You all know and are familiar with the amount of energy that it takes to play sports, exercise or even just perform our jobs. And how eventually you need to take some time to recover and rest from what you were doing. Rest is an essential component of a healthy lifestyle. It enhances performance, lowers the risk of injury, and helps you recover faster when you do get injured or ill.
Speaking of illnesses, our body is home to countless immune cells that work tirelessly to keep us protected from diseases and sickness. In fact, have you ever thought about whether our immune system gets tired? Do the individual cells ever need a break now and then, just like we do. In fact, could rest benefit our immune cells? And if so, in what ways?
Hi folks, my name is Cole and I have a Masters of Immunology. Today on Investigate Explore Discover, we’re going to be looking at how to give CAR-T cells a boost. So hang around with me throughout this whole video to get all of the relevant background information so we can dive into some exciting experimental results.
Now before diving directly into how we can enhance CAR-T cell activity, we first need to understand exactly what CAR-T cells are and in what context they’re used. Now our immune system is made up of many different cell types that are derived from the bone marrow, which perform various functions to keep us healthy. These cells play a key role in preventing cancer from growing but ironically can fall prey to developing cancer themselves. Now when these cells develop cancerous qualities, it is called leukemia. Leukemia originates from the bone marrow and is subdivided based on the cancerous immune cell type. Furthermore, it does not cause distinctive solid tumors. Now these cancer cells do not function normally, crowding out and causing problems for functional immune cells. Leukemia is one of the most common childhood cancers, but it is more often diagnosed in older adult populations. In the US alone, there are over 61,000 new diagnoses per year. And even though there is a five year survival rate of 65%, over 23,000 people still succumb to it yearly.
Now, there are multiple ways that cancer can be treated, which contribute to this longer survival rate. There is surgical removal of the extra cells, you can use chemotherapy, or there is immunotherapy, which we’re going to focus on today. In particular, we’re focusing on a growing field of immunotherapy, which uses modified T cells to fight cancer as leukemia cells. T cells are part of our adaptive immune system, which gives us tailored responses to cancer and infections. And T cells can be genetically equipped with Chimeric Antigen Receptors or CARs, which are meant to specifically target cancer cells.
Now to create CAR-T cells, they are isolated from the patient, modified, expanded and then reintroduced into the body where they can start to attack and clear the cancer. These T cells are known to be activated because they secrete Proinflammatory Cytokines like IL2, Interferon Gamma, and TNF alpha, alongside destroying cancer cells by recognizing them with their Chimeric Antigen Receptors.
Now, when T cells are activated, they expand and transition to further differentiated cells. This transition can be seen through multiple different cell surface markers and genes that are upregulated on and inside of the cell. Now these genes that get specifically turned on are not always available to the cell. Some of them are only available through epigenetic modification, which is mediated by methylation of the DNA.
Now this situation uncurls the DNA for expression of targeted genes. When there are small amounts of cancer or infections, the T cells are able to clear the problem and turn into memory T cells. These cells are self-proliferative, and are better able to clear the problem if it ever returns. However, just like how we cannot continue to exercise forever, T cells start to get exhausted after being continuously stimulated and activated. Now this results in the T cell starting to lose some of their anticancer activity. This exhaustion is recognized through the upregulation of inhibitory receptors on the cell surface, and certain epigenetic and genetic transcriptional changes inside of the cell. This becomes an issue in CAR-T cell therapy because the cancer cells don’t really want to be cleared.
Because CAR-T cells are targeted against one specific cancer pattern, this leaves them vulnerable to how cancer avoids clearance. This is done by the cancer cells expressing diverse receptors and actively suppressing immune cells. This leads to exhaustion of CAR-T cells and tumor escape. Over the years, we recognize this and come up with multiple strategies to thwart the cancer. We have the capability to give CAR-T cells additional tools to utilize in fighting cancers like targeting multiple cancer cells and blocking the exhaustion markers on the cells, giving them a second wind.
Now when we block exhaustion markers like PD1, this allows T cells to be functionally reinvigorated and continue to exhibit cytotoxic activity towards cancer cells. Though, this works some of the time we need to continue to develop strategies to further enhance T cell function and prevent or reverse their exhaustion.
Now, I want to take a moment and really highlight why CAR-T cell therapy research is so important. As it stands, CAR-T cell therapy is primarily only used for treating non solid tumors like leukemia, and less than 50% of patients receive long term cancer control after treatment with CAR-T cells. Another issue when using CAR-T cell therapy is that excessive continuous T cell signaling can induce exhaustion, which limits their effectiveness. Therefore, preventing or reversing T cell exhaustion could lead to more effective cancer treatments.
This brings us to the paper that we’re focusing on today. This paper is called Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling by Weber et al. from Stanford University in Stanford, California, USA. In this paper, the authors investigated how preventing T cell receptor signaling could affect the function and transcriptomic profile of T cells that are targeted against leukemia.
Now to start investigating CAR-T cells, the authors needed to use Chimeric Antigen Receptors that they control, whether they were on or off. So, they just went and designed their own receptor against leukemia cells, but they included one essential piece – a destabilizing domain. This causes the CAR to not be expressed unless it is in the presence of the stabilizing drug shield one. Removal of the stabilizing drug led to a rapid decrease in CAR expression, which was reversible with the addition of the drug once more now to determine the dynamic range of CAR expression and assess the relationship between CAR expression and function, the authors co cultured these CAR-T cells with leukemia cells and found that the CAR-T cells were able to be sufficiently activated and kill the cancer cells that they were then in contact with.
Now to explore how controlling CAR expression works in an in vivo setting, the CARs were modified slightly to respond to an antibiotic instead of a drug. These cells were controlled to have variable CAR expression at different times, and were then injected into mice that had leukemia to assess their characteristics. The authors found that cells that were situationally turned on just before antigen challenge, i.e. they were rested, proliferated more than the other cell cohorts and greatly increased the lifespans of mice with leukemia. The authors next looked at exactly what happens to cells that have been activated and then allowed to rest. So the authors looked at these cells outside of live animals, allowing them to determine how rested cells might be different from always activated cells. The gross changes that the authors found indicated that instead of transitioning to exhaustion due to continued receptor activation, rested cells expressed a more memory T cell phenotype, expressed less exhaustion markers, and had cell populations that were diverse, meaning that this is a cell population wide effect. Now since halting of continued signaling altered CAR-T cell fate during the transition to exhaustion at day 11 in their experiments, the authors hypothesize that perhaps rest could also be programmed T cell populations on which exhaustion was already imprinted.
So they compared cells at day 15 that were rested at day 7, 11, or were always rested with cells that were always on or that were given a checkpoint blockade therapy. Now, over the course of the experiment, the cells that were rested for 4 days proliferated more than the always on cells. And in fact, when cells were allowed to rest even longer, they proliferated more. Now, when all of these cells had their transcriptomes examined, it was found that the rested cells had decreased exhaustion related gene expression, and they also had upregulation of genes that were also found in stem cell like memory cells. Through unbiased grouping of gene expression patterns, the authors observed commonalities through separate clustering of rested cells and non-rested cells. But to figure out exactly what this means when cells are exposed to their targets, the authors first normalized CAR expression on the T cells and exposed them to leukemia cells.
They found that all of the cells, but the always on CAR-T cells, could kill leukemia cells. What is particularly interesting to note though, is that only the rested cells were able to secrete multiple Proinflammatory Cytokines. Now to see if this holds functional significance in live models, mice with leukemia were injected with rested cells, and the authors found that rested cells killed more of the cancer cells, corroborating the in vitro data. Now to determine the impact of transient rest on the epigenome, the authors analyzed the differences in chromatin accessibility between all the different cell treatments in vitro. They found once again that rested and non-rested cells clustered separately from one another. And most of these epigenetic changes occurred in the first 7 days. Now when looking at what these changes were, it was also found that the rested cells exhibited increased accessibility of memory associated genes. The epigenetic modifier, EZH2, effectively prevents hematopoietic stem cell exhaustion and is critical for T cell differentiation, maintenance of T cell memory and anti tumor immunity. The region that is normally methylated by EZH2 was unmethylated in always on cells, contributing to their exhaustion, but not in the rested cells.
So the authors thought to test whether this protein was functionally important in restoring T cell function through CAR blocking rest. Now, when blocking this protein, there was little effect on always on and always off CAR-T cells. However, when looking at the rested CAR-T cell, blocking EZH2 resulted in loss of protective effects that resting the cells conferred, thus indicating that EZH2 was important to restoring T cell functionality through rest. Now, it has been previously demonstrated that Dasatinib, an FDA approved Tyrosine Kinase Inhibitor suppresses CAR-T activation via rapid and reversible antagonism of proximal T cell receptor signaling kinases. With this information the authors hypothesized that Dasatinib mediated receptor rest could reverse exhaustion. So the authors gave Dasatinib to cells for multiple lengths of time, just to see if it would do just that. The authors surprisingly found that the longer that these cells have their CAR inhibited, the more they proliferated and were functionally invigorated and we’re better at killing leukemia cells in live animals.
Of note, cells treated with Dasatinib for 4 days did not function as well as the cells treated for longer. So the author’s wanted to see whether there was a point of maybe irreversibility in CAR-T cell exhaustion, or perhaps there’s just a minimum adequate amount of time for rest. To assess this, the authors instigated a longer time course experiment, going up to 25 days. They found that the longer they gave cells Dasatinib, the more rest they had, and the more they exhibited behaviors of functional anti cancer cells, indicating that restoring full function requires a minimum period of rest.
Now, finally, because rested cells exhibited greater anti-cancer function than exhausted cells, the authors wanted to determine whether there was an optimal dosing schedule to facilitate this function in live models. The authors found that giving a 3 day on and 4 day off treatment with Dasatinib resulted in increased cancer clearance. And when these findings were tested on solid tumors, which is important because these are normally harder to treat with CAR-T cells, the 3 days on and 4 days off treatment also resulted in tumor shrinkage. In fact, in this model, treatment with Dasatinib for 7 days resulted in a more functional T cell response, corroborating previous results, that more rest proves to be more beneficial for CAR-T cell function.
To quickly summarize everything altogether, the authors of this paper found that through resting CAR-T cells by preventing signaling through any of their T cell receptors, they were able to induce transcriptional reprogramming and epigenetic remodeling to restore T cell function and promote a memory phenotype that helped to kill cancers in vitro and in live animals.
They also found that this response is dependent on the duration of rest, where more rest functionally restores the T cells better. Essentially, this means that instead of going into situations exhausted and angry, T cells are going in calm and refreshed to better fight off cancer. Not only do I think that these findings and experiments are exciting to investigate and learn about, they’re also significant in a broader context.
This information is significant because the authors show that after becoming exhausted, T cells can become functionally reinvigorated by resting in vitro and in vivo. Furthermore, this rest can be facilitated by readily available drug treatment Dasatinib. This then gives us another avenue to pursue when exploring anti-cancer treatments. In fact, this strategy could be used to promote long lasting cancer clearance.
Now, all science is basically a stepping stone for new knowledge. And these steps are driven by questions. And I had a few questions on my own after reviewing this information. The first question that I had has to do with the T cell populations undergoing the epigenetic remodeling. Are there specific T cell populations that are doing this? Or is it a more generalized phenomenon throughout the whole population? And if it is only certain cells, can they be isolated further to be used for future treatments?
The second question I have revolves around the dosing and timing of the Dasatinib treatment. What is the optimal timing and dose that will be necessary for effective long term cancer clearance? My third question has to do with administering the Dasatinib itself. There’s a worry that by broadly inhibiting T cell function, this would allow the cancer to grow unchecked for a period of time. So is there a way that T cell populations might be sequentially targeted to rest in waves, perhaps by using lipid nanoparticles to target specific T cell populations?
And, as always, my final question revolves around you. What sort of ideas or questions popped into your head when hearing about this information. I would love to hear about them in the comment section below. Also, let me know if there are any topics that you’d like to hear about in the future. Ultimately, I hope that you learned something. But more importantly, I hope you enjoyed your time doing so. Well, that’s everything for today. Thank you for watching, and I’ll see you next time.
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