Episode 3

To listen to this episode, click on one of the following links.

Recommended for Background Information

• Immune by Philipp Dettmer (ISBN 9780593241318)

Texts Discussed in This Episode

• “Protein Synthesis and Metabolism in T Cells” in the Annual Review of Immunology, by Linda V. Sinclair and Doreen A. Cantrell, in Annual Review of Immunology, https://doi.org/10.1146/annurev-immunol-082323-035253

• “Enkephalin-producing regulatory T cells in the skin restrain local inflammation through control of nociception” by Alejandra Mendoza et al., in Science Immunology, https://doi.org/10.1126/sciimmunol.adz6869

• “Neuro-immune cross-talk in cancer” by Moran Amit et al., in Nature Reviews Cancer, https://doi.org/10.1038/s41568-025-00831-w

Transcript

Hello and welcome to “If Socrates Had a Podcast”! I am your host, Vinay Kalva. In today’s episode, I attempt to answer some unanswered questions from Episode 2. I also correct an error that was made in Episode 2, and examine its implications on that episode’s conclusion.

Before proceeding further, I would like to make an important announcement about the podcast’s release schedule. Due to a recurring issue with publishing on alternate Fridays, I have officially changed the release schedule such that I publish every alternate Saturday, starting with this episode. This will ensure that I can release episodes reliably and consistently for your benefit.

Proceeding to today’s episode, a few notes. First, if you have not already listened to Episode 2, I highly suggest that you pause here and come back after you do so. This brief dialogue picks up directly where Episode 2 ends, and I believe you will find it more helpful and interesting if you see the questions, the error, and the implications of both first. Second, this dialogue is based upon the two papers discussed in Episode 2, as well as one additional review article: “Neuro-immune cross-talk in cancer” by Moran Amit and colleagues in Nature Reviews Cancer. This paper, unfortunately, is paywalled. However, you can use any institutional access you may have been legitimately granted, or contact the authors to request a copy. Please act considerately, civilly, and legally when attempting to access this paper. Finally, as said previously, please make sure to read Immune by Philipp Dettmer if you need extra background. This episode, like Episode 2, will be more enjoyable if you are able to recognize the terms being used. As always, I have linked all the resources mentioned just now in the podcast description.

There are three characters in this dialogue: Socrates, Marie Pasteur, and Louis Pasteur. I will play all three parts, speaking as follows when I am Socrates: (slowly say “I am Socrates”); as follows when I am Marie Pasteur (rapidly say “I am Marie Pasteur”); and as follows when I am Louis Pasteur (normally say “I am Louis Pasteur”). With that, let’s get started.

SOCRATES: Thank you for letting me come for dinner, Madame Pasteur.

MARIE PASTEUR: It is our pleasure, Socrates. We have some extra food with us, so do not worry. As you must be going right after dinner, why don’t we start right away?

SOCRATES: Certainly. Here are my bags, if you wish to store them by the door. Will Monsieur Pasteur be eating with us tonight?

MARIE PASTEUR: Thank you, Socrates. And yes – Louis will be coming.

SOCRATES: Excellent! I am curious to hear from him.

LOUIS PASTEUR: Well, if it isn’t the world-famous Socrates!

SOCRATES: It certainly is. How do you do, Monsieur?

LOUIS PASTEUR: I do well. How do you do?

SOCRATES: No better than you, and certainly no worse than you.

LOUIS PASTEUR: Very well, Socrates. Now, you and Marie, what did the two of you discuss? I saw both of you in the café.

MARIE PASTEUR: We were discussing two papers Socrates had read, about regulatory T cells and how a certain subset of them control responses to harmful stimuli.

SOCRATES: I didn’t know how they related at first, and one of those articles took some time to understand.

MARIE PASTEUR: Now he does. He understands them and how they relate.

LOUIS PASTEUR: Personal scientific development? Count me in. 

SOCRATES: On that note comes my big question. I was reflecting on my discussion with Madame Pasteur, and it seems I made an error.

LOUIS PASTEUR: What sort of error do you think you made?

SOCRATES: I think I was incorrect about tumors.

MARIE PASTEUR: Yes, I remember this point. Socrates said that tumors, being outgrowths, do not respond to neural signaling.

SOCRATES: That’s quite close to the way I said it.

LOUIS PASTEUR: Well, it is wonderful happenstance that you made that error. When I saw both of you in the café, I was reading several review articles about that very subject. There were so many that I needed to get some coffee myself, just to pay attention and get through it.

MARIE PASTEUR: You don’t seem fatigued to me.

SOCRATES: I cannot disagree.

LOUIS PASTEUR: Don’t worry, Marie and Socrates, I didn’t drink that much. Just a cup, like you both did.

MARIE PASTEUR: Thank goodness.

SOCRATES: Well, with your newfound energy, what did you learn?

LOUIS PASTEUR: Well, I found this very fascinating review article that I wanted to share. It was called “Neuro-immune cross-talk in cancer”, and was written by what seems to be a diverse and international team of authors led by a person named Moran Amit.

SOCRATES: That seems most interesting. If you will, Monsieur Pasteur, would you tell me briefly what was discussed?

LOUIS PASTEUR: I suppose, Socrates, that it will be more constructive if we go through the questions you had after talking to Marie. I am sure the review answers some of them.

SOCRATES: Certainly. We could start out with the first one, posed by the review article that Madame Pasteur and I discussed: How do exhausted T cells control protein synthesis? It is noted in the review that this is relevant to tumors and the specific areas that surround them.

LOUIS PASTEUR: From the review by Amit and colleagues, it seems there are two factors that can cause exhaustion: calcitonin gene-related peptide (CGRP) and β-adrenergic receptors. They note that CGRP and β2-adrenergic receptors specifically affect T cells with CD8.

SOCRATES: Let us start with CGRP, in that case. How does the review describe CGRP as causing T cell exhaustion?

LOUIS PASTEUR: The review mentions that the answer to your first question is not fully known, but they hypothesize that it involves signaling through the CGRP receptor complex, regulated by a protein called Gαs. The end result is the interference of T cell receptors, which reduces the production of certain proteins. As a result, T cell exhaustion occurs.

SOCRATES: You mentioned two proteins that are not produced. Perhaps that is where our answer lies. What are those two proteins?

LOUIS PASTEUR: IL-2 and interferon-γ were their names.

SOCRATES: From what I recall, receptors cause signaling cascades, which regulates gene expression and thus the production of proteins. As protein production changes, so too does the cell’s activity.

LOUIS PASTEUR: What follows, then?

SOCRATES: It follows, then, that exhausted T cells see changes in their general activity.

LOUIS PASTEUR: I don’t quite see where protein synthesis comes into this.

SOCRATES: Well, perhaps consider this: cell activity is a broad term. Protein synthesis would be included in that, arguably.

LOUIS PASTEUR: Certainly. In that case, exhausted T cells would have different protein synthesis levels than regular ones.

SOCRATES: Then we have an explanation: the protein synthesis of exhausted T cells is controlled not by themselves, but by external stimuli. That is CGRP for CD8-expressing T cells.

LOUIS PASTEUR: Of course, there are also β2-adrenergic receptors, for which the explanation is quite similar, if not the same.

SOCRATES: Indeed, Monsieur Pasteur. Thus, exhausted T cells do not control their own protein synthesis, but are prevented from synthesizing proteins.

LOUIS PASTEUR: Now, Socrates, what is the next question?

SOCRATES: Does T cell protein synthesis play a role in exhaustion? I suppose the answer is yes, because it is the lack of protein synthesis that results in exhaustion.

LOUIS PASTEUR: I find it difficult to disagree with you in this regard.

SOCRATES: The next question, then: What pathways control amino acid transporters, ribosomes, and general activity in regulatory T cells?

LOUIS PASTEUR: I don’t think the paper extensively discusses this, short of the β-adrenergic receptor pathway. When the β-adrenergic receptor is bound, a phosphate group binds to GSK3 and thus prevents it from acting. This causes regulatory T cells to survive longer.

SOCRATES: I assume you are arguing in the latter case that survival influences general activity.

LOUIS PASTEUR: Precisely. If regulatory T cells survive for longer, then they would have more general activity overall, even if within a given timeframe short of survival the level of general activity remained the same.

SOCRATES: That does not seem right. I suppose it will be useful if we know what “general activity” actually means.

LOUIS PASTEUR: I would assume that “general activity” refers to any action which is undertaken by a cell.

SOCRATES: In that case, survival would ensure more “general activity”, for the cell would undertake more actions.

LOUIS PASTEUR: It follows, then.

SOCRATES: Additionally, from my discussion with Madame Pasteur, we established that the enkephalin production pathway is an example of a pathway that increases general activity of regulatory T cells.

LOUIS PASTEUR: We thus have two pathways, indeed. However, I do not believe that, at this point, we can proceed further with an inquiry into this question, and I thus suggest that we do not use our valuable time going in circles.

SOCRATES: Certainly, Monsieur. Let us proceed to the issue at hand, with our background knowledge at least somewhat established. 

LOUIS PASTEUR: Socrates, would you mind telling me the question and your provisional reasoning, so that I can accurately diagnose the error which you have made?

SOCRATES: Yes. The question at hand was this: “How does nociception affect inflammation?” The answer to this question was not completely apparent after discussing the Mendoza et al. paper, so here is how we attempted to reason the answer. 

First, we developed the following pathway based upon the paper and background knowledge regarding cellular function. It starts when T cell/glucocorticoid receptors are activated, causing a signaling cascade which results in expression of the Penk gene. Penk mRNA is transcribed, and the mRNA is translated into proenkephalin proteins. These are converted to enkephalin proteins, which proceed to bind to opioid receptors on neurons. This results in reduced activation of neurons, and therefore inflammation is reduced.

After examining this pathway, I saw that there was no clear indication of where exactly nociception (response to harmful stimuli) was implicated. When I thought about the paper again, I realized that the pathway applied to the nociceptive neurons. However, I did not recognize this at the time, so Madame Pasteur and I tested two explanations. The first was that the glucocorticoid and T cell receptors were stimulated by neurons. I argued that this was implausible for T cell receptors, which were bound by antigens. This still left glucocorticoid receptors, and when Madame Pasteur suggested that neurons could release glucocorticoids that could bind them, I dismissed the possibility. My reasoning for this was as follows: tumors produce Penk in response to glucocorticoid binding, as the paper established. However, tumors could not possibly respond to neural signaling. Thus, the cause could not be neural signaling.

On this basis, we rejected the first explanation. The second explanation – neuron-neuron communication occurred, and the regulatory T cells had some role in this – was what we came up with, and we ended there.

Thus, Monsieur Pasteur, I have rendered to you an account of how I arrived at the error.

LOUIS PASTEUR: I must make a most troubling diagnosis. You have inappropriate reasoning based upon an incorrect premise.

SOCRATES: Yes, that is correct. However, now that I know that the pathway actually involves nociceptive neurons, it is clear to me now. 

LOUIS PASTEUR: What precisely is clear to you?

SOCRATES: The pathway occurs within nociceptive neurons. This means that the regulatory T cells act upon these neurons directly.

LOUIS PASTEUR: Certainly. What does that entail for the inflammation?

SOCRATES: The nociceptive neurons are inflamed, and the regulatory T cells calm them down.

LOUIS PASTEUR: How, then, are nociception and inflammation related?

SOCRATES: Nociception causes inflammation, but activation of the regulatory T cells reduces it.

LOUIS PASTEUR: Now, moving to the specific understanding of the pathway, where exactly is the point of uncertainty?

SOCRATES: Well, it seems the neuron-neuron communication theory is incorrect, as the nociceptor neurons are themselves the subject of inflammation. The point of uncertainty, then, moves to the start of the pathway.

LOUIS PASTEUR: I would suggest, Socrates, that to resolve this matter, it will be useful for us to examine the methodology of the paper, and compare it with the review which I read.

SOCRATES: I agree with you, Monsieur Pasteur. However, as we have finished dinner, I think it might be appropriate for us to take some time off before proceeding. After all, the paper was long and complex, and I must take some time to bring all the information together.

LOUIS PASTEUR: I understand, Socrates. We can all wash the dishes, and Marie and I will show you around the house.

SOCRATES: We can certainly do that.

This regularly scheduled program is interrupted for a short break.

Dear listeners,

As Socrates and the Pasteurs clean their dishes, and the Pasteurs show Socrates around their home, I will tell you another anecdote that remains in my memory. While on the ship bound to France, Socrates and the Pasteurs met a most peculiar fellow Parisian – someone who may be relevant. I will let you, the listener, decide how relevant the person is, but before that I implore you to open your ears and eyes as we return to the train, past the British Isles and Le Havre and on the way to Paris.

It was near the end of the ship ride, and both Socrates and the Pasteurs were packing up their belongings. The journey was all fine and good thus far, excluding a major thunderstorm in the middle of the Atlantic. At that time, the three needed to stay in their individual quarters, and Socrates managed to re-read the entirety of the Iliad and Odyssey. He had read them well, of course, but it never hurt to read them again. Regardless, on the train, Socrates and the Pasteurs decided to sit together on the way back. Shortly after departure, a most interesting person entered their compartment and asked to join them, to which they agreed. The man then asked: “Who might I be meeting on this fine day?”

Socrates replied: “Across from me are the Pasteurs, Monsieur and Madame, who I surely hope you know.”

“Of course I know Monsieur Pasteur! I am a Parisian, after all – oh, my apologies, Madame, my sincere apologies. Of course I am not worthy of sitting in front of the eminent scientists. And who might you be?” replied the mysterious man.
“Me?” said Socrates. “I could tell you, but perhaps you could guess.”

“Might you be Odysseus? Surely you should be joined by Penelope, but you seem not to be.”

“While I did read the Odyssey on that ship as it tossed and turned the other day, no, I am not Odysseus. Why would I commit such blasphemy to put myself at the level of a Greek great? Forget Zeus – even the lowliest god would never let me be here were I to do that.”

“I am incorrect, then, and shall guess again. Are you Homer?”

“Who reads their own work for leisure? I certainly do not.”

“I see I have failed. I will take your leave.”

“With respect, do not despair. I am Socrates.”
“Socrates! The famous man from antiquity. I am afraid you will have a lot of questions to ask me.”

“Why might you say that?”

“I am involved in producing terrible things. Weapons, you see. I make what causes hurt to so many people.”

“For what purpose might you do this?”

“For my living! What else could I make it for?”
“You live by making weapons which end life? That’s one mighty contradiction.”
“Such is life. In any case, I shall write a will to restore my legacy. Before that, however, I must use the toilet, so I will momentarily bid you farewell. We will continue when I return.”

The man, yet unnamed, left to go to the toilet. A few moments after the compartment door closed, however, the train suddenly stopped. Everyone assumed a train was ahead, for the train waited for a few minutes. After that several more minutes had passed, but the train did not move one centimeter. The unnamed man returned and said: “I see the train has stopped. I opted to examine both sides of the carriage – curiously, the couplers have failed, and the car is detached from the others on both sides. It would be a dangerous job to put it all back together, but there is no chance of this train moving until it is done. Might you all be willing to render assistance?” 

Socrates and the Pasteurs readily agreed, and went about the train looking for anyone with equipment. A person who worked for the railways provided their supplies, and all worked together to re-couple the train – Marie and Socrates on one end, and Louis and the unnamed man on the other. When they did it, they embarked once more, told the conductor, and proceeded to Paris. It was at this point that Socrates wondered what this man’s name was, and he told him. It was none other than Alfred Nobel, the man whose last name became the most notable prize. 

Quite unfortunately, Socrates and the Pasteurs never met him again after that ride, although they certainly discussed plenty more on the remainder of the journey to Paris. Why, I am not even sure how the conversation ended up here. Was it a camera? I don’t know. Let us continue, unimpeded, onward.

We now return to our regularly scheduled program.

LOUIS PASTEUR: What do you think of the place? It’s reasonable in size according to me.

SOCRATES: Certainly. I cannot disagree.

LOUIS PASTEUR: Now, what were we discussing before?

SOCRATES: I remember now. We were planning to discuss the methods, and their scientific validity, from the Mendoza et al. paper. I remember them now.

LOUIS PASTEUR: I will allow you to go ahead and tell us the methods.

SOCRATES: I recollect that there are six experiments, but there are some common methods used. From the paper and my discussion with Madame Pasteur, I recall that there were several: application of capsaicin and diphtheria toxin together; imiquimod treatment; resiniferatoxin treatment; usage of a Penk reporter involving Cre recombinase; a specific type of RNA sequencing; generating specific mice with specific genes; and tamoxifen treatment.

LOUIS PASTEUR: That is indeed a lot of methods. Perhaps it will be most useful if we go at it one at a time. Let’s start off with capsaicin. Why was it used?

SOCRATES: The paper said that capsaicin was applied to mouse ears in order to activate the TRPV1 receptor. What does the review say about this?

LOUIS PASTEUR: From what I recall, the review says that both heat and irritants – irritants like capsaicin – activate the TRPV1 receptor.

SOCRATES: I suppose, then, that this part of the method is a valid and justifiable choice.

LOUIS PASTEUR: What about the diphtheria toxin addition that was done in combination? The review does not discuss the diphtheria toxin receptor at all.

SOCRATES: The Methods section of the paper does not go into much detail about the treatment, although the Results mentions that the toxin prevents protein synthesis in regulatory T cells which have a receptor. This causes their inactivation and death.

LOUIS PASTEUR: I suppose that, if we take this to be true, we can add this to the pathway that you have developed. Surely diphtheria toxin would prevent the synthesis of enkephalins just as much as it prevents the synthesis of other proteins.

SOCRATES: I am inclined to agree, although a full analysis of the evidence would be needed to determine if this hypothesis were true. I suppose we can accept this conclusion for now, however.

LOUIS PASTEUR: This seems reasonable. Now, if we put capsaicin and diphtheria toxin together, what is the expected combined effect, assuming that no interaction occurs between the two?

SOCRATES: The combined effect would be TRPV1 activation and no regulatory T cell action. Indeed, that was the purpose of using it in the experiment – to see how sensory neurons acted in response to regulatory T cells.

LOUIS PASTEUR: In that case, could we add this to the pathway, and where?

SOCRATES: It makes sense to add this to the pathway. However, this occurs within the neurons, and there is no clear connection to the regulatory T cells yet.

LOUIS PASTEUR: In that case, let us continue with the experimental techniques. What was the purpose of imiquimod treatment?

SOCRATES: Imiquimod is used in a mouse model to cause a skin disease similar to psoriasis. The condition is inflammatory, so we can assume that imiquimod results in inflammation.

LOUIS PASTEUR: How is this relevant to developing the pathway?

SOCRATES: Well, it is an inflammatory agent. That surely is helpful in developing a better explanation. Does the review mention it?

LOUIS PASTEUR: It does not. What about resiniferatoxin? The review mentions that it can be used to remove nerves with TRPV1.

SOCRATES: These abilities of resiniferatoxin are described in similar terms in the paper, and the paper adds that it also removes TRPA1 and TRPM8, the latter of which detects cold sensations.

LOUIS PASTEUR: TRPA1, for its part, is mentioned in the review, but TRPM8 is nowhere to be found. TRPA1, in any case, is said to be activated by chemical irritants.

SOCRATES: I suppose all of these are useful for our pathway. Now, let us discuss the reporter with Cre recombinase, the RNA sequencing technique, and the generation of different mice – all genetic techniques.

LOUIS PASTEUR: Discussing three techniques at once seems excessive. I will say, however, that there are many types of RNA sequencing. We can start with that one. What is the type used, and why is it used?

SOCRATES: They used single cell RNA sequencing to analyze the transcript product of regulatory T cells in mice with inflammation and the control mice. The transcript is mRNA, so I suppose it works.

LOUIS PASTEUR: I cannot disagree. What about Cre recombinase?

SOCRATES: Cre recombinase is used to determine Penk expression in the paper. What does the review say?

LOUIS PASTEUR: The review discusses Cre-Lox systems and seems to validate what you said before about chemicals which remove the proteins that detect harmful stimuli.

SOCRATES: I don’t know how essential this will be for our pathway to be developed, but we can determine how important mouse generation is.

LOUIS PASTEUR: The review says mouse models are very important for study, but again, that does not help us very much in our efforts to develop a pathway.

SOCRATES: I agree. However, tamoxifen seems like a possible element that could be important.

LOUIS PASTEUR: The review does not mention it at all. What does the article say about the substance?

SOCRATES: The article mentions that it is applied to mouse ear skin through corn oil, but there is not much else about it. I assume that it is an inflammatory agent.

LOUIS PASTEUR: In that case, I think we have two areas of uncertainty: whether diphtheria toxin prevents enkephalins from being produced, and what the purpose of tamoxifen is. However, neither is an impediment to developing the pathway further.

SOCRATES: In that case, here are the additions we need to make. TRPV1 is stimulated by heat and irritants, TRPA1 is stimulated by irritants only, and TRPM8 is stimulated by cold sensation. This stimulation results in the nociceptor neurons with these receptors being activated. Wait…where exactly does the pathway come into this?

LOUIS PASTEUR: Did you not say that T cell receptors and glucocorticoid receptors start the inflammatory regulation pathway?

SOCRATES: I did say that.

LOUIS PASTEUR: We also know that stopping T cell receptors causes certain proteins to not be produced, specifically IL-2 and interferon-γ, and this leads to exhaustion.

SOCRATES: I remember something. In the paper, T cell receptor activation when IL-2 was present was insufficient to start the pathway. This was the basis for adding glucocorticoid receptors as a relevant condition.

LOUIS PASTEUR: Then, perhaps, we can add a condition. When IL-2 is sufficiently present, activation of T cell and glucocorticoid receptors starts the pathway.

SOCRATES: Indeed. This also suggests what happens to inflammation response when regulatory T cells are exhausted: inflammation is exacerbated.

LOUIS PASTEUR: Certainly. These are the unavoidable conclusions.

SOCRATES: I am happy to have resolved my questions. The pathway, then, is this: Stimuli cause protein activation, resulting in nociceptor neuron action. Separately, antigens bind to T cell receptors, and glucocorticoids bind to glucocorticoid receptors. When there is sufficient IL-2 content, this causes the pathway: a signaling cascade which results in Penk expression. This results in proenkephalin production, and eventually enkephalins. These bind to opioid receptors on the nociceptor neurons, and temper inflammation.

LOUIS PASTEUR: A most satisfying conclusion, I must say. 

SOCRATES: Indeed. However, it is about time that I get moving. Thank you both for hosting me today.

MARIE PASTEUR: It was our pleasure, Socrates. I really enjoyed speaking with you.

LOUIS PASTEUR: It was mine, too, and an honor, to be sure.

SOCRATES: Thank you both. I will now take your leave.

This concludes the third episode of “If Socrates Had a Podcast”. As noted in the beginning, you can find the papers and other resources in the podcast description. If you have any questions or would like to suggest books or notable people that I should feature on this podcast, reach out by sending me an email at ishap.podcast@gmail.com. That’s i-s-h-a-p.podcast@gmail.com.

I hope that you learned something new and exciting today, and enjoyed the brief intermission. If you like what you heard, stay tuned for my next episode, coming up in two weeks. Thank you very much for your time, and have a wonderful day.