Scientist Stories: Laura Mackay, Memory T-Cell Responses

This is episode 32 of the Immunology Podcast, Memory T Cell Responses with Dr. Laura Mackey. Hey everyone, this is Dr. Jason Goldsmith and Dr. Brenda Raud. Welcome back to Immunology Podcast. We have conversations with immunologists. The 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. Laura Mackay on from the Peter Doherty Institute for Infection and Immunity at the University of Melbourne to talk about her research on memory T cell responses. We've also got our usual roundup of recent highlights in immunology news coming up, but first—

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All right, how are you, Brenda?

Pretty good, pretty good. I have not had to prepare cells in a while, so I guess my life is easier.

How'd you swing that one?

I've been thinking a little bit more and doing less experiments, but that's going to end soon.

Back to working then and less thinking?

Yeah, yeah. I mean, that's how it is, you know, mindlessly pipetting predetermined experiments. It's kind of something therapeutic about it. You just like, You don't know what you're doing, but it's just like, this is what Brenda of 3 days ago thought it was necessary. And I'm just going to follow her lead. And it's great.

But if you weren't going to be stuck pipetting all the time, Brenda, what would you actually do with your life? Got to ask. We always ask our guests this.

Probably something outdoors. You know, I come from, I grew up in the mountains in Argentina. My dad for a long time was like a guide, like a tourist guide. He would go on hikes and fishing trips. These things with tourists. And I always thought it was pretty cool. So, you know, just do like hikes and like be a, yeah, like a nature guide in the mountains. What would you do, Jason, if you weren't an MD-PhD?

If I threw out all the degrees in science, I would be a board game and pen and paper role-playing game designer. Okay, which I already am, but that's a side gig.

Oh well, that's, that's cheating. You cannot do— you cannot choose something you're already kind of doing.

But I do it like just a little bit for funsies. Okay, well, well, paper time. All right, this one is called The Metastatic Spread of Breast Cancer Accelerates During Sleep in Nature, published the 22nd of June. So very high-level parts of this paper. When you're sleeping, your cancer metastasizes more.

Wow.

And not only do your primary tumors shed more metastatic cells, those cells, if you take them out and shove them in later, or take cells shoved out during the daytime, doesn't matter, right? At night, whatever, whatever cancer cells you have, whether they're daytime or nighttime cells, they grow more and seed better during your rest cycle as well.

That is evil. That is such an evil thing.

So sleep causes cancer? Not exactly, uh, but, but what they find is melatonin, testosterone, and glucocorticoids, those signaling pathways not generated by the tumor but your innate pathways, right, for your circadian rhythm. Those hormones influence the cancer cells, and because of the spikes in those hormones, right, and the timing, the net aggregate effect is that at nighttime you shed more of your cancer out to do things. And they, and they do this in mouse models. They have human patient data from people in the hospital. They recapitulate multiple mouse cancer models. People pick it. It's reproducible. It's a really slick paper.

So is there any specific immunological component here? Because, for example, glucocorticoids can be, you know, immune depressing.

It's, it's too fast. It seems to cause cell cycle changes.

Okay.

What's the immunology angle? There are effects on immune surveillance. Um, because immune surveillance changes with circadian time too.

Well, you know what they say, there is no rest for the wicked. So it's not a consequence of sleeping, it's just the same signals that induce the same—

the signals that induce sleep and induce cell cycle change. You tend to wound heal at night, right, when you're resting, and turning on all those pathways for multiplication of cells makes them more cancer—

more—

makes the cancer go to happy land. So while you're asleep, the cancer's at play in your body.

Great. Another thing to be scared of at night. Anything else to add to this scary, disturbing—

No, that's enough. That's enough nightmare fuel for now.

Yeah, thanks. Thanks. Well, you know what? As we are on the cancer topic, I'm going to bring like a more positive cancer story because I feel like this This is a little bit— the kind of the end of this conversation is a little bit dissatisfying. So in this paper titled Harnessing Anticytomegalovirus Immunity for Local Immunotherapy Against Solid Tumors, published in the Proceedings of the National Academy of Sciences, PNAS, on the 24th of June, first author Nicholas Shuburu from the lab of John Schiller at the NIH. And basically in this work, what they look into is whether preexisting T cell responses against viruses can be harnessed to modify, to change the tumor microenvironment, and maybe that help break kind of immune inhibition in the tumor microenvironment. And they make the point that there's a particular virus, CMV, cytomegalovirus, that is very highly prevalent. So most people go around having asymptomatic infections of this virus. And the infection results in a kind of a very large and very prevalent and durable CD4 and CD8 T cell responses that often inflate with age. So the older the person is, usually the higher the percentage of CMV-specific T cells you have in your repertoire. And these numbers can go up to 10% of circulating T cells. So they are very prevalent. And we know that tumors can be infiltrated by CMV-specific T cells that will be stained by tetramers, again, that are loaded with particularly immunogenic peptides. So they have a model of mouse. They have a mouse CMV. That is similar to the human and is used widely as a model for human CMV infection. And they have specific epitopes that are known to be immunogenic. And they say, look into— well, they have mouse. If we infect these mice, you can evaluate particular epitopes derived from EA3 proteins, M45. Those are known to be highly immunogenic. And there's one particular difference between IE3 and M45. Of immunity is that in the case of IE3, it shows to be more inflationary. So the percentage of T cells specific against this antigen increase in time. And this is not the case for every antigen. For example, not the case for M45. So what they do basically is they have a tumor model, a TC1 tumor model that expresses human papillomavirus type 16, so HPV16. And it has some of the oncogenes and also has a mutated HRAS. But so it has E6 and E7 oncogenes that are kind of neoepitopes that can be recognized by an endogenous immune response. And what they say is basically that in these mice, if they establish a subcutaneous tumor, if they inject in this tumor MHC I restricted or MHC II restricted peptides, And on top of that, so they— and they also show that— so they have these peptides, and they also do experiments where they do adjuvant. They put polyIC to induce cytokines, in particular type 1 interferons. These can really result in a high activation, in immune activation in the tumor, necrosis of tumor material in the case of MHC I type restricted. MHC I restricted peptides, they can really end up with tumors that have large necrotic tissues. And in the case of MHC II peptides, although they see infiltration, they don't see such a strong necrosis of the tumor. But in any case, they do show that by activating the resident CMV-specific cells, they can modify the tumor microenvironment in a way that reduces tumor growth, in many cases results in mice surviving the tumor and regressing the tumor completely, and that the addition of polyIC really improves this effect. But it can be accompanied by toxicity that is just not, well, surprising given that you're going to have CMV resident cells all over the mouse. I thought it was very interesting because this gives you the idea that if you could treat patients with this kind of generic peptides and you can already break tumor microenvironment inhibition using this, it would be really good. And they do show that if you have the mice that are treated with these peptides and polyIC, you can end up inducing for example, responses against the antigens that are specific to the tumor, in this case E7. So they show that you're going to have activation and expansion of E7-specific T cells, CD8 T cells in mice that are treated with a combination— particularly good when you have a combination of MHC class II restricted and MHC class I restricted peptides. And the combination really works the best. So in general, I think there's a lot of details that they look at, but I think that in general it's a really nice approach that maybe has potential for therapy. They also have a little bit of a test with other tumor models. It doesn't work as well in, for example, a B16 melanoma model, but they do have another MC38 model, which is a colorectal tumor model that they also reduce the growth of the subcutaneous tumors in these mice.

And as a cancer nerd, you think the models are pretty relevant for what we're going after here, pretty translatable?

Well, I like the one from the first one, this, the TC-1 tumor model. This is with a human papillomavirus. I mean, you know, in all cases, these models are— they're injected subcutaneously and they grow very fast. So yeah, there's always the question, how much is this relevant to the human situation and to the real tumor that would spontaneously arise? And I think BCC melanoma is kind of famous for being really non-immunogenic despite having a high tumor mutational burden and for being kind of— MC38 is also kind of this weird colorectal tumor that is injected subcutaneously. So I think that's a question that is not— this, this paper does not really dive into.

All right, well, time to shift gears a little. I don't have a good segue here. I guess repair regeneration cancer, I don't know. Anyway, it's called the gut metabolite indole-3-propanoate promotes nerve regeneration and repair in nature, first off of this Author is Elizabeth Surger. Last author is Simone Di Giovanni in Nature, also published the 22nd of June. So I was really excited about this paper because it combines all the awesome things. It combines the microbiome and wound healing and a little bit of immunology, mostly through neutrophil chemotaxis, and it combines everything I love in life. So If you intermittently fast, that's known to induce repair states, right? Because, you know, creatures evolved not to have constant calories, although we're bad at that now in society. It's long and short, if you take mice, intermittently fast them one day on, one day off, they do better regenerating from sciatic nerve injury without hyperalgesia. So they heal but don't get oversensitized. Pretty cool. They find that this is due to a gut metabolite, IPA, or indole-3-propanoic acid. And they do this through some combination of metabolome analysis, looking at RNA-seq data to see what genes would be regulated by this. So they looked at the serum first, found genes that thought, you know, found metabolites that they thought could be differentially expressed in the two states, you know, fasting, not fasting, saw this, knew it was a bacterial metabolite, then went after the bacteria with bacterial depletion, found that it's mostly driven by Clostridium sporangens, and that's required for it. If you get rid of the microbiome through antibiotics, you lose the effect. If you make— if you FMT them with a variant strain that doesn't produce this, that loses the enzyme to make this, so on and so forth. You know, you lose it and you lose the protection, right? They knock it all down. Then they show that this metabolite is really important for neutrophil chemotaxis. So, or, or so that this, this pathway, this IPA-dependent regenerative pathway requires neutrophil chemotaxis because if you blockade that through antibodies, you lose the regenerative function. So properly tuned increased neutrophil chemotaxis due to gut metabolites driven by an intermittent fasting state enhances wound repair and nerves.

Why, why do these bacteria like the fasting?

Well, presumably because there's not other food in their intestinal tract that they can use instead, so they shunt to this.

Okay.

So they're also a little bit hungry, and instead of dying, they shift to make this downstream metabolite.

Can you get the metabolite on its own?

You can give people this and— or mice this, and it causes the same effect.

Okay.

Yeah. So they can exogenously introduce it and it works.

I mean, that can be therapeutic.

Yeah, right? Yeah.

Is that the point that they're making in the, in the paper?

Uh, kinda.

It's so cool. I mean, well, yeah, I, I'm not surprised that the story—

like, you like the story because it has microbiome, it has neural signaling, which I used to do a little bit of opioid stuff. It has immunology, neutrophil chemotaxis. It has regeneration, which is also my previous life. It has all the things.

Nice.

Well, it completed my Maslow's hierarchy of science needs.

Anything else to add to this beautiful work?

I'm good.

Okay, okay, then I'll take over because I think you're also going to like this. You as a middle-aged man, um, reminds me— and that's why I dedicate this paper to my father, because when he— when, you know, being a scientist, he's told me there's two things you need to take care of in this world. You need to get rid of mosquitoes and you need to prevent hair loss. He was very, you know, he was very focused on the important things. And the mosquitoes are not my thing, but apparently you can help with hair loss using immunology. So let me, let me tell you more about it. The paper is called Glucocorticoid Signaling and Regulatory T Cells Cooperate to Maintain the Hair Follicle Stem Cell Niche. Published in Nature Immunology, first author Shi Liu from the lab of Ye Seng at Salk Institute in La Jolla, California. And basically, uh, yeah, it's— I think it's a really cool story connecting T. rex and very specific homeostasis, um, functions of T. rex and how important they are for little things such as hair repair, hair regeneration. So, the authors start with showing that there's a very high expression of one particular— of the glucocorticoid receptor. The gene is— well, glucocorticoid receptor, and the gene is NR3C1. And basically, this glucocorticoid receptor binds to its ligand, which are these steroid hormones that are produced mostly in the adrenal gland. And in response to stress. So probably a lot of us have them in our blood. But it's also apparently synthesized locally by tissues, particularly including skin, particularly as a response to some kind of insult, some kind of damage. And so there's one particular known glucocorticoid receptor agonist, which is dexamethasone, which is known to be an immune suppressor. And they— so they show that skin resident Tregs have a high expression of this, of GR, of this glucocorticoid. I'm going to just say GR is too long. And when they— and they also know that Tregs are important for the growth of hair after depilation with, for example, they use this cream, this depilating creams like you can buy at the pharmacy. And so they kind of look like at the connection between these two things. And they show that if they depilate mice with this cream, what they see, they see a local increase in the glucocorticoids in the first 48 hours. And then usually mice normally, they regrow the hair within 2 weeks. It's back to normal. So given the fact that there's this particular T. rex under these circumstances, they have this high expression of GR, they generate T. rex-specific GR knockout mice. So they're basically, they just don't express the GR only on FOXP3-positive cells. And when they test then depilating these mice, they have a very severely impaired regrowth of the hair compared to wild type. And, you know, when they look into, they do, you know, microscopy stains on the follicles and they really show that the follicles don't grow, they don't change the morphology, they just stay there quiescent. They don't, activate in order to generate new hair. So these stem cells that are part of the follicle, they don't start proliferating like normally. They measure with Ki-67, with EDU stain, and they show that basically they stay quiescent. And so they try to think what is then about GR on T. rex specifically that seems so important. So they look into RNA-seq and they compare T. rex from wild type and knockout cells after depilation. And they zeroed in particularly a particular pair, which is TGF-beta 3 and the TGF-beta receptor. So on T. rex and on the hair and the follicle and the cells from the hair follicle. And they show that there is this elevated expression of TGF-beta 3 in skin T. rex and not in conventional T cells of the skin. And they show that if they have dexamethasone as— what's the word— treatment of Tregs, they produce TGF-beta 3 in response to this stimulation. So basically, they show that there is this cooperation between FOX3 and the glucocorticoid receptor for the production, amongst other things, TGF-beta 3. And this TGF-beta 3 on the hair follicle stem cells promotes a change of programming that involves the activation of phospho-SMAD2 and SMAD3, which are part of the TGF-beta downstream signaling. This initiates proliferation and the function of the hair follicle to grow new hair. They can also show that if you inject TGF-beta 3, this improves the hair growth. They show that if you have an in vitro transwell system in which you have Tregs, if you block TGF-beta 3, then you don't have— so you have transwell on one side, Tregs on the other side, stem cells, hair follicle stem cells. You see again that this TGF-beta 3 is necessary in order to stimulate proliferation of these cells. And they also, to some extent, reproduce the phenotype by having TGF-beta 3 knockout Tregs, which they're mostly normal. So TGF-beta 3 doesn't seem to be critical for other aspects of Treg function, but it does seem to be very important for the hair follicle regeneration induced by Tregs in response to glucocorticoid production on damaged skin. So there you go. No, you— maybe that can help, uh, you know, with hair loss.

So I'm trying to remember if glucocorticoids are also induced during stress responses. I think so. So once stress causes you to have more hair, uh, this only works if you actually have, um—

I think it's more on a response if you take the hair out. I don't think it makes a huge difference in like hair that is growing normally, but if you have Um, so if you're gonna add on top, you need to get them back, this works, but not if you have a receding hairline like mine just due to the testosterone killing your hair. Yeah, I think it's probably not for the hormonal version of, but maybe, maybe for, uh, people that have, uh, what, how is it now called, um, alopecia, alopecia maybe. And also they say that apparently glucocorticoid, but they, I think they mentioned in the paper that now I'm thinking about that glucocorticoid, like long-term glucocorticoid treatment, is associated with higher hair growth. Let me just check because they mentioned it in the discussion. Um, but I thought it was, yeah, kind of cool. It's like my dad would be very interested to hear those. It's probably not for him either, alas.

All right, well, we're going to be speaking with Dr. Laura McKay at the University of Melbourne in just a moment. But before we get to that, if you're looking for tools to optimize your T-cell workflow in addition to your hair growth, and get high yield of viable T cells, we have a webinar for you. Stem Cell Technologies and Precision Nanosystems have teamed up to produce a webinar on how to optimize T cell isolation, cell culture, and gene transfer methods all in one place. The webinar covers best practices and technologies to cover high yields of viable engineered T cells for your research. You can watch the webinar at www.stemcell.com/webinar-tcell-research. All one word.

We are talking today to Dr. Laura McKay, who is a laboratory head at the Peter Doherty Institute for Infection and Immunity at the University of Melbourne, so several time zones away. Thank you so much for joining us. Thanks for, for stopping by.

Oh, thanks, Brenda and Jason. It's such a pleasure to be part of this episode. Thank you.

Thank you for being here. All right, Brenda, it's T-cell time, so do you want to just jump in?

Well, maybe yes, maybe we can start talking about the amazing research that Dr. McKay does, because I personally think, I mean, I love T cells of all shapes, sizes, locations, but I do feel that tissue resident T cells, they have this special thing. And the fact that we are only understanding them now thanks to all these advances in technologies that allow us to really look at very small amounts of cells and really find them in all these tissues. I think it's really a big kind of frontier in our understanding of the adaptive immune system in the barrier tissues or in like outside of blood, outside of lymph. So maybe we can get started. Lara, you can just share with our guests what does it mean to be a T cell that does not live in the blood, that is like resident, that is what are they doing there? How do they stay there? How do they survive in this? Environments?

Yeah, Brendan, that's exactly kind of our lab's focus. How do these T cells get there, stay there, survive? What are they doing, and what is the tissue doing to them? So we know that tissue resident T cells, they have a lot of distinct properties from those cells that circulate in the blood. Their cardinal feature is that they don't recirculate. So if they're in that tissue, they are in it forever. You can transplant tissue across you know, and it's now been shown in humans, in mice, the T cells stay put. There's lots of— of course, this is immunology, right? So we're starting to subset tissue resident T cells. So there are some that are more resident than others, really sort of experimental methods like parabiosis, conjoining the blood circulation of two animals. That's your kind of, you know, your main procedure to demonstrate tissue residence. But with regards to what they are doing, these T cells that just stay put, The way they were first found is that at sites of previous infection. And so it's the immune system's really nifty way to say, we had an infection here before. Yes, we're gonna get our circulating armies kind of ready to fight, but we're also gonna put, you know, troops at the site where we had it before. And so it's a really kind of smart way to put memory exactly where you would need it if you would see the same infection again. So that's where they were first found, sites of previous infection. And we think that's their major function is to fight off recurring infections.

So we can't talk immunology without talking about cell surface receptors or markers, usually subjected to flow.

Of course.

What are the widgets, for lack of a better word, that make a resident T cell a resident T cell?

Oh, it's becoming convoluted because the field's enlarged. So back in 2009, when the kind of tissue resident papers started to come out in. And this was identifying CD8s in epithelia. CD69 and the integrin CD103 were the two hallmark features of you are tissue resident. That was amazing for the field because then people could go and say, hey, they're expressing those markers too in the lung and they're different to the blood. And, you know, and so that was so important for the field. But, you know, now kind of using transplantation parabiosis, it's been shown that not all TRMs express CD103 and CD69, so they can be used. They are largely, you know, CD103 is a fantastic marker, you know, in epithelial cells, but it doesn't denote a T cell as resident, say, in a solid organ like the liver or the kidney. So now there's a real cohort of molecules that need to be used in unison to define the cell as resident. And a lot of them are transcriptional. And so there's no good antibodies. So certain reporter mice are being made against certain transcription factors like HOBBIT, which is also really good at defining residency in the mouse.

And what does a T cell need to survive in the tissue? Like, I mean, I think there's one thing is like markers that you find, but then which of these markers you have actually established a function that allows the cells or like induces the cells towards this phenotype?

Yeah. So both CD69 and CD103, the molecules I mentioned, they're both relevant in the survival. So, CD103 and epithelia allows T cells to tether and hang on into epithelial tissues. CD69 has a relationship with S1P, which allows the shutdown of tissue regress alongside the transcription factor KLF2. But there's been various transcription factors that have been shown. The major thing that you need with all the genes that have been shown is to shut down tissue regress. That's step one. If you want to stay in a tissue, program everything that's going to, you know, shut down tissue egress and stop T cells coming back out the blood. So, you know, shutting down CCR7, shutting down KLF2, and S1PR1, S1PR5. So that's your first step. Um, so this is step 1 in, you know, the road to tissue residency. Well, actually, step 1 is there might be some sort of committed precursor cell at step 1. Step 2, get into the tissue, right, chemokine receptors. Step 3, shut down tissue egress. Step 4, survive there. Long-term survival, it's different in different tissues what you need. But say some universal factors that operate in the majority of tissues are things like a lot of alpha-Dean in the tissue and TGF-beta in the tissue are good for these cells.

So you said maybe there's a committed precursor cell. Maybe. Does that mean that maybe not all T-cell or T-cell precursors can become resident T cells and only a subset can, and you have to now go down the path of lineage tracing in ways you weren't hoping not to?

Yeah, I feel like I've been working on this for such a long time now, trying to work out whether there's a real kind of precursor. And there's, you know, studies starting to show out that kind of give an indication that there's sort of a break in decision. But yeah, I'd put my money on there being, say, even within the— of course, we know there's an effector and memory T cell break, you know, cells that express certain things, they're going to give rise to long-lived memory, which of course is what it's all about. We all want better T cell memory. But for tissue resident memory, we think we can break it again on, I'm gonna rather go to the tissue, I'm gonna rather be in the circulation, I'm gonna rather be a central memory cell. And we're getting there, but it's been a long and arduous task. We had to develop a lot of new techniques to try and get there, but we're still going. We're still going.

All right. One more follow-up then. So when I think resident memory, resident T-cell, memory T-cell, a lot of CD8 in the literature on this, right?

You know, CD8.

Yeah. What about CD4 cells?

Oh, that's such a good question.

And Tregs.

And Tregs. Yeah. Of course there's tissue resident Tregs too, and tissue resident ILC-1s. Yeah. CD4, the literature started to come a lot later. A lot of this also comes down to tools that you have, you know, which is, you know, just such a— is such an immunology— immunologist thing to say, isn't it? You know, using, you know, the transgenics were there, the OT1s, the p14s in certain viral infection models. CD8s all came first. CD4 transgenics and tetramers, you know, came a little bit later. It's now been shown in various infection models that CD4 T cells are resident too. But I think what we've recently shown them— we've got a paper that's about to come out that really shows that CD4s, they operate by different rules even in the same tissue. So the types of transcription factors they need to program residency, the things that they need to survive, they're all different even if it's within the same tissue. So I would say that they're resident, but I would say they're not as good at it as CD8s. I think CD8s are the real stayers.

Do they at least have the same don't-leave or block egress signaling, or is even that different?

Yeah, no, some of them are the same. Oh, but some of them are different. But things like the downregulation of KLF2, I have— we haven't done it really. It's Steve Jamieson's lab at University of Minnesota. He's been working on that. But I said I would and we'd have to ask him. But I suspect that CD4s need the downregulation of KLF2 as well to stay in tissues like the skin and the gut.

I mean, it's fascinating, I guess. Is— do you think in a kind of a profound way, is this a consequence of, you know, CD4 cells have— they're very, I mean, they're very different than CD8 cells. That's an understatement. But we know that because they have this different type of plasticity that CD8 cells have. They have an access— I think they have a much wider range of potential transcription factors and epigenetic changes than CD8. And do you think that that's what makes them— is that related to their different ability or their different ways they establish themselves or they associate themselves with tissues.

You're absolutely on the money. Certainly in the skin, they're more plastic. And so CD4 T cells, certainly within the skin, which is like, I guess, my favorite tissue, if I had to, you know, give a, give a favorite hat to one. And the CD4s can leave and become other things. The CD8s cannot, that we find. Right. And so I think CD4s are more plastic. And also, if you kind of take a step back and you're like, well, why would you want a CD8 to be more resident and stick around more than a CD4? CD8s after activation, they really downregulate a lot of homing receptors, so they just lose the capacity to get in, whereas CD4s don't seem to do that sort of as stringently. They still have, you know, sort of receptor expression, so they can still home around the skin, they can still patrol, whereas CD8s, you need to keep them there because they downregulate everything they need to keep circulating around. So it's that sort of like, you know, that kind of like— that kind of makes sense in my mind a little bit of why you might want the CD8s to really stick.

Okay. Well, I have one more question related to, you know, how you make cells survive and, like, really stay. I have, in my past, I was an immunometabolism aficionado. And I know that you published some work in which you're looking into, for example, fatty acid binding proteins in resident lymphocytes. And I wonder whether you have an opinion regarding or whether what your research says regarding how do these cells survive metabolically in this environment? Because, you know, usually our standard view of lymphocyte metabolism is very glucose-centered in a way. And I think this has been changing, but it's really hard to measure, right, in this, especially in tissue-resident cells. But where do you think we stand when it comes to what are they How are they maintaining their metabolism in this, in these areas?

Yeah. So we had, we had a little kind of dip in the ocean here just looking at their dependence on fatty acid binding protein. So we think it's fatty acid oxidation. But of course then to work on tissue residency, it's hard because you need to get the cells. It's very difficult to go and do seahorse on TRMs in the skin. What I can say is that these cells appear to be very quiescent. So they certainly seem to be quite held in check, suppressed. Their proliferation is very low. So I think that also fits with their sort of longevity of same place at the same time. So they really seem to be very in shutdown compared to their circulating memory counterparts.

I'm taking us back a little bit, but you also mentioned ILCs, which I find very interesting because those also hang out in tissue. But don't have any antigen specificity. I'm wondering if you guys have started to see— because let me take a step back. I think one of my hypotheses is that all of the— oh, this is this differentiated pathway and this is this one— that's true, but also somewhat human artifact of our desire to bin things, and things are much more of a smear in reality. Do you any see any of that smear or You know, resident memory T cells look a lot like an ILC, but with one receptor that's been selected, you know, one antigen process selected for.

They have a lot in common, and a lot of the sort of major transcriptional regulators of residency are shared between resident NK T cells, ILCs, CD8 T cells. I could also say almost less for conventional CD4s, but there's a lot in common transcriptionally that programs residency across the board in these cells. And say, you know, we think about antigen specificity and how kind of, you know, various, you know, antigen-specific, you know, TCRs might preferentially give residency or not. You know, I can't really speak to that. That hasn't been shown. But one thing I can say is that certainly to be resident, survive, you don't need antigen. You don't need TCR. You know, once you're in the tissue, you know, you can do it in a Rag mouse. You can downregulate the TCR. You can do it in a Class I knockout. The CD8 T cells, once they've got what they need in the environment, they'll stick around. And again, it's those major residency programming regulators that kind of set that process up.

I work a lot on cells in the context of cancer and solid tumors. And sometimes I see a lot of overlap between, for example, tumor-specific cells that are living in within— I mean, after all, that solid tumor is kind of a tissue of a sort. Certain markers like CD103, for example, that is always associated with tissue residency, now it's also been associated with tumor specificity in infiltrating lymphocytes. Do you think— where do you think the similarities or the overlaps, if you've ever thought about that, come? And what can people that are studying you know, survival in tumor microenvironment can learn from what has been done on tissue residence.

Yeah, that's— it's a great point. And it's becoming an incredibly complex area. Say, you know, 10 years ago when we were working on tissue residency in the skin and the gut in after acute infection and everything's resolved, it was sort of like easy days. Now, to the complex areas of like having chronic antigen around in the context of tumors, there's a lot of overlap. On, you know, if you were to call a, you know, a tissue residency gene signature, if you just take that gene signature of a select number of genes, which is generally quite a small number of genes, right? And you overlay that onto what's in a cancer microenvironment, you can see that there's some— there's something in common. And that makes sense. They're sort of in a tissue, and you're sort of— to say whether they're resident or not, it's not been— it's not really been— it's not been shown. There's— there is a large amount of overlap, but of course there's going to be huge amount of differences. And, you know, we've looked into this a little bit about the differences between cells that are really exhausted and seeing antigen and tissue resident. And there's a whole host of differences as well. It's one of those, if you look hard enough, you can see a lot of similarities. Unfortunately, you know, one of those tissue residency markers that I mentioned, CD69, it also is— if you see, if you're in the tissue, you see chronic antigen, that's going to go up, of course. And this is where these markers really become unstuck if you use them in isolation too stringently, because all of a sudden everything's tissue resident. And, you know, you want to go back to the cardinal property of what is a tissue resident cell— it's something that sticks and doesn't circulate. But of course, where some of the kind of, you know, knowledge in the tissue resident field can then be applied is, say, you know, if you want to get, for example, you know, CAR-T cells into the tumor microenvironment, how do you do that? How do you get them in? How do you get them to persist better? You know, how can you deal with how functional T cells might be within a tissue? Because, you know, TRMs are different, their function Say in certain tissues it's different, what sort of cytokines modulate that? And so that information, if you want to get better T cells functioning in tumors, regardless of residency or not, a lot of the same rules can then sort of be applied to inform that, that space. But yeah, it's a very, very complex area, the sort of crossover between TILs, tumor-infiltrating lymphocytes, and those that are really tissue resident.

So, so really quick before I jump to another question, Can resident T cells become exhausted?

That's a great question. I think if you, in the right setting, yes. No one's really shown that, I think. But I think if you have resident cells and then you were to turn on chronic antigen, you could, you know, of course it's sort of how stringent you want to be about what type of exhausted cell it is and, you know, what was its precursor, you know, whether it's a TPEX or a terminal TX. Who— what bag— again, it's the problem of, like, categorizing your subset. This is the issue. So if you want to say by this set of criteria it was resident and expressed this, and now it expresses some TIM-3 and it's downregulated, you know, marker XYZ, now it's exhausted, you know, I'm sure that that could be done in a context. But I think with— of course, with any cell, I think if you slap it hard enough with antigen, it'll start to exhaust its functionality.

That's a good way to put it. I'm going to use that, slap it hard with antigen. All right. So, so to shift gears now to the follow-up, you started talking about cancer and therapy and, you know, this makes me want to bring up the, the cool work you're doing with Pfizer, which is, you know, one of the more kind of atypical industry-academic partnerships in a good way. I was wondering if you could kind of give us the highlights of that.

Because that's pretty neat. Yeah, so that's been a really, um, like just a really tremendous experience for the lab to partner with Pfizer. So it is, um, we're working on, um, basic cancer immunotherapy discovery research, which is really rare. It's, it's, you know, it's before there is a sort of tangible target to, you know, go forward and, you know, make a drug against. It's sort of preceding that. It's still very basic discovery.. And Pfizer had an interest in cancer immunotherapy and kind of saw the value in, you know, potentially exploiting tissue-resident T cells for better cancer immunotherapy. You know, what are the rules? How do we get more of them? You know, what are sort of genes that may regulate their function so we can get better protection against cancer? That's the whole premise of how they were thinking. We were asking similar questions. And so we partnered with Pfizer and, you know, tremendous group of scientists largely based in San Diego, who we meet with and share ideas, data, and work together. It's really the sort of collaboration that I didn't think existed until I'm in it. At least in Melbourne, I hadn't heard of anything similar. It's quite a sort of rare thing in Melbourne to have such a partnership. I know more common in the States, but I didn't realize that you could do such creative basic discovery in partnership with industry. Of course, now looking on it, it makes sense, you know, that, you know, we're all interested in the same goal, but I love the idea of working together for faster outcomes because then, you know, something that we find can be just drugged by Pfizer and move so much faster. And I never really want to do science in isolation. You know, you have to work together to push things faster. So then it might happen in our lifetime, you know? So I love that partnership. It's a dream partnership.

I think that often it is, especially outside of the US, a lot of countries or places have a harder time like establishing really dynamic collaborations with industry. I think it's, it's important also to keep, I think often the question is how do you keep, you know, the, the ethos of a university or of a public research environment and make that really complement what a company does that in the end, you know, publicly traded companies have, they have a purpose, then they have an obligation to make profit. How do you think, what do you think is the best way around this, these issues? I mean, now I also think the research that a lot of pharma and biotech companies do is extraordinary. I think more and more it becomes a really attractive place for scientists to go. Um, what do you think is like the future of these types of collaborations? I mean, I know maybe it's a very broad question, but I, I assume you've thought about it.

Yeah, I think, um, partnerships between academia and industry is the future. Um, you know, I think that ultimately, um, you know, our team and the team at Pfizer have exactly the same goal. You know, we both ultimately want better patient outcomes. In academia, we can't do that in isolation. And so this is why it's the best. It's a perfect partnership because we do what we do. They also, they care about what we do and they can take it one step further, which is something that our lab, you know, doesn't do. You know, we are in basic discovery, you know, we, you know, the discoveries that we make, I want them funneled into somewhere, you know, quickly. But at the moment, you know, the goal is exactly the same for, you know, both sides, you know, industry and academia. You know, we want to find out what molecules regulate a certain type of cell that we think is going to be really kind of important for better outcomes against cancer. And so the goal is aligned. And so I think we're all on the same page. So I must say, I don't think about the profit margins. I just want the science to be done.

Right. And I think, you know, just hearing how good the relationship is, I'm assuming that you guys just set terms to begin with, like you're going to let them know cool things you find. They have a little time before you publish it, but then you can at a certain period. So I think, I think it's communication, right? Like, know, know what the rules are ahead of time, be open and transparent, and then work together.

Exactly. And, um, and you know, that also of course comes down to, you know, who you're working with, you know, in, you know, whatever team you're partnering up with, you know, who are those people? And, you know, it's all very open. Um, we, we meet every month, you know, we talk offline all the time as well, um, freely send data and um, and, you know, everybody's working towards the same goal. So, um, I think in that scenario— and I'm sure that not all scenarios are as, are as open— but it's a very beautiful thing. And, and I, uh, and it's something that I would like to keep, you know, continue to do throughout my career, the industry partnerships.

And did your institution make it easy? Because I, I took 2 months with an institution to get a CDA in place, which is— yeah. Agreement to talk to one scientist?

You know what, that was the holdup because there wasn't a similar agreement at University of Melbourne. And so that actually did hold things up because, you know, it was kind of a very off-the-books arrangement. You know, I had met people from Pfizer at conferences, you know, we started talking, we started to meet, um, and so it was just all something that happened so organically, but it did take a long time to to actually kind of, you know, sign the deal. That did take some time, which I wasn't prepared for because I want everything done yesterday. So I was very confused. Why was that with lawyers?

I had that too, like this institution for me to talk to, someone wanted anything I said that was confidential to be written down on a piece of paper as confidential. But I'm like, I want to talk about ideas that are confidential.

Why are they going on a slide? I am probably naive and not business-minded, but I just wanted to do the science and talk about science. Freely and, you know, let the chips fall where they may.

I think it's great. And I think that it's great also for Australian science. Sometimes I think, is it hard to be so many kind of time zones away? Do you feel— sometimes I feel like Australia is far. I mean, there's so many prestigious, especially in immunology, there's some seriously good research. But sometimes I feel, man, they are so far. And I come from Argentina, so, you know, I'm a southern I'm also from the Southern Hemisphere, but from the wrong side of the Southern Hemisphere.

Yes, the Earth. And I didn't really feel that until COVID, but then I was like, I'm stuck on this island. I mean, I'm British originally. I went for a postdoc and now I'm like, I'm on prison island now. So yeah, it's really far away and you're always stuck with the time zone because when there's meetings and there's Europe and there's the US, Australia, it's just never going to work out. It's going to be 4 AM. But, you know, if you're going to— if you're going to live in Australia, you got to pay the price.

What are the perks then? Do you go surfing in the morning?

So, oh no, I'm in Melbourne. It's cold. It's winter. It's so European. Like, I was in the UK last week. Amazing. And then I come back here and it's cold and grey and the beach is very far away. So, no. But what the good thing is, is that Melbourne really has this epicentre of immunology in Australia and there's a lot of institutes that are really on each other's doorsteps, and there's a lot of collaboration and sharing. It's a very open community, and that suits me really well. I like to talk about science. I like to work with people and collaborate very openly, and Melbourne is just fantastic for that. You can walk across the road to neighboring institutes, and especially when you're starting up a lab and borrow reagents, and people very kindly will lend you said reagents. So it's a really nice place to work.

Okay, that's very good for anybody thinking of moving to Australia. There's the— here there's always a good Yelp review views from our guest.

All right, well, we'll have to start wrapping up here, but we always try to get an interesting question from, from our guests before we end things. So the one I wanted to go with is, what is the biggest or best, most awesomest piece of advice that you've ever been given, professional or not?

Um, the one that always sticks with me, and it was, it was my PhD supervisor who You know, didn't say too much, but one thing he said to me super early was, "Laura, you'll never make it if you're a wallflower." I was really shy. You know, in Britain, it's very hierarchical, and you don't speak up, you know, as a young student. And actually, Melbourne really beat this out of me because the Australians are pretty outgoing as a people, I have to say. So I kind of really tried to take that on board forcing myself to say things when I really didn't want to and trying to break the shyness. Because science is such a team sport, you know, you want to work with people and, you know, you want to sort of put yourself forward if someone, you know, if someone's doing something and you want to work with them, or, you know, you want to go up to somebody, or you want to go and, you know, get a job, or, you know, speak to an editor, or anything that you might find scary and you don't want to. Or even things— asking for things that you might deserve, really hard conversations and you've just got to— even I just don't ever want to do it, but just biting it and just doing it. I, I think about the, uh, Don't Be a Wallflower, Laura, you'll never make it. I think about it all the time and I force myself to say things I don't want to say. And it generally, you know, I sort of wouldn't still be in science, I think, if I hadn't had that reoccurring in my mind all the time, because it doesn't come naturally.

And now you're on a podcast.

Yeah, exactly. But I think it can be hard sometimes to, especially if you're introverted and also if you are a little bit, maybe as a student or as, you know, as a kind of early stage researcher, you might find yourself intimidated by some other people. I think it's, and sometimes the environment is not very inviting for your opinions. I think you really have to be strong and also have some people on your team, right? If things cannot give you the strength or give you the confidence to speak your mind.

You do need that voice in the back of your head, "Don't be a wallflower, just do it." Yeah, exactly. And I wouldn't have got my postdoc job had I not had that, you know, "Laura, just go and ask!" You know, to be like, "Hi, I like your work. I want to come to your lab." Like, you've just got to do it, you know?

So. Okay, then, you know, listeners, you heard her here first. Just do it. Go for it. Don't stick Do well like a wallflower and show yourself. I think it's a good final recommendation for us.

What do you think? And of course, in science, apply for everything, right? If you don't think you're ready, if you don't think you're good enough, just apply. Just do it. Just break down the shame barrier of, I won't get it.

Just do it. Jason, what do you think about that?

No, I agree. For me, talking is a defense mechanism, so I never didn't talk.

I talk too much. I feel you. It's like if you talk, other people cannot, you know, there's no time for other people to, to contradict you if you don't let them. Exactly. Right? Perfect. I don't think that's what she meant, but good for you.

No, but I think it's good advice to go out there. Do. I think, I think I was just reading something that, you know, people say, you know, you know, you got to get motivated, then act. It's often that action or motivation comes from action because you start building those early successes, then you get motivated to continue. Yeah, for sure. It's kind of the same concept of you just got to seize it.

All right, then carpe diem. Thank you so much for joining us all the way from Australia and for sharing with us your research and your experience. And well, we wish you all the best with this, I know, upcoming publications that you have, and we're looking forward to seeing you around.

Oh, thank you so much for having me on, and thanks so much for the wonderful work you do bringing immunology to everyone. This podcast.

I think it's wonderful. That brings us to the end of our show. Don't forget to subscribe to our newsletter at www.immunologypodcast.com to get the show notes, including an episode summary and links to all of the interview and roundup papers. You can also reach out to us on Twitter @AminoPodcast or via email at immunologypodcast.com with feedback or suggest guests. See you next time.

Mm-hmm.