Scientist Stories: Leonardo Ferreira, Engineered Immune Cell Therapies

This is episode 36 of the Immunology Podcast, Engineering Immune Cell Therapies with Leonardo Freire. Hey everyone, this is Dr. Jason Goldsmith and Dr. Brenda Rapp. Welcome back to Immunology Podcast, where 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. Leonardo Freria from the Medical University of South Carolina on the podcast to talk about his research designing and developing engineering immune cell therapies for autoimmune disease, cancer, and aging. We've also got our usual roundup of recent highlights in immunology news coming up.

But first, first, we would like to remind our listeners about Cell Therapy News, which is a free weekly newsletter brought to you by Stem Cell Science News. Summarizing the latest research, news, jobs, and events in the cell therapy research. Use Cell Therapy News to stay current with the latest of cell therapy, gene therapy, and regenerative medicine research. Subscribe at www.celltherapynews.com. How are you doing, Jason? Looking forward to our interview today?

Yeah, I'm looking forward to it.

Do you listen to other podcasts besides our very good one that you co-host?

So I listen to a good amount of Freakonomics podcasts, the main Freakonomics and No Stupid Questions, which I'm a big fan of.

Oh yes, I love those. I haven't actually listened to Freakonomics lately, but definitely one of the first podcasts I listened to actually.

And then I've dived into Make Me Smart, which is a Marketplace podcast, which I started listening during the pandemic and that's been pretty good. Those are my mainstays. I do a lot of that when I'm mowing the lawn.

Yeah, you gotta, you gotta keep yourself, uh, entertained while cutting the grass. I, I commute to, to work, like, I go with my bike, so I have also like a half an hour time to, to listen to. I really like, you know, for especially for all people that are wanting to work on their English and listen to nice— the BBC has such nice podcasts, a couple of science-related ones. I'm listening to this one now called The Bomb, uh, is from first of a series about the, like, the Manhattan Project. And, uh, but like from the view of the scientist, it was very, very interesting. And this second part is actually— it's the second season— talks about, uh, this, um, spy who was a German kind of refugee, German, uh, exile, exilee from the Nazi times, and he was a communist, and he was all the time, he was a very, he was called Karl Fuchs, and he was a very accomplished physicist. And all the time he was in the US in the middle of the Manhattan Project, and all that while he was giving all the information to the Soviets, all that time. And that's part of why the Soviets could develop their bomb so quickly, because he was literally giving them like the blueprints of what he was literally doing in Los Alamos. So fascinating to see. They have such nice podcasts at the BBC. Well, let's continue with this podcast today. So why don't you get started?

You want immunometabolism or immunometabolism?

Oh man, I'll take the immunometabolism, please.

All right. We will do— we will start with a deep dive into lactate.

Okay.

It's called Carbon Source Availability Drives Nutrient Utilization in CD8 T Cells. It's in Cell Metabolism. First author is Iram Kaimak and last author is Russell Jones. It came out on the 17th of August. And this has a couple of interesting things in it. So one, they say that we know that the media we use when we do in vitro experiments is artificial, right? It's designed to get the cells work, may not exactly be what there is physiologically. So, they have a more physiological medium that they'll use, which is instead of the IMDM, so Isakov's Modified Dibelko's medium, there's a Van Ander Institute Modified Isakov's medium, which is also a defined medium that has more proximate amounts of the polar metabolite concentrations that are in mouse serum. And then you can then add into that. So that's VIM. And then they added into that the ones that are highly abundant in mouse serum but absent from most culture media. So acetate, beta-hydroxybutyrate, citrate, lactate, and pyruvate. So they call these physiological carbon sources or PCSs. And they wanted to see basically At a super high level, how do cells behave when you actually make them physiological at baseline and then under perturbed systems using a, if I remember this right, a Listeria infection model? And so what they found is that you don't actually, when you produce new metabolites that are different, you're not having— So going from Vim, so going from The standard media, the IMDM to VIM, does induce genetic changes in your standard KEGG RNA-seq pathway search for metabolism, but adding the standard, these physiologic carbon sources doesn't, either media. So there's a difference between the two main medias. When you throw in the physiological carbon sources, it doesn't change anything. But what they find, and they do a lot of radio tracing for all of these studies, is that lactate is actually preferred during infection. So if you put these other carbon sources, is use less glucose for the citric acid cycle, and then you have increased effector function of the CD8 cells downstream, it's not changing the metabolic metabolomic transcription genes. So it's not like they're upregulating something else, but they're more efficient with this and produce downstream increased effector gene signals. And during Listeria infection, T cells preferentially in mice consume lactate over glucose. And lactate enhances the cell's abilities to produce cytokines essentially. If you nuke lactate dehydrogenase, you have worse outcomes in a Listeria infection. So this does a couple of things. One, it says your media is probably altering your experiments, which we all know but don't really do anything about. We just keep doing the same thing. And then they get into it and show through And the way they do this is a bunch of mass spec with radioactive labels on it. You essentially affect its ability to be proliferative and have downstream interferon gamma, TNF, and so on. So they call it a physiological fuel, and if you get rid of its ability to process lactate, might have worse outcomes from infection. So that's the first one. I thought that was pretty cool, especially since lactate's viewed as the devil, right? Like it's a, it's a waste metabolite that, you know, causes muscle aching. It's actually seemed to be pretty important.

Yeah. I think lactate has been gaining recognition lately for the, yeah, especially for me, me, me, metabolism people, because there's so much of this being produced. Oh, and that it all makes sense that the cells would benefit from being able to use it. Also, T. rex have been also associated with being able to consume a lot of lactate preferentially. So I guess this is another added evidence of the importance of this metabolite. I have a question. I think I might have missed it when you started. So at what point are the cells The mice are fed with this metabolite?

No, they feed— so they'll feed them different things, right? So they can feed them, they can feed them lactate to trace, they'll feed them glucose to trace. What they're seeing is that lactate is preferred over glucose. That's—

but that's what they see on the mice when they're—

and in the cells.

Yeah, and the cells.

But lactate is a preferential fuel source during infection.

Okay.

If you kill lactate dehydrogenase, thus the ability for lactate to enter the citric acid cycle to produce energy during infection, you have worse outcomes.

Very interesting. Yeah. And also I think Russell Jones' group have really nice system with, um, with the isotopes and their, their ability to really follow, uh, the, the carbon throughout the, the Krebs cycle and glycolysis. They is really, they have really, uh, worked on this. On this whole workflow. And they have— yeah, they can make really nice, really nice metabolic measurements with them. All right, moving forward from immunometabolism, before going back in your second paper, I'm going to talk about another very interesting field, which is vaccination. Not against COVID, although yay for omicron-specific vaccines. They're coming apparently. Let's see if they help. Um, this is about a vaccination for a different disease that I think unfortunately doesn't get— I don't know, doesn't get enough attention, or we're just too slow to get through with proper advances, which is malaria. Um, so this paper was published in Immunity. It's called Vaccination with a Structure-Based Stabilization Version of the Malarian Antigen PFS48/45 Elicits Ultra-Potent Transmission Blocking Antibody Responses. And first author, Brandon McLeod, from the lab of Jean-Philippe Julien at the University of Toronto. And I really like this publication. I really like everything that has to do with, you know, diseases that sometimes are ignored, or they're diseases of a lot of people, such as malaria. And I was very interested in this paper because I had also followed a little bit the development and the initial clinical trials on a different malaria vaccine, namely the RTS,SAS/01, which was, I think, was a hot topic, I think almost like 5, 7 years ago when it was, the clinical trial was being run. And it was actually a little bit of a disappointment. So this vaccine, which targets an antigen from the— what is known as the circumsporocyte protein from the sporocyte of Plasmodium falciparum, which is one of the most important for malaria. And this sporocyte is the one that kind of gets into the bloodstream from the mosquito bite, and that The idea is that you can find blocking antibodies against this particular stage of the parasite, you can prevent infection. This vaccine, the RTS, the one— not the one I want to talk about, but this other one took 30 years to develop. So it was being in development since 1987, which shows how slow some vaccine development can take, even though the need for a vaccine against malaria is enormous. So many people around the world have it, have problems with the disease, especially children. And this vaccine, so the problem with this vaccine was that phase 3 testing actually was quite disappointing. Children can only, this only works for children, only gives some protection for children. Children must be vaccinated very young between, I think, 5 to 7 months, they need to receive 4 doses of the vaccine, and the efficacy in the end, uh, was about 30— it's like 36%. Uh, so it was actually not great. Better than nothing, but not great. And also it's hard. The logistics are hard, having to get such an early, um, yeah, injections. It's not easy. And so the author is talking about, they approach this as thinking, can we target something else? Is there, if we look at people that have been infected with malaria, what kind of antibodies are they making? What are their antibodies recognizing in the malaria parasite? And so they think that they can combine, which is maybe the little immunity they can get from this other vaccine with a different vaccine that blocks transmission, that blocks that you can infect the mosquito with malaria. So the malaria keeps getting transmitted. And so for this, they're looking at different antigen. They find a— there's one particular antigen that is highly targeted in immune responses, which is present in the surface of the gametes of the parasite. And this is PFS48. 45, which is a glycophosphatidylinositol-linked protein and is basically kind of the later stage of the parasite inside your blood and then gets taken by mosquitoes. So what they look is they look at this one particular domain of this protein, which is a 6— they call 6C domain. And they want to modify this protein in a kind of a very directed manner. To make it a good protein for vaccination, make it more stable because it's a very large protein. There's issues with the stability of the protein that prevents it kind of to— for being used in a simple vaccine approach. So they look very closely into how this protein is bound by this one particular neutralizing antibody that they have identified previously, which is known as TB31F. And what they do is they find, they characterize the epitope and the surface, the residues that are stabilizing the epitope. So they're inside and outside the epitope, and they predict residues, sorry, amino acid residues outside the epitope that would improve the stability of the protein without interfering with the actual epitope at the binding of a antibody to that area. And so, you know, and this is a very nice, very best paper on structural biology. And basically they find, by changing different antibodies, they actually find particular mutations that really substantially improve the stability of the protein. They reduce the aggregation, making it a better vaccine candidate. And they test this, they test this engineered versions of this domain. They do immunization and they also introduce to a platform which makes immunogenic liposomes to improve the immune response. And they actually get very nice, very nice results compared with the wild type version of the, if they use a vaccine with a wild type version of this protein, their stabilized versions have substantially improved functionality. And even the one, the best performing, actually generates a complete blocking of transmission. And they have this assay in which they feed, they like make like a kind of a serum imitation, and they put mosquitoes And they get them like blood meals. They have like a film, like a membrane, and they have this mix of serum and blood and the parasites. And they show that they can, by having this, by using serum from mice that had been, what's the word, treated with this vaccine, they can block the transmission of the gametes that are in this blood meal to the mosquitoes almost 100%. So I think it's very interesting because it gives you like, is a different way of looking at vaccination against malaria. Given that it has been so hard to come up with a good protective vaccine so far, this vaccine might be very important in reducing community transmission, maybe. And if everyone's vaccinated, then their mosquitoes have nowhere to get contaminated from. So kudos to, to this, uh, different approach.

So how long do you think it's going to be until this gets in people?

Oh my God, I hope less than 30 years. I don't know. I mean, they don't discuss it in the paper, um, and I guess it's very early stages, um, so I really don't know. And it's so hard because in malaria, it's so hard to get. I guess this could be easier to test. I don't know. I mean, it's so hard. This, this parasite has been evading, uh, vaccination for so long.

I know. Well, it also doesn't— like, you don't develop permanent antibodies to it normally, right? Like, immunization isn't a normal mechanism, right, for managing it in people?

I, I think— I don't think so. No, they have— there's a lot of— also, they keep changing their antigens and hiding. Also, they hide inside the cells of your liver. So I don't know, it's very difficult to— they also, I think, those will hide a lot with glycoproteins. They have all these sugars like hiding epitopes.

So peekaboo.

Yeah. So let's see. Hope maybe, maybe an advance is coming, but I don't know.

All right. I guess it's time for more immunometabolism.

Bring it.

All right. So here we go. The LACC1, which I'll call LACC1, bridges NOS2 and polyamine metabolism in inflammatory macrophages. First author is Zheng Wei, last author is Jason Crawford, and co-last author is Richard Flavelle. It is a 4-author Nature paper. It's only 4. That's pretty cool. So that's neat to see to start with. So there is a protein known as Laccase Domain Containing 1, LACC1 protein, that's known to be in macrophages. And all of these other genetic studies have shown that it is a regulator, plays an important regulatory role in IBD, arthritis, clearance of microbial infection, There's all these SNP studies and other stuff, and if you knock it out, bad things happen. And no one knows what it does. They know it's in macrophages, but they don't know what it does. So this group came along and what they did is they took mouse and human. So they first, they looked at the sequences and thought that it should have a copper binding domain based on its structure. But in fact, they found when they isolated M-Lac1, mouse Lac1 versus vector protein and found that it actually had zinc, not copper. So that was surprising. They took a Lac1 knockout mouse and extract metabolites from inflammatory BMDM model cells and then figured that substrates would accumulate in these cells, right? Because they don't have the enzyme to convert them. Then they incubated it with mirin lac-1 plus zinc and then used ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spec. So UPLC-QTOF mass spec. All right. And then looked at metabolites and put it on a volcano plot to figure out full change versus false discovery rate. And the biggest hit was L-ornithine. L-ornithine itself is a metabolite of R-L-arginine metabolism. I'm just going to start saying instead of the enantiomers, I'm just going to go ornithine and arginine at this point. Otherwise, it's going to get real rough. Normally, arginine is metabolized in macrophages to nitrous oxide, NO, nitrous oxide, which is that important macrophage molecule, right? And citrulline via NOS2. But if you, in IL-4-stimulated anti-inflammatory macrophages, L-arginine is metabolized to L-ornithine and urea via arginase. Okay. So, why the heck is ornithine so important here? So, they used, they put in assays, they put L-arginine in, argininosuccinate, L-citrulline, and found that citrulline was the substrate of this LAC1. So they confirmed the stereochemistry, they did all the things, and then they looked at mutant versions of this as well that were known to have damage, that were known to be physiologically relevant, right? Like there's these disease polymorphisms that they found, so they put those into the gene as well and tried to see what happened. And they showed that they had indeed worse enzyme kinetics. They did the basic Michaelis-Menten kinetics and got the Kcat and the Km and found that the mutants functioned less well. But they found that one, which was correlated with mutations, a mutation associated with disease actually had improved catalytic efficiency due to better binding, which accelerates the timing of the response inflammatory macrophages. That's why it comes sooner than normal. And they showed if you chelated away the zinc, the enzyme stopped functioning or stopped functioning as well. So they established that this LAC1 cleaves L-citrulline, L-ornithine. They couldn't find out what the second fragment was at baseline because of the experimental conditions. It was something small and it was kind of being buffered away or immediately converted to something else. So, they did mass spec with 13C-labeled L-citrulline C1 and with heavy water and then did NMR and found that LAC1 is a novel HNCO synthase. So, it makes this radical carbon source molecule, micromolecule HNCO from L-citrulline when it makes L-citrulline and L-ornithine. So, and its conjugate base is sodium cyanate. So they did more work to show that they basically used a carbonylation assay to capture it so that you could see it show up in the system. And they did, and they found that. And then they did the mutants and showed that it worked less well. So then they're trying to figure out how this whole thing works. They stimulated cells with LPS, interferon gamma, looked at cytokines and BMDMs and basically showed that LAC-1 has anti-inflammatory role at baseline. And then, if you supplemented with L-ornithine but not the other molecule, that OCN or HOCN, that fixed the problem of the LAC-1. BMDN knockout. So if you supplemented the downstream molecule, you mostly recover the problem. They did T. ferium, S. tiferium infections and showed that the mice lost a lot more weight in the knockout. If you gave L-ornithine back orally, they did better. So you didn't have to IP it, you could just put it in their water and showed— and so then, That is interesting from a therapeutic perspective, right? Because you can eat it. And then, they showed that the enzyme's also important for polyamine synthesis by mapping out a bunch of pathways and basically show that it's part of the— So, arginine can become ornithine with urea, but this argin— this NOS, you know, arginine to citrulline, the LAC1 also moves it to ornithine. And then that goes into the polyamine synthesis, which is putrescine, spermidine, and spermine. So normally L-citrulline is derived from NOS in inflammatory macrophages. They thought that LAC1 would be dependent on NOS2 for its function because NOS2 provides the substrate, which is true. So they knocked out, they did the knockouts and crossing the LAC1. And saw that the NOS2, that the function of LAC1 was dependent on the loss of NOS2, right? There's no extra loss of function because if you don't have the enzyme, it doesn't matter if you don't have the substrate and vice versa. If you fed them the substrate, if you fed them the downstream, it worked. So long and short, they map out the whole pathway. And basically find that this enzyme has a whole new function that no one knew about that basically is an anti-inflammatory shunt from inflammatory signaling in macrophages by converting the downstream inflammatory molecule to the anti-inflammatory one.

Is it clear when you said it what induces the expression of this enzyme?

Nope. They don't go into that here. That's other work that they don't really talk about. So this has been known for a long time because this is not— I guess I don't want to say a long time. This enzyme apparently has been known about for a while, but no one knew what it did.

Okay. They saw it as something was expressed in microglia.

Or like, and if you do GWAS studies in human early onset IBD, it's a polymorphism there. And if you knock it out, the mice do badly, but no one knew what it did.

Because no one was expecting it to synthesize ornithine.

Right. It converts citrulline to ornithine.

Yeah. So they, they're both things.

Well, you have to, right? Like, you, you know, you're cleaving, L-citrulline gets cleaved to make ornithine.

Okay. So do you think, yeah, I guess it gives a different, a new way of intervening in these pathways if you—

Well, exactly. Well, so for people that have this polymorphism that's associated with disease, you could just give them ornithine.

Right. The missing ornithine can be just compensated.

And that's it. And you can just orally give that because that's absorbed. Interesting. You don't have to inject it. You just give them ornithine.

Do you think that it would be worth testing in people?

Oh, sure. I mean, this is apparently a polymorphism that's associated in human disease. So you find those people that have that genetic quirk, it's a pretty easy trial, right? And you just give them ornithine.

Okay.

Not hard to get oral or anything.

Okay. Looking forward for the follow-up or, or maybe the startup company they make to test them.

I don't even know if this will be a startup. Like this, this is like, this is one of those that's so easy to do that no drug company, like.

Yeah, no drug company would do it.

Yeah.

Okay. We'll see. So maybe, maybe a clinical trial for, for the sake of humanity. Okay, then for the sake of humanity, I will continue with my second paper of the day before it's too late. So my second paper, it's also a little bit of a, I would say, a long story, complicated. So I hope I bring it on in an understandable manner. But it is about T cells, so I think it's very interesting. Uh, the paper is called Signatures of Recent Activation Identify Circulating T Cell Compartments Containing Tumor-Specific Antigen Receptors with High Affinity, uh, published in Science Immunology. And the first authors are Anna Porcaria and Sebastian Jarosz, uh, and this is from the labs, uh, of Dirk Busch and Kilian Schrober at the Technical University of Munich. And in this paper, I think the very nice thing about this paper is the way they go about studying the importance of avidity in antitumor responses and how difficult it can be sometimes to identify tumor-specific T cells in a polyclonal population of T cells that you find in a patient with cancer. So nowadays, especially given that there's so many opportunities for us to generate transgenic T cells or trying to specifically target mutations on the tumor, for example, new antigens, it is becoming more important to understand which are the T cells from the lot that actually have an antitumoral potential. They're recognizing antigens. And I think there's also very much interest in recognizing new antigens in contrast to tumor-associated antigens that are coded in the germline, because we expect them to be better targets, specifically in comparison to some of the tumor-associated antigens that can have more side effects on target of tumor-related. So one of the questions that is not clear, because there's a lot of maybe contradicting studies and I think there's still a bit of a muddy landscape is which are the teasers that you want for taking over an immune response? Do you want the very high avidity? There's also— because there's some papers that suggest that intermediate or even low avidity might actually also give you a more long immune response. Also, are the cells in the T— if you see a tumor and you see all these T cells are infiltrating this tumor, they have markers, particularly PD-1, that is highly used to kind of identify T cells that are tumor-specific. But then if you see T cells in a tumor, a lot of them have high expression of PD-1. Are they all tumor-specific? Are they all recognizing antigens from the tumor? You see some clones that are expanded and some aren't. Is tumor T cell expansion, or the size of the clone population, correlated with the chance of this T cell actually identifying specific tumoral antigens? And there's still a lot of questions all around this area. So in this paper, they tried to reductionist approach in which they have a lot of mouse models in which they have a specific neoantigen, which is OVA, and they try to understand what exactly is the— they characterize a lot about the amount of initial T cells that you would need to mount a response. And then to identify whether you can see those T cells as tumor-specific later on in the response, and whether the avidity of different TCRs interferes or affects how these populations look in a kind of after you have a tumor model. So I think the most interesting part, they do a lot of kind of groundwork showing that they can have a model in which if you have a T cell that has a TCR that has a high avidity for your target, like OT1, you can transfer very, very few cells, as little as— they show as little as 128 OT1 T cells are sufficient to mediate tumor protection in this mice. So you already see that that's kind of similar to what you expect the naive repertoire to have of a kind of polyclonal response against one particular antigen. So I think the most interesting part of their work is that they have a library that they have previously developed in which they have different TCRs. They're all against the syncytin peptide, so the OVA peptide, and that are— have different— have characterized different avidities. So they have a group of TCRs that have a high avidity, some TCRs that have intermediate avidity, And one TCR that has low avidity, but it is specific against this peptide in the right MHC. And they compare how these different clones perform in comparison. And of course, they show that in the case of an infection, they have— so the avidity of the TCR has a positive correlation with expansion of the T cells, of protection. In this case, they have a Listeria model, and you need a lot less cells to protect the mice from the Listeria infection. So something as 800 cells were sufficient to protect the mice from Listeria with a T cell with high avidity. But in the case of a low avidity, you needed about 5,000. T cells being transferred. And when they move into a tumor model, I think this is where I really want to get to, is that they again have these different avidities. And they show that if you transfer these TCRs as a polyclonal population, you don't see differences in how these TCRs expand or populate the lymph nodes, the tumor differences are very difficult to see. The expansion in the non-draining lymph nodes, and particularly in the blood, you don't see differences when it comes to the expansion of these clones, even though you know for a fact that they have very different protective capacity and very different avidities, which shows you that maybe looking for clonal expansion is not necessarily going to give you much information. And when it comes to another marker that is very highly used, which is PD-1, they show that in the case of cells in the tumor, cells that were isolated from the tumors in this mice, they have an MC38 tumor model, they show that for the exception of maybe the low, very, very low avidity, it's really hard to differentiate T cells for their avidity of T cells based on PD-1 expression. However, if you look in the cells that are in the blood, that's where this difference becomes apparent. And then you can identify to some extent the avidity of these TCRs and how it relates to the PD-1 expression, but only in the blood. And this suggests that PD-1 could be used as a surrogate marker for TCRs with high protective capacity because they have a high avidity, but only if you look at them outside the tumor, not necessarily the ones that are inside. Tumor because they— a lot of them expressed PD-1 high in general, possibly because of a high expression of the antigen in the tumor. They also look into human data and they analyze new antigen-specific T cells from 2 patients with melanoma, which were actually samples that came from my institute. And again, they have 2 particular new antigen mutations, they have 2 patients And again, they look, try to find, compare the avidities of different TCRs that are specific against this new antigen and how they identify the avidity by transferring these TCRs to T cells and comparing their functionality. And again, they see that they identify very subtle differences in the activation profile of these different avidities, but only in the T cells from blood. If they have cells that have been activated, for example, in a culture, and because these patients were treated with tumor-infiltrating lymphocyte therapy, so these T cells were collected from the tail from the tumor and they were expanded and reinfused. But what I think was very interesting is that they could identify based on activation signatures cells before the infusion of this tumor-infiltrating lymphocyte product. They can identify high-ability TCRs based on activation signatures before treatment. I think the paper was very interesting because they did a lot of very deep work, I think, was a little bit hard to read sometimes because they did so many things trying to understand whether PD-1 works as a marker for protection. And I do think that it's very interesting to see that they used this model with a very reductionist approach and they got interesting results in mice that they could correlate with how they see in these human subjects in melanoma. And I think it adds to the idea that tumor clonal expansion is not necessarily a good measure of tumor specificity, especially if you see T cells within the tumor, but that there might be a use for this marker in T cells from blood, that they might be actually useful for therapy.

So do you think this model would be something you'd want to think about employing, like something like this when you're in like car type of work or something else where they can rapidly get the avidity?

Well, I— well, they, they do. So for comparing the avidities, I think this is very interesting. They have, uh, so Killian Scrover, one of the senior authors, has a very nicely working system for CRISPR-mediated replacement of TCR with any transgenic TCRs at physiological levels. So they knock in the TCR that they're screening, and it gets expressed at regular kind of native TCR levels, which makes it easier to compare, I think, fine-grained kind of compare this small-scale, the abilities between different TCRs. And I think that's something very interesting. That I also like to use in my own research because it's a little bit different to using just transduction. So that's how he compares, for example, the avidities. And I think this idea of using PD-1 or they also have activation signatures that they have identified for identifying actually reactive clonotypes from the blood will be really nice because at the moment we don't really have a way of identifying active reactive clonotypes from the blood. We assume that there's some activation markers that do the job, but it's really hard to pinpoint. You have so many TCRs in the blood and just looking at the ones that are expanded doesn't seem to be the answer.

Interesting. All right. Well, we will be talking to Dr. Leonardo Freri in a few moments. But before we get to that, if you want to decorate your lab, you should do so with a Nature's Protocol wall chart. It outlines the production of therapeutic CAR T cells from apheresis collection and T cell enrichment to gene modification, expansion, and delivery. Request a free copy of the wall chart at Stem Cell Technologies T Cell Therapy Resource Center by visiting www.stemcell.com/t-cell-therapy.

Welcome today to our guest. We are talking to Dr. Leonardo Ferreira. He is assistant professor of microbiology of immunology at the Medical University of South Carolina, and he's going to to be talking to us about a topic that is very dear to my heart. So all those listeners, all listeners will know because Leo, just as I, is really into Tregs, aren't you, Dr. Ferreira?

Yes, the regulatory T cell. I even wrote a love letter to them at some point last year.

So we've talked Tregs before, but you have an interesting angle on it, which I, which I call as a CAR Treg. Essentially. So high level, when I think Tregs, I think specific antigen presenting recognition on them to tamp down things. How the heck does that work with the CAR Treg given that outside of some weird stuff like celiac, we really struggle to know the antigen that is needing to be generating tolerance for, for therapy. So, so how do you get there with CAR-Tregs as a thing?

Well, I think that's the beauty, is that I think you don't need to know the cognate antigen, the causal antigen of the disease, to design a CAR-Treg. Let's say you want to protect beta cells from attacking type 1 diabetes. What do you want, or one of the things you'd want, is to redirect to drive the Tregs to go to the islets and protect them. You don't need to know what antigen, what epitope Tregs usually see. You just have to make a CAR that drives them to the right place. So part of the beauty of Tregs, you know, they have bystander suppression and infectious tolerance. And so those two properties, once they are in the right place at the right time, they get to do the right thing. So I would argue that that's some of the beauty of the CAR chemokine receptor platform for Tregs, you don't need to know what the natural antigen is. Some people will fight me on this, but that's what I think.

So you just need to bring them to the right place at the right time, and as long as they're there, they're doing their job.

This is part of what I think. So, uh, for type 1 diabetes, for example, there are still studies undergoing, like what's the composition of islets in healthy versus type 1 diabetics. At different stages of the disease, right? As the disease progresses, you have less and less islets to look at, but even then, some islets have a lot of infiltrates, a lot of T cells in them, and others don't. So, there's some mystery there. Also, why, you know, now with the advent of single-cell sequencing, you can do all this analysis to show that, you know, sometimes people, you know, they've lived with diabetes for decades and they still have some beta cells. What makes those different from the ones that died? So there's also some research on the beta cell side. How are some beta cells different from others that some survive and some don't? Is it stochastic? Is there something there? But so in general, you know, the concept of bystander suppression is that if you have the Treg in the right place, it will secrete cytokines such as IL-10, IL-35, TGF-beta, and it will also reeducate antigen-presenting cells, sometimes even kill them to really reeducate, remodel that microenvironment.

So the idea then that without the cognate antigen recognition, it's not as good, but it's good enough, essentially?

That's a good question. So I'm actually trying to see this myself. So I'm looking to do a characterization of CAR Treg signaling versus endogenous TCR C28 signaling. And I think that's because in part, what you mentioned in general, the CAR looks very different from a T cell receptor. And one reason why is because the affinity of an antibody, which is what drives CAR recognition, is very different from the affinity of a natural T cell receptor. And so there'll be differences in affinity and in dwelling time as well. I think with a CAR, a CAR T reg might spend more time bound to a target cell than a natural T reg. And so there's stuff like trogocytosis, right? Comes from Greek. Trogos means to nibble. And so you'll hear about T cells and K cells or immune cells taking pieces out of other cells. And Tregs do this, right, with CTLA-4 attaching to CD8 and CD86 from the APC, and it kind of emasculates it, really educates the dendritic cell that now it becomes— instead of being pro-inflammatory, becomes tolerance-inducing. And so I wonder if the CAR is also doing something similar. And so I don't know if a CAR Treg is better or worse at suppressing a specific immune response than a natural Treg. I think that's an open question.

I saw that some of your research, you actually saw some unexpected behavior of CAR Tregs in which they actually seen in a model of a tumor model, they seem to actually be also directly inhibiting tumor growth. Can you maybe tell us a little bit about that, and were you surprised?

Yes, and I still am. So the idea here was we wanted to have a model as simple as possible to test CAR-T reg-mediated suppression. So what cells go really well? Cancer cells. We have cancer cells, we have a C19-directed CAR, and we have C19-expressing cells. So, okay, just put C19 CAR on a Treg, see if that works. And it did. You know, the Tregs, they remain FOXP3 and Helios positive, that they proliferate, that they suppress T cells proliferation in vitro. So, you know, you can cross all the T's, dot all the I's. But then when it came to the in vivo stuff, that's when the interesting part and the surprises really came. And one of them was, you know, if you put these CAR Tregs at C19, with SNF expressing tumor cells, they actually interact quite well to the point that the CAR-Tregs now, yes, they remain Tregs, but also have heightened production of interferon gamma, TNF alpha, granzyme B, perforin. So I think it goes a bit to the previous question, which is how is the CAR— are the CAR-Tregs as good as natural Tregs, better, worse? Do they have new functions even? 'Cause I was looking, I compared two signaling domains, 28Zeta, 4MBP Zeta. These are kind of the, you know, vanilla and chocolate flavors of the CAR T-cell therapy that is FDA approved. And if you compare those two and other people as well now published, you see differences in Tregs that you don't see in effector T cells. So that's also one thing I learned the hard way, if you will, is that whatever difference you see in different flavors of CAR T cells in conventional T cells, need not apply to Tregs. You have to repeat the work in Tregs and see what happens. And so, yeah, there's some findings that were surprising indeed. And so you'd imagine that not being great because, you know, let's say I drive a CAR-Treg to the islets to protect them. The last thing I would want is for this CAR-Treg to go to that site of inflammation and actually contribute with some inflammatory cytokines. So, for example, TNF-alpha, there are drugs, medicines that target TNF-alpha for diabetes. So we know that TNF-alpha is bad news in T1D, and we have Etanercept and others precisely that block that. So you wouldn't want to inject cells that make more of that bad molecule, if you will.

So along those lines then, with, with kind of where we're at in the knowledge, CAR-T cells are out, yay, huzzah. How far behind do you think are CAR-Tregs? Is this 10 years down the line, 5 years down the line? 20 years down the line? And the manufacturer is not going to be that hard because we can kind of already do it for, for regular Tregs. We have a regulatory pathway now, or for CAR T cells rather. But like, how much more work do you think needs to be done in that translational space before we can really start getting into the clinic significantly?

I want to be optimistic and say 5 years, but I'll tell you why. I was at this Treg Summit There's really a conference called Treg Summit, by the way, that I was honored to give a talk at in May in Boston. And there's actually not only academia, but also many companies, many startups that are working in this space. And I was very surprised how far they've come. Some of them even, they make synthetic Tregs. You force expression of FOXP3 and HELIOS. And if you do gene expression analysis, they kind of look similar. They work similar. But this was a lot of it was in vitro or undisclosed. But I really liked the talk by someone who is a German scientist. You know, we leave it to Germans to be blunt and cut through the chase. He basically says something that amounts to, if this field as a whole doesn't work in the next 5 years, I think we're all out of business. Because I remember I wrote the review with my postdoctoral advisor, Jeff Bluestone and Qizhi Tong at UCSF in 2019. When I posted on Twitter, you know, very happy, very proud. And someone commented, T-Rex, forever in phase 1. And so this is the feeling in the field. And I'm giving a talk with Everett Meyer next month, a webinar, and he has data on phase 3 T-Rex for Graef-Rezo's disease. So the work is being done, but it's few and far apart. But I think over the next half decade, something has to happen. I think there's been really an inflection point with new companies like Quell, and Sonoma and TxCell. And so I want to be optimistic and say next half a decade we'll see big differences in all these different targets for transplant, for IBD as well. So I want to say 5 years, but of course it's going to be more like 20 to see the full potential. But I hope that at least in 5 years we'll see either this approach works or not, because, you know, these cells can be fragile, as you know, they become T-factor cells or they just die altogether. So that there's— and then I'll stop talking, but just very quickly for the manufacturer, yes, the path has been laid, but one, Tregs are much more rare than T conventional cells. So I've seen myself, or been, yeah, I've been seeing that some trials actually stop because time after time you can't make enough cells to inject the people with. You don't meet the target dose. So there's actually quite some challenge there in things as simple as just growing enough Tregs.

But I can imagine, as you mentioned, the issue with the manufacturer, because it is, as you said, not that easy to expand T-Rex, like functional T-Rex, to millions and millions and billions of cells that we do with conventional T-cell products. And do you think that is something that has to do— what do you think is the main issue with that kind of the stability of these T-Rex? Why can't— why do we seem to lose them? As we try to expand them?

That's a good question, because people try different things, you know, the sorting. So one difficulty is that there's no unique cell surface marker for Tregs, and that might be actually the biggest difficulty, because if you don't know what to start with, you know, people say garbage in, garbage out, and then I don't think the Treg field is like that. But the sorting part, you know, we do CD25 versus CD127, and we try to be as stringent as possible. Even at CD45RA. So then we have naive Tregs which grow way better, it's like 10 times different. I've tested this, 100 labs have tested this, so nothing new here. But any case, so we are getting better at sorting them to start with, but still, yeah, they just have these dangers and difficulties that conventional T cells don't. Usually don't worry as much about your T cells in culture stop being T cells, but Tregs, yes, they do stop being Tregs. If you do your FOXP3 versus HELIOS 2D staining, then you start seeing a population creeping out that is double negative or at least single negative. And so why is it difficult? I think it's a mix. I think Tregs naturally divide slowly, and so all these in vitro artifices stimulate them to divide fast. And so that might actually be something— maybe we are violating the very nature of Tregs, and, you know, something gives at some point. So that could be one explanation. It's a bit hand-waving, And not very hopeful, but it could be that we're just, you know, somehow changing the biology of Tregs so much in vitro. Because one thing is that sometimes you see some autoimmune diseases, for example, they find mutations that make sense that would lead to Treg defects. And so, okay, these people maybe have the same number of Tregs or frequency or both, but their Tregs, each one of them works worse. But then we put them in vitro and they work just fine because we give them so much IL-2. An i6-12 round bottom with just the right number of effector cells to suppress. And so, yeah, I don't know, I think there's some biology there, but as technology just keeps improving, you know, better media composition— it's actually a shame it's so hard and expensive to make, you know, a select medium, but there's been some progress, right, in serum-free medium and so on. So I actually have quite some hope for that, for just making better media to grow these cells.

So kind of continuing on, and you talked about the struggles industry, are you thinking of getting involved in the biotech scene at all on the side while being in academia with what's going on?

It's a good question. I mean, I'm very academic in the way I am and the way I think, but at the same time, in ology, you have to try really hard not to be translational. And so I have, uh, 7 patents now, of which 4 have been licensed, but have I ever entered the company that licensed them? No. So I don't know what they're doing with them. I hope they're doing good work. But so I don't know, I just see myself as more— I always worry that the personal level, then in industry or in biotech, you kind of have a great idea that is very applicable, a product, and then you're married to it. And so if it goes down, you go down. And then even if it goes up, you know, you become like the next, you know, Elon Musk or something. But I think in academia, I kind of like the bouncing back and forth ideas. I have 10 ideas, 9 of them fail, 1 is all right, so we keep going on that one. I kind of like that freedom. I think by the time it gets to a product, I'm already over it. I'm already on the next discovery. I like discovery phase. It's a bit like you have the guy who discovered DNA and the guy who cloned the first gene. Both extremely important, right? But very different, right? If you clone insulin, that's very important in cell. But a guy discovered DNA, I mean, yeah, that's interesting, but only was discovered how important it was, you know, we're still working on DNA, right? And so I see myself as the guy who tinkers and discovers DNA than the guy who clones the first gene and makes a billion bucks. But that's for now. Of course, I think in the end, companies are what brings products to people. It is also the ultimate lab, you know, the lab is the world at that point. And so I kind of like— so again, from these T-REX summits I went to, I like the mix of academia and industry because there's actually a lot of rigor in science and industry because like, hey, there's $50 million on the line and people's lives. And so I really liked that really low tolerance for, you know, things that are nebulous, not clear. So I kind of like that rigor. So it is important. I appreciate the work and the rigor that goes into taking things into people.

Well, there's a lot of people already working on that, so maybe you can relax and keep working on the discoveries. I mean, as you mentioned, your former advisor, Jeff Bluestone, actually moved to work to—

Yes.

Actively working on synoma therapeutics. He talked to us about it back in some episode some time ago. But it's really cool, as you say, the rigor. And I also have some kind of experience seeing how companies work, and there is something very exciting about that, like very high-paced, very focused on the one problem. And there's, I sometimes, that can be also very, I don't know, very interesting. I have a question. So going back to the science then, because I had to say I'm very interested about how, as you mentioned, How do we bring the T-rex to the right place? You also have some work on— I think you also mentioned it— using T-rex to prevent transplant rejection. So in that case, you are— we use different types of targets. What other— so maybe we can discuss a little bit about what do you think is the promise, or how good do you think this might work targeting the HLA alleles of a transplant or something like that to prevent rejection as a different— you mentioned type 1 diabetes, but I guess there's so many diseases that can be targeted that way.

Yes. I think transplant is of course a prime target for Tregs. I think it will be a case where Tregs will be perhaps not sufficient, but a nice addition to it because there's such a large frequency. Of alloreactive T cells in anyone. It seems additive too. There's some work from Megan Sykes at Columbia University and others, and so I think there, no matter how many Tregs you inject, I think you also need to deplete effector cells. And so actually Sonoma and others, they have also, you know, T cell depleting agents. And so I think it'll be a combination of depleting, you know, aggressive T cells and then providing Tregs, but that would be That's how actually I got started in my PhD. I was in HLA lab. Jack Strominger would literally crystallize HLA-A2 back in the '80s, and so HLA always follows me throughout my entire career at this point, I feel like, because he crystallized HLA-A2, I worked on HLA-G when I was in his lab, which is a non-classical molecule at the maternal-fetal interface responsible for local tolerance, and my postdoc part of my work was being involved in HLA-A2 CAR. We made this graft of HLA-2 CAR fully humanized, and then we showed in the mouse model, humanized mouse model, the ND traffics to human islets and they prevent graft-versus-host disease. And so that field of transplants is obviously a prime target, and I know that TXL has a trial on this with CAR-Tregs in the transplant. And so I think this will definitely be a good example, and I think it goes back to the very beginning of our conversation, because in transplant we know exactly what the antigens are. There's no doubt that the HLA alleles are the target to go after. And from biology, I think it's also really interesting because Tregs are HLA class 2 restricted. But if I make an HLA 2 CAR Treg and actually I do a knock-in, so I replace TCR with a CAR, now I have an HLA class 1 restricted Treg, but it's MHC independent at the same time. But so I think it's just There's also some poetry there almost. I always like to think of synthetic immunology not just knowing the rules very well, but also bending them. So now we have T cells with B cell molecules expressed on their surface with a CAR. And now CD4 T cells are seeing MHC class I through some other receptor. And maybe we can do a payload that is usually found in macrophages. I really like that mix and match. Just to get into the color palette and try new things with the immune cells. That's what I like about the immune system in general. It's very programmable. It's everywhere in your body. And so I think those are two powerful properties that really made me fall in love with the immune system early on in my PhD.

Indeed. I also think I agree with the synthetic immunology also is very interesting because information, I think in a way It's so important to the information. It has such an important role. And then by changing information, one of the things you can really change with synthetic immunology, you get different signalings. If you can generate different signalings that give the cells particular signals, then you just change their whole behavior in a very predictable manner. It's very interesting. What do you think, Jason? I see you very—

Well, this is why the Treg is my second favorite immune cell.

Oh, is it? Is it? First time I hear this.

We've never asked past the first ever before. We never got in the rank-ordered list discussion.

Well, what's the third? Sorry, let's just get this over with.

The third would probably be a cytotoxic T cell.

Okay.

And then a fourth is the ILC-3.

Yeah, I guess cytotoxic T cells, they do get some merit.

They're kind of important.

Kind of.

You can't take the infantry and like say they're not important and not have them in a top 5 list.

Otherwise, come on, it'd be a very sad army.

It would be, right? I got all these cool specialized soldiers, no front line. Uh-oh.

Well, you always had like neutrophils, they go and kill themselves for the, for the cause.

I don't know, neutrophils are on the bottom of the list because I can't culture them worth crap. They always die whenever you look at them.

But that's what they do.

I know.

That's the whole point.

They die when you extract them, they die when you look at them, they die on Wednesdays more than Tuesdays, apparently.

Can we ask you, Leonardo, do you have a top 3 favorite, favorite immune cells you would like to share?

Uh, sure. I mean, I think the T-reg would have to go number 1. Fair enough. Otherwise, I really like the natural killer cell. It acts like a rheostat. There's always a balance of how much activation, how much inhibition in the KIR. Are they important or not? I used to work with a German postdoc that he said, I just want to knock out all the KIRs and prove that they're not important. That was a project in the back of our minds. Just knock out the entire KIR locus and figure out whether it's important or not. NK cells will come in at number 2. Also, at the maternal-fetal interface, natural killer cells are actually the most abundant. Lymphocytes. It's an inverted ratio from peripheral blood. And HLAG inhibits NK cells. It all kind of comes together nicely. And then the number 3, maybe— yeah, I think the cytotoxic T cell will come number 3 because it's just kind of the— I think it's just beautiful. These T cells, they make the immune synapse and they release their cytotoxic granules. I think it's just a cool function. That there is a cell whose function is to kill other cells. I don't know if there's any other. I guess the macrophage also engulfs, but like a cell that is a killer cell, right?

It's got some lasers that shoot out from it. Doesn't just eat things, it blows them up. Yeah. Yeah. Little missiles. What are your other two, Brenda? We know yours are T cells. T cells. Yeah. What are your top?

I read. Hmm. Well, I have to say, I also do like the NK cells very much. I haven't worked with them almost at all in my life, but they always seem to pop up in the most unexpected places. And they can do things differently. They do their own thing. And I am very kind of preaching, or I really like TCR specificity and tumor recognition. But then NK cells, they use a completely different set of signals to work. And I do respect that. I think it makes the system more redundant. I like it. So I would also go for NKs second. And then my third, my third cell, I think it has to be the macrophage. That's great. You know, they're just there taking care of everyone's leftover. You know, they are the ones that show up after everything else is done. And I respect that. So I guess that would be my third.

And they were the first immune cell, right?

Was— was— also indeed, they're even in sponges. So clearly They clearly have a long line, a very lustrous line of existence. So respect, respect to the phagocytes. Um, I wanted to ask you now that also— but I was very interested— you, you know, during your postdoc, you're also very active in kind of community outreach, and you had a program for, uh, teaching science in Latin America. I think—

is that—

am I correct?

Yes, Clubes de Ciencia Bolivia, Science Clubs Bolivia. So the movement itself started in Mexico and then it really spread like wildfire. So Mexico, Colombia, Bolivia. So Mohamed Mostajo was my classmate, was executive director for the Bolivian one. He's from Bolivia. And so I guess looking back, it was really great time and experience because it was— we're all classmates. And so some from Mexico, some from Bolivia, some from Colombia. And so on. And so it really went to almost 10 countries total, and it's really something I always looked forward to in January, because every January for 5 years, I would fly out of the United States— first year Boston, then 4 years San Francisco—

July—

January 1st, I would fly out down to Miami, and then Bolivia, there's no direct flight, and we'd spend there almost 2 weeks, and we went up to 400 students. So these are late high school, early college students. So up to 400 of those, and we had 17 courses. I was instructor of one of them, and the president of Bolivia herself came to the closing ceremony in the one in 2020. So it's actually hard to believe, but January 2020, before the whole lockdown happened, there were 500 of us, 500 people in the same building taking a group photo and doing all these, you know, team building activities and classes and lab. It's pretty intensive because it's only a week. So the morning is lecture and the afternoon is lab. So in my lab was— I call it rewriting life. It was about CRISPR-Cas9, also some immune cell engineering. And, you know, we got everybody's DNA, ran a PCR, ran a restriction digest with a CRISPR kit from Josiah Zayner from the Oden. He now moved to Austin. He used to live in San Francisco, so I knew him personally. Where, you know, can do CRISPR to edit antibiotic resistance gene in bacteria. So you can see very, you know, black and white results. So, you know, in one week we kind of did it all: PCR, DNA, CRISPR. So it was a really great experience. I think it's important because they say in Latin, right, that by teaching you learn. And for me, that couldn't be more true. I think the questions that these kids, I guess, ask you, they're very different from questions that reviewers ask you, that people at conferences ask you, that your advisor or now, you know, other colleagues ask you. So I think it's important. To keep that passion for science alive and being always open to new and fresh questions. So it's really great experience, those 5 years.

So are you able to do more of that now that the pandemic is winding down?

That's a good question. So I know that with the, with the, with the Bolivian version, it hasn't happened again. Uh, with the Mexican one, I know they did some online version of it, but I don't think it's the same. I mean, I hate to be this old person, but it has to be in person. We literally— the whole slogan of the thing is that we are flying scientists from, you know, Harvard, UCSF, you name it, down to Santa Cruz de la Sierra, Bolivia. They're going to teach you for a week. We had, you know, 2,000 applicants. We picked 300 and then more as we got more funding and more space. I think that that was a big appeal of it. I mean, maybe I'm self-serving, but the big thing was that they're all together for a week And you see all the scientists, they're people just like us, you know. They have interests, they, you know, they walk around us, they teach, also have fun, they do science, they have struggles, a lot of struggle. And so I think that in-person component is hard to capture if we're doing online only.

Yeah, that's very interesting. I, I— who, how did you guys fund this? Did you get funding from Who helped you like plan and finance all these trips and all these activities?

Yeah, it was a mix. And really my friend Mo, Mo Saho, he's really a genius at this. I mean, we got grants from the State Department, we got help from local banks and brands. I remember there'll be booths like Chocolikes and like local chocolate drink company and so on. So yes, we got funding through really reaching out to as many partners as possible. Also some government funding. We even had a session that I wasn't part of. I'm not an American citizen. There's a session in Washington, D.C. that was a mix of American citizens and Bolivian descendants, American citizens in D.C. And so, yeah, the program— and they also had the, whether hackathon actually or a version of it. So that was— I was also, again, not part of it. So it really was branching out. And then every— during the week, at the end of the week, with a series of seminars that were open to the public. So not only the high school and college students that were selected benefited from it, but also the local community could attend the talks. So those, I'll give one, and I think it was 10 talks total about different topics I work. So it was a program that became over half a decade, no small amount of time, but it got very mature. So it was really a shame that the pandemic kind of put some cold water on it, and then, you know, next thing you know, I moved, Mo moved, another Omar also moved, so he moved to New York City. Mo moved to Santa Cruz, I moved to MUSC, and so kind of the original 7, I think there are 7 of us at the beginning, we've all moved on to different jobs in different locations. But it was excellent programming, it should definitely be repeated. Let's see how this whole traveling— I think now the restrictions are over, but now there's monkeypox, and so I don't know if that's going to be a thing as well.

Just never ends. Oh my God, just never ends.

No, no, no.

You heard, you heard it here first, listeners. Monkeypox is gonna—

doesn't transmit that well. Stop.

All right, sorry, we live in denial. We just, we just don't want to know.

We're gonna have polio in the US.

Oh yes, yeah, only because apparently so. I, I was vaccinated for, uh, polio And so one of my lab members was not born in the US, but two of my other lab members were born in the US. And I know they're born in 2000s. I don't even know anymore. I don't keep track, but they don't have it. Right. So they were scared that there were cases in New York because they don't have the vaccine for it.

So, uh, I mean, I was vaccinated for polio in the '80s. Yeah.

That was ancient history.

Yeah. Yeah. I know I'm getting old. All right.

Well, that depends by country. I mean, my lab member got vaccinated, he's 21 years old, but he was not born here, right? So he got the vaccine elsewhere. All right.

Well, we're running out of time and I'm getting depressed and apparently I'm old. So we're going to jump to the, uh, the fun question at the end and avoid discussing my age. So the question for you is, what is the best piece of advice you've ever gotten? It can be professional, it can be scientific, it can be unrelated to science or your profession or anything, but the best piece of advice you've ever gotten that you want to pass on.

Oof, that's a hard one. Can I give— can I say two? Sure. I'll be brief. So one of them was from my PhD advisor, Jack Strominger. He turned 97 years old August 7th, this past August 7th, it was a Sunday. He still runs a lab, and he started this lab when he was 26 years old because he went to college during World War II and med school, so everything was accelerated. He was at the NIH, had his own lab, worked on whatever he wants. And so anyway, from someone who had a lab for I know, for 60 years. What he always told me is like, is it fun? Only do it if it's fun. And now, I think what he meant is that if you're not passionate about it, you know, the years will just go by and you're just living miserable and you don't feel satisfied because science is so hard. That's why I tell everyone who joins my lab, science is very hard. Also, science is a team sport macroscopically, but individually, macroscopically, it's one person has to keep track of things and drive the project. So anyway, so I think this whole like, only do it if it's fun, I think that was really my favorite advice. And the other one I was going to say is in the same line. Someone told me, you know, don't just survive, also thrive. So if you're just surviving just to buy yourself another survival cycle, then it's not worth it. Then step back and reassess. That is from my good friend Iosef Gersten up in Boston. And so I think those two, only do if it's fun and don't just survive, thrive, those are the best advice I've gotten, which sometimes we're so down in the weeds, we're stressed, deadlines, things to do. Yeah, that's important, you know, get the job done, everybody's got to survive, but also don't forget to— and I think that's why teaching is important because then in front of these people, they're like, okay, what do I do again? You know, what's my very broad job description? Why do I wake up every day? Where am I going to go next? That does not involve how many R01s I'm going to submit, how many papers I have in submission. It's It's much bigger than that. And so I kind of like those pieces of advice to just, you know, just only do things that are fun.

I think that's great advice that definitely gives, gives you the energy to keep at it. You need to take it from somewhere.

Indeed. Are you thriving, Brenda?

I am. I mean, right now I'm a little bit tired. It's kind of late under this, in this time zone, but I do try. I think it's important, but as you said, it's very easy to get kind of distracted by all the immediate things and all the things that need to be done. And all the— and academia nowadays, as many people point out, can be a little bit of a hamster wheel. That it really is, can be tough to— there's not a lot of space to stop the wheel to take a moment sometimes, I feel, as a young investigator myself. And, uh, but well, I think you are completely right. In the end, you have to enjoy it and you need to find something that helps you thrive. I think we're all smart people and we should use that smart in a way.

Well, on that point, thank you very much for coming on and talking to us about Brenda's favorite T-cell.

Thank you so much. It's been great. And I wish you a lot of success with your lab. So you've been assistant professor for over a year now. And how is it? How do you feel? Any short advice for other young PIs?

I think the team is very important. Like, I've been lucky that MUIC has many programs in place, so there's a PhD program, but also, you know, medical students get some time off to do research and even undergraduates. And so I've been lucky that people that I've come in contact with, they are pre-selected. And for me, as I, you know, because no one knows who I am, let's be honest, as a, as a PI anyway, and so for me to hire someone more senior or like out of the blue, I personally, I'm a bit scared. But if it's someone who like reaches out to me, hi, I'm so-and-so, I'm a first-year PhD student, could I rotate in your lab? Or like, oh, I'm so-and-so, I need to do my senior thesis. So I had one of my lab members who is now a technician, he did his college thesis with me and he was actually excellent. He worked in labs before. And so that would be one thing, really use and abuse your network and just previous, you know, validation, recommendation. You know, again, I think PhD programs They're great because these people, if they made it past the, you know, the exams and the application interviews, you know, they really— and then when they join your lab, they're stuck with you, quote unquote, for 4 or 5 years. And so that would be maybe my advice is like really try to surround yourself with people that are invested, have skin in the game. For example, if it's a technician or a shorter, that's okay. Do they want to go to medical school or PhD? Good, because they will want to do well to get a letter from you and to learn from you before their next step. So always pick people with ambition because the PI always has ambition. That's why they start a lab and like in the hamster wheel, as you mentioned, but also make sure your people also have ambition on their own. It shouldn't be just you telling them what to do or do this because I want it to. They also want to have to want it. So I think I'm from the line of thought that being dedicated and passionate comes from inside. It can be helped, right? It can be helped to flourish, it can be maximized, but it has to be there already, that seed. And so that'll be my maybe very broad advice. But when you surround yourself, also at the beginning, every lab member counts, 'cause they have limited money, limited time. So again, I think really pick out the most passionate and invested people you can get through connections, through programs, whatever it is, yeah.

People, people, people, people, people. Number one rule of any, any group: people.

Yeah, for sure. So, people, thank you so much for today, for our conversation. It's been great having you on the show, and we're looking forward to seeing your lab's research published. Have a great afternoon, in your case.

Thank you for having me.

That brings us to the end of our show. Don't forget to subscribe to our newsletter www.immunologypodcast.com to get the show notes, including an episode summary and link to all the interview and roundup papers. You can also reach out to us on Twitter @immunopodcast or via email at info@immunologypodcast.com with feedback or to suggest guests. See you next time. Mm-hmm.