis (FACS) and assessment of killing activity by xcelligence assay
• Small scale AAVLP production, characterization by western blot and Dynabead-assay (FACS analysis)
• Medium scale AAVLP purification by density gradient ultracentrifugation and titration by ELISA
• AAVLP-competition assay with CAR+ T cell line (Transduction followed by FACS analysis)

part III: Characterization of the stress response of stem cells

Date: 14.-18. December 2020

Location: DKFZ Teaching Lab

Hosts/Supervisors: Marieke Essers (m.essers@dkfz.de), Sarah Geiger


Topics:

Infection is a common, natural form of stress, with which the body is regularly challenged. During infection or inflammation, cells of the immune system are responsible for fighting the invading pathogens, leading to high consumption of blood and immune cells. Restoration of the balance of the hematopoietic system following successful elimination of the infection is a crucial part of the recovery of the body. Restoring the balance of the hematopoietic system depends on the replacement of lost immune cells by hematopoietic stem cells (HSCs). Over the years, the biology of HSCs and their progeny has been studied in great detail during homeostasis. However little is known about how stress scenarios, such as infection-induced inflammatory responses, impact on HSC self-renewal and differentiation, controlling emergency hematopoiesis.


Content:

• isolation of stem cells
• phenotypic characterization of the stem cell response (FACS analysis, immunofluorescence)
• analysis of transcriptional and protein changes using qPCR and western blot
• functional consequences of the stress response

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