Cancer immunotherapy and transplantation are basically the same field; one’s just the flip side of the other“. Dr. Fadi Issa

Have you ever been to Oxford? Situated in the gorgeous English countryside, 56 miles northwest of London, Oxford is the charming home of the University of Oxford, the oldest university in the English-speaking world and among the top 5 highest ranked universities worldwide. Walking its streets feels like being in an episode of the iconic British TV series “Inspector Morse”, with bikes leaned up against the Radcliffe Camera’s railing and students strolling by groups of tourists.

It’s therefore not a surprise that the grant winner for the GeoMx Digital Spatial Profiler (DSP) instrument grant is the leader of the Transplantation Research Immunology Group at the University of Oxford. In 2018 Drs. Fadi Issa, DPhil FRCS(Plast) and Joanna Hester were awarded a GeoMx DSP system. They profiled biopsies from patients receiving regulatory T cell (Treg) infusion during renal transplantation to assess the presence of an immune tolerance signature, with the goal of eventually weaning them off immunosuppression.

NS: How did you get interested in solid organ transplant (SOT)?

FI: It has been an interesting and unusual journey. About fifteen years ago, I became interested in transplantation as the field was expanding into upper limb and face transplants – creating a crossover with plastic surgery. Once I delved into the field, the immunobiology is what really ignited my interest. Understanding the mechanisms of allograft rejection and the methods to induce tolerance – mechanisms which are relevant to many diseases including cancer and autoimmunity – are what drive our research program today.

NS: One important aspect of your work is the effect of hypoxia on immune cells. How does this tie into your interest in regulatory T cells (Tregs) in SOT?

FI: Hypoxia regulates nearly every process in mammalian physiology. We know this from our understanding of the intricate mechanisms by which cells detect hypoxia and the downstream effects of activation of the hypoxia inducible factor (HIF) pathway. It is also likely that hypoxia alters the function of immune cells in multiple pathologies including infection, transplant rejection, and cancer. We recently identified a mechanism by which Tregs reverse their function in response to changes in these hypoxia sensing mechanisms. If we can modulate the hypoxia-sensing mechanisms in a highly specific manner, can we alter how the immune system responds to infection, cancer or other chronic diseases?

NS: What led you to investigate the infusion of Tregs to reduce the use of immunosuppressive therapy?

FI: The field of Tregs is quite well-established. In the field of transplant immunology, Peter Medawar demonstrated that one can induce tolerance to a mismatched allograft by modulating the way the immune system recognizes the transplanted tissue. Over time, it has been shown that tolerance is at least partially dependent on the activity of Tregs. Over the past fifteen years, methods to isolate and then expand populations of Tregs have been developed and standardized. This coincided with the development and use of CAR-T cells and cellular therapeutics in immuno-oncology, helping to develop the path for cellular therapeutics in transplantation.

NS: Given that Tregs are such a sparse population of CD4+ T cells, how are they expanded to the level needed for your trials (~5-10 million/kg)?

FI: In our study, we isolate cells that express CD4 and CD25. These cells are then cultured with high levels of recombinant human IL-2, TCR stimulation, and rapamycin. Rapamycin appears to reduce the overgrowth of effector T cells while preferentially expanding of Tregs. We repeat this protocol multiple times to generate the required number of cells. Other methods are looking at incorporating a CAR into Tregs. For our work, it was important to avoid that extra level of complexity in the clinical manufacturing protocol, particularly as it is a large Phase II trial. The cell populations are then assessed for population purity, suppressive activity, and contamination (by beads or bacteria) prior to being deemed appropriate and acceptable for use in our studies.

NS: Why did you select GeoMx DSP for your research into biopsies of renal transplant patients?

FI: We searched for a long time to find or develop methods to assess what is happening in the transplanted tissue. Standard immunohistochemistry (IHC) and immunofluorescence are fairly crude – while they can roughly identify the types of cells present in the biopsy, they can’t tell you what the cells are actually doing. For example, in clinical trials where we gave Tregs, we looked at the biopsies and found FOXP3-positive cells. Were these really Tregs or were they activated T cells? Were they native cells or the cells we transferred? There was no single technique (IHC, single cell sequencing, etc.) that could help us to determine their function or their gene or protein signatures in respect to their spatial location. Previously, we had to limit our questions to the techniques available and that hampered our research. With GeoMx DSP, we now have a technique that adapts to and fits our needs.

NS: How has spatial biology helped you understand the mechanisms of acute and chronic rejection in SOT?

FI: As an example, we looked at some kidney biopsies and then selected twelve regions of interest (ROI) that all had cell infiltrates. Leukocyte infiltrate is a sign of rejection, so these areas were important to us. What we found is that each of these twelve ROIs gave you a different protein signature. That tells us that these areas are not homogeneous and that there are dynamic processes at play. Some of the signatures look more like an effector T cell infiltrate, some looked less active. Now we’re asking if these are temporal changes, indicating different stages of rejection. Without the spatial resolution of GeoMx DSP, we would not have been able to see that.

NS: Tregs are friends for SOT but foes in cancer immunotherapy because they are involved in suppressing the cytotoxic response. How do you think discovery of a druggable target against SOT rejection will benefit research in cancer immunotherapy?

IF: Cancer immunotherapy and transplantation are basically the same field; one’s just the flip side of the other. The goal of cancer immunotherapy is to activate an immune response against the cancer to kill it whereas the goal of immunotherapy for SOT is to regulate the immune response against the graft to protect it. In both fields, that response needs to be specific—you don’t want to activate all effector T cells in cancer; this would lead to disastrous consequences. You don’t want to regulate all T cell responses in transplantation and then suppress everything. In both cases, you want to maintain homeostasis, a level of protective immunity, and a specific response to what you want to achieve. I think both fields have a lot they can learn from each other.

NS: Tell us about your involvement in the nCounter® Human Organ Transplant Panel.

FI: This is a very interesting gene expression panel. The development of this panel involved multiple different investigators from the Banff Consortium. It is the result of decades of work where different studies have looked for gene signatures of rejection, infection, or damage to an organ. All of those genes were then scored, and we came to a consensus regarding their importance and used that information to create the 770-plex Human Organ Transplant Panel. As someone who is interested in therapeutics and generating tolerance to a transplanted organ, my contribution was to try and insert some genes which were indicative of immune regulation rather than rejection or immune activation. I think it is a really useful panel not just for detecting rejection but also for detecting potential immune regulation. This is particularly important for all the ongoing clinical trials of new therapeutics in transplantation.

NS: What are the major challenges facing SOT researchers and how do you think the nCounter Human Organ Transplant Panel will help solve these?

FI: The biggest challenges in SOT remain poor long-term outcomes and scarcity of organs for transplantation. The first challenge of poor long-term outcome is principally the result of immunosuppression, which is cardiotoxic, nephrotoxic, and results in a poor metabolic profile; it does not target rejection specifically. Global immunosuppression causes global dampening of the immune response, resulting in infections or cancers. The second challenge is the lack of sufficient organs for donation, resulting in a very long waiting list for patients in need. So how can this panel help? If you have a more specific method to determine if a transplant is being rejected or infected, then perhaps you have a better chance at saving it. The earlier you can determine that pathology, the better you can protect the organ. I think that the Human Organ Transplant Panel provides you with that very wide array of genes that give you additional resolution to help you start making those decisions. I do not propose using the panel as a clinical diagnostic at the moment, but perhaps as more centers use it in their research efforts, we will be able to pool our data to come to a consensus.

NS: How do you envision the nCounter and GeoMx DSP being used together in the same study? How will one type of analysis inform the other?

IF: We are doing exactly that in our current studies. The ideal would be to do GeoMx DSP on everything, but you don’t necessarily have unlimited biopsies. The nCounter Human Organ Transplant panel can be used on blood samples and on small biopsies to add an extra dimension to the analysis. I think it’s useful to have a high-level overview of the transcripts present in order to tailor the spatial biology approach.

NS: What benefits of nCounter technology do you think will resonate the most with SOT researchers?

FI: When it comes time to analyze data in SOT research, you have very little to work with because the transplants and biopsies are precious; taking the biopsy itself is damaging to the organ. It’s not like cancer where biopsies are done routinely. So, anything that you can do using a small amount of tissue or blood is very useful. The nCounter analysis system provides a platform to do that and can amplify a small amount of RNA to give you a list of genes that are actually relevant. I think that whole transcript sequencing can give you a lot of information but not necessarily the level of detail you’re interested in. It can also highlight transcripts that may be abundant but not necessarily important mechanistically. When you perform a targeted approach, you’re actually looking at the immune-related transcripts that are really critical to the biological processes. The other thing I have to say about NanoString and the nCounter platform is that it is super easy! Everyone in the lab likes using it; it gives you data quickly, and analyzing the data is very straightforward.

FOR RESEARCH USE ONLY.  Not for use in diagnostic procedures

Posted by Laura Tabellini