Session 3: Bridging Innate & Adaptive Immunity
Transcript of Part 3: The Role of Toll-Like Receptors in the Control of Adaptive Immunity
00:00:14;28 Hello. 00:00:15;28 My name is Ruslan Medzhitov. 00:00:16;28 I'm a professor at Yale University School of Medicine and an Investigator of the 00:00:21;10 Howard Hughes Medical Institute. 00:00:23;11 And I will discuss, today, our work on characterizing toll-like receptors and their role in control 00:00:32;21 of adaptive immunity. 00:00:36;26 My story started in this building. 00:00:40;05 This is a Library for Natural Sciences of the Russian Academy of Sciences in Moscow 00:00:47;18 when I was a graduate student there from 1990 to '93. 00:00:53;05 At that time, the Soviet Union just collapsed and there was a profound economic crisis, 00:00:59;05 so there were no reagents to... to be able to do experimental work, and there were 00:01:04;14 not even periodicals available to read. 00:01:07;23 And this was the only library that still had a subscription to Nature, Science, Cell, 00:01:14;03 and other journals. 00:01:15;22 And so I would go there every morning and spend all day in this library on the second floor, and... 00:01:22;09 just trying to read the literature, since I wasn't really able to do much in the lab 00:01:28;05 at the time. 00:01:30;21 And during one of these trips to the library, I completely randomly ran into a paper written 00:01:37;22 by the late Charles Janeway that changed the direction of my career and life. 00:01:46;22 And this was a paper in... that appeared in the Proceedings of the Cold Spring Harbor Laboratory 00:01:53;20 from 1989. 00:01:57;02 And this is the paper. 00:01:59;02 It was entitled "Approaching the Asymptote? Evolution and Revolution in Immunology". 00:02:03;24 And this is Charlie Janeway, who unfortunately passed away in 2003. 00:02:09;22 But what he described in that paper was a new perspective, a new theory to think about 00:02:16;11 the immune system that, when I read it, I thought that this is... makes so much sense 00:02:23;20 and this is going to completely revolutionize our understanding of immunity. 00:02:29;20 And I got completely enchanted with this idea and this logical framework that Charlie proposed there. 00:02:39;19 And that was the time when I became interested in immunology. 00:02:46;14 And to put this into historical context, what the idea was about is how the immune system 00:02:54;21 knows when to be activated and when to respond to a challenge. 00:03:02;25 And what was thought at the time is that the immune system -- T cells and B cells -- 00:03:11;16 only react to foreign antigens but not to self-antigens, for example, 00:03:16;01 antigens that come from our own tissues. 00:03:19;02 And so on the top panel, you'll see here a situation where self-antigens that may be 00:03:26;27 presented by antigen-presenting cells -- so, APCs -- to T cells, but there would be 00:03:32;24 no response. 00:03:34;08 Even though T cells can see the antigen, they should not be responding. 00:03:40;13 And at the lower part, you see the situation where there is an infection. 00:03:44;18 And when APCs detect pathogens, then there should be response. 00:03:48;24 As we all know, we respond to pathogens. 00:03:52;20 But it wasn't clear what makes the difference. 00:03:54;18 And again, one idea was that the immune system distinguished between self-antigens and non-self-antigens. 00:04:01;13 So, pathogens would be non-self, the immune system will react; self-antigens are self, 00:04:07;26 and the immune system would not react. 00:04:10;22 But it... there was also known at the time that for T cells to become activated 00:04:18;17 they require two signals. 00:04:20;05 And one signal is the antigen itself, which is presented by MHC molecules, 00:04:26;18 major histocompatibility complex molecules. 00:04:29;15 So, it's a complex between MHC and antigen that is seen by T cells. 00:04:35;19 And what became known at the time, from the work Ron Schwarz and Mark Jenkins at the NIH, 00:04:42;22 is that when T cells see only signal 1, there is no response that is generated, 00:04:49;27 but when they see two signals -- the second signal also coming from antigen-presenting cells -- 00:04:54;21 then there would be a response. 00:04:57;11 And the identity of that second signal was subsequently characterized, and it's 00:05:03;02 known as molecules called B7-1/B7-2, or CD80/CD86. 00:05:09;27 So, that was the state of knowledge, and it was... many different views about what controls 00:05:18;12 activation of the adaptive immune response, of T cells and B cells. 00:05:22;25 And there were different sorts of schools of thought and... and it was still very much 00:05:28;10 unclear, for somebody who's coming from the outside of the field, what was going on and 00:05:34;22 how the system worked. 00:05:36;26 And that's what was changed with this article by Charlie Janeway, where he proposed that, 00:05:43;02 in fact, what might be happening is that in addition to T cell receptors and immunoglobulin receptors 00:05:48;19 there is a whole different set of receptors, that are shown here in blue, 00:05:56;01 that Charlie hypothesized would be involved in direct detection of pathogens, and that 00:06:04;08 these receptors would then control expression of the signal 2. 00:06:09;09 And in this manner, when antigen-presenting cells encounter self-antigens or any other 00:06:15;02 antigens that are not infectious, there would be just signal 1 and there will be no response. 00:06:21;04 But when they encounter pathogens, then there would be this detection, by these hypothetical receptors, 00:06:26;08 of common microbial structures and that would lead to the induction of signal 2, 00:06:33;13 and that would result in the immune response. 00:06:36;00 And that was a very profound insight and it was a very beautiful idea that made so much sense. 00:06:42;17 And... and Charlie further proposed that these receptors, that he called pattern-recognition receptors, 00:06:48;18 because they detect conserved patterns found in pathogens... things like lipopolysaccharides, 00:06:54;16 peptidoglycans, and so forth... he proposed that these receptors are evolutionary ancient 00:07:00;09 and they are involved in immunity in invertebrates as well as in vertebrates, where they control 00:07:08;04 the innate arm of the immune system. 00:07:11;05 But in addition, in vertebrates they acquire a second function where they control activation 00:07:15;15 of the adaptive immune system. 00:07:17;03 And that was a really revolutionary proposition. 00:07:22;15 And the only problem was that the receptors that Charlie hypothesized should exist were 00:07:29;19 not known at the time. 00:07:31;14 And... and so, after reading Charlie's paper, I started communicating with him, 00:07:36;23 initially through email, and eventually I was fortunate enough to be able to join his lab as a... 00:07:42;22 as a postdoc in 1994. 00:07:45;27 And then my focus of my research was on trying to identify such receptors that can 00:07:52;17 control expression of signal 2 to induce... to control activation of adaptive immunity. 00:07:58;28 And at that time, there was really little known about how microbes can be recognized directly. 00:08:05;15 There were a few proteins that were known to bind to microbial cell walls. 00:08:08;25 One of them was a protein called mannan-binding lectin from the complement system. 00:08:14;02 Another is CD14 molecule, which is a GPI-anchored protein on macrophages. 00:08:19;20 And there were a few proteins known in invertebrates that were involved in recognition of LPS. 00:08:27;21 But other than that, it was really not clear what these receptors are supposed to 00:08:31;17 look like and how they... how to identify them. 00:08:37;06 And a couple of things that changed, that played a role in the development of the story, 00:08:43;21 was that... one was that in 1994, the year when I joined Charlie's lab, David Baltimore 00:08:48;20 came to Yale to give a talk in our department, and at that time he just... his lab just started 00:08:55;20 characterizing the first knockouts for NF-kappaB, which is a critical transcription factor 00:09:00;26 involved in inflammation. 00:09:03;12 And there he talked about the phenotype of the first NF-kappaB knockouts, which had 00:09:08;16 both defects in innate and in adaptive immune response. 00:09:12;26 And I remember, after David's talk, Charlie and I were talking in the hallway, 00:09:19;15 and we both thought that whatever it is that we're looking for, it's... whatever these receptors are, 00:09:25;10 they probably work by activating NF-kappaB. 00:09:30;22 And then we thought, okay, so one criteria we should use is that they... they are likely 00:09:34;15 to be NF-kappaB activators. 00:09:37;07 And at that time, the only two receptor families known to activate NF-kappaB where the TNF family 00:09:43;22 and IL-1 receptor family. 00:09:47;01 And IL-1 receptor is the one that we got particularly interested in because of this remarkable conservation 00:09:58;05 in IL-1 receptor signaling portion with a receptor that at the time was already known 00:10:06;08 to exist in Drosophila. 00:10:09;08 And in Drosophila that receptor is called Toll. 00:10:11;20 That receptor was identified and cloned in Kathryn Anderson's lab by Carl Hashimoto. 00:10:19;00 At the time, they were at Berkeley. 00:10:21;11 And the signaling pathway was elucidated by several investigators, including Steve Wasserman 00:10:28;03 and Mike Levine, Kathryn Anderson and others. 00:10:32;10 And it was known that this pathway operates in fly development. 00:10:37;15 But what was interesting for us is that it worked through NF-kappaB in flies, and the 00:10:42;15 related receptor, IL-1 receptor, worked through NF-kappaB in mammals. 00:10:47;17 And what they shared is the cytoplasmic portion, shown here in blue, whereas their ectodomains, 00:10:54;13 involved in ligand recognition, were unrelated. 00:10:59;06 And because of this conservation with flies, and because IL-1 receptor is involved in inflammation, 00:11:09;10 we thought that perhaps the receptor that we are looking for would be... would have 00:11:15;03 this C-terminal signaling domain, and the ectodomain perhaps would be something that 00:11:21;06 can recognize microbes. 00:11:22;26 And the only structures that were known at the time to recognize microbial components 00:11:28;20 were C-type lectins. 00:11:30;22 So I thought that maybe there is another version of this receptor that has cytoplasmic signaling domain 00:11:35;15 from Toll and IL-1 receptor, and the ectodomain would be from C-type lectin. 00:11:40;08 And... and that was my strategy to try to identify it. 00:11:44;28 And... that... what I took advantage of is that, after many, many failed attempts 00:11:52;01 to do it through various types of approaches... degenerate screening or by hybridization or 00:12:00;22 through PCR... took advantage of the fact that, at the time, new types of genomic sequences 00:12:07;19 started to be become available -- these so-called expressed sequence tags -- that were 00:12:13;10 just short sequences that were generated randomly in multiple tissues. 00:12:17;22 And using bioinformatics approaches and using conserved consensus sequences for the cytoplasmic domain 00:12:23;26 for Toll and IL-1 receptor, as well as for C-type lectins, I started searching 00:12:29;04 these databases and found several clones that corresponded either to C-type lectins or to 00:12:35;12 this cytoplasmic domain. 00:12:37;21 And then I pursued both of them. 00:12:40;02 And the one that corresponded to C-type lectin domain, I found that it was most similar to 00:12:46;15 Drosophila Toll. 00:12:48;22 And this was in some... starting in January, February in 1996. 00:12:55;27 And the... the gel shown on the left here was one of the first clones that I got, 00:13:06;24 which was... later would turn out to be a human homologue of Toll. 00:13:11;15 And the gel on the right in this bottom band... 00:13:15;17 I still remember that band because this was one of those happy moments in the lab 00:13:21;05 when I got this portion of the receptor, which was particularly difficult to clone. 00:13:27;09 And then by doing standard library screening through hybridization, we pulled out a full-length 00:13:34;24 of the cloning... coding Toll receptor followed by 5' RACE technique, which was at the time 00:13:44;21 popular in gene cloning, and eventually got the receptor. 00:13:50;21 And... by about May of '96. 00:13:54;18 And then another event that happened that... in the... in the summer of '96, we had a meeting 00:14:00;21 in Charlie's summer house in Annisquam, near Boston, where several other investigators 00:14:06;09 participated that shared Human Frontier Grant, including Alan Ezekowitz, Fotis Kafatos, 00:14:13;00 Jules Hoffmann, and Charlie. 00:14:15;07 And... and Jules Hoffmann then presented work of Bruno Lemaitre from his lab about 00:14:20;13 genetic work on Drosophila Toll pathway, where they found it was involved in immunity. 00:14:25;01 So, that was... it was further confirmation for our expectation that this receptor 00:14:34;04 might be involved in immunity. 00:14:36;08 And so, then I characterized this receptor in terms of its ability to control NF-kappaB signaling, 00:14:46;22 shown here on the right. 00:14:49;09 And more importantly for us at the time, for its ability to induce inflammatory cytokines. 00:14:54;20 And the most important for us, the holy grail for us, whether this receptor can induce 00:14:59;02 the second signal, or costimulatory molecule, a channel known as B7. 00:15:04;11 And that was the... the probably... one of those lucky moments in the lab when I saw 00:15:10;01 that Toll receptor, which I rendered constitutively active because the ligand was not yet known 00:15:16;24 at the time, and transfected in this monocyte cell line. 00:15:22;04 And when I saw that it can induce expression of this gene, that was the moment when 00:15:27;09 we thought, okay, this... this whole hypothesis, now, we can connect from the beginning to 00:15:32;03 the end. 00:15:33;11 And I even called Charlie... this was late in the evening, I even called Charlie and 00:15:37;03 told him that that human Toll can induce B7 expression. 00:15:40;15 That was... we probably would be the only two people in the world who would be 00:15:44;14 excited about that, but nevertheless others also appreciated it. 00:15:50;03 And so we published this paper in '97, where... which is an extremely simple paper showing 00:15:55;08 just cloning, and showing these couple of functional assays. 00:15:59;28 And... and then, subsequently, in the same year, Ben Lewin, who was the editor of Cell at the time, 00:16:07;26 asked Charlie to write a mini-review about innate immunity. 00:16:12;14 And so in that mini-review we discussed questions about evolution of innate and adaptive immunity, 00:16:23;05 and put this schematic together to... to show the parallels and differences between 00:16:28;24 Drosophila Toll and mammalian Toll pathways, where we suggested that recognition of pathogen-associated 00:16:37;13 molecular patterns -- things like LPS and teichoic acids and so on -- is detected by Tolls, 00:16:44;02 either directly or through some intermediary, as is the case in Drosophila. 00:16:49;05 And that leads to induction of NF-kappaB and various innate immune response and genes 00:16:54;07 genes controlling adaptive immunity. 00:16:57;04 And subsequent work indeed demonstrated that the Toll that I worked on is part of a bigger family. 00:17:06;23 There are about a dozen different Toll receptors in mammalian species. 00:17:13;04 And it's now known as TLR4 -- Toll-like receptor 4 -- and it's a receptor for LPS, and the 00:17:18;06 receptor part that detects LPS... actually, a protein called MG2 that complexes with Toll. 00:17:25;09 And subsequent work with... by many investigators elucidated specificities of different 00:17:32;24 Toll receptors. 00:17:33;24 And Shizuo Akira from Osaka University, in particular, played a major role in this 00:17:39;11 set of investigations. 00:17:42;02 And what we know now is that... if we... our main interest was not so much in microbial 00:17:50;15 specificity of receptors but in this idea that microbial receptors can control activation 00:17:56;02 of adaptive immunity. 00:17:58;08 And this is what is schematically illustrated here. 00:18:00;21 So, we have a dendritic cell, which is a cell type that normally activates T cells. 00:18:07;13 And when dendritic cell encounters pathogens, what's shown on the left side, here, 00:18:11;24 there is an endocytic receptor that will take up pathogen, take it into lysosomes, and then 00:18:16;17 proteins will be cut into peptides and loaded into MHC molecules and presented to 00:18:23;19 T cell receptors. 00:18:25;22 But that information by itself is not sufficient for T cells to know whether they should become 00:18:30;12 activated or not, because this peptide can come from pathogens or it can come from innocuous 00:18:37;26 food antigens or it could be even a self-antigen. 00:18:40;22 So, T cells have no way... 00:18:42;20 no way of knowing what the origin of the antigen is. 00:18:46;22 And the reason for that is because T cell receptors are generated at random. 00:18:50;13 And each T cell has one single specificity, but they are random, so it doesn't know 00:18:56;12 what it's specific for. 00:18:58;16 So therefore, there is a need for another signal, so-called signal 2, which will provide 00:19:04;09 information about the origin of the antigen that T cell is specific for. 00:19:09;15 And that is provided by this detection of microbial structures by Toll receptors 00:19:14;25 -- and now we know, several other families of pattern-recognition receptors -- 00:19:18;25 that detect those structures and induce expression of costimulatory molecules. 00:19:23;15 And now a T cell that has specificity for peptide derived from pathogen will also sec... 00:19:28;28 have a confirmation signal from a costimulatory molecule -- CD80/CD86, also known as B7-1/B7-2 -- 00:19:38;04 and then the T cell will become activated. 00:19:40;16 And in addition, Toll receptors and other pattern-recognition receptors will 00:19:45;03 induce production of cytokines such as IL-12 that will tell T cells what kind of effector response 00:19:51;11 to generate. 00:19:52;21 And that is now, of course, a well-accepted view of how immune response is activated. 00:20:03;20 And... and it's a... it's very satisfying to see this confirmation of the proposal 00:20:11;22 by Charlie Janeway, which was at the time largely ignored. 00:20:16;03 And that now it's become... it's a... it's a textbook knowledge in the... of the immune system, 00:20:22;25 that that's how activation of the adaptive immune system is controlled. 00:20:27;15 And Toll receptor was one of the first receptors to be demonstrated to... to be involved 00:20:34;17 in this process. 00:20:35;17 So, that's the story of this discovery. 00:20:38;24 And thank you for listening.