Immunology Exam Debrief PDF

Okay folks, good afternoon. Let's get going. I promised you an exam debrief, right? right? Let's talk about a graph of antibody teeter versus time and you introduce antigen A. introduce antigen A. So what happens in terms of antibody teeter at the start? You're a champion, lag, you know, put that...

Okay folks, good afternoon. Let's get going. I promised you an exam debrief, right? right? Let's talk about a graph of antibody teeter versus time and you introduce antigen A. introduce antigen A. So what happens in terms of antibody teeter at the start? You're a champion, lag, you know, put that lag, lag phase. Thank you for that. Who was that? Who was that? I want to give the credit to the person. Thank you. Is her? Is her? Right on, thank you. Lag phase, all right, and then what happens? We get a primary immune response, right? We get a bump, which wanes very quickly. What I should talk to anybody is that, ooh. How many, how many isotopes we got? Five. Five. What's the acronym? Gamut. If you don't know that by now, but you didn't know it when we first, a lot of you didn't know that when we first started the course. All right, so which isotope predominates in the primary immune response? Why? Why? Why would it be IgM? IgG's better at everything. Why wouldn't the body select IgG just to knock it off straight away? It's got something to do with the genes, the way the genes are arranged, right? What is special about the way immunoglobulin genes are arranged? Champion, absolutely. They're always produced first. And how are they switched? Is it more genetic recombination between IgM and IgD? It's not, right? What's the other mechanism that it can swap between the two heavy chains? transcriptional splicing. So yes, I would have given you the mark. given you the mark. So it is. The primary immune response is predominantly IgM isotide. Did you know that? You do know. Second time, we introduced the same antigen, antigen A, right? So do we get a lag phase? lag phase? Nope. We get a huge response that wanes very slowly over time. which anybody predominates in the secondary immune response? Why? It's a hard question, yeah. Two degrees, not really the answer. So the answer is it's predominantly IgG at this point. But guess what, there is some IgM still. So secondary immune response, primarily IgG, this huge bulk is IgG mostly. and it's because we've reintroduced the same antigen, we've potentiated the antibody response, potentiate to sort of stimulate, re-stimulate, and so originally the lymphocyte was quite happy, the B-cell and the differentiated form, which as the plasma cell is quite happy making IgM or IgD in secreted form and then all of a sudden we trigger the parent cell to undergo further rearrangement and the most commonly selected heavy chain isotype with that second rearrangement is usually IgG unless it's getting signals to go to a different function such as IgA. So, pointing out the importance of understanding the logic behind all these parts, likewise, in those tables of the attributes of each isotype. I put circles on them, right? So, you know what to study because that's where the exam question is going to be. The exam questions, it's 45 in 50 minutes. Some people finish it like 20 minutes, stand up and walk out, right? They're not pharmacists I want to go to. I want to go to the guys that are there scrutinizing every answer until the last minute. So don't feel bad. And the majority of them are just the one correct answer. So you'll pick it out. You'll sort of cross off other ones and you'll pick it out and it'll make sense. So they're all derived from the lecture notes, my lecture notes. And I know you're not going to buy the textbook for a one credit course, why would you, right? So all you need, all the information's in there, and you know that there's a lot of pictures in my lecture notes. It's the text where the questions are derived from. So you scrutinize text. And for every lecture, you would question, you would develop questions based on that text about 10 questions per lecture as a rule of thumb, right? And that's how you study for my exams. And, you know, don't decompose yourself because there's another exam. You're going to have so many of these exams, right? Find, I think you guys are all incredibly successful already. You wouldn't be here if you weren't. So you've got to find that balance of the anxiety, just managing any form of anxiety with reality. And that is, so what? Blow an exam. Screw that. Move on to the next one. I'll do better in something else. I've got skills in different areas. Some people study best by doing concept maps. Have you heard of those? It's where you take the lecture notes, And you say, what are the big picture items that were introduced in this lecture? And you write those words out on a blank piece of paper. And then you start drawing arrows and connections, and you start putting lists of other terms that you think are new and examinable. And then you sort of do a snapshot with your brain as you study. And when you're sitting in the exam, you can remember that where you wrote it. You remember where it is. They say that memory and, you know, creating knowledge, it's like it's the repetition that does it three times. Write it down, say it out loud, watch a video. When you sort of do it three times, you basically commit that to memory, something to that effect, right? All right, this one was great. You're going to get a lot of free marks there. All right, SMAM, what does that refer to? Specificity, M, I just can't think of, ooh, memory, right, A, amp, thank you, I did hear that, amplification, M, modulation, right on guys, all right, where does that fit into that picture? Oh my, I think it's not, whatever. What is the specificity in this picture? Well, the antibody is being made specifically against antigen A, right? And then it also increases the amount later because it recognized it again, so that's the memory. So it recognized the same one again and the specificity was the same because it's got a much greater antibody teeter. So it's potentiated. That's the amplification. And what about the modulation? Where's the modulation in that graph? It went from IgM to IgG isotype. Modulated the response, right? So all of these foundational concepts, all feeding back into each other and then informing your choice of that question. All right, I did mention that we've We've got some exam questions on E-Class. Let's just look at this. There's our syllabus, immunology and biotechnology. Course weight, one credit. What the ****? All those lecture notes, right? All that stuff, one credit. This is foundational. Like, you're healthcare professionals. You'll sit on the pharmacy. Anybody can push a product over to the patient and tell them to read the monograph but do you have the core of knowledge do you have a deeper understanding where you can read a monograph let's say a monoclonal antibody that you are giving to somebody and you're aware of aspects of that monoclonal antibody why it's for that particular condition or not and then some of the pharmaceuticals cold chain principles that will go in into detail in pharmaceutics. This course provides students with an understanding of immunology, molecular biology, and biotech processes and point-of-care tests that support therapeutic and diagnostic application and patient care. It sure does. It meets those needs. We go through each section, elements of the immune system, in particular innate and adaptive or acquired immunity, and then cells and the fluid- based factors. And then we go into fluid responses and cell-mediated responses, and then how cell-mediated immunity, today's lecture about how the T cell does things. So last lecture, we did B cell in the adaptive immunity. We know that they make antibodies. So these are the fluid-based factors that are most important for adaptive immunity. Today, we talk about the cell-based players, which is T cells. All right, you get info about exam locations, right? Exam locations from everywhere. And again, let's just put this behind us. The midterm, I think you'll feel a lot better. If you don't do well in the midterm, you should come and talk to me and we should get a strategy together for how to help you through this course. Because as I mentioned, almost everybody does well in the midterm. So if you're not doing well, it means you haven't read anything at all. You're just winging it because I've given you as much, as many tips as I can without giving you the exact answers, which I've already given you some anyway. All right, we'll do those trial questions at the end. Let's just get going with today's lecture. if PowerPoint, it seems to be behaving. Today's lecture, cell mediated immunity. We're going to talk about the T cell receptor, how antigens are processed and then presented to that T cell receptor to get an immune response by something called MHC restriction. And then the T cell will become activated. And we'll look at some of those activation steps and effector functions. Lymphocytes, both B and T cells are derived from the same lineage made in the bone marrow. If they're instructed in the bone marrow, they become B cells that make antibody. If they migrate from the bone marrow to the thymus, they become T cells. So T cells express the T cell receptor. They'll rearrange genes in the thymus and express a receptor, which is similar in structure in some ways to immunoglobulin in that it will express a unique variable region by gene rearrangement in order to interact with an antigen. And likewise, it has a variable domain that interacts with the antigen and a structural constant domain or region below that, which, again, is organized through very similar processes to the antibody. I'm just going to try and remove this there. So we're aware of where antigen binding sites on the FAB regions of an antibody are. Antibody have the two binding sites that can cross-link. In contrast, a T-cell receptor only has the one binding site to interact with antigen. A T cell receptor is comprised of an alpha chain in yellow, which is anchored in the transmembrane region of a T lymphocyte or a T cell, and a beta chain, and they're both internally disulfide bonded so that they stay, they don't float away from each other. And we have a coded domain, one of these green blocks as a variable region, and that is joined with another domain of protein as the constant region. So the T-cell receptor interacts with something called the MHC, the major histocompatibility complex, histo-tissue. This is your tissue compatibility factors on cells. So we know that immunoglobulin antibody will bind a wide range of antigenic epitopes. Those epitopes can be proteins, carbohydrates, lipids, mixtures of those. However, even the surface of organisms, soluble toxins, they can bind those, but the T cell receptor will recognize mainly peptide antigens. That's a big difference, not the other macromolecules. and only if that peptide antigen is bound and presented by an MHC molecule. So MHC molecules are found on the surfaces of every cell in our body except erythrocytes. There's a diverse variation in MHC molecules in the human population and MHC incompatibility is the primary cause of tissue graft rejection and graft versus host disease. rejection and graft versus host disease. What does that even mean? That a graft The simplest graft may be a bone marrow transplant, bone marrow full of leukocytes, and so some of those leukocytes may have been trained against self-tissues in the donor. Suddenly we receive it as a recipient, and this graft of lymphocytes starts to damage our own tissues because it finds itself in a foreign environment. That's a graft versus host disease. T cell receptor diversity is created similar to gene rearrangement by rag enzyme complex. So lymphocytes do it, be they B lymphocytes or T lymphocytes, they can do it. The genes incurred in the alpha and beta chains are fragmented in the germline configuration located on different chromosomes. Again, I'll remind you, I'm not testing you, examining you on any chromosomal location. But you notice which chromosomes are important now for your immune library of molecules that are created, 14, 7, 14, 2, 22, they're all the same players. And the gene segments are rearranged by rag enzyme complex to produce functional gene and T cell receptors with high specificity. And here we have a diagram showing that organizational and rearranging of the T-cell receptor genes. They are fragmented in the germline DNA for the alpha chain at the top, and they're fragmented in the germline for the beta chain at the bottom. We notice that in brackets for variable red regions, we have many choices of gene segment alleles, up to 70 or 80, and again, many choices that we can choose just one variable specificity for each of these T cell receptor chains, which will then become recombined through RAG enzyme activation, rearrangement, transcription, and splicing to bring those exons together and then basically create the protein chains that find each other and assemble through a disulfide bond in the endoplasmic reticulum when they're secreted. You know that surface immunoglobulin has accessory proteins that become activated when it grips the antigen, specific antigen, Ig alpha, Ig beta chains, right? So T cell receptors are similar, they've got accessory proteins that are critical to facilitate signaling function. Those additional membrane proteins are termed the CD3 complex, and basically the CD3 complex are a gamma chain, a delta chain, two epsilon chains, and two zeta chains. Let's have a look at how they work, and I put in the figure legends, so that'll help you study, because you can read it, and oh yeah, I totally get this. We've got the phospholipid membrane in gray on the surface of the T cell here, and what we see is the T cell receptor, the alpha- beta chains, and then we have a gamma chain in and two epsilon, and then an intracellular two zeta chains. And basically, when it will bind an antigen, this complex will reassemble and will signal transduce that and activate the lymphocyte. We'll look at that shortly. So just when we think we got it, T cells have T cell receptors. There's a second type of T cell receptor, the gamma delta T cell. It's composed of two different protein chains to alpha and beta, but makes that same type of receptor. The gamma-delta T-cells, which are less than 5% in our body, are believed to be involved with cancer surveillance responses where they can tell whether a mutated cell is still a friend or if it's a foe and be eradicated. T-cell will express only one type, never both. So, it's either an alpha beta or it's a gamma delta T cell with those primary chains. The delta locus out of interest is located within the alpha gene locus and T cells will select and express either alpha beta or gamma delta. But what's interesting why it's within the alpha locus here, here's the second class of receptor. A gamma chain and a delta chain textbook is shown in different colors but primarily got an identical structure, less than 5%, mostly produced when we're in our fetal stage, and then only after birth, the alpha beta tends to predominate, but this is an interesting bit that the gamma delta T cell receptor gene locus is here on the alpha chain amongst the fragmented genes and alleles, you'll find the delta chain locus there, and why is that important because if a lymphocyte chooses to make an alpha chain, it will use rag enzymes to cut that genomic DNA and so it will break apart the ability for the delta chain locus to ever be expressed. All right, so we get T cell receptors. So how does antigen interact with that T cell receptor? Well it needs to be processed and then presented to the T cell receptor. The T cell receptor can only recognize antigen in the form of a peptide bound to an MHC molecule on the surface of cells. We said every cell in the body has MHC molecules. How does it load it into that molecule for examination by our T cells? Pathogen-derived proteins must first be degraded into peptides, that's called antigen processing, and sampled by the host cells. Host cells will assemble a peptide MHC molecule complex and display this on the cell surface. That's what we mean by that term antigen presentation. And the type of MHC molecules, there are two types of MHC molecules that are going to interact with two different types of T cells. So I'm just going to try to clarify this part here by showing you the following. I have a T-cell, and we know that T- cells are going to have a T-cell receptor, TCR, but guess what? but guess what? T-cells also have another receptor called a co-receptor, co-RC. So there's a T-cell receptor and the co-receptor, and then all the other cells in our body are going to present antigen. Presenting. Presenting cell. Because every cell in our body has MHC. I'm just going to draw it as a cube right now. MHC. So, the MHC is going to process antigen and load it for presentation to the T cell and the co-receptor will stabilize that presentation. That's just something that I think will help you get through the next set of slides here. And the next set of slides now start to talk about what type of co-receptor is being expressed on the T cell. what type of co-receptor is being expressed on the T cell. Is that co-receptor a CD4 ligand or a CD8 ligand? co-receptor a CD4 ligand or a CD8 ligand? CD stands for CD cluster of differentiation. So that's a molecular biology term that means certain genes were expressed to make these ligands better functional on the surface of cells, in this case lymphocytes. So there are two primary classes of circulating T cells, the T helper or the cytotoxic T cell. The T helper is called a CD4 T cell, because that's the sort of glycoprotein you'll find on it. And the cytotoxic T-cells called the CD8 cell. So along with the T-cell receptor, T-cells also express this cluster differentiation glycoprotein, CD4, CD8, to stabilize the T-cell receptor MHC interaction. And these are called the co- receptors. That's what they look like structurally. CD4 has one stalk, S-T-A-L-K, one stalk anchoring it in the lymphocyte membrane, and then one, two, three, four domains that will now lean over and interact with MHC to stabilize the presentation of antigen, and we'll see that shortly in a diagram, whereas CD8, the cytotoxic cells have the two stalks, which again are disulfide bonded so they don't float away on that fluid phospholipid membrane, the ocean of the cell. Have you ever seen blowing bubbles with children, right? And the sunlight hits it, and you see the spectrum, sort of white light being diffracted and then swirling all over the surface of this bubble. and it's just showing us the fluidity of membranes and that's how these receptors self-associate, assemble, and are triggered to bring subunits together that can bind an antigen or a ligand in our fluid environment, which is our body. So the T helper cell, CD4, actually has two types. There's T-helper 1 that activates macrophages, T-helper 2 that activates B cells to differentiate into plasma cells and secrete antibody. And the CD8 cell is basically the one type, it's always cytotoxic and it's primarily there to kill virally infected cells. Here are your major T-cell populations. A CD8 cytotoxic cell, it recognizes a virus infected cell because of antigen presentation after cell contact, and then it will apoptose that cell. It's cytotoxic. It's got cell killing mechanisms. We'll look at them shortly. CD4T helper 1, so they're helper cells. What do they help? Helper 1 cells help macrophages. They activate macrophages with cytokines to become phagocytic and secrete pro-inflammatory cytokines in that region itself, so recruit more cells. So T-helper 1 works with macrophages, a T-helper 2 primarily triggers B cells to synthesize antibody. So there's the three types of T-cells that you really need to know well. Let's look at the MHC complex. There's two classes, class one and class two. Class one, we said, is expressed on all nucleated cells of our body. Class two is found only on professional antigen presenting cells of our immune system. What are professional antigen presenting cells? Phymus epithelial cells, because they're the ones that instruct self, non-self. They've got both class one and class two. lymphocytes, both B and T cells, actually have class II as well. And the professional APCs, antigen-presenting cells, are the dendritic cells, the macrophages, the B cells. And in order of importance, keep them always organized that way, what's a professional antigen-presenting cell that will process antigens and present them to lymphocytes all the time? The dendritic cell, our tissue resident cell of the immune system is one of the major players. And then after that macrophages are very effective, and then after that B cells can also do it. The B cells have a specificity of antigen binding, so it's a very limited repertoire of antigens that are presented from a B cell because it's related to what antigen the immunoglobulin binds. and here's how the whole process assembles. So the T-cell co-receptors CD4 or CD8 are specific for either MHC class 1 or class 2. So at the bottom here we have the lymphocyte in blue with its T-cell receptor and then when a target cell if it's expressing a CD8 cell it will interact with MHC class 1. If it's expressing a CD4 co-receptor it will interact with MHC class 2. So the co-receptor will determine what type of MHC it examines on the target cell. And remember that MHC class 2 can only be found on antigen presenting cells. One way I find that's easy to remember MHC class 1 or 2 is MHC class 1 only has the one stalk, whereas MHC class 2 has two stalks from the two chains. And likewise, when they associate side by side, I remember that CD8 interacts with class 1 because the two little subunits or protein domains sort of line up with this circular domain known as beta 2 microglobulin. So I'm always looking for that alignment with my eye, whereas here the CD4 tends to lean over and stabilize attraction to the class 2 receptor. We'll look at this again and again and I think it'll become more clearer for you. The way that class 1 and class 2 MHC differ in structure is only the fact that class 1 has the one peptide binding groom that's formed by the one protein chain, and that protein chain is stabilized by association with this beta-2 microglobulin, but the MHC class 2 molecule basically will create a peptide binding groove from the two separate chains. So they both have peptide binding grooms, and that's what it means, their variation on the same theme. Either structure is trying to create something that a peptide can be presented to our lymphocytes. And that T cell co-receptor, CD4 or CD8, well, that binding will stabilize the MHC antigen presentation. CD8 binds the alpha 3 domain of the MHC class 1. So target cell with the MHC, this is a class 1, so that's found on every nucleated cell. It's got an antigen it's presenting. And then a T lymphocyte with the correct CD8 co- receptor can line up and stabilize presentation to the T-cell receptor and potentially make this cytotoxic interaction, where the lymphocyte would say the target cell is infected with a virus. This is a viral antigen. I need to apropos that target cell before the virus replicates any further in that cell. So that's how Now, the co-receptor will dictate the type of interaction whether it's cytotoxic or whether it's a helper function, helper one or helper two. Helper one activates macrophages, helper two makes antibody. It's already starting to make a lot more sense when we repeat it two or three times. And again, the antigen- presenting cell with an MHC class 2 molecule, so only professional antigen- presenting cells can help them. What are professional antigen-presenting cells? Dendritic cells, macrophages, B lymphocytes, the surface immunoglobulin that's bound some antigen. That's your professional antigen-presenting cells. They found something, loaded it into an MHC class 2 molecule, and they're presenting it to a T cell that's got the CD4 co-receptor, and the CD4 domain, first domain, leans over and stabilizes this receptor complex and presentation. And now we're going to get those effector functions of an enraged macrophage, triggered and rage, sphagocytic macrophage versus helper 2 function where all of a sudden we're instructing B cells now to create synthesized specific antibody. Something interesting is that MHC have degenerate peptide binding specificity. So the MHC molecule, it's not called a receptor, it's called a molecule because it's got no downstream functions in terms of second messengers or anything. So the molecule will load various different shapes and charges of antigen. And that ability to bind this range is called a degenerate specificity. And think of this peptide binding group with the supposed colored antigen in it. Think of these alpha helices as coils. And you know, if you sort of put some sort of object into these two coiled springs, they'll spread apart and then they'll snap back on top of it, right? So having this ability for degenerate peptide binding specificity means that the way that they can offer, they can present a diverse range of peptides. So, they don't really have to be highly specific, like antigen-antibody attraction, or like T-cell receptor and peptide antigen attraction. Here's the text so that you can help with your study. The peptide binding groove of MHC class 1 and class 2, that's the T-cell receptors view of that groove that looks like coils with a peptide bound in the middle. and again it's just talking about that degenerate specificity. But the next figure legend 316 is an important point here that there are two major compartments within cells separated by membranes and this is the secret to how we sample antigen and load it onto MHC be it class 1 or class 2. So one One compartment in the cell is the cytosol. So that's the cytoplasm and the nucleoplasm, the liquid inside those areas, regions of the cell. That's called the cytosol. So it's the fluid-based internal environment, and that's contiguous with the nucleus via the pores in the nuclear membrane. The other compartment's the vesicular system. What is the vesicular? A vesicle is like a membrane-bound bubble, right? And we know vesicles in the cytoplasm, like lysosomes are the first ones you think of. But guess what? It consists of the endoplasmic reticulum, so those tubes of protein synthesis, but also as the proteins made, it'll bubble off at the Golgi, like a lava lamp, right? creating these endosomes, these vesicles that traffic towards the surface and on the way will meet a third type of vesicle, endocytic vesicles, where the cell membrane has scooped the extracellular environment, the liquid, and brought it in as a bubble. So the vesicular system consists of endoplasmic reticulum, which which is contiguous with the Golgi that bubbles to create endocytic vesicles, sorry, to create vesicles and lysosomes, but also extracellular endocytic vesicles that are coming in, and the vesicular system is effectively contiguous with the extracellular fluid. Let me show you what all that means, endocytic versus secretory vesicular traffic in a cell. So, we've got the nucleus and the cytoplasm, and then the blue-colored, blue-colored nucleoplasm and the light blue-colored cytoplasm are joined together by these nuclear pores. And so therefore, that can be thought of as a membrane-bound compartment of liquid known as the cytosol. There it is, cytosol. Meanwhile, in yellow, the external environment is being endocytosed or sampled into vesicles, which are moving inside the cell, but it's captured fluid and factors from the extracellular microenvironment. Why is that important? That's where bacteria are. Because bacterial antigens, this is the system that's going to scoop in some of those foreign antigens and bring it into the cell. And now somehow the cell has to load that on an MHC and put it back on the surface to interact with the T cell. So other than the endocytic vesicles are the natural vesicles formed from the endoplasmic reticulum here in yellow. And we see that the endoplasmic reticulum at the Golgi apparatus starts to create these vesicles which are meant to be for secretory purposes, but at the same time they also include lysosomes which will fuse. Here are lysosomes that can fuse with these endocytic vesicles and again now we're starting to see where some of the antigen can be presented. MHC class 1 and 2, class 1 expressed on all nucleated cells, will complex with endogenous antigens within the cells. Every cell has it, so the function of a class I molecule is to sample its own microenvironment. If I ever get infected as a cell, the class I will start to express those viral antigens or cancer antigens on the surface of the cell because I've sampled my own microenvironment versus, and you know it's going to present a cytotoxic T lymphocytes which will remove that compromised cell. versus class 2, which is only found on professional antigen presenting cells. They're the ones that sample the exogenous microenvironment, because where are they found? They're found in lymphoid tissue. There's another exam question. Got to wake up. Exam question type. Primary and secondary lymphoid organs. Pretty easy. There's only two primary, right? Bone marrow thymus and secondary. belt-melt-gelt, but also all the little lymph nodes and other lymphoid aggregates of lymphocytes. So I'm pointing that out because that's where these exogenous antigens are processed. That's where it will scoop it and bring it in in order to present it from the MHC class molecule with a CD4 co-receptor to a T helper, the T helper cells. And in this beautiful diagram it shows you both of those. This is potentially a macrophage, a large macrophage, and clearly as a macrophage like every cell in our body it's got MHC class 1 because it's a nucleated cell. But being a professional antigen-presenting cell, it also has MHC class 2. So if we look at MHC class 1, which we know samples and dodge in this antigen, what we see is that somehow a viral proteins, virus gets into the cell and starts to synthesize these red star- shaped viral proteins, but these proteins are always being degraded by an enzyme complex in our cytoplasm called the proteasome into little peptide fragments, which are transported into the endoplasmic reticulum where they can interact with an MHC class one, a single stalk, right? And the MHC class one that's being transcribed, translated as this molecule has the ability to interact with whatever's endogenously being processed by the proteasome, load it, and then through the vesicular trafficking, express it back on the surface waiting for interaction with a CD8 lymphocyte. The flip side of that is MHC class 2 where extracellular antigens are being brought in through endocytic vesicles and basically the phagocytic lysosome, when it fuses with the lysosome, those enzymes chop up that bacteria creating peptide antigens that have the ability to meet MHC class 2 which is coming from vesicular traffic and vesicular traffic is bringing it where they bump into each other and has the ability to load and then continue to the surface to present that peptide antigen to basically a T helper cell. There's the figure legend of what we've just discussed. And I know this is heavy material, but there are two beautiful textbook videos that I will show you shortly. And then we'll take a break after that. So this is describing what I, this is the text of the description I just gave to you from that picture of MHC class one processing. And again, the proteasome degrades cytosolic proteins and loads them via a transporter known as TAP onto the MHC class 1 in the endoplasmic reticulum. That process of loading the antigen is known as peptide editing. In the absence of infection, MHC class 1 will in fact carry self-peptides. But hopefully our immune system has been trained well to ignore self-peptides in the thymus. They do not usually provoke an immune response, self-peptides, due to that T cell negative selection in the thymus. Occasionally, a self-tolerance mechanism fails, resulting in an autoimmune disease. We'll look at that in one of our last lectures, lecture seven. What about class two? And this phagolizosomes, some microorganisms that are ingested from the extracellular environment in the phagosomes, will become acidified when it fuses with lysosomes, which are full of acid proteases and hydrolases. And so it chops up the microorganism to create a whole bunch of antigens in that phagosome. And then basically there is something known as the invariant chain. So, the invariant chain will block premature binding of peptides in the MHC2 cleft, and it's only dislodged, broken down and dislodged in the phagolyzosome by the action of another immune ligand player, HLA-DM, that exposes the MHC2 cleft for antigen presentation. This is what I'm talking about. The invariant chain, shown in green here, blocks binding of peptides to MHC class 2 in the endoplasmic reticulum. So it's not going to sample self-antigens because the binding site, the presentation site, is blocked by this green invariant chain. However, now that it's in a vesicle and it starts to become acidified, we get some cleavage, but this small remaining fragment, clip, remains lodged in there, still blocking any sort of peptide loading or presentation. And it will require this additional player, HLA-DM, to interact and attract and release clip so that antigen peptides can be loaded and put to the surface in order to present class 2 to CD4 helper cells. Let's have a look at these videos and we'll take a break. And I'll make the video available after the old lecture. The two chains of the MHC class 2 molecule assemble in the endoplasmic reticulum, the ER, with the third chain, the invariant chain, shown here in green. Let's have a look at class two because we just went through that with the clip. The video will mention other factors that help loading. And again, we can't cover everything in a one-hour, two-hour lecture, so the major point is the fact that class two MHC does not bind anything in the endoplasmic reticulum because of the invariant chain and in particular that clip fragment that blocks the site. Let's check it out. Well, that's impressive. There's no sound. Pause it. You know what? We're going to take our break now and I'll sort this out. This is just like doing a pregnancy test in my youth. It's haunting me. All right? We'll meet in 10 minutes at 2 o'clock. Thanks. the two chains of the MHC class 2 molecule assemble in the endoplasmic reticulum the ER with a third chain the invariant chain shown here in green a portion of the invariant chain binds to the peptide binding groove of the MHC class 2 molecule and prevents peptides or unfolded proteins present in the ER from binding the invariant chain also guides the transport of the class 2 molecule. The two chains of the MHC class 2 molecule assemble in the endoplasmic reticulum of the ER with a third chain, the invariant chain, shown here in green. A portion of the invariant chain binds to the peptide binding group of the MHC class 2 molecule and prevents peptides or unfolded proteins present in the ER from binding. The invariant chain also guides the transport of the class 2 molecule out of the ER and through the Golgi apparatus into a vesicle that eventually becomes part of the endocytic pathway by which pathogens and foreign proteins are taken into the cell. Progressive acidification of this endocytic vesicle activates proteases that cleave the invariant chain in two places, leaving a small peptide that leads to associated invariant chain peptide bound to the class 2 molecule. Engulfed pathogens or their proteins are also degraded by acid-activated proteases into peptides, but these cannot immediately bind to the class 2 molecule because the clip peptide still occupies the peptide binding groove. The removal of the clip peptide is the function of a specialized MHC class 2-like molecule, HLA-DM, which is also present in these vesicles. It functions as a catalyst, coordinating both the release of the clip peptide from class 2 molecules and the binding of pathogen-derived peptides. The MHC class 2 peptide complex is then transported to the cell surface, where it can be recognized by the antigen receptors of CD4 T cells. The folding and assembly of MHC class I molecules takes place in the lumen of the endoplasmic reticulum. The initial folding of the class I heavy, or alpha chain, is aided by the chaperone calneuxin. The partially folded chain is transferred to a second chaperone, calverticulin, which aids the further folding of the chain and the association of beta-2 microglobulin. Other proteins, ERP57 and tapasin, associate with the nascent Class I molecule, which binds to the tap transporter via tapasin to form a peptide- loading complex. The peptides that bind to the MHC Class I molecule are generated by a large protein complex, the proteasome, which is found in the cytoplasm. The proteasome degrades proteins within the cytosol of the cell to produce short peptides, which are then transported through the endoplasmic reticulum membrane by the TAP transporter. Some peptides do not bind to the MHC molecule at all. Others can bind, but are unstable. These are released from the MHC molecule, a process called peptide editing. Finally, a peptide binds to the MHC molecule with high affinity to make a stable complex. This causes the final step in the folding of the class 1 molecule to take place, and the dissociation of the peptide loading complex. The peptide-loaded MHC class 1 molecule is now free to exit the endoplasmic reticulum and be transported, via the Golgi apparatus, to the cell surface, where it can be recognized by the antigen receptors of CD8 T cells. And we're clear on that now, that class one is endogenous sampling of antigen. It samples antigen it finds within the cell. And I had the question during the break, how does, how do you get like bacterial antigens inside the cell? Rarely it's bacterial. Usually it's viral antigens, or like I said, cancer antigens. There are some bacteria that you can get inside cells and divide within cells. Mycoplasma, I think, is the species. So, you know, often that's missed in a lot of our microbiology. And we've got a problematic patient that's not responding to anything because it's got one of these microbes. But you'll learn more of that in microbiology. So, the clinical segments. So let's go back to finish up today's lecture. And what we're going to look at now is tissues that express class one and class two. And I think we're clear, class one is on every nucleated cell. So that includes in the lymphoid tissues, T cells, B cells, macrophages, other cells, epithelial cells of thymus, they've all got class one. What about class two? It's only found in professional antigen-presenting cells, including, what, dendritic cells, there, dendritic cells, macrophages, because I'm telling you to do it in the same order always. So macrophages and then the lymphocytes, both T and Bs, have class one. But moving on to other nucleated cells in the body, yeah, they've all got class one. All nucleated cells in the body, and in fact, look at that, neutrophils have lots of class 1. So after they've done their function, they've bound a lot of antigen, they're going to be presenting a lot of that antigen. Class 2, it's absent. The dagger bullet is saying macrophage-like microglia and the brain are positive. The class 2, that's not an exam question I'm going to ask you. class one on every nucleated cell. Clearly red blood cells don't have it. Maybe when they were nucleated they have it. Maybe, that'd be interesting to follow up. But then when they spit it out and there's no more genes being transcribed, translated, whatever's on the surface sort of stays there. There's a lot of endocytosis and removal of other stuff. So anyway, we know that mature erythrocytes red blood cells don't have it. Alright, MHC have some variability too. They have isotypes. MHC genes code for these isotypes that differ in the range of peptides they bind and the extent of genetic polymorphism in each of those isotypes. These are conventional stable genes. They've got a conventional gene block an exon, just one that's going to code for the MHC, and they do not rearrange by rag enzyme mediated rearrangement. They've got another way of shuffling and creating a bit of diversity. That diversity is provided by what's called genetic polymorphism, alleles, so you get multiple alternative forms of that same gene, for example, the two parental chromosomes. So you get one chromosome from your mom and the other chromosomes from your dad and the pairs. And so therefore you've got identical gene segments on both known as alleles. And so you can get some diversity by meiotic recombination and inter-allelic conversion. Also point mutations like we spoke before. But remember there's two ways that cells can divide. Mitosis, so in the mature adult like all of us, our cells are dividing by replicating their DNA and then organizing them into chromosomes and pulling apart into two daughter cells, right? That's mitosis. But meiosis, meiosis is germ cells, like in our parents when their germ cells divided and then divided again to give only half the number of chromosomes, one set in the sperm, one set in the egg, that when they join create the two sets again. But that's where we get a lot of meiotic recombination through these mechanisms. Here are the mechanisms. Why is this important to mention? That's where the MHC diversity is generated, and that's why it's unique from person to person to person. On the left panel, we see that it's recombination between alleles of the same, so two parental chromosomes, for example, and we get segmental exchange, shown by the colors here, of a segment of that gene versus inter-allelic gene conversion, where it just wraps around on itself and swaps some material between alleles. And I saw this wonderful explanation of this process once, that MHC antigen binding diversity is genetically determined from one individual to the next, like shuffling a deck of cards. And so we've all got similar genomes. So very similar, almost identical genomes. We all get the card of, a deck of cards, 52, right? Kings or queens, everything. But then everybody gets a different shuffle through these myotic changes. And that results in unique MHC specificity or ability of a range of specificity for peptides that it's very, that it will interact and present with. Here's some definitions of the terms we've used. Alleles, different forms of any given gene, which when reshuffled will code for genetically determined allotypes of the MHC receptor. Look up these terms, allotype referring to yourself. And then, so a unique type, MHC protein products, are highly polymorphic. There are many isoforms produced from combinations of multiple genes and alleles. And here's, put an asterisk next to these last two bullets because you need to know this stuff. It's examinable. What you need to note is that there are six MHC class 1 isotypes and five MHC class 2 isotypes. And I've got an easy rule to remember these again. If it's a class 1 isotype, it's only going to have a single letter, because class 1's got a single stalk. But if it's a class 2 isotype, we know that it's got the two stalks in the MHC, and it's got two letters to represent it. So class 1, it's A, B, C. There's no D, because the Ds are in the class 2. And then it's E, F, G. So what are the six MHC class I isotypes? MHC A, B, and C, E, F, and G. Easy. What are the five MHC class isotypes? So we know it starts with D, and there'll be two letters. DM, there is no N, DO, DP, DQ, DR. This will become important in lecture seven when we talk about allergy and autoimmunity. autoimmune disease, and the fact that our own MHC class two molecules, or class one molecules, are creating false recognition of self and treating them like foreign antigens and creating antibodies, self-antibody or autoimmune disease. So six class one, five class two, here they are in pictures. and what's important to notice are the ones that are in red because there's a high polymorphism in the binding region of these isotypes of MHC in red. So that's class 1 A, B, and C isotypes have a lot of polymorphism. They can bind a wide range of different peptide antigens, whereas EF and G are very limited in the specificity that they can sort of bind. Likewise, class II isotypes, it's really the beta chain of HLA-DR. That's highly polymorphic and binds lots of exogenously derived antigens. And we've heard of HLA-DM, its importance, in terms of not so much binding the peptide group but dislodging the clip in order to load fragments there. So this could be an exam question, for example, where what's the most polymorphic isotopes of class one or class two? Because you're going to see autoimmune diseases that involve HLA A, B, and C and HLA DR. Finally, we'll talk about this process of MHC restriction. T-cell receptor not only recognizes the presented peptide, but guess what? It will also recognize the receptor edges, the lips of that MHC receptor. And I've called it a receptor here, MHC molecule, right? So that co-recognition is termed MHC restriction, and it's highly specific. So, one specific T-cell receptor will not recognize a different class one molecule lysotype or different antigens, and this is what we mean here. MHC restriction, there's the T-cell with a T-cell receptor. It's bound a specific peptide antigen, and the triangle shape matches the receptor binding shape. but the rest of the T cell receptor is looking for the class one molecule, the edges of these subunits, and it's a congruence and a match of these subunits on the antigen-presenting cell. When we get recognition of both the peptide antigen by the T cell receptor and of the HLA isotype, we will have recognition will have activation of that T cell. If we get no recognition of the MHC by the T cell receptor, it will not bind and react to the peptide, even though it was specific in this example, but because you need both factors having specificity for the receptor, there's no recognition here. And in this This third example, what they're showing, is that an antigen presenting cell is putting up perhaps some self-peptide, which this T cell receptor has no affinity for, even though it has a good affinity for the HLA molecule. But in this case, it will not recognize it either. So both have to match in order for the T cell to be activated. Dendritic cells and macrophages are present, so let's talk about antigen presentation in cell mediated immunity. Dendritic cells and macrophages are present in all tissues of our body. They carry antigens from sites of infection, by lymphatics, to secondary lymphoid tissues where the lymphocytes are, and that's where they're going to interact with the T lymphocytes, showing them what they've brought from all these tissues of our body after homing to these secondary lymphoid tissue. And that process of homing for the lymphocyte is facilitated by adhesion molecules. Many of you probably heard of those. It's the fact that we look at four structural classes with our immune cells. So we have the ones on leukocytes, the selectins. We have vascular on the endothelial cells around lymph nodes, addressins, which are sugar-mutin-like, and then clearly the selectants have an attraction for the adresins, and as they grip they'll extravasate into the lymph node. So how come they can float through the bloodstream, ignore all other locations, but then it comes close to lymphoid tissue, the bloodstream and lymphoid tissue, and it'll get gripped by some of these vascular adresins and brought in. There's also titer binding integrin receptors that then facilitate cellular movement at that region. And we have intracellular adhesion molecules, or ICAMs, which can make endothelial vessels leaky, for example, to facilitate that movement of the cell into the lymph node. And likewise, CD2, we're going to introduce a lymphocyte function associated antigen LFA in the following. So T-cell homing to lymphoid tissues. A circulating lymphocyte in blue enters the endothelial venule in the lymph node. So lymph node tissue, but that's the endothelial blood vessel barrier. And as it's there expressing some of these adhesion molecules, we basically get binding of the selectin, L-selectin, the yellow sugar molecule on the surface that begins a rolling motion. You can imagine if you're floating by and somebody grips your arm, you'll spin around to that arm. And as you spin, you start to roll. And so therefore, these lymphocytes start to roll across the surface of this endothelial cell and then basically grips very tightly through other adhesion molecules and once it's tethered tightly and immobilized in a location, it will start the process of diopedicis or squeezing between that into the tissue. So migration into these lymphoid tissues. The other side of the story is where your professional antigen presenting cells in our tissues, such as this dendritic cell in yellow here, is sampling antigen at the side of an infected tissue back to, by the lymph, that it's back to the lymph node. So the lymphocytes and the antigen presenting cells are meeting in the lymph nodes. lymph nodes. And this is just showing that the red bacterial antigens in the skin of an infected wound to enter a draining lymphatic vessel in that area and basically drains into the regional lymph node and therefore these dendritic cells arriving and bearing the antigen will now settle in the T cell lymphocyte areas where they will start to present the antigens, class II presentation, to helper lymphocytes. Let's see the final part of the puzzle. What actually happens with an activated T cell? How does it kill cells? What does it do? So professional antigen presenting cells, they meet or reside in these secondary lymphoid tissues. We mentioned several times now it's Anagen-presenting cells have a costimulatory CD molecule called B7. So not only does the T cell have a T cell receptor and a co- receptor, the anagen-presenting cell has an MHC molecule, but it also expresses a costimulatory molecule. Here, costimulatory molecule. So, both sides have two major players and we're introducing this co-stimulatory player on these professional antigen presenting cells. Antigen presenting cells present antigen to naive T cells that have not encountered antigen before and that may trigger their proliferation and differentiation. They're active in different regions of the lymphoid tissue and their effect on the antigen presentation we've covered a number of times, dendritic cells the master, macrophage is pretty good too, B cells quite limited in what it can do. So professional antigen presenting cells, be they dendritic, macrophages or B cells, and what we see is dendritic cell picking up viral antigens moving into T cell areas, the macrophage is picking up exogenous bacterial antigens, bringing them throughout the lymph node structure, including to interact through helper function in the follicles, and then the B cells, primarily in those germinal centers, once they've been activated. We'll look at these now, shortly. Just an electron micrograph, that's the nucleic acid stained in a dendritic cell in the middle, but you can see these dendritic-like processes reaching out into all the adjacent lymphocytic cells, which are all nucleus, right? cells, which are all nucleus, right? So they're lymphocytes. So three lymphocytes in this picture with one dendritic cells with lots of dendrites or processes. So increasing surface area of antigen presentation through that fluid membrane to multiple cells at once and hopefully interacting and creating a recognition. The importance of this costimulatory molecule on the antigen-presenting cell is what will define it as an immature or mature antigen-presenting cell. When it's immature, these antigen-presenting cells will express very few MHC molecules and virtually no co-stimulatory molecules. So they can pick up antigen, but they're not really going to be good at presenting it until we get maturity forming through this co-stimulation. Now often innate immune responses help us up-regulate these co-stimulatory molecules and it's actually the innate immune response receptors present on these antigen presenting cells that recognize microbial surface components for example, So foreign bacterial sugars and cell wall proteins and that will induce B7 or this co-stimulatory molecule expression and lots more MHC upregulation of the MHC molecules to endocytose and bind and present these bacterial microbial components. So the point being is that antigen presenting cells are mostly quiescent, scooping a little bit, presenting a little bit, but in the presence of microbial surface components in particular, it's going to trigger a lot of upregulation of these components. And the B7 ligand is important because it will trigger a receptor on the T cell to activate that T cell. This picture is showing that in an immature dendritic cell, even though it's got class one and class two, it's got no costimulatory molecule, which is shown in green here, B7. So again, even though it can bind and present, it's not readily gonna trigger a T lymphocyte until some other cytokine factor or bacterial factor triggers adjacent cells to create and upregulate B7 and lots more class one and class two. So this is a mature dendritic cell which is loaded with antigenic burden and now effective at activating T lymphocytes. How does the macrophage do it? It'll phagocytose the bacteria here, break down the bacteria in the phagocytic lysosomes, and then it will trigger through those related cytokine cell surface factors, it'll trigger upregulation of the green costimulatory B7 molecule. So now we've got MHC presentation, we've got costimulatory molecule that the macrophage can now signal the T cell to recognize this antigen. And so it's shown here by your textbook that there are two signals that are critical to activate a T cell. The first is specific recognition of not only the peptide antigen shape for that promiscuity of the MHC haplotype that you might have, but sorry, allotype that you might have, but also the edges of that presented MHC receptor. And then the second message is the costimulatory signal, which is on the antigen-presenting cell is a B7 molecule and on the T cell is a CD28 receptor that will give the second message and that activates the T cell. It'll now proliferate, differentiate to T cell specific for that antigen. So a whole amplification of cells that can eradicate that type of pathogen antigen. How do B cells do it? They're a little unique. B-cell, you know, has the B-cell receptor on the surface, so it will bind, the specific immunoglobulin will bind a specific antigen, but then it will in turn, that will trigger Sir IgA and beta chains to internalize, degrade with the phagocytic lysosome in order to load on a MHC class II and put back on the surface. So that process again can trigger costimulatory molecules, B7, and now we get the two signals, the first being antigen recognition and receptor recognition, and then the second being the B7 costimulation by B cells to activate a T cell. So in summary, again, T cell activation requires signals from the T-cell receptor and also a costimulator receptor. These activated T-cells will then synthesize and secrete interleukin 2. For autocrine or paracrine action, induces T-cell proliferation differentiation, antigen recognition, and absence of costimulation often leads to a non-responsive state termed energy. And this is important because we've introduced two important things here. when the T-cells becoming activated its interleukin-2 is the cytokine that does it. Interleukin-2 activates T-cells. You've got to remember that. It's the special juice and if you can recognize an antigen but you don't get the costimulatory signal then you'll end up that lymphocyte will no longer ever be responsive to that sort of antigen, and it's termed an anergic state, and that's how we protect against recognizing our own tissues often. We develop energy from interaction with self-tissues. Just talking about how interleukin-2 activates the lymphocyte. A naive T cell will express only a low affinity interleukin-2 receptor, so this green low affinity two subunits, but it also contains the genes in that lymphocyte for a higher affinity receptor subunit. And so therefore, when an activated T cell, the T cell becomes activated, it'll start to transcribe and translate, that's what the squiggly mRNA means, these additional two receptor subunits in yellow, so it's an autocrine, or self, triggering, and binding of the IL-2 now to the third subunit, the two green and the one yellow, high affinity receptor will now send the signal to the cell for T cell proliferation. So, just showing that autocrine activation of T cell proliferation is from IL-2 and also the expression of a high affinity IL-2 receptor subunit. And here's that process of energy where the T cells at the bottom in the blue, it's looking for the two signals and an antigen presenting cells coming with a specific antigen, it's giving the first signal, and then it's giving co-stimulation. So there's a second signal that will activate that T lymphocyte. But if you've only got the specific signal alone without any co-stimulation, without any B7 on the antigen presenting cell, it'll become anergic. It recognized specificity, but there's no co-stimulation. I will no longer react to this antigen whenever it's shown to me, even though it's got receptors that will bind it. And finally, we also see that we can get costimulatory signal alone if somehow B7 was upregulated, but no antigen was specifically bound for this receptor. The one signal by itself, even if it bound this lymphocyte receptor, would not give any effect on activating that T cell. T cell activation requires signals via the antigen T cell receptor complex and co-stimulator receptor CD28 that binds V7 ligand. There's also another one, another player, CTLA4, that will also a receptor that will bind V7. And the importance of introducing CTLA4 is it's a mechanism whereby we can after an immune response has happened and the lymphocytes have destroyed a lot of cells or made a lot of antibody we can slowly shut down those lymphocytes so that they no longer cause this response in our body and the mechanism we do that is by putting up a competitive ligand that can also bind the costimulatory B7, but it's got way more affinity. It'll bind it 20 times more strongly. And so therefore, it'll start to out-compete and shut down CD8 binding on the T cells. Diagram shows it really nicely. CTLA-4 binds B7 more avidly than CD28 and delivers inhibitory signals to the activated T cell. So, we've got an antigen-presenting cell that's upregulated the costimulatory B7 green molecule and it's triggering lots of CD8, CD28 on the T cell to activate it. But basically what's happening now is we're seeing the upregulation of CTLA-4 in pink color here. and CTL-A4 has got way more affinity for the green B7, and so it's going to preferentially bind, and it does not send a second signal, so it's gonna start to reduce the activation frequency or the activation ability of this cell, this lymphocyte, because the lymphocytes are no longer getting the secondary signal through co-stimulation. And helper T-cell activation, CD4 T-cells can acquire different helper functions, helper one, helper two. Helper one is macrophage, helper two is B-cell activation and antibody secretion. So a naive CD4 cell becoming mature and either differentiating to helper one or helper two. Helper one being primarily macrophage can do other things, but we always think of it primarily macrophage activation, a help or two primarily activating B-cells to make antibodies. Cytotoxic, on the other hand, we see these professional antigen-presenting cells, dendritic cells, and even T helper cells, which will co-stimulate, will activate a CD8 T cell into a cytotoxic effector cell. So to get a cytotoxic effector cell, you can have three different mechanisms. There's autocrine IL2, there's costimulatory action from a helper cell joining in, or paracrine from an adjacent cell. We'll look at that in the next picture. So activating the cytotoxic T cells will program the apoptosis of a target cell through membrane bound ligands known as FAS and FAS ligand, the death domain on a cell that, when it's triggered, it will apropose. And it's all part of the TNF family activator of Caspase enzymes, are the ones that chop up the DNA. TNF stands for tumor necrosis factor, and so clearly it does the same when activating this death domain to a necrose these target tumor cells, for example. They also, cytotoxic T cells can release lytic granules. So these granules contain enzymes, perforins, as their name implied, they will perforate a target cell's membrane, and granzymes, granular enzymes that can destroy structural protein. So you can imagine if a cytotoxic lymphocyte comes up to an infected, one of our bodies are infected, virally infected cells, It's going to trigger the death domain ligand to start up with toes and caspases that are getting up-regulated in that cell, but it's also punching holes in its membrane to destroy its osmotic patency, and it's going to enzymatically degrade that membrane as well in order to destroy the infected cell, so it can't propagate that virus anymore. Here are those three mechanisms, autocrine, paracrine, and the third, autocrine, self, auto. So dendritic cells express high levels of B7, so it's a mature dendritic cell, it's virally infected with lots of antigen, and can activate a naive CD8T cell through the two signals, two arrows, first being receptor recognition and antigen recognition, and second being co-stimulation. So that's an autocrine standard mechanism, and therefore the activated CD8T cell makes its own interleukin 2 to drive its own proliferation, differentiation, and destruction of all these virally infected target cells in a tissue. The costimulatory mechanism, however, is where, for example, an antigen-presenting cell, which showing the class one but in the absence of costimulatory molecule there's no B7 on this antigen- presenting cell and so the cytotoxic cell is not responding but in the meantime the antigen-presenting cell has stimulated an effector CD4 T helper cell and this T helper cell activates the antigen and presenting cell to finally upregulate B7. And now the B7 was activated through this stimulation, the co-stimulation of the other response, recognizing endogenous antigen that's specific, will give the two levels of activation. And as you know, it will now upregulate autocrine interleukin II production for that clone to multiply into multiple effective cells. So a costimulatory mechanism where CD8 and CD4 cells work together. And finally, a paracrine mechanism. And so again, the antigen presenting cell here activates the CD4 T cell to make interleukin 2. And then this naive CD8 T cell starts to express the receptors. interleukin-2 secreted by that activated CD4 cell is bound there and basically will upregulate MHC as well as B7 in order to trigger paracrine mechanism of CD8 T-cell activation, cytotoxic cell activation. That's what happens when cytotoxic cells mediate apoptosis. We can see the cytotoxic lymphocyte, which is basically triggering the death domain on the target cell and also releasing these lytic granules at the sites of cell contact. An electron And a very important point about cytotoxic cell mechanisms in our body. They do not create inflammation. So here's a healthy cell. It's all nucleus, it's probably a lymphocyte. And we can see distinct chromatin in the nucleus. However, in a necrotic cell in this top picture, a necrotic cell, so one that's died because of lack of oxygen, for example. A necrotic cell will spill out its enzymes internally and undergo autolysis, right, self-digestion. And it will create a lot of pro-inflammatory molecules in that process of necrosis. However, an apoptotic cell is cleanly disposed of without all these pro-inflammatory signals, such as a necrotic cell gives out. And so therefore what we see here is that the chromatin is being condensed and chopped up with the activation of those caspases and likewise the membrane forms is shed in terms of vesicles that bubble off the apoptosing cell rather than blebbing is the word on the necrotic cell and triggering inflammation. And again, we see from this early-stage apoptotic cell to a late-stage apoptotic cell how effectively it's almost removed the entire volume of that cell by activating these death domains, caspases and the following. So effector T cells produce distinct sets of effector molecules, CD8 and CD4 cells. CD8 are any cytotoxic, CD4 can be helper 1 or helper 2s, and helper 1s to macrophages help or two do antibody secreting cells. CD8 do cytotoxic through phas, phas ligand, death domain interactions as well as cytotoxins. And what sort of molecules, I mean, would you recognize cytotoxic molecules, such as a perforating perforin, or a granulitic enzyme, a granzine, and phas ligand as a death domain, all being important cytotoxic effector molecules cells of a CD8 cell. It's sort of logical. Likewise, a T helper 1 cell is going to work primarily by enraging that macrophage through pro-inflammatory cytokines, TNF alpha, some colony stimulating factor for triggering more leukocyte development. leukocyte development. T helper 2 cells, different interleukins. I'm not going to examine anything here, okay? here, okay? Delete it right now. Don't waste your energy. We only talk about T helper 1 and T helper 2s. In fact, there are some other lymphocytes too. T helper 17 are known to be involved in inflammatory responses. T regulatory cells, important suppressing T cell responses, they're also follicular dendritic T cells, but what we're most interested in is a cytotoxic, that kills infected cells, mostly virally infected cells, T helper 1 to activate macrophage, T helper 2 to make antibody production. Oh my gosh, we got through it. That is very hard material. This is a very high- level lecture. We're introducing a lot of terms and we will examine from this lecture in the midterm. I told you, let's have a look at a few questions just before we leave. just before we leave. We've got five minutes. Which antibody isotype has the longest plasma half-life? longest plasma half-life? You got it. Somebody wants to get out. wants to get out. Why IgG? Which Each isotype has the longest plasma half-life. We're just clear. There's gamet, so we've got all the different isotypes. So we know that M and D are synthesized first in the primary immune response, but IgM is a huge lumbering molecule that gets cleared pretty quickly around a 10-day half- life, IgG a lot longer. And so therefore, you're going to find most therapeutic antibodies we use with patients are going to be IgG isotype because they've got a better profile for half-life. All right, where would you expect to witness the clonal deletion of T cells? We just did it today, Prof. Dostagas, so unfair. T cells, so T lymphocytes are in the thymus, right? Where would you expect to witness clonal deletion? Why would we be deleting T cells? Because maybe, perfect, absolute correct answer. You plucked it out of my thought process and said it in a co- stimulatory manner. Thank you so much, that's exactly right. So it's cleaning off thyroid, lymph nodes are not going to delete the T-cells, belt, bronchial associated lymphoid tissues, these loose aggregates of lymphocytes, is not deleting T-cells, that's where they're active. They're active in lymph nodes and belt. They're active in the spleen. But in the thymus, that's where you're going to see it. Yeah, you have to think about that one. But it's really only one good choice there as an answer, if you know the processes. This one you know now after today's lecture. Exogenously derived antigen, like from a bacteria that gets scooped in. Is that class one, class two? Is it going to a CD8 cytotoxic cell? Is it presented to a T cell receptor? Is antigen presented to a T cell receptor exogenously to the MAC class one after proteasome digestion? So it's exogenous. It can't be proteasome. That's endogenous, right? Class one's endogenous. So scrap A and scrap E. What about C and D? All right. So, to a site, a toxic cell works with class one. That's endogenous, scrap C. What about, so my choices are B and D. So, an MHC class two molecule will definitely interact with exogenous antigen. MHC class two. And exogenous is presented by the T cell receptor. No, no, it's presented to the T cell receptor. That's why I can't do that one. Only one correct answer. I had to think about it. I knocked out the other ones, but I got it. You are a higher level immunologist, okay? All right, guys. I think the exam's here. I'll see you in here, okay?