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[Audio] Welcome to the second part of the lecture, Generation of diversity in T and B cell Receptors..

[Audio] In this part we are going to talk about the T cell receptor structure and the generation of diversity in T cell receptors. This part is shorter than the first one because the general mechanisms of the generation of T cell receptor is the same as, as the one for B cell receptors..

[Audio] Here you see the T cell receptor structure compared to antibody. T cell receptor is made up of two polypeptide chains, just like the B-cell receptor. But when it comes to the T cell receptor, the two chains are called the alpha chain and the beta chain. Just like the B cell receptor, they have a variable region, the lower part here is the cellular membrane. The variable region of the T cell receptor is the outer part. There are two immunoglobulin domains. Which of the one of the alpha chain and one of the beta chain that make up the variable region of the T cell receptor. And then the two immunoglobulin domains that are closest to the membrane make up the constant region C And then if you see here, there is also a small hinge region and then a transmembrane Membrane region, just like for the membrane bound antigen receptor, the B-cell receptor. And also here you see very short cytoplasmic tails. And those two chains, the alpha and beta chain are also joined together by a disulphide bond. To the right you can see, similar as you see the terminology that we showed before for antibody where you the FC fragment. And actually here you can also see the outer fragment, which is the fab fragment where you have the antigen binding site. Uh, and this so this arm, the arm of the antibody which contains the four immunoglobulin domains, two for the light chain and two for the heavy chain are corresponding to the T cell receptor with its antigen binding site in the outer part..

[Audio] Next slide describes T cell receptor complex. Similar to the B-cell receptor, the T cell receptor doesn't have a any intracellular portion. Instead, the T cell receptor is also associated with a signalling polypeptide chains. And this complex of different chains is called the C-D-3 complex. When the T cell receptor is engaged, the C-D-3 complex will signal into the cell that there is an activation. The T cell receptor binds M-H-C--. So in this case there is an M-H-C class two which is which is the molecule that is recognised by the C-D-4 positive T lymphocyte. The little red ball here illustrates the peptide. So the T-cell receptor will bind the M-H-C class two molecule that presents a foreign peptide, and at the same time that the T cell receptor binds the M-H-C class two molecule. In this outer region, there is also a molecule called C-D-4 that will bind to the inner part, the more membrane proximal part of the M-H-C class two molecule. And it's this interaction of different surface molecules that will lead to activation of the CD4 T helper cell. So the C-D-4 molecule will also bring in some intracellular activating proteins and stabilise the binding between the T cell receptor and M-H-C class two complex..

[Audio] Nex move to the organisation of the T-cell receptor genes. The upper part is the locus of the T-cell receptor alpha chain. And below that is the locus for the T-cell receptor beta chain. And you can see here actually that the alpha chain corresponds to the light chain. Because the alpha chain has a number of V regions here in red. They are 70 to 80 different regions illustrated here in this complex in this locus. And then there are a number of J regions, the joining regions. And here it's 61 illustrated, in this complex. So the alpha chain only has V and j regions or v j segments, while the beta chain in the lower part you can see has the red segments, the V segments. It also has uh, the green uh D for diversity segments and the yellow J segments. And then for both alpha and beta chain, you also have the blue, constant uh gene segments. The variable antigen binding sites of the T cell receptor are also made up of the D and J segments. And then you have the constant region which is C in the illustration. And so the alpha chain is made up of the V plus the J together with a constant segment. And the beta chain is made up of a rearrangement between v, d and j plus the constant segment..

[Audio] The next slide shows the stepwise recombination of the T-cell receptor genes. And we start here in the lower part where there is first a rearrangement of the T-cell receptor beta chain. Just like the heavy chain is first rearranged in B-cell in B-cells. So the very low lowest part here you can see the germline D-N-A--. So germline in this case means the non rearranged T-cell receptor beta chain locus. And you see that indicated the different V segments the different segments and the J segments. And so this again is a stepwise rearrangement where the d and the j first rearranges and then the DJ rearranges to the V segment so that above that you can see the rearranged D-N-A where you have a V and a D and the J joined together. Now this is then, uh, transcribed and spliced and translated into the T cell receptor beta chain, which you can see here indicated in the membrane. And then in the upper part you see the corresponding process for the alpha chain where. Now in this case there is a v that is directly joined together with the j And then there is a transcription, splicing and translation to form the T alpha chain protein that is then combined with the receptor beta chain protein on the surface of a T cell..

[Audio] On the this slide you see the diversity of the T-cell receptor. We can calculate the diversity, the possible diversity of the alpha beta T-cell receptor by first looking at the number of segments and then the combinatorial diversity and the junctional diversity. So you see here the number of segments of the alpha and the beta chain. And you see that there are two d segments in the beta chain, none in the alpha. And then you have the joining the J segments of the alpha and the beta chain. And those together amount to about three times ten. If this variability together will amount to about three times ten to the six different receptors. But if you then on top of this add the junctional diversity, which we talked about before, before the nibbling and the addition of nucleotides, you can see that the variability of the T cell receptor is actually as high as ten to the 16. So the the theoretical theoretically possible this cell diversity is around ten to the 11. And when it comes to T cells this is actually even higher. So both the T cell receptors and the B cell receptors have an enormous diversity that can be expressed on on on the population level..

[Audio] So the variation in the binding sites of the T cell receptors is similar to what you see in the B cell receptors. You have a recommendation of different V, D and j segments. You have the addition and removal of nucleotides at the edges of this gene segment. And then finally you have a combination of different alpha and beta chains. So these three levels will make up this enormous diversity that you find in the T cell receptor repertoire. And this variability is created in the thymus independently of foreign antigen. This creates the T cell receptors that will then remain the same throughout the life of a T cell. T cells do not undergo somatic hypermutation which I mentioned for busy cells before. T cells are always having exactly the same. T cell receptors throughout its life, and this is very important to avoid autoreactivity actually..

[Audio] Now just like for B cells there are also. T cell subsets that have less variable receptors. On the next slide you'll see that there. There are a couple of examples here. One is gamma delta T cells. And this is a type of T cells that expresses, a gamma and a delta chain in their T cell receptor. So that's a homologous but different, protein chains in their T cell receptors. And there are also natural killer T cells, which is a subset of alpha beta T cells. And those two have T cell receptors with limited variability. They don't have the same diverse repertoires as conventional T cells. And also this T cell receptors don't bind to antigen presented on normal M-H-C molecules. And I will talk more about those types of T cells in the lecture. Well both in the thymocyte differentiation and also later on in in one of the later parts of the course where we talk about tumour immunity. And I will also talk about N-K-T cells, you can see here illustrated on the picture to the left is the normal or the common more frequent alpha beta T cell receptor that we see on the conventional T cells that consists of an alpha and a beta chain. And then to the right you can see the corresponding chains uh homologous to the alpha and beta chains, but they are actually encoded by a different gene loci. And this is the gamma and the delta chain that are expressed on the gamma delta T-lymphocytes. then we have N-K-T cells that are a subset of cells, that where you have one subgroup that have quite, low variability in the T cell receptor, while another subgroup has a little bit more diverse T cell receptors. We will talk more about those, in a later lecture. and the gamma delta T cells, like I mentioned, the T cell receptor is homologous to the alpha beta T cell receptor, but less variable. And the gamma delta T cells, are not as frequent as alpha beta T cells in lymphoid organs, but they are instead mainly found found in the skin and in the mucosa. So they have a little bit different functions as compared to conventional alpha beta T-cells..

[Audio] So I will just finish this part by going through very superficially the processes of thymic development, which we will hear much more in detail in the coming lecture. So you can see here on top of the picture where the thymus is placed. So it's above the heart and the lungs. And in this structure is specialised for supporting the maturation of T lymphocytes. You can see here in the illustration a cross section of the thymus. The upper part is the outer part of the thymus which is called the cortex, and the lower part here is the medulla or the inner part of the thymus. And as the maturing thymocytes or the maturing t-lymphocytes move through these different regions of the thymus, they will go through these different processes of thymocyte development. And they will then recombine the T-cell receptor genes, and they will go through positive T-cell receptor selection and negative selection. And in this process, they will also decide there will be a lineage decision, whether they will become C-D-4 positive T helper cells or whether they become C-D-8 positive cytotoxic T lymphocytes, or if they will be regulatory cells or natural killer T cells, or if they become gamma delta that it sells. So all of these steps or selections will occur within the thymus. And if we look at this in in a time scale like we did for the lymphocytes, that is illustrated on the next slide..

[Audio] Again we have a stem cell or a pluripotent lymphocyte precursor that decides to go to the thymus to become a T lymphocytes. And it will make a first choice of whether it should become an alpha beta T cell or a gamma delta T cell. In this case, the green line here indicates the selection or the lineage choice of an alpha beta T cell, and they will first be a rearrangement of the T cell receptor beta chain. Once this has rearranged, it comes up on the surface together with what's called the T Interceptor Alpha. And then, this signals to the cell that the heavy or the the T cell receptor beta chain is successfully rearranged. And then the cell will move on to rearrange the alpha chain. Uh, and once the cell expresses the complete T cell receptor on the surface, the alpha chain together with the beta chain, then the cell is ready to go through positive and negative selection. if this is successfully passed, the cell, becomes an immature T cell, that has either C-D-4 positive or C-D-8 positive, and it will, uh, mature, uh, and then be ready to leave the thymus and go out into the periphery to be ready to be engaged in an immune response..

[Audio] the T-cell selection will ensure that T-cell receptors are functional and that they are non harmful. The positive selection ensures that they are functional in the sense that it will select the T cells that have a T cell receptor that can bind to embassy peptide complex complexes. So this selects for functional T cell receptors. And that's a positive selection in the negative selection. T cells that bind too strongly to M-H-C plus peptide in the thymus. Then they will die by apoptosis and be eliminated. And so these two processes together will shape the final T cell receptor repertoire. And if this is successfully done the cells will be allowed to leave the thymus and go out and be ready to be engaged in immune responses. And so this is again illustrated on the next slide..

[Audio] Here you see the life of a T cell where a precursor cell moves from the bone marrow to the thymus, where it will start to recombine the T-cell receptor genes. Once they have successfully rearranged the the thymus site, the immature T-cell will go through positive and negative selection, and those cells that are successful and survive the selection steps will move out into the periphery and recirculate through the blood and lymphoid organs and be ready to meet their antigen. And once this happens, they see their antigen presented on a dendritic cell. The cell will be activated and into different effector functions and leave the lymphoid organs and go back to the site of infection..

[Audio] finally on the next slide. to summarise for these two parts of the lecture on the rearrangement of B and T cell receptors. So the antibody or B-cell receptor consists of heavy and light chains that are built up by variable and constant regions. Correspondingly, the T cell receptor consists of an alpha and the beta chain, and that's also in the same way built up by variable and constant regions. The variable antigen binding sites of T cell receptors and B cell receptors are in turn made up of A, V, D, and J segments, so the heavy and the beta chain have the D and the alpha and the light chain have only the V and the J The enormous diversity of this variable antigen binding sites of the cell receptors and T cell receptor is created in the bone marrow for B cells and the thymus for T cells by the recombination of this gene segments of the different V and J gene segments, and the unprecise ligation of these segments, which occurs by nibbling and addition of nucleotides, and then finally the combination of the two types of receptor chains. So that's the alpha and beta for the T cell receptor and the heavy and light chain for the B-cell receptor. And these processes the creation of these receptors, they are not dependent on the presence of foreign antigens. So that's all for today..