March 2010 - Issue #9
Immune system fails on video
Story by Graeme O’Neill
View articles in related topics: Health & Medical, Optical Physics
As we live and breathe, millions of T-cells that are part of the human body’s molecular defence force patrol the blood and lymphatic systems, seeking out and destroying cells harbouring viruses and bacteria or mutant cells that could turn cancerous.
T-cells are assassins and fortunately, in the main, they are on our side. The trouble is, these immune-system bodyguards can also turn treacherous.
In some individuals they attack healthy tissue, causing autoimmune disorders such as rheumatoid arthritis, type 1 diabetes, multiple sclerosis and systemic lupus erythematosus.
To try to find out why, immunologist Dr Sarah Russell, of Melbourne’s Peter MacCallum Cancer Centre and Swinburne University of Technology, has teamed up with Professor Min Gu’s research group at Swinburne’s Centre for Microphotonics to develop new imaging tools to study T-cells in vitro.
Dr Russell – the recent recipient of a prestigious Australian Research Council Future Fellowship – and Professor Gu are hoping this close-up exploration of the T-cells’ structure, as they develop their specialised forms and functions, will provide clues to what goes wrong and triggers an autoimmune disease.
It is a complex and fascinating process at the heart of human biology.
As part of the project, the researchers are creating a biochip to study the development and behaviour of T-cells, individually corralled within microscopic ‘paddocks’ formed by a microgrid of silane polymer ‘fences’ deposited on silicon.
“The biochip will allow us to cage single cells as we observe their individual development, instead of trying to monitor 20-plus cells at a time,” Professor Gu says.
The window to this extraordinary research is a video camera linked to a laser confocal microscope. This will deliver high-resolution, dynamic images of the confined cells responding to biochemical signals piped into the ‘paddocks’ through a network of microfluidic channels etched into the chip’s surface.
Dr Russell says the apparatus will provide the first real-time video images that monitor T-cells over multiple generations, with precursor cells moving, changing shape, dividing and differentiating into mature T-cells with specialised forms and functions.
“T-cells form from bone marrow stem cells that migrate to the thymus, where they undergo a complex process of differentiation and selection,” she explains.
The thymus gland, high in the chest, is ‘T-cell university’, where cells progressively differentiate into naïve, functionally specialised T-cells (‘naïve’ because they have yet to be activated by exposure to alien antigens).
As they ‘graduate’ they are ready to be activated in the event of an external or internal threat. Once activated, mature T-cells undergo final transformation into:
- effector T-cells – programmed to kill any cells advertising their infected or pre-cancerous state by displaying unfamiliar, ‘non-self’ protein antigens on their surface; or
- memory T-cells, that can linger in the body for decades, ready to reactivate and rapidly repopulate the body with new effector T-cells should they detect the familiar antigenic signature of an old enemy.
In addition to conventional laser confocal microscopy, they will use a powerful new technique called photo-activated luminescence microscopy (PALM), capable of resolving individual cells in nanoscale detail. PALM can even track individual protein molecules.
It works by attaching DNA sequences for luminescent protein ‘tags’ to genes for native cellular proteins. These ‘tags’ cause the hybrid proteins to glow green or cherry red under laser light, allowing researchers to observe their movement and interactions.
Dr Russell’s Swinburne research focuses on a suite of polarity proteins that have been conserved across the billion-year evolutionary divide between simple nematode worms and humans.
Polarity proteins play integral roles in almost every aspect of a T-cell’s life cycle and function and are a key focus of the research.
In three-dimensional space, polarity proteins aggregate at one ‘end’ of the cell, providing an internal reference point that allows the cell to orient and link to its neighbours to form the highly organised, layered structures of bone, cartilage, soft tissues and organs.
T-cells are motile and fluid in form and investigations by Dr Russell and other international investigators have shown that the same suite of polarity proteins found in static cells is involved in nearly every aspect of T-cell development and function.
Dr Russell explains that polarity proteins underpin T-cells’ ability to move, to orientate towards biochemical cues in their environment, to change form and function, to recognise alien protein fragments (antigens) presented to them by sentry cells, and to undergo clonal expansion.
She says polarity proteins are believed to form complexes that manipulate the cytoskeleton, the internal network of microtubules that stabilises and shapes the cell, and allows it to move and make contact with other immune-system cells.
From this, and as part of the probe into why T-cells sometimes turn against us, Dr Russell hopes to detail what happens within a structure called the immune synapse, which is involved in activating T-cells to attack cells displaying unfamiliar antigens.
The immune synapse forms when a naïve T-cell ‘docks’ with a specialised antigen-presenting cell that is displaying an alien antigen in a groove on its surface.
There is still much to be learnt about how the cells signal each other and come together to form the synapse, or how the antigen is subsequently transferred to the T-cell for use as a template to recognise and destroy infected or mutant cells.
Dr Russell also wants to investigate the role of polarity proteins in asymmetric division, a process crucial to T-cell development, maturation and activation.
In the face of threat, the immune system must create a host of new T-cells – and it creates these from non-specialised precursor cells, without depleting its reserve of precursor cells. Precursor cells are cells that are incapable of self-renewal and instead differentiate into one or two closely related final forms.
When precursor cells divide they can either produce twin clones of the original cell (symmetric division), or a non-identical pair – a single daughter clone and a cell that has taken the next step towards differentiating into an activated T-cell (asymmetric division).
Dr Russell and Swinburne researchers are developing automated systems to capture and analyse the high-resolution images of these processes. “We’ve come a long way in developing the software, and in constructing microgrids on the biochips,” Dr Russell says.
“But we still have a long way to go to develop the microfluidic system to manipulate the biochemical signals to the cells.
“We’ve already obtained some images without manipulating the signalling environment process. We’re pretty good at imaging with standard fluorescence microscopy, but we have to learn a whole range of new skills to do PALM imaging.”
Dr Russell says much of the Swinburne team’s work will depend on being able to obtain images at the level of individual protein molecules.
By studying T-cells undergoing normal differentiation in vitro, Dr Russell says they should be able to identify errors that unbalance the process, potentially resulting in lymphoma or leukaemia blood cancers.
“I suspect any defects in polarity and asymmetric cell division will be apparent long before any autoimmune problem,” she says.
“Our work is likely to make a difference to understanding how polarisation develops, and how it influences each step in T-cell differentiation and activation.
“For example, when the immune system has eliminated an infection, most of the T-cells involved die off, leaving just a small population of memory T-cells to keep watch for any future infections by the same microbe.
“And we hope to define the key processes that determine whether precursor cells will differentiate into effector or memory T-cells.”
Dr Russell says that by providing the first comprehensive picture of T-cell development and maturation, the Swinburne project should provide clues to the origins of autoimmune disorders and help inform the development of a new generation of vaccines against infection and cancer.
