13_14 Immunology Dr.

Leitenberg 01/17/14 2:00-3:00pm Notetaker #30 Slide 43: We have an un-rearranged receptor locus it rearranges its beta chain gene it makes a protein and goes on a develops further, but one of the other things that might happen to that double negative t cell is it might rearrange its beta or its delta chain and if it does that they will pair and its going to leave the thymus and migrate to peripheral tissues with a distinct function. Slide 44: And so these gamma delta t-cells are unusual in that they tend to be cells that have an innate like function have a limited diversity in how they recognize things, they recognize things in weird ways one of the things wed like to talk about is we like to think of them as being evolutionarily primitive. So early in embryologic development, early embryos look like fish, so it mimics early evolutionary stages, so to mimic that and so gamma and delta t-cells are one of the first types of t-cells to develop. And they kind of come out of the thymus early in development in waves that have relatively distinct end receptor usages and specificities and you see too they are largely mucosal sites. A lot of these t-cells have a relatively limited receptors repertoire, but as you develop, you get receptors that are slightly more diverse. They dont go through the same development. The weird thing about these guys is they violate one of the rules I told you about t-cells is they often recognize non-peptide antigens and sometimes these molecules are presented by non-classical MHC molecules. The commonality between all of them is that they seem to form similar regulatory functions; they get activated easily on antigen exposure. Slide 45: So this is just a graph showing you they make up a lot of the t-cells early in embryologic development, even thought theres not many to start with, but as you go on and develop further, ultimately in the adult, these cells will make up a smaller portion of the t-cells in the adult and the alpha/beta form make up about 95% of the cells in your body as an adult. Slide 46: This is just a cartoon in a mouse, but similar things happen in people. The T cells develop in waves. The waves correspond to different parts of the body. So this wave of t-cells goes to the skin, subsequent waves goes to uterus, lung and tongue dont memorize this, the whole point is just to show you this is cool and there are different stages of development which in turn regulate where the go and survive in the animal or person.

Slide 47: Any questions? Slide 48: The next steps in conventional alpha/beta t-cells. Signals to cell neuron go on to become a double positive cell and signal to undergo TCR alpha chain gene rearrangement. If it does that right it can express the mature form, but these cells arent ready to leave the thymus yet, they are double positive. Slide 49: The next major checkpoint in development is really the checkpoint associated with what happens when the cells express the mature form receptor, how does that regulate whether or not the cells survive and determine whether or not you will be classified as a single positive CD4 cells or single positive CD8 cells. The second check point is expression of mature form TCR receptors. Slide 50: 1) Once the cells express, three things can happen. One of the things that can happen is the cells can die because the T-cell receptor doesnt recognize anything at all and that happens allot. So the cells are randomly expressing and so they die by apoptosis or die by neglect because the receptor is not being engaged in any functional way at all. 2) They can also die by a different mechanism, they can die because the t-cell receptor engages with self-antigen relatively strongly, we call that death due to negative selection. So the cells that express receptors get negatively selected and die by apoptosis. The third thing that happens is that in those that are functional, a kind of special signal is given off in the cell and it is allowed to survive and undergo further maturation and that signal is called positive selection. All of this is dependent on the specificity of the T cell antigen receptor. Slide 51: So heres the cartoon, showing the three things again. One of those phases is lack of positive selection and it doesnt recognize anything in the thymus and so the cell dies by apoptosis. Here is the negative selection sample this is when the t-cell suppresses the receptor it recognizes self-antigen presented by the thymus in a relatively strong way, high avidity recognition and that also results in cell death and then the third thing that can happen is recognizing low-affinity and it signals the cell not to die but to survive and it will then further differentiate into single positive t cells. Slide 52:

Avidity model of T-cell development or the goldilocks model. SO if its too hot, and it recognizes something too strongly, then it dies by deletion. If its just right, medium to lowing affinity, it undergoes positive selection. But if its too cold then the cells will die by neglect because they arent getting that survival signal through the antigen receptor. Slide 53: Another way of thinking about how this process is regulated in part by interaction with other t-cells mature in the thymus. Remember I told you the earliest T cells, in the cortex of the thymus, they express the CD4, CD8, the antigen receptor here, and when they are at that stage they are able to interact with positive selection cells. The other thing that might happen is they might develop further and they may encounter additional cells such as medullary cells, epithelial cells or other antigen presenting cells and these might be slightly different with the types of antigens they present and they might be better at mediating negative selection where as cells when they are at this stage might be more likely to go into positive selection. Notetaker aside: (It was difficult to interpret this slide but I think the main point is: there is more positive selection in the cortex, while there is more negative selection in the medulla) Slide 54: Positive selection is important for developing T-cells. The T-cells develop and come out of the thymus are going to work and we depend on those cells to recognized MHC complexes so if they cant do that then they are worthless. We dont want them. And that is partly what we mean when we say T-cell antigen receptors are MHC restricted, but they are also restricted to recognizing only the MHC that they grew up with in the thymus. And so this process ensures the receptors recognize the peptide expressed by MCH. Also as youll see later on is that it also ensures that the cells will express the proper co-receptor CD4 to interact with MHC class II complexes or CD8. CD8 cells recognize class I. Someone pointed out a mistake in the outlines of these notes. So in the outline of these notes I had that backwards!! Slide 55: Here is a famous experiment that recognizes the importance of the MHC molecules that are expressed in the thymus and regulate how they will develop. You take bone marrow from a mouse that expresses two different MHC molecules a and b and you put it into a mouse that expresses only a in the thymus and when they grow up in the thymus that is type a. When they go into the periphery and what they generate an immune response it is restricted to be MHC antigen presenting cells will be predominately type A. If you do the same experiment and put it in a mouse with type B the opposite results will occur. Slide 56: And so this is a cartoon illustrating that process. T-cell receptors come out of the thymus and are restricted to recognizing peptide presenting by MHC molecules that

are expressed in that thymus. The TCRs are recognizing both peptide and MHC molecules. Slide 57: CD8 T-cells recognize MHC I complexes where as CD4 t-cells tend to recognize MHCII complexes. Now how does that happen? Slide 58: So you have these cells that are double positive, expressing both CD4 and CD8 that are sampling their microenvironment. Again, in this case you have a double positive cell expressing both CD4 and CD8. This cell happens to express a t-cell receptor that recognizes MHC class I complexes. When that happens the CD8 protein helps facilitate t-cell activation in this context. These cells are signaled and they ultimately develop in to pseudo positive CD8 cells. They will down regulate their expression of the CD4. Where as these here express a different TCR that happens to recognize MHC class II, and the CD4 protein helps this signal and will down regulate the CD8 protein, maintain its expression of CD4 protein and the cell becomes a positive CD4 cell. Slide 59: And so we know that experimentally. One is by genetically manipulating mice to express receptors of a single specificity, a single t-cell antigen receptor. Force the cells to express only this single receptor that recognizes MHC class I and the cells that come out of that thymus will be single positive CD8 cells. But if you do the same thing so that the cells only recognize MHC II, the cells that come out will be CD4. Slide 60: In people we can do similar experiments. Where people have mutations that affect either MHC class I or MHC class II. Such as in TAP I deficiency patients, which means they wont express MHC class I very well, but you will be able to express a lot of MHC type II. In those peoples thymus they only have MHC II complexes to interact with and so they only will express CD4 cells. And the revers its true in people who have mutations in the MHC class II. They will still be able to develop CD8 protein cells. Slide 61: So positive selection is kind of linked to CD4, CD8 lineage. Now lets talk about negative selection. The negative selection the idea is that you let those t-cells develop and go into the periphery that might be bad because you might develop an autoimmune disease. So this prevents that from happening. This only works well if the self-antigen that you are worried about is expressed and present in the thymus. So this isnt always true and so in real life you do have a lot of T and B cells that develop and skip this negative selection until they go into the periphery where they will undergo other peripheral tolerance mechanisms as opposed to central tolerance mechanisms, but I will come back to that in a later lecture.

Slide 62: For now here is a question: If a T cell receptor on a double positive thymocyte binds to a self-peptide presented by MHC class I with low affinity, the most likely result is: A. Negative selection and apoptosis B. B. Cell proliferation C. Rearrangement of the second beta chain gene locus D. Positive selection and development of a CD4_CD8-T cell E. Positive selection and development of a CD4-CD8+ T cell T - cells dont actually undergo receptor editing thats a b cell thing. The answer is E. I ask questions like this on the test Slide 63: So her is putting it all together, this is a little complicated but I USUALLY ASK QUESTIONS RELATED TO THIS ON THE TEST Force all the t-cells that develop in the thymus to recognize a peptide that is presented by this particular molecule we will just call it b. This is a funny t-cell receptor. Its a T cell receptor that recognizes peptide thats only expressed in boys, in male mice. We refer to this peptide as being an Hy peptide, encoded by the y chromosome, and so the t-cell receptor has H-y specificity. So you are making the receptor grow up. Its specific for boy stuff, boy antigens. And so there is 3 possibilities on where its growing up, it can grow up in a boy that expresses a boy that expresses the b, a girl that expresses the b or a girl that expresses the k. You are forcing the t-cells to express a receptor that recognizes boy antigen in the context of MHC b. So if it grows up in a boy mouse it will recognize self-antigen relatively strongly so its going to be positively selected because its going to recognize some other antigens weakly, but its also going to recognize that boy antigen strongly and that will cause it to be negatively selected. So youre not going to get t-cells that will develop in that mouse. If these t cells are trying o grow up in a girl mouse that expresses MHC b, the cell is going to recognize MHC b it in a low-affinity way, those are going to be positively selected, but because its a girl they wont be negatively selected and it will develop into single positive CD8 positive cells. If they grow up in a girl that doesnt have HMC b, the cells will die by neglect. They wont interact with anything in a functional way and all the cells will die. This experiment illustrates all three of the fates that can occur during cell development. So this is complicated, you should look at this and hopefully understand it and apply it. Slide 64:

So Im going to add one additional wrinkle to this. I told you negative selection only works well if the cell peptides are expressed in the thymus. There are lots of cell peptides that wont be expressed in the thymus. We have evolved away from that problem and the way to define this is to express a particular transcription factor, especially the medullary endothelial cells in the thymus to express pieces of proteins that you wouldnt think would be expressed in the thymus for example you only express a piece of the insulin protein that would normally only be expressed in the Islets of Langerhans. The expression of the AIRE transcription factor that would normally only be expressed in peripheral organs. We know that this is expressed in a rare disease and they get this weird, multi-systemic, autoimmune disease and they get autoimmunity to insulin, diabetes all sorts of stuff and thats because they have a mutation in this AIRE protein and it limits their ability in the thymus to express these proteins and so there is a defect in negative selection, the recognized thyroglobulin where as they would normally be deleted. Slide 65: So this is what happens normally and that allows transcription that recognizes thyroglobulin to leave. But if you do not express the AIRE protein you are going to get development of a whole bunch of t-cells that recognize self-antigens that expose you to autoimmunity. Slide 66: So summary of everything we have talked about today. T cells develop in the thymus. As well as patterned and antigen receptor gene rearrangement. The developmental fate is driven by the specificity of the TCR and you get these different outcomes. They can be positively selected, negatively selected, or neglected. Slide 67: So to put this all together, you have 3 major checkpoints. The first checkpoint is regulated by the ability to express the pre-t-cell receptor. The second checkpoint is regulated by the mature t-cell receptor. The 3rd is the specificity of the antigen receptor and whether or not It recognizes self antigen.