Leucocyte Membrane Protein Laboratory

Overview

The Leucocyte Membrane Protein Laboratory studies molecules expressed at the surface of white blood cells. These receptors play a critical role in the biology of white blood cells as they serve as molecular antennae. Their role is to receive signals from outside the cell and allow the cell to respond to and interact with its environment. This laboratory focuses on two complementary, but distinct fields of interest. Firstly, we are fascinated by a particular type of cell surface protein known as the Tetraspanins, and we aim to determine the precise role that these molecules play in the immune system. Secondly, we are interested in the identification and characterisation of novel molecules expressed at the surface of a rare and important type of white blood cell – the dendritic cells (DC). DC expressed surface molecules may represent exciting new targets for manipulating the immune system.

Tetraspanins

The Tetraspanins are a type of membrane protein characterised by four highly conserved membrane spanning domains. White blood cells are known to display up to 17 different tetraspanin molecules at their cell surface. The precise function is not known but there is now evidence that the tetraspanins may be “molecular organisers”, and their major role is probably to organise complexes of other transmembrane and signal transduction proteins into "microdomains". This allows the tetraspanins to influence a number of key cellular processes such as proliferation, adhesion and migration.

We are addressing the question of tetraspanin function with an in vivo approach using the power of gene targetting technology. Our aim is to produce mice that are genetically incapable of expressing specific tetraspanins at the cell surface and to examine how the immune system functions in the absence of the tetraspanin in question.

CD37

We have characterised mouse CD37 and produced mice that are deficient for this gene. It is clear that the immune system does not function normally in the absence of CD37, and both the T and B lymphocyte lineages are affected. Although development of these two lineages is unaffected by the absence of CD37, antibody responses to model antigens are clearly dysregulated. T cell independent antigens elicit an abnormally high response whereas T cell dependent antigens elicit a poor IgG response. In vitro analyses of lymphocyte function show a striking hyperproliferation of CD37-deficient T cells in response to mitogenic stimuli. CD37-deficient T cells on average enter first division earlier than normal T cells. This hyperproliferation is preceded by an enhanced production of the T cell growth factor IL-2. The results are consistent with the idea that tetraspanins are organisers of multimolecular cell surface signalling complexes. Thus the absence of CD37 may disrupt the composition of a multimolecular signalling complex in the T cell membrane leading to a dysregulation in T cell receptor signalling, and enhanced transcription of genes, such as IL-2, involved in proliferation. It will be of major interest to immunochemically determine which signal transduction complexes in T cells are affected by the absence of CD37 (see Figure1.)

CD151

CD151 is a tetraspanin which is thought to have an important role in controlling biological processes such as cell migration, cell adhesion, wound healing and cancer cell metastases. CD151 has a robust molecular interaction with the cell adhesion integrin molecule a3ß1, and has been described as the third protein chain in this important complex. The fundamental importance of a3ß1 to biology is underlined by the fact that mice that are deficient for this protein die 10 days after birth. In collaboration with Prof. Leonie Ashman and colleagues (University of Newcastle, NSW), we have produced mice that are deficient for CD151. To our surprise, the mice are developmentally normal. Further investigation of the CD151-deficient mice, particularly their immune system, is underway.

Dendritic Cells

Dendritic cells (DC) are a rare type of white blood cell with a critical role in presenting foreign material to the immune system and thus inducing immunity. DC play a critical role in vaccination. Recent research has revealed that DC are not a homogeneous population, rather there are distinct sub-populations of DC which vary in their function. For example, in the mouse spleen there are three distinct DC populations which can be distinguished by the expression of the surface molecules CD4 and CD8: CD4+DC, CD8+DC and CD4-CD8-DC. These DC differ in their function. For example, CD4+DC and CD4-CD8-DC are particularly strong stimulators of T lymphocytes, whereas CD8+ DC are relatively weak stimulators and might have a regulatory role. We wish to understand the molecular differences that exist between the differing DC populations as these molecular differences may explain the functional differences that exist between the dendritic cells. Given the differing types of immunity induced by DC populations, molecules that are differentially expressed on the surface of DC are potential targets for developing new drugs to manipulate the immune system.

FIRE and CIRE

In collaboration with Dr I Caminschi and Prof. K. Shortman (WEHI) we have identified two novel membrane proteins that are expressed on the surface of CD4-CD8- DC. FIRE is a novel member of the EGF/TM7 subfamily of membrane proteins and has a fascinating and unusual structure of seven transmembrane domains with an extracellular domain containing two EGF domains. The function of the EGF/TM7 molecules is not known, however the presence of EGF domains in the extracellular region suggest a role in mediating specific molecular interactions at the cell surface. CIRE is a type II integral membrane protein with a C type lectin domain in its extracellular region. A number of C- type lectin domain membrane proteins expressed on DC are known to mediate molecular interactions with the T cells surface. Thus both CIRE and FIRE are molecules likely to mediate interactions between DC and other cell types. Future work on FIRE and CIRE aims to understand the contribution of these molecules to DC biology. The production of knockout mice by gene targeting and the identification of FIRE and CIRE ligands is our first priority.

New Dendritic Cell Molecules

In a collaborative project between the Leucocyte Membrane Protein laboratory, Mireille Lahoud and Ken Shortman (WEHI) and KIRIN; we have initiated a program aimed at identifying additional DC molecules differentially expressed between populations. We have already constructed a normalised cDNA library generated from a pool of dendritic cell populations and used robotic technology to construct micro arrays of cDNA clones. These micro arrays have been interrogated cDNA generated from highly purified DC populations. We have identified several candidate genes that appear to be differentially expressed by other DC populations by the CD4+ or CD8+ DC. A major focus of our future work will involve the molecular and immunological characterisation of these novel genes.