The Immune System in Regenerative Medicine

I'm going to talk a little bit about a winding path in tissue engineering that actually starts by focusing on stem cells and ends in the

immune

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immune

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. As you are all familiar, tissue and organ loss remains a global challenge and while organ donation is increasing, we have synthetic implants and options for some tissue and organ losses, such as hip or knee implants. There is still a great desire to have a true tissue substitute. The field of tissue engineering evolved to essentially provide this solution, using a three-pronged approach. Where you have a biomaterial scaffold that serves as a three-dimensional framework for the growth of new tissue and also as a framework for stem cells or just normal cells from a biopsy and various factors that could be used to stimulate the growth of that tissue.

What we noticed when I started is that there was a lot of enthusiasm for the field, but there wasn't as much translation. We were very interested in getting something to the clinic as soon as possible while also studying the basics of stem cells. One of the key things that will come up is what are the key therapeutic factors if you want to translate? If you want to develop the best technology, what are the levers that we want to move up and down to really promote tissue growth and how might they be different in different scenarios and different people?

Our attention was focused on the cartilage. Initially, it is a tissue that covers the surfaces of the joints. Unfortunately, when it is damaged, it cannot be repaired very well. I mentioned hip and knee implants and they are widely used. But ultimately what we really want is a biological solution. When I started 20 years ago, it was around the time that stem cells were gaining ground in their research focus. This image here of the mesenchymal stem cell was something that was shown at practically every conference when I was starting out. Of course, this was of particular interest to us who analyzed cartilage.

We don't want to take a biopsy. Can we take these cells and make cartilage? We build materials to do that. Then there were embryonic stem cells, so we spent a lot of time trying to understand how we can take an embryonic stem cell and get it to make cartilage and, in fact, also bone and adipose tissue. Finally, the last type of stem cells we looked at were induced pluripotent cells. Okhee Jeon did some work with bone-producing osteoclasts and osteoblasts from iPS cells. But it's been a while since we worked with stem cells in the lab and I want to tell you a little more about that.

As we looked at translational technologies, we wanted to take the information we learned from our work with stem cells. With these synthetic hydrogels and with which we would encapsulate cells, we can take chondrocytes, the cells that make cartilage, adult stem cells, embryonic stem cells, and provide various signals to induce the growth of that tissue. But at the same time we wanted to get something into the clinic right away. It was clear that delivering cells, living cells, to patients in general was going to be difficult. We wanted to start with something that connected with current surgical practice.

To translate what we learned from hydrogels in working with stem cells to the clinic, we ended up working with microfracture, a procedure in which you basically take a photograph and drill the bone, cause some bleeding and mobilize cells, and the real objective is to mobilize endogenous progenitor cells. This was the basic paradigm. We had to develop an adhesive to help keep the hydrogel in place, so we painted that adhesive, we did the microfracturing and then you have the hydrogel and you can see that essentially that hydrogel enriches and concentrates the things that come up. of that drilling process.

Additionally, it discourages the growth of fibroblasts or scar tissue. Helps in different ways. There were two clinical trials in this regard. The first trial lasted one year, the second focused more on efficacy at two years. If the microfracture is taken alone and is shown in red, it begins to degrade at approximately 12 months. This has been seen in the literature, as well as in our patients. But when we have that hydrogel to help redirect that healing process, the cartilage is more robust and lasts longer. One thing we noticed is that in this trial, that cartilage repair trial, we were redirecting the wound healing process.

The biomaterial did not serve as a three-dimensional framework for the growth of new tissue, but rather redirected the wound healing process. We also had another clinical trial that we were looking at for soft tissue fillers and we did a clinical trial where we did the implants and it was in patients who we will undergo a tummy tuck procedure. But three months before they received the implants, we could recover the biomaterial. We noticed some interesting things; Depending on which tissue the same biomaterial was adjacent to, whether subcutaneous muscle, adipose or dermis, the immune cells were different. There was a tissue-specific immune response to the same material.

This is really interesting looking at the immune system and maybe rethinking the response of the biomaterial, and maybe what was the first target that we could use to promote tissue repair. I went on sabbatical to Switzerland. Luckily, Jeff Hubbell and Melody Swartz welcomed me to EPFL there, so I was at least able to learn a little bit of a language. Immunology is not easy. Then when I came back to Hopkins, I didn't know that right next to me, in the adjacent buildings, were the cancer immunotherapy people who are really doing innovative work in that area. While they focused on dissociating tumors and looking at what immune cells were there, we can apply those same techniques to dissociate tissue spaces where wounds and biomaterials were implanted to understand what was happening in the repair process.

The immune system is interesting and tissue repair, sometimes it helps and sometimes it hurts. I have some document snippets here that show how these cells are required. Macrophages are known to be necessary for tissue repair. But there are things that are associated with negative tissue repair, whether it's CD8 T cells or certain immune signatures in the blood that can predict rapid or slow recovery. Now, in the case of tissues like muscle, type 2 signals, immune signals, were found to be important. I'll talk about that first. Type 2 signals are characterized by interleukin-4. An immune response is not always bad, and this was a little different when it came to how we thought about biomaterials.

At first, the materials were supposed to be hidden and stealthy, and then when we started to consider putting stem cells in there or promoting tissue repair, we wanted to have a positive interaction with the surrounding tissue and cells. Ultimately, we want the immune system to recognize what we're doing here, but we need to be specific about how it recognizes what's happening. Before we get into that, I want to quickly go over some of the basic immune classes that I'll be talking about. There is the innate and adaptive immune system and different types of cells in each.

The innate immune system responds quickly, but is generally less specific. The cell types that will be important here are neutrophils and macrophages and then there is the adaptive immune system, which may take a little longer, but is very specific. You'll have antigen-presenting cells that present signals and antigens to things like T cells and B cells. We'll talk a lot about T cells and maybe another time about B cells. Then there are some interesting cells that live on the line between the 2 innate lymphocytes. As the name suggests, they have innate behaviors but are derived from the lymphocyte lineage.

Then there are Gamma Delta T cells, which I won't talk much about today, but we have published some interesting things about Gamma Delta T cells in the foreign body response. All of these different types of cells are not only there, but they communicate and work together. By the end of the presentation, I hope to understand how all of these cells communicate and work together to make a tissue a healing or non-healing environment. In addition to cell types, there are certain phenotypes. I mentioned the type 2 immune response. This is characterized by the production of cytokines, interleukin 4 and others such as IL5 and 13.

They can be produced by several types of cells: eosinophils, innate lymphocytes and T cells that express IL4, or are called Th2 T cells. . Now, I always like to look at what the normal classical functions of these cells are. These cells were associated with host defense, like most cells of the immune system, but in particular parasites, helminths, and extracellular microbes. It is also associated with allergy and asthma, some negative consequences of host defense. Then finally I'll show you some evidence and there's also a lot of published literature on the importance of these cells in tissue repair, particularly liver and muscle.

Another group I will talk about are type 17 or type 3 immune responses characterized by the production of IL17 in different forms, a and f. These are produced by Gamma Delta cells, again, innate lymphocytes and T cells that express IL17, or they are called Th17 cells. Again, these are associated with certain types of host defense. So, the negative sequelae of this pathway is autoimmunity, which is why the IL17 type is associated with a series of autoimmune diseases. Additionally, fibrosis, which makes sense with the foreign body response, would be important. Let's now return to tissue repair. As we were moving forward to try to understand the immune system and how we might want to target it therapeutically to promote tissue repair, we first wanted to look at what the natural immune response is after tissue injury and what it looks like in a patient. wound that is healing, and how is it similar or different from a non-healing wound characterized by fibrosis and chronic inflammation, and can we use this information to change the resolution of that non-healing wound and redirect it to tissue healing?

The patterned tissues I will talk about today are muscle tissue. We are constantly damaging our muscle if, for example, we exercise. Of course, if you have a very large defect, you will need additional help to fix it. But in general, little damage to muscle tissue can be repaired. That's the opposite of the cartilage I mentioned before. The reason it was an important durable target for tissues is that it doesn't have much repair capacity. There are now some biomaterials that can help shape or even exaggerate these environments. I will talk about biological supports, some of which are used clinically, and then about synthetic materials, which I will not talk to you about today.

We're just going to talk more about the cartilage environment. To understand these responses, we really want to map the responses here, defining the specific tissue injury and biomaterial response when we use them as a model. We want to understand who was there, what they are doing and who is talking to whom. What I'm going to show you, then, is that you get the same types of cells there, but the way they communicate with each other is different and that results in a different outcome in the repair process. We take these cell types individually, isolate them, and then also analyze them together, trying to characterize who is there.

We have these tissue maps, they will be useful for designing therapies, targeted therapies, but also relevant for a number of tissue pathologies in general. We will use a variety of techniques. Flow cytometry, the standard immunology, has only grown, grown, and grown for many years. The number of cellular markers that can be observed is constantly increasing and of course single-cell technologies are an interesting area. So even some traditional areas, such as massive sequencing, sorting and sequencing of specific cell types, can be very useful for rare populations and simply confirm results. Starting with the muscle injury, Caitlin Sadler, who now works at the NIH, began placing biological supports in these materials.

Biological scaffolds had already been shown to work from an immunological point of view, so macrophages were important for the therapeutic response. These are also used clinically and are considered pro-

regenerative

. We implant a scaffold in quadriceps defects. You punch it and you take out a lot of the quadriceps, and what we can see is an increase in the production of interleukin 4. I mentioned that interleukin 4 was important for tissue repair and that we could increase it with the scaffold there. Now, you may have some questions about the composition of the scaffold. We evaluateseveral different sources of that tissue extracellular matrix.

It can use small intestine submucosa, bladder, urinary bladder matrix, bone matrix, and even cardiac tissue matrix. Virtually the same biological results can be obtained depending on the source. You could simply process them differently for different applications, whether you want particles, powder or sheets. If we implant these biological scaffolds, if we do, first, a characterization of the myeloid populations, the labels here are down with the different colors, but generally speaking, you can see that there are different types of cells there. Just muscle injury, we have those types of cells. For many macrophages, we have different ways of characterizing them.

Then we have the scaffolding there. First of all, we have a lot of eosinophils, but we also have scaffold-associated macrophages that are a little different than standard macrophages. Now, if you look at the standard markers of macrophages, they are quite classic: the 86 pro-inflammatory and 206 surface macrophages are more pro-regenerative macrophages or, alternatively, activated macrophages. The scaffold decreases the expression of the pro-inflammatory macrophage marker on the surface of the cells. It doesn't change much with 206 except for a scaffold that induces more fibrosis. But here I want you to look at the pod field, this is 206 and 86, most cell types, macrophages, express both.

They are present together; It's just the ratio of one to the other. I'll talk later about how we've used individual cells to get better surface markers to actually characterize what's happening with macrophages. Now, let's look at the adaptive immune response. We notice that CD4 and T cells are increasing. CD4 T cells are considered T helper cells that secrete cytokines including interleukin 4. If we look at the wound alone, which is treated only with saline water compared to the wound with the ECM biological scaffold, from the beginning we get gamma interferon and also IL 17 is expressed by CD4 T cells.

But then IL-4 tends to take over and increase over time. Again, moving towards resolution and regeneration. Now, if we look at that IL-4 a little more, this is with a mouse that has to be connected to the IL-4. Again, we can see that you have a little bit just in the wound, but when you put that scaffold down, you essentially give an energy boost to the TH2 T cells. You get more of them and you get more expression of interleukin 4. Now, I already showed you what happened with the macrophages. Now, if we don't have T cells there, if we remove those T cells, the macrophages shown here in red then in the Rag knockout animal significantly increased their expression of that proinflammatory macrophage cell surface marker and we lose the CD206. , then double whammy, more inflammation and less resolution.

Now Kayla, notice something very interesting. She took that draining lymph node and noticed that when there was an implant, it was larger and also had interleukin 4 expression there. This was particularly interesting for two things. Number one, it means that the local lesion, the local implant, could be having systemic, regional and systemic effects. Then she also introduces the possibility that systemic aspects of the organism can affect the local response to whatever is happening with the wound or material. Now, to investigate the T cell response a little more, Jonathan Powell had Richter knockout mice and we can take the Rag knockout mouse and put in normal T cells or T cells that were deficient in the Richter protein, which is part of of the TORC2 complex, which is essential for TH2 differentiation.

The T cells and those mice were unable to differentiate. What we found is that you can see that there is additional fibrosis and epigenesis instead of the good repair tissue. Those T cells were important in directing those macrophages, but it also highlights that the innate and adaptive immune systems are working together, talking to each other. Let's look again at some types of cells. We have been exploring single-cell techniques. I mentioned the diversity of macrophages. We took out macrophages and processed a single cell and essentially now have a better definition of the different subsets of macrophages, those that are more fibrotic and those that are more regenerative.

We've also done it with fibroblasts, so there's a lot of interesting work being done with fibroblasts, but we're particularly interested in immune regulation. We have many different cells that communicate with each other not only in traditional immune cells, but also in a tissue-resident fibroblast. We have a macrophage data set here. Here we have a fibroblast data set and we also take CD45-enriched single-cell data sets. There are many different types of cells here. We first run the single cell and you can see that there are many different groups, many different cell types, and when we compare it to the group composition between groups, these different treatment groups, whether it's the biological scaffold, the wound or not. injury, we don't see big differences.

We don't see the same differences that we would expect based on different biomaterial implants, so we looked at building a program, Chris Cherry created a program that makes an estimate of how cells communicate with each other. There are some programs out there, but he did it a different way. We first look at transcription factor activation and then how it correlates with certain receptors that we are signaling. This is important because many times we do not capture the expression of the ligands. For example, eosinophils that produce IL-4 will be difficult to capture with a single cell, but we can capture the receptor and activation of pathways associated with IL-4 and transcription factor activation.

This Domino program does it in an unbiased way. It is independent of the clusters typically seen with single-cell data sets. This is what it looks like when we have a pro-regenerative muscle environment versus a fibrotic muscle environment. You can see that the communication pathways are quite different and then we can look at what the specific activations of the transcription factor are in a healing wound and in a non-healing wound. This is what it looks like. Now, the interesting thing is that you have an immune module divided into specific modules. In tissue specific modules where you would put things like stem cells and then we have a fibroblast module.

The way they communicate is different and we have some particular predictor receptors and transcription factor activation that you can look at and then investigate further using this program. This will be important when we analyze that wound that does not heal. In particular, looking at those non-immune cells that are immunologically active. When we were trying to make cartilage, I showed them data on a focal cartilage defect, which is usually more of an athletic or traumatic injury and if you're in a healthy environment, you can get a decent repair. But most patients who need cartilage repair have more diffuse degeneration related to chronic inflammation, so there is an immunological component.

What we found is that when we take these cells out of an arthritic environment or expose them to cytokines like interleukin-1 Beta, tissue production is decreased, so that main part was important. At the same time, we discovered, in collaboration with Unity, that senescent cells are not present in any way, so it was published for a while and we looked at what is the actual active component of these cells and are they positive in arthritis? What is senescence? Senescence was first discovered by Hayflick in the context of replicative senescence, and then telomere shortening and sorting attrition is another area that is important for senescence, as cells age and can no longer proliferate. , telomeres shorten, then you also have pure or stress-induced premature senescence.

Oxidative stress, oncogenic stress, and what I'll show you, immunological stress can induce senescence. Just like the immune system, sometimes they are good and sometimes they are bad. Judith Campisi demonstrated the importance of senescent cells for wound healing. She characterized fibroblasts and endothelial cells as the primary senescent cells and were required for efficient repair. Then I'll talk to you about how chronic senescence or having senescent cells for a long time that are not eliminated actually inhibits regeneration. What are senescent cells and how do they work? They are making proliferating arrests, so they are no longer divided, but they are far from inactive.

They are actually quite active and secrete a senescence-associated secretary phenotype or SASP. This SASP is involved in its pathologies in promoting tumors or various age-related diseases such as, of course, heart disease, arthritis and diabetes. But, as I mentioned, there are positive factors in tissue repair. We first used a mouse model developed by Judy where she had a p16 promoter connected to something that allows us to visualize the cells, but then also an off switch with ganciclovir so that we could selectively eliminate these p16-positive cells. When we did a joint injury and a joint injury, Sham just opened up the joint.

Very minor injury versus a section of the ACL and this is a vehicle. An increase in senescent cells or at least bioluminescence can be seen. Then, if we give ganciclovir and kill those senescent cells, we can get rid of them. This is a good model to see, that these senescent cells simply correlate with the disease, or are they causative? If we use that genetic model or some drugs, what we notice is that when we eliminate the senescent cells after that injury, we significantly reduce that inflammation. You can see a number of factors that are suspected to be SASP factors, interleukin-1 Beta, interleukin-6, and many MMPs.

Then functional results such as pain can also be observed. If you eliminate those senescent cells, you not only reduce inflammation, but you also reduce pain. Then, surprisingly, we saw that when those cells were declared senescent, better tissue repair was achieved. You can get resolutions. I think this is important because we do not conserve any growth factors, we do not provide stem cells. We are simply removing the inhibitory factors that block the tissue repair process. Ultimately, eliminate senescent cells and various sensory factors and you can reduce the secretary phenotype associated with senescence, which includes many inflammatory factors, reduce pain, and increase tissue repair.

We were quite interested in some of these SASP factors, such as IL-6 and IL-1 Beta. Because when we look at how things might communicate, these factors are known to promote the differentiation of immune cells through particular pathways. In particular, IL-6, IL-1 Beta in the presence of TGF-Beta induces an interleukin 17 or type three mediated immune response. We went back to the joints and looked for that and what we found was, in particular, that CD4 and IL-17 ANF gamma/delta expression increased significantly with that ACL injury. IL-17 was also secreted by things like innate lymphocytes. Several different cell types produce that IL-17.

Now that is a trauma. Osteoarthritis is considered a local disease. They simply wear out and the joints wear out. But like the muscle where I said we had looked at the draining lymph node. We also look at the lymph node that drains here. What we found was a really significant increase in both overall gene expression and in specific cell types. These are CD4 T cells that produce IL-17 or TH-17 cells that are significantly increased in the lymph node. Especially in the joint that does not have much vascularization. You don't have too many immune cells there. That draining lymph node provided almost a magnification of what was going on.

We can really see these immunological changes very well. What about a senolytic? Again, it's about establishing that connection between senescent cells, fibroblasts, and IL-17 immune cells. What we found is that when we administer that senolytic, which is sought in the lymph node, those IL-17 signatures can be significantly reduced. This is very exciting because it is a way we can establish a connection between the two of us. If we neutralize IL-17, we can reduce the expression of factors related to senescence. In particular p16, and this is p21. We went in vitro to validate this a little further and this is what we found.

If we artificially induce senescence by radiation and expose those cells in vitro to naïve T cells, activate those T cells, and put them in the presence of TGF-Beta, we get a significant increase in IL-17. Now, vice versa, if we take Th-17 cells and co-culture them with healthy fibroblasts,we can induce senescence in those fibroblasts as seen by the amount of SASP factors that increase in p16. If you look at the expression profile of these cells, it is quite different. This inflammation-induced senescence has different characteristics compared to standard classical ways of viewing senescence with oxidative damage.

However, an important factor is: can we still repair when we are old? Many people who arrive with this joint degeneration are not 10 or 12 week old mice. The reviewers actually ask us this first and Jan Van Deursen published a paper using another mouse model that clears out senescent cells and looks at lifespan. I asked him, do you have any joints left? He didn't look at the joints and fortunately there were some joints left. What we saw was amazing. When you clear out the senescent cells, beautiful cartilage appears, and without it, you can barely see where that joint space was.

Now, if we look at senolytic only in wild-type animals, the first thing we see before surgery is that the joints don't look as good and the injury makes them look even worse. Senolytic doesn't really do too much. Inflammatory markers can be decreased, but not much tissue repair is seen. Now, another collaborator, Daohong Zhou, has focused on bone marrow and understanding bone marrow senescence and how bone marrow can be rejuvenated. One thing we noticed in old animals, if we look at the CD4 T cells in the lymph nodes. There aren't that many. There really aren't too many T cells left in the lymph node as we age.

We have analyzed the use of local senolytic as we did with young animals. Then also the systemic senolytic that Daohong used to rejuvenate the bone marrow. This is what we saw. Again, IL-17 decreases with senolytic in young animals. But in old animals, we can also make IL-17 decrease. It's not a great deal. But what was different was interleukin four, that same cytokine that was important for muscle. When we gave that combined inter-articular and systemic senolytic, that was the only time we got an increase in interleukin four in the joints and in the old animals and saw good cartilage repair.

You can repair it when it's old, but you'll probably need help. You will need some additional senolytic or additional treatment of the systemic immune changes, which will eventually affect the muscle as well. This was in clinical tests conducted by Unity. They did the testing, phase one looked good, and they saw a good correlation in phase zero between senescence markers and disease severity. Phase one and phase two. But here's a tricky little part. They gave a single injection and the patients were up to 85 years old. What I didn't tell you in our regimen was that we did multiple injections daily and I really needed at least three injections every other day, so that dosage was really important.

Then I showed them aging. P16 in older animals represented here by a is much higher. Dosage matters and then patient population. This raises a few more questions: can regenerative therapies still work in aging? We did the newly published cartilage work last year and now we have some muscle work, which I hope to publish as a preprint very soon. But this is exciting because it's quite intriguing this article from several years ago that with aging, the muscle stem cell population still wasn't really functioning. But if you take it out and restore it, it may work again. The stem cells were there, but there are things that simply block them.

Again, thinking about senescence-inhibiting factors, what are all those things that block tissue repair? Here is the same model of muscle injury, the same biological scaffolds. What happens in older animals? Well, I know there's a lot of data here, but the main factors we saw with aging decrease eosinophils and CD4 T cells. This is a high-level parametric flow cytometry that analyzes young and old animals. Here, if we select a few pieces here, these are the increase in CD4 T cells. I showed them that in young animals it does not increase the same in old animals. On the other hand, CD8 cells increase.

CD8 cells are those that normally secrete proinflammatory factors. A reduction in helper cells, an increase in pro-inflammatory cells, and then a decrease in our number of eosinophils. CD4 T cells and eosinophils were important in IL-4 production. We did single cell again and as I showed you with dominant, we didn't have a large number of samples per group. It's quite expensive. If you just look at the differential expression in the groups between the young and the old, you don't see much, but we can use techniques like this non-negative matrix factorization and we find that the collagen markers are higher in the older, some more things related to fibrosis in old animals and in younger animals, there is more activation of macrophages in the presentation of antigens.

Then when we apply the ripple effect, so cell interaction, we see some really interesting differences. Again, these are the different modules talking to each other. This is unbiased and they are simply grouped together. The types of cells that seemed to be interacting with each other. You have your immune tissue, fibroblasts and then the antigen presenting module. There is a lot of connection here in young animals. But look, we're losing connection in old animals, in particular, this fibroblast module is really out of place on its own. We are really dysfunctional in some cellular communication. Jin Han, who worked on this, looked at some string analyzes to look at protein-protein interactions, to see what was going on, and all 17 signals were predicted to increase just with injury and the aging environment.

This was pretty cruel because if you just compare the young and the old with these various cytokines that were predicted by this analysis, you don't see any difference. It was only after that injury that the old animals had this crazy response. We analyze this a little more. Again, looking at interleukin 4 and IL 17, IL 17f in muscle tissue is another example of one of the factors that only increased with treatment. Gamma Delta T cells were a major component that produced IL 17. If you look at just the overall gene expression, you'll see all of the IL 17f. If we do flow cytometry and staining for IL 17a, you can see that in Delta Gamma it is increasing.

Oh, did I forget the most important key part? Sorry, I missed a slide. This is new data that just came out. What we can do then is administer neutralizing antibodies against IL 17 and recover part of that therapeutic response. Actually, this type of analysis offers new therapeutic objectives to pay attention to. I really think we will need combination therapies. We have all these different types of cells, communicating, working together. You have environments that change, like the aging environment. There is no way that a single therapy works for both the young and the old. We are going to need these combination therapies, whether analytical, systemic and local or an immunological factor together with the biological scaffold.

What does this concept of regenerative immunology mean? We are connecting regenerative

medicine

, immunology in tissue engineering. But there is still much to be done to map this immune response to injury. People think the response is innate for quite a long time, but the adaptive response is quite interesting and now we're looking at antigen specificity. Even a memory of his injuries and even biomaterial implants and many clinical samples coming from that. There is much to do to map these immune responses. Then understanding how immune environments affect tissue repair, including things like stem cell activation, cell types like fibroblasts and senescence, how they might affect vascularization, things like that.

Then use this information to design immunotherapies to create pro-regenerative environments. Whether it is autotherapy, neutralizing antibodies, or small molecule assays or biological scaffolds, we can use information from this mapping and communication across different tissue environments to promote tissue repair. My cancer immunology collaborators like to say that the immune system is therapeutically accessible, making it a good target for regenerative medicine. Many people thank the lab members, both current and alumni involved in this work, collaborators at the Bloomberg Kimmel Institute for Cancer Immunotherapy. In our computational collaborators as we move towards single space senescence, collaborators and, of course, our clinical collaborators who help us with clinical samples.

That being said, thank you for the invitation to speak today and thank you for your time. It was fantastic, Jennifer, thank you very much and very inspiring. If anyone has any questions, please write them in the question and answer section at the bottom of the screen. They are starting to arrive. Curiously, there is a mix of very specific and also very philosophical themes. Maybe I'll alternate between them. The first question is: do CD4 T cells form immune synapses with any of the macrophages? Are CD4 reactions specific to any particular antigen that may be present? Fantastic questions.

First, I didn't mention what the scaffold-associated macrophages look like. When I showed the myeloid reactions to those scaffolds, I have those macrophages associated with the dark blue scaffold. These co-express CD11b and CD11c and our MHC at high levels. It suggests that they are doing some antigen presentation. I think it makes sense, especially when you think about tissue damage and all the pieces that need to be cleaned, tissue damage would involve some antigen presentation. In fact, we also see some tertiary lymphoid structures. Where you have macrophages, T cells and B cells. You see those tertiary lymphoid structures.

Your last question is antigen-specific T cells. Two pieces. Number 1, we are in the process of performing a TCR analysis with a single cell data set. I'll have information on clones next time. Hopefully not for long. We are pursuing the understanding of that clonal response. The most specific data we have is found in Liam Chung's Scientific Translational Medicine article. It was specific to the foreign body response, but I think it is relevant to tissue repair. He made a bone marrow chimera from wild-type bone marrow and bone marrow from an OT-II mouse. That OT-II mouse can only respond to ovalbumin.

We were curious: Would you see the same increase in IL 17 in this case associated with fibrosis, a response to a foreign body around the material? Essentially, in wild-type mice the production of CD4 T cells increased. Then in the OT-II mice, you don't see any upregulation in IL 17. There's no ovalbumin there, so there doesn't seem to be any non-specific activation of those T cells. I think it will be antigen specific and we will have TCR clones. and we are trying to solve it. I'm excited. Excited, great. Thank you. Some of the questions are actually a little more philosophical, but...

I'm curious what a philosophical question might be. . It's meta. Since the initial clinical reaction to most injuries, particularly to the musculoskeletal system, is to administer anti-inflammatory agents and said steroids, things like that. Their findings that the inflammation is of the genus cara. It does some good things and some bad things. What are your feelings? What standard of care is most of the time for the orthopedist? That's a great question. I think Steve Battle Lake posted that looking at the response to a biological scaffold and frankincense suggests that you would get some reduction in the therapeutic response of that biomaterial with the enzymes.

However, with common injuries, I think it's a good question. I think there should be some data published at least from a physical therapy exercise training perspective. But if you think about the joint where there is not a good immune response. You don't have the types of cells you usually want. You can imagine that an anti-inflammatory would be helpful. We did an experiment on the cartilage of a steroid injection in addition to the sentalytic. What we did notice is that it blocked all immune cells, even those producing interleukin 10, which is increased in sham surgery and appears to promote repair.

I think this is also consistent with clinical data that steroids tend to cause more joint degeneration. Yes, I think we need to be smarter about addressing inflammatory attacks. Excellent. First of all, congratulations on his fantastic work, which I agree with. Thank you. Did you expect an almost complete reduction in interleukins with the removal of these senescent cells? Aren't there other types of cells that contribute tointerleukin levels? Absolutely. This is just a snapshot in time. I think this analysis is going to be temporary, first of all. If we look further ahead after this analytical treatment, we expect other time points, we see the increase in joint degeneration.

Yes, there are other types of cells besides T cells. We are also looking at co-culture of senescent cells and macrophages, and there is a lot going on there. Many times, when a T cell is eliminated, innate lymphocytes can compensate for cytokine production. We show that innate lymphocytes are producing that. You may have a temporary reduction in those cytokines and it's not completely zero, the cytokines. But I think the presence of those cytokines will induce greater senescence. It is positive feedback. It is necessary to address both the stromal cell and the inflammatory or senescence cell, in addition to the immune inflammatory cell.

Another argument in favor of combination therapies, which I think presents a unique challenge from a clinical trial perspective and a regulatory perspective because combination therapies are going to be a challenge. I certainly agree with his last comment, almost identical also to the end of my talks. The regulatory hurdle facing combination therapy has to... The next question actually starts with the difference between mouse models and real human systems. There are significant variations in Gamma Delta T cell populations between mice and humans. For example, the homotopic receptor on dendritic GDT cells in the mouse epidermis has no equivalent in humans.

Have your human wound repair studies correlated with your mouse models? Also great presentation. Thank you very much and that's a fantastic question. I will point you to Liam Chung's Scientific Translational Medicine article on IL-17 in senescence in the foreign body response, and there we can obtain many clinical samples of breast implants, or essentially the tissue expanders that are placed before the implant permanent. We get that tissue sample when they are exchanged and we can extract the cells from there. That's where we see a lot of Gamma Delta T cells. Much more so when we show this data to our immunology collaborators, they say, "What an incredible amount of Gamma Delta cells there are." That's just in that tissue around the breast implants, right?

Not where one would expect to have a ton of Gamma Deltas. I think we are going to find new functions of Gamma Delta cells and, as I mentioned, we have TCR analysis from single-cell data sets in mice. We are also doing the same with human samples. We will try to look at antigen specificity as well as understand more about Gamma Delta cells in clinical samples. Yes, they are very different on the skin. I don't know where they come from in this fabric sample. Again, this is a different scenario in mouse studies. When we place an IL-17-inducing intestinal infection in the intestine, we see more Gamma Deltas in the lymph node draining the implants.

I think they potentially act as an environmental sensor and in that way impact what's happening in the local fabric. I think it's a super exciting area. They're in human samples and we're trying to figure out what they're doing besides producing IL-17 A and F. Okay, this is one of the more philosophical questions. Taking into account the use, not only in unregulated clinics, but also in some orthopedic departments, of MSC therapy, injections for arthritic joints and the like, have you, based on your work, formulated an opinion on this? ? Well, I'd say I had an opinion even about chondocs when I was in grad school.

Oh, sorry, that wasn't the product, Carta cell. Autologous chondrocyte implants, right? What I had always thought somewhere is that we are delivering dead cells. The interesting thing is that there are papers now being published that apply them to cardiac lesions, and I think another one came out recently that essentially kills MSCs, right? They are injected to get those stem cells, many of which die, and can actually provide a pro-regenerative immune response. I talked about tissue damage and damage-associated molecular patterns that can induce an immune response. Many of them become trapped in the spleen, an immune organ.

Not because of intra-articular injections, but simply because of the idea that a dead cell can have a therapeutic impact. I'm not passing judgment, I'm just saying that one dead cell, especially if you have millions of them, can have a significant therapeutic impact. Are you mobilizing more immune cells, changing the immune phenotype? I can imagine many mechanistic scenarios where that could lead to some result. How is that? Well. Perfect. In your experiments, when you injected senolytic, presumably systemic, did you see any impact on other organs besides the musculoskeletal system, for example, the heart or the brain, or did you not have the opportunity to look at those organs?

For young animals, we injected the senolytic locally into the intra-articular joint, and then for older animals we injected the systemic senolytic navitoclax, which Daohong had published. It focused primarily on the bone marrow and the changes that occur in it. Essentially, with aging, the bone marrow becomes more myeloid, so you can see the replenishment of lymphocytes in the marrow and rejuvenation of the marrow, making it appear younger per se. Periodically, we have seen senescence of the kidneys, liver, and lungs, and typically with these systemic senolytics, they will subside, but we use the same regimen as Daohong, so we don't spend too much time looking at that.

That said, I think the bone marrow is really important as a place where you have some memory of an injury or something. One of my favorite articles recently is by Catherine Moore discussing myocardial infarction and breast tumor growth. That tissue injury to the heart impacted tumor growth at a distal site, and they found that epigenetic changes in the bone marrow, monocytes were partly responsible for that, and could be transferred to another animal. I think that's amazing. I think it's amazing to think that if there is any injury or implant in the body, you can have a memory of it imprinted in your bone marrow.

That's philosophical, right? We're getting close to the end of the hour and the last question is a bit philosophical, so we'll make it the last question. Given that you indicated that senescent cells also seem to be a bit Janus-faced, and that, for example, as you pointed out, Judith Campisi talks about their positive impact, I think I know where it's going to fall. in this. Is it allowing senescent cells as they become senescent to stop producing good things or start producing toxic things? We have some data on this and are working to put this document together. I think we have started to find the good and bad senescent cells.

We have done this using a transfer learning technique in which we can obtain bulk signatures of a senescent cell by separating specific populations of a transgenic that allows us to label them brightly enough. We can do full cytometry to understand what types of cells are senescent and then specifically classify them and do massive sequencing. Take that massive sequencing signature and apply it to a single cell to understand which clusters are most similar to senescent cells because you can't capture senescence in a single cell for a variety of reasons that I don't have time to explain, but it's hard to see. over there.

We have some groups that we think are associated with looking at the signatures of those groups and then fishing to see where they are. We think that some of them are specifically associated with angiogenesis, and some of the senescent cells with a different phenotype actually have a cartilage-like phenotype, but they are in the area of ​​fibrosis. I think there are different types of senescent cells and we are going to learn which ones are good and which ones are bad. There's probably a kinetic, right? How long they remain and whether they are reversible or being eliminated. But I think there are different ones, and we will define the good and the bad, then we can develop really good drugs.

Keep the good ones but get rid of the bad ones. That's the dream. Anyway, one last topical question arose. It's not so philosophical. Maybe we'll end it. Well. Fantastic presentation. Could you comment on the potential impact of the extracellular matrix on stem cell senescence in vitro? Does the extracellular matrix act on stem cell senescence? Probably. I guess you can light it up. Is there any relevance to the substrate? That is, in which they grow or in what could be found in situ. Yes. In vitro you do not have the immunological aspect. So you depend on specific factors in the ECM, either like Steve Butler talks about Matrix-bound vesicles that have important factors in them, or certain components of the ECM that can help proliferation.

But what I think is also interesting is that cultures potentially eliminate senescent cells as you go, because essentially those senescent cells that are left there can spread the senescence or cause problems with the other cells, and it's a domino effect. If you can grow them and while you work to eliminate those senescent cells, I think you can keep the healthy ones longer. That's a great answer. It delves into another completely different territory of the senile brain that we do not have time to enter. Thank you so much. We have reached the end of the hour, in fact, we have reviewed, you have been very generous if you look at the hour, and it was a great and stimulating presentation.

Thank you so much. Thank you so much. Have a good rest of your day. Bye bye. Thank you so much.