PI3K signalling at the immune synapse asymmetric division and immunological memory.

T helper cells are master cells of the immune response. T cells are responsible for detecting infectious agents, be they bacteria, viruses or parasites; they are responsible for assessing the potential damage these infectious agents can cause, and to mount proportional and selective immune response to get rid of the infectious agent while causing minimal damage to the host. The T cells make this decision as they form conjugate with antigen presenting cells - that is cells that are specialised in devouring foreign particles, digesting them in to smaller fragments, and presenting these fragments to T cells. T cells respond by stimulating other cells of the immune response to secrete antibodies that eliminate the pathogen. Cytotoxic T cells act by killing infected host cells, thus incapacitating the pathogen inside. Both these cell types will be investigated as part of this proposal. T helper cell makes their decisions over several hours during which the T cell corresponds with an antigen presenting cell though a structure called the immune synapse. The synapse is a term immunologists have stolen from the field of neurobiology. Neurons transmit signals from one nerve to another through synapses, which in fact is what immune cells do as well. However, there are important differences. Immune cells are highly motile and travel through the lymph nodes and through different tissues, such as the skin and gut where infections agents may be found. This constant movement places additional constraints on T cells to form stable synapses - a bit like parachutists trying to grab each others hands while in freefall. p110delta belongs to a family of enzymes called phosphoinositide 3-kinases (PI3Ks for short). We have engineered mice in which the gene for p110delta is modified such that the enzyme is no longer functional. T cells from such mice are poor at forming conjugates with antigen presenting cells. Moreover, we have evidence that p110delta-deficient T cells fail to organise themselves in the right conformation to maximise their ability to read and interpret the signals provided by the antigen presenting cells. The first purpose of this grant is to more fully characterise these defects at the molecular level as it is at present not obvious why p110delta should be important for these functions. Next, we will determine how important this actually is for the ability of T cells to respond to infectious agents. We will also monitor how T cells respond after infection with a bacterium called Listeria monocytogenes. This bacterium is sometimes found in unpasteurised milk products, such as cheese, and can cause disease in humans. We will observe in T cells taken from infected mice whether they make functional conjugates with APCs and if they are capable of responding appropriately. In addition, we will test if p110delta-deficient T cells can divide in such a way that one cell becomes an effector cell (destined for a short life dedicated to immediate elimination of the infectious agent), whereas the other cell becomes a memory cell that waits in the background and is prepared to raise an even more immediate and effective response should the particular infectious agent be encountered again. This process is the basis for how vaccines work. Together, these experiments will examine how p110delta, a target for drugs being developed by the pharmaceutical industry, affects fundamental immune responses that protect from recurrent infections.

The PI3K p110delta is a key signalling protein which is implicated in T cell receptor signalling. We have found that stimulation of p110delta-deficient T cells with antigen presenting cells and cognate peptide results in reduced proliferation and differentiation both in vitro and in vivo. However, stimulation using antibodies can rescue proliferation and cytokine secretion responses completely. These results indicate that p110delta-deficient T cells do not form stable or productive conjugates with antigen presenting cells (APCs) and that this affects their further activation. In addition, recruitment of the TcR to the contact area is impaired, as is the recruitment of lipid raft-associated proteins. Thus, the p110delta deficient T cells may not appropriately reorganise their cytoskeleton upon contact with an APC. We will measure the frequency and duration of the interaction between p110delta deficient T cells and APCs in vitro and in vivo, whether proper supramolecular activation clusters are formed and whether the TcR is internalised normally after activation. We will investigate how p110delta regulates Small GTPases such as Rac, Rho and Cdc42 and their downstream effectors. We will also investigate cytoskeletal rearrangements by monitoring changes in actin polymerisation and microtubule formation. The potential functional consequences of defective conjugate and synapse formation in p110delta-deficient T cells will also be investigated. It has recently been proposed that asymmetric division of T cells in conjugates forms the basis for the generation of short-lived effector cells and long-lived memory cells. We will test whether key differentiation proteins segregate during mitosis of p110delta-deficient T cells in conjugates. Moreover, we will determine if these T cells differentiation to become competent effector cells or memory T cells after division in response to infection with Listeria monocytogenes.