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Binding Kinetics of Proteins at Immune-Cell Contacts

Chouliara, Manto LU (2022)
Abstract
Protein-protein interactions are crucial in numerous cellular functions and biological processes that take place inside our body. It is therefore not surprising that these interactions also govern the response of our body´s defence mechanism, the so-called immune system, towards an infection. Understanding how proteins interact entails studying the binding affinity (strength) and the lifetime (duration) of the protein-protein interaction to better decompose how an immune response is initiated and how we can explore this knowledge to treat diseases. In this thesis, total internal fluorescence microscopy (TIRF) and single-molecule imaging were used to observe and characterize protein-functionalized supported lipid bilayers (SLBs) interacting... (More)
Protein-protein interactions are crucial in numerous cellular functions and biological processes that take place inside our body. It is therefore not surprising that these interactions also govern the response of our body´s defence mechanism, the so-called immune system, towards an infection. Understanding how proteins interact entails studying the binding affinity (strength) and the lifetime (duration) of the protein-protein interaction to better decompose how an immune response is initiated and how we can explore this knowledge to treat diseases. In this thesis, total internal fluorescence microscopy (TIRF) and single-molecule imaging were used to observe and characterize protein-functionalized supported lipid bilayers (SLBs) interacting with immune cells to obtain the binding kinetics of various protein-protein pairs.

In the first part of this thesis, the interaction between the rat CD2 (rCD2) adhesion protein and its ligand rat CD48T92A (rCD48T92A), a high-affinity mutant of the wild type rat CD48, was used to establish a new method of obtaining single-cell binding affinities of T cells interacting with SLBs using imidazole titrations. The results showed a relatively small spread in the rCD2-rCD48T92A binding affinity values despite the considerable spread of receptor densities within the cell population. The lifetime of the rCD2/rCD48T92A interaction was also investigated using single-molecule imaging and tracking displaying a similarly small lifetime spread within the cell population. Using both these methods, the single-cell binding affinity and lifetime of the cell population can be investigated and their spread can provide information concealed with
population-average techniques.

The second part of the thesis focused on the CD4 co-receptor whose role in initiating an immune response is ambiguous. Even though the CD4 co-receptor increases the sensitivity of T cell signalling manyfold, it binds to its ligand, peptide major histocompatibility complex II (pMHCII), with the lowest binding affinity known to this day. The CD4-MHC II interaction is so weak that adhesion molecules are needed to ensure a successful CD4-MHC II contact formation. For this reason, the influence of an adhesion molecule, rat CD2, on the obtained binding kinetics of the human CD4 co-receptor was initially examined showing that the accumulation of CD4 was influenced when having a high concentration of bound CD2 inside the cell-SLB contacts. Later, the studies focused on the CD4-TCR-MHC II ternary complex where it was demonstrated that the presence of L3-12 TCR strongly supported the CD4-MHC II interaction by increasing the local density of MHC II inside the cell-SLB contacts. However, the presence of TCR did not seem to significantly influence the specific affinity for CD4 to MHC II. Lastly, CD4 binding studies showed that the co-receptor did not noticeably affect the TCR-MHC II binding at physiological levels of hCD4 in the SLB. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • PhD Ganzinger, Kristina, AMOLF, Netherlands
organization
publishing date
type
Thesis
publication status
published
subject
keywords
T cells, Binding Kinetics, Affinity, Lifetime, CD4, CD2, TIRF Microscopy, SLBs
pages
110 pages
publisher
Lund University, Faculty of Science
defense location
KC:A, Lund. Join via zoom: https://lu-se.zoom.us/j/67035915755
defense date
2022-09-28 09:15:00
ISBN
978-91-7422-877-9
978-91-7422-876-2
project
Binding Kinetics of Proteins at Immune-Cell Contacts
language
English
LU publication?
yes
id
e9eee4ad-d756-4eb8-bd66-d92e35fbe554
date added to LUP
2022-08-21 16:53:19
date last changed
2022-08-30 11:35:39
@phdthesis{e9eee4ad-d756-4eb8-bd66-d92e35fbe554,
  abstract     = {{Protein-protein interactions are crucial in numerous cellular functions and biological processes that take place inside our body. It is therefore not surprising that these interactions also govern the response of our body´s defence mechanism, the so-called immune system, towards an infection. Understanding how proteins interact entails studying the binding affinity (strength) and the lifetime (duration) of the protein-protein interaction to better decompose how an immune response is initiated and how we can explore this knowledge to treat diseases. In this thesis, total internal fluorescence microscopy (TIRF) and single-molecule imaging were used to observe and characterize protein-functionalized supported lipid bilayers (SLBs) interacting with immune cells to obtain the binding kinetics of various protein-protein pairs.<br/><br/>In the first part of this thesis, the interaction between the rat CD2 (rCD2) adhesion protein and its ligand rat CD48T92A (rCD48T92A), a high-affinity mutant of the wild type rat CD48, was used to establish a new method of obtaining single-cell binding affinities of T cells interacting with SLBs using imidazole titrations. The results showed a relatively small spread in the rCD2-rCD48T92A binding affinity values despite the considerable spread of receptor densities within the cell population. The lifetime of the rCD2/rCD48T92A interaction was also investigated using single-molecule imaging and tracking displaying a similarly small lifetime spread within the cell population. Using both these methods, the single-cell binding affinity and lifetime of the cell population can be investigated and their spread can provide information concealed with<br/>population-average techniques.<br/><br/>The second part of the thesis focused on the CD4 co-receptor whose role in initiating an immune response is ambiguous. Even though the CD4 co-receptor increases the sensitivity of T cell signalling manyfold, it binds to its ligand, peptide major histocompatibility complex II (pMHCII), with the lowest binding affinity known to this day. The CD4-MHC II interaction is so weak that adhesion molecules are needed to ensure a successful CD4-MHC II contact formation. For this reason, the influence of an adhesion molecule, rat CD2, on the obtained binding kinetics of the human CD4 co-receptor was initially examined showing that the accumulation of CD4 was influenced when having a high concentration of bound CD2 inside the cell-SLB contacts. Later, the studies focused on the CD4-TCR-MHC II ternary complex where it was demonstrated that the presence of L3-12 TCR strongly supported the CD4-MHC II interaction by increasing the local density of MHC II inside the cell-SLB contacts. However, the presence of TCR did not seem to significantly influence the specific affinity for CD4 to MHC II. Lastly, CD4 binding studies showed that the co-receptor did not noticeably affect the TCR-MHC II binding at physiological levels of hCD4 in the SLB.}},
  author       = {{Chouliara, Manto}},
  isbn         = {{978-91-7422-877-9}},
  keywords     = {{T cells; Binding Kinetics; Affinity; Lifetime; CD4; CD2; TIRF Microscopy; SLBs}},
  language     = {{eng}},
  month        = {{08}},
  publisher    = {{Lund University, Faculty of Science}},
  school       = {{Lund University}},
  title        = {{Binding Kinetics of Proteins at Immune-Cell Contacts}},
  url          = {{https://lup.lub.lu.se/search/files/122935523/Thesis_Manto_Chouliara_WEB.pdf}},
  year         = {{2022}},
}