T Cell Biology PDF

So, I've been introduced many, many times, it looks like, that I'll be talking about this, I'll be talking about that, but I will talk very little. So, don't worry. Just to introduce myself, I'm the Graduate Program Director and Professor and the Vice Chair of Education in PMI. So, which means that...

So, I've been introduced many, many times, it looks like, that I'll be talking about this, I'll be talking about that, but I will talk very little. So, don't worry. Just to introduce myself, I'm the Graduate Program Director and Professor and the Vice Chair of Education in PMI. So, which means that anything that you have in terms of issues, even with Dr. Greeta, you can come to me. I know my research interest, however, is in cancer biology. As I mentioned right in the introduction that most of the people that will be teaching you or will stand in front of you to go through the sessions, have been experts in the area, kind of. I mean, we've been involving the wheel to some extent, and like, for example, I mean, this morning Dr. Greeta talked about MSC. He did his post-doc, this is about 30 years, he's been involved with MSC for the last 30, 35 years, with Stanley Nathanson in Albert Einstein. So, when I'm teaching you T cell biology, I was purifying T cells back in the early 80s, when there were no markers and you'd be talking about cluster of differentiation and CD markers and the complaints that will come up, do we need to know all the CDs? Some of them that are important, you need to know. And I've been involved in cancer biology for the last 35, 40 years. So, obviously, we've seen a lot of changes that have come up and I hope that we can convey those changes and the passion that we have for this basic science that I think will be the basis of your clinical work that you will do in your life. And that is a great privilege for us to do that. So, T cell development is what I'll be talking about. And why are they called T cells, by the way? Time was wonderful, that is Dr. Liria's magic. So, time must, because they are T cells, they are time must derived. Nothing happens with respect to T cells until and unless they get into the time must. Prior to the time must, they are very similar to many different cells. Once they get into the time must, something, some magic happens. And it is because of that magic that we are alive. Because if there were no T cells, severe combined immunodeficiency diseases. So, that's an important aspect of our structure and what we do. We were always not like that. If you go through evolution, T cells have a different role. But you know, that's a story for a separate time. But basically, if you think about it, evolution of T cells and the B cells happened as a result of one gene and that Dr. Suriano mentioned RAG1 and RAG2. We'll talk about all that. So, our objective is define the origin structure and function of T cells. These are time must derived T cells. And I've been talking about a branch of immunology called cellular immunology. Why do you think it is cellular immunology? Because once you know the terminologies and how they are connected, you will remember concepts. That's why I bring that up. Cellular immunology. And B cell, everything with respect to B cell is called humoral immunology. Why? Everything happens with the B cells in the humus, the bone marrow. Everything happens with T cells in the time must, that's the way time must derived. But in the context of two cells, always. So, that is why it is cellular immunology. So, what are the two cells that are important in this? One is the T cell that will have a receptor and if it has a receptor, it has a ligand. The ligand is the other cell with which it interacts. And what it interacts, and Dr. Gallipra has gone through this many, many times and I will repeat it at least 50 times today, T cells recognize only processed antigens. Processed antigens means processed proteins. Processed proteins means peptides. Peptides in the context of MHC and nothing else. MHC peptide complex is what the T cells recognize by virtue of their receptors. So, we'll examine the mechanism by the generation of the specificity and diversity of cellular immunity. And the most important feature of T cells is related to thymic education that we will discuss. By the end of the session, you should be able to understand the difference between self and non-self. That is the most important job of T cells to distinguish between self and non-self. Non-self is basically anything that we get bombarded with and self is all the antigens that we're talking about. Now, if you think about it, T cells recognize only proteins, not proteins, peptides, processed peptides. That means processed through the whole protizoma degradation and presentation. MHC on the cell surface cannot come without the peptide in it. And Dr. Ghidipra just mentioned all the mechanism by which peptides are put into the MHC cleft and the rules for it, right? So, MHC cannot come up to the surface without a peptide. The peptide has to come in the cleft and then that is the MHC peptide complex. That is the ligand that the T cell receptor interacts. And that is a part of what we call cellular immunity and the humoral immunity is a part of the adaptive immunity. Why? Adaptive immunity is extremely important to why? Through evolution, we have evolved a system of diversity and specificity that is extremely important. Adaptive immunity does not come up unless an innate immunity has laid the foundation for an adaptive immunity. What is that foundation? If you think about it, it's an inflammatory response. So, if you think about clinically, when you will look at inflammation and inflammatory response is what gives you an idea that an immune response is occurring. Cleaver is also an immune response as a result of cytokine induction and sort of changing the thermostat. Such that it is not conducive for infectious diseases to propagate. So, with that introduction, what are the things that we will be talking about? We are talking about an adaptive immune response that is the cellular immune response. Centered to this cellular immune response and adaptive immune response, innate immune response has to come in. That means there has to be a baseline inflammatory response by which the adaptive immune response sets in. And so receptor in B cell also you talked about receptor is a very important aspect. B cell receptor, which is the antibody, when it is soluble, it can be soluble as well as it can be onto the surface of the B cell. But a receptor ligand recognition is key for the induction of an immune response. So receptor or recognition, correct? Recognition is important. Once it has recognized also, immune response has to be regulated. And no one has taught us more about regulation than COVID. That immune response needs to be regulated. The pathogen itself is not the problem. A regulation of the immune response is a major problem. So regulation is an extremely important aspect that, of course, and all of these three things that we will talk about. So if that is the case in terms of cellular immunity, today's session, and I just want to preface that again, is a very conceptual session. Once you understand that, then all the other facts that will come in subsequent to today's session will be much easier and will be easier to understand and to remember as well. So center to that is the T cell receptor. So we should understand the T cell receptor structure and function. So we will talk about the T cell receptor, how it is made, and what its function is. And the most important function that you will, understand of the T cell is these two functions. One is MSC restriction, and the other is elimination of auto-reactive T cells. That occurs in the thymus as a result of two processes. And the first process is positive selection. You will hear this a lot, and this has become almost a colloquial language in the clinic as well. MSC restriction is something that, it is a colloquial language, but you should understand what it is. And Dr. Ghidipra alluded to this in his session early in the morning. MSC restriction is the ability of the T cells to recognize your MSC. Your MSC repertoire is very individual. So the T cells that recognize the MSC are restricted to your MSC are very individualized. And that is why they cannot be transferred from one person to the other. Although the same pathogen will be eliminated by each one of you. And the other thing is MSC restricted, yes. But how is this done? How are the T cells educated so that, basically they are MSC restricted. That means they can recognize only peptides in the context of your MSC. That's important in transplantation as well. So that is positive selection. And of course we know that the distinction between self and non-self is important. So elimination of the auto-reactive T cells, T cells that will recognize self antigens, they need to be eliminated. So that you should have a repertoire of T cells that is diverse enough, but specific to the host. So that means it will recognize when a pathogen comes in and your MSC presents that particular pathogen, it will recognize it, it will eliminate it, and that will be the effector function that we will talk about. Today we'll just talk about the development. And tomorrow morning I will talk about how the effector function is generated. So that is the specificity and diversity of this immune response, and it is mind-boggling as to how we can generate this amount of diversity for things that we have not even encountered. Or what we have encountered, we generate all these memory cells and all, and we will get into all of that when we get into regulation. And that also is mind-boggling that for everything that we have encountered, we have generated memory cells and everything exists within our body. So it's fascinating. So let's move on to our, it's important in terms of why is the generation of this particular education and what happens in thymus is important, because you have several diseases that are, that you see a common acute rheumphoblastic leukemia is a disease that even for a progenitor cells. Thymoma is a thymic stromal epithelial cell disease, acute rheumphoblastic leukemia, again it's a thymo, and these are some of the markers that distinguish the T cells that harbor these diseases. So they are very specific to these diseases, hence those markers are important. And that is why what these are CDS, what are CDS cluster of differentiation. So the way that these cells differentiate and the markers that come up, those are the ones that have been sort of marked as a marker of that diagnostic marker of that disease. So you have a stem cell, which is CD34 is the marker CLL, CD10, 19, 20, and of course these are targets for therapy as well. Antibodies to these can be used as therapeutic agents. And so you have these thymic stromal cell, cytokeratins and Caesare syndrome, adulte cell, leukemia, which is a CLL, you have CD3, TCR, CD4 and CD8 that is in the periphery. These are the progenitor cell markers and these are ones in the periphery. So you've seen this slide, I will not go into great details of it, but fundamentally the way that the diversity is created is very similar in the B cell as well as in T cell. So you have the heavy chain and Dr. Soriano, Robert mentioned the heavy chain is similar to the beta chain, the light chain is similar to the alpha chain and you have these variable and constant region genes and you have transmembrane region with a little bit of it in the cytoplasm and keep in mind that this is on the surface of a cell. PZ is on the surface of a cell. And what is this surface is a plasma membrane, is a fluid thing with lipids moving around. It needs, if it interacts with the ligand, it needs to be stabilized. So this is the T cell receptor and I will come to the T cell receptor complex when you have other proteins that come in, not as interacting with it, but by a wonder walls and by charge interaction, they're just holding the receptor up there so that it can give the time to interact with this ligand. So that's why you need the T cell receptor complexes. That's why you need the B cell receptor complexes because it's a fluid membrane cell interaction. You need the time for it to interact and that's why Dr. Gilipta mentioned core receptors that are also important in stabilizing this interaction. Okay, so one of the big things that you should remember in this is that, let me hide this float. So yeah, so down, go on. Okay, so cell surface bound, the ones that are in blue are important for your exam as well as things that, you know, in your green boxes that I have on the slides, high yield and all of that, you know, you guys are experts in finding out what will be the question. So there's always a surface bound, never soluble, very short cytoplasmic tail, no somatic hypermutation. That means once a receptor, T cell receptor is made, it is made. The affinity maturation takes place with B. It does not take place with B cells, T cells. So, but what is similar and that's the most important thing is the gene recombination mechanism is this. Why? Because the enzymes that mediate it are the same, rag one and rag two. And I'll just say, you know, not go into details. If you come into my graduate class, then we go through the evolutionary aspect. It was a transpose on that hit the immunoglobulin genes and separated them. Rag one and rag two were those transpose on genes that actually got separated. So that's why when rag one and rag two functions as a dimer, it recognizes the same sequences that it split, heptamer and nonamer that Robert Soriano talked about. The 1223 rule that he talked about. So those are the spatial regions by which, because everything in biology happens as a result of conformation. It doesn't recognize the sequence. But it is the folding of that protein that recognizes those sequences is based on that conformation. So the way that the gene was split, nonamer and heptamer were there. That is the recognition signal. And these two dimers go in and bind to the area that is compatible with the 1223 spacer region. That is why the gene recombination event is not a random event. It is a very orchestrated event which is guided by these sequences. So how is the diversity created? So just to mention that basically there are two kinds of receptors. One is the alpha beta receptor, which is 95% of the T cell receptors that you see. 5% of this is the gamma delta T cell receptor. What you need to know and remember for your exam as well as for your board is this 95% of the human T cells are alpha beta. Gamma delta T cells home to the skin reproductive tract and epithelial tissues and are not MHC restricted. Underline that. They do not go through thymic education and they are not MHC restricted. We know a little bit of its function, but not much. Now, this is how the diversity is created. Why is this important for you guys to know that you know how much of this, much of molecular biology and I'll show you the next slide, how important this is to define tumors. So you have 70 to 80 of these regions that combined with 61 of these. Each one of these will combine to form one alpha chain and you have these constant region genes. So then alpha receptor, alpha polypeptide chain is made onto the surface. So obviously you go through permutation and combination. Dr. Soriano went through, Glifter went in greater details today. Basically you can, and then you have the beta chain that has go through the DJ joining first and then the V beta chain. Now you have 52 of these that can combine with each one of these six. And so when you do the permutation combination, you get and keep in mind that one of these alpha can combine with several of the beta ones. So you have a different receptor with a different affinity. Each T cell has only one receptor on its surface. So with this permutation and combination, you have about 10 power seven, 10 power eight. 10 power eight, basically a kind of distinct discrete T cell receptors on a single T cell surface. So far, so good. And this is all done through this 1223 spacer regions. And it is important that the gene that actually recombines has this either the heptamer or the non-nomer sequence. The heptamer sequence is the CAC AGTG and AC followed by five A's and C is the non-nomer sequence that the RAG1 and RAG2 recognizes. Very important, RAG1 and RAG2 are present only in lymphocytes and all the other genes that enzymes, DNA repair and recombination, et cetera, they are present in all nucleate cells. RAG1 and RAG2 only present in lymphocytes. That's why you get the B cell and T cell. Diversification and diverse, right? How would you remember this? Somatic gene recombination. Now, you see, okay. And then you have this, what we call the P and N nucleotide diversification where they have the terminal deoxidone nucleotide transferase that is an important enzyme that opens up that hairpin loop structure and then you have these random nucleotides that come in and pair and then you have, this is the additional diversification that comes to about 10 to 100 fold and this is the hyper-variable region of the T cell receptor as well as the B cell receptor that you see as a result of this P and N nucleotide diversification. So important. RAG1 and RAG2 defects, you see in what? See recombine immunodeficiency disease. Why? Because this SCID patient with defective RAG have non-functional B and T cells and are prone to multiple infections or are thrust due to candida. This bone marrow transplant can rescue the infants from SCID, which is three times more prevalent in males than females, and you have this X-linked SCID and I'll come back and talk about fungi also to you guys and fungal infections and coliform bacteria, diarrhea, et cetera, is very common in patients that have defective RAG genes because they cannot make B and T cells. What happens if there's a partial, if there's a mutation or there's a partial function of the RAG and RAG2 genes? You have basically a disease called the Omen syndrome. The diseases that I talk about are very, are important in for your board exams, not necessarily that they come all the time, but they always are mentioned and I give you a big list at the end of all the immunological diseases that are important with the pathways that we have gone through with you guys. So the T cells present over here, are home to the skin and they're activated to attract what we call these inflammatory cells and you see the Omen syndrome is characterized by high IgE levels and eosinophilia and we will talk about this. It has a TH2 phenotype which secretes IL4 and IL5. When I said the recombination stuff that I talked about and why is that important? When you have T cell clones and T cell receptor gene rearrangement that characterize individual clones of T cells. So individual clones of T cells are characterized by the alpha and beta chain that is as a result of the recombination, right? So now if you have a tumor, look at this, this is a normal patient, this is a Southern blot analysis, but what is important is that you see these additional bands which means that these are clones of T cells that are proliferating a lot in the patient. Now as a result of, you can even monitor that before and after treatment to see whether those T cells are being targeted or not by your therapy. And that is why, and this is as a result of, you can sequence it and do a PCR and go into which V region gene or the J region gene or the beta gene was actually targeted in these clones. So you can go through, so that is why it is critically important for you guys to know a little bit of the diversity of the genes by which the somatic gene recombination brings about the different kinds of T cell receptors on the surface. So now we have the main actor in today's play has been defined, the T cell receptor, right? And we now know the way the diversity is created and we also know if these T cell receptors have very unique receptors on the surface, they will recognize what? What will they recognize? What will these T cell receptors recognize? Only MSC, MSC peptide complex, the answer that I'm seeking for MSC peptide complex. T cell receptors, MSC peptide complex, that's it. So the ligand is always the MSC peptide complex. Dr. Raria mentioned CD3 many a few times in his thing. This is basically, if you look at this, these are the CD3 areas. The T cell receptor, the polypeptide alpha beta is put together as a T cell receptor complex by these invariant proteins, epsilon, delta, gamma and epsilon as well as zeta that actually compensates for the short cytoplasmic tail. And now this is important for signal transduction. What is the consequence of a receptor ligand interaction? This is brain biochemistry. The consequence of receptor ligand interaction is gene transcription. Why? Because the ligand and the receptor as a result needs to induce certain genes to change certain phenotypes or to make certain things that is necessary for that particular cell. This is what is signal transduction. And what is signal transduction? Signal is basically a receptor ligand interaction that goes through some second messenger signals, second messengers that do some alterations by which transcriptional factors can get into the genes. This is all. And most of the times it's a very simple mechanism of phosphoridation and dephosphoridation. Phosphoridation is a very important mechanism in biochemistry by which everything happens. A lot of things happen. And for you, that becomes important. Why? Because you will see because all of the immunological events that are mediated as a result of signal transduction are the ones that you will target, cytokines, et cetera. So that's why it's important. So this, we talked about the T cell receptor with the alpha and beta polypeptide chain. And then you have the T cell receptor complex which has these invariant proteins. And these together are called CD3. And so you can use an anti-CD3 antibody to histologically stain things to determine if you have those T cell areas or not. So far, so good. Crystal Crea, wonderful. So once we have defined the act, and now let's look at the play. This is act one. We will go through act two tomorrow. And Dr. Rady has already mentioned what happens. But let's look at it from an immunological aspect. So Thymus is a primary organ. Bonmaro is another primary organ. Rest, he talked about the secondary lymphoid organs. Secondary lymphoid organs, let me define them also, are the arenas where the battle occurs, they're the choroseum. But this is not one of those gross things that we would see in a Roman choroseum, but basically things that are extremely important. Those are areas where T cells and B cells, they interact with their compatriot to see whether we need to get activated or not, whether we need to become effector cells or not. And that is why you have so many different kinds of secondary lymphoid organs that is distributed all over your body. Mod, guard, et cetera, et cetera. And he talked about so many different cells that are important. And that's extremely important for an immune response to happen. Primary organs do not induce immune responses. Immune responses happen only in secondary lymphoid organs. So what does the thymus do? I mean, Dr. Deria went through a lot and he told you exactly what it does, education of the T cells. So it's a primary organ, and the thymus does not receive lymph and is involved only in T cell education and is not concerned with the activation. Activation is secondary lymphoid organs. So the T cells, everything that happens with respect to T cells happens in the thymus. We just talked about gene rearrangement. Where does that happen? I heard thymus. Does everybody agree or you have a dissenting view here? Bone marrow. Whoa, that's a real dissenting view. I would go with thymus. Everything regarding the T cells happens in the thymus. Just always remember, it's a T cell, thymus. That's the first sentence that we spoke. T cells, thymus. So you have these progenitor T cells that come from the bone marrow, but the progenitor cells come from the bone marrow. The bone marrow makes all the different kind system cells. And when they come into the thymus, they are educated. What does education mean? They differentiate and they are taught a few things. They're taught only two things, but they remain loyal for life. They're taught positive selection and negative selection. Positive selection is, hey, you guys should not recognize anything else until unless your peptide is presented by the MSC, MSC restriction, positive selection. You should not recognize self. You should, so all the T cells that will recognize self, antigens are all eliminated. That is negative selection. So these are the only two processes by which things, the T cells are educated, right? But for the education, they need to interact, the T cells have to interact with the MSC ligand. They need the T cell receptor on the surface. So what they, when the progenitor T cells come in, what do they do? They first rearrange and make their receptors. So the progenitor T cells, they come in, enter the thymus with the blood, and they come in into what Dr. Raja just mentioned into the cortex. And they interact with the cortical epithelial cells. They are of thymic origin. And this is where most of the positive selection takes place. There is a syndrome that is extremely important for you, which is called the de Georges syndrome. The de Georges syndrome, the thymus fails to develop, and you have a severe combined immunodeficiency disease. And clinically, what it leads to the loss of chromosome 22Q11, and it leads to the loss of terminal epithelial cell differentiation. And what are the clinical symptoms that you see? Cardiac abnormalities, C, abnormal fascia, A, chymic aplasia, T, cleft parrot, C, H, hypocalcemia. The mnemonic is catch 22. Why? Because it's 22 chromosome. Loss of chromosome 22. Catch 22, chymic epithelium. So you see how important the cortical epithelial cells are in the thymus. The progenitor cells first interact with the cortical epithelial cells. But what will they interact? T cells cannot interact with anything else. They need the receptor first, right? So the receptor is made. So this is what happens, commitment of the T cell lineage. Now, I'll go through this slide. It is not a very difficult slide. We talk about double negative. We talk about, there are two terminologies that we'll talk about. One is called double negative and double positive. Everything refers to CD4 and CD8. These are molecules on the surface that Dr. Griepter mentioned to you. Double negative means cells do not express CD4, neither CD4 nor CD8. Double positive means that the cells, T cells, they express both CD4 and CD8, right? So you have, first, if you think about it, you have an uncommitted progenitor T cell. Double negative is CD4 and CD8 negative. So you have an uncommitted progenitor T cells that is CD34 and CD44. This is the stem cell marker. It tells you that you have this lymphoid progenitor cells that is a stem cell mark, a stem cell, which, what does the stem cell mean? They are pluripotent cells. They can become anything. They were going to differentiate and go through this whole differentiation process. So the adhesion molecules come in first and why the adhesion molecules are important? Because they have to interact with the cortical epithelial cells. So they have to adhere first. And these uncommitted progenitor cells become double negative thymocytes that are now getting committed to the T cell lineage. Differentiation is happening. All this is happening in the thymus. And what happens when they become double negative? CD2, CD5, CD127, and CD1A, together with a rare complex gets upregulated. Why is a rare complex getting upregulated? Because it has to start recombination. These are all adhesion molecules. And these are cytokines that are important in terms of their differentiation pattern so that now these T cells can be selected for the education process, right? So basically you have these uncommitted progenitor cells that will start rearranging their gene such that they become there. And it starts with a double negative. All of a sudden through the differentiation process when the rearrangements have taken place and alpha, beta is on the surface, they become double positive. That means CD4 and CD8 are upregulated. So far so good? So progenitor cells, stem cells, adhesion, goes through the rare complex and a lot of things are upregulated. Double negative becomes double positive. And these cortical epithelial cells have MSC class 1 and 2 and they are presented presenting peptides. There is no infection, but they are presenting peptides. Why? They are presenting self-peptides, normal proteins of your body that have been broken up. Proteasome is doing that all the time. So they are presenting it with the MSC, class 1 and class 2, right? The ones that will, so let's go through a little bit of more details of this fundamental process, double negative, double positive, and then it becomes single positive. Not that all of this is extremely important for you, but what I want to sort of categorize this and then you will understand this better that while you have this, from double negative to double positive, or this gene rearrangement is taking place, right? Bear with me. Then you start having these signaling molecules that come up and the whole gene rearrangement process is complete. Then you have the signal transduction molecules. You will understand that a little better after tomorrow's lecture, why they are important and they are activated. And all of these transcriptional factors that are induced are the ones that differentiate the different lineages of T cells. CD4, CD8, T regs, et cetera, et cetera. All of this is as a result of these transcriptional factors. So, but the net process is that basically a T cell receptor has to be put onto the surface. So that it can interact with the cortical epithelial cells that are presenting the normal peptides in the context of the host hemisphere. So this is the early development. You have progenitor cells come in double negative, become double positive and mature double positive cells. They become single positive cells. So, yeah. So this is positive selection. So this is what happens. This is you have your cortical epithelial cells and your T cell receptors have been made. They are interacting with the MSC peptide complex. Only the cells that will recognize MSC peptide complex with a moderate or strong binding are given the signal to proliferate. The ones that don't recognize MSC peptide complex are of no use. Why? Because they have to be MSC restricted. So this is the positive selection process first. Basically T cell receptors that are T cells bearing the T cell receptors that interact with the MSC peptide complex presented on the cortical epithelial cells with a strong or a moderate binding actually are MSC restricted and given the signal to proliferate. One second. 95% of the progenitor T cells that make the T cell receptor die. It's only 5% of this that actually recognize the MSC class one and class two that are given the signal to proliferate. And yes, question. One more question. From double positive you become single positive. CD4 reacts with MSC class two. Four times two is equal to eight. CD8 reacts with MSC class one. You have a number eight. This is also according to the Hindu mythology. Number eight is the most auspicious number that you can think of. So if you remember that four times two is equal to eight. Eight times one is equal to eight and everything is great and you will live. Yes. No, it's a ligand receptor interaction. I kept talking about this ligand receptor interaction. Of course, there is a lot to it but the ligand receptor interaction is the most important because that is what recognizes what in ultimately you will recognize. No, MSC, it is all different. What is happening with this particular red dot is all coming from different kinds of proteins, normal proteins, right? This is coming from breakdown of normal proteins and these peptides are being presented. All different kinds of only the ones that actually will recognize the MSC peptides with moderate binding are going to be going to be positively selected. The rest will be, rest will die. This is 95% of the population, weak or no binding. Okay, let me answer your question in a different way. When the T cell receptor is made, each is a unique receptor. It has its own affinity. So if that affinity is moderate or strong in the beginning for the T cell receptors that actually bind to the MSC peptide complex that are presented by the cortical epithelial cells, you have positive selection. So far so good? Questions? Yes. Yes, yes, yes, yes. Yes, they do. But that is my short answer. But the fact is that you have two times more CD4 T cells than you have CD8. So obviously there is some bias there. And that bias is very beneficial. The CD4 T cells, you have a big repertoire. CD8 is only one kind, yes. So the dictum is that it is a random, random event. But how can it be random when you have two times more of CD4 than you have CD8, right? But the fact is that it is a random event. It all, again, depends on affinity. And Dr. Glitter went on into great details of how the affinity of the different peptides and the different MSC alleles can change affinities. And that is exactly what happens in this case. Okay, so let's go to what happens. So now the ones that are positively selected that are MSC restricted, they are in the cortex. Now they move into the medulla. And the medulla is where another magic happens. That is the negative selection, yes. So CD4 and CD8 are proteins, right? So they are there in that differentiated cell when double negative or double positive, both of them are upregulated onto the surface, providing the T cell different, that differentiated T cell an equal opportunity to either become a CD4 or a CD8. Once it becomes a CD4, once it interacts with the MSC class two, MSC class one is downregulated, and now that cell remains to be a CD4 cell. Yes, yes, yes, why? Because you have all different kinds of regulatory T cells. CD8 T cells have only one function. Let me find the guy that I need to kill and kill him. That's it, okay. Negative, now they get into the medulla. Yes, question, yes, yes, yes. The short answer is yes. It is a chance event, but fortunately for CD4, the chance event favors it two times more, three times more than that, yeah. So now it comes into the medulla. And in the medulla, it encounters another group of cells. And these are the professional antigen presenting cells. This is your dendritic cells and macrophages, et cetera. And in this case, negative selection of alphabetic cells by dendritic cells, T tight binding, eliminated. You are recognizing you are MSC restricted and is great, but now you are recognizing self antigens. If they are going to be presented by professional antigen presenting cells, you are no good for me, so you have to die. So anything for tight binding, but a moderate binding lives on, and this is the process by which auto-reactive T cells are eliminated. And so let me just go through this. Double negative becomes double positive. That is the time we go through positive selection and then double positive go through the medulla and go through the negative selection and they're eliminated. In the end, what do you have? You have a mature self restricted. Self restricted means MSC restricted. Self restricted means MSC restricted. Self tolerant means they will not recognize self- products. Single positive CD4 or CD8 T cells, they are naive cells waiting to become effective T cells. And let's go through an animation, I think, and... T cell development takes place within the thymus, a specialized organ where T cell progenitors mature and differentiate. The thymus is divided into several different anatomical locations, and as thymocytes mature, they pass from one environment to another. T cell progenitors enter the thymus through high endothelial venules and migrate to the subcapsular region where these thymocytes start their process of maturation by rearrangement of their T cell receptor genes. As maturation proceeds and the cells begin to express a specific receptor, they move further into the thymic cortex. Interactions between the T cell receptor and MHC peptide complexes displayed by the thymic cortical epithelial cells play an important part in the fate of the thymocyte. At this point, the developing thymocytes express both CD4 and CD8, as well as an alpha beta T cell receptor. A cell that has a receptor able to recognize an MHC class one molecule receives both a survival signal and a maturation signal. Eventually, the cell stops expressing CD4 and maintains expression of CD8. Thymocytes that obtain a survival signal are said to undergo positive selection. A cell that has a receptor able to recognize an MHC class two molecule also receives a survival signal and a different maturation signal. Eventually, this cell stops expressing CD8 and maintains expression of CD4. Thymocytes whose receptors are unable to recognize either MHC class one or MHC class two molecules fail to receive any survival signals and die by programmed cell death or apoptosis. Thymocytes that are able to recognize MHC class one or MHC class two peptide complexes too avidly receive a strong signal that drives them into cell death. In this way, thymocytes capable of responding to self-peptide antigens are eliminated in a process known as negative selection or central tolerance. The surviving T cells migrate from the thymic cortex to the medulla. At this stage, they remain capable of recognizing self-peptide antigens expressed on other cell types such as dendritic cells or thymic macrophages and receive a sufficient signal to cause them to undergo programmed cell death. The remaining thymocytes, now mature naive CD4 and CD8 T cells pass out of the thymus either returning to the bloodstream directly, passing into venules or via the lymphatic system. So basically, I bring you back to the original first slide where some of these diseases that important, I brought about skid and I brought up omen syndrome that is important. I brought about some of the reasons, the ways by which you can look for T cell tumors. I think the last slide that I want to talk about is auto- reactive regulatory T cells. So we talked about a central tolerance mechanism of negative selection by which all the T cells that recognize self antigens, self peptides coming from self antigens are eliminated. You also have a group of CD4 T cells that become what we call T regs and you will see this a lot in your clinic now because the T regs are extremely important in maintaining what we call peripheral tolerance. So central tolerance is what has happened in the thymus negative selection. This is, and these are a group of T cells that are CD4 positive and CD25 positive. So you remember, you know now why the CDs are important because they classify a particular cell type. So CD4 positive, CD25 positive, FOXP3 positive is a T reg cell. And when they are absent, you have an immuno- peripherative immuno-disease that is extremely important, which is called IPEX. And we'll talk about this, but I want to just end up with central tolerance and peripheral tolerance. We will talk about peripheral tolerance in details also in terms of activation, but all of these tolerance mechanism is protecting us from attack within. So that's an important concept that I just wanted to raise up. These are a group of CD4 cells that have a very important regulatory function, but they have also have a very good, very important therapeutic function that I will talk about in the cancer biology regulation of immunology because this is also important. What if supposing this particular antigen was a cancer antigen and you have an improperly, you had a regulatory cell that suppressed activation. So these are also important subsets of cells that I have gained therapeutic importance. Anyway, I will end today here and thank you very much. And we will talk again tomorrow. Yeah, give me one second. Let me record, stop recording. Why didn't it say? You just have to say yes. Oh yeah.