Co-senior author Dr Terry Strom, co-director of the Transplant Institute at Beth Israel Deaconess Medical Center (BIDMC) and Professor of Medicine at Harvard Medical School (HMS), and colleagues describe their new imaging system in the June issue of Nature Medicine, which appeared online on 23rd May.
The success or otherwise of a transplant, after the recipients stops taking drugs to suppress the immune system, depends on whether the body tolerates or rejects the new tissue.
This appears to depend on the balance between destructive and protective T cells, said Strom in a statement, explaining that:
"With this new system, we can actually visualize this balance."
The correct term for the destructive T cells of the immune system is destructive effector T cells (Teffs) and the protective cells are called protective regulatory T cells (Tregs). The delicate balance struck between subsets of these two groups of cells determines whether the new tissue is eventually tolerated or rejected.
Strom explained however that this is not as straightforward as it may seem, for even when transplants are rejected, there may still be some protective Treg cells present, and conversely, when the tissue is accepted, there will be some Teff cells present.
However, because until now it has not been possible to differentiate these cell types in living tissue, the importance of the balance has not been well understood.
With co-senior author Dr Maria Koulmanda, Associate Professor of Surgery at HMS, and colleagues, Strom set about to address this problem by first genetically engineering two types of lab mice: one that expressed natural Treg cells (called nTreg to distinguish them from induced Treg cells) in a fluorescent green protein, and another that expressed natural Teff cells in a fluorescent red protein.
There was also a third color effect: yellow. This occurred when the red Teff cells commited to the induced Treg type, which was expressed by red and green proteins and thereby appeared as yellow; offering a useful way to distinguish between natural and induced Treg cells, said the authors.
By working with co-authors based at Massachusetts General Hospital, the team then developed a new imaging technique that brought together in vivo flow cytometry (a way of counting individual cells) and endoscopic confocal microscopy (a way of creating high resolution 3-D images of the inside of a specimen), so they could clearly see in real time the ratio of protective T cells to destructive T cells.
To test the method, they observed what happened to the T cell ratio when they transplanted genetically unmatched tissue into two groups of mice. One group was first given treatment to make them immune tolerant, while the other was not. The untreated mice vigorously rejected the tissue, while the treated mice accepted it, said the authors.
"Infiltration of T cells into the transplant was then visualized in untreated recipient mice in which vigorous rejection occurred," said Koulmanda.
The tissue they transplanted comprised genetically mismatched insulin-producing islet cells, which they placed beneath the thin capsule surrounding the mice's kidneys.
The new imaging process allowed the researchers to see clearly the ratio of protective to destructive T cells, which differed markedly between the two groups of mice. In the mice that rejected the transplant, the fluorescent red cells (Teff) rushed into the transplanted tissue much sooner than they did in the mice that accepted the transplant.
Fourteen days after the transplant, the researchers observed what they described as "costimulation blockade-based therapy inhibited infiltration by [the fluorescent red] Teff cells" in the treated mice.
Strom said that by enabling them to visualize the process, the new imaging system helped them understand more clearly the "quantitative and qualitative characteristics of the CD4 T cell response to allografts in rejecting and tolerized hosts".
"A picture really is worth a thousand words," he said.
Koulmanda explained that:
"As the events of rejection proceeded, the number of transplanted infiltrating T cells vastly exceeded those present in the tolerant transplants, even though the numbers of 'protective' yellow and green cells were equal in these groups at a later point in time."
"This is the first time that we have been able to monitor transplanted allograft tissue in a live lab animal. While static images of cells have been captured in the past, our new method captures much more than just random snapshots of the process," she added.
"Given the effectiveness of these tools, we hope to construct a road map such that we can create drug-free transplant tolerance for our patients in the future," said Strom.
Grants from the National Institutes of Health and the Juvenile Diabetes Research Foundation helped pay for the study.
Source: Beth Israel Deaconess Medical Center
