Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

5-22-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Paul M Allen

Abstract

The T cell is a critical player in the adaptive immune response. T cells function by stimulating antibody production by B cells, secreting cytokines to attract other immune cells, regulating the response of other T cells, and directly killing infected or damaged targets. The role of the adaptive immune response depends on the specific recognition of foreign antigenic epitopes by the T cell.

A T cell's specificity for antigen is conferred by the T cell receptor: TCR). The TCR is designed to bind a peptide antigen presented on an MHC molecule by an antigen presenting cell: APC). The strength of this interaction is determined by the ratio of the rate of dissociation: koff) and the rate of association: kon) between the TCR and the pMHC. The overall affinity of the complex must be sufficient for productive transmission of signals through the TCR to activate a T cell. However, the precise regulation of this event is not fully understood. Structural changes are thought to occur once the TCR encounters pMHC. Because of the energetics involved, kinetic and thermodynamic parameters have been correlated with the pattern and strength of T cell activation but these relationships do not explain all TCR:pMHC. The contact duration or dwell time of the TCR with pMHC takes into account the potential for rebinding events, which can enhance the signal strength and are related to the kon and koff of the complex. A faster kon can balance out a fast koff to have sufficient interaction between the TCR and pMHC for complete T cell activation. The kon also controls the number of rebinding events that can occur. Even so, the role of kon in T cell biology has not been explored independently of changes in koff.

Presented in this thesis is a system using two TCRs with specificity for the same antigen to compare how changes in kon alter T cell development and function. The n3.L2 and a mutant version, M2, recognize the Hbd(64-76) antigen presented on the I-Ek MHC class II molecule. M2 had a 3.7 fold stronger affinity for Hb(64-76)/I-Ek due solely to a faster kon. As a consequence, the M2 TCR responded more strongly to a broader range of altered Hb peptide ligands: APLs). While this presumably was due to an overall increased association with the MHC molecule, which could result from increased kon, the M2 TCR still retained antigen specificity and did not respond strongly to all Hb APLs. N3.L2 hybridomas and double positive thymocytes responded more strongly to two APLs of the P2 TCR contact residue. Therefore the changes between the n3.L2 and M2 TCR structures only allow certain residues to productively bind. By measuring the kinetics of n3.L2 and M2 in association with APL/I-Ek, the maximal IL-2 response is accurately predicted by the koff. No kinetic parameter correlated with the amount of APL needed to stimulate IL-2 production, suggesting other factors may be involved.

Since the response to APLs can mimic the ability of a TCR to recognize selecting self-peptides in the thymus, peripheral T cell responsiveness may be developmentally controlled. TCR transgenic mice were generated expressing either the n3.L2 or M2 TCR. M2 thymocytes had stronger recognition of endogenous peptides and were deleted through negative selection when exposed to Hb(64-76) as a self peptide. N3.L2 thymocytes underwent full development and were not completely deleted by Hb(64-76). Interestingly, this difference in T cell selection led to functional consequences in peripheral T cells. Ca2+, an early activation signal, was more sustained in n3.L2 CD4 T cells and more oscillatory in M2 CD4 T cells. Interestingly, M2 CD4 T cells failed to proliferate in response to antigen. Therefore, the TCR sensitivity set during T cell selection leads to qualitatively different signaling cascades in the periphery and can generate an anergic population with increased kon for pMHC recognition.

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html.

Permanent URL: http://dx.doi.org/10.7936/K70Z7190

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