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Thesis project proposal
Role of CTLA-4 in Regulatory T Cell function – Mechanisms involved in CTLA-4 interactions that
induce Treg cell activation and proliferation
Aditya Murthy
Dr. Gerald Prud’homme, MD
Abstract
CTLA-4 is a costimulation receptor constitutively expressed in regulatory T cells (CD4+CD25+
cells), and has been shown to play a critical role in the development of T cell anergy. Some
studies suggest that engagement of CTLA with its ligand B7 on the APC leads to the activation of
the regulatory T cell, resulting in release of several inhibitory cytokines such as IL-10 and TGFβ.
Furthermore, proliferation of regulatory T cells may be induced, leading to a positive feedback
mechanism that ultimately results in the downregulation of effector T cell response in an antigen
specific manner. The mechanisms involved in these interactions have not been elucidated so far,
despite the extensive studies on CTLA-4 function in an immune response. Shedding light on this
mechanism would allow us to further clarify the much-debated role of CTLA-4 and regulatory T
cells in immune responses.
Our project attempts to resolve the mechanisms that lead to the activation and differentiation of
Treg cells (regulatory T cells), and the role played by CTLA-4 in their generation. In some
experiments, CTLA-4 interaction with its ligand will be blocked using specific soluble anti-CTLA-4
antibodies as inhibitors, and quantitative changes of CD4+CD25+ T cells (these are classified as
regulatory T cells) will be measured. The ability of CTLA-4 engagement to induce T reg cell
differentiation will be studied in vitro. CTLA-4 engagement with monoclonal antibodies will be
combined with signals that activate T cells, such as CD3 and CD28 ligation, and the media will be
supplemented with cytokines (IL-2, IL-10, TGFβ). Naïve T cells that have been selected by
magnetic sorting for a non-Treg phenotype (CD4+CD25-) will be stimulated under a variety of
conditions with the reagents listed above, and followed for the development of a regulatory
phenotype (CD25+, FoxP3+, CTLA-4high) and function in Treg assays. ELISA will be used to study
changes in cytokine expression levels as a result of CTLA-4 interaction with its ligand B7 in
activated T cells. Theoretically this interaction should result in the release of TGFβ and IL-10, and
induce regulatory T cell development. The purpose of these experiments is to establish whether
CTLA-4 engagement contributes to the differentiation of T reg cells from non-suppressive
precursors.
Significance
Studying the effects of CTLA-4 interaction, inhibition of this interaction and changes in cytokine
expression patterns as a result of this interaction will shed light on its relevance in the generation
of Regulatory T cells, and its influence on the development of anergy and effector cell
inactivation. Understanding of the mechanisms behind Regulatory T Cell functions have lead to
the development of therapeutic benefits such as generation of treatments towards autoimmune
diseases (eg. Type I Diabetes) via gene therapy, and clinical procedures such as inhibition of
alloantigen specific transplant rejection.