Cell Biology of the Immune Response
Transcript of Part 1: Cellular Basis of the Immune Response
00:00:04.01 Hi, my name is Ira Mellman. I'm Vice President of Research on Oncology at Genentech, 00:00:09.09 a large biotechnology company here in San Francisco. Prior to that though, 00:00:13.12 in fact up to 2007 I was a professor at the Yale University School of Medicine, 00:00:17.29 chairman of the Department of Cell Biology and a member of the Ludwig Institute for Cancer Research. 00:00:22.01 During that time and in fact up until today, my laboratory has been 00:00:26.15 very interested in understanding the fundamental mechanisms of membrane traffic. 00:00:30.02 In other words, understanding the movement of cells and membranes 00:00:33.27 and the proteins and lipids that go to comprise those membranes 00:00:37.00 allowing them to work together to make cell shapes and cell functions of various sorts. 00:00:43.00 The last, more recent period of time though, we've become increasingly interested 00:00:47.27 in trying to understand how membrane traffic acts in highly specialized cells to allow them 00:00:53.16 to carry out the variety of important and specialized functions that are associated with specific 00:00:59.05 and more complex systems. The system that we have chosen to work with 00:01:03.02 over the last several years is the immune system, trying to understand how membrane traffic conspires 00:01:09.06 to get cells of the immune system to work and work together to generate an immune response. 00:01:14.02 It's a terrific problem of cell biology which almost inexplicably to me, at least, 00:01:18.05 has been relatively inaccessible to most cell biologists almost as inaccessible 00:01:22.17 as cell biology has been to immunologists. But nevertheless, there is an enormous amount 00:01:26.29 of very important cell biology to be learned by looking at cells of the immune system, 00:01:30.20 but it also provides a lot more. It provides you with the opportunity to actually understand 00:01:35.27 how events that occur at the molecular level and at the cellular level 00:01:39.22 can integrate with each other at the systems level to understand a very complex 00:01:44.09 and important phenomenon, which has a great great deal of relevance with respect to 00:01:49.13 understanding important and very devastating human diseases. 00:01:53.02 Now in order to introduce you to the topic, and let you know what it is you can indeed find out, 00:02:00.00 I have to first review with you some of the basic features of what the immune response is, 00:02:06.05 and how it's organized in its cellular level of organization. 00:02:10.03 So, what is the basic function of the immune system? It seems to be to be able to provide us with 00:02:15.14 protection against a limitless number of pathogens and toxins 00:02:19.07 that one finds in the environment. It's basically conserved in it's most elemental forms across evolution. 00:02:25.16 Invertebrates have a type of immune system and certainly we vertebrates 00:02:29.23 have a type of immune system, which provides us with a terrific amount of protection 00:02:34.16 under normal circumstances to viruses, bacteria, protozoa, and other environmental poisons and allergens. 00:02:42.00 Now, the immune system is extremely good at this and it's also extremely clever. 00:02:46.02 It can kill incoming viruses or incoming bacteria, but it does so in a fashion which avoids the injury 00:02:53.13 to the host, in other words, us, which provides a really important conundrum in 00:02:59.09 understanding how the immune system works. How is it that cells in the immune system 00:03:03.15 can distinguish self from non-self? How can they distinguish 00:03:07.01 the normal cells and tissues of our bodies from the proteins and components 00:03:13.02 that are associated with the protozoa and bacteria and other microbial invaders 00:03:17.07 that are coming in from the outside. This is probably the major fundamental 00:03:21.29 and conceptual problem in understanding the immune system 00:03:24.25 and it's one that undoubtedly will have the cell biological solution associated with it. 00:03:30.02 Now is this complicated? It's absolutely very complicated, but in my view it’s not 00:03:35.03 really any more complicated than anything else, 00:03:37.00 and if you try and understand how the immune system works in cellular terms, 00:03:40.29 through the eyes of the cell biologists, in fact I believe it does become quite accessible. 00:03:45.02 Now, it's worth the effort, as I've said already, because it's not only interesting, 00:03:50.28 but it's of profound importance to understanding and controlling a wealth of 00:03:55.03 important human disorders, infectious disease, is obvious, arthritis, 00:03:59.10 Crohn's disease, asthma, autoimmunity, and quite possibly even cancer. 00:04:04.01 Now the immune system, or immune response rather, consists of two interconnected 00:04:08.08 arms, the innate immune response, or innate immunity, and adaptive immunity. 00:04:14.02 Well innate immunity refers to that portion of the immune response, 00:04:18.15 which is responsible for detecting components shared by all pathogens 00:04:24.27 that we have to deal with as invaders. 00:04:28.00 An interesting concept, in terms of how this might work, and not immediately 00:04:32.15 obvious how it works, but indeed it turns out to be the case. 00:04:35.01 Adaptive immunity is something different. Adaptive immunity 00:04:39.24 refers to the type of immune response that is molecularly crafted to mold and detect 00:04:45.21 individual antigens that are specific to individual pathogens and individual types of pathogens. 00:04:50.21 These two work together in some rather interesting and mysterious ways that we'll 00:04:55.14 go into later, but nevertheless it is important to keep it in mind that there are 00:04:59.10 these two equal and equally important aspects of the immune response: 00:05:03.04 innate and adaptive immunity. Innate immunity was really first discovered 00:05:07.01 by this great scientists of the past, Elle Metchnikoff, shown here working in his 00:05:12.20 laboratory about 100 years ago at the Institut Pasteur in Paris. 00:05:16.00 Metchnikoff's great conceptual advance was the understanding that inflammation, 00:05:22.27 which is what happens after you become infected by the introduction of a bacterium or a virus, 00:05:30.02 this is why your cuts become red and inflamed and hot and painful if they go untreated, 00:05:37.09 this process of inflammation is a protective process of immunity 00:05:41.26 and not a manifestation of the tissues destruction. In other words, as bad as it looks, 00:05:46.01 this is actually a representation of how the body is responding to invading microorganisms, 00:05:52.05 to rid the body of those microorganisms, rather than a reflection 00:05:57.00 of the microorganisms capacity to meet out damage on the hosts tissue. 00:06:03.01 Prior to this point, this idea was completely foreign to scientists. 00:06:10.01 Now the story goes that one of the most important observations that Metchnikoff 00:06:17.09 made was actually made while on vacation on the beach with his family, 00:06:22.03 and like many modern day scientists, who bring their computers with them on vacation, 00:06:27.03 Metchnikoff in the age before computers brought his microscope, 00:06:31.10 and studied the reaction of starfish larvae to the introduction of foreign materials 00:06:38.16 such as bacteria introduced experimentally into the larvae. 00:06:42.02 What Metchnikoff found was that a series of cells would come in 00:06:46.00 and surround the bacterium and in fact, quite visibly, engulf these microorganisms 00:06:52.00 shown here as these red oblong creatures, contained within this cell. 00:06:56.02 He named these cells macrophages, because they were large cells that ate bacteria, 00:07:02.08 macrophage: “large eater". The bacteria were taken into the interior of the cell, 00:07:08.00 and Metchnikoff further found that if he stained these cells with a dye 00:07:10.02 that partitioned into acidic elements within cells and other things, 00:07:17.00 the bacteria stained red. Further indicating to him, and in fact correctly to us, 00:07:22.26 that the bacteria after they entered into the cell would be delivered to 00:07:26.28 an acidic intracellular compartment, which we now understand and know as lysosomes. 00:07:32.01 Now bear in mind, this was well over 100 years ago, 00:07:35.24 and long before even membranes were truly discovered. Now these bacteria eventually stopped growing, 00:07:42.18 they died after being taken up by the cells, and eventually disappeared 00:07:49.00 and Metchnikoff further understood that as a process of degradation. 00:07:51.02 So what actually happens when a macrophage eats a bacteria or encounters a bacteria is shown here. 00:07:58.01 The response is not only limited to the uptake and intracellular killing of the bacteria, but also 00:08:05.01 these cells secrete a variety of important cytotoxic agents, 00:08:09.03 degradative enzymes as well as protein hormones referred to as cytokines. 00:08:14.01 Cytotoxic agents include important mediators, such as hydrogen peroxide, 00:08:18.28 which is a common disinfectant that is used, but macrophages do this on their own, 00:08:25.05 as well as other types of active forms of oxygen. They secrete an enzyme called 00:08:30.06 lysozyme, which is quite adept at degrading the cell walls of incoming bacteria, 00:08:34.17 and a series of lysosomal enzymes contained within the lysosomal structures of 00:08:39.09 these cells that basically degrade all sorts of other things. As I mentioned, 00:08:43.11 the cytokines that are released by cells, by macrophages after encountering 00:08:48.17 bacteria, some of which you may already know, such as the molecule interferon, 00:08:54.03 macrophages are a prime source of interferon have the effect of recruiting more 00:08:59.03 macrophages and other cells of the immune system to the site of bacterial 00:09:03.01 invasion, increasing the overall level of inflammation, but again, 00:09:06.24 not for the purpose of tissue destruction, but rather to recruit 00:09:10.24 more and more cells of greater complexity and of increased levels of sophistication 00:09:17.00 that help deal with the infection and hopefully eradicate it before it 00:09:20.27 gets out of hand. Now why is it that macrophages and other cells 00:09:26.07 of the innate immune system, such as neutrophils, 00:09:28.17 know to respond to microorganisms? What is it that they are recognizing? 00:09:33.18 So here the story turns to a relatively recent observation 00:09:38.27 having to do with a class of membrane protein receptors found on macrophages 00:09:43.02 as well as other cells, called Toll-like receptors. What are these? 00:09:47.18 The story actually starts a little bit earlier than that, in the work of 00:09:52.07 Catherine Anderson and Carl Hashimoto, who identified the activities associated 00:09:56.25 with a well known gene in Drosophila called Toll. Now Toll was originally identified 00:10:05.26 as acting in early embryonic development in Drosophila, specifying the dorsal 00:10:11.13 from the ventral pole of the early Drosophila embryo. Toll was known to bind to a ligand, called Spaetzle, 00:10:19.07 which activated the Toll-receptor, sending a signal to the embryo 00:10:23.01 allowing this dorsal ventral polarity axis to be established. Here you can see some embryos 00:10:29.16 that either do or do not express wild-type Toll, or members of the pathway. 00:10:35.01 In panel A, all the way on your right, you can see what the normal embryo looks like, 00:10:39.26 and in the absence of functional Toll-receptor or an active Toll-like pathway 00:10:43.03 you can see that these embryos completely disorganized and they don't develop 00:10:49.01 and it is known as a lethal defect early in embryogenesis. Now Toll it turns out also 00:10:57.17 has a role in the adult fly, a quite different role, but one that is no less important 00:11:05.06 in so far as the fly is concerned. Here a different group of investigators, 00:11:10.23 lead by Bruno Lemaitre and Jules A. Hoffmann found that Toll was absolutely 00:11:16.18 required in order to protect adult Drosophila from their pathogens, 00:11:21.20 in this case what you are looking at is a poor dead fly that was unable to combat 00:11:26.00 the fungal infection that you can see here, growing almost as a beard, or a beard of death 00:11:34.01 around this fly's torso because of the absence of a functional Toll-receptor 00:11:41.05 in the fly as the adult. This is really one of the very very first indications 00:11:46.19 that Toll has a role outside of early morphogenesis and also plays an important role in host defense. 00:11:55.01 Now how does it do this? Here is a diagram of what the Toll-receptor looks like in flies, 00:12:01.24 it has a nice extracellular domain with some repeats, 00:12:04.22 it has an intracellular domain that one can presume has signaling molecules 00:12:09.01 or signaling features associated with it, but one of the most interesting features 00:12:14.06 that fell out of the analysis of what this receptor looked like, 00:12:17.18 was that it was very very similar to a receptor for cytokine, called IL-1. 00:12:22.02 Again, another product of the macrophage. And specifically where it was similar 00:12:27.17 was in the cytoplasmic domain, here in the case of Toll, and here in the case of IL-1 receptor. 00:12:34.03 Since it was known that stimulation of IL-1 receptor turned on an inflammatory 00:12:38.21 response from other sorts of studies, it was surmised that 00:12:42.21 indeed the Toll-receptor did much the same thing. So in subsequent years, 00:12:48.11 when Charlie Janeway and his colleagues started looking for the expression of these receptors 00:12:54.21 in vertebrates, they found indeed that there is a whole family of Toll-receptors, 00:12:59.08 and indeed well over a dozen of them are now known that have the same basic organization as 00:13:03.26 Toll does in Drosophila with an extracellular domain that is similar in many ways, 00:13:09.07 but most importantly the cytoplasmic domains that have a large degree of homology 00:13:15.14 to the Toll-receptor in Drosophila as well as to the IL-1 receptor. 00:13:20.01 Now what these receptors do, and the reason they are so many of them 00:13:24.17 is that each one has a specificity for a different microbial component. 00:13:29.01 In each case, the microbe itself needs those components to survive. 00:13:34.00 So here Toll-like receptors are expressed on the plasma membrane, 00:13:40.05 such as Toll-receptor four, or TRL-4 and TRL-5, see various important 00:13:46.00 components associated with bacteria. So TRL-4 sees components associated 00:13:52.15 with the peptidoglycan, a lipopolysaccharide component of bacterial coats. 00:13:58.01 TRL-5 sees a major protein associated with bacterial flagella, 00:14:02.01 both of which are components that are absolutely needed by these bugs in order to survive. 00:14:07.03 They are shared by great many types of bacteria, hence these Toll-receptors 00:14:12.01 recognize these conserved molecular patterns and act as the innate immune system sensor. 00:14:18.03 Another set of Toll-receptors are actually found in intracellular vesicles, endosomes 00:14:23.09 and lysosomes found within macrophages and other cells, examples here are TLR-7 and TLR-8, and -9. 00:14:30.01 And they have a tendency to recognize nucleic acid components 00:14:34.09 such as double-stranded RNA, DNA, and single-stranded RNA, 00:14:38.29 which are associated with other types of incoming pathogens 00:14:41.03 such as both enveloped and non-enveloped viruses. So here you have a complete pattern 00:14:48.20 of conserved receptors that have this remarkable capacity to be able to detect, 00:14:54.19 again, the shared components that can be found with a wide variety 00:14:58.13 of different types of incoming and invading microbes, turning on the macrophages 00:15:03.26 and in fact any other cell that happens to be expressing them. 00:15:06.26 Now the way that this turn-on takes place, or the way that this signaling occurs, 00:15:13.06 perhaps not surprisingly, is similar to the way that stimulation of IL-1 receptor, 00:15:19.26 the cytokine receptor, does it, turning on a series of signaling intermediates leading into the nucleus 00:15:27.03 activating I think the most important and simplest element, the so-called Nf-kappa-B pathway, 00:15:33.01 which leads to in turn a great number of events that prime 00:15:38.27 almost everything important that happens in the context of an innate immune response. 00:15:44.02 So intracellular signaling pathways are all activated by TLR receptors, 00:15:49.21 the pathways themselves can be different from TLR to TLR as they can from cytokine to cytokine, 00:15:56.13 but this really summarizes the basic feature of how the system works. 00:15:59.02 Now the consequences of it are shown here, here what you are looking at is a video 00:16:04.24 of macrophages that have been given yeast to play with, and what you can see 00:16:11.11 is that the macrophages move towards the yeast and then engulf them. 00:16:16.00 So here is one that is moving directly towards it, and engulfs it. Here is another yeast 00:16:20.19 that is here, which will eventually be attacked and eaten by another macrophage, 00:16:24.10 shown right there, so in much the same way that Metchnikoff found 00:16:28.03 these invading microorganisms activated the macrophage, turned it on, 00:16:35.28 increased its capacity for migration, its capacity for cytotoxic killing, 00:16:41.14 and as shown here its capacity for actually killing and eating the invading microorganisms. 00:16:49.00 This is another movie showing that the process of phagocytosis of these yeast, 00:16:57.17 or in fact any other large particle that a macrophage encounters, 00:17:00.15 is intimately associated with the ability of the macrophage to polymerize actin 00:17:08.03 as a motile cytoskeletal component directly underneath where the particle binds 00:17:14.02 and turns on itself, growing out long pseudopods that engulf the particle, 00:17:20.12 capturing it and bringing it inside the cell, and eventually getting it to fuse with lysosomes, 00:17:25.09 again shown here. So what you are looking at in this particular video is a particle 00:17:31.02 that is entering a macrophage whose surface is stained green and shortly after internalization, 00:17:38.12 this green membrane turns red as a consequence of fusing with these 00:17:43.06 red endocytic vesicles that are found inside the macrophage. 00:17:47.02 Again, showing that very high tech components which Metchnikoff knew over 100 years 00:17:53.22 ago, which is that macrophages can detect, can bind, internalize, 00:17:58.24 and to deliver to lysosomes for purposes of degradation those incoming bacteria and 00:18:05.03 other sorts of pathogens that are found in the outside world against which we need protection. 00:18:09.02 So to summarize then, Metchnikoff really has to be credited with providing us 00:18:16.02 a great deal of not only conceptual understanding, but in many ways even 00:18:19.25 experimental detail that has defined for us the innate arm of the immune system. 00:18:25.03 The system that recognizes shared pathogen-derived components that are recognized 00:18:30.04 by Toll-like receptors as well as a variety of related receptors that 00:18:33.27 we won't have a chance to talk about. And it is powered to a very large extent by cells 00:18:40.16 which are referred to collectively as phagocytes and these include 00:18:44.19 the macrophage, which are the large cells that Metchnikoff first identified 00:18:48.02 as well as neutrophils, or granulocytes, polymorphonucleocytes, 00:18:53.15 which are actually smaller than macrophages, and indeed Metchnikoff had seen these too, 00:18:58.12 he referred to them as microphages. These mediated protection by 00:19:02.05 activating cytotoxic mechanisms, and as you have seen, quite graphically, 00:19:05.19 by actually physically eating, clearing, and degrading extracellular bacteria 00:19:10.24 and other types of microorganisms. Now, almost at exactly the same time 00:19:15.05 that Metchnikoff was working, elsewhere in Europe another 00:19:19.07 terrific scientist, Paul Ehrlich, was also hard at work 00:19:24.01 and you can tell from this image that Ehrlich was a terrific and great scientist 00:19:29.06 because he has one of the hallmarks of great scientists, which is an incredibly messy office. 00:19:34.00 So here he is sitting, reviewing some notes, and what Ehrlich's contribution was, 00:19:41.20 was to show for us how the other main arm of the immune system was organized and what it did. 00:19:49.03 This is called "adaptive immunity". What Ehrlich identified was that individuals, 00:19:56.10 whether it be the humans, it be the animals, that are immunized with various types of microorganisms 00:20:02.03 or bacterial derived toxins, make a response in the blood that is inherently protective, 00:20:10.06 and in fact Ehrlich coined the term "antibodies" to describe this response, 00:20:15.01 which has the ability, obviously, of protecting us against whatever it is that had entered 00:20:20.27 into our systems or into our bloodstreams.Antibodies see individual antigens 00:20:26.27 that are specific to individual pathogens types, so if you immunize with one type of bacteria, 00:20:33.02 you will not necessarily make antibodies that will recognize and protect a second individual 00:20:38.02 against another type of bacteria. So in this way, the system is fundamentally different 00:20:43.21 from the innate immune system. It's crafted in a molecular fashion 00:20:47.22 to the molecular entity that happens to be coming in. The way the cellular 00:20:54.00 mechanism, whereby the adaptive immune system works, is using not so much 00:20:58.11 macrophages, as we know now, but rather making use of antigen-specific 00:21:02.21 lymphocytes that indeed make these antibodies that recognize and kill the infected target cells 00:21:08.03 either separately from, or as you will see in a minute, in conjunction with the macrophages. 00:21:14.01 This is what an antibody molecule looks like in crystal structure. It consists of two major parts 00:21:21.04 a Fab fragment and a Fc fragment, both of which are linked together, 00:21:26.01 the molecular weight is about 150,000 Daltons and consists of four chains: 00:21:31.28 two light chains, shown in red, and two heavy chains shown in yellow and in blue. 00:21:37.01 The antigens themselves actually bind to the so called Fab regions of antibody 00:21:45.26 molecules, demonstrated in this diagram, out near the tips. So here you find a 00:21:50.26 great deal of sequence variation that allows the antibody to form complementary structures 00:21:56.02 allowing it to interact quite specifically with whatever antigen comes into an organism, 00:22:04.03 against which an antibody can be made. Now how antibodies work. 00:22:10.01 After recognizing their antigen, they are made, and as shown here they'll bind 00:22:16.03 to the antigen and this particular example that antigen is present on the surface of the bacterium, 00:22:21.20 and one of the simplest ways in which the antibody can work to kill 00:22:25.29 the bacterium is to recruit another set of proteins that are found in our plasma 00:22:31.00 called complement, which actually is a number of proteins, not just one. Complement is 00:22:37.00 recruited by antibodies, inserted into the bacterial membrane causing 00:22:41.23 the bacterium to lyse. Now in addition, these antibodies will work together 00:22:46.02 with element of the innate immune system, macrophages in particular, 00:22:49.26 but also neutrophils. Macrophages are probably more important 00:22:53.00 since macrophages actually have receptors for the Fc domains of these antibodies, 00:22:58.04 which have some of the same effects that Toll-receptors do, 00:23:02.01 in other words binding of antibody coated microorganisms to macrophages will trigger 00:23:06.24 the release of the same types of cytotoxic compounds that we've already discussed 00:23:11.01 and also trigger the ability of the macrophage to mediate phagocytosis 00:23:15.17 and intracellular killing and destruction of these antibody associated 00:23:21.09 or antibody coated microorganisms. So here you have this nice example of the innate system 00:23:26.12 working together with the adaptive immune system. Now antibodies are not made by 00:23:31.20 macrophages, although that was an idea that was originally thought of by 00:23:36.01 Metchnikoff and by Ehrlich who by the way shared the Nobel Prize 00:23:39.11 for their discoveries back in 1908, 100 years ago this year. Antibodies instead 00:23:45.16 are made by lymphocytes, a particular class of lymphocytes, as many of you know I'm sure, 00:23:49.27 called B lymphocytes. B lymphocytes exist in great number found throughout 00:23:57.22 a variety of lymphoid tissues in the body, and have a repertoire 00:24:04.03 of potential interactions, in other words, due to inherent and preexisting variability in 00:24:10.11 that small Fab region, or complementarity determining region 00:24:15.01 found in the Fab region of antibody molecules, incoming antigens can find or B cells 00:24:23.13 rather with the right specificity can find and actually bind to 00:24:27.00 antigens associated with bacteria, or antigens that are simply with soluble proteins, 00:24:32.09 and the simple act of binding of the antigen to the antibody, 00:24:37.03 which at this point in a B-cell's development is actually a membrane protein receptor 00:24:41.02 on the B-cell, is sufficient to simulate development of the B-cell and finally the 00:24:46.22 production of an amplification of more and more of these antibody molecules. 00:24:51.03 Now this is a video that shows you how the system works, when you get a sore throat, 00:24:57.27 which is indicated here in red. What that's again a manifestation of, 00:25:02.27 as Metchnikoff told us, a protective response, not a destructive response. 00:25:07.01 So here you see bacteria lining the surface of your throat following an infection 00:25:14.02 and these bacteria are putting off a variety of protein antigens that are associated with these bacteria, 00:25:22.03 so not only have you generated an innate immune response, 00:25:25.24 but now these bacterial specific antigens are being released and what happens to them is that they begin 00:25:33.13 to drain into the intercellular spaces, that one finds in all of your tissues 00:25:39.08 referred to as the lymph. The lymph drains into the lymphatics, 00:25:42.25 which then comes into these nodes, or specifically lymph nodes that monitor every 24 hours a day 00:25:52.04 what proteins, what antigens, what bacteria have entered into the lymph. 00:25:57.03 So here you see our bacterial protein entering into the lymph node, and what they 00:26:02.07 encounter here are the wide array of lymphocytes and other cells and here you can 00:26:08.01 see them in a higher magnification and as our bacterial derived proteins come in, 00:26:12.26 they filter through these millions or billions of cells as you can imagine, 00:26:20.22 and cells that are B-cells that may detect a component of that antigen will bind to it, 00:26:29.23 based on these antibody molecules or via these antibody molecules 00:26:33.07 that are intercalated as membrane proteins on the surface of these early B-cells shown here 00:26:38.19 clicking on, and this will then hit onto another B-cell receptor, and a third 00:26:45.05 and a fourth, until enough of them come together to actually generate a signal 00:26:50.00 to the B-cell, which says that I have now detected the antigen I was born to detect 00:26:57.10 and as a result, it is now time to start making more of myself. So these B-cells 00:27:03.23 become very active and begin to develop and divide. 00:27:10.29 The affinity that they have for the individual antigens, in essence, increases as B-cells with increasingly 00:27:20.24 large affinities, increasingly great affinities for antigens, 00:27:24.07 compete with each other to bind more and more. So here you see a clone of B-cells that gives rise 00:27:31.07 to a larger number of itself and then as you can see here, in this representation, 00:27:39.13 antibody molecules begin to be released into the extracellular space 00:27:44.19 and here is just an indication of the secretory event that takes place very similar 00:27:51.10 to that which is described in another of the iBioseminar science series given by Randy Schekman. 00:27:59.01 Now here the antibody molecules at this stage in development are actually pentamers 00:28:04.00 coming back homing to the bacteria, coating them with antibody molecules, 00:28:09.11 recruiting complement, and, as shown here also recruiting macrophages, 00:28:13.28 which again, just as Metchnikoff has already shown us, will come and eat those 00:28:18.08 bacteria that are coated with antibody taking them up by phagocytosis, 00:28:21.27 killing them and degrading them. Now it turns out that things are not that simple, 00:28:29.28 of course. What I've just shown you in that video isn't in every sense correct, 00:28:37.20 but it turns out that B lymphocytes make the best antibodies when they are helped 00:28:41.02 by another type of lymphocyte, referred to T, or thymus-derived, lymphocytes. 00:28:46.18 And the way that works is that here you can see an extracellular antigen coming, 00:28:50.28 binding to the surface antibody molecule of B-cells and then it turns out that 00:28:56.09 the antigen is internalized by endocytosis into the B-cell, and a small fragment 00:29:01.23 of this antigen is cleaved off and is bound to a molecule referred to as a major 00:29:07.02 histocompatability class II molecule, or MHC class II molecule. This complex of the 00:29:13.17 peptide plus the MHC class II molecule is then recognized by a preexisting T-cell, 00:29:19.26 which has a different type of antigen receptor referred to as the T-cell receptor, 00:29:24.11 again which has a great deal of diversity built into it as a consequence 00:29:30.24 of the development of T-cells in the early development of the immune system 00:29:37.09 and what the T-cell then does, is to secrete again our friends the cytokines, 00:29:41.06 in this case a class of cytokines, which helps the B-cell develop, helps them 00:29:47.28 divide even more, and helps them create antibodies with every increasing affinity. 00:29:54.22 Now the way the T-cell system works, is illustrated in a little bit more detail here. 00:30:00.08 T-cells come in two general flavors, and we'll come back to this later, 00:30:04.28 CD4 T-cells, as well as CD-8 T-cells. We've been talking about CD-4 T-cells, 00:30:09.27 which recognize bacterial or other derived peptides bound to these so-called MHC class II molecules, 00:30:16.23 and they do this by the virtue of having these T-cell receptors, which again have 00:30:22.14 their own specificities associated with them, such that by random chance, 00:30:28.08 the appropriate T-cell receptor is made for this particular complex of peptide and MHC class II. 00:30:36.00 Diversity in the T-cell receptors, rather than being so much generated by mutation, 00:30:41.00 which is what occurs in the antibody in the B-cell system, occurs by 00:30:45.11 rearrangement of the T-cell receptor gene, again rearrangements that cause a greater number of 00:30:53.15 potential combinatorial sequences that can recognize a wide array 00:30:57.27 of these peptide MHC complexes. So that said CD4 T-cell sees MHC class II molecules to a large 00:31:06.04 extent because it makes the second membrane protein called CD4, 00:31:09.17 which also recognizes the MHC class II molecules, but recognizes an invariable portion 00:31:16.01 of those molecules. CD8 T-cells express exactly the same type of T-cell receptors, 00:31:22.02 but they're targeted to a different type of MHC molecule, in this case a MHC class I molecule 00:31:28.01 and the reason this particular targeting takes place is because the CD8 membrane 00:31:33.01 protein found on the CD8 T-cell, recognizes the class I molecule and not the class II molecule. 00:31:41.01 So very complicated and this is obviously a simplistic view of how this works, 00:31:47.10 but at its most elemental form, this is how T-cells cooperate with B-cells 00:31:52.27 and in fact with a variety of other cells as well. Now CD8 restricted T-cells 00:32:00.28 or those T-cells that see antigens bound to class I in general don't see antigens 00:32:07.07 that are coming in from the outside. In much the same way, 00:32:11.11 or at least I should say, analogous to what happens in the case of Toll-like receptors 00:32:16.06 that are expressed either on the surface of a cell or inside, the CD4/CD8 system, or class I and class II 00:32:23.08 MHC systems are adapted to incoming pathogens that reveal themselves 00:32:28.04 either on the outside world or on the inside world. The CD8 system and the class I system 00:32:33.28 take care of those things that are going on in the inside world. 00:32:37.01 So when viruses infect our cells, those viruses almost invariably work by binding to 00:32:43.14 receptors on the surface of the cell and fusing, or entering, the cell 00:32:48.25 either at the level of the plasma membrane or following entry into endocytic vesicles, 00:32:53.22 and then breaking out into the cytoplasm. That type of endogenous pathogen, 00:33:00.16 because you can now refer to it as endogenous because it's found inside the cell, 00:33:06.15 that type of pathogen is treated by the immune system to generate peptides 00:33:11.29 that are then loaded onto class I as opposed to class II molecules. Now the way 00:33:17.13 these T-cells work, again, is somewhat analogous to what one finds in the 00:33:22.09 innate immune system, except that it is all very specific and very antigen driven. 00:33:26.01 So here you are looking at a cytotoxic CD8 positive (+) T-cell, 00:33:30.15 this is work of Julian Griffin at the University of Cambridge in England, and what Julian 00:33:36.18 has noticed as well as others over the years is that these cytotoxic CD8+ T-cells, when they recognize 00:33:44.07 a virus-infected target in fact polarize all of their lysosomes, 00:33:50.08 which actually now are differentiated to contain a wide variety of cytotoxic compounds, 00:33:55.27 towards the infected T-cell. These granules line up at the interface, or synapse, 00:34:02.19 between the T-cell and the target, and are eventually released, killing the target. 00:34:08.01 This is what you are looking at here in an electron micrograph also from Julian's work. 00:34:12.12 Here you can see these cytotoxic T-cell, or CD8 T-cell, or CTL granules 00:34:18.01 in the region of the cytoplasm that is very very close to the infected target cell. 00:34:24.02 These granules contain lysosomal enzymes, various lytic enzymes, ligands 00:34:30.04 such as Fas and receptors that will induce apoptosis or cell death in the target, 00:34:36.01 and also perforating molecules called perforins among others that will punch holes 00:34:43.10 in the target cell. All leading to its rapid death. So in other words, the virus has 00:34:48.15 infected this cell, think its safe by existing within the cell and then the cytotoxic 00:34:52.16 T-cell comes along and recognizes a peptide derived from the virus, 00:34:56.17 releasing the granules at the site of the infected cell, thereby killing the cell. 00:35:02.02 This is just the diagram showing how this process works. In fact over the last several 00:35:07.16 years, as a consequence of interrogating some important mutations 00:35:11.13 that lead to defective CD8 T-cell responses, which Julian and others have shown, 00:35:17.00 is that these granules actually have to physically polarize by moving on microtubule tracks 00:35:22.19 from diverse places within the cytoplasm to this interface, this synapse, 00:35:27.14 between the T-cell and its target. In the absence of genes that are required 00:35:33.10 for the translocation of these granules to this point in the cell, Rab proteins, 00:35:40.02 microtubule binding proteins, etc. these T-cells are incapable of docking 00:35:47.11 with or incapable of fusing with the plasma membrane of the T-cell close to the target. 00:35:54.02 Again, emphasizing one critical element of where membrane traffic 00:35:58.25 plays a major role in allowing the immune system to conduct its job. 00:36:03.00 Now how does this all work, we've discussed already a bit how TLR's activate 00:36:11.16 cells such as macrophages in the innate immune system. T-cells become activated 00:36:16.20 in two very important ways. The T-cell receptor itself is a signaling molecule, 00:36:23.01 recruiting tyrosine kinases to its cytoplasmic domain after it recognizes its ligand, 00:36:29.22 which as we've been discussing is a complex of peptides plus MHC molecules. 00:36:34.00 But also a variety of other interactions take place so-called co-stimulatory receptors 00:36:41.02 which are other receptors that are present on the surface of T-cells, 00:36:45.02 recognize a variety of other molecules that can be found on the surface of infected cells 00:36:49.28 or on B-cells or on other cells of the immune system, that send further signals 00:36:54.24 to the T-cell, enabling it to more effectively do its job and also amplify its own 00:37:01.03 numbers in a way that is selective and antigen driven, 00:37:05.12 quite analogous to what happens in the case of B-cells. Further, selected cells in the immune system 00:37:12.05 which are referred to as antigen-presenting cells or APCs, 00:37:15.22 which we'll come back to in a later lecture are capable of secreting their own sets of cytokines, 00:37:22.13 which have the effect of further accentuating and enhancing T-cell responses. 00:37:27.01 So here we have two aspects, two critical aspects of the immune system. 00:37:32.04 Innate immunity, which is characterized by direct cellular responses 00:37:37.09 to pathogens by detecting shared microbial patterns that is not antigen specific, 00:37:43.09 working together hand in glove with Ehrlich's observation of the adaptive immune response, 00:37:50.20 which generates specific antibodies to variable antigens, 00:37:54.01 and occurs as a consequence of an antigen stimulation of T-cells and B-cells. These two things, 00:38:01.09 as I've already intimated to you are connected, but they're connected in even a more intimate 00:38:06.01 fashion, which we'll turn to in the next lecture, provided by a recently identified 00:38:11.10 cell type referred to as the dendritic cell, which really we now understands 00:38:15.16 provides the long sought after missing link between the innate and adaptive immune system.