ONTOGENY AND PHYSIOLOGY OF "NATURAL" REGULATORY CD4 T CELLS

The transcription factor Scurfin, also known as forkhead/winged helix (Foxp3) is a specific marker for regulatory activity and is essential for the development and function of regulatory CD25+CD4 T cells. Regulatory activity and low amounts of Foxp3 mRNA are also found in the CD25- CD4 T cell population. Recently, it was shown that the aeb7 integrin (CD103) expressed at high levels in T cells seeding mucosal tissues, identifies a small subset of splenic CD25-CD4 T cells that displays regulatory activity. CD103 expression also distinguishes two types of regulatory CD25+CD4 T cells secreting different cytokines and showing different regulatory capacities. Whether these three populations express Foxp3 and belong to the same lineage is not yet determined.

Development and homeostasis of the peripheral pool of Foxp3-expressing CD4 T cells

It has been proposed that regulatory CD25+CD4 T cells are only significantly exported from the thymus after the third day of post-natal life. This hypothesis is based on the fact that mice thymectomized between days 2 to 4 after birth, develop organ-specific autoimmune disorders, which are prevented by the early transfer of adult CD25+CD4 T cells. However, the pattern and variable incidence/severity of these diseases, the absence of other types of inflammatory diseases such as inflammatory bowel disease, and the chronic state of lymphopenia, are in sharp contrast with the syndrome resulting from the inactivation of the Foxp3 gene. In this case, the complete absence of regulatory CD25+CD4 T cells leads to generalized lymphoproliferation and massive lymphoid infiltration of several tissues, death occurring within four weeks after birth.

To resolve this paradox, we recently found that the peripheral immune system of day 3 old newborns is endowed of a remarkable regulatory potential, and a well-represented compartment of Foxp3-expressing CD25+CD4 T cells. This potential can fully develop in the absence of further thymic supply, thus explaining the absence of generalized autoimmune or other inflammatory diseases in neonatally thymectomized mice.

We will determine whether the Foxp3-expressing CD25+CD4 T cell population present in adult neonatally thymectomized mice represents the progeny of those present in the periphery of day 3 newborns or whether they derive from CD25- naïve CD4 T cells. Next, we will analyse the TcR diversity of this regulatory population and determined whether the regulatory deficit in these mice is due to discrete “holes” in the their repertoire. We will attempt to identify the natural ligands recognized by the CD25+CD4 T cells that control autoimmune gastritis and protect from inflammatory bowel disease.

Lineage relationship between the distinct populations of Foxp3-expressing CD4 T cells

Our first results show that similarly to the canonical CD25+CD4 T cells, CD103+CD25-CD4 T cells express high levels of Foxp3, and that the expression levels of these two genes are differentially controlled in the newborn and the adult thymus. We will determine the lineage relationship between the distinct subsets of Foxp3-expressing cells as defined by the CD25 and the CD103 markers and whether these thymic populations represent the precursor pool of their respective peripheral equivalents. Given that both the CD25 and the CD103 are not constitutively expressed, we will study the dynamics of interchange between the distinct peripheral compartments of regulatory CD4 T cells.

Diversity of the TCR repertoire of the natural pool of CD25+CD4 T cell sin mice and humans

The TcR repertoire of these cells appears polyclonal. Using the Immunoscope approach, we have sequenced TcR hypervariable regions from CD25+CD4 T cell, and found that their diversity is probably of the same order of magnitude as that observed for naïve CD4 T cells. This implies that the overall clonal size is small and that these cells may recognize a very diverse set of natural ligands. Similar studies are in progress using human peripheral blood CD25+CD4 T cells.

Regulatory CD4 T cell sin the human melanoma

We recently observed an over-representation of Foxp3-expressing CD25+CD4 T cells in metastatic lymph nodes of melanoma patients. We will develop the technique of single cell PCR for Foxp3 to evaluate whether all CD25+CD4 T cells are indeed regulatory cells. In parallel, we will generate clones of these cells to make a more refined functional characterization (e.g. the pattern of cytokines that they produce) and to identify which antigens (tumor or non-tumor) they recognize. For this purpose, we will use cDNA expression libraries of autologous or allogeneic melanoma and dendritic cells. Moreover, we will analyze the role played y the IL-10 and TGF-b produced by the melanoma cells in the local induction of regulatory cells of the Tr1/Th3 type, as suggested by our previous work.

Web site created by Marie-Christine Vougny (04/2004)