Research

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.