T-cells are central to our success as a species. They confer specific and long-term immunity in a process known as adaptive immunity. During adaptive immune response, pathogen ingested by peripheral sentinel cells are brought to the local lymph nodes and presented to T-cells. T-cell recognizes the antigen via its receptor complex (TCR-CD3). The high affinity binding primes the cell for activation. With a positive costimulationary signal from CD28, the T-cell is fully activated, resulting in IL-2 secretions and cellular proliferation. Clinicians are increasingly harnessing the adaptive immune system to combat diseases such as cancer. Specifically, T-cells are activated and expanded ex vivo for adoptive immunotherapies. The ability to modulate T-cell activation is crucial in engineering appropriate effector cell populations for therapeutics. The focus of this thesis is to address the functional impact of CD28 spatial organization on T-cell activation.
It has been observed that the spatial segregation of CD3 and CD28 by a few microns has resulted in poor activation of human T-cells. Lck, a Src family kinase (SFK) emerges as the instigator of the phenomenon. The kinase is associated with both CD3 and CD28 signal cascades. We propose a reaction diffusion model to describe the delicate balance between protein mobility and Lck de-activation. The work in this dissertation describes two probes to investigate Lck kinase activity, which permit real-time imaging of both the initiation of pLck activity and its duration. A FRET reporter is constructed to study the spatial and temporal initiation of the kinase activity. Embedded with the Lck membrane domain and contained a substrate for pLck to phosphorylate, the FRET biosensor reports the Lck kinase activity in real-time. Using microprinting to control CD3 and CD28 spatial organizations, the FRET reporter reveals that while T-cells require CD28 for significant IL-2 secretion, CD3 engagement is essential to initiate cellular activation through a spike in pLck kinase activity. Spatially, the reporter shows heightened kinase activity concentrated at the center of the cells upon CD3 engagement.
To study the duration of pLck activity, a recruitment reporter is made. CD3 is found ubiquitously throughout the cellular membrane. And its activation by pLck induces the recruitment of a pair of tandem SH2-domain. The recruitment probe (also containing a pair of tandem SH2-domain) revealed curtailed pLck kinase activity due to CD3-CD28 segregation. Ultimately, understanding CD28 modulation of T-cell activation is clinically relevant as it provides new opportunities and targets for the development of therapeutics.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8VH5NVX |
| Date | January 2016 |
| Creators | Chen, Haoqian |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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