Dan Salomon Embraces the Complexity of Transplant Medicine

Medicine could be so easy—a matter, mostly, of swapping old and broken parts for new ones. But although doctors have been doing transplants for more than a century, they still haven’t solved the problem of transplant rejection.

“The half-life of a kidney transplant today is the same as it was a decade ago,” said Daniel R. Salomon, MD, professor in The Scripps Research Institute (TSRI) Department of Molecular and Experimental Medicine and program medical director of the Scripps Center for Organ Transplantation. “That half-life is about 12 years—which means that about 30 percent are gone by 5 years, 50 percent of transplants are rejected after 12 years, and the majority of all transplants are lost by 20 years.”

This progressive loss of kidney transplants is also true for liver, heart, lung and pancreas transplants, and all this loss represents a tremendous personal price for patients and families and billions of health care dollars for society.

Transplant immunologists aren’t entirely sure what lies at the root of this problem. They already have a substantial arsenal of anti-rejection drugs, from antibodies that wipe out circulating T cells to powerful small-molecule immunosuppressants such as cyclosporine, FK506, mycophenolic acid and rapamycin.

Should they be making better use of this arsenal? Or do they need a better arsenal? In principle, all they need to do is knock down, once and forever, the immune system’s sensitivity to an individual donor’s tissue—leaving the rest of the system intact. But so far, the sheer complexity of human immunity has defeated all comers.

Toward an Understanding of Rejection

Salomon long ago decided to embrace this complexity—or rather to corral it, using the tools of functional genomics. In one recently completed project, he and his colleagues monitored the expression levels of hundreds of different immune-related genes in patients undergoing kidney transplant rejection and in those with healthy, well-functioning transplants. Comparing the two datasets, his team was able to find robust signatures of acute and chronic rejection, both in biopsied tissue and in blood samples.

One payoff of this will be a diagnostic test that clinicians can use to detect kidney rejection early, before serious damage has been done. Such a test can also be used to monitor the adequacy and safety of immunosuppression in individual patients, in other words, to personalize immunosuppression. Another payoff will be a better understanding of the rejection process from the molecular level. Immunologists already know that classical or “acute” rejection is a relatively swift and powerful T-cell-mediated response, provoked by the recognition of “foreign” proteins on donor cells. But there is a subtler, slower, chronic rejection response that they have understood far less well—they haven’t even been certain that it is an immunological response.

“What our new gene-expression data show is that chronic rejection is also immunological and, like acute rejection, represents a failure of immunosuppression,” said Salomon. In fact, Salomon’s group created a bioinformatics tool to map more than 60 functional molecular networks established in the immune literature and demonstrated that many are activated and shared between acute and chronic rejection.

Inadequate immunosuppression can result from attempts to avoid immunosuppressive drugs’ side effects, which include kidney and liver toxicity, and hypertension, plus increased risks of cancers, diabetes and heart disease. Doctors, and patients on their own, tend to ease up on the doses after a while, especially after the first year when acute rejection becomes less of a worry. Nothing may change suddenly, particularly as the current measure of kidney function, the serum creatinine, is so insensitive that more than 50 percent of kidney function must be lost before the level increases.

The danger, Salomon said, is that the chronic rejection process just smolders silently, so that a cutback of immunosuppression after the first year can lead to the loss of the transplant years later—through an immune-mediated process so slow and subtle that cause and effect have been hard to connect until now, using global gene expression profiling and molecular mapping.

Plugging the Hole

Salomon’s work also points to a class of T cells, known as memory T cells, as key culprits in the chronic rejection process, at least for kidney transplants. Salomon demonstrated that the population of these memory cells is activated and expanded in the first three months after a new transplant, despite the artillery barrage of anti-T-cell antibodies and other immunosuppressants that doctors use in that early phase to quell acute rejection.

“We think that there’s obviously a ‘hole’ in the efficacy of current immunosuppression therapy, and we’re trying to figure out how to plug it,” said Salomon.

One way to figure out the rejection process is to see how gene-expression patterns change in immune cells. Another—a “proteomics” approach—is to track the levels of rejection-related proteins. Salomon’s lab has done both, the latter in collaboration with another TSRI investigator, Professor John Yates, and his laboratory group in the Department of Chemical Physiology.

But gene expression patterns and protein levels don’t tell the whole story. For example, after a gene’s nucleotide code is copied into an initial strand of RNA, the latter may be diced and spliced together in various alternative arrangements, each of which eventually results in a distinct version (“isoform”) of a protein.

“This alternative splicing process can have powerful effects on proteins,” said Salomon, “because it can remove or add regulatory regions that affect message lifespan, where the proteins go and with which partners they interact.”

Remarkably, this process has stayed under the radar in transplant immunology because traditional gene expression and proteomics don’t readily distinguish alternative splicings of a given gene transcript or the resulting protein isoforms. Can transplant immunologists afford to ignore it anyway? Absolutely not, said Salomon.

Salomon’s laboratory reported evidence in a landmark paper in the journal PLoS ONE in 2009 that alternative RNA splicing may transmit many otherwise-hidden signals during immune cell activation. “A third of the genes that change during T-cell activation actually show no change in their net expression level,” Salomon said, “but rather change significantly only at the level of alternative splicing.”

All of this information has been effectively ignored until recently. However, the Salomon laboratory has launched a new collaboration with Jamie Williamson, dean of graduate and postgraduate studies, and Ian Wilson, chair of the Department of Integrative Structural and Computational Biology and Hansen Professor of Structural Biology. The team has been studying the assembly and structure of multiprotein splicing complexes on RNA molecules in the nucleus and how these complexes are regulated during T-cell activation. The goal is to determine how alternative-splicing changes occur and what role they play in T-cell biology and transplant rejection.  

Peeling Back the Onion

There are still more genomic layers that Salomon’s lab has had to reckon with. One involves microRNAs, snippets of ribonucleic acid that evolved to regulate gene expression by preventing some gene transcripts from being translated into proteins. Another consists of the methyl groups and other alterations to DNA that serve as high-level “epigenetic” controls of gene expression in any cell.

“We’re trying to determine, for example, the epigenetic signatures of memory T cells and to correlate these with activation-induced gene and protein expression to better understand why current immunosuppressive regimens are relatively ineffective against them,” said Salomon. “A key point is that a working understanding of the molecular basis of rejection, T-cell activation and the selective failures of immunosuppression will require tying all these complex pieces of information together.”

Adding to all this complexity is a wild-card factor from outside the immune system. Several years ago, leading transplant immunologists drew attention to the fact that activated B-cells—the immune cells that produce antibodies—are frequently found infiltrating transplanted kidneys with chronic rejection.

To some, this suggested that B cells mediate chronic rejection just as T cells mediate acute rejection. Salomon and his laboratory launched a series of studies, working in a partnership with Richard Lerner, TSRI’s Lita Annenberg Hazen Professor of Immunochemistry, Staff Scientist Rajesh Grover of Lerner’s group, Professor Nicholas Schork and Assistant Professor of Molecular and Experimental Medicine Ali Torkamani. These studies used deep sequencing of transplant biopsies to demonstrate that these infiltrating B cells and antibodies are in general not directed against donor kidney cells. Instead, their characteristics suggested that they are provoked by a chronically infecting pathogen.

“We asked ourselves: what’s the most common infection in a kidney transplant patient? The answer: E. coli urinary tract infection,” said Salomon. “And lo and behold, Drs. Grover and Lerner found that these clonal antibodies lit up E. coli cells.”

A New Suspect

In particular, the studies lit up an E. coli molecule called lipopolysaccharide, a toxin that is notorious for its powerfully provocative effects on the human immune system. Grover then used an elegant system of recombinant E. coli to map the epitope definitively.

Salomon and his colleagues now are following up to determine the prevalence of suspect E. coli strains in chronic kidney rejection cases using both protein biochemistry and deep sequencing of microbial DNA. But the finding already raises the tantalizing possibility that a big improvement in kidney transplant survival could be brought about simply by eliminating some bad bacteria.

“There’s another door that just got opened on the whole field of transplantation immunity: the microbiome,” said Salomon. “And what we stumbled on may be a mechanism driving immunity and inflammation in many diseases, not just chronic transplant rejection.”

Solving transplant rejection would be quite enough for one career, and Salomon’s embrace of complexity seems to be bringing that solution closer. He hopes soon to be using profiles of gene expression, alternative RNA splicing, epigenetic marks, protein levels—the whole functional genomics mix—not just to diagnose ongoing rejection responses but to predict them before they start, and maybe even before the transplant occurs.

Unsurprisingly, he also plans to use these data to guide the design of better immunosuppressants and to enable doctors to fine-tune the dosing of current drugs for any given patient; in other words, precision medicine for transplantation.

Salomon came to TSRI 20 years ago, after a distinguished early career with stints at Harvard Medical School, the University of Florida and the National Institutes of Health. “I’ve worked hard to link my medical interests to basic science, all the way down to cell biology and chemistry,” he said. “TSRI’s science-focused, collaborative environment is perfect for that approach. Doing the best basic science possible and taking every opportunity created by good science and outstanding collaborators to translate the findings to a better understanding of health and disease is why I’m still here.”

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