Feature: The structure of immunity

Since 2003, Professor Jamie Rossjohn’s team in the Protein Crystallography Unit at Monash University has been mining a rich seam in the field of antigen recognition by looking at the shape and structure of proteins – and what happens when they malfunction. This work has netted them papers in Nature, Nature Immunology, Immunity and other leading journals.

One of the avenues they’ve been exploring is antigen recognition by cytotoxic T-cells, the key event in cell-mediated immunity. For the immune system to clear a viral infection, cytotoxic T-cells must be primed to recognise and destroy virus-infected cells that continue to churn out newly replicated virions.

As well as studying the mechanisms of T-cell signalling, Rossjohn and his collaborators are exploring the fundamental mechanisms of major histocompatibility complex (MHC) restriction in cell-mediated immunity. They are trying to understand the workings of the MHC, the most polymorphic locus in the human genome, and what goes wrong to cause pathological T-cell responses like T-cell mediated graft rejection. “We’re focusing on the nitty-gritty of what happens when T-cell receptors engage MHC antigen-presenting molecules,” says Rossjohn.

The central event in cell-mediated immunity occurs when the infected cell presents peptide antigens from dismembered viral proteins to T-cells for recognition, an act that will mark it for destruction. The infected cell mounts the peptide fragment within a groove in an MHC class I receptor on its surface, which is possessed by all nucleated cells.

Natural killers

Rossjohn and his colleagues, particularly Professor Dale Godfrey from the University of Melbourne, have also focused on Natural Killer T-cells (NKT cells) a subset of T-cells that specialise in recognising lipid-based antigens. These antigens appear to pose a problem in antigen recognition because natural selection has created a special family of MHC class I-like molecules, called CD1d receptors, to present them.

According to Rossjohn, while the mechanisms by which T-cell receptors interact with MHC-bound peptides have been known for a long time, it was not known how the T-cell receptor (TCR) on NKT cells interacted with lipid-presenting CD1d receptors. Then in a Nature paper in 2007, Rossjohn presented the first description of how the NKT cell’s specialised lipid-antigen receptor interacts with a CD1d-bound lipid antigen.

Rossjohn says NKT cells are the most studied group of T-cells known to interact with lipid antigens. All NKT cell types express a semi-invariant receptor, dubbed NKT TCR, that recognises a specific MHC class I-like receptor, from the CD1 family. The NKT TCR protein is highly conserved and semi-invariant, placing it at the very intersection of innate and adaptive immunity. Rossjohn’s group showed the receptor ‘docks’ parallel to, and at the very end of, CD1d’s antigen-presenting groove, facilitating a lock-and-key interaction with the lipid antigen mounted lengthwise in the groove.

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Methods and mycobacteria

Rossjohn specialises in structural biology and the application of biophysical techniques to studying the molecular interactions between pathogens and the host, and the specialised immune system cells that deal with them. He is also interested in the basic mechanisms underpinning T-cell mediated autoimmune disorders.

As part of the research program within the ARC Centre of Excellence in Structural and Functional Microbial Genomics, Rossjohn’s group is also interested in the cell-wall components of mycobacteria, which are unique among bacteria. As models, they use the bacterium primarily responsible for tuberculosis, Mycobacterium tuberculosis, and several mycobacterial relatives, including the pathogenic Escherichia coli strain O113:H21 STEC, agent of haemolytic uraemic syndrome or ‘hamburger disease’; and Epstein-Barr virus, the agent of infectious mononucleosis, or glandular fever, one of the most common viral infections in humans.

“Mycobacterial proteins tend to be highly insoluble, making them very difficult to study structurally,” he said. “So we take a pragmatic approach of casting our nets quite widely in terms of the Mycobacterium species we work with and are presently in collaboration with Dr Travis Beddoe, Dr Paul Crellin and Professor Ross Coppel, focusing on an important group of enzymes termed glycosyltransferases.”

Beddoe, an NHMRC CDA Fellow, works with husband-wife team Professor James Paton and Dr Adrienne Paton at the University of Adelaide’s Bacterial Pathogenesis Laboratory. Two years ago they made an important discovery about what makes the AB5 toxin of E. coli O113:H21 STEC strain such a potent cell killer: the toxin is potentially lethal, especially in young children infected by inadequately cooked hamburger mince. It attacks the kidneys and destroys red blood cells, causing bloody diarrhoea.

Rossjohn says the AB5 toxin is structurally similar to toxins secreted by the bacterial agents of cholera (Vibrio cholerae) and whooping cough (Bordetella pertussis). The Beddoe-Rossjohn-Paton collaboration revealed the toxin’s A subunit is a protease, which targets the binding immunoglobulin protein (BiP) protein in the endoplasmic reticulum. BiP is a chaperone that protects other proteins at times of cell stress.

The toxin’s B subunit mediates its entry into the cell by binding to an unidentified glycan on the cell’s surface. Once inside, the A subunit destroys BiP, and without the shape-stabilising influence of the BiP chaperone, vital proteins go awry, inducing rapid cell death.

Working with the Consortium for Functional Glycomics in the US, they found that the infected cells put out a welcome mat on their surface of non-human origin: a glycan that can be translocated to human cells via milk and meat. Part-cooked contaminated hamburger mince is thus doubly dangerous: not only does it harbour the bacterium and its deadly toxin, but it provides the means for the toxin to gain entry into human cells. “It was the first example of how a bacterial pathogen has evolved a strategy to enter human cells from which it would otherwise be excluded,” says Rossjohn.

“We had a 12-month odyssey with the reviewers before Nature published it last December; they kept on coming back asking for more data, including that from animal models. When it was finally published, Travis and I flew to Adelaide for a celebratory dinner and a few drinks with James and Adrienne.

“Travis spearheads investigations of mechanisms of infection, while my lab focuses on understanding immune receptor mechanisms essential to adaptive and innate immunity.”

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The kiss of EBV

Rossjohn’s team has also recently begun a collaborative study with Dr Bob Anderson at the Walter and Eliza Hall Institute exploring the possible role of deamidated antigens in coeliac disease.

Coeliac disease is caused by an aberrant immune response to gliadin, one the gluten proteins responsible for the remarkable elasticity of wheat flour. In the digestive tract, glutamines in bread dough are converted to glutamates, allowing them to associate with certain variants of MHC molecules like DQ2 and DQ8 that trigger an auto-immune reaction that destroys the cells lining the small intestine, impairing nutrient absorption.

“Coeliac disease is a T-cell disorder and we are providing an understanding of how deamidated gluten peptides – but not the native peptides – are bound by DQ2 and DQ8 receptors, triggering an aberrant T-cell response.”

Rossjohn says the Epstein-Barr Virus collaboration, with Professor Jim McCluskey from the University of Melbourne and Associate Professor Scott Burrows at the Queensland Institute of Medical Research, is an ideal way of studying MHC restriction in naturally outbred populations of humans.

Around 95 per cent of humans contract EBV-caused glandular fever or ‘kissing disease’ within the first three decades of life; some are only mildly affected, but others are debilitated for months. “We’re studying the antiviral immune response, T-cell repertoire selection and the impact of receptor polymorphisms on the course of the disease,” he says.

“It was originally considered that MHC Class I receptors can only present small antigens of eight to 10 peptides, but about 10 per cent of them can present antigens larger than that. We’ve provided an understanding of how the T-cell receptor recognises these longer epitopes.

“The TCR is very adept at interacting with challenging molecular landscapes and these snapshots help us understand the fundamentals of MHC restriction. T-cell receptors are adapted to recognise specific antigens, but they can also react with things they are not adapted to recognise. The T-cell receptor’s plasticity can cause auto-immune problems.”

Fellows

Rossjohn considers he has been fortunate as a researcher. A native Welshman who graduated from the University of Bath and obtained a PhD from the same university, he won a Royal Society Travelling Fellowship to undertake post-doctoral research in Australia in 1995. “Australia enables a great lifestyle, and opportunities to do great science, so I stayed on after my fellowship,” he says.

He continued his post-doctoral work in Professor Michael Parker’s laboratory at St Vincent’s Institute of Medical Research, studying bacterial toxins. That led to an Australian Research Council postdoctoral fellowship and then an National Health and Medical Research Council Career Development Award.

“What really launched my career was a Wellcome Trust International Senior Research Fellowship,” says Rossjohn. “In 2002 I relocated to Monash University to establish and head a protein crystallography unit. The university has been very supportive and, moreover, I have been fortunate to work alongside many very talented and dedicated students, postdocs and colleagues.

“Currently, we’ve nurtured eight NHMRC Peter Doherty Australian Biomedical Fellows, three NHMRC CJ Martin Overseas Biomedical Fellows, two NHMRC Career Development Awards, and one ARC Research Fellowships Queen Elizabeth II Fellowship. Many of these people have used our success to establish their own careers.”

Rossjohn was also an ARC Professorial Fellow and is currently an ARC Federation Fellow. But he’s not taking his success for granted. “I was saying to the people in my lab the other day that while papers may be made from trees, they don’t grow on trees. We’ve been fortunate to have some big results but we’re competing against major international laboratories, which means hard work and involves pressure.

“You can’t escape that if you want to play in the major league. We’ve been fortunate to have published Nature papers in 2006, 2007 and 2008, but those successes stem from work we started four to five years ago. Our researchers work very hard and we spend an awful lot of time ensuring the questions we are addressing are important enough to give us a chance of playing in the major league. We’re constantly asking what the next big challenge is, and where the field is heading.”