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Title: Defining mechanisms of co-inhibitory receptor Lymphocyte Activation Gene-3 (LAG-3) in T cells
Author: Alhumeed, N. A.
ISNI:       0000 0004 7428 4421
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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The lymphocyte activation gene-3 (LAG3) inhibitory receptor was first discovered on natural killer cells but has since been shown to also be present on other cells such as activated T cells and B cells. LAG3 is similar to the CD4 receptor and shares a 20% sequence similarity. The ligand of LAG3 has also been determined to be MHCII. Although there is similarity between LAG3 and CD4, LAG3 can bind to MHCII with significantly higher affinity. Therefore, functionally LAG3 can inhibit proliferation, cytokine (IL-2, IFN gamma) secretion, and plays an important role in tolerance and also acts as a checkpoint in the activation of lymphocytes. In recent years, LAG3 has been recognised as an important target for immunotherapy and several studies and clinical trials utilising blocking antibodies are taking place to help It has to be noted that the molecular mechanism of action of LAG3 remains unclear and this study aims to reveal the cellular activity and intracellular pathways that are modulated and altered by the LAG3 receptor. Furthermore, this study aims to develop a better understanding of the effect of LAG-3 receptor in metabolism and to screen for any change in the cytokines and chemokines produced. Results from this study suggests that the F5 TCR transgenic mouse could be a suitable model to investigate the role of LAG3 on CD8 T cells in an antigen dose-dependent manner when presented by mDCs. The key findings described in this thesis is in the field of LAG3 signalling and metabolism. We observed altered signalling pathways during LAG-3 blockade. Blocking LAG-3 can increase the tyrosine phosphorylation of a significant number of kinases proteins and adaptor proteins, which include LCK, Zap70 and the adaptor proteins LAT and SLP76. There was also a reduction of serine phosphorylation on the feedback loop driven by HPK1, and an increase in Akt-S61 phosphorylation. Further investigation also shows that effector CD8 T cell metabolism is altered depending on the concentration of the restimulating peptide after blocking LAG-3. This observation sheds light on the role of LAG-3 as cellular function relies substantially on metabolic respiration. Interestingly, blockade of MHCII binding also alters the CD8 T cell metabolism but in a high-low-high pattern when restimulated with increasing concentrations of NP68 (0.1 nM, 1.0 nM, 10.0 nM). One of the main functions of CD8 T cells is to produce cytokines and chemokines that influences the immune system and this was investigated in the context of LAG-3 blockade. There were significant changes in the production of mainly IL-2 and IFN gamma after LAG-3 blockade but this did not have a significant effect on number of cytokines. These results indicate that blocking LAG3 receptor alone makes cells behave differently during stimulation and encourages cells to be more active which may alter the downstream immune response. Overall, the combined results from this study suggests that LAG3 can be targeted for use in immunotherapy and the F5 TCR transgenic mouse is a useful model to study LAG3. The experimental work described in this thesis illustrates the early changes induced by the LAG3 receptor on F5 TCR T cells and helps to define signalling and metabolism changes, as well as to broaden the knowledge of using anti LAG-3 antibody as an immunotherapeutic.
Supervisor: Cross, Michael Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral