The long term aim of the proposed research is to understand the mechanism of signalling through the T-cell receptor (TCR) complex, which comprises the antigen-specific TCR heterodimer together with the CD3 signalling components delta, gamma, epsilon and zeta. Defective signalling through the TCR complex has been implicated in clinical immunodeficiency and as a cause of impaired T-cell function in cancer patients, while normal signalling is required for T-cell development and for mature T-cell function. An intriguing aspect of TCR-mediated signalling is the capacity to transmit alternate signals in response to different ligands. This leads to entirely different functional outcomes, for instance, in the case of T-cell development positive versus negative selection. The objective of the proposed project is to determine whether individual CD3 components have unique roles in mediating alternate signals. To test this possibility for the CD3 delta component, the principal investigator has engineered a mouse line that specifically lacks the CD3delta chain (delta-/-mouse). delta-/- mice show a specific defect in development of alpha beta T-cells. In addition, the small number of peripheral alpha beta T-cells that do develop show marked phenotypic and functional abnormalities. This project seeks to further define the essential role of CD3delta in the development (aims 1-3) and mature function (aims 4,5) of alpha beta T-cells. In the first aim, biochemical means will be used to test the hypothesis that TCR alpha, which normally interacts directly with CD3delta, is specifically uncoupled from the CD3 complex in delta-/- mice. In the second aim, the nature of the TCR repertoire expressed on the rare peripheral alpha beta T-cells in delta-/-mice will be examined by expressing TCRs from such cells as transgenes in normal mice. In the third aim, experiments to distinguish whether normal development of gamma delta T-cells as opposed to alpha beta T-cells in delta-/- mice reflects more efficient TCR surface expression or different signalling requirements during development, the principal investigator will coexpress gamma delta and alpha beta TCR on developing delta-/- thymocytes through the use of an abnormally regulated gamma delta TCR transgene. In the fourth aim, experiments to determine whether alternate usage of the CD3delta and gamma chains can represent an in vivo means of modulating the TCR's signalling capacity will be done by analyzing the phenotypic and functional characteristics of naturally occurring CD3delta-negative peripheral T-cells in delta-/- mice reconstituted with a human CD3delta transgene. In the fifth aim, experiments are designed to determine whether defective mature T-cell function in delta-/- mice reflects a true requirement for CD3delta or rather an outcome of aberrant development. This will be addressed for CTL by engineering a transgenic mouse in which CD3delta is expressed during development but specifically silenced in the CD8+ lineage.
