# T cell Development

https://www.youtube.com/watch?v=4ZERgtF2JcU

[00:00] I already made a video for b-cell development and so this is gonna follow a similar pattern.
[00:11] So just so that we're all on the same page, there's another video for b-cell development that's already been made.
[00:17] There's going to be a video about the five mechanisms of genetic diversity, and this video is really gonna go into how do we make these incredibly diverse antigen receptors that create the b-cell receptor and the t-cell receptor.
[00:39] There is also a T and B cell development overview video that I actually recommend you watch kind of after you've digested everything to see if you can put the whole story together.
[00:50] And I'll kind of cue you as to when to watch that video in your notes.
[00:55] This is really difficult stuff, so if you're watching this and feeling like I'm giving you an alphabet...
[01:00] Soup of too many stages and too many things to know.
[01:02] That's okay.
[01:04] This is something that we're gonna come back to over the course of two years, and you're gonna have time to digest it.
[01:09] That's why there's kind of all these different videos and resources that you might choose to use as you try to figure out how we create these cells.
[01:19] All right, so just like I did in the B cell video, we're gonna talk about T cells now.
[01:23] I want you to remember what the T cell receptor looks like.
[01:26] So remember, if this is my T cell, it's just two chains: an alpha chain and a beta chain.
[01:35] The antigen binding portion of this, this peptide binding groove there, is going to have a V segment, a D segment, and adjacent on the beta chain side, and just a V segment and adjacent on the alpha chain side.
[01:52] The beta chain is going to rearrange first, and then the alpha chain.
[01:56] So we're going to go through this process now.
[01:59] We have the same goals that we had when we were creating B cells.
[02:02] Want to create a diverse antigen receptor population so that we can bind lots of different antigens.
[02:10] Now there is a slight difference here.
[02:13] B-cell receptor are capable of finding all sorts of antigens, right?
[02:18] They can bind peptides, they can bind lipids, carbohydrates.
[02:23] I guess fats are lipids, but you see what I'm saying.
[02:26] They can bind anything and everything.
[02:29] They are non-discriminatory that way.
[02:32] The T-cell receptor is much more discriminatory.
[02:35] T-cell receptors are only able to bind peptides, and they can only bind peptides that they see in the context of MHC, and not just any MHC, your own MHC.
[02:49] So if you have questions about that, go back to the MHC videos that were associated with the previous case, and that'll clear that up completely.
[03:00] So the first thing we need to do is to generate this diverse antigen receptor.
[03:02] We also...
[03:05] Need to eliminate any self-reactive receptors.
[03:08] We didn't really talk about this much in the B cell video because I didn't really get into B-cell selection.
[03:14] But we will in future videos.
[03:17] In this video, we are going to talk about elimination.
[03:19] We're going to talk about a process known as central tolerance.
[03:24] Central tolerance is actually one of it.
[03:29] Central tolerance is how we get rid of self-reactive lymphocytes while they're still in their primary lymphoid organs.
[03:37] So for T cells, that would be the thymus because that's where T cells develop.
[03:42] Okay, and that's part of the reason why developing T cells are known as thymocytes.
[03:46] And then, just like we did in B cells, the final step is to promote foreign-reactive cells that are beneficial to us in fighting a particular pathogen.
[04:00] So we're gonna follow a similar format to develop T cells that we did when we were developing B cells.
[04:07] That we did when we were developing b-cells.
[04:08] It's not important that you know every single stage of t-cell development.
[04:14] And I'll kind of point out the ones that it's more important that you know versus the ones you don't necessarily need to know.
[04:18] Okay, remember with b-cells that I said that b-cells and t-cells both come from the lymphoid progenitor which can be found in the bone marrow, right?
[04:26] And as b-cells develop, they move through the bone marrow and are assisted by the cytokine IL-7.
[04:41] Well, the story is pretty much the same for t-cells.
[04:44] Common lymphoid progenitors from the bone marrow move from the bone marrow into the thymus.
[04:56] Okay, and the thymus is this rather fatty organ that sits kind of right above your heart, around you know just below your throat there.
[05:04] And just like b-cells moved.
[05:08] Through the bone marrow, T-cells are going to move through the thymus as they're developing, or rather thymocytes.
[05:19] Well, now I want to go over just a concept here.
[05:24] We often hear that as people age, they become cell-mediated immunity deficient.
[05:31] You've heard me talk about it in various microbiology videos.
[05:37] And part of that actually has to do with our friend the thymus here.
[05:42] So I already mentioned that the thymus is a fatty organ, and much like our waistlines, as we age, our thymus only gets fattier.
[05:56] So it's not that we lose our thymus; it's that the space in the thymus that was reserved for T-cell development, that wasn't fat, gets made smaller and smaller and smaller and smaller as the thymus.
[06:09] Itself gets fattier and fattier, and this is kind of a very simplified view of thymic invasion, which I'm sure your histology professors will spend a little bit more time on.
[06:25] So in thymic involution, it's not so much that we lose our thymus; it just becomes less useful.
[06:30] It doesn't work as hard, and therefore it's also fattier, okay?
[06:34] But let's get back to what we're actually talking about with the thymus itself.
[06:40] So here I'm showing, in an electron micrograph, which has kind of these developing thymocytes, and that's all of these like spherical cells here.
[06:48] If it's spherical, that's a developing sinusite, and they're gonna occupy the space within the interspaces of this extensive network, just really huge, of epithelial cells.
[07:01] These epithelial cells are the thymic stroma, okay?
[07:05] And the thymic stroma is largely made up of kind of two different types of cells, m tex.
[07:11] And c Tex okay, the EM Tex are your medullary thyroid panel cells.
[07:16] And the C techs are your cortical thymic epithelial cells.
[07:20] They're both gonna play a huge role in signaling our thymocytes in what to become.
[07:25] And these are gonna be really important for the central tolerance mechanisms that will basically figure out which of these cells are non-self reactive and get to leave the thymus to be a mature T-cell one day.
[07:40] And which of these cells need to be killed because they're actually going to attack us and cause damage.
[07:48] Just as in the bone marrow, IL-7 plays a huge role in development of these cells.
[07:54] And in fact, I didn't mention this in B-cells, but patients who lack receptors for IL-7 or are unable to make IL-7 have significant immunodeficiencies where they can't make T cells or B cells.
[08:06] So this is a really important cytokine for lymphocyte development.
[08:09] Alright, first just a quick.
[08:12] Refresher, remember there are two main types of T-cells.
[08:14] Your CD4 positive T cells are your T helper cells, right?
[08:20] They're gonna secrete cytokines that makes other cells better at their jobs.
[08:24] And then your cytotoxic T cells, your CD8 positive T cells, and these guys are going to be cytotoxic.
[08:30] They're going to kill cells that are intracellularly infected or maybe precancerous cells, things like that.
[08:43] MHC class 1 is going to bind to CD8, and MHC class 2 is going to be bound by CD4.
[08:51] And it's CD4 that kind of differentiates T helper, and CD8 toxic.
[08:58] All right, so let's talk about the actual generation of these antigens specific cells in the thymus, okay?
[09:05] So just like B-cells, we're gonna depend on signals that are received within the thymus, okay, when the common lymphoid...
[09:15] Progenitor cell arrives in the thymus.
[09:19] From the bone marrow.
[09:22] Nothing has rearranged, okay.
[09:25] There are no rearrangements.
[09:27] There is nothing that is being expressed yet, okay.
[09:31] That means all of its DNA is in its germline state, okay.
[09:37] That means it's untouched completely, you know, as new and complete as it was, okay.
[09:44] So this is known as double negative, okay.
[09:47] What I mean by double negative is that it is not expressing CD4 and it is not expressing CD8.
[09:58] That is what I mean, okay.
[09:59] It's also at this point probably not expressing CD3.
[10:02] This is you know as bald and new, as like a newborn baby.
[10:04] It's double negative, okay.
[10:07] So no CD molecules, yeah, three, four, eight, nothing.
[10:10] The thymus itself is divided into peripheral cortex and central medulla.
[10:14] Most T-cell...
[10:17] Development is going to take place in the cortex only mature single positive cells.
[10:25] So cells that are expressing either CD4 or CD8 are really seen in the medulla.
[10:31] Okay, that means that as a cell is moving deeper, so it first arrives out here and as it moves through from the subcapsular region to the cortex, so the core Tacoma jewellary Junction all the way down to the medulla, it's going to eventually go from being a double negative cell to down here a double positive cell.
[10:54] Double positive meaning it expresses CD4 and CD8 to then choosing which side it wants to be on where it'll express only CD4 or only CD8.
[11:07] So those are kind of the stages I want you to grossly understand, okay?
[11:13] So double negative means it's got nothing.
[11:17] Double positive means we're on our way.
[11:19] Single positive means we've completed this journey to create an antigen receptor.
[11:26] And once it's a single positive t-cell, we sometimes refer to it as a naive t-cell.
[11:34] This just means that it's ready to see antigen, but it hasn't yet.
[11:38] So they've finished development.
[11:41] They'll differentiate later.
[11:43] All right, just like in b-cells, we're gonna break things down into different stages.
[11:45] I'm gonna say this right now: you only need to know that the cell is double negative.
[11:51] You don't need to know if it's DN1, DN2, DN3, DN4; that's really, really confusing.
[11:57] So DN is actually four stages.
[12:00] So the first thing that happens is that basically IL7 causes the cells to proliferate.
[12:06] Remember I said that was a really important step.
[12:08] So it's gonna cause the CLP to proliferate, but there have been no changes, no rearrangements, just growing cells.
[12:17] So since no rearrangement of the giant germ...
[12:20] Line has happened nothing is being expressed.
[12:22] And that's why what we're seeing here during this whole process there's one important molecule.
[12:29] And that's known as notch, notch one on the cell surface of the thymocyte interacts with transmembrane ligands on up at the by McCarthy lleol cells.
[12:41] And this basically drives the cell along the path of T cell differentiation.
[12:46] So you can see that notch is kind of expressed throughout the various stages.
[12:51] And this is what's basically going to tell the cell to begin trying to rearrange first its beta chain and then its alpha chain.
[12:59] And remember, by rearrange we're literally talking about taking these various gene segments.
[13:07] So if this is a beta chain locus for variable diversity and joining, so that we've got like maybe V 3 D 2 J 1 as our beta chain by the time all of this is done and they're all.
[13:21] Nicely arranged together and then that can be put together with an alpha chain.
[13:26] Okay, so let's go through the various stages.
[13:29] All right, in DN1, a common lymphoid progenitor cell leaves the bone marrow and travels to the thymus.
[13:37] It's devoid of anything, right? No CD3, CD4, CD8.
[13:40] So it's DN1.
[13:41] All right, IL-7 signaling in the thymus basically leaves it to proliferate at the DN1 stage.
[13:50] No rearrangements have occurred; we're still looking at germline.
[13:54] All right, but now we're gonna move on to DN2.
[13:58] Okay, in DN2, the beta chain is gonna try to rearrange.
[14:03] Okay, so just like we did in B-cells, we're gonna take a D beta and a J beta on the first chromosome and we're gonna try to put them together.
[14:11] If it's able to do that, it will go on to being a DN3.
[14:14] If it's not able to do that, it'll go to the second chromosome, and again if it's successful, we go on to DN3.
[14:19] If it's not...
[14:21] Successful it dies, okay, all right.
[14:27] Once we get to DN 3, the V beta chain rearranges again to basically take this successfully made DJ beta segment and pair it with a V beta segment.
[14:39] Any sale cells that fail to make a successful rearrangement will die.
[14:45] Any cells that are able to make a successful rearrangement will move on to DN for the DN 4 stage.
[14:53] Basically means that we successfully made a beta chain, all right.
[14:58] So now we've got this beta chain, and just like in B-cells, we want to make sure that this beta chain is functional.
[15:05] So what we're going to do is we're basically going to pair it with a surrogate chain, and in this case the surrogate chain is the invariant pre T alpha chain, okay.
[15:19] This is just to make sure that we made a functional beta chain that can bind to what will be an.
[15:25] Alpha chain at some point, so if we go ahead with this and we make an alpha chain, will it actually work?
[15:32] Okay, so at this point we're going to have CD3 on the surface, and that's going to provide signaling.
[15:38] So we already talked about the CD3 complex.
[15:40] So we have our beta chain, we have a pre-T alpha chain, and they can come together to form a super dimer.
[15:51] And when this happens, it basically tells the cell, "You did it, you made a beta chain."
[15:56] We're going to go ahead and make an alpha chain, and that means it's going to move on to the double positive stage.
[16:04] All right, so we made our beta chain, we paired it with the pre-T alpha chain, it was successful, yay!
[16:17] And then now it's gonna be a double positive cell.
[16:19] What does that mean?
[16:19] Now the cell is going to express CD3, we already knew that, CD4 — oops, that's new — and CD8, more new.
[16:28] Stuff so it doesn't know what it wants to be yet.
[16:29] So it's time to now do a couple things.
[16:32] One, we need to test it so it learns what it should be, and also so that we eliminate any self-reactive T-cells.
[16:42] So now, boys and girls, it is story time.
[16:44] I'm going to tell you the story of Goldilocks and the three bears.
[16:46] Now Goldilocks right here is a precocious young thing who enjoys breaking into people's houses, eating their food, and sleeping in their beds, and then running away when they deign to ask her why she's there.
[17:01] These days they now have various services for that like Airbnb, but that's not what she was into.
[17:06] So instead, what she does is she tests if something is too hot or too cold, right, like I'm showing here with the porridge.
[17:15] Our T-cells are going to do the same thing, and this is known as central tolerance.
[17:19] You need to be able to recognize self enough that you can see your own MHC molecules, because remember...
[17:29] We only recognize peptide in the context of our own MHC.
[17:32] However, that MHC molecule is going to have an antigen in it.
[17:37] And that antigen is likely going to be a self antigen.
[17:43] So we want to recognize the self-MHC but not recognize the self-antigen.
[17:52] If you recognize the self-antigen too much, than you die.
[17:55] So this is a tricky game we're playing, right?
[17:58] So if you recognize self too strongly, that's negative selection.
[18:03] You die by negative selection.
[18:05] But if you can't recognize self at all, if you can't recognize the self-MHC, you still die.
[18:11] And that's known as positive selection.
[18:13] So just like Goldilocks was trying to find that just right bowl of porridge, we're trying to find that just right level of self recognition.
[18:24] Okay, so we made it to our double positive stage, right?
[18:26] At this point, the TCR alpha chain.
[18:30] Is going to begin to rearrange now just like the B cell light chain the alpha chain can go through multiple rounds and this is somewhat controversial some people think it can happen some people think it can't it's not that big a deal either way it's going to try to rearrange a V region to a J region on the Alpha chain now in the B cell there was Kappa and there was lambda and there were all these Greek letters in this one it's just beta and alpha beta is kind of our quote-unquote heavy chain and now we've got our quote-unquote light chain which is alpha it's going to be paired with the beta chain if it's able to bind then it moves on to the next step which is where it's going to determine what kind of naive T cell it wants to be and be tested by positive and negative selection if it can't find it'll try the next low sigh or the next chromosome and eventually if it's unable to do so it will die but let's be positive and think that it actually can bind to the beta.
[19:33] Chain okay, so the TCR alpha chain rearranges binds to the beta chain.
[19:39] We have a fully formed T cell receptor, tada.
[19:42] Okay, but now we have to test it.
[19:46] We need to make sure that A, it's not going to react with self, and B, that it can see the MHC molecule.
[19:56] These are the two things that absolutely has to do.
[19:59] So this is what we do.
[20:02] We have our MHC molecule here.
[20:04] I'm gonna use a different color and see the MHC molecule actually does touch the TCR.
[20:10] So we're gonna try to make it see that, all right.
[20:12] And this MHC is gonna be expressed by some cortical medullary epithelial cells, something like that, some M Techs or some C tax, all right.
[20:23] Now if the cell recognizes the self antigen with moderate to low strength, it's going to survive, okay.
[20:29] If it recognizes self MHC with moderate strength, it's going to...
[20:35] become a cd4 positive cell if it
[20:38] recognizes the self MHC with very weak
[20:41] strength it's going to become a cd8
[20:43] positive cell if it doesn't recognize
[20:47] the self-mhc at all it is not selected
[20:51] so it dies if it recognizes the self
[20:56] antigen with too much strength it will
[20:59] die and that's known as negative
[21:01] selection pretty much all roads lead to
[21:04] death unless it recognizes the MHC well
[21:08] but the antigen not really at all
[21:12] alright so I mentioned earlier that
[21:14] these MHC molecules that we're making
[21:17] were probably expressing self antigens
[21:21] right now we don't want just any self
[21:25] antigens we want self antigens from
[21:28] various parts in the body okay there are
[21:31] a whole bunch of antigens that this
[21:34] little t-cell is gonna see from all over
[21:36] the body that aren't normally found in
[21:38] the thymus things like myelin proteins
[21:41] that are found in the brain or ovarian
[21:44] proteins women there's milk ducts
[21:49] pancreatic islet cells these are all
[21:52] things that we don't have in our thymus
[21:55] that theoretically t-cells could react
[21:57] to and cause damage too right so there's
[22:00] actually a really cool process for this
[22:02] and it is handled by air the auto immune
[22:06] regulator gene
[22:08] air basically gets turned on in our
[22:12] medullary fire Kappa filial cells
[22:15] and leads basically to the expression of
[22:18] these distal peptides so things like
[22:21] myelin and pancreatic islet cells and
[22:24] things like that
[22:25] so these distal peptides are expressed
[22:28] and the t-cell can be checked against
[22:30] them and if it responds too strongly to
[22:32] that self antigen it will die if it
[22:35] doesn't see the self antigen it'll live
[22:38] so it's a very cool process that
[22:40] actually modulates this whole negative
[22:42] selection process and for patients that
[22:46] actually lack air it winds up being a
[22:50] really severe condition patients who
[22:54] lack air are not able to undergo
[22:56] negative selection and if you can't do
[22:59] negative selection you're gonna have a
[23:00] lot of self reactive cells that are
[23:02] released into the periphery so this
[23:05] leads to a condition known as apec II D
[23:09] which is autoimmune Paulo poly
[23:12] endocrinology candidiasis dystrophy it's
[23:15] also known as a PS and over time these
[23:18] patients will develop significant immune
[23:20] responses against self tissue as they're
[23:23] not able to clear self reactive T cells
[23:25] and these responses are autoimmune
[23:27] responses and classified as
[23:29] hypersensitivity reactions okay so as I
[23:32] showed in the last page once you've
[23:34] gotten past negative selection and
[23:36] positive selection now you're going to
[23:38] become a single positive cell so that's
[23:40] going to be either a cd4 cell or a cd8
[23:44] cell and at that point you're ready to
[23:46] leave the thymus and go out into the
[23:48] world there's two other things that
[23:52] could potentially go wrong with t-cells
[23:54] first off I think I've made it really
[23:56] clear that t-cells absolutely need a
[23:59] finest in order to develop they don't
[24:02] have a thymus you can't develop t-cells
[24:05] and there actually is this condition
[24:06] it's known as to George syndrome it is
[24:08] not very common but it is a favorite of
[24:11] the board's it's this is surrounding a
[24:13] thymic individuals these are patients
[24:16] who lack a famous or who have poor
[24:18] development of a thymus or some other
[24:21] form of thymic hyperplasia or thymic
[24:24] dysfunction it's a genetic disorder due
[24:27] to deletions
[24:28] in chromosome 22 I think it's 22q 11.2
[24:34] they tend it's actually not so much
[24:37] about the thymus it actually has to do
[24:38] with poor development of the pharyngeal
[24:41] pouches so they also have a lot of other
[24:44] issues that go along with this syndrome
[24:48] not just lacking t-cells the other thing
[24:51] that I think have been pretty clear
[24:52] about is that MHC is absolutely integral
[24:56] to the development of t-cells if you
[25:00] lack MHC you're gonna if that's gonna
[25:03] impact the type of t-cells you develop
[25:05] okay and this is known as bare
[25:07] lymphocyte syndrome and what it really
[25:09] means is that you lack the genetic
[25:11] material to express either MHC class 1
[25:14] or MHC class 2 so if you lack the
[25:20] genetic material to make MHC class 1 it
[25:23] means the only cells you'll make are cd4
[25:26] cells right because you will not be able
[25:29] to make cd8 if you lack the genetic
[25:33] material for MHC class 2 the only cells
[25:35] you'll make are cd8 cells because you
[25:38] won't be able to make cd4s
[25:40] right because you have to have that
[25:41] interaction between the appropriate MHC
[25:44] and the appropriate Siddhi molecule in
[25:46] order to select it again this is a rare
[25:50] one but just one of those ones that the
[25:52] board seemed to love