Research reveals new insights into transplant rejection and new drug development targets

Graphic abstract

image: Researchers at Cincinnati Children’s and the University of Cincinnati discover cellular signatures of organ rejection that appear detectable in a urine test. The results were published in the Journal of Clinical Investigation.
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Credit: Journal of Clinical Investigation, Cincinnati Children’s and University of Cincinnati

CINCINNATI — Imagine a day when a urine test could inform a doctor exactly why a kidney transplant patient was experiencing organ rejection and suggest the best medication to specifically address the problem.

He leapt closer to reality that day thanks to a remarkable series of single-cell analyzes that identified the most specific cellular signatures to date for kidney transplant rejection. The results were detailed on May 25, 2023, in JCI The Journal of Clinical Investigation.

The study results reflect eight years of teamwork by experts from Cincinnati Children’s and the University of Cincinnati College of Medicine with contributions from researchers from the University of Notre Dame and Novartis.

Tiffany Shi, an MD/PhD student with the Immunology Graduate Program and the Medical Scientist Training Program at Cincinnati Children’s was the first author. Senior coauthors were David Hildeman, PhD, interim director of the Division of Immunobiology at Cincinnati Children’s, and E. Steve Woodle, MD, professor of surgery and William A. Altemeier Chair in Research Surgery at UC College of Medicine. Hildeman and Woodle co-direct the Center for Transplant Immunology at Cincinnati Children’s.

“The treatments available to stop a rejection event have not changed much in recent decades. These cellular signatures open the door to creating an entirely new set of anti-rejection therapies,” says Hildeman.

“Having a precision medicine approach to treating organ rejection has the potential to greatly reduce the threat of rejection for transplanted organs,” Woodle says. “More follow-up research will be needed, but these findings have implications beyond kidney transplantation to potentially apply to liver, lung and other transplantations.”

Organ rejection affects 10% of recipients

Kidney transplant is the most common form of organ transplant; provided after organ failure from diabetes, infection, injury and other factors. In 2022, surgeons performed 25,498 kidney transplants in the United States, according to the United Network for Organ Sharing (UNOS).

Over the past 30 years, gradual improvements have allowed kidney transplants to last longer, so that now the “half-life” for living donor kidneys exceeds 20 years and approaches 12 years for deceased donor organs.

“For an older person, these survival rates reflect quite a long time,” Hildeman says. “But for young adults and children, the chances of needing a second transplant remain high.”

However, once a kidney transplant recipient experiences acute rejection, many lose their transplant and return to dialysis within 1 to 3 years. Also, once a patient’s immune system rejects one organ, it is much more likely to reject a second transplant.

Unfortunately, the tools available to effectively treat rejection – corticosteroids and antilymphocyte globulins – have remained largely unchanged for over 60 years. Evidence accumulated over many years has indicated that these treatments inadequately or incompletely address rejection.

Discovering clues one cell at a time

In the new study, researchers used powerful single-cell genomic analysis technologies to painstakingly compare biopsy samples from transplanted kidneys that found acute cell rejection. The studies also compared rejections that occur under the commonly used maintenance immunosuppressive agent (tacrolimus) and two new alternative drugs (belatacept and iscalimab).

The analysis was so detailed that the team was able to track how gene expression changed within specific populations of cells driving the rejection damage, which the authors termed T-cell clones. expanded allospecific CD8 (CD8EXP).

The researchers say this study is the first to apply a combination of single-cell RNA analysis with single-cell T-cell receptor (TCR) analysis to explore acute kidney transplant rejection.

“The power of what we’re doing comes from being able to look at cells at the single cell level. We can look specifically at those that are responsible for rejection, and we can see how rejection changes over time as T cells are shifting their response to different drugs,” Shi says.

Woodle describes CD8EXP cells like the “spearhead” in waste.

The work revealed three key findings:

First, even when an acute rejection event has been stopped, research has revealed that treatments often aren’t thorough enough to eliminate all the T cells that had cloned themselves to attack the graft. In some cases, hostile T cells persisted for months after anti-rejection treatment.

This suggests that multiple rejection events, previously thought to be completely separate, may actually be one longer, steaming rejection event. Addressing lurking cloned T cells that have escaped initial treatment will likely require improved testing techniques and the adoption of more consistent standards of practice.

Second, the team found around 20 CD8 ‘clonotypes’EXP T cells – potentially thousands – that have reproduced to mount attacks on a transplanted organ. The types differed according to the receptors carried by the T cells. The relatively low number of clonotypes excited the researchers because it will make it easier to find potential new treatments to stop transplant rejection.

By studying these rare but efficient cells, the team found distinct cellular signatures that occur during a rejection event that varied depending on the maintenance immunosuppressant drug used. The different genes involved raise the possibility of using other drugs not typically associated with the treatment of organ rejection as new weapons for specific situations.

For example, this team also recently reported success using an mTOR inhibitor called everolimus to help patients who have not benefited from belatacept treatment. But the same drug appears to offer no similar benefits when tacrolimus treatment is involved. This work led to an ongoing clinical trial led by Woodle for treating patients with belatacept and everolimus for maintenance immunosuppression.

Third, the same types of T cells that cause rejection events can also be detected in urine samples.

Why a urinalysis is important

Currently, obtaining the crucial details behind the rejection of a transplanted kidney requires the collection of a tissue biopsy, a surgical procedure that requires a visit to the hospital. Conducting multiple biopsies over time to monitor treatment outcomes is expensive and potentially risky for patients.

However, urine tests could be collected more frequently noninvasively and potentially without the inconvenience of going to the hospital. In addition to directly supporting patient care, a viable urine test would help expedite the research work needed to evaluate new anti-rejection treatment protocols. Research has also shown that CD8EXP The T cells that were found in the organ of rejection were also present in the urine.

“This finding indicates that a simple urine test could replace a more invasive kidney transplant biopsy and thus make it much safer and easier for patients to monitor the effectiveness of their rejection treatment,” says Hildeman.

The key challenge in achieving practical clinical urine testing will be establishing a process that can produce test results in 48 hours rather than the research-focused process used in this study, which took several months to complete.

Award winning presentation

As first author, Shi presented the results of this study at ATC2023, the annual scientific meeting of the American Transplant Congress, held June 3-7 in San Diego. Shi’s presentation was awarded the “People’s Choice Award”, which means that it was selected by the more than 5,000 attendees as the best plenary presentation of the meeting.

Shi also received a Young Investigator Award, and several other Cincinnati Children’s and UC researchers were honored at the event.

Next steps

More research work is needed to explore treatment possibilities related to the T-cell signatures revealed in this study. Work conducted by the co-authors has already demonstrated that tacrolimus was an excellent treatment for some patients with resistant rejection cases.

In the long run, the benefits may go beyond expanding the “half-life” of donated kidneys.

For example, finding alternatives to tacrolimus could help people with liver and other organ transplants avoid kidney complications from their anti-rejection regimens.

And finally, a new understanding of the key cellular mechanisms involved in transplant rejection could lead to ways to reduce the risks of xenotransplantation (using genetically modified animal organs in humans).

“Most people who need an organ transplant never get one because the supply of donated organs remains so limited,” Woodle says. “With insights like these, we may be able to substantially reduce the loss of transplanted organs to rejection, thereby freeing up donated organs for new transplant recipients.”

About the studio

In addition to Shi and Hildeman, Cincinnati Children co-authors who contributed to this study included: Ashley Burg, PhD, Krishna Roskin, PhD, J. Timothy Caldwell, MD, PhD, and Cyd Castro-Rojas, PhD.

In addition to Woodle, University of Cincinnati collaborators included Rita Alloway, PharmD, and Adele Shields, PharmD.

Funding sources for this study included a grant from Novartis and Public Health Service grants from the United States Department of Health and Human Services (AI167482, AI142264, UH2AR067688, UL1TR000077, and AI169863).

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