Scientist Stories: Henrique Borges da Silva, Extracellular Nucleotides

This is episode 39 of the Immunology Podcast, Extracellular Nucleotides with Dr. Enrique Borges da Silva. Hey everyone, this is Dr. Jason Goldsmith and Dr. Brenda Raud. Welcome back to the Immunology Podcast. We have conversations with immunologists. Immunology Podcast is brought to you by Stem Cell Technologies. A global biotechnology company supporting life science research and fostering communication and collaboration in science. Today we have Dr. Enrique Borges da Silva from the Mayo Clinic on the podcast to talk about his research investigating how extracellular nucleotides regulate the immune response. We've also got our usual roundup of recent highlights in immunology news coming up.

But first, are you participating in an immunology journal club? Stem Cell Technologies is offering you an opportunity to win $300 worth of refreshments to fill your journal club. And the discussions there. To find out more and enter this sponsorship contest, visit stemcell.com/journalclubcontest. Sounds fun.

Yeah, we have journal club, but I don't think I get— I can apply for that. But that's okay.

Probably not. I mean, I also would say what we're doing now is kind of a journal club, don't you think? Should we be getting refreshments from Stem Cell?

We should. We should ask them. There we go. All right. You have a paper that you want to cover first up that is a hot one that I think we're going to end up talking about quite a bit.

I think so.

Why don't we do that and talk about it and then we can get into the, into the controversy?

I mean, I don't know if there's controversy yet, but the results of this paper are just kind of weird. And I have to say that this reminds me of like the start of those papers. And then, you know, down the road they show that they were not as reproducible as expected. So without meaning to mistrust the first author, let me just talk to you about the paper that I'm going to call the rise of the zombie T cells, maybe. So this paper is called Intracellular Transfer of Telomeres Rescues T Cells from Senescence and Promotes Long-Term Immunological Memory. And it was published in Nature Cell Biology. And the first author is, let's see, Lana. And he is the founder and CEO of a company that has clearly been set up in order to kind of cash in the results of this paper. So the company is called Sense Cell and was founded by this first author. And what first caught my attention, besides obviously the, the, the, the kind of the, the title and the what it kind of conveys, is the fact that it has also an interesting kind of combination of authors, uh, including a person called Brandon Bloomer, who is a CEO of a blockchain company. Um, I assume it's kind of a strange addition to the author's list. People from several different labs have collaborated into this publication, but it seems to really be the brainchild of our first author. The idea on this paper that I think is crazy and I really don't think has been suggested before is that upon synaptic T cell synapse with APCs, the APCs undergo expulsion of like vesicles that contain telomeres, telomeric sequences in there that can be transferred to the T cells they are interacting with. And these T cells not only pick up these vesicles but incorporate these telomeres into their own chromosomes. And I think that's just mind-blowing. So that's why I call, you know, the rise of the zombie T cells, because basically these T cells are increasing their kind of longevity at the expense of the telomeres of the APCs. So maybe more than a zombie, it would be like a vampire, vampire T cell. So I have to admit that this paper, I did a little bit of digging because I thought this is kind of an interesting situation because obviously this is a Senescence and kind of longevity, it's a huge interest field nowadays. So there's obviously an economic interest also, I think illustrated by the fact that a company was set up by the first author. And I feel a little bit kind of skeptical about this being so perfectly, all perfectly kind of coming up together. I don't know how they're planning on cashing in this particularly. There's probably going to be some kind of product, something that is inducing synapses and favoring telomere transfer to T cells. And I guess the idea is that you can keep your T cells young for longer, maybe improving vaccination responses or improving the overall longevity of your immune system. We know that throughout age, there is a substantial decrease of cells that have— there are certain phenotypes that are associated with kind of non-senescent T cells. And also T cells do get their telomeres reduced throughout their proliferation. They proliferate a lot. And so quick overview of the data presented in this paper. First, the author shows that the length, they'd have this kind of this blot in which he can look into the telomere length. And I'm not sure exactly, I don't think it's completely clear how he exactly isolates the cell types. But the first figure of this paper is basically APCs after being part of a synapse, they have ended up with reduced size of telomeres. So you see a smear of a different size. The T cells from that interaction end up with almost 3 kb more of telomeres, which is something that is not mediated by telomerase, which we know is a little bit activated in T cells, but this level of telomere extension is not explained only by telomerase. They do an experiment which is do a CRISPR knockout of telomerase. I'm a little bit skeptical about the technique that they use because I do CRISPR knockouts all the time. And it feels like they do viral— they do, I think, plasmid transfection of CRISPR. And it's a bit— that doesn't work that well in primary T cells. So I'm surprised that it had such what they seem to be almost 100% knockout of telomerase. That seems a little bit strange. But if we follow this data, they show that their telomeres. So APCs get shorter, T cells get longer. And then they do a lot of FISH experiments. So they have these special probes and they can pick up the telomere TTAGGG. I forgot what is the specific sequence that characterizes telomeres. And they have these kind of microscopy images in which they show that there's these vesicles that are staining for this fish in this fish assay. They're vesicles containing telomeres that are found in the APCs upon synapses. Then they see that these vesicles are expelled from the APCs and then they're taken up by the T cells. Then if they, for example, do a BrdU stain of the genomic DNA of the APCs beforehand, they can find that this fluorescence is transferred also to the ends of the chromosomes of the T cells. So like that's how they show that there's actually kind of a horizontal transfer. And this is very strong. They say that about, in their numbers, about 8% of T cells, they look at the T cell chromosomes in metaphase, they see incorporation of BRD or, sorry, EDU from the right, from APC telomeres. In 8% of T cells.

So they don't do like, like a labeled test, like take radioactive or heavy nucleotides, feed them to the APCs, have that incorporated in their DNA after replication or whatever, and then, and then watch it move to the T cell?

So I guess they, they do that with the EDU stain.

So they EDU incorporate in the APC and then show it on the telomere.

Yeah. Yeah.

Okay.

That is the way they do it. And I think what's also like, and so they move on and they show the, they have, so basically you have this telomere, these vesicles, the telomeres, and they don't have only, so, and they also look at some certain proteins in the APCs that are associated with kind of telomere extension and protection. So they have a particular enzyme, a particular protein, this they call TZAP, telomeric zinc finger-associated protein, which kind of is a binding protein that binds to telomeres. And that activity, they show that it's upregulated upon activation of the APCs and that this stimulates them to cut, they trim the telomeres, and this allows these telomere fragments to be encapsulated into vesicles. And these vesicles also have RAD51, which is a recombination factor which is involved in telomere elongation. So it's kind of these little packages, they come with the telomeres or with the enzymes required to incorporate them into the T cells. So it's like, this is insane. I find this insane. And it's weird that we never seen this before because it's— I mean, the data they show is really kind of very eye-catching in a way. So basically—

Maybe no one's looked before.

Maybe. I mean, it's such a weird idea to start with, right? I mean, this is not the first time something like this would happen, right? Often we miss, completely miss huge things. I think no one's looking. Like, if you're not looking, maybe you're not going to see it. So he also does some experiments in which he looks into— so they have basically cells that are activated in the presence of telomere vesicles or not, and then they show that cells with its vesicles. They have reduced senescence upon proliferation. They can proliferate for longer. They also have some in vivo experiments in which they transfer cells to mice. And so, they have these OVA-OT2 cells, and then into mice, they pulse the mice with OVA. And then the T cells that were expanded with telomeres from APCs, have kind of better proliferation expansion. They're going to have less signs of senescence compared to those that were not, were transferred without this kind of telomere boost before. So I think it's a very, very intriguing result that we see here. And I wonder how this, yeah, will play down, like, down the road. Looking forward to see people trying to kind of, uh, yeah, reproduce this or like work on, on this idea. What do you think, Jason?

Well, I think someone needs to replicate it and soon.

Yeah.

And then just wonder if it extends to any other cell types. And then if it does, then I start believing it. It's, it's interesting. It could be something no one looked at. It's weird enough it could have been overlooked, right? It's not like something that they got something new and fancy that they're going to sell a company on. It is weird. Which is actually a favor for them in my mind, because it's something you wouldn't expect. But it's just weird.

It's weird. It's cool, but it's like, wow. So I'm looking forward to see the future of this paper. Um, this is one of those things I thought, what? So I don't know. I don't know. I do not want to, you know, I want to keep my cool science head. Not get too excited about the shiny new thing. Uh, I do want to— well, kudos to the author, to Alessio Lana. Let's see how his company goes. So I'm want to wait to see what product they are, uh, releasing to, uh, cash on this, um, discovery. And, and we'll see how it, how it, uh, plays out, right?

All right, well, okay, I don't have anything that's interesting, but I do have an accelerated article. Uh, it's in Nature. It is Common Human Genetic Variants of APOE Impact Miring COVID Mortality. So APOE is that lipid gene, and there's APOE2, 3, and 4. These are known isoforms. APOE3, you know, there's APOE and then there's 2, 3, 4. APOE2 and 4 known to be bad actors associated with Alzheimer's, and particularly APOE4, um, is known to just be associated with disease, in particular, as I said, Alzheimer's. Well, they— these authors, I don't know why they did this, but probably they just went on a fishing expedition and found something pretty cool. And about 3% of people are homozygous for ApoE2 or 4. And ApoE variants are associated with immune process dysregulation, anti-tumor immunity, atherosclerosis, and now it's COVID. So people with ApoE2 or 4 have worse COVID outcomes. So, uh, I'm going to go to the last part of the paper where they look in humans in a UK database and find this is true. They have worse mortality and outcomes than ApoE3 or other ApoE people.

Okay.

Um, but it's associated all the way with survival benefit. They do this in mice because they have humanized mice that have the ApoE gene that's been flipped over. And they basically recapitulate that, um, E2 and E4 mice have increased COVID-accelerated progression with worse histological damage, but that's in the lungs, not in the interstitium or vessels. And, um, this has to do with antiviral immunity is delayed in them, but eventually ApoE4 covers. So early immunity ApoE2 and E4 genes are downregulated, but ApoE3 are not. And then they deep dive in and show that they have more myeloid cells and lymphoid cells in the lungs. And then they see hyperactivation of coagulopathy pathways in ApoE2 and E4 mice. And, and recombinant ApoE3 significantly reduced infection in a cell system, but not ApoE2 or E4 when it's introduced. And then, um, but later on ApoE have more robust antiviral T cell responses than ApoE2, but early on they're both screwed, and it's the early response that seems to be causing everything. So ApoE2, E, and E4 lead to decreased adaptive immune responses, which leads to worse COVID outcomes in mice, and also increased hypercoagulability responses. And then in people, this holds true, and people with these isoforms die more from COVID That doesn't sound very nice to those people that have the isoform. Nope. And then if they survive, they'll get Alzheimer's. ApoE4 is the single most monogenetically associated gene with Alzheimer's.

Oh, wow. How is that mediated? Is this a similar thing or do we not know?

It's no, no one knows how this is mediated either. This is like, it affects the immune system and immune genes and cells. No one knows how the lipid protein does what it does.

Much immune system, many T cells, much myeloid. Yeah, I see. Okay, maybe it's something related, right? So in this case, it's affecting the ratio of myeloid cells to T cells, and maybe it's something similar in the—

Yeah, I mean, so we know it affects those things, but I don't think anyone's figured out how it works very well.

I guess, well, this is very interesting, you know, because to, to start teasing out those genetic components of COVID response. So it's very, very interesting result. And then they could kind of better model it in the mice, in the mouse model. Yeah. Thanks. Thanks for sharing. Okay, so for my second, less controversial paper, I have a little nice story from immunity. That talks about another of our favorite topic of ours, nasal vaccination and nasal immunity. So this paper, Mucosal Plasma Cells Are Required to Protect the Upper Airway and Brain from Infection, first author Sebastian Welford from the lab of Ashley Moosman at Duke University. Very nice story in which they kind of differentiate what happens in an infection of kind of the nasal cavity. And they differentiate between different areas in the nasal airways. And there's two main regions that are important to keep in mind. And one is the respiratory epithelium, and the other one is the olfactory epithelium. And this is important because in the olfactory epithelium is where all the olfactory sensory neurons are, which are projecting the sensory dendrites into the airway. And that's how they pick up all the scent or the information about scents. And they have kind of a direct highway to the brain, to the olfactory bulb in the brain. And this is very critical because many pathogens actually are capable of hijacking this highway and finding their way all the way to the brain. Among these, among some of the famous, the Hall of Fame for doing this is this Naegleria fowleri, this brain-eating amoeba that sounds horrible and does horrible things to the people they infect.

You're never going swimming in a lake again, are you, after reading this paper?

I know.

I— well, you know, I heard about— it was like a case recently in somewhere in the US. Like, now climate change is making things warmer and something like that. It's terrible. I mean, I grew up in a place of cold waters, so I feel quite safe there, but Damn. So brain-eating amoebas. Several viruses also are capable of infecting the neurons in the olfactory mucosa and go up, including SARS-CoV-2. So here's that SARS-CoV-2 segue just for you. But this paper does not focus on SARS, but they do have a figure at the end. Of course it is, after all, after post-2020. But so they have this viral model. They have a VSV, a vesicular somatitis virus infection in mice, and that is capable of kind of infecting the olfactory pathway all the way to the CNS. And usually mice, there are certain strains of VSV and the strains that they use, mice can kind of really mount a robust response. And they're mostly protected against reinfection. But they show that the neutralizing antibodies that these mice generate in the serum are not mediating any protection against infection on the olfactory mucosa. So the respiratory mucosa seems to be kind of served by the neutralizing antibodies in the blood, but not higher than that, sort of like closer to the olfactory nerves, that area around the olfactory nerves. And they even do parabiosis experiments, and again, they show that having neutralizing antibodies in the serum alone is not protecting against kind of infection of the olfactory mucosa. And So what they ended up showing is that antibodies don't reach those cells right up there. And so they kind of end up defining what they call it the BOB, the blood olfactory barrier, showing that it's similar to the blood-brain barrier. The soluble, these large proteins from the bloodstream cannot go through. But if you get antibodies right inside beyond this barrier, then there's kind of free flow of things within this area. And so the question is, if you have— if you do see antibodies there, where are they coming from if they're not coming from the blood? And those antibodies that are protecting, because these mice are protected somehow, are actually coming exclusively from B cells that are resident in the olfactory mucosa. So they have these plasma cells, mostly IgA positive, that are there inside and are making the antibodies inside this particular olfactory mucosa. These B cells, these plasma cells, they are— So they do a bunch of experiments and they show that these B cells, the differentiation of these B cells happens in the lymph nodes close to the nose. They require CXCR3 and they are recruited by kind of with the help of CD4 cells. Within the lymph nodes, and then they acquire a phenotype that kind of shuttles them all the way to the olfactory mucosa. And they home there, and that's where they make their antibodies, and they're specifically protecting that area. What is interesting, they end up kind of— the story, they end up showing that there are ways of inducing these olfactory mucosa resident B cells in a way, But not every adjuvant. So if you have, like, vaccination strategies, not every adjuvant works in the same way. And they have this adjuvant they call— that is called DMLT, which is a double mutant toxin. So from— they're derived from E. coli. And they showed that it's capable of inducing these mucosal kind of B cells if they vaccinate the mice in a kind of double— that would do two doses. And in this way, they can actually kind of give the B cells the right signals to migrate to the olfactory mucosa. Unfortunately, they're not really good at showing exactly how this happens. So that's where they're a little bit fussy in that part. But they really seem to kind of bring the point that there are certain types of vaccinations or adjuvants that will induce the right signals in the lymph nodes that might make these B cells that go to the nodes. And they do one experiment with SARS-CoV-2 model. They actually have a VSV with SARS-CoV-2 envelope proteins. And they show that actually with the mRNA vaccination from— in this case, they use the one from BioNTech, from Pfizer-BioNTech, they seem to show that they can induce resident B cells in the mucosa with this particular vaccination type. But this is in mice, suggesting that, I don't know, in a way suggesting that maybe it's not that critical to do nasal vaccinations, that the B cells are still going there. But it is hard to tell because as these B cells are resident in the tissue, I don't think people take nose biopsies to look into B cell activity in there. Must be very difficult to evaluate in humans whether you actually have kind of resident plasma cells making IgA in that particular region. Also, I would not like anyone going there touching my olfactory nerves, so I'm not sure if you can actually test it better in humans. But it was a kind of an interesting— I think it was an interesting, um, an interesting idea, this kind of this olfactory barrier and the idea that you have— you really need specific B cells that are resident there. Doesn't matter for the respiratory epithelium, but does matter for this part, this mucosal part higher up closer to your brain.

Interesting. One of the treatments they were having for— that became popular was this nasal gel of zinc that was really effective at preventing infections.

Mm-hmm.

But then they found the zinc was destroying your sense of smell. Oh, but it actually was working, probably by preventing this other pathway from being hijacked.

Maybe. Maybe all your cells— all the cells are dead, and then where there's nothing for the virus to infect, well, your epithelium there is not receptive. It's not, uh, it's not receptive to any virus.

Now we know more about the nose, or we need to vaccinate the nose, clearly.

Yeah, or make ways of making the B cells go to the nose. Apparently you can do that.

You don't want to, you don't want to go there, it's smelly.

Yeah, I mean, I wouldn't want to go to my nose either. I don't know. Okay, what you got?

I got one more here. Nature Microbiology: Inflammation-associated nitrate facilitates ectopic colonization of oral bacterium Vianella parvula in the intestine. First author is Daniel F. Rojas-Tapias. Last author is Ramnik J. Xavier. It's coming out the 22nd of September. So I like this paper because it gets past the chicken and egg problem, the microbiome. We're like, oh, you have a disease, and then is the microbiome causing it or not? So they noticed that this one bug, Viennella parvula, or VP as I'm going to call it, It is mostly associated in the mouth, but it's been associated in IBD patients before. Like, it's just there. They're like, well, this is weird. What's going on here? So they, instead of trying to see is it a bad actor now, they're trying to figure out how it happens. What they basically find is that inflammation in the gut results in INOS activation, right? And that releases nitrate. Well, this bug is a preferential nitrate bug. And when you have a food source that's compatible with its life, it will live. So it will normally die. We swallow it all the time. It's in our mouth, right? But normally doesn't survive very well unless you give it nitrate. And that nitrate and other lets it shift its metabolism allows colonization then. And so they show this in mice with DSS colitis, which is a chemical monocolitis. But importantly, they do a good amount of work mapping out the metabolic pathway and show these bugs— this bug really likes nitrate in certain ways to live off of. And I'm not going to go in all the details here, but they basically do a whole bunch of different genetic deletions and use a whole microbiology knockout strategy and knock-ins and show that what it likes., and it can formate lactate. But basically, it's able to respirate in the inflamed intestine when nitrate's present. They show that, hey, if you remove nitrate, doesn't grow. If you add nitrate and it grows, if you do this, you do that, they establish ex in vitro— it's living conditions— and how much it likes nitrate and what pathways and what operons it's involved in, which is not really the focus of the podcast, which I'm ignoring that part. But then they show that the inflammation in the gut releases nitrates through INOS, right? And so they knock out INOS in the mice and then give it the bug, and it doesn't colonize even with colitis. So the DSS still made the colons more inflamed in the INOS knockouts. They still have the same colon length and other inflammation markers, but the immune cells driving INOS, when it's gone, there's no nitrate there for the bug, and then the bug doesn't show up.. So that's what I thought was cool. It's showing that this host microbiome relation's due to a metabolite of the immune system when it's doing its thing, and then this bug likes it and it comes, hangs out. Now, that could be pathogenic and induce a pathogenic environment or not. That's a separate question, but it's getting at the relationship much more in depth than, oh, this bug's here with this condition, maybe it's causative.

Yeah, it's a really good show of correlation is not causation.

Right.

Let's get rid of that chicken and egg thing that's been driving me nuts in the microbiome. That's pretty good. That's a pretty cool story. I like it.

So, so I like that too. And, you know, we're going to be talking more about metabolites here, uh, with Dr. Enrique Borges da Silva at the Mayo Clinic in just a moment. But before we get to that, uh, Stem Cell Technologies would like to introduce you to Human Immunology News. It covers everything from immunotherapy, autoimmunity, and adaptive and innate immunity. Human Immunology News keeps readers current with the latest news, research, policy, events, and jobs relevant to the immunology community.

Subscribe for free at humanimmunologynews.com. Hi everyone, we are talking to Dr. Henrique Borges da Silva. He is assistant professor at Mayo Clinic in Arizona, and he's going to talk to us about the lab, the work his lab does to understand how extracellular nucleotides regulate the immune system. Very interesting. Professor Da Silva, thank you so much for joining us today.

No, thank you. Thanks for the invitation. I'm actually a listener of the podcast. It's good to know that somehow you guys came across my name and wanted to hear a little bit about my research and myself.

We're so happy to have, like, people that are listeners, like, coming on the podcast now. So it's fun.

I enjoy that. I'm going to kick it off. Alright. Good question. So the way I wanna frame it, I'm a T-cell, I'm a rapper, and I go ATP. Yeah, you know me. So what, what, what is this love affair you've discussed, discovered between T-cells and ATP?

Well, this story actually started, I mean, I, I gotta tell the historical context, right? When I was a grad student in University of São Paulo, I was in a laboratory that worked with trying to understand how T cell immune responses are formed against malaria and tuberculosis. Because as you're probably aware, if, if you are in South America, a lot of the immunology research is focused on infectious diseases. So, uh, in my laboratory, that laboratory was transitioning to study more the role of purinergic receptors, which are receptors that recognize nucleotides, including ATP, in immune response. But I was the remnant of the past, let's say. So I was the only person not working with ATP sensing. T cells. But again, the focus of that lab was CD4 T cells. So I, I kind of in the back of my mind was interested in understanding, well, is there something related to ATP sensing in CD8 T cells? And I knew by going to, for example, Imgen.com that some CD8 T cells do express P2RX7, which is a receptor for ATP. So I came to University of Minnesota to Steve Jameson's lab to do my postdoc. And my initial project was not going to be ATP at all, was going to be something related with KLF2 and S1PR1. That's what Steve does. But then I mentioned that to Steve and Steve was like, well, that's funny because we are trying to see if we block this pathway, we can rescue better numbers of tissue-resident memory CD8 T cells. Because the idea initially is that, well, tissue-resident memory CD8 cells express very high levels of P2RX7 and ATP can induce cell death of many CD4 T cell subsets. So, The assumption that we had is that, well, this is gonna be the same for CD8 cells. There are TRMs and we have, we in the field have a chronic problem in recovering optimal numbers of tissue resident T cells whenever you do harvest, right? Because you have to do mechanical disruption of the tissue, enzymatic digestion. I mean, Dave Mazepust had showed that pretty nicely in a Cell paper in 2015. I'm gonna try to summarize, but But, but the idea is that initially we, we thought, well, if we block this pathway, we're gonna have more TRM cells being rescued during harvest, which in some ways is true. It did publish that in 2019, but then Steve is always trying to push you a little forward. He's like, well, what happens if you block this for a longer period of time? I mean, is this detrimental for the maintenance or the establishment of tissue as the memory CD8 cell? So initially we thought the opposite was gonna be true, right? That if you block this pathway, you're gonna have more TRMs, but then I came to Steve and say, well, Steve, this is the result. He told me to repeat that using other models and the rest is history, right? I mean, we didn't notice that T cells express varied levels of these receptors and that is important for the establishment of memory CD8 T cells in a very weird way. And I've been working on that since then.

Very interesting. Just, just to maybe take a step back, I have a very basic question to ask. Why is there extracellular ATP and where is it coming from?

Oh, that's a very good question. And that's part of the research that our lab is doing right now. So ATP in the extracellular environment is traditionally understood as what we call danger signals, right? So in situations where you have a threat to the host. So you have sterile injury, you have cancer, you have infectious diseases, you have the release of these molecules that are usually inside the cell. They are part of the host, but they're not supposed to be outside. And ATP is one of those molecules. So in a lot of cases, what happens is that you have tissue damage and cell death of infected cells, for example, and What happens is that you have passive release of extracellular ATP to the extracellular environment. There's other parts, and that's a work that we're about to submit, that there's active release of ATP by channels. But the notion or the idea, if you think about, is that these molecules, they usually typify a situation that, well, there's something wrong with the host. So, Usually innate immune cells are the ones that sense those signals. And a lot about ATP is known on innate immune cells. So that's the work of Vishva Dixit on the NLRP3 inflammasome. But it has been appreciated in the past few years, and that includes our work, that T cells also express receptors for this ATP and other danger signals, as a matter of fact. So, the idea is that they're not usually out of the extracellular environment, but when they're out, usually that means that there's a situation that things are not okay for the host.

So do we know what, I guess, intracellular concentrations or concentration tuning is needed to generate a signal in these CD8 cells? Like how much ATP does there need to be?

That, that is a question that will depend on the receptor that we're thinking because there are 7 receptors for extracellular ATP. PTORX7 is the one that we study because it's expressed in a more abundant way on T cells. Usually what you need to activate the PTORX7 receptor are considerably high levels of extracellular ATP. This is a low affinity receptor for ATP. So you need at least 50 micromolar of extracellular ATP to engage this receptor. The thing that is important to note is that if you think about extracellular concentration, ATP is not uniform. It's not like every single portion in the extracellular environment, you're gonna have similar concentrations of ATP. What you usually have, and that is true, works from Wolfgang Junger, which is another specialists in the pure energy field have shown that the concentration of ATP in what we call the pericellular space is much higher than the concentration of ATP that you find away from the cell. In a way that if you take the entire supernatant of a cell culture, for example, you're gonna have nanomolar concentrations, very low concentrations. But if you somehow get the supernatant that's only in the pericellular space, you'll have actually pretty high concentration of ATP enough to activate PPARγ7. So to summarize our question, you do need quite high concentration of ATP to activate P2RX7, but usually that occurs during infection for some many reasons. I mean, because of this unequal distribution of ATP, there is multiple sources of ATP that is not only passive release, you also have active release by T-cells themselves as a matter of fact. But, but, but, but usually to activate the Pituric 7 receptor, you need quite high concentrations of ATP.

And is ATP stable enough in tissue circulation elsewhere?

Yeah, that's an important question and it's something that we want to track. We have some probes that we can transduce T-cells. We are gonna study that and we can basically in vivo track how much ATP the cell is sensing at a determined point. What I believe it happens is that ATP is not stable at all. You have a lot of ecto-ATPases present. But what I think happens during infection, you have a dynamic state where you have ATP being released and degraded, but the ATP release is to some extent constant, but not necessarily all the time, but it occurs in an enough amount of time that you can promote the activation of these receptors because you're gonna have ongoing tissue damage. You're gonna have ongoing activation of channels that are present on innate immune cells and adaptive immune cells that are gonna release ATP. So I don't think it's stable, but I think the fact that the release of ATP occurs in a span of, in a longer span of time helps making the activation of Pitrexin feasible. That's what I believe. Of course, I haven't—

I don't have proof of that yet. Yet. I think just kind of to wrap up that idea of where the ATP is, and because if you say that this receptor P2RX7 is required for the tissue residency of the CD8 cells, then you would assume that it's because it's signaling in a way. So it's been activated by sufficient amounts of ATP to— or how would you— then you would expect that there is some kind of release of ATP, this kind of basal levels of ATP, otherwise the existence of the receptor would not make a difference.

Yeah, yeah, those are good points. And, uh, uh, and actually this is one thing that we were puzzled about because we, we have a Pitrex7 conditional knockout mouse where we can knock out this receptor during the effector stage immune response and after memory CD8 cells are already formed. And basically we found in 2020 that the T cells, the memory T cells, they need Pitrex-7 also for the long-term survival of memory CD8 cells where you're not gonna have tissue damage, right? So your idea of basal levels of ATP I think is spot on. And there is one particular channel that caught our attention that is expressed by T cells. And it's called Panaxin-1. So there's a lot of work done by researchers such as Cody Ravichandran, which is now at Washington University, showing that T cells and innate immune cells and also epithelial cells at certain sites, they express this channel. And Panaxin-1 is a nucleotide release channel. So it sounds counterintuitive, but what it may be occurring, and we are about to submit a paper on that, we know that it occurs, right, is that the T cells, CD8 cells, express this channel called Panexin-1, and it induces the release of extracellular ATP, and it is present at constant levels throughout the immune response. And if you knock out Panexin-1 at the memory stage, the CD8 T cells have the same problem with survival that we have if you knock out Pitrex-7. And we know based on double knockout systems that this is true Pitrex-7. So, What happens is that there's always a basal amount of extracellular ATP around the CD8 T cells, and this is being dictated in a populational level by the CD8 T cells themselves actively exporting ATP. Now, this sounds very weird, and that's something that we're still trying to understand. What is the evolutionary reason why this is occurring? I mean, why would CD8 T cells pump ATP out of the cells, right? But the basal amount of ATP is something that we believe it does happen. And we believe at least in part is through this release via this mChenylcopanexin-1.

All right. So I guess the next question then, we've talked a lot about the ATP getting out there and it being important for CD8 cells, but you have a pretty recent paper.

What does this ATP do to CD8 cells? So traditionally what we know about P2RX7 is that it is an ion channel. So basically when ATP binds to P2RX7, it induces calcium influx and in some cases potassium influx. And in many cases that will ultimately induce NFAT activation and proliferation. For CD8 T cells, we know that this is one of the pathways that is important for the function of P2RX7. So if you block somehow, or if you go on Pitrex 7 knockouts and you forcefully increase intracellular calcium levels, you kind of rescue the phenotype of Pitrex 7 deficient CD8 cells. And ultimately what it seems to be doing is it is favoring the expression at a transcriptional level of factors such as TCF1. And, and, and this is happening already kind of early in the effector stage of immune response. If you, if you're talking about the JI paper that we published in 2022, and what happens is that it helps CD8 T cells, I mean, if we help CD8 T cells to, to have their fate decision toward longevity versus short-lived. Now, how is this happening? It's something that we're still trying to understand. I mean, how we get from Pituric 7 and calcium influx all the way to TCF activation. Part of it seems to be related to alterations to the immunometabolism or the, the intracellular metabolism of T cells. So we, we have done this experience before. And if you think about memory cells or cells that are already committed to memory, if you knock out Pituric 7, they have a problem. Engaging oxidative phosphorylation. And this seems to be partly related to the inability of Pitrexin-deficient cells to activate the AMPK pathway. But how does this affect TCF expression, TCF7 expression, is something that we still trying to understand. And this is gonna be one of the projects that actually we got funded to do that, try to understand how Pitrexin induces this pro-longevity phenotype in CD8 T cells. But to summarize your question, I think it is absolutely because, or is absolutely related to calcium influx. It is absolutely related to an increase in the ability of these cells to engage oxidative phosphorylation. It does induce TCF7 and other transcription factors associated with pleural prolongevity such as God, back to— I mean, I have— there's a kind of a long list. TCF7 is the one that usually pops up more correlated with Pituric7, and it does have a relation with the AMPK activation. But of course, we still need to do more research to tie those things together.

I'm thinking of AMPK activation. I think, well, AMPK also can sense ATP, ADP. I don't know if that But I guess it's two different interface, one of what happened outside and another thing was what was happening inside.

No, actually that's a good point. And there are three ways by which AMPK, the AMPK pathway can be activated. I never remember the third one, but it's TBK1. But you have calcium-induced activation of AMPK, but you also have AMPK activation if you have a skewing toward increased AMP-ATP levels., right? If you have the ratio between ATP and one of the things we, we have done recently, we, we're about to submit this paper probably in a couple months related to Panexin-1 is that P2RX7 activation promotes the recruitment of Panexin-1 to the immunological synapses. This is known by, by other researchers. And what happens is We did metabolomics and we know that the AMP ATP ratios intracellularly are skewed in a way that if you knock out P2RX7, you not only have lower levels of ATP extracellularly, but you have increased levels of ATP intracellularly early on during the infection, during activation. And then what happens is that this is somehow hindering the activation of the AMPK activation. Makes sense. So, so part of the reason why AMPK is activated is likely because of the fact that P2RX7 is recruiting PON1. PON1 is promoting the export of ATP, which by itself induces sensing of P2RX7 again. But also this export is not only to serve as a source of ATP. That's what we are trying to make the point in this next publication, but also because it is also skewing the levels of AMP ATP inside of the cell. So AMPK activation occurs in a lesser way, on a lesser extent.

Interesting. Yeah, I mean, also AMPK has activation and the downstreams of that have been associated with memory, with longevity. I mean, so that makes— that sounds kind of reasonable, a reasonable framework. I have a little bit of a slight change, but related to the topic. But I work a lot with cancer and T-cell immunology in the tumors. And one of the molecules that comes around a lot when it comes to characterizing this exhausted, overactivated phenotype, it's CD39, which is this hydrolase that is extracellular as well and degrades ATP outside of cells. At least that's the understanding I have about the importance of CD39. And CD39 is very widely used to kind of apparently to identify tumor-specific T cells. So there seems to be a lot of like importance put on this molecule. Do you have Given what we just learned, what would you— do you have any kind of comments on the consequences of this particular enzyme?

Oh yeah, me and a lot of people have. And actually, the idea is that the CD39 together with the molecule CD73, which is expressed on T cells and other cells that are present in the tumor microenvironment, is going to ultimately induce production of adenosine. So that's the ATP gets cleaved, forms ADP, AMP, and adenosine. And adenosine is immunosuppressive. So if you— we have started looking at the role of PTORX7 in CD8 T cells in the context of cancer. We published a paper early on this year showing that if you have activation of PTORX7 during the priming of CD8 cells in an adoptive cell therapy model, DCD cells tend to survive better and promote anti-tumor response in the context of melanoma. This is published in Cancer Immunology Research. But it is also true, and this was shown by Fabio Grassi, which is another researcher on the field and others, that PTORX7 can be detrimental once the T cells are in the tumor. Now, the idea here is that you have kind of a weird balance in the tumor. And if you think about a tumor that's immunogenic like melanoma, I think that's the example that I, I keep more in mind because I work more closely with melanoma. It's a hot tumor. You have a lot of ATP being induced by cell damage. I mean, you have, you have up to 300 micromolar, so you're gonna have enough to activate PIK7. You also have a lot of adenosine. Which is immunosuppressive. So what a lot of researchers believe, and I also believe in that in the same extent, in some extent, is that if you manipulate the balance between ATP and adenosine, that's gonna somehow skew T-cells toward more function versus less function. And at the same time, it's interesting because in a lot of cases with human cancers, expression of CD39 on T cells is correlated with better prognosis. So there, I remember looking at posters from folks from the Wery lab suggesting that this is the case. So it gets a little complicated because to what extent what they're seeing is just a readout that you have more T cells in the tumor microenvironment versus you need somehow to curb excessive ATP Because there's another characteristic about Pituric 7 that I perhaps I, I, I kind of glossed over is that it's a weird receptor, right? It induces calcium influx and potassium influx if we have intermediate to me mildly high concentrations of ATP. But if we have too much ATP, it forms these non-specific pores and it can induce apoptosis of T cells. So what we believe happens once the T cells enter the melanoma microenvironment, and again, I'm just saying about melanoma, is that you may have enough ATP sensing to activate these T cells, but there's two signals that could be happening at the same time that could be bad for the T cells. One is adenosine, which is immunosuppressive per se, and one is just excessive ATP sensing, which is inducing apoptosis of T cells. So to summarize, it's a very complicated scenario, but the idea is that— the general idea is that the balance between ATP and adenosine is going to dictate whether T cell is going to respond better or not to tumor.

How complex. I would just add to that, that because it's very close to my field, I would see around me in my research group that CD39, maybe the signal of CD39 is related to CD39 being correlated with tumor specificity in contrast to kind of bystander T cells. And that's maybe the signal that they're seeing is maybe is not a causality, but just a correlation in that sense.

But man, it's so complex. That's a good point because to what extent, if this is true, then that brings back something that we don't fully know about this, all these purinergic sensors and manipulators, et cetera. CD39, Pitrex7, adenosine receptors that what is the regulation of the, what is the upstream regulation of expression of this? Yeah. Is this TCR induced? Is this induced by hypoxia? I mean, a lot of people believe that expression of adenosine receptor, for example, is regulated by hypoxia. Okay. I'm not entirely sure if this is true for T cells, but this is certainly true for other cell lines. Yeah. So based on what you're saying, it would suggest the CD39 is somehow regulated by TCR, right?

Yeah, I think that is the idea. That is the— from what I understand, a little bit of the consensus, because if you look closely into populations that are expressing their specific markers, like high amounts of PD-1, CD103, CD39, I think in the last years have really kept coming up as the ones that are characterizing the T cells that are recognizing antigens on the tumor in contrast to those that were just like hanging out there and they're not tumor-specific. And I think we've discussed this in the podcast a couple of times already. So really, this seems to be like a signature of some kind. But well, you know, it's really difficult to conclude much from that.

That is a correlation.

And it can be very context-specific, right? For sure.

Yeah, I also think so.

Yeah. So this is kind of a tangent, but makes me think we're talking about these patterns. We talked also about additional ATP of the adenosine receptor, right? Well, most famous adenosine receptor, I think, or the most famous— not receptor, famous adenosine antagonist is caffeine.

Oh yeah.

And there's a lot of studies that keep coming out that like drinking 3 more cups of coffee a day or 3 more or more cups of tea a day all reduces all-cause mortality. In multiple studies.

It's just like broad-based. Yeah, I know. It's good to know because I drink a lot of coffee.

Are you trying to justify your coffee consumption, Jason?

But it's also true of tea. The UK came back and said, hold my tea and showed this. But I wonder if there's something there when we're talking about how central these very poorly understood receptors are that, like, Pounding down and antagonizing that adenosine receptor.

I, yeah, but, but, but see, see, that's the thing that is interesting for me because, okay, we have this general idea if you think about cancer that blocking adenosine receptor is good. But I always try, I'm a biologist, so I always try to think about the evolutionary reason why is this thing there? Why, for example, why do T cells evolve to express adenosine receptor? Receptor. There got to be some level of advantage. And for instance, we have done some pilot experiments. We should publish that at some point that if you knock out the adenosine receptor 2A, you actually have less tissue resident memory CD8 T cell. I mean, I have never published that. This is one of those thousands of experiments that you do as a postdoc that you never follow up. But evolutionarily speaking, it is the same for CD39. CD39 and other inhibitory receptors are expressed to some extent extent without the outside of the context of exhaustion by tissue-resident memory cells. I mean, I think Laura McKay showed that in a recent paper that you have this low-grade expression of these inhibitor receptors. So my question is, I shouldn't be asking the questions, right? But this is a rhetorical question. I mean, is this gonna be always advantageous to block an adenosine receptor? I mean, like, but I mean, if that justified coffee consumption, I'm all for it. I drink a lot of coffee.

That's my question, right? Like, this data is like some of the most robust all-cause mortality data they've ever found. Like, all-cause mortality is the holy grail of anything right now. The good news is you have huge ends of people who drink coffee and those who don't, and they drink tea and don't. It's not controlled for like soda and stuff. Maybe soda has enough bad things in it that you, it ablates anything good, right? But like coffee and tea are plant phenols, which maybe have some effect too, and caffeine. And it, both of them now seem to drastically like have real effects on everything from liver elasticity in some diseases, just straight up you don't die as much.

Yeah, yeah. But that is an interesting thought. I mean, maybe I could go back and start looking. I mean, that's one of the reasons why I made a clinic, right? I have access to a lot of patient samples. I can start trying to see if there's any correlation between overall survival of patients of every single disease and expression of these receptors or coffee consumption and God knows how.

Yeah. I mean, the coffee, the coffee consumption's pretty well nailed. It's like 3 or 4 big studies now. And the tea has one, but like what that, why, that's where I'm getting at.

Why The thing that could overcomplicate things, the caffeine probably doesn't only hit adenosine receptor, right? I mean, I could probably block many other receptors or induce many other older ones.

Yeah. I don't know how specific it is.

I haven't, I haven't looked at the Kds. Yeah. And, and actually there's a very, very anecdotal something. Yeah. Way back in the days in 2011, I was doing part of my PhD project which was on dendritic cell phagocytosis of Plasmodium parasites in Portugal. And I was doing intravital microscopy. And I was helping this PI, uh, he's not a PI, I won't make it anymore, uh, to do some intravital microscopy of the uterus of the mouse. And, uh, one of the problems we had is that mouse uterus keep moving. And now, and this PI, this researcher, he's back in Brazil now. He found out in an obscure paper that if you administer caffeine topically in the uterus, the movement slows down, the movement of the organ slows down, probably due to hitting some sort of nervous system cell. And it worked. I mean, I got the uterus is moving very slowly. So this is just something that crossed my mind right now is that caffeine must be hitting other receptors that can be present in every single organ. And, God, I mean, this is just, I love digits. So I mean, do you do that? Because now we're gonna be here for 5 hours. No, just joking. But, but, but I don't think caffeine hits only adenosine receptor. I am working with adenosine receptor and how it affects T cell responses.

So At the Immunology Podcast, we ask the hard questions. Yeah, for sure.

How does coffee save your life? I love those weird questions, man. I feel if you, if you keep asking these questions, we're gonna be here forever.

But, uh, but no, let's not do that. I wonder if anybody did— you know, Jason, uh, in Latin South America— I don't know if you, uh, Enrique, you drink cimarron, uh, but you know, in Argentina we drink all this mate.

Uh, infusion.

In Brazil, they drink a lot of it. Yeah, right. You're too far north for this.

Are you from São Paulo? Yeah, so we're more like coffee, but I have drank that before.

I've been like, uh, for those who don't know, what is this thing of which you speak? It's a type of infusion that it's made with a very similar process, similar to tea. But it's a different plant. It's a plant that is native to South America, to Paraguay.

It's called mate.

And it's very bitter. I mean, it's nice.

It's like a strong green tea.

It's an acquired taste, right? But it also has caffeine to some extent. So it's our version of coffee. Anyway, I have to say, I don't think a lot about, you know, the ATP outside the cell, mostly focusing on the one inside. It's very interesting to see how the cells are sensing their environment in that way.

And I have a feeling that this is going to be way more complicated than we're describing now, right? Because all our first papers when I was a postdoc and now as an assistant professor, we're looking at very, very contrived systems, right? LCMV infection, which is a very benign infection of the mice, or melanoma mouse models. So now here at Mayo Clinic, we're going to go a little crazier. I mean, like, we have access to patient samples, uh, we are now studying in more relevant infectious diseases such as influenza. Ironically, I have the flu right now, but, uh, uh, but I think, uh, then it's going to become a little bit more complicated and context-dependent.

I mean, we already know some weird things about it, but I mean, that sense— just one comment, uh, you also show in the case of the two— and the tissue resident cells that it's about TGF-beta signaling and how it helps the cells respond to TGF-beta, which is very important for residents. So there's a— and also, that is—

is that also mediated by the calcium influx? Ah, good question. I mean, that, that's something that we still need to look at. I mean, the most we know is that what it was published on the, the immunity paper, that Pituric7 is promoting the upregulation of TGF-beta receptor to, to some extent already in the outside of the tissue. But certainly inside of the tissue, that's going to be some additional signals. Now, whether is it calcium influx, whether is somehow the MPK activation inducing, that's something that we need to look at a little bit more closely. I mean, again, this is going to be a grant proposal that we're going to send out. I got to send out at some point, which is transcriptional regulation of Pituitary 7 and induced by Pituitary 7 because it's kind of a black box right now. I mean, like we are in that part that we know point A and point B, but we don't know much what's happening in the middle. We know a few dots, but about TGF-beta, you know, we really not sure how is Pituitary 7 inducing that.

So I guess my last question to wrap this up is do we know if extracellular vesicles carry ATP?

That's a good question. And theoretically, this is possible. I mean, like every time I present a seminar, I put that in my slides, but we haven't got the guts to study that yet. But theoretically, this is possible. I mean, like there, there are 3 ways how nucleotides can be exported actively, right? Via channels such as Pannexin 1. Via connexin channels like GJA5 or whatever, and via extracellular vesicles. We, I mean, this, this is one of those things that the limitations of being an assistant professor, right? You have to think about, you have to prioritize certain studies because you have a limited amount of people, you have a limited amount of money, and you gotta get your pub, your first papers published, your first grants submitted. So You don't get fired, right? But, but this is something that we need to study. I mean, it is theoretically possible that extracellular vesicles can export ATP. We, we will do that in the future. So keep, keep up, I'll keep you guys post updated about it. But I don't know, I don't know to what extent this is gonna be important, but theoretically it is possible.

So, all right, so there seems to be a lot of work in your future. Uh, maybe is this a good time? Are there any open positions in your lab that you would like to advertise? Are you—

any maybe listener would be interested in? Yeah, I'm always open for, for people to, to come. Uh, my lab is kind of full right now because I just hired 3 new people. I got funded recently, so it was time to expand the laboratory. But I'm always open, for example, to people that are from outside the United States and want to do a visiting PhD or visiting position. And then eventually, I mean, you know how science works, right? I mean, yeah, it's kind of filled up right now, but eventually people leave, people get jobs. I mean, like, I already have alumni weird. Someone that's in industry now. Um, so, so, so I'm always open for, for people that are interested to talk to me, but I don't have physical space to put more people right now. That's the point. So I need to be responsible about that.

But hey, congrats on a full hiring suite. That's been a struggle for a lot of people lately.

Oh, I know, I know. I, I, I'm lucky because I have a Brazilian connection. I mean, like, uh, uh, one If you are from another country and you become a PI in the United States, you realize, A, that it's almost impossible to get postdocs from the United States because not even the folks like Raf Yaman or Richard Flavell, even they are struggling. And B, you realize that you always have kind of a bond with your alma mater in your country of origin. 3 of the people that are in my lab now are from Brazil. And for them, it's easier to hire them because you have that channel of communication. So, so I got lucky on that. I mean, it's funny actually, because I was in a conference in a Gordon research conference in June and Hongbo Qi from St. Jude was a, he knows me from before, right? And he was chatting with me. I was like, did you get postdocs? It's really hard to get postdocs. It was like, oh yeah, I have this connection with some Brazilians. Like, can you please advertise in Brazil?

I'm looking for postdocs, please send them to me. Is it that bad? I mean, here in Europe we hear about all the struggles that Americans are having, American labs are having, uh, lately to get postdocs.

The biggest struggle is that the industry is being such a I shouldn't say a sink, but like a, yeah, very attractive now in hiring people from PhD. And I understand the grad students then end up going to industry, right? I mean, the pay is better. Academia is tough. I mean, like, uh, the next step from postdoc to PI is, is not easy. I mean, there's not enough PI jobs. I actually think this is going to get better in the near future, uh, for many reasons that I don't need to discuss now, but, uh, it's been really hard.

So, yeah. All right. So I think our conversation is coming to an end, but it has been super interesting chatting with you. We like to ask our guests a non-science-related question to get to know you a little bit better. So our question to you is, if you were not a scientist, what would you be?

Oh, that's a, that's a good question because it depends on the context, right? Because as a scientist, you become more and more in your field, right? So you learn more and more what you do and less and less about everything else. So if that question is relating to right now, I think I'll be homeless if I'm not a scientist because I don't think I know how to do anything else. But when I was a kid, I used to love— I used to love— I still love sports, right? I mean, my hobby is to basically watch soccer, and World Cup's coming, by the way. But the thing is that I'm usually, I'm generally not good at practicing or playing. I'm very bad at it. I mean, I think the only sport that I was really good at playing was volleyball. I was a good setter in school. But then when I asked my teacher if I had a chance to be a pro, he said, no, you're too short. That's so rough. So, so probably I'll be a sports journalist because I like to read and I like to read about sports. I mean, like, I probably memorize more stuff about sports than about science, to be honest. So, so I think I'll probably be a sports journalist. I'll be those guys that work on the, those morning sports shows, provoking each other or something like that.

That would be good. You can always ask the losing team right after they lose how they could do better to not lose next time. Yeah, that's always my favorite interview. It's like, so you just sucked, how would you not suck?

And I'm like, by sucking less. Yeah, that's a great— and that's a great answer, very, very, you know, applicable, uh, plans. So yeah, did you, did you see Ted Lasso talking about sports?

I like that. Did you see that show? That much? I saw a few episodes. It seems pretty good. I mean, like, I need to to watch a little more.

Um, that's the only sports-related show I see.

I've seen a few episodes too.

It seemed hysterical. I loved it. He's— it was such a nice, like, feel-good story, you know? I, I like that after watching a Game of Thrones, when every— whatever— where everything goes to just trash, at least that last show gives you a, you know, warmth in your heart.

Yeah, but I, I need to watch more often. I mean, I'm a little late on shows. Ain't nobody got time for that. Yeah, I was way too out of TV recently, but now we're— me and my wife are coming back.

Yeah, we need to catch up.

You'll never catch up. With that in mind, we should let you get back to the TV. All right, well, thank you very much for coming on.

Thank you. Thank you. It was a pleasure.

It was a pleasure. Bye, guys. Well, that brings us to the end of our show. Don't forget to subscribe to our newsletter at www.immunologypodcast.com. You can get the show notes, which includes an episode summary and also the links to all the interview and roundup papers as well. You can also reach out to us on Twitter @immunopodcast or via email at info@immunologypodcast.com if you have any feedback or you would like to suggest a guest.

See you next time.

Mm-hmm.