Meeting 7

Meeting 7 - NICOLE Extra OH Wednesday MSB 1601 Jeff OH...

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Unformatted text preview: NICOLE Extra OH Wednesday MSB 1601 Jeff OH 3:30-500 next to CHS18-111 M____ conference room If the cell gets lysed, it breaks apart, the radioactivity comes out. If you take antigen recactive cells and put it on the same target cells, you get no lysis. If you hput the target cells that ARE __- it wiill lyse. If you take target cells .. you will get no killing. KNOW that this is 2-3 hour assay. That's important because there is another assay that takes a couple days and that's different. This is an immediate killing assay. You're lysing cells that do'nt have the sa... How does this happen? We have two types... We have a famous antigen, the KLH that ... It's arestricted to IAf cluster mmolecule. You don't have to memorize IAf but just realize the top left is different from top right. it's restricted for a different MHC molecule but this is IAf and the other is IAk. If the dual receptor model is working, if you fuse these two together, yo9u should get reactivity of KLH in the context of IAk and OVA in the context of IAf. Now youdo the experiment and you only see preserving of the original you don't see the other one. Therefore, it's the altered self... It doesn't see ... You can immuno precipitate the proteins. In order to get the genes for this, they taok a CD4 TCell clone, extracted the DNA, made a cDNA and ... Any RNA that is lymphocyte specific is eliminated. You only have T cell specific RNA. they isolated the gene for Tcell receptor beta and ythey found TCR gamma. The alpha beta chains that have a couple of domanis, the cytoplasmic domains between the leaf ar e very short. That's not really .. there are no signal domains in the cell. Ify ou immunopreciptiate the TCR proteins, you get kilodaltons signals and they are known as CD3. They transport the signal to the cell. If you express a TCR in a cell,without CD3, is no signal. So if you stain with CD3 in exams, you're looking at T cells. What does this CD3 complex look like? It's multiple chains. These guys convey the signal, we do'nt have to know which combination, as long as you have CD3. You require crosslinking to get a signal. That birngs these guys together and you get a signaling.. You can crosslnk them on the outside. I want to introduce the other receptors with detal TCR. Not really imp for this class. That's all i really want you to know. Slide This is the gene layout for TCR genes. I won't give you too much material just tell you the things I think are important and what is different with the genes for Ab. if we cover it on the midterm we won't cover it on the final. We should understand the mechanism for rearrangement for this midterm. There are about 100 Valpha genes. it's a huge locus. In the middle of that locus are the delta genes. You hhve alpha variable then variable and then constant delta and J genes. the beta locus has a whole bunch of J regions. Both TCR beta and delta ahve D regions much like heavy chiains and Ig ahve a D subunit. If you have arrange the variagle alpha gene and the J region of alpha, you'll llopp out the entire Delta. You can't be a gamma delta cell. that's controlled by an element called the alpha silencer. They can't tell where it's located. if that silencer is on, there's no alpha rearrangeement and you get gamma delta rearrangement and if it's off then it will rearrange and you delete the delta. Again, you've had most of this before. To rearrange the receptor chains, the Va dn J rearrange... You get combinatorial diversity you get D regions and rbut there's more than one D region and because of the 12-23 rule, they can loop out you can make a whole bunch of sequcneces (B) because the 12-23 rule allows it to do so. The other thing that is dfiiferent from BCR is that there are no genomic rearrangment in T cells. They retain the recteptors and don't spit them out. There's no alternative splicing to a soluble component and there's no class switching. There a quite a few things that are different but the mechanisms are similar. Most of the mechanisms of diveristy are ... and the alternative splicign of different sublcasses .. those don't happen in t cell.s Understand the diffreence between TCR and BCR. I want to talk about the T cell activation. We're atlking about how you turn on a TCR. Some are important, some just know they exhist. Thi s is your TCell. yellow is APC, blue is T cell. Thisis a class II MHC. when these guys dock, the adhesion molecules come into contact. There's rearrangment and the TCR comees in contact. CD4 which is on CD4 cells binds a conserved cell of CD.. you've got some adhesion bindings, you got receptors binding... The way it gets going is CD45 (imp) and binds to CD22? This guy is a phosphotase, when he binds, he activates a phosphotase and it dephosphyroalyate several moleucles. KNOW LCK! When this guy binds, he activates a couple of these signaling molecuels. These guys are bound and you get the m closer and you get crosslinking. These kinases Lck and Fyn because CD45 has taken the phosphotase off. It starts off with dephosphorylation and then tyou get his nasty signal into the cell. You're getting activation of CD4 component.s Another protein wihc is Zap70 but i'ts not improtatan to memroize. So you get interaction etween TCR, CD45/CD22, crosslinking gets ckinases to phosprhoriylate... Here's the TCRMHC complex with CD4 bound. In a nutshell, when this signal happens, there are several phosphorylation evetns leading to two different signals, so you don't hneed to knowa ll these playser but do know there are 2 signals. You get two major .. calcium blocks and activateion of TFs. Calcium flux ends up causing a TF NF-ATc(IMP!) leads to dephoshporlylation of ... Anything that has a NFATc .... The second event is activation of protein kinase c PKC that turns on NFkB, sitting ing in plasma in inactive form with a bound inhibitor. It phosphorylates the inhibitor and removes it. NFkB turned on and binds to things to turn it on. Recap: Calcium flux that ends up activating NFATC by celaing off phosphate. Know calcuim flux and NFAT. Activate PKC to uninhibit NFkB and that goes on to turn on signaling. There's a phenomenon known as co-stimulation. you need two signals to activate a T cell. 1. TCR/MHC (what i just showed you) binding The problem is signal 1 alone leads to anergy. It's specific shut down of that cell. There's a reason why it's set up for this. If you have a autoreacitve receptr and even binds on to low affinity, you will kill your own cells. The only thing that is if a profession APC gives signal 2 and signal 1. 2. CD28/B7 binding It activates and turns off anergy. It stabilizes the mRBNA for IL2. It allows for DNA synthesis. If you don't get IL2, you'll get anergy for a very long time. Thi sis a mechanism for a cell to be tolerized by receiving only one signal and not two. IL2 is an autocrine GF. Understand not the mechanism but that you need to have that second signal or you shut the cell down. Skip this slide before the blue slide. Blue grey white slide CD28 that is the second receptor. it's constitutively expressed, all the time. Where a professional APC has a B7. It's gonna have, an activated APC will express B7. A non-activated won't. This will cause it to be activated. If this is not present or is not activated you don't get this molecule. CTLA4 also binds B7. But it's not constitutively expressed. When this guy binds B7, it sends an inhibitory to prevent over activation. CTLA4 is turned on a day or so after the T cell is activated. When CTLA4 binds, you shut this system down. It's a lot of exquisit controls of checks and balances. Three things. If you crosslingk CD3 with Ab, do you also have to stimulate CD28? Yes. REMEMBER Signal 1: Calcium flux and PKC turns on NFkB Signal 2 CD28/B7 binding CLA4 is not turned on right away. Clcium flux is very reactive but most of thes things take a couple of hours to get activated. CD45/CD22 ... CD45 is to hlep strengthen the activation. In the absence of CD45, it still turns on. T cells don't class switch but they can help B cells class switch. Lipid Rafts: There's a new observation. In the cell membrane, outside, is a dynamic thing. I thas mechansims that can augment signaling. What happens is there's Signal transuctisons LDK and FYN that are associated with structure.s.. When youligate TCR (CD3, same thing) it moves into these rafts and get coallescence of these rafts. You're brinign components of signaling cascades into jusxta position owith TCR . Reallingning cell to bring them together. What allows them to contactare adhesion molecules. CD43 is big. When you bind,y you sget a sloppy fit. As you reararange the membrane, you move aaway these larger adhesion molecueles allowing the smaller molecules like TCR to bind. You're also concentrating tyour signal transuction components here. You start activating. CD45 doesn't get into raft and gets pushed away. Why? It takes Pi off LCK and FYN, once it gets going is phosphorylation, you want to push it away because you don't want it to dephosphorylate the cascade that is phosphorylating. You need more than one interaction to get this to happen. there are going to be multiple MHC molecules in the cleft and they're gonna coallexce to make this mess. any important concepts. Another concept that I dearly love!!!! MHC tetramers! They're pretty new, they're a way of specfically identify ing T cells. They're a fluorescent bead, and attached to the bead are 4 identical MHC class 1 with an identical peptide. When you get this tetramer,with 4 molecules, gives it higher avidity... 4 receptors will bind... You get one to tone binding. It will shine in a flow cytometer. It's easier to get a tetramer.. Imp that this will only attach the right moleucle. It's a way to quantitate. ...
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This note was uploaded on 12/31/2009 for the course MIMG 185 taught by Professor Zack during the Fall '06 term at UCLA.

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