Muaz Rushdi

Biomedical Engineering Ph.D. Thesis Defense

Date: Thursday March 21st

Time: 11:00 AM

Location: Petit Institute room 1128, Suddath Room

Thesis committee:

Cheng Zhu, PhD (Advisor)

Antony Chen, PhD (Co-advisor, Peking University)

Lou Ann Brown, PhD (Emory University)

Melissa Kemp, PhD

Khalid Salaita, PhD (Emory University)

Title: Toward Contextualizing Receptor-Ligand Interactions in T cells and Macrophages

Abstract:

Immune system response to pathogens is mediated by triggering of receptor-ligand interactions such as the T cell receptor (TCR) engaging with peptide-major histocompatibility complex (pMHC) expressed on infected cells, which can be facilitated by the CD4 coreceptor. However, detecting CD4-pMHC binding has been elusive due to its purported weak affinity. Additionally, there are no models that describe cooperativity between more than two molecules. To address these limitations, the first aim of this thesis was to characterize CD4-pMHC binding and mathematically model trimolecular cooperativity. Using an ultra-sensitive micropipette adhesion frequency assay and biomembrane force probe, the complete set of CD4-pMHC kinetics was experimentally detected for the first time. Surprisingly, when controlled mixtures of TCR and CD4 were probed against pMHC, binding frequency was greatly enhanced clearly demonstrating synergy between TCR, pMHC and CD4. These results were used to develop the first documented analytical solutions for trimolecular cooperativity, which imply a conformational change in TCR-bound pMHC that greatly enhances CD4 binding. A cell system corroborated the model by confirming that the ratio between TCR and CD4 dictates synergy observed in binding kinetics and intracellular signaling, suggesting that CD4 is not a passive facilitator of activation as previously thought.

As T cells travel to sites of inflammation, the trimolecular complex is exposed reactive oxygen species (ROS) generated by other immune cells also seeking to clear pathogen. ROS has been suggested to inhibit T cell function, especially in cancer microenvironments, but in other scenarios, it has been shown to improve T cell function. The dual-role of ROS has long convoluted study of its effect on immune cells which has notoriously limited clinical intervention. Therefore, the second aim of this thesis was to monitor TCR-pMHC binding in response to ROS in real-time using a novel modification of the micropipette assay, in order to deconvolute how the many molecules involved in binding are each affected. TCR-pMHC binding was reduced by the introduction of ROS but could be partially rescued by the coreceptor. The degree of rescue was modulated by the surrounding cholesterol content of the membrane as well as the catalytic activity of proximal kinases. By parsing out molecules in a holistic in situ system, it was concluded that TCR-pMHC binding is weakened due to the disruption of the trimolecular complex by ROS.

Similar to T cells, the macrophage TLR4 receptor requires other surface receptors to robustly recognize its ligand LPS, and the relationship between oxidative stress and macrophage function is mired with conflicting results. Correspondingly, the framework developed in the first two aims of this thesis was applied to a third aim which sought to understand how macrophage function can be both enhanced and impaired by ROS. Unique morphological changes of macrophages induced by LPS engagement were dependent on ROS and correlated well to previously described inhibition of macrophage phagocytosis. At the same time, ROS enhanced cytokine secretion by macrophages and TLR4-LPS binding did not display differences between ROS-treated and untreated cells. Lastly, actin reorganization appeared to be reduced when treated with ROS. These data suggest that unlike T cell activation which is dependent on TCR-pMHC binding, macrophage function is diverse in its regulation as exemplified by ROS improving TLR4 signaling but also inhibiting movement by disrupting actin.

Overall, by introducing biophysical and biochemical contexts to receptor-ligand interactions in tandem, a better understanding of how our immune system sensitively detects pathogens was achieved. The novel results described herein inform rational design of therapeutic interventions including protein engineering enhancement of antigen recognition as well as acute and compartmentalized antioxidant protection.