Principles of clonal selection in T cell populations undergoing antigen-driven expansion in vivo

The immune system protects the body from harmful micro-organisms, such as viruses, and the
development of certain cancers; however, it can also malfunction to cause autoimmune diseases,
such as multiple sclerosis, and allergies. Many of these effects are caused either directly or indirectly
by T cells, which react to small fragments of the immune target known as antigens. However, while
many advances have been made in understanding how these cells work, the factors that determine
whether T cell responses are successful or unsuccessful remain unclear. The aim of the work
described here is to study, in detail, individual T cells responding to specific antigens. This will be
accomplished using a series of novel and highly sensitive techniques that, for the first time, allow
analyses that even recently would not have been feasible. These studies hold the potential to
enhance our understanding of how the immune system responds to antigens, and to identify the
inherent strengths and weaknesses of T cell immunity. Such knowledge is critical for the rational
design of effective vaccines to both micro-organisms and tumours, and for the development of novel
immune therapies for many different diseases.

Adaptive T cell immunity is critical for host defence against intracellular pathogens and tumour
surveillance, yet potentially autoaggressive and aberrantly responsive to inert antigens. Quantitative
measures that characterize the magnitude of antigen-specific responses have largely failed to reveal
consistent correlates of T cell-mediated immune protection or immunopathology; thus, new
approaches that examine qualitative aspects of individual responses are required. The aim of this
proposal is to deconvolute the clonotypic architecture of specific T cell populations responding to
defined antigens in vivo under a spectrum of conditions in order to clarify the basic biological
principles that govern antigen-driven T cell selection in the periphery and to establish the biological
relevance of these processes with respect to the underlying pathology. This will be accomplished
directly ex vivo using a series of novel and sophisticated technologies including point-mutated
peptide-major histocompatibility complex (pMHC) molecules to assess antigen avidity, polychromatic
flow cytometry to determine functional and phenotypic properties at the single cell level, and
quantitative molecular clonotyping with a template-switch anchored RT-PCR to amplify all expressed
TCR gene products in a linear manner without bias; in addition, surface plasmon resonance and
crystallography will be used to characterize TCR/pMHC interactions of interest biophysically and
structurally. The following types of antigen-specific T cell responses will be studied: (i) virus-specific
(dangerous foreign); (ii) tumour-specific (dysregulated self); (iii) alloreactive (foreign self); (iv)
autoreactive (self); (v) allergic (non-dangerous foreign). The resultant data will be mined for
pathophysiological correlates. Additional experiments will investigate new approaches to antigen
delivery that might enable the manipulation of clonotype usage in response to given antigens with
beneficial effect. Ultimately, it is envisioned that this work will inform clinically-relevant translational
vaccinology and immunotherapeutics through a detailed understanding of clonotypic ontogeny
during antigenic challenge and the identification of molecular signatures that characterize both
successful and unsuccessful antigen-specific T cell responses.