Successful T cell activation depends on the recognition of antigenic peptides in the context of a Major Histocompatibility Complex molecule (pMHC) by the T cell antigen receptor (TCR), together with additional signals from co-stimulatory receptors such as CD28. Despite their importance, a thorough understanding of how TCR-pMHC binding properties relate to T cell functional responses remains unclear. In addition, there are no consensuses to the exact mechanism leading to CD28 receptor triggering. Activation of CD28 is dependent on the phosphorylation of key tyrosine residues within its cytoplasmic domain by Src family kinases. Just like the TCRs, CD28 receptors are susceptible to perturbations of the local kinase: phosphatase ratio. The K-S model postulates that upon ligand engagement, large RPTPs such as CD45 are segregated from the area of close contact, resulting in increased relative kinase concentration and CD28 receptor triggering. This hypothesis was tested in chapter 3, where elongated forms of CD80 were examined for their ability to costimulate primary T cells. CD28 costimulation was indeed diminished and there was reduced CD45 segregation from the elongated CD80 molecules. Additionally, CD28 habouring key Y170F tyrosine mutations were less susceptible to CD28 signal abrogation by elongated CD80 molecules. Interestingly, elongated CD80 molecules remained much less effective in mediating costimulation even when pMHC molecules were also elongated, suggesting that TCR-pMHC and CD28-CD80 size matching is not critical for costimulation. Despite the well-documented MHC-restriction requirement for TCR recognition, the relative energetic contributions of peptide versus MHC in TCR-pMHC interactions remain elusive. To address this question, the energetic footprints of four TCRs (1G4, JM22, A6 and G10) to HLA-A2 were determined via systematic alanine scanning mutagenesis on the HLA-A2 heavy chain in chapter 4. By targeting exclusive TCR contacting residues on the MHC, we conservatively estimate the contribution of MHCs for the four TCRs to range from 15% to over 70%. Several models have been formulated in an attempt to relate TCR-pMHC binding properties to T cell activation. Validity of the models was tested in chapter 5 using a supra-physiological TCR. By mutating key residues within the cognate pMHC, we generated a panel of TCR-pMHC with affinities that varies up to 105-fold. These reagents were used to stimulate Jurkat and primary T cells transduced with the supra-physiological TCR. Results in the Jurkat T cell system demonstrated the presence of an optimal off-rate (k<sub>off</sub>) for TCR-pMHC interaction at low concentrations of pMHC concentration. The results argue against affinity models and the basic kinetic proofreading model for T cell activation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647609 |
| Date | January 2014 |
| Creators | Lim, Hong-Sheng |
| Contributors | van der Merwe, Anton |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:17bb3aa2-02fd-4f48-a4ad-1dd5c6ee67aa |
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