(upbeat music) - From Micro-Up TV, this is Immune. Episode number 84, recorded on August 20th, 2024. - The American Association of Immunologists, also called AAI, has a wonderful resource on several immunology topics, including vaccines. The AAI created, Immunology Explained, to help the public better understand how their immune system works, so they can feel confident in their health decisions that they make for themselves and their families. The website is immunologyexplained.ai.org, and it covers topics from allergies to autoimmune diseases, to how our immune system changes with age, to how vaccines work, how they're tested, and how they're approved, and there's much, much more. All of the information you find on that website is from Immunology Experts. The site provides information backed by science and scientists. AAI's Immunology Explained is a one-stop shop for trusted information. Visit immunologyexplained.ai.org for more information. The link is in the show notes. - I'm Vincent Drakenyllo, and you're listening to the podcast about the body's defenders against disease, joining me today from Ithaca, New York, Cindy Leifer. - Hello, welcome back. How are you doing, Vincent? - I'm okay, early morning, early ish. - Yeah. - Time for our, across the ocean guests, also joining us from Cleveland, Ohio, Steph Langel. - Hey, hi there. Great to be here. Excited for another immune episode. - From Madison, New Jersey, Brandt Barker. - Hi, great to be here. I'm really excited about all the things we're gonna be talking about today. - Our guest is from the Caroliska Institute and Imperial College London, where I believe he is at the moment, Petta Broden. Welcome back. - Thank you so much for having me. - Petta Broden. - We have five, six, eight, a couple of years ago. Let's see what year was that. It was pre-pandemic for sure with that number. October 2019. - Wow, look at that. - She's a monster. - It's just like a lifetime ago, 2019. - It does, it is, it's an era. And then I tried to see you in June this year, but we didn't make it. But here we are on immune, so that's great. Thanks for joining us. If you enjoy these programs where we discuss science of all kinds, the micro-TV is the production company, we'd love to have your support. Micro-TV is a non-profit entity in the US, therefore your donations would be US federal tax deductible. Micro-.tv/contribute. So Petta, let's, we're gonna talk about a paper of yours today. But before we do that, you give, tell us, get a little bit of your history. I know we did it on Twitter, but that was an era ago, so let's do it again. - Things have changed. - Yeah, yeah. So I'm originally Swedish, I'm from Stockholm, actually itself. I did my MD PhD program at the Kerlinsk Institute. I graduated with both PhD and MD, actually the same year, in 2011. My PhD work was on NK cells and the regulation of NK cells, particularly the missing cell hypothesis, which is famously discovered by my then supervisor, class Sherry, when he was a PhD student, actually, you wrote that hypothesis as a part of his PhD thesis, which is incredible. - Anyway, just for our audience, could you expand a little bit on that hypothesis of what you found? - So missing cell hypothesis describes the killing of cells that lack MHC molecules, MHC class one, which is paradoxical because T cells typically use that to bind their targets and attack them and kill them. NK cells in contrast kill cells that lack this self-antigen, the self-tag, if you will, MHC class one. And the inspiration, as I've heard it from class Sherry, for discovering or proposing the missing cell hypothesis, was inspired by the Cold War submarine chase that was going on in the Baltic Sea at the time. Because the Swedish military actually told the public to keep an eye out for foreign submarines, but it was really difficult to describe every possible foreign submarine and what they would look like. So what they instead, they describe the Swedish submarines and said, if you find something that doesn't look like this, you should let us know. - That's great. - And I think this is a true story, and that was the inspiration for class Sherry's missing cell hypothesis. - Wow. - I like that. - It's a great story. - Yeah. - Already editing my lecture about that. - Oh, there you go. - So we started off with your MPHD, but we're also curious. - Tell us about your childhood was science and medicine something that was an early interest, or did you grow into it? - Yeah, so I didn't grow up in an academic household. I was the first one, actually, to go to university, in my family, my family, my friends were, basically nurse assistants working in a mental institution, they actually met in a mental institution, but they were working there. And then, so I always had sort of, I always had stories of patients and clinical work, around me and both me and my sister chose a clinical path. So medicine and she's a nurse. And so that was always there. The whole care for patients and caring for people. But science, basically, the one thing that I reflected upon previously is that I've always enjoyed challenges. I've always tried, I've always enjoyed and thrived in challenges. And I met this person when I was doing my military service who told me he was this sort of hero of mine, sort of navy seals kind of person, and I looked up to him and then he said, "Now I'm done with the military, now I'm going to go and get our proper challenge." I said, "What could that be? You've been in the military doing everything." And then he said, "I'm going to be a doctor." And that sort of really sparked something in me. He said, "You will never learn everything. You will continue to be challenged and learn new things throughout your career. You can never perfect your craft." And anyway, so that really sparked something in me and I thought, "Okay, I should proceed medicine." That sounds like a real exciting area and field. And I already had that with me from before. And then science, basically, class sherry gave a lecture on end-case cells and immunology in general in my second semester of medical school. And I can't say I understood much, but I was so, I was mesmerized by this whole field. This whole really complex system. And I just wanted to learn more. So I told him, "Hey, can I just start hanging out in your lab and doing some science and learning some things?" And that's where I started second semester of medical school. And then I sort of did my PhD in parallel for the rest of my studies. - Great. And it's interesting about the military service. I'm just curious. So this is required for all Swedish men. - So it was then. - Okay. - It was required to do the initial screening. But then not everyone did the service. I did a year of military service. And now, then it sort of went away. And then now people are more and more people are doing it. As you probably know, we've now recently joined NATO. So we need to beef up our military a little bit. - Right. - And so now there's more of that in Rome, both for men and women, actually. - Sure. - Okay. - So when you worked on end-case cells, were you working on human end-case cells, or mouse end-case cells, or what organism? - It was all mouse models. All mice. And it was all sort of transgenic and congenic MHC variants that we were playing around with to see how the end-case cells would be regulated, depending on their HLA, or MHC class one background. And actually, this is the fact that I did all my studies on mouse models really inspired me to go to Mark Davis' lab, as opposed to how I could stand for it. Because in 2008, Mark Davis wrote a review for opinion paper in immunity, which there are a lot of interest and some fury, which basically said that we need to be paying more attention to human immunology. And we need to focus more on human immunology. And although the mouse has been an enormously viable tool for studying all kinds of processes, it's not an ideal tool for everything. And there are certain types of conditions and certain type of influences that are really difficult to recreate in a mouse. And that really, again, inspired something within me that I really felt I needed to explore that. So I reached out to Mark Davis and that's how I ended up doing my postdoc there. That's the answer. - Do you think that focus on mouse was simply because it was really hard at the time to do human immunology? - I think that's definitely a big part of it. But there's a lot of researchers like any people are creatures of habit, right? And you get used to doing something and it's really convenient to work with mice. If something fails, you kill another mouse and you try again. If there's much less variability, you can control them, you keep their environments stable, which is the whole purpose. And then there's the genetic diversity which you're basically taking away in most cases. So all of these things makes your experiment easier to interpret, right? And in somewhat more reproducible, but also the drawback of it is that they're less relevant to humans, obviously. And the human diversity is lost there. And also the human environment. And that was sort of my focus when I was a postdoc to try to understand the contribution, the relative contributions of environment to genetics in determining healthy humans' immune systems. - Can you say a little bit more about what that research was? - Yeah, so when I went to Mark Davis's lab, we talked a lot about what we wanted to do. And this was sort of in the early days of trying to apply systems immunology, which has now become sort of a buzzword. And people use it for different things. For me, it's about focusing on the different components of the system and how they interact and depend upon each other. So in my case, it's typically blood, blood samples, because that's what we can get most easily. And then systems immunology means capturing all of the different cells and ideally many of the proteins and other factors in the blood. And then trying to understand how these things co-vari and affect each other. So that's systems immunology, in my opinion. And we were trying to do this sort of thing in a human twin cohort study, based on the classical approach of comparing mono and di-psychotic twins, and then trying to estimate how different, how much more similar with a mono-psychotic twin pair be, as compared to a di-psychotic twin pair, which share about 50% of their genome, right? On average. And then that difference can inform you about how much of the genetic contribution is and how much the environment contributes. And so that's what we did, basically, based on thousands of measurements in the blood samples from twins. - So you mentioned a second ago that you were looking at thousands of blood measurements from the twins and you talked a little bit about doing systems immunology. And so I wondered sort of how did you transition into some of the computational things that I see showing up in your work? Was that sort of an easy thing to add on? Was that some, or how did you, how did you make that addition in your skills? - So that I'm not a trained computational biologist in the sense that I have a strong computational quantitative sort of science background, but I'm very interested in it. And I guess I've taught myself and learned from more experienced and more knowledgeable people in that area. And this started actually before I went to Stanford for my postdoc, I was starting to do programming of just some basic data analysis tasks that I needed to do instead of, in those days, this was 2010, 2011. We were often just give our data sets away to a data scientist and told them to solve the problem. And I always felt that that was the fun part. So to give away the data and then you hope to get an answer, but the fun part is digging through that data and looking at it from different angles and playing with it. So I wanted to learn that myself. And I started doing a bit of flow cytometry data and some gene arrays, which I think some of the kids listening don't even know what they are. (all laughing) - So I guess you're right. - Yeah, so we started playing with those types of data and then I explored that a lot more at Stanford. - So let's move on to this paper, which is currently in press, right? And what's the journal again, remind me? - It's nature. - Okay. - Maybe by the time this episode comes out will be published. But the title is a immune system adaptation, doing gender affirming testosterone treatment. Maybe you could, there are a lot of authors here. Maybe you could tell us what some of them are doing. - So this is a long, this has been a long process. This project was actually initially in 2016. When me and my co-senior author, Niels Landegrand, who's now in Uppsala, we had an idea that we really wanted to try to distinguish the effects of sex hormones from the effects of genes, similar to the twin study I was talking about earlier. But in this case, in their contribution to the differences seen between males and females with respect to their immune systems. And the problem has been hundreds of studies have compared men and women. And hundreds of studies have tried to do these kinds of studies in mice comparing either hormone treated, non hormone treated, et cetera. But if we stick to the human, if you compare males and females, they will be always different both from a chromosomal point of view with X and Y chromosomes and their hormonal profile. And so to distinguish those two, we thought following individuals that undergo this gender affirming hormone treatment in both directions, actually, which is presenting the first one here, would be an interesting way to try to understand how much of the immune system will be adapted when, in this case, a someone assigned female sex at birth transitions to a hormone profile that is typical for a male. Well, obviously, the genome is fixed, right? So the genetics will not change. So that was the idea. And then the other thing since then, this topic has become massively controversial in the US, in Europe, in many countries, about transcare. And so it's a very sensitive topic. And my take on that is that I think we as physicians and as researchers have not done a good enough job of caring for these individuals in a science-based fashion. We haven't considered and followed up what these hormone treatments actually do to people. And they've been sort of treated mostly, I'm generalizing, but this is generally true in my experience. They've been treated in smaller clinics or sort of outside of the university hospital systems and not always in a research facility and not always followed up. And I think this is, we owe it to these individuals to follow up and see what happens 20 years later. Do they develop cancers or do they have autoimmunity or do they have severe infections? And we really don't know. There's very little data on that. So as we went along with the study, this became another sort of really important aspect of it. I thought of exactly those types of things when I was reading this paper. And I was so glad that you had done them and I'm so glad that you mentioned them. But that also made me wonder, did you have difficulty recruiting subjects or were people excited to participate? So because of the whole social stigma around this treatment and this group of individuals, it's a really sensitive topic. So it's been incredibly difficult to recruit these individuals and not just because the individuals themselves have been unwilling to participate, but also because clinicians that care for them have been sensitive about asking to participate in studies because they are worried about their alliance and the trust, it's a massive trust issue, right? So it's been really difficult. And I'm really grateful to all the clinicians that are co-authors on this paper for their enrollment of these individuals and caring for these people. It's a really important job. So the cohort here, tell us a little bit about that and these are people who are female at birth and then had a four month area. Like at what age roughly do they have that? - Yeah, so they are between 20 and 40. So these are adults. So it's important to keep in mind that these individuals have undergone puberty and their birth sex. And that's a really important thing to keep in mind when we're thinking about the results. And I'm putting that out there right now. And anyway, they're born as assigned female sex at birth. We then treat, they've been treated with testosterone. We follow them. We have a blood sample before treatment and then a three and 12 months after the treatment has started. And we basically monitor in the blood what's going on with the immune system, how they're changing, et cetera. - And it's really great that you're able to get those before us because I'm sure that that is the most challenging part of this entire study, right? - Yeah, that's the, so in all of us, I should just comment on that in respect to human immunology in general. This approach of monitoring people over time is absolutely essential when we're doing human immunology because each and everyone becomes their own control. And as you would see in this study, we're comparing every individual to their own baseline. - Right. - And that's absolutely essential when doing these types of studies. Because the inter individual variation is so huge here. And especially this is a rather small cohort. I mean, it's the biggest one that's been reported to date, but it's still only 23 individuals in this case, it's small cohort. But with this longitudinal profile and the changes that each individual undergo, we can actually see significant adaptations that we would have never seen if we would have compared different individuals before and after, for example. - Were there specific things that you anticipated or that you were looking for? Were you just generally saying, let's just take a look and see what's there? - Well, we were obviously biased by what's been published before. Testosterone has been, if you ask 100 immunologists, so I think a few, the majority would say that testosterone is immunosuppressive. And, I mean, what does that even mean? It's obviously a very simplified term, right? There are so many different processes and some are suppressed, some are enhanced, as we find. But we were obviously, we had those glasses on when we started looking at this data, right? And then the other thing is, there are stark differences in certain infections in vaccine responses, in autoimmune disease, obviously, most not all. So we were also obviously biased by our ideas of what these processes might be, which processes might be involved in those conditions. - So you've had blood from these patients. Tell us what you did with the blood. - So as soon as we get the blood, we did a few different things. We stored viable cells for later stimulations, et cetera. - And do you do mononuclear cell isolation? Are you just getting the whole cell pellet, or how are you? - That's mononuclear cell isolation in this case. In many of our other studies, we prefer to stimulate at the time of blood draw and then stick to the whole blood as much as possible, given that that's the more physiological tissue, if you will. This wasn't possible here for logistical reasons, because these patients are cared for in many different places, et cetera. So it was mononuclear cells, we preserved plasma, as quickly as possible, after blood draw. We preserved nucleic acids in HACC gene tubes, which is one of our true, yeah, true come true, these kinds of RNA and DNA stabilizing solutions. And then we preserved the whole blood cell populations for psychometry assays. So that includes the neutrophils and the platelets, et cetera. - When you were mentioning sort of things you had expected before you did the study, one of the things that I always really impressed by were the differences or the sort of speeds of change but at time, three months versus 12 months. And so what were you expecting to see with those different time points? - Yeah, so this goes, if we're sort of jumping ahead a little bit, this was one of the reasons for our interpretation of sex hormones, particularly being a dynamic regulator of the immune system as opposed to the genetic differences between males and females, which would be fixed. And so we expected it to be dynamic but we didn't expect it to be very quick. But when we think about it nowadays, after having done the study, it makes a lot of sense because if you need to regulate something during the menstrual cycle, for example, you need to regulate things within that month. And so it makes sense to me now, in hindsight, that things are regulated that quickly. - Is a lot known about the fluctuations of sex hormones besides puberty or at menopause for women? - Well, there's the cycling, obviously, with large effects on the reproductive age. For testosterone, there's mobilization with certain stressors, such as exercise and other things that leads to quick bursts of testosterone release. And then there's pulsatile release triggered by hormones from the central nervous system. But a lot, you know, this is a super complex biology that I am by no means an expert yet. And it's a really interesting area, actually. There's also so many different forms of the androgens, just as an example. And they do different things. They bind the receptors at different affinity, they're more abundant in certain tissues than others, so it's massively complex biology here. - And, you might as well as the apologies, it's daily testosterone treatment. And what's the form? What's the, it's a tablet. - Okay, okay. Synthetic or are they extracting it from an animal? - Synthetic. - Okay. - So let's go through some of the findings. You found some interesting differences in cell populations, right? - Yeah. Yeah, so in the paper, we talk about the broad sort of swath, which is the mRNA transcriptome in whole blood without taking cells into account. And then we see these two shifts where things related to type 1 interferomes, which is a large group of genes seems to suppress, seems going down after testosterone. And then everything related to pro-inflammatory pathways, like TNF guided things, IL-6, as such, are going out. And then in keeping with that, we saw a contraction, not a massive, you know, depletion, but a significant contraction of basmoslytoid and tridic cells, which in the blood is a very, it's a small population of cells, but they have very important functions and environmental infections, such as SARS-CoV-2 infections, they're really important. So that was intriguing, and it fit with this idea that the type 1 interferon responses were suppressed. It was also really interesting, and that became our first clue here, because diseases that are much more common in women, such as lupus, is believed to at least have a very important contribution from these cells in the type 1 interferon pathway. And so that was also interesting that that was suppressed after testosterone. - And that lupus disease is perpetuated by things of my interest, which are the nucleic acid sensing TLRs. So you went and looked at TLR responses, right, in these cells, and what did you see? - Yeah, so we stimulated, again, these live cells with a TLR 7 and 8, agonist R8 for 8, to mimic the nucleic acid response, and found that that response was suppressed in the PDCs, but also in the monocytes after testosterone. And that was done by single cell RNA sequencing. So we could detect specifically which cells, what the strength of the response was in individual cells. So I think that was a very powerful finding. And then we had trends that also side to kind of secreted were showing the same trend. So I think sort of orthogonal methods pointing in that same direction. - But you didn't look at TLR 9, so no DNA responses. - Yeah, we did not look at TLR 9 in this case, and we just didn't have enough cells to do with these things, that would have been very interesting to do. - Yeah. And so so far your results are agreeing with a hypothesis that was formed from the literature that testosterone in this context is suppressing these responses are downregulating these responses. - Absolutely, and it fits with the disease prevalence and the infectious disease susceptibility, which is going in the other direction. So yeah, that was all in agreement, and very interesting to us. It wasn't entirely known before that testosterone was the mediator of that, right? - Sure. - Because there's also important chromosomal differences, the receptor I just mentioned TLR 7 is on the X chromosome. There have been discussions about gene dosage effect differences between males and females. Another layer of really complex biology between sexes. - So when you find these differences in cell populations, was it always in each donor, or did some not show it? - No, my impression is that this trend was similar across all, we didn't have any outlaw here that behaved in the completely different way. - Yeah, this was quite uniform. - And there were individuals who were taking different forms of testosterone or different levels as well, right? - There were different doses, and that was due to some of the subjects had to have doses reduced because they had no blood counts or very low VMI, and so then clinicians would adjust the doses accordingly. But that didn't really show a clear impact on the hormone levels in circulation. - There was CD4, CD8 ratios, you did not see a difference. - Yeah, no, in general, the T cell populations showed much smaller differences. And then I think that's an intriguing aspect of itself. And it goes back to this idea I mentioned earlier about these individuals having going through their whole development in their birth sex, then most of the populations of T and B cells, at least the T cells here would be mostly memory cells, they would have seen their antigen, they have been polarized in a certain environment, et cetera. And so I think that's my favorite theory about why we didn't see so much in the T cells. - Because in females versus males, the CD4 or 8 ratio is higher, correct? - Yeah, but testosterone treatment did not cause that. - No, we couldn't see any evidence for testosterone being the reason for it. It could be genetic differences, it could be developmental things if we would have looked at young individuals instead. - But you think that it has to do with these individuals having gone through puberty already, right? So you've already set that ratio. - We think so because it's very well known even since there's hundreds of years old studies showing that castrated rabbits have enlarged thymine. And there's a well-known effect of testosterone inhibiting the thymus. So if this would have happened earlier during the sort of main thymic activity, if you understand what I mean, early in life in the childhood years, that would have been maybe another effect. - So the main difference was really on the PDCs and the monocytes. - And the monocytes. - So the balance did. - Right, and TLR signaling was affected and one of the main players to connect TLR, at least seven and eight in PDCs to the interferon that you're seeing is IRF7, right? So you started looking a little bit at these mechanisms, including IRF7, can you say a little bit about how you think testosterone is influencing the function of PDCs? - Yeah, so that's a great question. IRF7 is one key regulator, which is well-known to impact the interferon response in PDCs. So we looked and saw that that's decreasing in its expression level. We can't do sort of full mechanistic work here because that would require us to knock out genes or-- - Yeah. - And we just can do that with these XVV samples from these individuals. But yes, we think that's one of the mechanisms at least, impacting IRF7 expression and effect secondarily on type-on-it-fian responses. - And there were also some effects on NK cells, or no, 'cause you started out your career starting NK cells. - Yeah, yeah. - I know you should be looking at them a little bit here. - Of course, yeah, we look at everything. And then, you know, sometimes we don't see anything and it could be, you know, for various reasons. But with the NK cells, it was intriguing because there's a sort of potentiation of certain functions, particularly the release of gamma interferon seems to be affected. And then activation of this NFKAPA-B pathway, this was the second part was found in an epigenetic analysis, looking at chromatin accessibility of these cells after testosterone. We saw that the NFKAPA-B binding sites were opening up in T cells and NK cells. So that could imply at least that that pathway is more active. But then we could show directly that the function of NK cells could be modulated, specifically the gamma interferon release by testosterone. - And then the opposite. - Oh, yeah. That was with like cisgender people, right? So that was taking their normal blood cells and then adding testosterone, based on the timing of things, like how representative do you think is that of what you're seeing in the individuals that go under go hormone treatment? - Yeah, so this is a really great point and something that we explored a little bit. We, because of the fact that we have observations in these individuals, but we don't have much sample from them. And we want to do mechanistic work. So the idea is always to try to set up an in vitro system to try to dig a little bit further, rather than going to a mouse and doing the work there. And at least that's our favorite approach whenever possible. And so here we set up an in vitro system where we could pre-treat cells from cis females or males and treat them with hormone prior to doing simulation experiments. And in this way, what we found was pretty intriguing was that if you took blood from a female during their first seven days of the menstrual cycle, when the estradiol levels are rather low before the ovulation phase, when the estradiol and progesterone increase. And then you pre-treat the blood with testosterone. You can see this effect, which we report in the paper. If you would have taken this sample, which we did a few times later in the menstrual cycle, after the ovulation phase, when the higher, when the levels of endogenous estradiol was higher, that effect was sometimes not seen. And so I do think there is a balancing effect between the different hormones, such that sometimes a female endogenous female estrogen would cancel out some of the effects that we try to induce with testosterone. But again, this is a whole complex biology and the interplay between these hormones that would require a lot more additional studies to work out. - Right, because I was thinking, is that due to the estrogen receptor already being found in those sick, that signaling cascade is already stimulated or alternatively the testosterone receptor is down-regulated during that time? - Yeah, so that leads to another really interesting type of biology here, which has to do with a hormone receptor expression, right? - Right. - And if you look that up in the literature, there's a lot of contrasting reports of what cells express the endogenous receptor, what cells express the estradiol, the two receptors. What we found using both protein assets of staining for flow cytometry and mRNA analysis was that plasma cytolidendritic cells actually express more endogenous receptor than any other cell in the blood. And they also express high levels of estradiol receptors. So you could even, you could actually think, and we speculate a little bit about this, that the PDC serve as sort of a sex hormone sensory cell and then convey its sort of regulation further. Because if you look at other cell types, the expression levels are much lower, maybe they can be induced in certain contexts, but at sort of resting state, they're very low levels. And so that's all really intriguing. And to answer your question, Steph, I don't know what molecular mechanism between the sort of cross the balance in effect is. And that's something we're trying to figure out right now. - From a technical perspective, are you, are these cells being allowed to rest for a time? And would you find that the hormones, you know, I'm just trying to think about recycling of the receptor and is there a washout period that would make those cells more responsive to the hormone? - So you mean hormone pretreatment, then rest, and then stimulation? - Or more, you're pulling them out of the person, you're letting them rest, and then you're treating them with the exhaustionous hormone. - So what we did was, because these were cisgender females, they have endogenous levels of hormones. - Right. - And then we measured their endogenous hormone levels in the culture, so in the supernatant of the culture that we set up in vitro. - I see. - With or without the supplemented testosterone. And so we could then basically calculate ratios and do things in relation to those ratios. What we saw was that the females that we use as donors had very uniform levels of endogenous hormone. And then the supplemented hormone had a relatively similar effect. But that was not always true. If they were a sample later in their menstrual cycle, just younger than me. - I do. So the key is normalizing the endogenous levels. And really that's just syncing menstrual cycle. When you're collecting it during the menstrual cycle, that's how you're able to normalize for circulating levels. - That's what we think. And these are all in females of reproductive age, right? And obviously if you're on certain hormone contraceptives, or if you're older or if you're younger, that whole thing, equation changes, right? - Yeah, that was exactly what I was thinking as I was making me wonder about immunological effects of different contraceptives or hormone replacement therapy and women of sort of older ages or things like that. So I think those are all really interesting questions that come up based on this. - No, I agree. - I was actually really excited about your sort of distinction between what was going on in the PDCs and what was going on in the monocytes. - Yeah, me too. - And the sort of cross-regulation that you talked about with the type 1 interferons and with the TNF, yes. - The TNF, yes. - Yeah, exactly. So can you tell us a little bit about what you saw there and was that something that was expected or that surprised you? - So it definitely surprised me, but when I read up on it, because it was such a, you know, sometimes we make these observations again and again and again in different contexts. You look at different data sets and you see the same thing from different angles. And that's usually because it's true. Usually that's a good thing, you know? Rather than if you see something in one type of data and then you look at another one, you see something opposite, that's worrisome. But anyway, here was one of these observations that came up again and again. So we went to the literature and was really interesting. And I actually knew some of these things, but I didn't think about it is that this cross-regulation is described all over the place. I mean, if you think at severe COVID, why do individuals have life-threatening COVID in the beginning prior to vaccines? Well, men, mostly men, had severe COVID because they mounted a, they failed to mount a strong type 1 interferon response. And instead, they had a viral replication that went out of control. And then the immune system tried to catch up with more pro-inflammatory responses, like TNF and IL-6, leading to this type of kind of storm. That cross-regulation is already there, right? So females tended to have really strong type 1 interferon responses and then have lesser TNF responses. Men on the other hand had sort of vice versa. And then in some patients that I care for, because I'm a pediatric immunologist, so we give TNF therapy to some of our patients. And we all, as physicians, know that about 10 to 15% of patients that go on TNF will develop a lupus-like disease with elevated type 1 interferons. And obviously, this is, again, this cross-regulation coming into play, right? And then there are papers from people like Virginia Pasquale, who's a professor in New York, pediatric rheumatologist, and Carolina Paluka, and others who've studied this cross-regulation in lupus and shown similar things. And so there is precedence for this. This is not something that we have just uncovered. But what's novel here is that sex hormones play a role in particularly testosterone in modulating this balance. To me, that's not been reported before. And I find that quite intriguing. And in your patients that you're giving the TNF therapy, they're pediatric patients. Yeah, both pediatrics or adults could be there. And the same has been reported for both. Okay, I was just thinking about potential differences pre-puberty. And if there are sex differences, do those, are those falling away? If it's pre-puberty or post? I don't think, yeah, that's a great point. I don't think that the clinical data that have reported this condition is granular enough to actually look at free or post-puberty or sex. But I think that's a really good point. I should dig a little deeper on that. So could I ask you, you have a cohort of cisgender individuals that you compared with the individuals who had been treated? So overall, what did you find from that comparison? So in the cis individuals, we basically pre-treated them in vitro with testosterone, right? And this was a way to try to replicate the effect. And the main reason for doing that was to be able to, number one, become a bit more mechanistic. But number two, one of our reviewers suggested a really, or brought up a really important point, which is that in vivo, when we give testosterone, the endogenous estradiol decrease, right? They stop cycling their menstrual cycle. So how can we say that the effect we see are mediated by testosterone increase and not estradiol decrease? So shout out to this reviewer. This was a very good, you know, an example of a really productive review, actually. So very good. And this was obviously really difficult for us to study in vivo in these individuals. We are currently conducting analyses in the opposite direction. So individuals that are born assigned male, sex, at birth, and then giving. But I don't want to give that away too much. We thought we could put them together, but it became impossible because they are not each others. So you promised to come back and talk about that. They are not each others mirrors. That's the short story. It's a bit more complicated than that. And it probably has to do with menstrual cycle and other things. Anyway, but so we had to set up an in vitro system to try to disentangle these effects. And that's why we did. This was the best we could come up with. The pre-treatment with hormone, with or without the blocking of a receptor for that hormone, to try to control that the effect was actually hormone mediated directly. And so with that, we could see that the induction of the NFKAPA B pathway seemed to be directly mediated by testosterone. While some of the other effects seems to be more loss of estradiol mediated. So yeah, that's the short story of that. And I think the other really interesting, applicable part of this is that testosterone is not only given in women transitioning in this cohort-- in this case, this cohort. So postmenopausal or perimenopausal hormone therapy usually includes some amount of testosterone in addition to estradiol. So I think the dose effect-- you didn't see a dose effect, although maybe there were enough people to understand that. But I didn't know if you have any thoughts about testosterone treatment in periopostmenopausal women. Yeah, I think it's a really important point again. And we are doing this way too lightly, in my opinion. We are giving these hormones left and right because people feel like they want a little bit more of energy for other reasons like that. But if you have increased someone's risk of a life-threatening infection or increased someone's risk of a cancer potentially, I don't know that. That's a serious thing. And I can give you an example from women who have a metabolic condition, PCOS, polycystical variant syndrome. These are individuals who have higher than normal levels of antigens due to metabolic perturbation. He has nothing to do with-- it doesn't only come from the ovaries. That's a common misconception. But anyway, these women have higher levels of testosterone and other antigens. They have more T and F in circulation. And when infected with SARS-CoV-2, again, early in the pandemic, they had much more severe SARS-CoV-2 infection than age and BMI matched women without PCOS. So that tells you even a subtle increase because it's rather subtle increase in testosterone can have those kinds of effects, I think, should be careful. So people-- women who are being treated with testosterone is in this study. We have enough numbers to know if they had more severe COVID. In our cohort, we couldn't see that. And it's very difficult because of vaccines coming out. And when did they actually have their primary infections, et cetera, et cetera? And there have been a few reports from-- there have been a few reports from clinical centers reporting early in the pandemic their experiences. But these studies are not great. And it's very difficult to draw any farm conditions, I think. I think the PCOS literature is more reliable in that case. I just wonder if the results of these studies and the other one you're doing, do they help formulate recommendations for individuals who are undergoing gender affirming treatments? This could happen to you so you have to be on the look at for it. Yeah, I think that's a great point. And this is also another important take home message of this study, not only that we should be cautious and be careful with what we do and follow up what happens. But I do think if these individuals should be considered, many of them, they are all young, relatively young, 20 to 40. But I do think we should be a little bit more vigilant with flu and other infections in these individuals and consider the fact that they have this form of therapy as a risk factor, I think. Because we don't really know. We have basically perturbed the normal regulation, not only of their sex hormones, but also their immune system. So they should, for example, be up to date with their vaccinations. I think that's a recommendation that these clinicians-- yeah, should bring with them. And also potentially antiviral therapy, I think. Yeah. So I find this aspect fascinating, because people who want to change their gender probably don't even think about the immunology. No. Oh, my gosh, I can do this. I can be what I want to be. But they're always consequences, right? Yeah, no, absolutely. And the immune system is-- it's really a dynamic system, right? And it's finally regulated. It can kill you in a minute as an anaphylaxis, right? But without it, we die with a simple minor infection. So it's a really potent and powerful system, but it needs to be kept in tight regulation. And I think that's the regulation we are playing with here, without really knowing what we're doing. Yeah, I think that this has some big impacts for making sure that individuals who are getting gender for immunotherapy are getting the best care across the board medically, not just thinking about endocrine care. So thinking about relative risk, would these individuals be at the same risk level as men? And so it's not like they're specifically so much higher risk that they shouldn't do this, because men are at the same-- I guess talk about our men at the same risk and this is men are a vulnerable population that we should be worried about for infectious disease. Well, it depends on what infection you're talking about. But for some, yes, I mean, just like pregnant women are a risk group for certain infections like flu. I definitely do think that we should be more generous of including people in those risks groups. But the problem is we don't always know beforehand, right? We now have a lot of discussions about monkey-- sorry, Mpox, former monkeypox, Mpox. And their children are more vulnerable, it seems. Why is that? Whenever this new infection, or not a new infection, but an infection comes up on the radar, we start asking these questions. Whether they're more or less likely to have severe infections than men, we don't have the data. I don't think anyone has the data yet. But the difference is that here we have perturbed a normal regulated immune system, normally regulated immune system. So we have played with something that was regulated in a certain way. And that's not the case for generally for men. Yeah, because evolutionarily we've evolved with our chromosomes and our hormones, right? And they're matched to provide us with some level of protection. And now you're missing with the balance between the genetics and the hormones for regulating the immune system. And what you're showing is that, yeah, there are some things that now make a female undergoing hormone therapy look more like a male. But we don't know deeply what else might be changed that we haven't seen yet. Yeah, no, I agree. Exactly. So that point that Cindy makes about the fact that all of these things have evolved together does also remind me of a section in this paper that I absolutely loved. And maybe this is because I'm in a general biology department, which is where you talk a little bit about life history theory and sort of trade-offs in different sexes and/or in different organisms in general. And that whole section of your paper is fascinating. But I was wondering if you could talk a little bit about the life history theory and kind of what types of hypotheses or ideas came from that. So life history theory is one of my absolute favorites, sort of frameworks, I would say, for understanding biology. And what's interesting for those of you who are not familiar with this concept is that we are dependent upon limited resources. And these limited resources need to be prioritized for different things. That would vary across our lifetime from a growing child to a sexually mature male or female to an older individual. And in all of these phases, there will be different priorities necessary to maximize our chances of survival and reproduction. And this is true for every species. And life history theory talks about the prioritization or allocation of limited resources towards a few different traits, where growth is one of them. Reproduction is another one. Maintenance where the immune system is included is the third one. And then just sort of think about how others have written about this way before me. There are papers talking about, in other species, how testosterone could serve as a mediator for this resource allocation. And to me, it makes intuitive sense that that's the case. We know that the immune system requires massive amounts of energy, particularly during a systemic inflammatory response, the cytokine storm that we talk about infections is incredibly costly from an energetic and resource point of view. So to prioritize that differently in different individuals makes a lot of sense. And if I have written before, for example, during the pandemic in an opinion paper and immunity, along the same lines to try to explain why children might have a mild resource co-2 infection, then an adult, is because a growing child would prioritize resources towards growth. Statutory growth is a massively important trait for a child. And probably that would mean that they would withhold some of the resources from their immune system at certain ages. And therefore might not present with a vigorous inflammatory response that would lead to symptoms, et cetera. And I think it's the same here that if what's the purpose of regulating your immune system with hormones? Well, it's because it needs to be dynamic, I think. While chromosomes are fixed, those are the evolutionary shape differences that are important. So if there's dynamic regulation, why do you need that? Well, because things change over the course of our lives from childhood to sexual maturation to the elderly. And some of the things that change could be new pathogens in sexual maturation, you could see new pathogens, like sexually transmitted infections, for example. But also, as a female, you need a menstrual cycle and you need to allow for implantation. So you need to regulate your immune system to maximize the chances of successful reproduction and implantation. And so these are the sort of along some of the lines in which we interpret this data and the dynamic regulation of immunity by hormones. I thought that stuff might also have enjoyed this. I don't know why I hadn't thought of this before. But the sentence that you have that said, evolutionary pressures from pathogens have shaped human immune systems. And the risk of vertical transmission is a selective pressure unique to females. A sort of was a little bit of a light bulb moment for me that I don't know why I never thought about the fact that vertical transmission was only a problem in females explicitly in that way before. But I was very interested in thinking about it. Yeah. And type one into fear on this is a really important factor for limiting especially viral, surgical transmission of viruses. And so to potentiate type one into theorems in a female, particularly during pregnancy, is makes a lot of sense intuitively. While some of these inflammatory responses like T&F gamma interferon have been shown to be associated with failures of implantation in IVF settings, for example, and can lead to early abortions, et cetera. So it makes sense to suppress those things to maximize in a female the chances of implantation and reproductive success. There's a really interesting field of study that I think relates a lot to this. And probably there's some literature there. But those who study animal genetics, so this would be domestic animal species that we use. We breed them for higher levels of milk production, a greater ability to produce higher levels of progeny per litter. I think are really informative for this life history theory, because one, we have a lot of numbers. We raise a lot of animals, mammals, that you can gather information from. But it's a very fine balance economically that you are making sure that the energy of the animals being used for the things you want, which related reproduction, immunity against infection. And I know that there's people who have written about this. And of course, I don't know if they have gone in the immunological molecular depths that you are. But I think it's being outside that group, I think that that's an interesting field that would cross over to what you're talking about. Fascinating to read up on that literature. So I like this discussion of the metabolic costs of the immune response. And it just makes me realize that I've always thought that immunology is exhausting. [LAUGHTER] So for all the trainees out there, when you fail exhausted during class, it's OK. We all do. It's a trade-off. It's also fun. It is fun. It's very important. Oh, that's why we have a podcast devoted to it. And again, I'm taking notes. I have my first day of class. But I'm teaching next week. You guys are giving me so many great things to say to my immunology class already. It's going to be perfect. I did have one question about unique cohorts that might inform your work. So have you looked at individuals with Androgen insensitivity syndrome? So these are individuals who are called Morse syndrome. They have a mutation in the Androgen receptor. And so they can present its female. They have female genitalia, but they have high levels of testosterone. So we are enrolling such individuals. We don't have any samples collected yet. But that's a great point. Absolutely. We're interested in that. Anything else, folks, before we say goodbye? Sorry, I didn't have another question. So curious, you were-- So the postdoc, after your postdoc, you went to care at Linska. That was your first-- can you talk about that job and then what made you move to Imperial? And you're in both-- you have a presence in both universities in that balance. So I was recruited back to Sweden. I wasn't intending to move back. I loved Stanford. I had a fantastic time there. I was learning a lot. It was an amazingly dynamic environment. But I was given an offer to start my own group. And I had two children near my wife at that time. And we thought, you know, there's no better place in the world to raise kids than Sweden and Scandinavia. And also-- Yeah, you guys got to go and stop my own lab. And then just start my own lab. And that was what I always wanted after I sort of realized that research was amazingly exciting for me. I knew I wanted to combine the two clinics and research. And so to get that opportunity, you know, it doesn't come around all the time. So I was asked to come and set up my own lab. And I took it that opportunity. And then, you know, having been at the Kalinska, that was 10 years ago. Actually, we celebrated 10 year anniversary of my lab in this spring, since I started my own lab. And then, a few years ago, I was going to say 2020 or so '21, I was approached by some colleagues at Imperial, some really amazing scientists at Imperial, who asked me if I was interested in considering setting up a lab at Imperial College and moving to London. And I think, as I go back to this idea of being challenged, this idea of not getting comfortable and trying new things, exploring new avenues. And this was intriguing to me, to see, can I continue growing, learning new things, testing a larger arena, if you will, in terms of populations of study subjects and patients and more rare conditions. And so London is amazing for that. And Imperial College is a phenomenal institution with amazing science across different disciplines, engineering, computer science, and so on. So it's a great place to be as well. So you go back and forth, right? Yeah. So tell me, which place has better food, London or Stockholm? London is probably the best food place in the world, I would say. So the whole world is in London. You can find any kind of food from a particular neighborhood in your favorite city. You could find food somewhere in London. So it's incredible. I love both places. All right. Anything else, Steph? Last chance. That was it. You're good. I could go on. Listen, you have a great body of work on pregnancy and neonatal mean responses, but that's for another podcast, I think. Yeah, still growing on, Steph. I'm not giving up on the babies. I'm not. I don't. I love that area. Good to hear. Well, you know, you can come back any time and talk about any. Thank you. We love to have you. That's immune number 84. You can find show notes at microbe.tv/immunity. If you have any questions or comments
[email protected]. And if you enjoy these conversations, please support us so we can continue microbe.tv/contribute. Our guest today from the Carolenski Institute and Imperial College. London, Patrick Broden. Thank you so much. Thank you. Cindy Leifer is at Cornell University at Cindy Leifer on X. Thank you, Cindy. Thank you. This was great. I learned a lot. Steph Langell is at Case Western Reserve University at Stephanie Langell on X. Thanks, Steph. Yeah, thank you. Thanks all. This was great. Brianne Barker is at Drew University Bioprof Barker on Blue Sky. Thank you, Brianne. Thanks so much. This was fascinating. I learned a lot. Yeah, please definitely come back. Yeah, definitely. Thank you all so much, Brianne. I'm Vincent Rackeniello. You can find me at microbe.tv. Music on Immune is by Steve Neill. Thanks for listening to Immune, the podcast that's infectious. We'll be back next month. (upbeat music)
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