----start---- biochem basis of dz chris hunter 9/4/98 The role of costimulation in the regulation of T cell mediated diseases [question: Describe in detail the main elements involved in the activation of T cells. In the context of costimulation, how would you use this knowledge to enhance the survival of skin grafts? describe the process of graft rejection and where you might intervene.] Things we'll discuss: Events that occur during activation of T cells Major players involved Molecular events during T cell activation Using this info to design disease therapies Diabetes and a model of transplantation in diabetes Using costimulation factors to augment transplantation therapy fig 1 handout: the T cell interacts with an APC via the MHC on the APC and the TcR on the T cell. So the MHC-TcR interaction is one you should be familiar with. Now, if you just have MHC-TcR interaction, nothing will happen -> you have anergy. The T cell will not proliferate. You need a second signal. Second signals are provided by other molecules on the APC - this allows the T cells to proliferate, make factors, etc. These events are required for generation of effective T cells and T cell mediated immune responses. There are exceptions - if you give a T cell enough stimulus you can induce some responses without the second signal - but costimulation lowers the threshold for T cell activation, so T cells are activated at a lower level of peptide/T cell interaction. So - the players: B71 and B72 - the B7 molecules. These are structurally similar molecules found on the APCs. On the T cell is a molecule called CD28, which interacts with B71 or B72. Stimulation of CD28 is a positive signal for the T cell. Also on the T cell is something called CTLA4. This is a dimer like CD28. It also interacts with B71 and B72 but it produces a negative signal. SO it's like an accelerator and a brake. You have to combine these signals to regulate T cell activity. For everything that turns on a T cell, you hvae to be able to turn it off. yOu do not want hyperactive T cells. There have to be constant checks. These molecules are involved. Well, why don't they cancel out? CD28 is constitutively expressed on mostT cells. it's already there. CTLA4 isn't. after T cell activation, CTLA4 is upregulated, then it is expressed, then it can give the negative signal. So that's a good way of working it. The CD28 is always ready to give the + signal, which induces the receptor for the negative signal. CD28 has a low affinity for B7 molecules; CTLA4 has a high affinity for them! that's another thing that downregulates the T cell response. so, molecular events of costimulation: if a T cell is activated via the TcR,that alone induces transcription of several genes and induces production of RNA. IL-2 is produced and this is a T cell growth factor. So, stimulation of TcR induces this IL-2 production - but it is a weak stimulus and doesn't do much. The CD28 stimulation enhances IL-2 production, by stabilizing the mRNA for it, increasing 1/2 life from 2 hrs to 8 hrs. So it upregulates transcription and production. so you make more IL-2. you also affect production of other factors. so the costimulation increases transcription and has strong induction and enhancement of the mRNA half life, so you get more product. The factors are secreted and act in an autocrine fashion to stimulate the cell. we don't know how costimulation increases mRNA stability. regarding the B7 molecules, they are also regulated. the B7 mols on the APCs - B71 can be expressed constitutively - but B72 is not expressed on these cells. B72 is upregulated in diseased animal. this is confusing me. functional consequences of these molecules: the best way to look at importance is to look at gene knockout studies. when the gene for CD28 is removed, and the T cells are studied, we find that T cell proliferation is affected in certain ways. When the T cells are given a generalized stimulus, they still proliferate normally. WHen stimulated with a mitogenic stimulation using normal stimulatory pathways, animals lacking one CD28 gene have reduced T cell proliferative responses. Animals lacking both CD28 genes have really bad responses. When you do the same experiment and look at IL2 production, you see a similar thing - lacking one CD28 gene, animals make less IL2, and lacking both genes,they make even less IL2. and we know you need IL2 for T cell growth. Adding IL2 back into the first group increases T cell proliferation response. So the problem is that you need CD28 to produce IL2 response. what about CTLA4? when they knocked out those genes, the animals died of lymphoproliferative disorders at young ages. without this molecule, the T cells were hyperactivated and killed the animal. This is important - CTLA4 is really needed to control the normal response. without it the animals die. a ++ wild type animal will proliferate in response to normal TcR stimulation - in animals that lack CTLA4 there is proliferation without stimulation. the knockout animals severely overproliferate, even without stimulation. the levels of cytokines produced in absence of CTLA4 also dramatically increase. the IL2 actually decreases - why? it is consumed. It's used up as fast as it is made, that's why. If you block the receptor you can see it is really being elevated too, but it is just used up inthe proliferative response. we know that in CD28 knockouts proliferation is suppressed CTLA4 knockouts die of overproliferation how do you use this knowledge to intervene inimmunemediated diseases? design antagonists to costimulation. CTLA4-Ig was developed - it's an antibody made of CTLA4 that binds specifically to B71 and B72. This is a great thing. It binds B7 molecules. Remember CTLA4 has a higher affinity for B7 than CD28. how do we know this works? well, using this CTLA4-Ig, adding it into the culture, then if you try to stimulate the T cell, there is no IFN-g production. realize - you are blocking the B7 from interacting with the TcR. There is no greater response from the cells in the presence of the CTLA4-Ig thanf rom the CD28 knockout cells. Ok, what else? T cells help B cells,right? T cells have to be activated to help B cells make antibody. So if you immunize an animal with KLH, so they make Ab to it, and then treat them with this other stuff - they get a high titer of antibody. If you give CTLA4-ig after immunization, you can inhibit the antibody response because you inhibit the activation of the T cells, so there is no help given to the B cells. this shows that CTLA4 Ig interferes with development of B cell respose. now what? models - cells make insulin, insulin goes to liver, gluconeogenesis decreases, uptake of glucose increases, etc. there is a molecule called STZ that kills pancreatic cells that make insulin. So you lose insulin, gluconeogenesis increases, uptake of glucose decreases, hyperglycemia occurs, ketogenesis occurs, metabolic acidosis occurs. Right? ok. In this model, after STZ treatment, no one knows how this works, but it is T cell dependent, immune mediated. people took mice and gave them STZ - in wild type aniamls, 5 days later there is hyperglycemia. but in CD28- animals, you do not get hyperglycemia occuring. also, using CTLA4 Ig you can prevent the onset of this diabetes. so this form of diabetes is an immune mediated event. you can use CTLA4-Ig to block the interaction leading to the development of the response. so this model helps us look at transplantation. you kill the pancreatic cells. now you try to transplant them back. autograft - self to self- not rejected syngeneic graft - related donor and host (identical twins are best) allogeneic graft - unrelated donor and host - more difficult, more rejection xenograft - different species successful transplantation has increased, because of recognition of the important role of MHC in rejection, leading to MHC typing, and development of immunosuppressive drugs. But, we still need to make new ways of preventing rejection. So, our proteins are all the same - your actin looks like my actin. But we have a lot of individual variation in MHC. our T cells recognize foreign MHC and reject it. this is the problem. MHC typing is really important for organ transplantation. even with MHC typed grafts, there are other differences that can lead to rejection. we need to find ways to prevent this immune activation. think of what happens during rejectoin: foreign cell with foreign MHC. 1 to 10% of your T cells will recognize the strange MHC . costimulatory molecules on the donor cells (B7 molecules, same as host B7) will costimulate the host T cell, which will then help B cell, and the donor cells are treated as nonself and are attacked. Antibodies will bind the donor cells, activate complement, and lyse the cells. but that depends on t cells which depend on costimulation. also T cells activate effector cells which attack the donor cells, or which make cytokines to activate other cells- but this all depends on the activation of the T cells that recognize the foreign MHC. so, now what? recently a study was done. Monkey treated with STZ, developed hyperglycemia. insulin was given so it would survive. then pancreatic cells from another monkey were transplanted in. glucose returned to normal. (they also measured C peptide levels to see for sure if the new pancreatic cells were working). but, within 7 days rejection occured. hyperglycemia recurred. had to treat with insulin again. this is classic graft rejection. monkey number 2 - same thing. gave STZ, it got hyperglycemic, thenn they treated with CTLA4Ig for 2 weeks after transplanting in new pancreatic cells. now they didn't have to give insulin for almost 60 days. this prolonged the interval before rejection set in. would repeated doses of CTLA4-Ig prevent rejection? we do not know. question: is the CTLA4-Ig immunosuppressive? it prevents the T cell activation. BUT, an infection is not a subtle thing. remember, a STRONG stimulus doesn't require the costimulatory factor. CD28 lowers the threshold of activation of the T cell. blocking it keeps the threshold high. it doesn't prevent all activation. sean asked a good question that I could not hear. dana asks - what about other costimulatory or regulatory elements on the cells? maybe some antigens use other costimulatory factors? he says that is a good question. there are other costimulatory factors and interactions that are less dominant than the ones we are discussing. These are the oens that are really dominant,that is why we are focusing on them in this class. what about a xenograft? human =--> mouse here the mice are being given human cells. after 5 or 6 days, cells are rejected and mice get hyperglycemic again. when treated with CTLA4Ig the rejection doesn't occur so soon. in some animals it doesn't occur for a long time. so the same questions apply. can you give repeated doses to prevent rejection? significance of this: what immune mediated diseases are important in veterinary medicine? IBD - seems to be T cell mediated autoimmune thyroiditis -big thing in dogs rheumatoid arthritis diseases in which interference with costimulation affects the outcome of disease: EAE, diabetes, graft rejection/transplantation, allergy, Ab mediated immune disease, infection, vaccination, cancer. ----end----