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Immune Sequencing Pinpoints Mechanism of Kidney Transplant Tolerance


NEW YORK (GenomeWeb) – Researchers at Columbia University have used an immune sequencing strategy to describe a mechanism responsible for kidney transplant tolerance.

Using immune sequencing services from Adaptive Biotechnologies, the team discovered a set of patient-specific T cell receptors that react to the donor organ and disappear over time in those patients who do not reject the organ. They published their results this week in Science Translational Medicine.

"It's a new window into what's happening in the recipient anti-donor T cells," Megan Sykes, director of the Columbia Center for Translational Immunology and senior author of the study, told GenomeWeb. The technique has "practical applications," she added, "but also, using it to study transplantation will give us a lot of new information about what goes on after transplantation."

The team used the immune sequencing strategy within a clinical trial of combined kidney and bone marrow transplantation (CKBMT). CKBMT is a relatively new transplantation technique by which a kidney transplant is performed in conjunction with a bone marrow transplant. Studies have found that the combined transplantation produces a mixed chimerism state combining elements of donor and recipient immune system. This mixed chimerism state induces tolerance of the transplanted kidney without requiring immunosuppressant drugs. However, the mechanism of how this tolerance occurs was not known.

Sykes was previously part of the Harvard University group that developed the CKBMT technique, which was first performed at Massachusetts General Hospital in 2002.

Functional studies gave some evidence that regulatory T cells were involved in inducing tolerance, but, looking longer term, those T cells seemed to disappear, Sykes said. The functional studies did not provide enough clarity to determine whether those regulatory T cells were actually deleted or if they were still present but inactive, so the team turned to next-generation sequencing.

First, the group examined donor and recipient cells from patient/donor pairs prior to the transplantation. Sequencing was done at Adaptive Biotechnologies using the firm's ImmunoSeq assay. After sequencing, the team irradiated the donor cells so that they could not proliferate, fluorescently labeled them, and then used the donor cells to stimulate the recipient cells. The recipient cells were still able to proliferate.

Next, they collected the cells that had divided, sorted them, and sequenced the CDR3 regions, comparing them to the unstimulated pre-transplant cells to pinpoint which cells responded to the donor antigen. Those few thousand T cell receptor cells comprised the recipient fingerprint. Following transplantation, that fingerprint can be tracked to see how it relates to graft tolerance or rejection. To validate the protocol, they confirmed in healthy individuals that alloreactive T cell clones could be detected in blood.

The research team then tested the protocol on four patients that had undergone CKBMT more than five years prior and had stopped taking immunosuppressant drugs eight months after surgery. In three of those patients the graft had been accepted, and one had graft failure. The team also tested their technique on two additional patients that had received standard kidney transplantations and were both on immunosuppressant drugs, one of whom failed.

They first identified the donor-reactive T cells in each patient before transplant, and then ran the sequencing assay at six months, one year, and 18 months after transplant.

In the four patients that underwent the CKBMT protocol, they saw that deletion of the donor-reactive T cell repertoire correlated with tolerance. Compared to the pretransplant cells at the subsequent time points tested, the number of donor-reactive clones declined. In the patient with rejection, the donor-reactive cells did not significantly decline over time.

For the two patients with standard transplantation, the team was able to see expansion of the donor-reactive T cell receptors in the patients' blood.

Sykes noted that although the study was small, it has implications for not only better understanding of how and why transplant organs are accepted or rejected, but also for early detection of rejection.

She said that her group is now planning to study patients that have received standard transplantation to see whether the assay can better predict rejection. Another potential use of the assay is in conjunction with a clinical trial where patients are being weaned off of immunosuppressants, to see if it can detect early signs of rejection.

Immunosuppressant drugs cause a whole host of side effects, including hypertension, diabetes, and an increased risk for infection. In some cases, patients who have received transplants can be weaned off these drugs without having graft failure, Sykes said. For instance, approximately 20 percent of adults with liver transplants can be successfully weaned of the drugs, but there is not a good way of predicting who that 20 percent is.

"We're interested in finding out whether the expansion/deletion of this fingerprint can predict who can be weaned," she said, and "likewise, whether expansion of these donor-reactive cells can predict rejection before it happens."

The assay is "a new window into what's happening in the recipient anti-donor T cells," Sykes said.

Another facet she is studying is the fate of the deleted donor-reactive T cell receptors. To do this, she is looking at biopsy specimens to try and figure out whether those cells during a rejection event increase in the graft itself or just in the blood. That work is still unpublished, she said.