B-cell Development and Function PDF

Despite just everything, I'm truly sorry about all of this. There is a Dean's Hour, I believe, next week or the week after where Ray Stanley, who is a director of IT at Marion, will be speaking to students, so feel free to attend and ask questions. So anyway, we left off discussing B1 and B2 cells,...

Despite just everything, I'm truly sorry about all of this. There is a Dean's Hour, I believe, next week or the week after where Ray Stanley, who is a director of IT at Marion, will be speaking to students, so feel free to attend and ask questions. So anyway, we left off discussing B1 and B2 cells, and so this is again just a reiteration of this previous slide. So again, it's the same material, it's just presented in a different way if that helps you learn it this way, but we talked about how B1 arrives from the fetal liver by the eighth gestational week, so very, very early on in embryogenesis, and it provides some protection to the developing fetus while it is in utero. And so again, they do kind of argue that it's possibly a transitional lymphocyte that bridges the innate and adaptive systems, because again, it provides some protection to birth. It does have some slight immunological memory, very limited isope switching, and very, very limited repertoire, because again, it's looking for those conserved sequences. It is not a very well or fully trained B cell. It does make up a very, very small portion of the B cell repertoire, like the overall one. It's a lot less than B2, your conventional B cells here too. And its B cell receptors and antibodies are often directed against conserved antigens like lipopolysaccharide on bacterial cell walls. And we do frequently see it at tissues where pathogens can get in, so like the pleural and peritoneal cavities. And I highlighted it in some class on Friday, and then verbally today, even though there was no recording, I do want you to know natural antibodies. This is a definition term, and I know it's poorly named because technically any antibodies that your body makes, you could be argued to be natural. But in this specific case, it is specifically referring to immunoglobulin M molecules, or IgM, antibodies basically that are directed against blood type. And so they've had no previous training to it, like guarantee you right now, like I have A negative blood type. I guarantee you I have antibodies in my body from B1 cells that know what B blood is, even though it's never seen it. So it's already primed and trained for it, and that's why I call it natural, because it's like kind of tie into the fact that mothers and infants can often have different blood types overall. And I went to a little, you know, rant about that in class today, not really relevant for the exam, but please know what natural antibodies are. B2, again, are your conventional ones, and pretty much everything else we're going to be talking about for the most part with the rest of this module. And so these do arise during and after the neonatal period and widely distributed throughout your body, and they are continually replaced from the bone marrow throughout your adult life. They do require interaction with T cells for activation and proliferation. They have a broad and vast range of epitopes that can be recognized because, you know, they go through development and rearrangement of the VDJ of their heavy chain and VJ, you know, the variable junctional and constant of their light chains here. And again, upon repeated exposure, they respond more quickly with increased antibody quantity and quality due to that affinity of antibody produced known as affinity maturation, where they get better primed at responding to that specific antigen over time. We also see that accompanied change in immunoglobulin X type, known as ice type switching. And all of these do contribute to the hallmark of immunological memory because these B cells are what form your memory B cells overall. And so, you know, that pretty much sums up what we talked about kind of with all that B cell receptor editing all falls under phase one. And that we've reminded you of what B1 and B2 are. Now we're going to talk about phase two and phase three of B cell development, which is negative and positive selection. And if you notice, dogs are trying to fight right now, if you notice here in what I pointed out in class, negative selection actually occurs before positive selection in the case of B cells. It is different from T cells. And so the immature B cell population is purged of cells bearing self-reactive B cell receptors, because again, they need T cells to activate them. And so it's not really as critical for them to know what self or non-self is because, you know, again, the T cells are going to be directing them. And so in this case, you know, we don't want them to have, you know, self-responsive B cell receptors. So in this case, like to compare and contrast, T cells go through that positive selection first and then negative selection. B cells actually go through that negative selection first and then positive. And there's makes a little bit more sense because when we talk about B cell negative selection, it's basically saying we are removing any cells that react to self antigen and then we positively select them just to make sure that they bind on two antigen directly. So it's basically just one, make sure they don't respond to self and then two, make sure that they can, you know, function and bind onto antigen and then cool, they're good to go. And so that makes it a little bit easier in that, you know, we are negatively, in this case, removing them. And in this case, you know, positively selecting ones that work. So it is a little bit different based on T cells, because again, T cells, we were picking them with positive selection based on their ability just to recognize self molecules. And then a negative selection, we got rid of those T cells that bound MHC2 strongly. So it is a little bit different, but I would be able to compare and contrast positive and negative selection with B cells. They also differ in terms of what happens to them, you know, they respond to self versus non-self. And so with T cells, we talked about ones that respond too strongly or not at all, B cells have a few more, you know, tricks up their sleeve. And so in this case, in this specific picture, it's just showing you on the left, if there's no reaction with a self antigen, which is what we want, those cells are allowed, you know, they're known as immature B cells, they are allowed to continue in the developmental pathway, and they will move to the blood and start expressing IgD and IgM on the surface. So they're moving towards their maturation pathways. They're allowed to leave the bone marrow if they don't react to self. Now, if they do react to self antigen here, it is then kept in the bone marrow. And so that is different from T cells, because again, we just straight up killed them if they, you know, didn't respond at all, or they responded too strongly. And so the difference is here, if they are not specific for self antigen, they're allowed to continue their maturation and leave the bone marrow versus if they react with self antigen, they are retained in the bone marrow. And so another unique thing about B cells is that if they do respond to self antigen, rather than just killing them directly, like we do with T cells, we can actually go back and actually edit that B cell receptor known as receptor editing. So it's very, very well named in this case here too. And it does depend on whether the B cell binds what's known as multivalent with multiple points or monovalent self antigen. And so this is going to talk about your multivalent self antigen here, which means, you know, it has many pieces to it here. And so if you have an immature B cell receptor that binds to multivalent self antigen here, that B cell will then be kept in the bone marrow and will be triggered to undergo and rearrange its light chain genes. This is what is receptor editing, we can't really mess with that heavy chain, but since the light chain was the last thing to be made, we can actually go back and take the pieces of whatever's left from this, you know, original light chain that we've created, and we can go through and try to mix every possible combination here. And so there are two potential consequences here. So this picture continues on to the very next slide. That's where we'll pick back up here. There are two possible consequences here. And so if the new receptor that we make is still self reactive, we'll continue to rearrange those genes until all possible combinations have been exhausted. If we've done that, and they are still self reactive here, if all successive new receptors are self reactive, obviously, we've exhausted all of our possible options of new light chain. And so at that point, we will then kill the cell versus if we do finally do a new rearrangement and the new light chain and the combination of that light and heavy chain and that immunoglobulin molecule is not multivalent, self reactive, we will then allow it to leave the bone marrow. And it will start to then express as an immature B cell both IgM and IgD on the surface, and it will travel to continue its development and activation. And so again, receptor editing is unique to B cells. Now this is the case with multivalent self antigen because I feel like there's a lot more at stake, you know, of having multiple pieces versus monovalent. This is just responding to one specific antigen in the body here too. And so it's basically just showing an IgM of immature B cell binds to a soluble univalent self antigen like highlighted by this little red diamond here. Univalent is the same thing as monovalent basically here. And so if you do have a B cell that recognizes monovalent self antigens, we do not receptor edit these. Unfortunately, we also don't let them, you know, do their job either. And so what we actually do is we make them energetic. So basically, the analogy I use in class today is basically chopping off someone's arms. They're still alive, you know, it's a lot less energetically exhausting. It's like it takes a lot to kill a cell. And you know, I joke today about like, it takes a lot to kill someone and then hide the body versus with this when you just chop their arms off. So they're still alive, you know, they'll die, you know, their normal little life spans, there's no extra energy exerted. However, they can't do their job here. And so, you know, that's the consequence of if it binds to monovalent self antigen, we do not receptor edit, we allow those cells to leave into the periphery. However, we've made them energetic, so they are unable to respond to that antigen, and they cannot function. And so, again, they will die off with their normal lifespan, which is usually not long after this. And so that's pretty much what we talk about when we go through positive and negative selection. Again, with B cells, negative selection occurs first, and then positive selection is again, we're just allowing those that can bind antigen to leave into the periphery. And so again, you know, it is unique with B cells that we can do that receptor editing, and particularly with that multivalent self antigen, monovalent, we just make it energetic. And that's it. Now, we talked about the other phases of B cell development, now we're going to other phases here, which is out in the secondary lymphoid tissue, that's why we made it red, because again, look at the big picture bottom titles, searching for infection, finding infection, and attacking infection here. And so right here, we're talking about recirculation of mature B cells between the lymph of blood and the secondary lymphoid tissues here. Now, as you can see, this is another chart, like kind of how we looked at earlier with B cell development. So all of these kind of tie in, you know, to these sections here, although they're not as like clean cut organized as before, because they do kind of overlap just a little bit here. But as you can see, the big picture takeaways here, your immature B cell, which we've talked about before, you know, we now know that it doesn't respond itself, we're allowing it to leave, it's just expressing IgM at first. And so that's why it's highlighted IgM positive and IgD negative. And so it will leave the bone marrow and enter peripheral circulation. And once it enters into peripheral circulation, there we go, alternative splicing will then also allow us to express IgD on the surface. And so these immature B cells are now known as IgM high with IgD low, once they've exited out into the periphery here. And so there's some theory that, you know, IgD might be necessary to help them access primary lymphoid follicles and mature. But it is critical, at least, you know, it has a role that are also wouldn't really be there here too. And then notice, once we switch from this immature to this mature, there is a change here. IgM is low, even though it's not really pictured well here. But IgD is high, so we have a lot more IgD on the surface of a mature B cell. And again, some people argue it might be because it's a chaperone molecule, and that it's there to signal to the body that this B cell is now mature, and it's able to function, you know, and potentially encounter antigen and respond to it as needed. And so you'll see a lot more IgD on the surface of a mature naive B cell. And again, that naive means we're talking about it has not encountered its antigen yet. The opposite of that naive word is activated, which would be the next step here. Because again, notice, you know, at this point here, we have encounter antigen cells become activated, and it's kind of showing now that we, you know, are kind of probably going to progress towards a plasma cell that's highlighting with these extra like little lines here. And so notice again, IgD is gone, you don't really know where it goes, whether it's cleaved or reabsorbed by the cell, who knows. But at this point, once the cell becomes activated, it will divide into a memory cell and a plasma cell. And at this point, once once it's become activated, you can see isotope switching, somatic hypermutation, which helps with that affinity maturation. And eventually, you know, obviously, we'll produce plasma cells of various, you know, isotopes, depending on that isotope switching to help us fight that infection. And we also produce those memory cells. And so these are the consequences of once we activate, you know, those B cells here. And so we do see that final maturation step occurs in the lymphoid follicles. And so we talked about the movement of cells. And I asked you guys in class, like, obviously, B cells leave the bone marrow that's highly, you know, you know, innervated blood vessels tissue, they're going to exit out into the blood, right? And so how would they then enter into your secondary lymphoid tissues? And if they're coming from the bone marrow, they're going to enter via those high endothelial venules or HEV here. And once they're here, they'll kind of do kind of a tour, and they'll circle out just like they say hi to all the T cells in this area here, and they'll circle out into that follicle. So remember, please review lymph node anatomy here, but that's that primary follicle here, we call it that primary follicle first, because we don't have a germinal center just yet, but the germinal center would develop within that primary follicle. And so that B cell will actually do a little loop through. And if it does not encounter their specific antigens, they will then leave the follicle and exit out via that efferent lymph down here. And that's important, because again, once you get into that lymphatic system, it's more like a direct, you know, direct interstate to directly where you want to go versus if you exit out to the blood, you know, you kind of have to travel along the circulation, it's not like you can hop out and be like, oh, we're headed to the head, but I actually really want to go in the foot. That doesn't really work. And so when you're in that efferent lymph, you can jump from lymph node to lymph node to lymph node a little bit more faster than if you were doing the blood vessels here too. And so the circulation route will be the same for both immature and mature B cells, which will all compete with each other to enter into those primary follicle areas here too. So it is pretty competitive between, you know, cells that are actually ready to respond to their antigen and those immature B cells, which may not have fully reached, you know, full maturation yet too. And so, you know, how we know for them to mature, they first must pass through a primary follicle and a secondary lymphoid tissue to become mature. And so how do they know where to go from those blood vessels to get into that secondary lymphoid tissue? That's where chemokines come into place, because remember, they're chemo attractant, they recruit cells to specific areas. And specifically, when we talk about B cells, chemokine CCL21 is what actually attracts B cells to those high endothelial venules. And it's actually CCL21 in combination with CCL19 that will actually then pull those immature B cells into your lymph node area, where you can see here, these guys here are follicular dendritic cells here. And then within that primary follicle, it's like, you know, saying, hey, this is a B cell club, you all guys, you all want to be here in this area, in this primary follicle here, chemokine CXCL13 will pull those B cells and attract them into that primary follicle area. So it's seeking chemokines that are pulling them from the HEV up into that primary follicle here. So that's kind of what this picture is zoomed in on here. And then when they have interaction with follicular dendritic cells, that drives that maturation. So that's when we start to see that co-expression of IgD on the surface, and then higher levels of IgD. So notice here, it's higher IgM, you can argue that this is then an immature B cell, but interactions with that follicular dendritic cell first drive it to start expressing higher levels of IgD, at which point we call this a mature B cell. So a mature B cell is characterized by high levels of IgD and low levels of IgM. I'm not going through kind of all the rest of these pathways here. So I'm not entirely sure we truly know all of the steps involved in this process here. But it is, you know, if you know that which chemokrines help drive to the different areas and that follicular dendritic cells, you know, help signal that final development into a mature B cell, that that is the critical information here. Other that I forgot to mention in class today too, is that immature B cells that are unable to enter a follicle will continue in recirculation, but soon die because they haven't fully matured. And you need that maturation step so they don't die. And so then those mature B cells here that do not encounter their specific antigen will leave the follicle. And so again, we talked about that before, they don't encounter their antigen, they then travel out and leave the follicle via the efferent lymph to go hop to the next lymph node and look for their specific pathogen that can activate them. And so they can recirculate between the lymph, the blood and secondary lymphoid tissues as they are looking for their specific pathogen that they can respond to. Now, when they do encounter their specific path or antigen, this will lead them into differentiation of activated B cells, and it will form one will be a plasma cell and then will be a memory B cell. Because think about it, when a cell splits, it splits into two mitosis. And so with this one cell becomes a memory B cell and the other one will become a plasma cell. And we'll elaborate more on that in the next PowerPoint slide I'm going to go into here. But again, it is just kind of highlighting what happens here with that antigen mediated B cell activation and differentiation here. And so what's showing you is again, we had that B cell come from those high endothelial venules, you know, it can kind of travel through the T cell area looking for its antigen here. And if it does encounter its specific antigen, and again, think about it, antigen is coming with dendritic cells as well as like fluid from sites of injury. So again, when you get a cut swelling, things like that, it's not just dendritic cells that are traveling in the lymph, you're also seeing that lymphatic fluid, you know, plus all those other dead cells, things like that, but pieces and parts of antigen and pathogen can actually make it to the lymph node, you know, nearest draining lymph node, and they're going to enter via that afferent lymph. And so pieces of it are going to also come into this area here too, and those B cells can pick it up and recognize it. And so at the boundary between a lymphoid follicle in the T cell area, so it's basically on the border of these two spots, T cell area here and that lymphoid follicle, they're going to meet up in the middle, like where their joint space is here. And those B cells will be activated by a CD4 helper T cell, and that will form that primary focus of dividing cells. And so that will form your germinal center, which is featured here within that primary follicle. We have those rapidly dividing cells, this is where you'll also see somatic hypermutation for that affinity maturation, as well as isotope switching occurring in those germinal centers, which are now forming within that primary follicle here. And some of these B cells will actually migrate to the medullary cords, which is, you know, down the medulla here, and they will differentiate into antibodies secreting plasma cells, which can then either be released out, or they can secrete antibodies, which can also be released out and travel via that efferent lymph to travel to the site of infection, to fight the pathogen where it's at. Others can migrate to that primary follicle to form that germinal center, obviously where they divide, and it differentiates, so there's two main pathways. They split, they can, you know, travel down to the medullary cords to release plasma cells in the antibodies to sites of infection, or they can travel back and form these germinal centers here. And so B cells from those germinal centers can migrate either to the medullary cords, or they can even travel to the bone marrow to complete additional maturation into plasma cells in the bone marrow too. So there's just a lot of movement of these B cells, but it's really nice, because again, it's kind of like producing a lot of different waves of soldiers to fight the infection, because, you know, again, infection can last a while, and so it's really nice to kind of have this, you know, mission control center here with those memory cells, and even plasma cells that we form here, isotope switching, all that fun stuff, so that if we ever encounter the pathogen again, they are ready to exit out in the medullary cords and travel to the site of infection. And then our also scientist was Deborah Donniak here, and so she was an immunopathologist here, and a professor of clinical immunology, and she partnered with chemists and immunologists, Ivern Roet, and they discovered that Hashimoto's thyroiditis is an autoimmune disease in which antibodies target thyroglobulin proteins, so self-protein. They also found a separate antibody cytotoxic to thyrocytes, which targets thyroid peroxidase, and then specifically here, and it actually contradicted current scientific belief that antibodies only targeted non-self antigens, so they helped change this whole premise that once B-cells become activated, that they don't respond to self at all. They're the first to actually show that unfortunately, you know, we can target self-antigens in the forms of autoimmune disorders especially, and we'll talk about Hashimoto's in module four. So it was our first, you know, it was the rest of the rest of B-cell development too, and so then we were going to move into the B-cell activation today. So these are not really dogs, but they do like Alex sometimes, but only when he has food, so apparently they may not actually be his friends, or they may not even really like him, but these are his chickens that he wanted to share with the class. There's a Halloween costume contest on Friday, as in it's faculty dressing up in Halloween costumes for charity, but you do get to vote for your favorite professors, so hopefully you have some favorite green professors who will be there on Friday, but it is an LH2 from 12 to 1. I still get there early, you can snag your spots before lecture, but Dr. Hum, Dr. Osell, Dr. Sedding, and I always usually participate, and it's a lot of fun, so I do hope you guys can attend. Unfortunately, there is a ton of objectives for today, but I did this on purpose because the material for activation can appear a little dense, and so I included more objectives because I wanted you guys, like I really wanted to direct you guys where I'm kind of going for the exam and what I'm looking for, and so that's why it looks like there's a lot more objectives for this one here, and it can seem overwhelming, but it is to help prepare you and, you know, guide you in the right direction, so you can best prepare for the exam overall, and so a critical piece of information is that B-cell activation requires cross-linking of the B-cell receptor, and we'll talk about what that means, so in case you guys haven't figured out by now, the gray slides usually provide the factoid piece of information, and the pictures and slides after support that piece of information here, so obviously we're going to see pictures of cross-linking at, you know, this slide now, and so notice again there's an arrow here because the picture does continue onto the next slide. Cross-linking of B-cell receptors by antigens will actually initiate that cascade of intracellular signaling, and so again, you remember how that B-cell receptor needs that Ig alpha and beta on the side because they have those long cytoplasmic tails with itams that actually provide that intracellular signaling in order to activate truly the B-cell here, and what it's showing is you have this B-cell receptor binding onto antigen on the bacterial cell here, and so that's that cross- linking first shown here, and so the B-cell receptor complex on a mature naive B- cell is composed of, as you know, that B-cell receptor and Ig alpha and Ig beta here because they need those intracellular signaling, and this point obviously naive means it has not encountered its antigen, but once it does obviously it will become activated, so we're showing that this activation is occurring here, and so some examples when you have that multivalent antigen with regularly arrayed epitopes on the bacterial surface IgM can bind onto that and it's showing it here too. Once IgM binds you'll have what's known as cross-linking and clustering, and so actually a lot of these immunoglobular molecules on the surfaces in these B-cell receptors will become closer together so you have a lot more of them in a close space, and so because of that it makes it very very easy for phosphorylation of these tails in a, you know, close area to then provide a stronger signal down into the cell to activate them here too. So we do have those receptor associated tyrosine kinases BLK, FYN, and LYN help phosphorylate this tyrosine residues on your ITAMs of those cytoplasmic tails of Ig alpha and beta because again the B-cell receptor doesn't have Ig alpha and beta for signaling. And so continuing on with this, SYK will first bind onto the phosphorylated ITAMs of the Ig beta chain, so it's kind of shown here, and so because Ig beta are close together because of this clustering of them being, you know, brought closer together with B-cell receptors becoming closer together here, they activate each other by what's known as transphosphorylation which leads to further signaling. So when they become activated they activate each other too which provides an even stronger signal which then allows us to fully activate the cell. So those intra- nuclear signals are then relayed to the nucleus where obviously we induce changes in gene expression to start activate the B-cells, you know, the cytokines, you know, start isotope switching, all that other fun stuff, you know, telling it to start becoming a plasma cell, etcetera here. And so again that cross-linking or bringing of multiple receptors close together to provide a stronger intracellular signal is critical for B-cell development here. We also require signals from the B- cell co-receptor, and so this will bring back some terms you guys kind of already know, but B-cell co-receptor is made up of CD81, CD19, which you guys know from CAR T cells because it is found on all B-cells, and then CR2 which is a complement receptor here too, and so that's, you know, these three molecules make up that B- cell co-receptor here too, and so CR2, again complement receptor too, you guys might remember that because it has complement fragments IC3B and C3D which will be fixed on pathogen surfaces, you know, usually in that classical complement pathway. The extracellular portion is just this very long tail composed of a series of 16 CCP structural modules, this is just the definition of what it is, and so it's just kind of defining the structure of CR2. CD19 cooperates with your antigen receptor to produce activating signals, because notice which one of these has the longest tail, and it is CD19. So again it has long tail into the cytoplasm of the B-cell, typically means it helps with intracellular signaling and there should be itams on the surface, and so, you know, that is important to help with additional signaling here too. And then CD81, because it's kind of within the plasma membrane, is usually considered a structural, you know, protein and stabilization, stuff like that, and it helps bring CD19 to the surface, it helps organize and keep this B-cell co- receptor together within that membrane here. And so first, you know, obviously CR2 is the second complement receptor, there's obviously a first one here too, and so what it's kind of showing here first is that when we're activating this B-cell and this co-receptor is kind of binding on here too, and we notice remember C3B, part of that classical complement pathway and other parts of the complement pathway too, but gets bound onto the pathogen surface, CR1 actually comes in and along with C, I'm sorry, factor I, breaks it down into, you know, sub pieces here until we eventually reach this C3D. I like to think of it as, you know, this looks a little bit more oval, you know, this long chain won't really stick well to an oval thing, but once we make it this nice little rectangle of C3D, look at that, they line up perfectly and they touch and they become activated. And so that CR2 component of the B-cell can now bind onto C3D, so you can't just do it directly, you need the help from CR1 to prepare that C3B molecule to look like something that CR2 will recognize. But again, because they're all connected, you can then allow a signal to travel down that CD19 to help with that activation of that B-cell. And so again, you know, this is just the same slide as before with more words describing out what's happening, even though I just went through all of that with the picture here too. And so it's, the combination of the B-cell receptor and the B-cell co-receptor will combine to activate B-cells in response to both surface and soluble antigen. So this picture looks a little confusing, but again, break it down very easy, look at the top description here. The B-cell receptor and co-receptor cooperate in B-cell activation by a pathogen. It's talking about a whole pathogen here, notice it's a bacterial cell on the wall. Versus over here, it's talking about a soluble antigen, because the takeaway from this whole slide is it can bind to both surface, which means it's still bound on to a whole pathogen and soluble, which means it's pieces of a pathogen broken down, which is like this little red, you know, rose-shaped tulip, tulip-shaped, you know, molecule here that has T3D bound on the surface. And so all this picture is showing, it's the same thing as before. You have that B-receptor, B-cell receptor, it's complex, B-cell receptor complex with Ig alpha and beta and it's cytoplasmic tails, and your co-receptor complex here, also with that CD19 with cytoplasmic tails, and you'll be working together to send a stronger signal into the nucleus of the cell to induce gene expression and activate the cell versus with the soluble one. It's just a little bit different because CR2, you know, because it's made up of these little chains of things, can actually bend a little bit more and actually attach the very top and kind of hold that soluble antigen in place so it can't go anywhere, thus activating this complex here. So it's just takeaway again, bound onto a pathogen or free-floating a B-cell receptor and its co-receptor complex could actually combine then and send these synergistic activation signals into the nucleus of the cell to activate it. And so when you have effective B-cell mediated immunity, it will depend on help from CD4 T follicular helper cells too. And so basically, you know, we have this activation, but you also need signals from T cells in order for this to occur. And so in primary immune responses, the activation of most naive B-cells requires conjugation, which means it's joining together with a CD4 T follicular helper. So again, T-cell that's in the follicles and it helps. So on the border of, you know, the follicular area, the primary follicle cell that recognizes the pathogen drive peptides presented by MHD class 2 on the B-cell. So the B-cell can also pick up antigen, you know, it's coming in from that afferent lymph from the nearest straining lymph node, present it to a T-cell, activate the follicular T-cell, which then in turn signals back and activates the B-cell. So it's a very, you know, collaborative friendship overall here too. And so it is kind of showing you this picture here. It shows that the B-cell receptor is binding onto antigen. It's anisotosing it and bringing it inside of it. You know, one can also argue phagocytosing it, breaking it down into tiny pieces, presenting it on that MHC class 2, which it makes because it is a professional antigen presenting cell, will present it to that CD4 T-follicular helper cell, in which case that activates that T-follicular helper cell, which then sends cytokines to then be secreted and bind onto that B-cell, which can in turn then activate that B-cell. And so it's a really nice collaborative friendship here. And so it triggers the secretion of the cytokines aisle 4, 5, and 6 by that T- follicular helper cell, which in turn can activate this B-cell here too. And notice again, we talked about CD40 with CD40 ligand on the T-cell, which activates with CD42. Again, you had multiple signals, you know, with the T-cell activating it. So it's just a really, really nice kind of full circle, you know, moment to kind of tie it back to T-cells. And so a lot of times, I think you saw it here, it did say a cognate pair in this definition. And so cognate pair, you know, since I haven't really explained that fancy term before, it's basically a CD4 factor T-cell bound via its antigen receptor. So it's T-cell receptor to its target cell. In this case, binding onto peptide MHC on a B-cell. You can also use the term cognate pair to, you know, refer to any type of interacting cells, especially if one of them is a lymphocyte. You can also see this called a conjugate pair too, but I did want to introduce the word cognate so that you guys didn't say you never taught us that. It is on this previous slide, even though it's flagged as not being a word, even though it's actually, I promise it's a real word. So that is the definition of cognate pair. It's just a very fancy sounding term here too. This one is showing too. We talk about how CD40 is important. It's kind of like that third signal almost, you know, with T-cell, especially CD4 T-cell activation. It's 4-1BB, 4-1BBL with CD8. But it is critical because again, if a person does not have CD40 ligand, they're actually unable to form germinal centers. It's an autoimmune deficiency or an immunodeficiency known as CD40 ligand deficiency. We'll actually talk about it a little bit in module four, but your textbook did include some pictures of it in this chapter just to show you that this is what a normal lymph node should look like when you stain it. And it actually shows germinal centers, but this is actually from a patient with CD40 ligand deficiency. You actually won't see the formation of any germinal centers within their lymph nodes, which is actually horrifying here. And so again, the consequences of that, you know, you might not have full B-cell activation, but particularly you definitely won't have germinal centers to be able to do isotope switching and that somatic hypermutation and affinity maturation. So the patient would be considered immunocompromised overall. And so our next one, effective B-cell mediated immunity depends again. So we are still continuing on that T follicular helper trend, but it does depend on helping those CD4 T follicular helper cells. And so we do talk about it that for the most part with most naive B-cells, we require conjugation with CD40 cells. However, because again, I've already introduced that exact same thing up here. However, cue the, you know, unique feature. And so it is possible in a, you know, minority in a small group of B-cells that it is possible that signals generated from those B-cell receptors and co-receptors, especially cross linked together and in close contact with many of them can sometimes be sufficient enough to activate a minority of B-cell population without T-cells. So this is the exception because again, we say most B-cells need that assistance from the CD4. And so you guys can kind of guess, you know, which population of B-cells we're referring to. And again, it's predominantly these B1 cells, which don't need T- cells to help them. And so we call this T-cell independent activation, but there are some things that need to be met and some criteria that need to be met in order for this to occur. And so the epitopes usually must occur in dense and regular arrays. So it means they have to be close together, like in dense, like packed in and regular arrays, meaning they're close enough on the surface here, which would then allow for a lot of B-cell receptors to cross link and get really close to each other on that B-cell surface here. Because again, it produces that dense clustering of B-cell receptors and co-receptors, which again, if you have enough of these close together, can actually have a strong enough signal just due to all of these intracellular pieces together to then signal to the nucleus and activate those B-cells to proliferate and differentiate without the help of T-cells. And because of that, these dense and regular arrayed antigen on the surface that can activate some B-cells, particularly those B1s, we call them thymus independent antigens because of that. And again, because the response doesn't need T-cells to help them and activate those B-cells, we call those antigens specifically Ti antigens or thymus independent here. And so again, the second panel is just a more zoomed in version of what's happening at the surface. But again, if you have those dense and regular array antigens or Ti antigens on the surface, you'll get enough cross linking and clustering of these B- cell receptors and their co-receptors to trigger a strong enough signal to activate the cell without T-cells. And again, it is primarily B1 cells just to know. So going back to our classic conventional ones though, follicular dendritic cells in the B-cell area. So again, primary follicle will store intact antigens and display them to B-cells. This is a really beautiful picture here too. It's showing the dendrites of a very branched out follicular dendritic cell showing that it's taken up pathogen to these little nodules that you kind of see on the surface here. Those are pathogens and antigens bound to complement receptors on the follicular dendritic cell surface. So it can present it to pathogens in the area here too. But it is just a beautiful SCADA electron micrograph of this, especially when you can see the bound pathogen on it as well. But your 90 B-cells will recognize antigens captured by sub-capsular sinus macrophages. So again, sinus is right below the capsule here. Macrophages and follicular dendritic cells. So like I mentioned before, it's not just dendritic cells that travel in from the afferent lymph. You can also have antigen and even parts of pathogen come through into the nearest draining lymph node via that afferent lymph. And here it's already shown that it's antigen bound on by complement and particularly some of the classical complement proteins here. Well, classical, but also you can see the C3D with any of them technically. But anyway, when the complement bound antigen travels through the afferent lymph, it can actually get captured because macrophages also have complement receptor too. So it can actually bind on and grab onto that antigen labeled with complement that as it travels through here too, but then it can also present it to B-cells. So B-cells can see it displayed on the surface of macrophages and even follicular dendritic cells. So it is showing a follicular dendritic cell with that complement receptor too, holding on to that antigen, in which case B-cells can also kind of bind on and recognize it and encounter that antigen presented by either macrophages that are tissue resonant in the lymph node or even follicular dendritic cells that are resonant in the follicles here too. And so it allows those B-cells to screen it and they kind of just basically hold it in place and the B-cells can check it out as they're kind of doing their travels throughout the various lymph nodes here too. And so if they encounter a particular antigen as they are surveying, so all these different B-cells will be surveying it, some will respond, some don't, you know, they don't recognize it, they don't like it, they'll leave and travel to the next one. Some of them do like it and say, hey, this isn't the bad guy I'm proud to fight, they're going to stick around. And so at this point, those antigen activated, so they've encountered the antigen, they need that second signal from that T-cell and so they'll move close that T-cell area to then find a follicular dendritic cell for those final steps. So it seems a little complicated, we'll break it down here, but B-cells activated by antigen in the B-cell area will move to the area boundary where they are then helped by an antigen specific effector T-cell known as that T follicular helper cells coming from the T-cell area. So again, they're coming to this border of this T-cell area and that yellow area, which is that primary follicle here. So what it's showing, both naive T-cells coming from the thymus and naive B-cells coming from the bone marrow will enter for the first time via that high endothelial venule. Those naive B-cells will first travel to that primary follicle, those naive T-cells will travel to the T-cell area to look for dendritic cells. And so these B-cells here will go and encounter, you know, the follicular dendritic cells and it happens to have their antigen, they will then travel to this border of the T-cell area here. And at this point we're showing T-cells that are specific for that antigen presented by, you know, other dendritic cells in the T-cell area here, you know, that will trigger those T-cells to proliferate and differentiate, especially when you have those T- follicular helper T-cells, those guys will travel, so showing them traveling here, up to that, you know, joining point here where they can see their buddies and their friends, their cognate pair here, they're forming their cognate pairs. And those antigen-activated B-cells will now present the antigen to those effector, you know, activated T-cells here, thus forming those cognate interactions and cognate pairs, which can then allow those T-cells to fully and completely activate the B-cells to start differentiating to plasma cells and memory cells and actually, you know, fulfill their lifelong purpose. It's a lot of more detail here, but people actually figured out kind of what's happening in terms of like the signaling between the two cells here. And so this is really cool scan electron, a micrograph and even just fluorescence that they've shown, how they know that the cytokines are actually being expressed on the surface, which is just wild here. But those T-follicular helper cells will help at antigen-activated B-cells through their cell surface interactions using that CD40 ligand and CD40 and by targeted delivery of secreted cytokines, the B-cell surface. So again, we have, you know, the first and second signal, they're interacting here, that CD40 with that CD40L, particularly here too. So we have this conjugate pair here, the T-cells are starting to synthesize cytokines in their Golgi apparatus, because obviously they're going to pack it up and ship it to the surface here too. And the T-follicular helper cell will actually reorganize its cytoskeleton using a protein known as an MTOC, which stands for microtubule organizing center. But MTOC will actually help move the Golgi apparatus closer to the side where the T-cell is bound onto that B-cell here, that's allowing it. So once the cytokines are released, they're already close to the surface, and they can release them directly into that kind of bound area with that B-cell so they can directly bind on and activate and, you know, target specifically that B-cell here. So that's kind of showing what's happening here. But it is really, really cool overall that they've done these really, you know, in-depth microscopic experiments to be able to see all of this. I honestly, oh, I'm at 41 minutes. Oh, I've been talking really fast. Well, I'll keep going then. We'll see how much we can get through so that you guys have access to everything we need, since we have been able to move a little bit farther ahead nicely. I know this is a lot and pretty fast, but just break it down and go through it slowly. You guys got this. I have full and utmost confidence in you, and if at any point you get stuck, obviously office hours are a place to be. So we talked about a couple different focuses with the clonal expansion of B-cells here, too. And we'll talk about one being the primary focus, our first focus of clonal expansion in that those medullary cords will produce plasma cells first secreting IgM, because again, IgM is the first immunoglobulin molecule these activated B-cells are going to produce. And so it's showing here B-cells activated by antigen and T-cell cytokines can differentiate the plasma cells in two waves occurring at different sites in the lymph node. And so this first primary focus that we'll talk about for expansion of the antigen activated B-cells is in the medullary cords. So we first have that interaction in, you know, that's the border of the primary follicle and that T-cell area. And with that first interaction, they will travel down to the medullary cords, in which case those B-cells will differentiate into plasma cells, which can secrete that IgM looks like a little snowflake, but that's our pentameric IgM here. And so some members of each clone of those cognate pairs differentiate to plasma cells, which secrete again that antigen specific IgM, while the other cognate pairs will return to the primary follicle in the cortex. So this first primary focus is again, this movement towards the medullary cord and the production of these plasma cells to secrete antibody, because again, you want to start producing antibody as quickly as possible. The secondary focus for expansion is actually within that primary follicle. So, you know, one focus is traveling here to produce plasma cells, which can then immediately go out, you know, help secrete antibodies and really start fighting the infection early on. But then part of it that returns here, you know, to form those memory cells, as well as the germinal centers, that's the second focus. And so that second focus here is when we form those germinal centers, have more division of those cells. And within that germinal center is where we see the B-cells undergo affinity maturation and isopep switching of their B-cell receptors. And so that second focus, that first focus is again, send soldiers out to go fight in the war and help keep everybody safe. The second focus is again, now let's create super soldiers and like the super specialized response and ninjas and all the cool stuff here so that we can still mount an even stronger response if we ever encounter the pathogenic pathogen again, or even if we have to send like a second wave of cells to go help fight the infection. And so again, that primary focus is that first response where we're traveling to medullary cords and differentiating the plasma cells to secrete antibody. That second focus is when those cells travel back to the primary follicle and form that germinal center with that somatic hypermutation and affinity maturation and isotype switching overall. And the somatic hypermutation and isotype switching occur in that specialized micro environment of the primary follicle, which is now known as that germinal center, which we talked about. We're focusing on that here now. And so this is showing you the anatomy of the germinal center. It's a little bit more than we've ever elaborated on before, but you've seen this picture before. Again, it gets larger as it grows and there's more proliferation here too. And again, it's right on the border of that T cell zone, but within that primary follicle here. It is showing you this really, really nice morphology within this germinal center here. Within it, you will have follicular dendritic cells because they're still going to help and support the B cells within here too. You'll have T follicular helper cells in the border here too. And we split it up into what's known as your light zone and your dark zone, which I'll talk about in a second here. But I want to elaborate first on some terms and it's centroblasts and centrocytes. By definition, a centroblast is a rapidly dividing B cell and that forms the dark zone center here versus centrocytes are the ones that once we've gone through this rapid proliferation, they're kind of on the outside here, but these are now mature and non-dividing. So they're not dividing anymore. And these are B cells that are now out in this light zone interacting with the follicular dendritic cells. So when they're rapidly proliferating, they're known as centroblasts in this inner, deeper circle known as dark zone. And then when they've matured and they're not dividing anymore and they're out here in that light zone, they're interacting with those follicular dendritic cells. Here's a great frosted image showing that you can see how densely populated the dark zone is versus the light zone is a lot less. Mantle is just kind of the excess space around there, but the T cell area is surrounding this here too. And so this is just showing staining to determine where everything was here too. And so the centroblasts were stained green. That's why you can see them densely packed here. Your FDCs were kind of a red stains within this light zone area here too. And they have T cells also in a blue stain and you have a smaller number of T cells in the light zone area. Those are the T follicular helpers, which do help activate the B cells too. And so you're rapidly dividing B cells in a centroblast or in this dark zone. Your centrocytes when they stop dividing and become those small, centroblasts stop dividing and become small centrocytes. Centrocytes are, you know, why we joked that you can't tell the difference of a small lymphocyte because again, they're tiny cells. Nucleus takes up a large portion of the cell. You really can't, you know, determine it from a T cell here too. But again, it's because they are small and they are within this kind of light zone area at first. This is just another stain of it. It's a light micrograph of the germinal center here too. You can see some of the follicular dendritic cells here. I know you can't distinguish a centricite, but it is the smaller, smaller tiny dot here versus a centroblast. It's a little bit larger. Again, I won't show you this histology by itself and expect you to answer it by yourself. It might be supplemental to a question, but you need a lot more information because you guys are not pathologists and this is, you know, rough without a lot of practice. This is just another stain they did, but it uses DAB to be able to show the follicular dendritic cells in this kind of brown goopy stain kind of throughout here too. But again, germinal centers is where those B cells undergo affinity, maturation, and isotype switching. And that antigen mediated selection of centrocytes drives the affinity maturation of the B cell response in the germinal centers. Again, you know, the centrocytes are all just rapidly dividing. I'm sorry, the centroblasts are rapidly dividing. You have this massive variety of centrocytes out in this light zone here and it's the centrocyte that drive the affinity maturation of the B cell response. Because again, we're going to test all of the B cells, the centrocytes within this light zone area to see which are the best affinity to select them to continue on. So remember that little light popping bubble video we watched a few days ago with a very, very light blue, how it's arguing lighter and lighter and lighter over time as a somatic hypermutation produced cells with better affinity. That's specifically what we're looking for. And so it is that antigen mediated selection of the centrocytes that drives affinity maturation of the B cell response in the germinal center. And so after undergoing somatic hypermutation, centrocytes that have high affinity antigen receptors will be rescued from apoptosis. So again, I go back to watch that bubble popping video. I think it's a great visualization of what's happening here. And so again, you know, the B cell becomes activated. It undergoes that somatic hypermutation of its immunoglobulin variable regions in those rapidly proliferating centroblasts in the dark zone of that germinal center here. And it can have, you know, two, and again, it's completely random. So there are two possible outcomes. You can have low affinity surface immunoglobulins and high affinity surface immunoglobulins here too. And so that picture will continue on in a second here, but it's showing that centrocytes whose B cell receptors have acquired mutations that reduce affinity for the antigen are induced to die by apoptosis. We'll continue on to that next picture here. Again, it's the same slide twice. It's just to zoom in on this next portion, just a little bit more. I had to continue on, but the text is the same between both slides. And so again, you have this low affinity production. Because of that, the B cell receptor really won't cross link as well. Because remember that horrific video with like, it's the same video with the popping bubbles, but like how the B cell kind of just like flattened out. And there was just this really awful sound with that. It helps me remember it. But the B cell receptor is not cross linked and the centrocyte can't present antigens to the T follicular helper cell. So that will actually induce the cell to die. That's how we select the ones that have better affinity overall, that affinity maturation. And so, you know, if you have this terminal center centrocyte with high affinity, it'll lead to the B cell receptor cross linking and more of them binding onto that surface, thus leading to signals allowing that centrocyte to survive and divide. And because of that, it can then differentiate into plasma cells and continue on. And so it triggers these cells to express a protein known as BCLXL, which helps them become plasma cells. And it's an intracellular protein, by definition, so it should be bolded, that helps prevent apoptosis and ensures that cell survival. So I'll stop here. But next class, I will pick up with that video again, so you can put this back into that context of that video that we watched before with that affinity maturation and that effector function. And that really kind of ties it all together now. All right, so we will pick that up with next class, but it looks like we're getting through the stuff pretty good. So I appreciate, again, all of your patience with just the chaos with everything that's going on. Obviously, you guys know it's 1am right now. So it will not be a bright and early morning for me tomorrow, but when I have chances around my meetings tomorrow, I will rerecord Friday's lecture for you guys as well.