Cell Migration Group

Our research interests lie in lymphocyte trafficking, specifically in cell adhesion molecules and the regulation of their activity and function.

T cells traffic around the body in and out of secondary lymphoid organs, searching for antigen which becomes sequestered there. Following activation, T cells redistribute to sites of infection; trafficking to both lymphoid organs and sites of infection is directed very much by initial recognition between T cells and the lining of the blood vessels - the vascular endothelium.

How it began

We started culturing endothelial cells from lymph nodes, the specialised high endothelial venule cells, where trafficking occurs, and we set-up an in vitro model of lymphocyte binding and trans-endothelial migration.

This was before we knew about the adhesion cascade. We still don’t really know much about how the gradient works; how the cells undergo directed migration into tissues; how they get across endothelial layers; how they penetrate the basement membrane and get into tissues; and then how they target within tissue.

So we started-off modelling the first step, which is trans-endothelial migration, either between the cells or transcellularly - there’s evidence for both pathways. It may well be that both pathways operate, but that one is preferential and a homeostatic condition, and another is recruited, say, during the inflammatory condition.

A proteolytic step in transmigration - parallels with tumour metastasis

We found that transendothelial migration was associated with a proteolytic step, such that the adhesion molecule involved in tethering and rolling, the molecule CD62L, otherwise known as L-selectin, undergoes proteolytic cleavage as the cells cross the endothelial layer - this is what we found in vitro - and it turns out that the proteolytic event is mediated by a particular type of protease which belongs to the ADAMs family (related to matrix metalloproteinases).

If these sorts of enzymes are being activated during transendothelial migration, perhaps they’re activated for penetration of tissues. These are the sorts of enzymes that have been implicated in tumour cell metastasis for years; the matrix metalloproteinases degrade components of the basement membrane, collagen and laminin.

The way T cells move around the body is very much the way tumours metastasise. T cells and B cells are born in the bone marrow, and then T cells go via the thymus, and then they recirculate - they are distributed around the body via the bloodstream and lymphatics.

Similarly, a primary tumour in one organ, in order to metastasise, has to break away, gain access to the bloodstream and then be distributed to secondary sites. Of the enzymes that have been implicated in proteolytic shedding of CD62L, one of them is the same enzyme that cleaves cell surface TNFa (pro-TNFa) and TNF receptors, so this looked to be a difficult pathway to pursue because, obviously, if you knock out that enzyme, you knock out processing of a number of substrates, therefore it would be difficult to interpret the biology of the consequences of deleting or inactivating that enzyme.

The approach we took was to inactivate the substrate; we mutated the cleavage site in CD62L to prevent it undergoing proteolytic shedding, we then had basically a mutant form of CD62L which we expressed on T lymphocytes in a transgenic approach - using the human CD2 cassette which Dimitris Kioussis had worked with at Mill Hill.

So we engineered a protease-resistant form of CD62L on T lymphocytes, made transgenic mice and backcrossed them to the knockout so the only cells that expressed CD62L were T cells and they have either a normal CD62L or a shedding-resistant CD62L.

The present and the future

Now we have what has been hoped for, and that is an in vivo model where we can ask “what are consequences of interfering with shedding of CD62L?” and there are two processes that are implicated in this - one is the transendothelial migration step, which is what we found originally in vitro, and the second is after T cell activation, one of the very early events is the shedding of CD62L - and we know that activated T cells were the hallmarks of CD62L - low expression, and this is what many immunologists use as a marker for antigen-experienced T cells.

That’s the lymph node homing molecule - so the prediction is that it would be implicated in the altered homing of activated T cells to sites of infection. That’s what we’re doing now.

Awen Gallimore has various in vivo models of T cell mediated immunity. One of these was the ‘influenza model’. We look at the mouse model of infection which is as close to a natural infection as possible, as opposed to using peptide-primed dendritic cells to activate T cells.

We prime mice with influenza intranasally, and then we challenge the immune mice for their memory responses and ask whether they can clear a secondary challenge. What we’ve found is that the shedding-resistant mice are defective in clearing a secondary flu challenge - we know that they can make memory T cells, so it’s not that their memory is defunct, we think their homing is defunct. They’re not getting to the site of infection.

That’s what we’re looking at now - to try and understand the mechanisms. A major goal is to establish methods for in vivo imaging of antigen-specific T cells as they migrate from the bloodstream into lymphoid organs and sites of infection. We will start by collaborating with Professor Sussan Nourshargh of Barts and The London School of Medicine who has pioneered in vivo imaging of neutrophil extravasation using intravital and confocal microscopy by applying the methods that she has developed to image T cell trafficking.

The regulation of T cell-dependent immunity by CD62L shedding

In collaboration with Dr Awen Gallimore, we are using a mouse model of influenza infection to study the role of CD62L shedding in controlling the migration pathways of antigen-activated T cells.

The prediction is that the inability to shed L-selectin will prevent activated T cells from homing to peripheral sites and, therefore, prevent clearance of infection.

The ability to track the migration pathways of antigen-specific T lymphocytes in experimental animal models will allow this hypothesis to be tested directly.

Identification of zinc-dependent metalloproteinases that regulate CD62L shedding and lymphocyte transendothelial migration

The effects of inhibitors of MMP-related enzymes on lymphocyte transendothelial migration were not wholly reproduced by T cells expressing non-cleavable L-selectin, which suggests that proteolysis of substrates other than L-selectin may also regulate this stage of lymphocyte migration.

ADAM17, which is known to regulate protein kinase C induced shedding of L-selectin from lymphocytes, is a possible candidate. Using genetically deficient mice and natural and synthetic ADAMs and MMP inhibitors we have shown that T cell receptor-induced CD62L shedding is regulated by ADAM17 but endothelial-induced shedding (which regulates transmigration across HEV) is ADAM17 independent.

We will use expression profiling and functional analysis of related ADAMs family members and a proteomics approach to identify candidate enzymes and their substrates that regulate lymphocyte extravasation.

Molecular basis of VLA-4 integrin activation associated with transendothelial migration

We have previously reported that VLA-4 (a4b1 integrin; CD49d/CD29) is activated on lymphocytes that have transmigrated HEV (Hourihan et al, J. Cell Sci., 104: 1049-1059, 1993).

Analysis of activated VLA-4 on transmigrated lymphocytes has shown that it is distinct from that VLA-4 activated by all other known activating agents, i.e. PKC, divalent cations or chemokines. In collaboration with Professor Gillian Murphy, Cambridge and Dr Peter Newham, Astrazeneca we have found that ADAM28, which has been reported to bind to VLA-4, is able to activate VLA-4.

We are currently determining whether ADAM28 induced activation of VLA-4 is implicated in lymphocyte transendothelial migration and whether VLA-4 activation is metalloproteinase dependent.

Lead researcher

Ann Ager

Dr Ann Ager

Reader

Email:
agera@cardiff.ac.uk
Telephone:
+44 (0)29 2068 8872