Hedgehog signalling in T cell receptor (TCR) repertoire selection in the thymus.

T-cells are white blood cells that enable us to fight infectious disease. They can both attack infectious pathogens themselves and they can organise other types of white blood cell to attack infections. To do this, T-cells must be able to recognise what is ?self? and what is ?non-self? and so should be attacked. T-cells are produced in an organ called the thymus. When they are mature they leave the thymus and patrol our bodies looking for infections that they will attack. During the time that they are in the thymus, T-cells are selected so that any T-cells that might attack ourselves (attack ?self?) do not complete their maturation and die, but T-cells that are able to fight infections are allowed to leave. This selection of T-cells in the thymus is very important to our health because if T-cells that recognise ?self? leave the thymus they might attack our bodies causing autoimmune diseases like diabetes and rheumatoid arthritis. This project will study how the thymus controls which developing T-cells are allowed to complete their maturation and leave the thymus because they recognise ?non-self? and which T-cells do not complete their maturation because they recognise ?self?. We will study how molecules called ?Hedgehog? proteins that are made by the thymus control the fate of developing T-cells.
Hedgehog proteins are molecules that are made by cells and secreted to tell other neighbouring cells to undergo a particular process, such as to divide, die, or change into a different type of cell. They do this by influencing which molecules are made in the target cell, and this is determined by which genes are active in that cell. Hedgehog proteins are essential in our embryonic development because they transmit messages to developing organs controlling how the organs form and grow. We have shown that after birth Hedgehog proteins are also important in our immune systems because they allow communication between different cells of the immune system. They determine how many T-cells are produced in the thymus and they can tell developing T-cells to survive, divide, or mature, depending on if the developing T-cell recognises ?self? or ?non-self?. We aim to find out what messages Hedgehog proteins give to developing T-cells by finding out which molecules are made and which genes become active when Hedgehog proteins signal to developing T-cells.

This project will investigate the functions of the Hedgehog (Hh) signalling pathway in the regulation of T cell receptor (TCR) repertoire selection and differentiation from CD4+CD8+ double positive (DP) to CD4+ and CD8+ single positive (SP) thymocyte, during T cell development in the thymus. Our general experimental approach will be to probe Hh function in mouse mutants in which Hh protein production and secretion by thymic epithelial cells (TEC), or the ability to respond to the Hh signal in thymocytes, are either specifically ablated/inhibited or constitutively activated. We will use these mouse models in conjunction with TCR-transgenic mice to investigate the impact of Hh signalling on TCR repertoire selection.

We will use Shh-/- Foetal thymus organ culture (FTOC) and conditional FoxN1-Shh-/- adult thymus to test the hypothesis that Sonic Hh (Shh) secretion by TEC provides a microenvironmental influence on the outcome of TCR repertoire selection at the transition from DP to CD4+ and CD8+ SP thymocyte. We will test the impact of Hh signalling on positive and negative selection of transgenic MHC Class I- and Class II-restricted TCRs, in Gli2DN2-transgenic thymus, Gli2DC2-transgenic thymus, Shh-/- FTOC, conditional FoxN1-Shh-/- thymus and T-lineage specific conditional Smo-deficient thymus. We will test the hypothesis that Hh pathway activation modifies TCR repertoire selection by modulation of TCR signal strength in developing thymocytes. In order to investigate the interactions between Hh and TCR signal transduction we will use biochemical techniques to assess TCR signal transduction in DP cells from Hh pathway mutants. We will use microarray analysis of gene expression in DP cells from Gli2DN2-transgenic, Gli2DC2-transgenic, conditional FoxN1-Shh-/- and control thymus to identify Hh-target genes in developing thymocytes.

In parallel studies, we will investigate the functions of Hh pathway secretion and activation in TEC. We will test the hypothesis that it is a discrete subset of TEC (situated in the medulla and corticomedullary junction) that secrete Shh. We will define this subset. We will use Shh-/- FTOC and conditional FoxN1-Shh-/- thymus to investigate the impact of Hh signalling on TEC function, thymus architecture, TEC organisation, TEC phenotype and medulla formation. We will use conditional FoxN1-Dispatched1-/- mice to investigate the function of Dispatched1 in Shh secretion by TEC and the establishment of the Hh gradient.