Antibodies Part 3 PDF

Really quick before we get started, it's come to my attention that you guys don't mess around with Canvas that much because all the pages are different. So I did wanna point out that at the beginning of every single module that I do, I do have an overview page that kind of talks about what we are go...

Really quick before we get started, it's come to my attention that you guys don't mess around with Canvas that much because all the pages are different. So I did wanna point out that at the beginning of every single module that I do, I do have an overview page that kind of talks about what we are gonna go through and talk about. And I do have like big objectives there that are the big picture of what we're gonna be covering as well as the weekly structure. And then if you go through to the individual weekly ones as well, you will see the same thing, weekly objectives that kind of cover the big picture stuff that we're gonna talk about that week. So for those that are a little confused on where we're going and where we're at, that's there. So that can be something to help you if you are struggling with kind of the arrangement of things but we are finishing off antibodies part three. And again, a lot of this is repeat on purpose because I want you guys to be really comfortable and to have seen it multiple times because the best way to remember things is through repetition. So you guys already do know this, the different structure of these. And for the most part where they're found we'll elaborate on those a little bit more today. Again, this is a repeat of a slide you've already seen so there's no point in panicking on this one. We've already presented where the kappa, lambda and heavy chain clusters are located in the different chromosomes. And it basically just gives you the overall math that there's over and it does the math in the bottom for you, but over 26 million possible combinations and rearrangements not including any kind of mutations after the fact or variations, et cetera, like somatic hypermutation, affinity maturation, stuff like that. And so it does give you an appreciation for the broad diversity of the adaptive immune response overall. And again, another refresher from earlier with immunoglobulins here, we do have both a lambda or a kappa light chain as well as the heavy chains which are all in chromosome 14. Bless you, this is just another picture. And again, this is using either an IgE or an IgM just to show you the structure here since that constant heavy four is optional. But again, just different view of things we have already learned in case this picture helps you remember kind of what's going on. We already talked about the five different heavy chains and the Greek letters that are associated with them. And this is what gives you the letter of the immunoglobulin molecule, like alpha is with A, mu is M, delta D, gamma G, epsilon E. Isotypes have used this term a lot, but again, isotypes are genetically different forms of the light chain, either lambda or kappa or of the heavy chain. So an isotype could be mu, delta, gamma, epsilon or alpha. And so the class or the subclass, again, is determined by the heavy chain. And so again, we're named based on these guys right here. Again, those pictures, because yes, you could see these pictures on the exam. You could see the picture by itself. You could see the picture with a description. You could see a feature of the picture and ask to identify a key point, whether it was like a constant or variable or antigen binding site, things like that. The other important thing to know that we'll talk about. In there, they all occur as monomers when they're bound onto the B cell surface, but when they're secreted, I-G-D-E-N-G are monomers. A can be both a monomer or a dimer. We'll elaborate more on that next week. And I-G-M will form a pentamer overall here. I do not expect you to memorize this, but again, it is showing you the different subclasses featured here. Again, you can kind of like, this is kind of a, oh, this makes sense. Like I-G-M forms a pentamer, right? There's a pentamer larger than a monomer. Yeah, so it makes sense that the molecular mass is massive because it's a lot larger than the rest of these guys here. I also don't expect you to memorize the serum levels. I think that's a little absurd. However, I would note ones that are really high just because it does kind of contribute. Like again, I-G- D, we really, I told you, we really don't see that in the blood. It's not a normal thing. And so it makes sense that we'd see very, very low levels of I-G-D ever. Same with a normal level of I-G-E. It's not something we normally expect to see unless there's something pathogenic going on. You have a severe allergic reaction or you have a parasitic worm infection. And so again, it kind of contributes to like what our normal serum levels are that at some point we always have some level of I-G-M and for the most part, I-G-G and I-G-A present, but the other guy is not so much. Half-life is also just unique just because some do last longer and it makes sense. Our I-G-Gs are very useful antibodies which we will continue to elaborate on. And so they last a little bit longer in the bodies. Different ones have different half-lives though. I-G-A-E disappears very quickly. Its full life is only about six days, so less than a week. Same with I-G-D, it really doesn't stick around that long. And so again, not memorizing, but looking at general trends with these guys can help you overall here. And again, it is just showing you that the molecular mass for I-G-A is in the monomer form. Molecular mass for I-G-M is in the pentamer. Here, I do expect you to know this. The best way to tackle this because it seems overwhelming is to focus on the big picture and like the heavy, yeah, the heavy duty extra pluses because that means that's its main function. So I think it's really that critical to be super nitpicking and be like, yeah, like I-G-M is really good at neutralization. No, that's not, it's okay. It can do it, but its primary function is activation of the compliment system. I-G-M is very, very useful to that due to its features of agglutination. And so it's kind of tying in all the pieces. You have to put the pieces together to figure this out. Again, for whatever strange reason, I-G-D, very, very good at sensitizing basophils. Not entirely sure how or why. It does. We know that that's a fact, but beyond that, it doesn't do anything else. Like we really just really don't know anything about I-G-D. I-G-G-1 is one of the more effective I-G's, I-G subtypes of immunoglobulins. I-G-A, we expect to see more neutralization and transport across the epithelium because think about it. Where is I-G-A found? Nucosal membranes. What barrier tissue is at mucosal membranes? Epithelial cells. Whoo! So it kind of makes sense when you put the pieces together here. This one is also good at just diffusing into extravascular sites because again, think about it. Your sinuses are heavily vascularized. Mammary tissue heavily vascularized. And so it does make sense that we would have movement. And this is another reason why this one can cross and diffuse into extravascular sites around it. And so again, it makes sense if you go through it. We talk about sensitization of mast cells and basophils. I-G-E is heavily involved in allergic responses. It is the primary mediator of anaphylaxis. And therefore as a consequence, mast cells are the ones heavily involved in anaphylaxis, dumping massive amounts of histamine. And so it makes sense that I-G-E has high levels of sensitizing mast cells and basophils, which also plays some role in allergy. That's why it's a little bit less here. And so again, focus on those big picture features and try to differentiate them that way. Don't try to memorize every single thing about it or you're not gonna be able to function. Go through and work smarter, not harder and focus on the key important features. Yes, Collette. No such thing as some questions. Yes and no, yes and no. I don't stress, I'm not, for this, I'm not gonna stress you out about that. That's too nitpicky, that's not something, yeah. Oh, if you're talking about this, yeah, that's the same thing in this case. I just mentioned, if you use it, if you ask me broadly, sensitization and activation, those are technically two entirely different things in this context when it's talking about IgD, no, it's the same thing. It's working with basophils. That's the gist. Basophils, IgD. Nope. Oh, well, your textbook, okay. I won't ask that. I forgot, on the previous slide, I'll say mast cells. It may not be as well known, but that textbook, I would not ask that question then. Okay, that's not fair. The focus would be heavily IgD on this one. IgE though, oh yeah, mast cells. You better think that with, like anaphylaxis, you're going to die of severe allergic reactions. I've had epinephrine shot me recently. Yes, IgE is heavily involved in that, so I wouldn't know that one. That one's the focus when we think about mast cells. The primary one is IgE. So focus on that for this part. I'm not gonna try to trip you up with, it did describe mast cells on like a previous slide, with IgD, good catch though. So we're talking about IgM. It is secreted as a pentamer of immunoglobulin monomers. It's attached. It's five of them stuck together by what's known as a J-chain, and that's holding it all together here. And so it is showing you that five of these will be secreted, and then they join together with this J-chain, and that's what it looks like under scanning electron micrograph. You can see one, two, three, four, five. And so it is for joining chain. That's what the J-chain stands for. Please don't confuse that with a junctional segment. Those are two entirely different things, unfortunately. They didn't communicate when they were naming things. They are cross-linked by disulfide bonds here to that J-chain as well. And because they lack a hinge region, it is much less flexible. Because remember, if we talk about those hinges, there's a little bit longer bond here. These guys are like frozen shoulder attached. They can't really move or bend as much. So it's compensated for by having this pentameric form, because look at it, like say, you know, with a normal immunoglobulin molecule, they can kind of bend and reach and try to touch two different things, versus this one because your pentamer, you have 10 different binding sites, total one, two, three, four, five, six, seven, eight, nine, 10, it could still grab something from over here and over here. So it does compensate for the fact that it's not flexible by forming this pentamer molecule here. And so, especially with pathogens having multiple identical epitopes on the surface, IgM can bind many sites. Like it can fill, it can keep binding until it binds up every single one of these 10 spots on the surface, which is really cool. And we talk about IgA, it's the only other one that can form a multiple form. And in this case, it forms a dimer featured here. And so you see this in mucosal lymphoid tissue, it is synthesized as a dimer in association with that same J chain. So J chain helps combine them if they form like their Power Rangers multi-combined form here. And so it is the exact same molecule here. So in it, again, you do have disulfide bonds to that J chain, but not directly to each other. So that's what the shape of it looks like overall. And this helps it with mucosal surfaces. Now IgG, we talked about IgG is like the cheerleader of the group, it is very flexible and or gymnastics or Simone Biles of the immunoglobulin world here because it can do a little bit of everything and it has a very elaborate floor routine as needed. And so it can move a lot of different ways in order to bind onto pathogens in different spots. It doesn't have to compensate like IgM here. And again, the most flexible region is that hinged portion, although it can kind of wiggle its tail a little bit, which is kind of cool. And so this does also allow it to, another cool thing is notice here. So if I was at a molecule, and this is like the arms here in the body, it can bend its elbows like this one, the bending of the fab elbow. So it can also rotate and twist. It can bend, it's like one of the most flexible, like make a cheer routine out of it. It can wave its arms, it can rotate technically, it can wag its tail and it can bend its elbows. So you've got some really nice movement of the molecules here. This one is just specifically showing you the IgG1 subclass for an example, but we will look at the other ones later here. And so the cool thing about this, and by having that wagging tail, it helps to actually accumulate the binding of C1Q, which is one of those complement molecules so it can also help with that complement pathway, like we talked about earlier here too. Because again, it can get compliments attention. Come over and say, hey, like I've grabbed onto this, like, come here, give me a hand. And so, it's like when your dog wants you to come place, it's wagging its tail, it's really trying to get you excited about playing ball. It can attract compliment via the wagging of the FC tail. But like that previous chart that you guys panicked over, it looked like we have four different subclasses of IgG and they have different but complementary, complementary meaning they benefit each other with their functions. And so IgG1 was shown earlier. Notice how IgG1 looks identical pretty much to IgG4. IgG1 has a slightly longer hinge region, but if I gave this to you on an exam without IgG4 next to it, I don't think you guys would be able to eyeball it and be like, yeah, that's one of the longer ones. So that would not be a fair question for me to just give you a picture without anything else and just say, hey, is this IgG1 or G4? There would have to be something else there like a description of its functions, how they're different for you guys to be able to determine what it is because picture alone really wouldn't do it and that's not really a fair question. So there would be something else. However, look at this giraffe over here, IgG3. IgG3 is very, very unique because it has many, many disulfide bonds in a very long hinge region. So it can do a lot of bending because of that. And so, you know, IgG2 does have a little bit more bending too because notice again, slightly longer hinge region that's more obvious by the number of disulfide bonds there here too. But again, it does contribute to some of their functions because again, it allows them to do different things. Like when we look at IgG3 and then go back to our lovely table, oop, one too far. IgG3, really good at neutralizing, obstinizing, it's good at a lot of different things. It can also activate the complement system because it can wag its tail because notice its tail is very far away from the top of its body. It can really signal too. C1Q, it can also diffuse into extra vascular sites. Most of the IgGs too can transport across the placenta. That's something else I wanted to mention. The other guys don't, but I wouldn't be too nitpicky that I'm like, it barely crosses. All of them can cross. IgG1 is the most common one though, but it is pretty cool. So you again, try to tie things together and connect them in any ways that you can because it does help overall. And yes, these pictures are fair game, but again, like if I did G1 and G4, those are similar enough that I would have to give you some other piece of information because they look very similar on an exam. Like if I just ball parked that, I wouldn't know if it was the longer one or the shorter one. I'm not giving you guys a ruler either. So I would have to give you additional information. Again, I don't want you to memorize this. This is a little bit too much information, but kind of take it into context overall. We do actually see a lot more IgG1 overall. And again, it does have a slightly longer half-life too because again, it's a very effective molecule. G3 is, you know, it's longer, it's more awkward. It doesn't live as long and it doesn't make up as much of the proportion in your blood. So this is actually if we're looking at just like normal blood concentrations like without any kind of active infection that we're aware of, things like that. Again, length of heavy chain hinge, amino acids, not important, but again, it makes sense that, you know, 62 would be IgG3 because it's the longest. It's the giraffe of the group. As a consequence, it has a really, really long neck. Don't you think it would make more sense that it is the most susceptible to cleavage and being broken down and damaged because it's, you know, kind of vulnerable in this very, very long giraffe neck. I mean, if you were gonna be aggressive and try to take out IgG, you could just, you know, beheaded pretty quickly. So again, it kind of makes sense when you put this into the context of the big picture. It also is one of the shorter half-lives because again, it makes sense. It's more susceptible to being damaged and broken down. So therefore it does have a shorter half-life. It is very good at activating the complement system though. And they're all pretty good at activating protein, or like responding to protein antigens. Some are a little bit better at responding to carbohydrate antigens. And they play a loose role in also contributing to the allergic response. But these are things that you're gonna see when we talk about allergy testing later. But you guys know, hopefully very briefly, if you have anaphylaxis, that is an acute allergic response. It is heavily IgE. But if you've ever done an allergy test where it's like, okay, cool, they did a quick skin *****, you didn't flag on anything, so they inject stuff into you. That's measuring your IgG response. And IgG does play a role in allergies, but this is gonna be more of your long-term delay. So you touched poison ivy. Five days later, you have a rash. IgG is to thank for that. So that's kind of the playing role there. But again, put it into the big picture of everything here. I don't want you to memorize it, but again, a lot of this makes sense. It's the longest one. Therefore, it has a longer neck. It's a little bit easier to be beheaded, proteolytic cleavage. Therefore, because of that, it's gonna have a shorter serum half-life. But it is very good at activating the complement system. So very, very good early on. You can imagine and link bacterial infections. So we do see changes overall. Another cool thing about IgG4 specifically, this is the only IgG molecule that can do this. It can become what is known as functionally monovalent. So if you look at this here, like this, imagine like this is for one. So this is for like a lipopolysaccharide on the surface. And this is for some other cell wall component. It can actually split itself down the middle and fuse with each other to create one where each antigen binding site only gets one. That's why it's functionally monovalent. It has one binding site for each of the two separate things. So it could create two of these molecules overall, because again, you're cutting it in half and fusing them together in some sort of like sick hybrid experiment. But it is cool because it does give these IgG4 molecules more diversity. And so this is something that separates it and sets it apart from IgG1 even though it looks similar. It can become functionally monovalent. Does that make sense? There's one branch that's red, one branch that's blue. This is not the same binding as this one. That's why the pictures are different. This has got like a weird little oval thing and this has got like a bunch of little circles. Makes sense? It's a really cool thing about it though. And so it basically interacts in the circulation in the blood and swaps parts to become like a more effective killing machine basically here too. And so it has two different binding sites in this aspect. So if I asked you like, if I were to ask you how many different binding sites it has, it has two overall, because that's one and this is one. Sounds good. And so, thus they only interact with the pathogen or protein through one binding site. So this other part could be hanging off or it gets lucky every once in a while and both things are from the same bacteria, like one's lipopolysaccharide or one's lipotychoic and the other's tychoic acid. Close enough. This is again, a summer slide of things you guys have already seen before. And so again, just to tie it all together, where we get these molecules is from that recombining and creation of the B cell receptor through merging this heavy chain and then that light chain. And so here again with the heavy chain, it always goes first, which we elaborate more on today with B cell development. The party doesn't start until the DJ shows up, always. So the D and the J join first, then the variable region joins in here and then it will fuse with one of the different constants. And if it's gonna be membrane bound, we cut it at that polyadenylated site after the membrane coding region and then it could get bound on. And again, if you were trying to count like this is the variable heavy and then we have constant from the top, one, two, three, four. And then we'll go in and we'll make the light chain. And that's always gonna be because it doesn't have the diversity chunk, the variable with the junctional first and then we'll fuse it with a constant and it's either going to be kappa or lambda. You can't have one kappa and one lambda. They're both kappa or they're both lambda for the chains. And that goes on and creates the light chain of your immunoglobulin molecule. So we talk about that some processes are reversible and some are not. For the most part, most of them are irreversible because once you commit to something like you cleave something out of the DNA, you can't go back. So when we're rearranging the variable segments, if we cut something out of that center and when we're combining the D with a J or the V with a J, that stuff gets cut out and it's gone. So therefore we cannot reverse that. Same with junctional diversity. We start joining those little rearranged nucleic acids and adding in, you know, you have your palindromic ones and you add in your new nucleotides. We can't reverse that. Unfortunately, you know, the body could technically come in and remove stuff, but it's not thinking enough to be like, oh yeah, that's new. We want to remove it. No, that's irreversible here. Transcriptional elements, we didn't really talk about that, but that is an irreversible process. Transcription activity with co-expression of surface IgM and IgD. We talked about alternative splicing and because there's no switch region between M or the mu chain and the delta chain that will express both on the surface. This is considered reversible, because yes, we'll express both IgM and IgD on the surface, but once the cells go out into circulation, eventually they will lose their IgD and that is why it's called reversible because we don't have IgD on the cells for the rest of their lives. Same with splicing, we're changing the splicing. So this would be cross with our isotope switching. If you're doing isotope switching here, it's not reversible, but it's regulated. Because again, if we do cut mu out to switch to IgG, it's gone. We've removed that chunk of the DNA. We can't go back and add it back in. So it's not reversible, but it is considered regulated because we do control when we are about to switch. And again, that's usually with subsequent encounters and anytime that cell is dividing, it will then shift to that new isotope as we're going through here too. Again, anytime we have point mutations of genomic DNA with that somatic hypermutation, the body actually does not reverse this back. I know we will probably talk about genetics. There are some instances where the body tries to correct things. It's screwed up. This is not one of those cases. It's gonna continue on making that, inducing the point mutations, but it's what we want because it provides more diversity at that antigen binding site, which helps with that affinity maturation of these cells over time. And again, if it changes and the affinity decreases, we're just not gonna select for that cell. It's gonna get removed from the population and we will just continue to like those little popping bubbles that got lighter and lighter as we went on. We are selecting more so that ones that bind on with better affinity here too. Isotype switching again is that irreversible because once we cut stuff out, we cannot go backwards here too. All right, cool. And then also Winifred Ashby is you're also a scientist. She was the first researcher to establish lifespan of red blood cells in humans was longer than two to three weeks. Didn't we already talk about her? Oh, you're welcome. You already know her. Could be another question again. But she used agglutination to determine that. And that's why she's relevant to this because that is a technique we use with, it's based on antibodies. We use antibodies to bind on to antigen to be able to cause this agglutination here too. And so this process, which was known as the Ashby method was used for treatment during chronic anemia and revolutionized blood transfusion during World War II. Cause at this point they could do blood matching which saved a lot of lives. Cause before that, they just, you know, I don't know what your blood type is, hook us up and see what happens. And then, you know, I have a serum reaction and I die. So we'll talk about blood rejection and transplantation in module four, but it is a very, very, very precise science which is pretty cool here too. So easy. You might see someone you guys already know. And again, we also mentioned Brigitte Escones, another person you guys already know. So you're on the multiples right now of hopefully some easy questions tossed in. All right, so this is Phil and his bat wings cause it's now Halloween. So feel free to send me pictures of your pets in costumes. Extra credit if they're struggling and trying to get away like cause then I know you really had to work for it to get them into it. But Phil clearly loves his costumes. He also likes sleeping, but Phil is adorable. So we have pictures of Phil. Thank you, Abby, for sending that in. So today we're gonna talk about the phases of B cell development. So we've talked about B cell receptor editing. We've talked about immunoglobulin. Yes, OFC. Yes. Yeah, that could be one or any of the differences like unique differences in that chart of what their functions are. There would have to be some of my piece of information for that one. Either I wouldn't ask it or there's gonna be something else cause you can't just put the picture alone really. Technically one is longer, but on an exam really can you tell the difference now? There would have to be more information. So we've kind of already talked about a lot of this stuff. Now we're gonna synthesize it all together and talk about the phases overall and where these pieces go. And so a lot of today is going to be going through phase one which is that receptor development. If we have time, I've already posted B cell development part two for Monday. We may actually get to some of that today just because I did look ahead and I forgot. Activation and effector functions of antibodies are actually a lot of slides. Some of them are about 40 slides total. So we might potentially spill over and move immunological techniques to module four. I will update you guys as we go through. But again, it depends on how much we get through today and next week. And I'll adjust you guys aren't like, I'll message you guys on Canvas with any adjustments that we make, but I can kind of shorten transplantation and kind of shift immunological techniques to a little bit later in module four. So we'll get to it. I'll make sure you guys have enough material to like you have access to all the material for the exam ahead of time. So you know what you're getting yourself into. You have time to study. You're not overwhelmed even though you guys will be overwhelmed. Fact. And so we will go through it. We'll talk about B cell markers today. So a lot of these guys are gonna be definitions to know and we'll talk about how they're dependent on stromal cells, much like how T cells are dependent on stromal cells in the thymus for notch one. A little different for B cells but we will talk about that here too. And then this is more so from development part two but we'll start talking about B1 and B2 lineages and actually how they start to differ a little bit more than just the very basic definition we've already talked about. We'll kind of compare and contrast this with what you already know about T and B cells and we'll get into like positive and negative selection too. So these are the six main phases. These three occur in the bone marrow. That's why they're yellow. And then these three occur in the lymphoid tissue. I think their categorization is a little bit different because bone marrow is red. So I'm like, why couldn't we flip it? But anyway, phase four, five and six are in secondary lymphoid tissue like your lymph nodes and spleen. Phase one, two and three are in the bone marrow and it makes sense because that's where they develop because look at this repertoire assembly. That's forming that B cell receptor which we've already kind of talked about. Then we undergo negative selection first. It's different, we'll talk about that. And then positive selection here too. And so it is important to know phases one through three occur in the bone marrow. And then obviously when we travel and recirculate out if you have to make an educated guess, clearly it's not still in the bone marrow because it says recirculation. And it's moved out into the lymph blood and secondary lymphoid tissues because we're looking for infection. So I will talk about this a little bit more but we will elaborate on how it is different from positive and negative selection with T cells. But firstly, you guys need to know negative selection actually occurs first. And so phase one, again, we are in the bone marrow and it proceeds through several steps inside that bone marrow. So we're in stages now, not those phases. We're in phase one, we have stages within phase one. You guys already know CD34. Oh, you've got this, it's not that bad. Phases are big things, stages are smaller. Like emos, well, actually you can't say emos, not a phase. I was gonna use that as an example, but it's not a phase. I don't know, maybe you went through a really, you're really into backstreet boys for a phase. Although that's another bad example because I still love the backstreet boys. I don't know, you guys went through a period where you really loved cabbage pass dolls, I don't know. Or you really liked the baseball and you're like, this is not my sport, but that's a phase. It's passing, stages are a little bit shorter within that because you're like, I don't know, you do backstreet boys karaoke at your middle school talent job. I didn't, it was Shania Twain, but it was different. Okay, so CD34, you guys already know that, yes, it was Shania Twain. CD34, you guys already know this, but this is one of the hematopoietic stem cell markers. Unlike the T cells, which leave to go to the thymus to develop it to stays in the bone marrow, which is really nice. So with that common lymphoid progenitor, we'll solve that CD34, but we'll also start to play a role and express CD10 here, which may actually start to like inactivate regulatory peptides that might direct it to going towards T cells. So we've got the common lymphoid progenitor. CD10 seems to be directing it a little bit more to that B cell, so just stay in that lymphoid, or the bone marrow here. And then CD127 is important because we'll talk about how IL7 is a cytokine critical for B cell development. 127 is part of the receptor for IL7, so it makes sense that we expressed it earlier, because again, we're in this B cell precursor, eventually we will need help from IL7, and so obviously we will need a receptor, or IL7 won't have anywhere to go. Eventually we get to what's known as a pro-B cell here, we'll start to express CD19, because where is CD19 found? All B cells. So that's important here, because it is a subunit of the B cell co-receptor, and so it does help, because if you do not have this, eventually you won't be able to activate your B cell too, down the line. So it is important that we start expressing that here. Don't panic. We will go through, you already know all of this. We're all, we're adding its names to it now. You already know this, you guys already know this. So you know a stem cell is a hematopoietic stem cell, or a pluripotent stem cell, it is the exact same. It's a stem cell, it has nothing else on it yet, so no, we don't have any H chains expressed, we don't have any L chains expressed, and we don't have an immunoglobulin molecule yet, right? Cool. So now we are slowly committing to this early pro-B cell. Pro because it's like, I really wanna be a B cell, I'm committing to this. You have early and then you have late, and then you have large and then you have small. This is the really crappy part. But anyway, you have early first. This is when the DJ shows up, because remember, we do heavy chain rearrangement first, and with that one, you have to combine the DJ segments first. You guys already know this. We're doing the heavy chain still, so obviously we don't have any light chain, and obviously we don't have an immunoglobulin molecule because we haven't done the heavy chain yet. So we're good. You guys know this. Then we move into the late. So you can assume that the pro-B stages are pretty much that heavy chain rearrangement, because at this point, we've already rearranged D and J, and so now we combine that V, D and J, and so we have that heavy chain combined. Pretty much in between, it is implied at this point that we're going through and kind of adding in that constant region and creating that change, because by the time we get to that large pre-B cell, we've at least made the heavy chain, which could technically function. We're gonna test it out, though, like we talked about with T cells. We tested them out here, and so at this point, we have already rearranged that V, D, J. The heavy chain is done, and then with that small pre-B, is when we start rearranging that V and that J segment here. And so, again, it's just continuing on. You already have that heavy chain. We're making that light chain here. We have that new chain in the endoplasmic reticulum, and then we'll add on that light chain to it, and so by the time we get this immature B cell, both heavy and the light chain have been rearranged, and we now have that mu heavy chain and either that kappa or lambda light chain on there, and so basically now we have that IgM molecule, which is the first one we create that's on the surface. So really, it's not that difficult. You just need to add the names to it now. So if you're a stem cell, then you're early pro-B, and we start doing DJ, late pro-B, which is that V, DJ. Large pre-B, we start to make that heavy chain. We've got that constant N, and with that small pre-B, we start rearranging that light chain and do that V to J recombination, and then eventually once we get to that immature, we'll have that IgM here. So it is very, very, it's just the stages. It's just the names for what's happening. We're just naming what's going on, so it's very, very easy. We want to name the pain, as my therapist says, we're gonna go through it slowly, but I'm just kidding. As you guys all look very pained right now. This is driven heavily by protein expression that's signaling and triggering the cells to undergo these different stages overall. Do you not panic? These are just memorizations right here. These are just definitions you will regurgitate. FLT3 seems to be important very, very early on with the stem cell signaling to B cells. There's a lot of research saying they think it's involved in driving that common lymphoid progenitor and potentially even committing it to that B cell. So again, it is just something to memorize. It is a tyrosine kinase definition here. We have KIT, so again, it's showing kind of as we go a little bit more on with this development here. The cool thing is they do kind of separate it to show you that these are later on in life, just a little bit here too, but especially with growth factor. KIT acts as a receptor for a signaling molecule known as stem cell factor, because it's helping to commit it to this early pro-B cell here too. Obviously we will need an IL-7, so that's where that CD127 comes in. It's part of this IL-7 receptor basically here. And we'll talk about IL-7 on the next slide and why that's so important. You guys know RAG, and because again, we're rearranging heavy chain, we'll test it and then we'll rearrange light chain and test it, makes sense that RAG turns off and on as we're going through it. Are we gonna have RAG on if we're currently testing a heavy chain? No, because we're not recombining anything, we're testing it. Are we gonna need it on if we are testing the light chain? No, because we're testing it, we're not rearranging anything. You guys already know TDT, it inserts nucleotides. You guys technically don't know this one yet, but we will talk about part of testing, but you guys will learn V-pre-B and lambda-5 today. These are basically, you guys remember the pre-T alpha when we tested the heavy chain? This is the synonym in B cells, you just have to switch the name up. So basically, it makes up that surrogate light chain which we use to test the heavy chain when we're going through it, so it makes sense that we will see it. Where is it? Oh, here. As we're testing that heavy chain, we'll start to see that there too. We do also start to express a little bit later on in development IG-alpha and beta. This is copy-pasted from previous slides where you guys have seen this, because again, you know that these are the molecules directly adjacent to the B cell receptor that have the little tails with the ITAMs that allow signaling and activation. You guys know most of these. They're only a handful of new ones. I promise you. Somewhere in your brain, you know these. I guarantee you guys do. CD19 found on all B cells. You guys know this one too. Bruton's tyrosine kinase is new and I'm introducing it now just because it is involved in development. You guys should probably already know about PAX5 with cancer, like Dr. Sedding will probably talk about PAX5 a lot. These two I'm introducing now because we will talk about them and refer back to them, we talk about issues in immunodeficiency, in module four. So they will be relevant and we will bring them back, but for right now, just a definition because we will talk about if we knock these guys out, we unfortunately take out B cells, which leads to some cancers and immunodeficiency disorders. So we're introducing them now, but we will refer back to them in the future. B cell development obviously has to start with stromal cells in the bone marrow helping them to progress on here too. And again, it's interaction with cellular adhesion molecules. Kind of the same thing, we talked about ICAMs with T cells in the thymus. They're very, very similar to these stem cells and early pro-Bs, we'll use an integrin, which binds onto these guys. This one unfortunately is really weirdly named. It's very late antigen four. So apparently there were three very late antigens before this one, but it binds onto V-CAM1. I don't know what I said and I don't want to know. Okay, let's hope it's not working out. I was like, the one day I hope it's not recording. Okay, this binds onto VLA4 will bind onto V-CAM1 on the stromal cells here. Additional cellular adhesion molecules because you need extra binding here, promote that binding of KIT on B cells, that stem cell factor. That's why those were important from earlier here. Activation of KIT will cause these early precursors to start proliferating because then we start editing all of these precursors in these early proliferative cells. We start rearranging the B cell receptors inside of them. And this is what gives us the army of B cells that we'll have overall in the end here too. Interleukin 7 or IL7 plays a critical role in the early development of B cells because it helps promote that proliferation of these early B cells as we're then going to go in and change their B cell receptors and modify them here too. And they also help with survival during transition from that pro-B to the pre-B stage. So T cells had notched. You have a lot of cell adhesion molecules in IL7. Helps overall with B cells staying in the bone marrow and also proliferating so that all of these baby B cells can then have all of their receptors modified, changed, rearranged like we'll do all of the recombining. And so we have all these baby B cells with different B cell receptors in their surfaces which give you your B cell army overall. And we already talked about this. Heavy chain, we're repeating it again. Heavy chain rearrangement occurs in those pro-B cells. Don't panic, this is very easy. Remember with T cells, how you have not. If you think of it as easy, it will be easy. If you think of it as hard, your brain's gonna go in automatically assuming it's a very difficult thing. We'll break it down, okay? We talked about that early pro-B and the late pro-B. You guys already know. It's pro-B as when we do that heavy chain rearrangement here. And so it's just kind of showing you that remember, you have options where sometimes you combine things and it doesn't work, right? The body moves on and tries the next one. That's all it's showing here is just giving you an example. In this case, we got very lucky on the first try. Boom, we added the D and the J and they worked out perfectly. Everything is great. So it was a productive yellow rearrangement, cool. So we go on to the next step. We have to combine that variable with the diversity. Well, what if the first variable we grabbed didn't work? Moves on or say it works and we got really lucky on both chances, it would continue on. And the cell would be allowed to progress versus if that first rearrangement didn't work, cool. We can move to that second one here and go through. And if that one doesn't work here, at that point, we don't have that heavy chain. It's non-functioning. The cells are going to die. They don't have a functioning heavy chain. You can't continue on. And so about 50% of cells die off at this point because they did not produce, you know, a productive heavy chain here. But say hypothetically, you know, the first one got lucky or your second rearrangement got lucky here. Those cells would be allowed to survive and continue on to those pre-B stages where we would then work on those light chains. And in between those late and those pre, when we're going through, or the pro and then the pre-cells here, we're specifically testing too. So once we've made that heavy chain, we want to make sure that foundation is solid before moving on and rearranging that light chain. And so we will go through and test it. And so we will use this, what's pre-B cell receptor, that's V-pre-5 and things like that, or the V-pre-B and lambda-5 polypeptides here. Again, we have made this heavy chain. We'll create the surrogate light chain and bind it on to see if it works. If they kind of function well together, and basically it mimics that light chain. And so it's just making sure that we can continue on. It's our first checkpoint to make sure that heavy chain is solid before we commit to continuing on here. And so if it works, we'll continue to move on, go back to rearranging, rag will turn back on, we'll rearrange that light chain, and eventually we'll have this B cell receptor here. I do want to point out here, pre-B cell receptor is actually a low abundance. It's largely retained inside of the cell in membrane-enclosed vesicles. And so it's involved in cessation of heavy chain gene rearrangements so we can test it overall here too. And kind of at this point here too, you will also start to see Ig alpha and Ig beta being bound up here too, because you'll need those eventually. So it's a little bit more outlined in this development versus T cells when we're just like, hey, now we have CD3 molecules on the side. Then we move into those rearrangements of the light chain. We've tested it. It works that, you know, surrogate light chain was nice, it's happy, everything functioned well, we will move on. And so then we will rearrange that light chain in those pre-B cells. You guys have already seen this. You already know this picture. We pick one of the variables and then we join it with one of the junctionals. The only difference is this one's showing the kappa light chain. If you guys remember the charts from a while ago, the only difference with lambda, lambda is junctional constant, junctional constant, so it's intermixed. Just know that if you use the first junctional, at that point you have to pick the first constant. It can't just, you know, randomly pick and choose which ones it wants, they go together. Lambda, or kappa's a little bit different because you can pick one of these guys here and say this one doesn't work. Cool, it'll test this one now. If that combination doesn't work, they're trying to third one. If the third one works, cool, then we have that productive light chain rearrangement. It's just saying it goes through a couple different options until we get one that is productive and we can move on. And this specific example just said it got lucky and the third chance was correct. And so again, it's just showing you that we have, you get different chromosomes from your mom and your dad. You have a couple different options because it's going through and I'll check both kappas first. So we're going there and trying to create a light chain. We'll test kappa from one gene first on one chromosome from like mom and then one from dad. If either of those are productive, obviously we'll move on and have now a cell that has a mu heavy chain and a kappa light chain. Or this first one doesn't work. Cool, so we test out the second one. Second doesn't work, now we move to lambda. We have backup options. So we'll check out lambda genes for mom. That doesn't work cool, lambda genes from dad. That doesn't work, boom, cell's dead. We're not going to continue on. But if either of these are productive, we'll move on and express now that mu heavy chain and the lambda light chain on the surface. Cool, you guys know this stuff. It's very, very similar to T cells. Very, very similar to T cells. And so we do have those two checkpoints because again, we already talked about that first one. We're making sure that heavy chain is solid first. Well, now we're going to talk about our second checkpoint. So we already went through this. We rearranged that heavy chain and used that surrogate light chain of V pre-B and lambda five just to test it out, make sure it worked, cool. So we tested that out here. If it didn't really work well, we didn't get a good functioning thing, then cell will be signaled to die. So that was that first checkpoint. And again, you're selected for that functional heavy chain. So then we did that light chain rearrangement if it did work. And then again, if it does and we test it about that second checkpoint and that whole thing works together, cool. That cell will be allowed to continue on and become that immature B cell. If we do all of that light chain gene rearrangement, we test it out and it doesn't work. The cell will again be signaled to die, but that's how we test for that functional light chain. Very, very similar to T cells. You guys know this. You guys got this. You have to believe in yourselves just a little bit more. You're also a scientist. And because we're finishing early, we can jump into next week's just to get ahead so I can get through as much of the stuff as possible. You're also a scientist, it's Purdy Manisha. It's he, him, pansexual transgender man. He's a postgraduate student in molecular biology and biomedicine. And he's a cancer immunology researcher. He grew up in a war-torn country and fled because he wanted to express who he was, queer and interested in science about fear of being killed. Even after fleeing, he faced many barriers because less than 1% of refugees globally are ever able to access tertiary education. He works to currently characterize the expression of a tumor- associated protein in lung cancer cells in hypoxia and designing and optimizing a novel immunotherapy for adoptive cell transfer. And so CIGLEC is helpful because it's present in B cells. So, pretty cool. All right, you guys got this. It seems like a lot. Your textbook is just circling back. It introduces a little bit of information and it goes back and adds a little bit more. Yes, Matt, not Matt Cole, sorry. You guys are kind of interchangeable though. You guys need like. I can't do this to you guys. I was like, mole. Matt Cole. Yes, Cole, sorry. Yes, it's not here at all. I said switching is in the periphery in secondary lymphoid tissues, which we will actually go through and go through activation again, so. No, no, that's, surrogate light chain is just testing. It's like that pre-T alpha that we had to make sure T cells were good before moving on. That's all it is, is just a surrogate. It's there to see if it works. If we like it, continue on with the process. Okay, B cell development part two. So I do have, I think, an announcement on here that obviously if we don't get super far ahead, yes. Think about it. So you have bacteria. It could activate, it could neutralize it. It could trigger complement pathways. It could cause agglutination. It could activate other cells. The reason you want this broad thing is it's still specific for whatever subtype of pathogen it is, but now it can activate different components of the immune system. Like, yes. Yes. And we will talk about something, so there is an immunodeficiency disorder, just to kind of put it into context. There's one called selective IgA deficiency. These are people who have some alteration where they cannot produce immunoglobulin A. And because of this, they lose a lot of their mucosal immunity, so they're more susceptible to upper respiratory infections, sinus infections. That's actually one of the most frequent things is just recurrent sinus infections. It's frequently seen in this. People in, like, it's a lot. It's actually one of the most common immunodeficiencies, and it's because of failures of isotope switching. Because again, if IgA is so good at protecting mucosal membranes, if you can't switch to an IgA, isotope, whatever protection you might have against E. coli, or MRSA, or whatever, if you get MRSA up your nose, IgA is not really there to help protect you. So, it's pretty cool. You want that same binding site, but because they all have different jobs, it's useful. I'm trying to think of, like, a good military analogy. Like, you want a sniper, but you also want, like, a marine to beat things. Like, you know, they have different jobs. Like, they all want to kill the thing, but, like, you need different creative ways of doing it, basically. So again, like, we'll see, oh, and the cat, oh no. Oh no, who's, yeah, Liza, please, or, no, Liza. Liza, what is the cat's name again? Winston, that's right. And you said it's a neighborhood cat. Oh, if he goes missing, then I suddenly have a cat. I have Winston. He's adorable. So, apparently, she's trying to study, and he's just never letting her study. So, if you guys ever need someone to cling onto you, Winston is there to save you. So, if I do shift any material, I will let you guys know. It depends, kind of, on how much we get through today, and, kind of, of next week, too. More than likely, what I'm leaning towards, just due to the density of it, is probably push. And I'll tell you guys when I do it, I'm probably gonna swap out immunological techniques to module four again, just to space out the rest of the material for module three. But I will post very detailed everything with enough time for you guys to adjust and accommodate and not panic. Like, you will know, before the Monday, before exams, what the schedule's gonna be like, okay? Cool, we're not gonna overwhelm you guys. Trust, it's easy, you've got, keep telling, it's easy, keep telling yourself, you know this. Today actually will be, kind of, easy for the next few minutes, because you guys already know most of this. Today, we're gonna elaborate more on B1 and B2, just a little bit, and it's slides you've already seen before here, too. And then we'll briefly mention positive and negative selection, overall. Before moving into eventually the next steps is to talk about antigen, or activation and encountering antigen here. And so, we do have B cells expressing the cell surface protein. CD5 have a distinctive repertoire of receptors, and they're known as B1. So CD5, we found out, is a unique marker of B1 cells, which means it is not found on B2 cells. And so, again, this is just a slightly different presentation of things you've kind of already seen before in verbal definitions. We've talked about your B1 and your conventional. B1 are very, you know, produced very, very early on in development. You guys both, they got mole over here. They got mole over here. They got mole over here. Conventional B2s are pretty much created after birth. Because of B1s, they have, you know, they still have a B cell receptor on their surface, but they have very, very few, like, cool, you know, modifications and rearranges in the BDJ here. Obviously, they still have a BDJ because they still have a B cell receptor. But again, it's very few and very restricted, because again, they have a very limited repertoire. They're looking for conserved sequences, again, because they're your very primitive B cells, most likely there to provide some protection between developing fetus and utero, because you can still have some immune response, you know, in the later months of development. So if the mother ever gets sick, and the infant is unprotected, there's still some immune functioning going on, very limited, very basic, but still there. And so we do see this primarily in peritoneal and pleural cavities, because this is where we expect to see pathogens entering at this level here. They are self-renewing, which means, once they come from the bone marrow and leave, they can divide themselves. They don't need more to come from the bone marrow. They do produce immunoglobulin very rapidly. They don't need any kind of additional signaling involved. That's why it's called spontaneous production. They can just produce immunoglobulins. They frequently secrete mostly IgM and some IgG. We'll talk about conventional. We focus a lot on isotype switching, because there's four different types of IgG. It makes sense that over time, we'll see more IgG than IgM, because you have four different subtypes overall. These do not require T cell help. Conventional do require T cell help in order to become activated. So these frequently don't see either memory or somatic hypermutation. Again, they're very, very limited, very, very primitive. It's like if a superhero kind of drew a Superman logo on a cape and sharpie, versus if you had actual Superman, it's very primitive, very basic overall. B2 obviously does have high somatic hypermutation, because this is your adaptive immune system. It's with you for the rest of your life, developing and trying to create the broad response to everything. And yes, you also have memory with this one here too. You guys have already seen this chart. It's just to compare it. B1 are the equivalent of delta gamma T cells, again, that very, very primitive, limited repertoire cell. B2 are your equivalent to your alpha beta. Yes, good job. So it is plasma cells eventually. Sorry, yes. It can differentiate into plasma cells and secrete from their plasma cells, yes. Sorry, it just says that, but I mean, eventually it means down the line. What can come out of it? The big picture takeaway though, is there's more G than M in B2. And this is because there are those four types, isotypes of IgG that eventually you will have a broader, greater response of IgG than IgM over time, versus this one really doesn't do I-type switching. And so for the most part, you just see mostly that's why it's a lot of big arrows. Mostly IgM, occasionally IgG. Again, same thing. B2 is the equivalent of alpha beta. B1 is the equivalent of delta gamma. Again, just another elaboration on it here too, same thing. Split, B1's on this side. Again, it's fetal. Respiratory and GI is just another word of saying plural and peritoneal cavities. Again, low diversity, limited memory, usually targets carbohydrate. So it's more primed for bacterial responses because you see carbohydrates on bacteria. Very limited isotope switching, almost never requires T-cell help, and they self- renew. So again, it's just a repeat of the exact same slides here. And so it arises from the fetal liver by about the eighth gestational week. So early in embryogenesis and it can protect the fetus while it's developing in utero. We discovered it only about 20 years ago. Oh God. Well, some of you, most of you are probably still alive. No one's under 20 in here, right? I had one student who was 20 once and I was like barely on the cutoff. Are you snitching? Okay. We were just looking to see if anyone was 20. And so it was only discovered about 20 years ago here. They did think potentially it may have been a transitional lymph site, but again, it's protecting the fetus during the last few months of development before we ramp up our full immune systems. It does have slight memory, but again, it's just very, very, very primitive overall. B1, they're way fewer in the body than B2. Because again, B2 makes up the vast majority of your cells here too. And again, those B1 cells are mostly for those conserved sequences, usually carbohydrates on bacteria. Again, it's that primitive coverage in the immune system here and again, predominantly in areas where we're going to encounter pathogens like your pleural and peritoneal cavities like upper respiratory and GI. Most may be all of what we call natural antibodies which is kind of not a well-named thing because technically anybody, any antibodies your body produces are natural antibodies here. But these are ones that have kind of directed against blood type without any training that you haven't seen. So if you've never, like if you have type A and you've never seen type B blood, how does your body know there's type B blood, right? So they call them natural antibodies here, but these are ones that are directed against other blood types. So it might actually protect, especially if you think about it, in utero, babies may be different blood types than the mother. So it helps prevent because every once in a while, the mom can try to reject, so I'm A negative. If I ever have a kid that's A positive, my body could try to reject it or B positive, anything like that. So this may actually protect the fetus in utero and fight back against the mom's immune system to prevent the mom from miscarrying the baby because it thinks the blood type is foreign because it's rhesus factor positive or B blood type, which is pretty cool. So we'll stop there, but I would just know this specific example because it's a bold, it is important, flag it, circle it. Cool, we'll stop there.