PhD project by Natasja Wulff Pedersen

Name: Natasja Wulff Pedersen
Project Title: MHC multimer-binding CD8 T cells: Optimized detection, automated analysis and identification of antigen specific CD8 T cells in Narcolepsy type 1
Group: T-cells and cancer
Supervisor: Sine Reker Hadrup

Project Description: 

Antigen specific CD8 T cells of the immune system are crucial mediators of immunity against intracellular infections and cancer and when dysregulated can be involved in autoimmune diseases. CD8 T cells recognize their antigen in the form of fragments of proteins, called peptides, on the surface of target cells. Here they are presented in the context of an MHC molecule and the interaction between the T cell receptor on the surface of the CD8 T cell and the peptide-MHC complex on the target cell is instrumental to the function of antigen specific T cells. This interaction can be investigated using MHC multimers and flow cytometry, although it’s complexity poses a challenge to the detection. In this thesis three different aspects of describing antigen specific CD8 T cells with MHC multimers are touched upon.

In the first study, the possibility to optimize detection of antigen specific CD8 T cells with MHC multimers was investigated. We found that detection of these cells was possible when using different tags to identify MHC multimers and we propose a simple way to optimize flow cytometry instrument settings in order to get the best performance and most accurate results.

In the second study, automated analysis of MHC multimer-binding T cells was tested. Automated gating tools have been developed to deal with the inherent subjectivity, low reproducibility and standardization of manual analysis of flow cytometry data, as well as the challenge to analyze increasingly complex data manually. However, these tools have not been implemented in the broad scientific community, despite efforts to describe their advantages and we therefore wanted to test the use of these tools by a non-computational expert. We found that the overall performance of automated gating tools was good, although only one tool was able to detect CD8 T cells of very low frequency. Furthermore, we identified and evaluated major obstacles to the widespread use of automated gating tools by researchers without programming skills.

In the third study, CD8 T cells with the ability to recognize proteins expressed by neurons that are involved in the sleep disorder narcolepsy, were described. Narcolepsy is caused by the loss of so called hypocretin neurons in the brain that play a role in regulating the sleep/wake cycle. The neuronal loss is thought to be the result of an autoimmune attack, although this has never been proven. We show for the first time a broad presence of CD8 T cells that recognize several different proteins expressed by hypocretin neurons, in both narcolepsy patients and healthy controls. Although these potentially auto-reactive CD8 T cells were present in both patients and controls, their levels differed between the groups. Even though these findings do not provide conclusive evidence for the autoimmune hypothesis of narcolepsy, they do represent an important step on the way to full understanding of the mechanism behind narcolepsy development.

Kontakt

Natasja Wulff Pedersen
Postdoc
DTU Sundhedsteknologi

Kontakt

Sine Reker Hadrup
Sektionsleder, Professor
DTU Sundhedsteknologi
35 88 62 90