Phosphorylation of RIPK1 serine 25 mediates IKK dependent control of extrinsic cell death in T cells.

The Inhibitor of Kappa B Kinase (IKK) complex is a critical regulator of NF-κB activation. More recently, IKK has also been shown to repress RIPK1 dependent extrinsic cell death pathways by directly phosphorylating RIPK1 at serine 25. In T cells, IKK expression is essential for normal development in the thymus, by promoting survival of thymocytes independently of NF-κB activation. RIPK1 undergoes extensive phosphorylation following TNF stimulation in T cells, though which targets are required to repress RIPK1 has not been defined. Here, we show that TNF induced phosphorylation of RIPK1 at S25 is IKK dependent. We test the relevance of this phosphorylation event in T cells using mice with a RIPK1S25D phosphomimetic point mutation to endogenous RIPK1. We find that this mutation protects T cells from TNF induced cell death when IKK activity is inhibited in vitro, and can rescues development of IKK deficient thymocytes in vivo to a degree comparable with kinase dead RIPK1D138N. Together, these data show that phosphorylation of RIPK1S25 by IKK represents a key regulatory event promoting survival of T cells by IKK.

NF-κB and Extrinsic Cell Death Pathways - Entwined Do-or-Die Decisions for T cells.

NF-κB signaling is required at multiple stages of T cell development and function. The NF-κB pathway integrates signals from many receptors and involves diverse adapters and kinases. Recent advances demonstrate that kinases controlling NF-κB activation, such as the IKK complex, serve dual independent functions because they also control cell death checkpoints. Survival functions previously attributed to NF-κB are in fact mediated by these upstream kinases by novel mechanisms. This new understanding has led to a refined view of how NF-κB and cell death signaling are interlinked and how they regulate cell fate. We discuss how NF-κB activation and control of cell death signaling by common upstream triggers cooperate to regulate different aspects of T cell development and function.

Using Lactococcus lactis as Surrogate Organism to Study Group A Streptococcus Surface Proteins.

The isolation of a single Group A Streptococcus (GAS) virulence determinant in functional investigations is challenging, as GAS employs a multitude of virulence factors. The redundancy between many surface proteins such as adhesins also adds complexity and difficulty. Lactococcus lactis is a non-pathogenic Gram-positive species related to GAS that can be an ideal surrogate organism to circumvent this problem. Genetic manipulation in L. lactis is easy, and the mechanisms for processing and cell wall-anchoring of surface proteins are similar to GAS. Lactococci have been extensively used to express heterologous surface proteins from other bacterial species, and modern molecular cloning tools and protocols have been developed. This chapter describes the workflow of generating recombinant L. lactis strains expressing GAS surface proteins and the validation and quantification of their surface expression.

PilVax - a novel peptide delivery platform for the development of mucosal vaccines.

Peptide vaccines are an attractive strategy to engineer the induction of highly targeted immune responses and avoid potentially allergenic and/or reactogenic protein regions. However, peptides by themselves are often unstable and poorly immunogenic, necessitating the need for an adjuvant and a specialised delivery system. We have developed a novel peptide delivery platform (PilVax) that allows the presentation of a stabilised and highly amplified peptide as part of the group A streptococcus serotype M1 pilus structure (PilM1) on the surface of the non-pathogenic bacterium Lactococcus lactis. To show proof of concept, we have successfully inserted the model peptide Ova324-339 into 3 different loop regions of the backbone protein Spy0128, which resulted in the assembly of the pilus containing large numbers of peptide on the surface of L. lactis. Intranasal immunisation of mice with L. lactis PilM1-Ova generated measurable Ova-specific systemic and mucosal responses (IgA and IgG). Furthermore, we show that multiple peptides can be inserted into the PilVax platform and that peptides can also be incorporated into structurally similar, but antigenically different pilus structures. PilVax may be useful as a cost-effective platform for the development of peptide vaccines against a variety of important human pathogens.