Transcribed by Ana Sangadala July 18, 2014 General Pathology Lectures 8 and 9-T Cell Activation and T Cell Function By Dr. McCutcheon Slide 24 (from T Cell Activation Lecture)-No Title [McCutcheon]-Ok, I might as well reward the people who are here at 8 oclock in the morning. Good for you, and get started. Ok, backtracking a little bit, the T-cell gets activated LFA binds to IFAM, which allows the t cell to roll around on the dendritic cell arms. If the t cell binds to HLA plus peptide, then the LFA changes conformation. The t cells tops rolling. The t cell moves receptors to the point of contact. The dendritic cell moves receptors to the point of contact. You get many receptors, t cell receptors binding to hla peptide complexes. And thats the primary signal, you have to have the cell receptor recognize it because youre only going to have 1-3 t cells for any given infection. Then, you have co-stimulation, dendritic cell binds to cd28 on the t cell. That is a crucial step. Once that happens, the t cell is now activated. Its going to do everything that it can do without any further help, so the t cell starts changes the expression of the IL 2 receptor. It starts making a third chain for the IL2 receptor. This high affinity form of IL 2 receptor is now on the cell surface. The same t cell surface is secreting IL2, so the IL2 from the t cell binds to this high affinity form of the t cell receptor. It causes t cell proliferation. All of the daughter t cells will have the identical receptor to the parent t cell and that is clonal proliferation. Slide 25- T cell Proliferation [McCutcheon]-So we now end up with an army of t cells. They are identical to the parent t cell. Slide 26-Differentiation Pathways of CD4 T cells [McCutcheon]-And they start to differentiate. Differentiation is driven by cytokines. So, if the t cell, at the beginning of the response, most of the t cells are going to be either TH1 or TH2. TH1 t cells differentiate. You get TH1 t cells if the t cell is bound by gamma interferon or IL 12. You get TH2 t cells if the t cell is bound by IL4. Later on in the immune response is when you start getting regulatory t cells. The function of a regulatory t cell is to suppress the activity of TH1 t cells. You want TH1 t cells at the beginning of the response. You need to get them shut down later on. So, in that case, the t cells are going to differentiate from TGF-beta. All of this works through second messengers. So binding to the cytokine receptor activates a certain second messenger. Different cytokine receptors activate different second messengers. The second messengers then go off and go through phosphorylation events and eventually you end up with something in the nucleus that activates transcription factors. Different transcription factors turn on different sets of genes. Its a cassette. So, T-bet, which is the transcription factor that gets turned on by binding through IL 12 or gamma interferon turns on a cassette of cytokine genes that include IL 2, gamma interferon, and some others. IL 4, binding through the IL 4 receptor, you eventually turn on a different set of second messengers. You eventually end up turning on the transcription factor GATA-3. GATA-3 then transcribes a cassette of genes that include IL 4 and IL 5 and some other things. Binding through TGF-beta, you eventually turn on, you go through yet a third second messenger system. You

Transcribed by Ana Sangadala July 18, 2014 eventually turn on Fox P3. Fox P3 then turns on a set of genes that include TGF-beta and IL 10. Ok? So binding through the cytokine receptors, you turn on or phosphorylate a second messenger. Each group of cytokines has a different set of second messengers. Similar cytokines, IL 12 and gamma interferon are pro-inflammatory cytokines. So, the pro-inflammatory cytokines share a phosphorylation set that ends up turning on pro-inflammatory cytokines. The anti-inflammatory cytokines like IL 4 use a different set of second messengers. They phosphorylate a different set of second messengers. They turn on different transcription factors that give you anti-inflammatory cytokines. Al right? Slide 27-Alternate CD4+ T Cells [McCutcheon]-There are also 2 other kinds of t cells. Theres a little bit of lack of clarity. Theyre in the thymus. Natural t regulatory cells, which are opposed to inducible t regulatory cells. Inducible t regulatory cells come from a parent CD4 t cell. These are also CD 4 t cells, but in the thymus, cells that come into contact with Hassells corpuscles become natural regulatory cells. So, there is a subset of t cells that develop in the thymus and those are nT regs. And then, there are also TH 17 cells. These are also alpha beta CD4+ t cells. Some data show that TH 17 cells come from this parent CD4 t cell. Some data show that TH 17 cells develop in the thymus. Right now, Parm is saying, hes not putting them in the coming from the parent t cells, so we will say they are developing in the thymus. Its not a critical point. When t regulatory cells, IT regulatory cells, and TH 17 cells come into play is when you have an infection that youre not clearing. So the t cells keep proliferating. You keep having TH 1s, you keep having TH 1s, you keep having TH 1s. At that point in time, since the TH1 t cells arent getting rid of the infection, you have high levels of cytokines and you start to differentiate off these other kinds of cells. The IT regs, which then suppress TH1 function, and the TH 17 cells and the cytokines produced by TH 17 cells can either help disease or get rid of disease depending on which panel they are and we will talk about that today. Slide 28-No Title [McCutcheon]-So weve got these 2 different kinds of t cells. So now, we have a whole bunch of t cells and where are all of these t cells at this point in time? What organ? Theyre in the lymph node. Where is the pathogen? Somewhere else. Yeah, not in the lymph node. So we need to get TH1 t cells from the lymph node to the site of infection. The way that happens is called lymphocyte trafficking. So our original lymphocyte, our nave lymphocytes, they have LFA 1 on their cell surface and they also have an antigen calledIm going to blank on thisL selectin. Ok? So LFA 1 and L selectin and other things. But the 2 you know about, LFA 1 is the adhesion molecule that lets the t cell roll around on the dendritic arms. LFA 1 is also found on normal endothelium. L selectin is found on the t cell and another molecule found on normal endothelium is called sialated Lewis X. Sialated Lewis X. So the short hand for that is SLex. And here it is up here. Sialated Luessex. So the L selectin on the nave t cell binds to the SLex on the unactivated endothelium. All nave t cells and this is also true for b cells, all nave t cells have L selectin and unactivated normal endothelium has S Lex. So thats how t cells, and I CAMS, and some other things,

Transcribed by Ana Sangadala July 18, 2014 thats how t cells are moving along thorugh the circulatory system. They roll, they always roll. When the daughter t cells start to differentiate, the TH1 t cells lose L selectin. When the daughter cells start to differentiate, the TH1 t cells lose L selectin. And they replace it with a molecule called VLA 4, V for Victor. VLA 4. Now, VLA 4, unlike L selectin cannot bind to sialated Lewis X. So, the t cell cannot go to places that only have S Lex as a protein on the cell surface. The infected endothelium, the activated endothelium at the site of infection has gotten rid of its S lex and its replaced it with a molecule called V CAM, V for victor. There is a concentration gradient of V CAM, so the farther away you get from the site of infection, the less V CAM there is on the surface fo the endothelium, but there is still going to be VCAM thats gotten to the endothelium all the way up to the lymph node. The activated t cell, because it doesnt have S lex, it cant go back to the high endothelial venule, because thats an S lex, sorry it doesnt have L selectin. Thats an L selectin, S Lex interaction. It cant do that so hows the other way to get out of a lymph node? The efferent lymphatic. The efferent lymphatic because this has been activated is going to have V CAM so the activated VH 1 t cells cannot go into the peripheral circulation. They have to go through the efferent lymphatic because thats the molecule that they have. So they go out into the efferent lymphatic and as they move back, so lymphatic all drain back into the vena cava. So they go out the efferent lymphatic, they drain back into the vena cava. Now theyre going to be able to roll only along endothelium that has V CAM. So the place theyre going to go is towards the site of infection. The closer you get to the site of infection, the more VCAM there is. That will steer them to the correct arterioles, which then steers them into the correct capillary beds that end up draining right at the site of infection. Nave B and T cells, they still have L selectin, and the only place they can go is back into the high endothelial venule and back into the peripheral circulation. The activated t cells, TH1 t cells, theyve lost L selectin. Theyve gained V..VLA4..Im not talking very well this morning either. Theyve gained VLA 4. The only place they can go are endothelium that has VCAM. So the VLA 4 on the activated t cells takes them out the efferent lymphatic and then they follow that concentration gradient so they end up in the capillary beds at the site of infection. This really lovely system. Activated cells go one place. Nave cells stay in the circulation. Slide 29-No Title [McCutcheon]- So heres our sialated Lewis X. Heres our L selectin. This is endothelial venule, this is unactivated endothelium. Slide 30-Activated vs Resting T cells have different Adhesion Molecules [McCutcheon]-So our resting, unactivated t cells. They have L selectin. They lose L selectin and they dont have VLA 4. Our activated T cells, theyve lost L selectin and theyve gained VLA 4. Unactivated endothelium has S Lex. Activated endothelium loses S Lex and gains V CAM. So keep the Vs together as you are remembering this. VLA 4 binds to V CAM, so that other thing must bind to S Lex. Or the L in L selectin binds to the L in S Lex. Al right? So our activated TH1 t cells are now going to migrate off to the site of infection.

Transcribed by Ana Sangadala July 18, 2014 Slide 31-No title [McCutcheon]- LFA 1 and I CAM are on APCs. VLA 4, V CAM on activated endothelium. Slide 32-Trafficking [McCutcheon]-skipped Slide 33-Activated Th1 cells leave the Lymph Node [McCutcheon]- So the activated t cells, they leave the lymph node. Slide 34- End Result [McCutcheon]- Now, the other kind of t cell, main kind of t cell, the TH2 t cells, they lose L selectin. They dont get VLA 4. So TH2 t cells, as they differentiate out, they lose L selectin, they dont get VLA 4. Where are the TH2 t cells going to be? They stay in the lymph node. So TH1 t cells, CD4+ Th1 t cells, are the generals that go off to the site of infection and make all the other cells work together. Thats how you get rid of extracellular pathogens. CD4+ Th2 t cells, they stay in the lymph node and their job is to get b cells activated. So the Th1 t cells leave the lymph node, Th 2 t cells also lose L selectin but they dont get anything. In the absence of a molecule that lets them leave the lymph node, theyre stuck there. So they migrate from the paracortex over to the germinal centers, the primary follicles. Slide 35- CD8 T cell activation [McCutcheon]-Now, weve been talking about CD4 t cells. For purposes of this class, CD8 t cells are activated the same way CD4 t cells are. Al right? For purposes of this class, CD8 t cells are activated the same way CD4 t cells are, except CD8 is binding to HLA Class 1 instead of HLA Class 2. And the reason, we are simplifying this a bit. There are actually several different mechanisms that CD8 t cells get activated and one of them is the same way that CD 4 t cells get activated. It seems to be pathogen dependent. Some pathogens, CD8 t cells can be activated just fine by themselves. Some pathogens, you have to have help from the CD4 t cells. For purposes of this class, were not going to worry about the other two. We are just going to say that CD8 t cells are activated the same way that CD4 t cells are, except they use HLA Class 1 instead of HLA Class 2. That finishes T cell activation. Did anybody bother to read the email that I sent out? So you know that conference is going to be where you guys are going to tell me the answers to the study guide questions. And everybody talks. (Somebody asks an inaudible question) No they do not. T Cell Functions Slide 1 (T Cell Functions Powerpoint)-Review [McCutcheon]- CD8 T cells, once theyre activated, and were going to start talking about this now. CD8 t cells, for purposes of this class. For purposes of this class means that Im lying but the real answer is way more complicated than you need to

Transcribed by Ana Sangadala July 18, 2014 know as a dentist. You need to know what CD8 t cells do, but the nuances, not so much. So, for purposes of this class, CD8 t cells are CD 8 t cells, are CD8 t cells. What they are is killing machines. Thats all they do; well, they release cytokines. But they kill things. Ok, so how do you activate t cells? Whats the first step? I know its early in the morning and its Friday. I CAM. Ok? Whats next? T cell receptor to MHC. Whats next? Elevate titins. Whats next? (Inaudible response) Ok. And then what happens? Now youve activated your t cell. Whats the most important step in that list? T cell receptor ok? If the t cell receptor doesnt bind, that t cell isnt going to do anything. Its going to go back into the high endothelial venule and leave. So THE MOST IMPORTANT STEP is the t cell receptor. If the t cell receptor binds, then you must have co-stimulation or you will end up with an energic t cell thats not going to participate. Thats a fail safe. You have to have the t cell receptor bind. If the t cell receptor doesnt bind, nothing else needs to happen. What about dendritic cells? What about dendritic cells make them specialized to activate t cells? Clearly I wasnt thinking grammatically when I wrote that. Why are DCs good at activating T cells? (Inaudible response) Ok, say it again? They have both Class 1 and Class 2, CCL 18, right, they secrete the chemokine that recruits nave t cells. What else? They can be infected by any virus, and 2 more things. (Inaudible response). Pardon? (Inaudible Response) Ok. Thats true but thats not one of the unique things in DC. (Inaudible response) Pardon? Thats kinda what she said, sorry. 2 More. (Inaudible response) Nope. They can put viruses in vesicles so they can present viral particles on both HLA Class 1 and HLA Class 2. And theres one more thing. Pardon? They dont get killed off because theyre making interferon alpha and interferon beta. Right, so the dendritic cell gets infected and it doesnt get killed. Its very hard to activate a t cell if its dead. Good. How does trafficking work? Nave t cells have L selectin. Unactivated endothelium- S Lex. Activated t cells-TH1 and NCD8. CD8 also drops L selectin and gets VLA 4. Ok? Activated endothelium? V CAM. Good. What are the differences between CD4 and CD8 t cell activation? One of them uses HLA Class 1 and one of them uses HLA Class 2. Slide 2-CD4 T Cell Differentiation [McCutcheon]-Ok, so T cell differentiation. The Cytokine Milieu, milieu-the cytokine environment. So the cytokine environment, milieu is going to tell you what t cell youre going to get. This is all driven through second messengers and certain second messengers give you different transcription factors. Different transcription factors activate different cassettes of genes. Thats what makes a TH1 t cell or a TH2 t cell a TH2 t cell. Slide 3-CD4 T Cell Differentiation [McCutcheon]- So, TH1 t cells. The original binding is through the IL 12 or the interferon gamma receptor that activates T BET. When you activate T BET, you get a second messenger pathway that causes the transcription of gamma interferon and the gamma interferon receptor. That also is acting through second messengers. Production of gamma interferon helps increase the amount of T BET and it also turns on the transcription factor STAT 1. That causes an increase in the IL 12 receptor. That causes more IL 12 binding. That gives you more T BET, which gives

Transcribed by Ana Sangadala July 18, 2014 you more gamma interferon and gamma interferon receptor which gives you more STAT 1 and STAT4 which is a maintenance. T cells work in loops and this is a loop. So t cell receptor binding and then the cell starts to proliferate and then it starts to differentiate. If it gets IL 12, you get T BET. T BET, by getting different second messengers. When T BET gets to the nucleus, it starts transcribing both gamma interferon and the gamma interferon receptor. Binding to the gamma interferon through a different set of second messengers then causes STAT 1 transcription along with TBET transcription. And then you get STAT 1, STAT 4, and T BET and that keeps that cell as a TH1 t cell. Slide 4-CD4 T Cell Differentiation [McCutcheon]- The original binding, t cell receptor binding, and you get IL 4 and that gives you GATA 3 through second messengers. GATA 3 gives you more IL4 production and IL4 receptor production. IL 4 uses a different set of receptors than this original binding. That causes the transcription factor STAT 6. STAT 6 then helps keep transcription of GATA 3, which gives you IL 4 and the IL 4 receptor. Binding through the IL 4 receptor gives you STAT 6, which gives you more IL 4 receptor to give you more GATA 3, which gives you more IL 4 receptor and IL 4. So the t cell secretes the cytokine it needs to maintain its phenotype. Slide 5-CD4 T Cell Differentiation [McCutcheon]-CD4 iTregs, indicible Tregs. TGF beta gives you Fox P3. And the way you tell these cells apart early on is you do something called intracellular flow cytometry and you look to see what transcription factor is turned on. Once the cell is differentiated out a little bit further, it will have receptors for gamma interferon and IL 12 where it will have receptors for IL 4 and then you can tell that way. Fox p3 gives you the anti-inflammatory cytokines, TGF beta and IL 10 among other things. Slide 6-Alternate CD4 T Cell Activation [McCutcheon]-Natural T regulatory cells are generated by Hassells corpuscles and they wont come into play until later on in an immune response if youre not getting a pathogen cleared and you continue with a heavy duty t cell response. TH 17 cells, if you have TGF beta and IL 6 or IL 21, you turn on the transcription factor RORc. That gives you a different set of pro-inflammatory and anti-inflammatory cytokines. They are a family called the TH 17 family, not surprisingly. So, IT regs, its TGF beta, you get Fox p3, TH17 cells, its TGF beta plus IL 6 or IL 21. That gives you RORc, which gives you a different set of pro and anti inflammatory cytokines. Slide 7-2nd Messengers [McCutcheon]-Now the reason that I am stressing second messengers is, and Im going to tell you a little story in this point of time. Autoimmune diseases, like inflammatory bowel disease, like Crohns disease is an example, or rheumatoid arthritis, are TH1 t cell diseases. For a very long time, the only drugs you could use to treat these diseases were massively immunosuppressant. You put people on steroids, you put people on methyltrexate. What these drugs did was prevented t cell activation, period. So, steroids have A) youve completely suppressed the

Transcribed by Ana Sangadala July 18, 2014 immune response. If somebody gets a cold, there are no t cells, then you get no b cells, then you stay sick. And both of those drugs have really significant, nasty side effects. So about 10 years ago, people started making what are called biological. Everybody has a general idea of what an antibody is right? Antibodies are part of our natural immune defense but theyre also tools. We use antibodies in the laboratory to identify specific proteins and to purify specific proteins. So we make antibodies against a certain protein and that lets us study it. Now were using them in medicine, so weve made antibodies against cytokines. You can have an antibody against IL 1 or an antibody against TNF alpha or an antibody against IL 6. You know these drugs. You here about them on television: Humera, anti IL 6 antibody. Remicade, anti IL 1, and TNF alpha antibody. So if you give these people, instead of just suppressing all t cells, they started just getting rid of the excess IL1 or excess TNF alpha, or excess IL 6. By getting rid of the IL 1, TNF alpha, or IL 6, you cause the disease to greatly go into remission so people are a lot better. The side effects are not nearly so significant. You can activate t cells. If you get sick, youre better at fending off a disease than people who are on steroids or methyltrexate. If you get rid of TNF alpha, and somebody gets an infection, or if you have a patient who has rheumatoid arthritis and they come in right after their TNF alpha treatment. You get in there and you do this deep and heavy scaling and push all this bacteria into their tissue, they cant occlude their veins. Instead of a localized bacteremia that goes away in 24 hours, they get systemic bacteremia and they die. As dentists, youre going to have to be really careful when you treat patients with these biological diseases. One of the big drawbacks of these is you have to give them as an infusion. So every 2 months, the person goes and sits in the doctors office for 4 hours and they get this antibody liquid mixture infused into their veins and they have to hang out because most of the side effects are immediate, the nasty side effects. So they have to stay there, so they can make sure that its not going to do anything immediate. And thats a pain in the butt. Its also, you know, youve already got the activated T cells. Youre getting rid of the symptoms by getting rid of the excess cytokines, but you havent really fixed the problem. So there is now a new family of drugs. What they do, is they inhibit the second messenger JAK that comes through the gamma interferon receptor. This is a pill. Its called Zoljans. Its a pill that you take everyday. Now instead of having the IL 1 secreted youre stopping the t cell thats going to try and produce IL 1 for making it in the first place. So no IL1, no TNF alpha, no IL6 and no gamma interferon. The reason you are learning this is you are now going to have patients who come into your office taking this drug. Again, they are not going to be able to produce the cytokines they need to get rid of heavy duty scaling. Youre going to have to, you know, its a very new drug. Nobody has thought about how this is going to impact dentistry, so youre going to have to think about this. You may have to have them be off their drug for a couple of days. A lot of rheumatoid arthritis patients-they have no hand eye coordination. They have rheumatoid arthritis in their hands. Theyve spent years on steroids and steroids are really had for oral tissues. They have terrible oral hygiene. They finally feel well enough to come into the dentist and youre like oh my god, Ive got all this calculus and bacteria to get out of there. Well, if they just had their infusion or if theyre taking something thats preventing them from being able to secrete TNF alpha,

Transcribed by Ana Sangadala July 18, 2014 youre going to do some serious damage to them. You need to be aware of this. The reason you are learning this now is theres a drug against it. Slide 8-IFN Activated JAK-STAT [McCutcheon]- So again, details are not important. So what we have is we have interferon gamma binding to the interferon gamma receptor. It activates JAK. These JAKs, they turn on STATS. The STATs then go off and turn on transcription of other pro-inflammatory cytokines. So IL 4 also works through a JAK-STAT pathway, but its a different set of JAKS. You dont suppress TH2 t cells at all. You just suppress the TH1 t cells and they get activated, they differentiate, but theyre not able to secrete the cytokines that they normally secrete. They can do their other things, but theyre not going to secrete their cytokines. Slide 9-JAK/STAT [McCutcheon]-Just to give you an example so again dont memorize any of this. Know that each cytokine receptor acts through different JAK-STAT pathways. Ok? Slide 10-Mutual Inhibition [McCutcheon]- Now, theres inhibition. So once you start making TH1 T cells, you actively prevent that t cell from changing its mind and becoming a TH2 t cell. So once you get T BET or STAT 1 and gamma interferon. Or gamma interferon and IR 12, you actively prevent GATA 3 transcription factor from being turned on. You hypermethylate that DNA. If you get GATA 3, that youve gotten through IL 4, you actively prevent T BET from being turned on by hypermethylating the DNA. So, TH1 t cells prevent differentiation into TH 2. TH 2 t cells prevent differentiation into TH 1. This is contrast to gamma delta t cells. Gamma delta t cells can switch back and forth. So originally, they usually secrete pro inflammatory cytokines, but then they can stop doing that and they can start secreting anti-inflammatory cytokines. Then they can go back to producing pro-inflammatory cytokines. CD4 alpha beta t cells cannot switch. They do one or the other. Slide 11-Th1 Effector Functions [McCutcheon]- What do TH1 t cells do? They are at the site of infection. Their job first of all, the macrophages that are at the site of infection, they are able to phagocytose. They can do some cleavage of bacteria that are in the vesicles or viruses in the vesicles, but thats it. They secrete cytokines, but theyre not really fully functional. The first thing that TH1 t cells do at the site of infection is they activate the macrophage to be a fully functional armed effector macrophage. That does not happen until the TH 1 t cells get to the site of infection. Fully activated macrophages then start secreting reactive oxygen species. These are things like nitric oxide and H202, and the oxygen radical then pulls ions off of proteins and that causes the proteins to fall apart. Theyre very effective. Unfortunately, theyre very effective on host as well as bacteria. Once the macrophages are end stage cells, they get ramped up. They do their thing for 2-3 days and then they run out of steam. So once those macrophages are out of steam, you need to get rid of them. The TH1 t cell using HLA class 2 on the macrophage, will then bind to the HLA class 2 on that old

Transcribed by Ana Sangadala July 18, 2014 macrophage and then kill it. It kills it through apoptosis so the contents of the macrophage get degraded. TH1 t cells cause B cells that get down to the site of infection to switch to IgG, which we will talk about on Friday of next week. Macrophages have receptors for IgG and they are much higher affinity than the receptors for the toll like receptors. Once the bacteria is bound by or pathogen, I guess virus too, is bound by IgG, then the macrophage can phagocytose it much more easily. Natural killer cells have receptors for IgG so once there is an IgG attached to a pathogen, then the natural killer cell can kill it. Natural killer cells dont have toll like receptors so they cant recognize pathogens without IgG. It causes alterations in HLA class 1. You get more HLA class 1, and the proteasome changes format so that you are better at binding viral peptides, cleaving viral peptides that will bind to HLA class 1. If its an intracellular pathogen, its now easier for the t cells, the CD 8 t cells to bind to their target cells and kill them. Then there is just an overall increase in effector cell function. Slide 12-Why do you need T cells at the site of infection? [McCutcheon]- So why do you need cells at the site of infection? Here we see new macrophages and these are old macrophages. Theyre called foamy macrophages and theyve run out of juice. These are the macrophages, where if they present peptide on class 2, those are the macrophages that get killed. They can have peptide from either viruses or bacteria because the viral peptides would be bound by IgG. The IgG can be recognized by the IgG receptor on the macrophage and that allows the macrophage to recognize the virus. No kind of antigen presenting cells, except for b cells, have receptors for viruses. Slide 13-No Title [McCutcheon]-So the only way dendritic cells can recognize a virus to phagocytose it is if you put an IgG on it. A dendritic cell or a macrophage or neutrophil. These are active ones again. Slide 14-No Title [McCutcheon]-So here we see an infection thats going on and so these bacteria are sitting on top of a macrophage. These bacteria are in the macrophage. These are sitting on top of fibroblasts. So we have these bacteria that are just all over the extracellular space and we need to get rid of them. And when we start binding IgG on these bacteria, it makes it much easier for the macrophage to phagocytose them. You clear them out. Slide 15- Th1 and Macrophages [McCutcheon]- So Th1 t cells and macrophages. The Th1 t cell binds the macrophage and it causes the macrophage to secrete more cytokines. It causes the macrophage to change whats in the lysozyme and you lots of proteases and low pH. Then you get reactive oxygen species and you get receptors for IgG. The macrophage before it was secreting cytokines and it had lysozymes that could cleave with proteases and low pH. After the Th1 t cells, you get more cytokines. Your lysozymes add enzymes to cleave bacteria. The macrophages secrete reactive oxygen species. They put up

Transcribed by Ana Sangadala July 18, 2014 receptors for IgG because now youre going to have IgG, which you didnt have until you got the Th1 t cell. Slide 16-No Title [McCutcheon]- So here we see our Th1 t cell coming to bind the macrophage. This has to be both a linked recognition so the Th1 t cell receptor has to bind HLA class 2 on the macrophage so it knows its in an infected macrophage. There is no point in activating a macrophage that hasnt eaten bacteria. And CD40 receptor is on the macrophage and the CD40 ligand is binding to the CD40 receptor plus interferon gamma is binding to the interferon gamma receptor is what gives you that fully functional activated macrophage that can kill anything in vesicles except tuberculosis. It can release reactive oxygen species to destroy bacteria that it hasnt phagocytosed yet. So we need CD40 ligand on the t cell to bind the CD 40 receptor on the macrophage plus HLA Class 2 peptide binding to the t cell receptor. Then the t cell secretes interferon gamma which binds to the interferon gamma receptor. BOOM. Macrophages activated and it does what it does really well. Slide 17-No Title [McCutcheon]-So here, the cytokines secreted by activated TH1 t cells, were going to ignore IL 3 and GMCSF because everything secretes IL3 and GMCSF so were not going to worry about that. So interferon gamma and CD40 ligand, they get macrophages going. Fast ligand or lymphotoxin, that will help kill the chronically infected macrophages. Fast ligand is a membrane bound cytokine. It kills through apoptosis. IL2 helps support t cell proliferation. TNF alpha and lymphotoxin do what TNF alpha and lymphotoxin do. They help destroy endothelium so you can have easier access for effector cells. TNF alpha causes activation of endothelium and it occludes the venules. And then, CXCL2 helps chemotaxis. Thats chemotactic for macrophages. So that helps recruit monocytes out of the site of infection. New monocytes out of the site of infection, they differentiate into macrophages so they can keep killing the bacteria as the old macrophages are ready to die off. Slide 18-TH1 Cytokines [McCutcheon]-Alright? So in writing, I have a slide with the function of cytokines so that means there are details that you have to know. And you will see that there are questions on the study guide that ask you about the functions of these. Slide 19-TH1 Cytokines [McCutcheon]-CD40 ligand and, do I have it? CD40 ligand and fast ligand are membrane bound cytokines. Slide 20-IFN- [McCutcheon]- How the t cell regulates. Its what the TH1 t cell does through gamma interferon. Gamma interferon plus CD40 activates the macrophage. It helps increase the killing ability in lysozomes and it also allows the macrophage to secrete reactive oxygen species. Gamma interferon helps the natural killer cell put up receptors for IgG. IgG bound to a bacteria or virus can then be killed off by the natural killer cell.

Transcribed by Ana Sangadala July 18, 2014 Gamma interferon causes the epithelia to put up more HLA class 1 so if the cell is virally infected, it is easier for the CD8 t cells to kill the virally infected epithelia. Gamma interferon causes B cells to isotype switch to IgG. IgG then has receptors on natural killer cells and that increases the efficiency of the macrophage for phagocytosis and it allows the natural killer cell to have a target. And then gamma interpheron, oops, sorry. And then gamma interpheron helps make CD8 t cells more active. They have to be activated in the lymph node by the dendritic cell, but at the site of infection, gamma interferon helps keep their activity high. So gamma interferon is how the TH1 t cell coordinates the immune response at the site of infection. What kind of t cells get infected by HIV? CD4 t cells. So what happens is you lose the TH1 t cells at the site of infection and you dont have any means of getting your macrophages going. You dont have IgGs so the NK cells cant play a role. You dont get activated CD8 t cells for most pathogens. And so, when you lose enough CD4 t cells, you lose this. Once you lose this, you die of infections. Because the only thing that can really run on its own is a neutrophil and if youre amounting in adaptive immune response, then it means neutrophils werent doing enough. Slide 21-Not Title [McCutcheon]-So well go through this. Ill give you a break and well go through this again. So, the macrophage at the site of infection, and that is the symbol for macrophage. It secretes IL 12. IL 12 by itself can activate natural killer cells but at this point in time, natural killer cells dont have a receptor for them to be able to bind bacteria or viruses. IL 12 causes differentiation of TH 1 t cells, of the t cells into a TH1 t cell. The TH1 t cell then secretes gamma interferon. It makes the macrophage more active. It causes it to become a fully functional armed macrophage. The TH1 t cell secretes gamma interferon that makes NK cells more active. NK cells secrete gamma interferon secretes gamma interferon which helps keep the TH1 t cells fully functional. The TH1 t cells secrete gamma interferon and that causes the b cells to isotype switch to IgG. IgG can then bind to pathogens and there are receptors on the natural killer cells and there are receptors on the macrophages for the IgG and that allows the NK cells something to target. It allows the macrophages to be able to phagocytose more easily. The IgG then binds to the IgG receptor on the macrophage. The gamma interferon secreted by the natural killer cell and the TH1 t cell then keeps the CD8 t cells more active. The CD8 t cells also secrete gamma interferon, so the gamma interferon then helps keep the macrophages going and helps keep the natural killer cells going and helps the TH1 t cells going. So each one of the natural killer cells and the TH1 t cells can secrete more gamma interferon to keep everything going. So at the site of infection, this is what happens. While youve got lots of pathogen there, this is fantastic. Though, you are damaging host tissue. Those reactive oxygen species are doing a lto of damage to host tissue. Tumor necrosis factor. Ok? Lymphotoxin is tumor necrosis factor beta, so theyre causing necrosis on cells that have receptors for them, and thats host. So youre doing damage to host tissue but you are doing more damage to the pathogen. Ok, so its a little bit early. I will give you 10 minutes now, so we will start at 8:56 and Ill go through this loop again.

Transcribed by Ana Sangadala July 18, 2014 Slide 22-No Title [McCutcheon]- Ok. Lets go ahead and get started. So, Ive said this before and Ill say it again. Conference is next week. The study guides for innate immunity, antigen processing and presentation, t cell development, and t cell activation. Well do the other 4 study guides for conferences the following week. If youre in the Monday group, the lecture for shutting off the immune response follows immediately after the conference. So, go and listen to last years podcast. Thats the one lecture Im really not changing from last year. This is an interactive conference. You do all the talking. I do answer questions, Ill explain things, but I do not provide the answers for the study guides. And EVERYBODY talks. So I will ask you to arrange yourselves into groups of 4. Make sure one of your 4 is somebody who is somebody who is willing to stand up and talk because everybody has to talk. Not every person, but every group has to talk, so nobody is allowed to just sit there and copy things down. The more work you put into this at the front end, the more you will have out of it at the back end. The better you will understand immunology, which is going to help you through the rest of your courses and your career. Also, learning to think this way is critical to becoming a good differential diagnostician. Its the same process for figuring out how this works that you use to figure out disease. So, our macrophage at the site of infection at this point in time is only partially activated. It is secreting cytokines, one of which is IL 12. IL 12 by itself can activate an NK cell, although at this point in time, the NK cell doesnt have any targets. IL 12 also causes CD4 t cells to differentiate into TH1 t cells. TH1 t cells then start secreting interferon gamma, which in conjunction with CD40 ligand, activates the macrophage fully. The armed effector macrophage is now able to rapidly lyse anything in a lysosome and its secreting reactive oxygen species. Gamma interferon also helps keep the NK cell active. Active NK cells also secrete gamma interferon. Gamma interferon from the NK cell does all the same things that the gamma interferon from the TH1 t cell does. Gamma interferon causes B cells to isotype switch to IgG. There are receptors on both macrophages and NK cells for IgG and this is when NK cells get a target. They can bind to whatever, they bind to the IgG, and they can kill whatever is bound to the IgG thats bound to the NK cell. Gamma interferon secreted by whatever cell is secreting gamma interferon also helps already activated CD8 t cells maintain their phenotype. CD8 t cells also secrete gamma interferon, which will do all of the same things that gamma interferon does regardless of where it is secreted by. Somebody said why do you have all these cells secreting gamma interferon. The answer is that redundancy is good. So, at the site of infection, you have lots and lots and lots of gamma interferon and gamma interferon makes all of the cells play together. That gives you a very effective means of destroying extracellular pathogens. You notice that the TH1 t cell is not binding to the bacteria. Ok? TH1 t cells DO NOT recognize bacteria. TH1 t cells do not recognize virus. TH1 t cells do absolutely nothing directly towards a pathogen. They act through gamma interferon, making other cells do things to the pathogen. B cells can recognize both bacteria and viruses. The b cell thats activated. The b cell secreting antibody, the antibody can bind and that can be used as a receptor for opsonization or antibody dependent cytotoxicity, which will talk about Friday, next week. But CD4 t cells do not do anything directly to a

Transcribed by Ana Sangadala July 18, 2014 pathogen. Not TH1s, not TH2s, not T regs, not TH 17s. They act through cytokines. CD8 t cells kill things. Slide 23-Treg Function [McCutcheon]-So T regulatory cells. They suppress conventional TH1 t cells. Interestingly, everybody thought, well if you suppress TH1, like if you get rid of T regulatory cells and you dont suppress TH 1 t cells, disease is going to get a lot worse. Some diseases do, but some diseases happen when you get TH1 regulatory cells out of the picture. So, T regulatory cells can both suppress things and they can prevent disease. So they can prevent some kinds of diseases and they actually help other kinds of diseases. So N T regs are a part of thymic tolerance. iTregs help prevent peripheral tolerance. If you have TH1 t cells or CD8 t cells that are active against things, the iTregs help downregulate the function of those TH1 t cells so they dont do too much damage. Slide 24-No Title [McCutcheon]-So T regulatory cells that are bound to an APC will then prevent the activation of the CD4 t cell. So once youve got an immune response thats going on long enough and you dont want to recruit new TH1 t cells into the system, the nT regs and the iTregs prevent that. Slide 25-Th17 Function [McCutcheon]-Th17 secrete a bunch of cytokines. IL 17, not surprisingly. IL 21, IL 22, and IL 23. Notice that not all of them are pro-inflammatory. So IL 22 is anti-inflammatory. IL 21 can go either way depending on the disease. Slide 26-Th17 Effector functions [McCutcheon]-Diseases that are involved with TH17. So IL 17 is very good against fungal infections and it happens when you have a massive inflammatory response. And again, when you get TH 17 cells into the mix, you had TH 1 and TH 2 t cells. Theyve been doing their thing and they cant get rid of the pathogen. You have high levels of TGF beta because you have TH2 cells, but you have high levels of IL 6 because the TH 1 cells are making the macrophages produce lots of IL 6. So instead of getting an inducible T regulatory cell, you start making Th 17 cells. SO since the original cytokine secreted by the TH1 t cells: the gamma interferon, TNF alpha, TNF beta, CD 40 ligand, fast ligand. Those arent getting rid of the pathogen so were going to try a new set of cytokines. Thats the Th17 family. These are all similar to each other in structure and relative function. So IL 23, if you have IL 23, you have inflammatory bowel disease and fungal infections. Slide 27- No Title [McCutcheon]-So disease inducing psoriasis. Some of the cytokines that cause psoriasis, which is an autoimmune disease: IL 17, IL 22, IL 23. Rheumatoid arthritis, you see a lot of IL 17, IL 21, and IL 23. Multiple sclerosis, you see IL 17 and IL 23. Asthma, we dont know whats going on there but Th 17 cells are playing a role. Inflammatory bowel diseases, you see IL 23. There are some data that suggest that

Transcribed by Ana Sangadala July 18, 2014 some peoples periodontal disease has a TH 17 cytokine family component and I think Dr. Craig will talk about that. But we have disease protective. So we can have disease causing IL 23, but IL 17 and IL 22 actually protect against inflammatory bowel disease. And IL 17, IL 21, IL 22, and IL 23 help the host defense against massive infection. So depending on the relative levels of the different cytokines, you can make things better or you can make things worse. Slide 28-CD8 T cell function [McCutcheon]-So what do CD8 t cells do? CD8 t cells are killing machines and thats all that they do. They are directly lytic to cells containing intracellular pathogens. So CD4 t cells do not do anything directly to pathogen. CD8 t cells do not do anything DIRECTLY to pathogen, but CD8 t cells bind to HLA class 1. Any cell that is infected by a virus is going to have viral peptides on HLA class 1. CD8 t cells, thats what they do. They bind to HLA class 1 and once the t cell is activated, it doesnt need any of those other co-stimulatory molecules. It just needs to bind to HLA class 1. So virally infected cells have lots of viral peptide on HLA class 1, the CD8 t cell binds to the virally infected cell and then if the infected cell has Fas on it, CD8 t cells can kill through Fas ligand. If the infected cell doesnt have Fas, it can kill through an enzyme system called perforin and granzymes. Slide 29-No Title [McCutcheon]-So cytotoxic t cell, it binds to HLA Class 1. Ok? It moves all of its lytic granules over to the point of contact. And so, unlike reactive oxygen species that kill whatever they fall on. CD8 t cell releases the perforin granzymes directly onto the surface of the infected cells, so only the infected cell gets killed. So its highly specific. But remember, what species is the infected cell from? Host. Yeah, so CD8 t cells are killing YOU. And actually, when you have a cold, you all know that you are actually most infectious the day before you know youre sick? So the symptoms of a cold are not from the virus. The symptoms from a cold are from when your CD8 t cells show up at the site of infection and start killing off all of your virally infected cells. And then they are secreting cytokines, IL1, TNF alpha. Thats what causes the fever. Once you lose the integrity of the epithelium, thats where all the mucous comes from because theres nothing to hold it back. So the symptoms of a cold are when your CD8 t cells are there. So youve been sick for 4 or 5 days because thats how long it takes to get your t cells activated, 4 or 5 days. So youve got all of these virally infected cells in your nasal epithelium. And the t cells come along and they start killing off all the virally infected cells and you lose integrity of your nasal epithelium. Thats where all the mucous comes from and then the cytokines are what are secreted by the CD8 t cells are what cause fever. So the first 2 days you feel crappy, thats after the immune response starts. Ok? And a little aside, green tea activates your natural killer cells to secrete interferon alpha or interferon beta. So the minute you start feeling that scratchy feeling in your throat, start drinking lots and lots of green tea. Zinc prevents viral replication, so take zinc tablets, dont use the nasal spray. And the best thing you can do is stay in bed. If you sleep for the first 24 hours or rest for the first 24 hours, the duration of your cold will be considerably

Transcribed by Ana Sangadala July 18, 2014 shorter than if you show up at school and infect everybody around you and try to study. Green tea, it will make a huge difference in the duration of the cold, ok? Slide 30-No Title [McCutcheon]- So here is my CD8 t cell. Its binding to peptide on HLA class 1. It induces apoptosis in that cell and moves on to the next cell. In fact, it takes 20 minutes of contact with the CD8 t cell to induce apoptosis in the cell. The apoptosis occurs more slowly, but 20 minutes of contact with a CD8 t cell, that cell will die. It moves to the next cell, 20 minutes, and that cell will die. It moves to the next cell, 20 minutes, and that cell will die. CD8 t cells, and remember, its gone through clonal proliferation just like that CD4 t cell, so youve got 10,000 CD8 t cells at the site of infection and they kill. And they kill. And they kill. Thats all they do. So one of the reasons its so important to make sure that you, remember what cells have HLA class 1? All of your nucleated cells. So if you get armed effector CD8 t cells activated against a host peptide, those CD8 t cells will show up at the site of wherever that tissue is and theyre going to kill and kill and kill. Its incredibly important to keep your CD8 t cells regulated, because once you have them, theyre going to kill. Thats actually the method in spinuloarthoropathies. The CD8 t cells get activated to a peptide on HLA B 27. Its either from salmonella or shigella. That peptide is one amino acid away from a host peptide found on cartilage, heart muscle, endothelium. Once you get the army of CD8 t cells, they kill, except now theyre killing you. So if youre HLA B 27 positive, dont get food poisoning. Slide 31-No Title [McCutcheon]-So CD8 t cells, they kill virally infected cells. So CD8 t cell is the only thing that can kill a virally infected cell except an NK cell, and were not going to worry about that. CD8 t cells are the only things that can kill a virally infected cell. Once the virus has been released into the extracellular space, because one of the mechanism of viral replication is that they cause lysis of the infected cell and then that spreads out 50,000 new copies of the virus. Once the virus is in the extracellular space, the only thing that can recognize it is an antibody. Thats the reason that when they look at viral diseases, they want to see how many antibodies you have. If you have antibodies against the virus, youre not going to be reinfected. If your antibody titers have dropped down too far, and you come across measles, mumps, or chicken pox, then you will get reinfected. In order to get an active immune response against a virus, you have to have CD8 t cells, but you have to have TH2 t cells in order to get activated B cells. Where to do TH2 t cells come from? What kind are they? (inaudible response) Say it loud. (Inaudible response) CD4. Right. In order to effectively get rid of a virus, you have to have HLA class 1 presentation for the CD8 t cells, but you have to have HLA class 2 presentation to get your CD4 t cells activated. The TH1s dont do a lot. But you have to have TH2s to get antibodies. So an intercellular pathogen requires CD4 t cells, it requires TH2. The TH2 arm of CD4 t cells. It requires b cells to produce antibody, and it requires CD8 t cells. An extracellular pathogen thats only presenting on HLA class 2, CD8 t cells never get activated because they have no role in this disease. TH2 t cells-their job is to activate b cells and we will talk about that next Friday after weve done b cell development.

Transcribed by Ana Sangadala July 18, 2014 Where does the virus get made? Inside the cell. If its being made inside the host cell, what are the proteins going to look like? Host. Do macrophages and neutrophils have receptors for host proteins? No. Do you want them to have receptors for host? No, you dont want them to. So what can recognize that virus when its in the extracellular space? (inaudible response) Nope. They recognize peptide and HLA Class 1. So what can recognize the virus when its in the extracellular space? Just antibodies. Where do antibodies come from? B cells. How do you get b cells activated? TH2 t cells. So, if you just have CD8 t cells, CD8 t cells they kill a virally infected cell, they kill a virally infected cell, they kill a virally infected cell. The cell, 3 cells down, now ruptures. Its lysed by the virus and 50,000 copies of the virus come out and they can all go out and infect 50,000 new cells. So the CD8 t cells can kill the virally infected cell, but how do you stop the virally infected cells from infecting new cells if it ruptures before the CD8 t cell gets there? So theres no way to stop new viral infection with just CD8 t cells. You have to have something to prevent the virus from binding and infecting new cells and that something is anANTIBODY. SO you have to have antibodies to get rid of viruses. To prevent viruses from infecting and its antibodies that give you immunity, secondary immunity. Its having antibodies in your system. You come home, and your kid is covered in red spots because they have measles. Well, the reason youre not going to get measles again is because you have CD8 memory t cells that are going to kill off virally infected cells and you have antibodies that will prevent viral infection from happening. So its the memory from that that keeps you from getting viral diseases. Without antibodies, you get the viral disease again. Slide 32- T lymphocytes [McCutcheon]- Gamma delta T cells live in the tissue. They are antigen specific. Al right? So they are antigen specific. So they are part of the adaptive immune response. They live in the tissues. Their gamma delta t cell receptor is very flexible like an antibody, b cell receptor. They recognize native antigens like b cell receptors, so you dont have to have HLA class 1. You dont need antigen presenting cells. They respond to native antigens, and so the minute you have a virus or a bacteria, the gamma delta t cell receptor can bind to that native structure. The gamma delta t cells then start secreting cytokines. At the very beginning of the innate immune response, you also have adaptive gamma delta t cells and they help shape whether you are going to have primarily a TH1 t cell response or a TH2 response by the cytokines they secrete. Over time the original CD4 t cell, originally you start out with kind of equal numbers of TH1 and TH2. Over time, youre going to polarize one way or the other. If you polarize toward the TH2 response, youre going have a lot of antibody and not a lot of cell mediated immunity. If you polarize towards the TH1 response, youre going to have a lot of cell mediated immunity, and not so much antibody. For many pathogens, it doesnt matter which way you polarize. For some pathogens, its critical. Mycobacterium tuberculosis, which causes tuberculosis: of you polarize towards TH1, so mycobacterium are obligate intracellular pathogens. If you polarize toward TH1, you keep the pathogen in check. If you polarize towards TH2, so you have HLA Class 1, and you get CD8 T cell responses, and you keep the infection from spreading. If you polarize towards TH2 and produce antibodies, well

Transcribed by Ana Sangadala July 18, 2014 antibodies can only fight the mycobacterium when they hop out of the cell to hop into the next cell. You die really quickly. The same is true in Tb. Mycrobacterium tuberculosis, if you polarize towards TH1, you wall off infected macrophages with a granuloma and you keep the disease in check. If you polarize towards TH2, mycobacterium can only live in cells and antibodies cant go into cells. You get really severe disease really quickly. For many pathogens, it doesnt really matter. Slide 33-No Title [McCutcheon]-So, Im in trouble here because I have all of this time to show you movies that arent working. Slide 34-Extracellular Pathogens (this section is a lot of Q&A, and I couldnt hear the student responses to Dr. McCutcheons questions) [McCutcheon]- Except, at the beginning of the lecture when nobodys here. So for extracellular pathogens. What are involved in getting rid on an extracellular response? Start at the beginning. Ok, thats not the first thing. Complement. Complements the first thing, ok? Once complement is there, what happens? They recruit neutrophils. Ok? What do neutrophils do? They do phagocytosis and they can yeah. What piece of complement do we need? C3B. What does that do immediately? Its a loop so you can get lots and lots and lots of complement that leads to MAC. What else does it do? Its an opsonin, so the receptor on the macrophage for C3B has a much higher affinity than toll receptors so the bacteria stays attached to the macrophage surface a little bit longer and that makes it easier to do phagocytosis. The other thing that complement does that we will talk about on Friday is it activates b cells. What else? Weve got neutrophils, macrophages and complement. What else? Yup. So, what do they do? They change the endothelium. Al right? We are losing S Lex and replacing it with what? V CAM. And then they make it easier for cells to have access and that also puts CXCL8 on the epithelium so when the macrophages come to the site of infection, the CXCL8 binding to the CXCL8 receptor on the endothelium is what causes the rolling to stop so that diapedesis can occur. Macrophages and neutrophils. Whats the next step? Thats the innate immune cell part of the innate response, but you also have adaptive cells: CD5 b cells secreting IgM and gamma delta t cells secreting whatever theyre going to secrete. So even at the beginning of the innate immune response, there are adaptive cells. Thats another reason thats not a good dichotomy. So now what happens? (Inaudible response) Its not macrophages, its dendritic cells. Macrophages are going to stay at the site of infection. Dendritic cells phagocytose the extracellular pathogen. Now what happens? (Inaudible response) You skipped a bunch of steps, skipped a bunch of steps. They have to change their shape so the dendritic cell goes from being an immature highly phagocytic cell to a mature non-phagocytic cell. It has to pull its arms in so it can get out of the tissues. Then, it does what? (Inaudible response) How did the MHC Class 2 get there? (Inaudible response) So you have to tell me how did the MHC class 2 get there? Youve got it up to the vesicle. How did it do that? What happened before it got to the vesicle? Synthesis on the ribosomes. Then, something blocks something. Invariant chain blocks peptide binding groove. Youre still in the ER. It goes to the Golgi, gets put into the vesicle. While its in the vesicle what

Transcribed by Ana Sangadala July 18, 2014 happens? The outside part of the invariant chain gets degraded leaving CLIP. Ok. The vesicle containing the HLA Class 2 does not go into the membrane. Why not? Because the invariant chain is steering it towards other vesicles. When the vesicle containing the Class 2 binds to the vesicle containing the bacterial peptides, HLA DM removes CLIP, bacterial peptide binds the HLA Class 2, and HLA Class 2 ends up on the cell surface. Ok? Now, you can present to the t cell. Along with copies of peptide bonding to HLA Class 2, what else is on the cell surface of the dendritic cell? I CAM. Pardon? B7. One more protein and something secreted. CCL18 and thats going to attract what kind of cells? Nave t cells. One more receptor. DC sign. DC sign is going to play a role in B cell activation. Ok? Where is the dendritic cell at this point in time? In the nearest lymph node. What part? Paracortex. Ok? So now, the nave CD4 t cells and actually nave CD8 t cells are going to come check out the dendritic cell. What will happen to the CD8 t cells? (inaudible response) Ok, so LFA will bind ICAM. Then what will happen to the CD8 t cells? What kind of peptides does the dendritic cell have on HLA Class 1? They have host peptides. What will happen to the CD8 t cells? NOTHING. It will find nothing to bind and it will go away. What will happen in 99.99999% of the CD4 t cells? There will be nothing to bind and they will go away. What will happen in that one t cell? T cell receptor will bind to HLA peptide. Then what happens? (inaudible response) Ok. Yes. But thats what t cells do. T cells are like teenagers in small towns with drive ins. They cruise the strip, they pull into the drive in, they get their soda, they go back out, they cruise the strip, they pull back in, they get their French fries, they go back out, and they cruise the strip. You know, thats how nave t cells work. They go into a lymph node, theres nothing there, the leave the lymph node. The go into the next lymph node, theres nothing there; they go back into the circulation. They go into the next lymph node. Thats what nave t cells and b cells are doing. The dendritic cell stays in the draining lymph node and lets all of the t cells come to it. So, LFA titins. (inaudible question) Yup, which doesnt do us any good for infections, so what happens? (inaudible response) Right, theres binding of multiple receptors to multiple copies of pathogen. Then, CD28 binding to B7, you get t cell activation. What happens? We havent gotten that far yet. We only have one t cell at this point. You can just shout out, you dont have to raise your hand. (inaudible response) How? Weve already done that. What about IL2? Where did the IL2 come from? The t cell, the activated t cell now changes the confirmation of the IL2 receptor. It causes secretion of IL2. IL2 binds to the IL2 receptor. Now the t cell, you get proliferation. You get this army, 10,000 daughter t cells. What happens? (inaudible response) Not yet. Its not activated. Activation is having that t cell put out IL2 receptors and proliferate. Activation means that the t cell is the one thats going to change its IL2 receptor so that when IL2 binds, it can proliferate. All the other t cells in the lymph node, and thats full of them. They have the low affinity form of the IL2 receptors so IL2 binding to them just gives them survival signals. They dont divide. (Inaudible response). No, weve got this army of cells. We gotta differentiate them. T cell runs into IL 12 through Tbet. Tbet transcription transcribes what things? (inaudible response) Yes and the interferon gamma receptor, so interferon gamma binding to the interferon gamma receptor causes what? STAT 1 and more Tbet. Then eventually you get STAT4 and the STAT1 and the STAT4 cause transcription of a cassette of pro-inflammatory cytokines. What

Transcribed by Ana Sangadala July 18, 2014 else is happening on that TH1 t cell? Where is it right now? Its still in the lymph node. What else has to happen? (inaudible response) How? What proteins? VCAM on the t cell, VLA 4 on the t cell, VCAM on the endothelium. So now the TH1 goes to the site of infection. There it secretes gamma interferon, which does boat loads of things. What happened to the cells that ran into IL 4? TH2 through GATA3. GATA 3 causes transcription of? IL4 and? Receptors. So IL4 binding to the IL4 receptor, you activate STAT 6. That gives you a cassette of anti-inflammatory cytokines that we will talk about on Friday. Where are TH2 t cells? In the lymph node. Why didnt they go off to the site of infection? They lose L selectin and they dont get anything else. Ok? So the TH1 t cells at the site of infection are getting, as soon as the b cells show up, are getting the pathogen eliminated. Hopefully. If not, then we start pulling in NT regs, IT regs, and TH17 cells. HEY!! That was an extracellular pathogen. Now, somebody has just sneezed in your face and you snort in a boat load of virus. What happens? Not quite. Whats the first thing that happens with that virus? It infects cells. What cells? (inaudible response) Among other things. It infects what its supposed to infect and then it infects dendritic cells. Where is complement? Except for So complement is not involved except for C3B. How did we get to C3B? C3 can spontaneously cleave. Its always spontaneously cleaving. If it lands on host cells, you have the membrane bound factors that prevent it from becoming a convertase. If it lands on a virus, it stays there. You dont get the rest of the complement pathway because the virus isnt big enough but it will help get b cells activated. So weve got C3, weve got infected dendritic cells, and weve got C3 on viral particles. Thats one of the ways that the dendritic cells can do micropinocytosis. They use the C3B as an opsonin. We can get virus in intracellular, we can get virus in vesicles, weve got virus infecting. If the virus infects, what happens to the coat proteins as theyre being made? Whats the DC doing so that it doesnt get killed? It makes interferon alpha and beta. Interferon alpha and beta binding to the interferon alpha and beta receptors stops viral replication. So we have some virus, but the dendritic cells dont get overwhelmed by virus. So now what happens to those viral coat proteins in the cytosol? Theyre degraded by the protease. Theyre pumped into the ER by? TAP(sp?). Class 1, which goes closely to the surface. Now, the reason you have to know TAP is because, this is an aside: the human herpes virus family, HHV1, HHV2, so human herpes virus is the oral herpes, the genital herpes that you get. That virus, one of the proteins that gets synthesized is a protein that can block TAP. It plugs up. TAP is a channel and it plugs the channel, so you cant get peptides cleaved from the cytosol into the endoplasmic reticulum. In the absence of peptide in the endoplasmic reticulum, HLA Class 1 does not get folded up. It doesnt end up on the cell surface. Eventually, that cell loses all of its HLA class 1. Can a CD8 t cell kill a cell that doesnt have HLA Class 1? No. So, TAP prevents CD8 t cells from killing virally infected cells. Thats actually the role that NK cells have. So natural killer cells have receptors that are inhibited by the presence of HLA Class 1. So cells with HLA class 1 can be killed by t cells, inhibit killing by natural killer cells. In cells that have lost their HLA class 1, they cannot be killed by CD8 t cells. Theres nothing to inhibit the killing by the natural killer cell. Natural killer cells kill when cells have lost HLA class 1. So, TAP 1 can block the outside, the cytosolic side. Ugh, not TAP 1. HHV 1 can block the cytosolic side of TAP. HCMV can block the ER side of TAP. HHV2 or 3 I

Transcribed by Ana Sangadala July 18, 2014 dont remember, makes a protein that looks almost like the TAP subunit 2. So, when TAP 1, the subunit 1, binds with the subunit 2, it doesnt form a pore. Then you cant put peptides into the endoplasmic reticulum. HCMV also makes a protein that looks a lot like beta 2 microglobulin. So it binds to the HLA class 1 heavy chain, but it doesnt cause the peptide binding groove to form properly. So, it cant put peptide on the HLA Class 1 and it cant get to the cell surface. All of the herpes family viruses do something to HLA class 1, so that it cant get to the cell surface. 3 of them do something to TAP. Thats the reason you have to know about that protein. As far as I know, the rest of the chaperone proteins that are in HLA Class 1 synthesis are not involved in disease. So weve got our peptide on HLA Class 1 in what cell? Where is the dendritic cell? Not at this point. At this point, it stopped being phagocytic, immature cell and its a non-phagocytic mature cell. Its either traveling to the lymph node or its in the lymph node. Whats it doing? Its secreting CCL18, that recruits in nave t cells. Where else does the dendritic cell have peptide? In vesicles. So where would that peptide end up? HLA Class 2. So we have peptide on HLA Class 1, peptide on HLA Class 2. What kind of t cells get recruited by CCL18? Nave CD4 and CD8. This time, what will the CD8 t cells be able to do? There will be one of the CD8 t cells, and we really dont want more than one. There will be one of the CD8 t cells that will recognize the peptide presented by one of the HLA Class 1 molecules. That t cell will get activated. Those t cells are where at this time? Theyre still in the lymph node. So what do we need to do? They need to go to the site of infection. How do you suppose they do that? They lose L selectin and they get VLA 4. They go out straight out the efferent lymphatic. Concentration gradient. The closer they get to the infected tissue, the more VLA4 there is. The CD8 t cells get to the site of infection. They come out of the circulation, and they start killing things. Ok. So, long review. Let me go see if the movies are only going to play at the beginning of the..(doesnt finish sentence). [VIDEO] See how much of a surface area is in contact between those two cells? [VIDEO] We dont care about that. [Video] So does that help the people who want to see in 3D? Now how do I get my movie back? So now if thats going to work, lets try these. [VIDEO] You dont need to know the names of those (referring to alpha, gamma epsilon chains in the video). [VIDEO] Ok? So thats what happens with the t cell receptor. If you dont get signaling from CD28, youre stuck at that state and thats energy. [VIDEO] Ok, so in this, what you need to know is that it happens through phosphorylation. The names of all of those things are not important, al right? It happened through phosphorylation. You do not need to know the details of that. You DO need to know JAK-STAT. The rest of these, and you know, immunology is wonderful. You get into

Transcribed by Ana Sangadala July 18, 2014 something called MAP kinase kinase kinase, which phosphorylates MAP kinase, which then goes off and does things. None of that stuff at this point in time is used for drugs. There are no drugs based on the absence of these, or at least people dont live long enough for them to be born with disease from the absence of these so you dont need to know them. Al right? So we will pick this up with b cell development on Wednesday.