Date of Award

Winter 12-15-2014

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

It is well established that self-peptide Major Histocompatibility Complexes (hereafter self-pMHC) are essential for the development of a broad repertoire of mature, self-tolerant CD4 and CD8 T cells. Despite clear knowledge that the pool of self-ligands is critical for positive and negative selection, the exact kinetics and dynamic nature of thymocyte interaction with self-pMHC class II (self-pMHCII) bearing antigen-presenting cells (APCs) during development is still largely a mystery. The enigmatic nature of selecting self-pMHC is not unique to the thymic environment; increasingly evident is the dependence of peripheral T cells on extra-thymic self-pMHC expression, specifically selecting self-pMHC, for their homeostatic maintenance and peripheral functionalities. Unequivocal resolution as to the exact nature of selecting self-pMHC engagement with thymocytes and mature peripheral T cells has been hindered by the lack of known TCR:self-pMHC pairs. Our laboratory has identified the first bona fide, naturally occurring self-pMHCII, which positively selects a known CD4 transgenic (tg) T cell. The gp250/I-Ek ligand is able to positively select the ANDtg CD4 T cell, which recognizes the agonist ligand MCC/I-Ek. Using this system, we have surveyed and quantified the endogenous presentation of a naturally occurring positive selecting ligand-MHCII complex in the thymus and periphery. The redundant presentation of gp250/I-Ek on positive selecting cortical epithelial cells as well as tolerance inducing APCs lends credence to an affinity model of thymic selection, dependent upon re-recognition of "public" self-pMHC complexes in the thymus. The peripheral expression of this public ligand is affected by the activation state of peripheral APCs, suggesting that the recent immune status can impact the homeostasis of T cell repertoires unrelated to the ongoing immune response. Importantly, the expression of functional self-pMHCII complex does not correlate with mRNA levels, indicating a need to functionally detect the presence of these important ligands in order to gain a complete understanding of how they orchestrate the critical processes of T cell development and peripheral maintenance.

In an ongoing attempt to more completely understand the early events and cellular interactions critical to thymic selection, we have developed a novel method of in situ, intravital, two-photon imaging of the murine thymus. This groundbreaking technique allows for the visualization of thymic selection events that occur with intact vasculature and innervation, providing an advantage over current explantation methods to view intrathymic events. Indeed, published data in regards to the post-positive selection motility of thymocytes in explant preparations appear to overestimate the intrathymic cellular dynamics when compared to intravital imaging. Not only can utilization if the intravital imaging technique give a complete view of ongoing selection events, it is currently the only method to visualize the earliest thymic seeding events. Within 15 minutes of intravenous administration, fluorescently labeled bone marrow cells can be viewed within the thymic vasculature. Over the time of imaging, bone marrow cells can be viewed at each stage of extravasation into the thymic tissue. The ability to visualize the earliest thymic seeding events will provide a novel method to address longstanding questions in the field of T cell selection.

A T cell's dependence on recognition of self-pMHC does not end in the thymus; mature peripheral cells continue to require self-pMHC signals for survival. The exact nature of this interaction continues to be a topic of active research. Curiously, it appears that the same ligands experienced during thymic selection play a unique role in naive T cell homeostasis. Investigation of gp250/I-Ek mediated homeostatic maintenance of ANDtg CD4 T cells suggests that this confirmed endogenous selecting ligand can augment homeostatic proliferation. How this weak TCR engagement mediates homeostatic responses is unknown. Hypothesizing that the strength of self-reactivity dictates the degree of homeostatic proliferation, we turned to a different TCRtg system, LLO. We observed that despite only a few amino acid differences in their TCR and equivalent affinity for the same agonist pMHCII, the LLO118 CD4 T cell underwent reduced homeostatic proliferation compared to the LLO56 counterpart. The main difference between these two TCR is the strength of self-reactivity; LLO118 is less self-reactive as indicated by CD5 expression, suggesting that self-reactivity is correlated with homeostatic capacity.

Language

English (en)

Chair and Committee

Paul M Allen

Committee Members

Deepta Bhattacharya, Chyi Hsieh, Mark Miller, Gwendalyn Randolph

Comments

Permanent URL: https://doi.org/10.7936/K78W3BFS

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