Cellular and transcriptomic analysis of regulatory T cells in streptococcal infection

This proposal is concerned with a population of white blood cells termed ?regulatory T cells? (Tregs). Their role in the body is to damp down other immune cells, preventing inflammatory ?overshoot?. We wish to examine what they do in a particular setting of bacterial infection: infections with Streptococcus pyogenes cause diverse conditions from sore throat to scarlet fever, rheumatic heart disease, sepsis and necrotising fasciitis (?flesh-eating disease?). We know that the interplay between the body and this bacterium is a complex one: on the one hand, a robust immune response is required to defend the body, while on the other, many of the disease manifestations are themselves a consequence of over-exuberant immunity. It is immediately apparent that there is likely to be an important role of Tregs. The Altmann lab has already shown (in mouse models) that there is a very rapid up-regulation of Tregs and molecules associated with them at the site of infection with this bacterium. The aim of this new proposal is to start building up an understanding of what happens in human cells. The proposal brings together investigators with highly complementary interests in this area in Singapore and London ? Altmann (Imperial) has been interested in models of streptococcal sepsis and the role of Tregs, Rotzschke (SiGN) has been involved in the detailed cellular and molecular analysis of Tregs, and Yang (SiGN) uses DNA microarray technology to describe immune responses. This latter approach is a very powerful one, allowing comparative analysis of cells that have been treated in different ways by looking at tens of thousands of human genes and asking, in a particular situation, which genes are turned up and which turned down. The 3 teams will build a close collaboration through annual workshops, monthly videoconferences, and by sending personnel between labs to work together. The central group of experiments depend on culturing cells from human tonsils, either in the presence or absence of Tregs, then infecting the cultures with various versions of Strep, of varying virulence. We will compare what happens, with or without Tregs, in terms of the presence of specific cell populations and gene by gene, though array anaysis. We will back up these human studies with experiments whereby we give the Strep to mice that either have normal Tregs, or have had them genetically knocked out. These experiments should lead to an improved understanding of the immunological events controlling these diseases.

This proposal brings together two groups in Singapore and one in the UK, each at centres of excellent and bringing together diverse and synergistic skills to look at the impact of regulatory T cells control on immunity to Streptococcus pyogenes. The study encompasses complementary backgrounds in models of group A streptococcal (GAS) disease, Treg biology, and immune system array analysis and bioinformatics. The initial aim is, through joint workshops and videoconferencing, to build up a close working interaction between the groups in these countries. The hypothesis that we wish to test, mainly in human tonsil cultures, and then also in FoxP3DTR-inducible knockout mice, is that (a) the acute response to GAS infection is tightly regulated by Tregs, (b) that the degree of regulation will be modulated by the virulence and/or superantigenicity of the bacterial strains and (c) from the in vivo experiments ? that the extent of this Treg control will have profound effects on the extent of bacterial sepsis. The experimental design exploits the strengths of each of the labs and involves a panel of GAS strains, differing in superantigens and in virulence, and that can be used to infect human tonsils, that are either in the form of single cell suspensions or intact histocultures. In some cases, Tregs will be reomoved from cultures or added back in defined numbers. Readouts of these assays will be at the level of T cell subsets, newly induced transcripts as determined by array analysis, and bacterial colon counts.