Lauren Truby is a fellow in cardiovascular disease at Duke University Medical Center. She is the lead author of a new publication that uses proteomic profiling to identify a specific protein as a biomarker of primary graft dysfunction after a heart transplant. She was interviewed by Alissa Kocer, communications strategist for the Precision Genomics Collaboratory.
Alissa: Will you tell me about primary graft dysfunction and why you decided to do this research?
Lauren: In the current era, outcomes of a heart transplant are really excellent. The median expected survival of a heart transplant recipient is between 13 and 14 years. For these patients, the most at-risk time, is the first year after transplant. This is because things like rejection and infection are common, but also because of this entity called primary graft dysfunction or PGD. PGD describes a phenomenon of failure in the new heart within the recipient. It happens quickly -- within the first 24 hours of transplant – and it’s devastating both for patients and for us physicians who cared for them.
We have yet to understand the exact reason for PGD. We haven’t had consistent clinical risk factors that we’ve identified for it, and, most importantly, we don’t understand it at a cellular or molecular level. So this particular project grew out of and interest and a need to better understand PGD at a deeper level.
As a third year cardiology fellow, I am incredibly fortunate to work in the lab of and be mentored by Dr. Svati Shah who is a world expert in using ‘omics data for biomarker discover and cardiovascular disease. We, along with Dr. Christopher Holley, another member of the Duke transplant team, decided to use proteomic profiling to understand whether there were particular proteins circulating in higher levels in the blood of transplant patients who go on to develop PGD even before their surgery. By focusing on proteins related to immune activation and inflammation, we hoped that any proteins we found to be significantly different between PGD cases and those patients without PGD would help to give clues about its underlying biology.
Alissa: What did find with this research? What were your results?
Lauren: We used technology in Dr. Shah’s lab called the Olink Platform. With it, we can measure the relative expression of 92 different proteins using just a microliter of sample. Using this platform, we were able to measure a total of 354 different proteins in these samples that were taken immediately prior to transplant.
Our results were really interesting. We identified a protein called C-Type Lectin Receptor 4, otherwise known as CLEC4C, which is a protein found on the surface of a type of immune cell called plasmacytoid dendritic cells. We found that recipients with higher circulating levels of CLEC4C were more likely to have PGD. In fact, the risk of PGD was almost three-fold higher in those patients with higher levels of CLEC4C than those with lower levels. We validated this signal in our same data set.
We also compared CLEC4C expression to other clinical risk cores for PGD, namely a score called the RADIAL score. We found that CLEC4C performed much better in terms of risk-stratifying patients for PGD, so we were really excited about these results.
Alissa: What do plasmacytoid dendritic cells do, and why are they important?
Lauren: CLEC4C is a protein that lives on the surface of plasmacytoid dendritic cells, which are really interesting cells. Plasmacytoid dendritic cells play a unique role in the immune system. They bridge the gap between the innate and adaptive immune responses, and they’re really interestingly built to identify viruses, which can be difficult to detect by the immune system. Viruses either live within our own cells, or they freely circulate outside of cells, which makes them difficult to identify.
Plasmacytoid dendritic cells, however, have special receptors that bind to circulating viral DNA. Once it binds, it initiates a rapid and robust immune response, releasing huge amounts of inflammatory cytokines. In fact, plasmacytoid dendritic cells can release 10 times more interferon – one type of potent inflammatory protein – than any other cell type in the body.
The reason we think this may be related to primary graft dysfunction is that there are other types of DNA that can mimic viral DNA and illicit a similar response. One of these types of DNA is donor-derived DNA, meaning during the transplant process, some of the DNA from donor cells is released into the blood in large quantities, which may activate these plasmacytoid dendritic cells in a similar way, and in so doing, generate a strong immune response that can release high levels of interferon and TNF alpha that we know can be transiently toxic to the muscle of the heart and may cause this transient failure of the new graft.
Alissa: So what’s next?
Lauren: We’re really excited to continue this thread of research to try to better understand the role of plasmacytoid dendritic cells in primary graft dysfunction, and we have a few exciting things planned.
First, we want to confirm that the relative expression of CLEC4C that we’re seeing in these patients is reflective of an increased number of plasmacytoid dendritic cells. To do that, we plan to use flow cytometry on samples collected in a similar fashion as in our first experiment. This will allow us to count the number of plasmacytoid dendritic cells present in patients with and without PGD in order to understand whether it’s a relative number that’s driving this phenomenon that we’re seeing.
We are also looking forward to partnering with the Duke Immune Profiling Core and using exciting new technology called the IsoPlexis Platform, which will allow us to isolate plasmacytoid dendritic cells and stimulate them with cell-free DNA. After stimulation, we can quantify the amount of inflammatory cytokines that are being released on a single cell level.
Then we will be able to take the results from these two experiments together to understand whether it’s increased number of plasmacytoid dendritic cells, increased function of those cells or both that predisposes patients that to an increased risk of PGD. Eventually, we hope to translate this work into practical treatments and prophylaxis for PGD with, of course, the ultimate goal of improving outcomes for patients, their families and the community as a whole.
Alissa: Thank you, Lauren. This is really interesting and obviously important research that is going to have huge implications for heart transplant patients.
If you want to learn more about this research or explore other genomics-related research happening at Duke, please visit the Precision Genomics Collaboratory website.
This research has been published in the Journal of Heart and Lung Transplantation