Affliation: Fellow, Department of Infectious Disease and Immunity, Imperial College London

Convener: A/Prof Thomas Dick

Abstract:
Objectives:   Neutrophils are the major phagocytic cells in the lungs of patients with active pulmonary tuberculosis (TB). A matrix degrading phenotype in TB results in tissue damage, where the activity of matrix metalloproteinases (MMPs) is unopposed by their tissue inhibitors (TIMPs). Factors regulating neutrophil MMP secretion in TB in vitro and in patients were investigated. 

Methods:   Human neutrophils were infected with Mycobacterium tuberculosis (Mtb) or stimulated with conditioned media from Mtb- infected monocytes (CoMTB). Analysis of MMP-8/-9 secretion and TIMP-1/-2 was by ELISA, Luminex array and zymography. Gene expression of MMP-8/9 was investigated using real-time PCR. Neutrophil granule formation was assessed by confocal microscopy. Induced sputum samples from 108 healthy controls and TB patients were analysed. Brain biopsies from patients with central nervous system-TB were studied by immunohistochemistry.  

Results:   Neutrophil MMP-8/-9 secretion is upregulated by Mtb over time and is dependent on TB multiplicity of infection. CoMTB stimulated neutrophils resulted in a 2 and 3 fold up-regulation of MMP-8/-9 secretion respectively (both p<0.001). TIMP-2 is increased 2-fold (p<0.001) but not TIMP-1.  MMP-8 and -9 is increased 16 and 160 fold respectively compared to TIMP-2. MMP-8/-9 gene expression was increased 3.5 fold and 7 fold respectively by CoMTB stimulation at 24 hours (both p<0.001).  Confocal microscopy demonstrated colocalisation of early endosome marker Rab-5 with MMP-8 and -9 indicating that MMP-8/-9 are newly synthesised. MMP-8/-9 is significantly elevated in induced sputum samples from TB patients compared to healthy controls and both correlate with the neutrophil markers neutrophil gelatinase associated lipocalin and myeloperoxidase.  Brain biopsy specimens from patients with CNS-TB demonstrated neutrophils surrounding TB granulomas with MMP-8 and -9 present. 

Conclusion:   Neutrophil MMP-8/-9 gene expression and secretion is upregulated following direct infection with TB or stimulation by monocyte-dependent TB networks. The increase in MMP/TIMP ratio will result in a proteolytic environment which may lead to patient morbidity associated with tissue destruction.

Affliation: Department of Immunology and Microbial Science, IMM1,  The Scripps Research Institute

Convener: Prof Mary Ng

Abstract:
Recent work has demonstrated that endogenous non-stimulatory peptides can enhance T-cell recognition when antigen is limiting, as occurs when viruses or tumors reduce MHC expression as a means of immune evasion. The relative importance of T cell receptor (TCR) or co-receptor (CD4 or CD8) interactions with the non-stimulatory ligands in this activation-enhancement is unclear. We have now found that the affinity of the co-receptor for the MHC determines the requirement for specific interaction of TCR with the non-stimulatory ligand.

Protein kinase C (PKC)q has been known for a long time to function in T cell activation within the immunological synapse, but until recently it was not realized that PKCh is also important in T cell biology. It acts redundantly with PKCq  in thymocyte development, but is required in homeostatic proliferation.

The mechanism for discrimination between positive and negative or agonist selection based on recognition of self MHC-peptide ligands in thymocyte development is unclear. It has been a focus of this lab for many years. We have used fluorescence resonance energy transfer (FRET) and other imaging techniques to investigate how the developing thymocyte can distinguish between positive or negative-selecting ligands, finding that there are kinetic differences in intermolecular interactions during ligand recognition, as well as subcellular localization differences in parts of the TCR signaling cascade. We and others recently discovered Themis, a T-cell lineage specific protein that regulates thymocyte development. It appears to be important in regulation of TCR signal strength in response to positive selecting ligands. In addition, we find that Themis-deficient mice have defective regulatory T cells and develop a type 2 diabetes-like disease.

Affliation: Department of Immunology and Microbial Science, IMM1,  The Scripps Research Institute

Convener: Prof Mary Ng

Abstract:
Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents requires proper regulation of TCR signaling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex proteins (MHCp)[1]. Low affinity TCR interactions with self-MHCp generate weak signals that initiate “positive selection”, causing maturation of CD4 or CD8ab-expressing “single positive” (SP) thymocytes from CD4⁺CD8ab⁺  “double positive” (DP) precursors[1,2]. These develop into the mature naïve T-cells of the secondary lymphoid organs. TCR interaction with high affinity agonist self-ligands results in “negative selection” and “agonist positive selection” of specialized T-cells including gut intraepithelial lymphocytes, iNKT-cells and nTregs[1]. The threshold between positive and negative or agonist selection occurs over a very small difference in TCR-MHCp affinity[3], but the mechanism is unclear. Positive-selecting ligands induce slower interaction than strong ligands between TCR and CD8, and induce activation of the MAP kinase Erk within the cell whereas strong ligands induce plasma membrane-localized phospho-Erk[3,4]. Recent evidence suggests that there are specific thymocyte proteins that regulate the weak signals involved in positive selection[2,5].