It’s crucial that the immune system is activated only when needed. Therefore, the body has evolved a series of steps that prevent unwanted activation. This session provides an overview of regulation of the acquired immune system and the crosstalk that happens between innate and adaptive immunity. It explains how, upon infection, a subset of innate immune cells known as antigen-presenting cells (APCs) are primed to present antigen via their MHC-II molecules to helper-T cells. Activated helper T cells then modulate the acquired and humoral immune responses. On the other hand, virally infected cells present antigens via their MHC-I molecules to activate cytotoxic-T cells, which release cytokines to kill infected cells. In addition to the binding of peptide-MHC complexes, T cells require additional signals for full activation. These signals arise from the activation of antigen presenting cells via Pattern-Recognition Receptor (e.g. Toll-Like Receptor) binding. Together, these systems ensure appropriate activation of the immune system.
Review Questions for Session 3: Bridging Innate & Adaptive Immunity (Educators only)
- Duration: 00:25:28
00:00:01.00 Hi, Ira Mellman again from Genentech. I'd like to continue our discussion
00:00:05.24 of the cell biology of the immune response, this time turning to specifically
00:00:09.22 the problem of antigen presentation and the role of dendritic cells
00:00:13.17 in linking the two aspects of the immune response
00:00:16.06 that we've already discussed, innate immunity and adaptive immunity.
00:00:19.00 Innate immunity having been discovered by Metchnikoff
00:00:21.29 and adaptive immunity by Ehrlich all about 100 years ago.
00:00:25.00 Now in order for T-cells to do their job helping B-cells respond by
00:00:31.15 making antibodies and also by helping to kill virus infected cells and other types of pathogens,
00:00:37.13 they themselves require a significant amount of help, which is provided
00:00:43.03 by the so-called antigen presenting cells or APCs. Now recognition of T-cell
00:00:49.17 receptors of their targets does not necessarily lead to cell killing.
00:00:55.00 In fact this is an interaction that has to take place in order to generate a sufficient number
00:01:00.01 of T-cells to mediate the immune response in the first place. So the signal
00:01:04.01 that is sent by the T-cell receptor to the T-cell is a consequence
00:01:08.22 of seeing its ligand serves to activate the cell to not only to kill but also to differentiate
00:01:16.21 and divide, again depending on the type of T-cell you happen to be.
00:01:20.00 These types of signals together with other types of signals provided by other types of
00:01:25.07 membrane proteins referred to as co-stimulatory molecules,
00:01:28.21 are provided by a class of cells referred to as APCs or antigen-presenting cells.
00:01:35.00 Now as we reviewed in the last lecture, T-cell receptors see two different types
00:01:40.29 of peptides, peptides bound either to MHC class II molecules or peptides bound
00:01:46.09 to MHC class I molecules. CD8 T-cells recognizing the class I molecules,
00:01:51.16 CD4 T-cells recognizing the class II molecules. This is a structural
00:01:56.20 representation of how the recognition of the peptide MHC complexes by T-cell receptors actually occurs.
00:02:04.00 So here in the upper level you see the T-cell receptor itself,
00:02:07.07 the two major antigen recognition elements of it, the alpha chain and the beta chain,
00:02:12.00 which form a complex that is specifically adapted to recognize that a particular peptide
00:02:17.03 bound to a particular MHC molecule so here you can see the peptide,
00:02:21.04 which usually ranges in 9 to 10 or more of amino acids in length,
00:02:26.21 nestled within a very specific peptide binding cleft found in this case in a MHC class I molecule
00:02:34.00 that is expressed by the antigen presenting cell expressing in turn the antigen from
00:02:39.18 which this particular peptide is derived. In order for this system to work,
00:02:43.23 it has to be amplified, it has to be selected, the T-cells have to be amplified
00:02:48.11 and have to be selected and have to be affinity matured in such a way
00:02:51.28 that they can detect their peptides with higher and higher affinity
00:02:55.14 continuously again sculpting these T-cell receptors. Now producing
00:03:00.23 the peptide MHC complex, which is obviously key to this entire process
00:03:04.00 is a job of the antigen presenting cell. Now how does the antigen presenting cell do this?
00:03:10.27 We've already talked about the fact that there is both a class I and a class II
00:03:15.28 dependent pathway or restricted pathway of antigen presentation.
00:03:21.00 The class II pathway is predominantly adapted to presenting those molecules
00:03:26.27 derived from extracellular pathogens such as extracellular bacteria or proteins,
00:03:33.11 toxins, whatever are released from bacteria. So in those cases what happens
00:03:38.16 as we already discussed in the case of B-cells, the antigens bind to the surface
00:03:42.26 of the cell, the antigen presenting cells are taken up and deposited
00:03:47.16 within endocytic vesicles within the cytoplasm of the APC.
00:03:53.11 Here the antigens are unfolded, unwound, and eventually degraded into peptides
00:03:59.04 that are loaded also in endocytic vesicles onto the class II molecules themselves.
00:04:03.00 Now how the class II molecules get there is a rather remarkable story
00:04:08.01 and a remarkable piece of cell biology and membrane traffic in its own right.
00:04:11.00 Class II molecules are membrane proteins and like almost all other membrane proteins
00:04:15.27 begin their lives at the level of the rough endoplasmic reticulum,
00:04:19.05 where they are synthesized and inserted across the ER membrane
00:04:23.00 and assembled together with a chaperone called the invariant chain in the ER
00:04:28.04 that then after assembly takes place gets transported from
00:04:32.06 the endoplasmic reticulum into the cis-golgi, through the golgi stack, emerging at the
00:04:37.13 trans-side of the golgi complex but unlike proteins that are destined for
00:04:44.10 secretion or destined to be inserted into the plasma membrane,
00:04:48.02 MHC molecules are diverted from this constitutive secretory pathway and instead
00:04:52.28 are taken into the endocytic compartment where the invariant chain is removed
00:04:57.15 and we'll come back to that in a moment, and the class II molecule rendered accessible
00:05:01.11 to the peptides that can be derived from the incoming antigen.
00:05:05.17 Following binding of the peptide to the class II molecule, this complex is taken to the cell surface
00:05:11.15 where it can now be recognized by CD4+ T-cells.
00:05:16.00 Now the class I pathway as we've discussed services predominately those antigens
00:05:21.24 that are self-synthesized by a given cell. This of course can include
00:05:28.01 any membrane protein or cytosolic protein that is synthesized as a
00:05:33.01 consequence of the viral infection. So in the case of cytosolic proteins, these proteins are made,
00:05:39.06 ubiquitinated, degraded in the cytosol by the proteosome and peptides
00:05:43.25 that are produced by the proteosome were transported into the ER, where they are then bound
00:05:48.07 to class I molecules that are again, transported from the ER to the golgi
00:05:52.05 but now rather than going to the endocytic compartment,
00:05:55.00 instead the class I molecules go directly to the cell surface. Now all antigen presenting cells
00:06:00.20 are not created equal. There are amateurs and there are professionals.
00:06:04.00 Amateurs can make the class I response in most cases, because MHC class I molecules
00:06:10.23 are expressed by virtually all nucleated cells in the body so therefore virtually
00:06:15.09 all nucleated cells are protected by the immune system against infection
00:06:19.22 by viruses, which is a good thing. The MHC class II system on the other hand
00:06:24.20 is synthesized and expressed only by a relatively restricted number of cells
00:06:29.14 in the body. In most cases our cells that are specialized cells in the immune system
00:06:35.05 B-cells, macrophages, and most importantly these dendritic cells
00:06:39.23 that we mentioned earlier. Now dendritic cells are really special
00:06:43.24 and in fact are the professionals professional. They are the Tiger Woods
00:06:48.05 of the antigen presenting cell universe. Why? Because they are by far the most efficient.
00:06:54.14 They can capture almost meaningless and non-existent amounts of antigens
00:06:58.22 and turn those antigens into small peptides that can stimulate T-cell responses. They have
00:07:05.28 the rather unique capacity to be able to capture antigen from
00:07:10.17 wherever in the body antigen is first introduced,
00:07:14.12 be it in the skin, in the lung, in the intestine wherever the antigen is captured
00:07:19.07 and then its not simply left to passive transport through the lymphatics to go back to the lymph nodes,
00:07:25.05 but rather these cells are adapted to hone in on the lymphatics and make a bee line directly
00:07:30.10 into the lymph nodes where the dendritic cells can find a huge accumulation
00:07:35.29 of both T-lymphocytes and B-lymphocytes and help their stimulatory events take place.
00:07:42.12 Perhaps most importantly, dendritic cells are the only cells in the immune system,
00:07:47.13 the only antigen presenting cell that can actually initiate the antigen specific
00:07:51.10 immune response. In other words, prior to the advent of the first of an antigens kind
00:08:00.15 coming in before you had your very first infection with influenza
00:08:05.12 you may have T-cells that are capable of responding to influenza virus,
00:08:09.00 but they are naÃ¯ve, they don't really know what to do. Only the dendritic cell can wake
00:08:14.16 them up. If you delete dendritic cells from a mouse using a variety
00:08:20.07 of genetic knockouts, you find those mice are almost completely incapable
00:08:24.12 of mounting antigen specific immune responses. Why? Because only
00:08:29.08 the dendritic cell can present an antigen at a sufficient level of efficiency and with
00:08:36.27 a sufficient amount of stimulation provided to the T-cell in order to wake the T-cell up
00:08:41.29 and get the T-cell response going. Now the other side of the coin here though
00:08:48.02 is the issue of tolerance in the sense that remember one of the key abilities
00:08:54.03 of the immune system is to be able to mount virulent cytotoxic responses
00:09:00.03 protective responses to invading pathogens, but somehow minimize
00:09:05.01 if not avoid entirely destructive components that are directly against our self antigens
00:09:12.00 in other words our own tissues and selves. Dendritic cells play also a key role
00:09:17.09 in ensuring that our immune system maintains tolerance to self antigens
00:09:22.21 and we'll come back at the very end to discuss some of the more recent ideas
00:09:27.21 on how we think that takes place. Now probably though the most important
00:09:33.02 key conceptual element of how the dendritic cell system works
00:09:37.16 and why dendritic cells play such a role that is so important in linking the innate and
00:09:42.27 adaptive response is that like cells of the innate response, innate immune response,
00:09:48.13 dendritic cells can respond to exactly the same types of microbial signals
00:09:52.12 as do the macrophages. Again, by virtue of the fact that they express
00:09:57.07 the same classes of Toll-like receptors that macrophages do, but instead of under
00:10:02.28 most circumstances emitting cytotoxic compounds as a consequence of that,
00:10:07.01 they turn the information and the advent of microbial pathogens
00:10:14.05 coming into the system into the peptide language that can be understood by T-cells,
00:10:19.08 thereby linking the activation of the adaptive response
00:10:25.09 to the activation of the innate response. So then as I'm mentioning in words,
00:10:30.18 as you can see here, dendritic cells do play this really important missing link
00:10:35.21 that intimately connects innate immunity with adaptive immunity responding
00:10:41.04 to the innate signals and turning those signals into the language of the adaptive immune response.
00:10:47.00 Now this is a concept that is relatively new and certainly not nearly as
00:10:54.13 old meaning 100 years, as the first discovery of the adaptive response
00:11:00.14 and the innate response. It took almost 80 to 100 years later to really figure this out,
00:11:05.26 and that was done by this gentleman with no beard, Ralph Steinman,
00:11:10.20 who works at the Rockefeller University who was really among the very very first
00:11:14.23 to appreciate that dendritic cells have this remarkable role in being able to have a critical element
00:11:23.15 in linking the innate and adaptive responses by being just so powerful and so special
00:11:29.23 and so adept at generating T-cell responses in response to antigens
00:11:37.05 and in response to adjuvants or microbial products. Now the basic logic of
00:11:42.11 the dendritic cell system is shown here, and this is terrifically rich and complex
00:11:46.23 but indeed quite understandable. The idea is as follows, dendritic cells exist
00:11:52.22 as immature sentinels in a variety of peripheral tissues, in fact all of our peripheral tissues
00:11:58.05 contain dendritic cells. Here we are looking at the skin, at the epidermis,
00:12:02.02 where dendritic cells are intercalated in various levels in the skin,
00:12:06.27 most interestingly in the epidermis itself where these long stellate cells
00:12:12.00 are intercalated among the far more numerous keratinocytes. Indeed, the fact that these dendritic cells
00:12:19.18 existed in the skin is actually a fairly old observation made by Paul Langerhans
00:12:25.15 also who was responsible for having first identified the islets of Langerhans in the pancreas,
00:12:31.07 but Langerhans didn't know what these cells did, but indeed he identified that they were there.
00:12:36.01 We now know that they are present in the skin for the purposes of immune surveillance.
00:12:42.02 They are there to capture incoming antigens, to capture incoming pathogens,
00:12:47.18 and after that capture takes place they migrate out from the skin, enter the lymphatics,
00:12:52.28 and eventually as I already mentioned find their way into lymphoid organs
00:12:58.26 where they also now start to intercalate together with lymphocytes T-cells and B-cells.
00:13:06.00 Now by the time they get to lymphoid organs under most circumstances,
00:13:10.24 these dendritic cells change in their characteristics, they become mature.
00:13:16.21 The difference between an immature cell and a mature cell turns out to be key
00:13:21.25 in understanding exactly what happens and we'll come to that in just a moment.
00:13:26.00 Immature dendritic cells and mature dendritic cells differ from one another
00:13:31.17 in some very very important ways. Immature dendritic cells are shown here
00:13:36.05 in an immunofluorescence picture. What you can see is that all the MHC molecules,
00:13:40.24 particularly the MHC class II molecules that are expressed by
00:13:43.28 immature dendritic cells are sequestered inside the cell in lysosomes and late endosomes.
00:13:50.00 They are not at the surface, so as a result these immature DCs are relatively speaking
00:13:57.08 incapable of stimulating T-cells. In addition they don't express co-stimulatory
00:14:02.27 molecules, they're very poor at secreting cytokines and they are
00:14:07.03 relatively non-motile, so as a result they are very poor at T-cell stimulation.
00:14:12.20 What they are good at though is antigen accumulation. So we view these
00:14:16.15 cells as the sentinels that are the first ones that encounter antigen in the periphery
00:14:20.18 and then as a consequence of detecting antigen and also having the ability
00:14:27.09 to detect the innate signals encompassed or encoded in those antigens
00:14:32.06 via Toll-like receptors and other inflammatory product receptors these
00:14:37.20 dendritic cells change their morphology and also change their function
00:14:41.10 dramatically and really can do so in a remarkably short period of time.
00:14:45.16 We find that only a relatively few hours is required to transform a cell
00:14:51.14 that looks like this to a cell that looks like this, one that extends out enormous
00:14:57.00 dendrites that give them their name, that now relocate all of the
00:15:01.12 class MHC molecules that were present in lysosomes to the surface of the cells
00:15:06.11 and also induce the expression of a variety of other important molecules
00:15:11.14 such as these co-stimulatory molecules that are necessary for optimal T-cell stimulation.
00:15:18.26 So the mature dendritic cell is the cell that is most adept at antigen presentation
00:15:24.00 and antigen stimulation. This flip from the immature to the mature state
00:15:28.27 is intimately linked to the fact that the immature dendritic cell expresses
00:15:34.14 these Toll-like receptors. Were it not for that fact, not for the fact that
00:15:39.07 these cells that are intimately associated with the adaptive response
00:15:42.22 also have the capacity to respond to the most fundamental and
00:15:47.24 elemental element of the innate response we would not have this link. So it's actually
00:15:54.14 maturation that does this. Now as cell biologists interested in membrane traffic
00:15:58.19 we've been very interested over the years as have been many other groups
00:16:02.16 in trying to understand what's responsible for this dramatic morphogenetic
00:16:07.00 change that dendritic cells exhibit and how does it relate to the function
00:16:11.18 of these cells and how to these functions relate to the overall control
00:16:15.18 of the immune response. So here on the left you are looking at a diagram
00:16:20.09 of the membrane traffic phenotype if you will of an immature dendritic cell.
00:16:25.07 These are cells that are highly endocytic, they take up lots of antigen
00:16:29.20 by a variety of different mechanisms of endocytosis, those antigens come in from
00:16:33.25 the outside, find their way to endosomes and finally to lysosomes
00:16:37.13 where quite remarkably they sit unlike most other cells that degrade
00:16:41.20 very rapidly proteins and nucleic acids and lipids and carbohydrates
00:16:46.04 that make it into lysosomes in immature dendritic cells the antigen that enters lysosomes
00:16:52.24 is protected and protected from degradation. At the same time,
00:16:57.24 these cells are making a large number of MHC molecules, particularly MHC class II molecules
00:17:03.17 which instead of being taken to the cell surface where they sit,
00:17:07.26 which is what happens in most other cells, now these molecules as well are targeted to the lysosomes
00:17:12.23 but basically nothing happens, the MHC molecules sit there, the antigen sits there,
00:17:17.10 a little bit of degradation, a little bit of loading of peptide onto the MHC molecules
00:17:22.12 but really not too much takes place until these cells are exposed to a TLR, a Toll-like receptor ligand.
00:17:31.26 In other words, one of the preserved molecular patterns that are conserved
00:17:36.13 from microbe to microbe, then everything starts to change and starts to change really fast.
00:17:42.07 One of the first things that occurs is that endocytosis, at least many forms
00:17:47.27 of endocytosis are shut off to a very large extent, this reflects
00:17:53.09 the fact that these Rho-family of GTPases particularly of the molecules
00:18:00.06 Cdc42 rather than being present in a constitutively active form
00:18:06.06 is now de-activated and present not as the GTP active form but rather as the GDP inactive form.
00:18:14.02 So antigen uptake is not cut-off completely but is diminished.
00:18:17.15 Next thing that happens is that newly synthesized class II molecules,
00:18:21.18 rather than being directed entirely to lysosomes are now taken from the golgi to endosomes
00:18:28.01 and like in other cells for other types of molecules are targeted to the cell surface.
00:18:33.06 There is a lot of reasons for this, we'll come back to one of the most important
00:18:37.01 in a minute but much of it has to do as well with changes in the metabolism
00:18:43.02 of this class II associated chaperone referred to as the invariant chain. As long
00:18:48.06 as the class II molecules associated with the invariant chain the class II molecule
00:18:52.17 is taken to lysosomes. If the invariant chain is removed, the class II molecule can
00:18:57.22 now proceed out to the cell surface. This happens for a variety of reasons,
00:19:02.06 not the least of which is due to down regulation of an antiprotease
00:19:06.13 called cystatin c, which turns off the proteolytic enzyme that is normally responsible
00:19:12.01 for degrading this invariant chain. In fact the lysosomes are activated
00:19:17.27 completely in this case for a variety of reasons, probably one of the most
00:19:23.06 interesting is the activation of lysosomal acidification. Normally
00:19:28.16 lysosomes are acidic vesicles, as Metchnikoff first told us, but in immature dendritic cells
00:19:34.26 they're less acidic than they need to be. The reason for that has been described
00:19:39.22 over the last two or three years as reflecting two key events.
00:19:44.04 One, the vacuolar proton pump or the ATPase that is required to move protons
00:19:50.27 from the cytosol into the lumen of the lysosome thereby dropping its pH,
00:19:56.01 is inactive in the immature dendritic cell. As a consequence of maturation,
00:20:00.24 as a consequence of TLR stimulation, this proton pump
00:20:07.00 is activated by turning on an assembly process, now allowing protons
00:20:12.14 to be translocated from the cytosol into the lysosomal lumen
00:20:16.05 in exchange for ATP hydrolysis thereby acidifying the interior of the lysosome.
00:20:22.10 Sebastian Amigorena in Paris has further found that in some ways like
00:20:26.21 macrophages, dendritic cells are indeed capable of generating active
00:20:30.27 oxygen species but one of the most important features here
00:20:34.23 is not so much to kill the incoming pathogen but rather to further regulate
00:20:39.11 the ability of lysosomes and incoming phagocytic vesicles to acidify their lumens,
00:20:47.02 again emphasizing how important it is to the dendritic cell
00:20:51.21 to ensure that the pH of the compartments involved in antigen presentation
00:20:56.14 and antigen processing are indeed carefully regulated.
00:21:00.08 So this is basically how it works, very simply the lysosomal pH of
00:21:05.19 immature dendritic cells is slightly acidic, it has a pH of 5.5.
00:21:10.07 The lysosomal pH found in organelles of mature dendritic cells
00:21:17.03 is more acidic by one whole pH unit, 4.5.
00:21:20.23 Doesn't sound like a lot but it turns out that most of the lysosomal
00:21:24.15 proteases and nucleic acid degrading enzymes and lipid degrading enzymes
00:21:30.00 that are found in lysosomes have a very sharp pH optimum
00:21:33.18 and they really don't work very well unless the pH that they are working in
00:21:38.08 is below pH 5, so the maturation process then drives the pH down
00:21:43.24 from a pH that's too high for optimal activity of the lysosomal proteases
00:21:48.17 to a pH that now is just right, the goldilocks effect allowing these
00:21:54.03 lysosomal proteases to do their work at optimal levels increasing
00:21:58.15 the efficiency at which peptides can be generated for association
00:22:04.12 with MHC class II molecules. Now this is a diagram just quickly as to how this works.
00:22:10.08 So here you can see a class II molecule that begins its life associated
00:22:15.18 with the invariant chain as you've seen in previous diagrams,
00:22:19.03 it consists of two membrane proteins, an alpha chain and a beta chain,
00:22:23.08 here is the invariant chain in green together with its lysosomal targeting signal.
00:22:28.00 The invariant chain is degraded by a series of proteolytic cleavages
00:22:31.26 most important of which is mediated by an enzyme called cathepsin s,
00:22:36.19 which is found most prevalently in professional antigen presenting cells such as dendritic cells.
00:22:43.24 This removes the lysosomal targeting signal from the invariant chain,
00:22:49.27 leaving a small segment of the invariant chain that is rapidly removed
00:22:53.14 by virtue of the activity of another class II associated chaperone,
00:22:57.26 not invariant chain, but something called HLA-DM,
00:23:00.28 that destabilizes the affinity of this remaining invariant chain fragment
00:23:05.04 to the peptide binding cleft of the class II molecule,
00:23:09.03 allowing the peptide to bind and displace the invariant chain derived peptide
00:23:16.24 and this then peptide MHC complex can go up to the cell surface.
00:23:21.19 I mentioned cystatin c before, it works at this stage by inhibiting
00:23:26.13 the activity of cathepsin s, it slows the cleavage, renders the cleavage less efficient
00:23:32.16 of the invariant chain making these molecules less accessible to peptide loading.
00:23:38.15 In a flow diagram in terms of what this means for membrane traffic,
00:23:43.05 here emanating from the golgi complex is the invariant chain class II complex,
00:23:48.06 enters endosomes and in immature dendritic cells nothing happens
00:23:51.27 because the level of cathepsin s is low, the activity of cathepsin s is low because
00:23:58.17 the activity of cystatin c is high and also the pH of these structures is not optimal,
00:24:04.17 and instead these class II molecules go to lysosomes. Maturation reduces
00:24:09.20 cystatin c activity, increases cathepsin s activity playing a role in helping
00:24:16.09 these class II molecules proceed on to the cell surface. Now the molecules
00:24:20.11 that had made it to lysosomes are there and also as long as the dendritic cell
00:24:26.15 remains immature not much happens. Here is a video taken by Amy Chow
00:24:32.28 in our laboratory a few years ago from a MHC molecule that has been linked
00:24:40.13 to the green fluorescent protein in immature dendritic cells, and what you
00:24:45.02 are looking at are lysosomes that are just sort of bouncing around, aimlessly
00:24:48.05 in these immature cells. So as I've mentioned earlier,
00:24:53.10 the class II molecules are there, the antigen is there and nothing is happening.
00:24:56.21 But very soon after adding LPS to this system, a ligand for a Toll-like receptor,
00:25:02.29 specifically TLR-4, you get a very different effect. Now what you can see
00:25:07.20 is that these lysosomes begin shooting out tubules and the lysosomes
00:25:13.17 start accumulating in a small dot depleting the amount of class II that is
00:25:17.29 associated with them and you can begin to see class II appearing on the surface
00:25:22.25 of the cell, all these events taking place really just over a time course of
00:25:27.20 a couple of hours.
- Duration: 00:14:37
00:35:01.22 Now class I as I've already mentioned is a pathway that is mostly adept
00:35:09.16 to dealing with endogenous antigens. So the best example I can give you
00:35:15.23 is if you have an influenza virus infection and epithelial cells in your airway
00:35:23.19 are infected by influenza virus, those cells are going to be making
00:35:29.12 lots of proteins that are encoded by the virus. Those proteins are going to be degraded
00:35:34.22 in the cytosol, again following a ubiquitination event they'll be degraded
00:35:38.18 by the proteosome in the cytosol, small peptides generated from
00:35:43.03 those ubiquitinated proteins and then those small peptides translocated
00:35:48.24 into the lumen of the endoplasmic reticulum by virtue of the activity of a rather
00:35:54.04 remarkable ATP-driven membrane peptide transporter called TAP, actually TAP1 and TAP2.
00:36:03.06 So the peptide that enter into the ER are loaded onto class I molecules
00:36:07.13 and then they make their way out to the cell surface by the constitutive secretory pathway.
00:36:13.02 Now there's a flaw in this logic. Remember I told you that only dendritic cells
00:36:17.08 can start an immune response. And I also just told you that the predominate cell,
00:36:22.25 in fact possibly the only cell that is infected after you become infected by
00:36:30.03 influenza are epithelial cells. How do we guard ourselves against
00:36:36.10 the possibility that the dendritic cell doesn't become infected?
00:36:39.00 The epithelial cells are incapable of generating a robust T-cell response,
00:36:43.10 only the dendritic cells are capable of doing that but the dendritic cells are not infected,
00:36:48.16 they're not making the influenza virus specific proteins. So how do
00:36:54.13 dendritic cells deal with this? They deal with it by having developed
00:36:57.26 a rather remarkable system of membrane transport,
00:37:03.00 which is classically referred to as cross presentation. It was really first described
00:37:07.08 by an immunologist, Mike Bevan. Here it was thought to be the case
00:37:12.04 that antigens coming in from the outside rather than being restricted
00:37:16.29 to the MHC class II pathway can cross over and in fact have access to the class I pathway
00:37:23.00 and do so by somehow breaking out of the endosome lysosome system,
00:37:27.03 entering into the cytosol and becoming accessible to the ubiquitination proteosome
00:37:32.06 degradation system that then is also responsible for servicing
00:37:37.16 those antigens that are endogenously synthesized by the cell. So the peptides
00:37:43.20 that form from these cross presented antigens can enter into the ER lumen
00:37:49.09 via the TAP1 TAP2 translocator, be loaded onto class I molecules
00:37:54.29 and then as I've been describing, make it out to the cell surface.
00:37:58.09 So the way this probably works in practice in the case of virus infections
00:38:02.23 is diagrammed here, immature dendritic cells will capture and take up
00:38:08.27 just as another phagocytic load a virus infected cell, which has been killed
00:38:15.08 by virtue of its virus infection. So dead cells, apoptotic cells, necrotic cells
00:38:21.06 can be nicely recognized by dendritic cells, enter into phagosomes,
00:38:25.09 these cells are then degraded and then antigens derived from the infected cell,
00:38:30.00 most importantly the virus encoded antigens now come out into the cytosol
00:38:35.04 and can be degraded by the proteosome system and be presented on the surface
00:38:39.23 of the dendritic cell on class I molecules. Now remember while all this is going on,
00:38:46.10 proteins that are also intrinsic to this self of the infected cell,
00:38:52.14 in other words our own proteins, are not going to be immune to this,
00:38:56.01 they will also be degraded in the phagosome and some portion of them
00:39:00.25 we have to imagine will also come into the cytosol and be degraded
00:39:05.18 and be loaded onto class I molecules and presented to now
00:39:09.15 CD8 positive T-cells on the cell surface. So how is it then that
00:39:13.24 the dendritic cell can distinguish between the viral antigen and the self antigen?
00:39:18.08 How does the immune system do this? How does the immune system do that?
00:39:22.29 Now it may be apparent to some of you, but I actually had to go off
00:39:25.29 and think about this for a little bit, but it turns out that to understand
00:39:29.22 how the immune system balances tolerance and immunity responses
00:39:33.18 to self versus non-self actually has a lot again to do with dendritic cells,
00:39:38.02 specifically the property of dendritic cell maturation which we now should
00:39:42.00 come back to and look at in a little bit more detail to understand just how
00:39:45.12 these cells via the process of maturation controls the progress of the immune response.
00:39:50.21 Now remember that maturation links the two major arms of the immune system,
00:39:55.24 the innate immune response to the adaptive immune response
00:39:59.13 by detecting microbial pathogens and then turning those pathogens into
00:40:04.03 the peptides that are required to be presented to T-cells to generate adaptive immunity.
00:40:08.05 Now the maturation process itself in its first instance is controlled by this family
00:40:14.15 of Toll-like receptors that I've mentioned, that essentially act as barcode readers
00:40:19.01 and I'll come back to that in a moment, that identify and deconstruct exactly
00:40:24.16 what type of pathogen happens to be present at the time the maturation event is induced.
00:40:30.28 Now there are a number of different Toll-like receptors. Some of them are
00:40:34.15 diagrammed here. About 12 or more, I've lost count since it changes periodically,
00:40:40.19 and what these Toll-like receptors are for is that they are specific for a variety of
00:40:44.13 different components that one finds in a wide variety of different pathogens.
00:40:47.19 You find some of the Toll-like receptors expressed on the plasma membranes
00:40:51.10 of cells, other Toll-like receptors are expressed in endosomes and lysosomes.
00:40:55.05 They are specific for bacterial proteins, bacterial lipids, one of the proteins
00:40:59.14 is one called flagellin, which is the major component of the bacterial flagella.
00:41:03.14 LPS is a major lipid species found in cell walls of many bacteria,
00:41:09.25 but many of the intracellular Toll-like receptors in particular actually react with specifically
00:41:15.28 and identify specifically nucleic acids, both single stranded and double stranded RNA and DNA.
00:41:22.27 In all cases, the general property that is elicited is this property of maturation,
00:41:28.17 but not all forms of maturation are created equal because not all
00:41:32.07 Toll-like receptors are created equal, and in fact they transmit different types
00:41:36.18 of signals to the dendritic cell. So as I said, these receptors work together
00:41:41.17 in the sense of a bar code. Some firing, others not firing, depending upon
00:41:46.12 what pathogen happens to be present, and the dendritic cell reacts to this information
00:41:51.22 and undergoes a path of its own maturation that adapts itself specifically
00:41:57.28 to the type of immune threat that is coming in from the environment
00:42:02.00 again and identifies the nature of the pathogen, the nature of the threat based
00:42:06.27 on the combinatorial array of Toll-like receptor ligands that happen to be present
00:42:13.06 and associated with that particular bacterium. Now as a consequence
00:42:17.27 of detecting these different arrays of Toll-like receptor ligands,
00:42:24.04 the dendritic cell matures and what it does as a consequence of that is
00:42:30.03 of course is to secrete a wide variety of different cytokines, which are again
00:42:33.16 essentially immunological hormones. Now T-cells are very smart, they are
00:42:39.27 almost infinitely capable of recognizing a wide array of different types
00:42:45.08 of antigens. But they have to essentially be told what to do by the dendritic cell
00:42:51.21 and this is not only by virtue of identifying the particular peptide MHC complex
00:42:57.01 that is presented by the dendritic cell that actually gets the T-cell responses going,
00:43:01.08 but this mixture of cytokines that is released by dendritic cell specifically
00:43:06.05 and in a customized fashion depending upon the type of microbe that was detected
00:43:12.19 is that which actually determines what the overall differentiation of the T-cell is going to be.
00:43:19.16 So its not enough simply to stimulate T-cells as a consequence
00:43:23.05 of having them detect via the T-cell receptor, the peptide MHC complexes
00:43:27.24 that are formed by dendritic cells, but the dendritic cells add to that process
00:43:32.21 by transmitting their experience of what type of pathogen had come in,
00:43:38.14 and they do this in this case by secreting the characteristic cytokines.
00:43:42.14 So one example of this is certain types of Toll-like receptors, or stimulation by
00:43:48.29 Toll-like receptors will cause dendritic cells to release the very
00:43:53.05 potent immunogenetic cytokine, IL-12, or interleukin 12.
00:43:57.27 T-cells that detect antigen exposed on the surface of
00:44:03.10 a dendritic cell that is secreting interleukin 12 undergo a type of differentiation
00:44:08.09 that allows them to become a particular sub-class of T-cell called Th1 T-cells,
00:44:14.04 which are highly inflammatory and highly immunogenetic cells.
00:44:17.16 Now the way this looks actually in situ is shown nicely here. This is a
00:44:22.06 scanning electron micrograph showing a dendritic cell in the background
00:44:25.26 with a number of T-cells that are attached very closely, as I've shown you
00:44:31.19 earlier in one of the video images, T-cells move around quite a bit across
00:44:36.07 the surface of dendritic cells, but when they finally find a good match,
00:44:40.17 a peptide MHC to a given T-cell receptor, they have a tendency to stay there
00:44:45.14 and stay there for a fairly long period of time, hours if not more,
00:44:51.08 and over this period of time they are literally bathed in the cytokine mix
00:44:56.04 that is being released by the dendritic cell, instructing them to become
00:45:00.22 a wide array of T-cells that all recognize antigen but all have a very very different
00:45:07.27 functional outcome with respect to how the immune response works.
00:45:11.09 I've lifted some of the more popular T-cell types that one can find.
00:45:15.08 We won't go through them in any detail at all, but just simply to say that
00:45:20.11 there are many many different possible T-cell outcomes that have different effects
00:45:25.03 on the so CD8 killer cells, or cytotoxic T-lymphocytes,
00:45:30.25 CTLs are ones that actually kill their target, so a cell that is infected by a virus
00:45:37.21 as we've discussed before will be killed by a cytotoxic T-cell of this particular type.
00:45:44.11 Not any T-cell will do this, but in fact only these will. CD4 helper cells help
00:45:49.19 generate antibody responses by working in collaboration with B-cells.
00:45:53.22 Inflammatory cells, central memory, effector memory cells are all T-cells that
00:45:58.14 circulate retaining the information of the immune account that had occurred,
00:46:03.16 retaining the lessons learned from the dendritic cell, and finally one
00:46:07.06 that we'll come back to in just a moment is the regulatory T-cell,
00:46:10.03 which rather than promoting immunity, actually dampens immunity and
00:46:13.13 probably assists or plays a central role in assisting the process of tolerance.
00:46:18.04 Again, often these cells are generated by dendritic cells, but not always,
00:46:24.17 and I think here I'd like to turn to just this very issue of tolerance and recognition
00:46:31.10 of self or non-self. How does it work? Basically it works in two settings,
00:46:36.10 before birth and early after birth, in the organ called the thymus where all
00:46:44.25 T-cells have their origin, prior to the exposure of the developing fetus or organism
00:46:51.07 to any exogenous antigens at least under normal circumstances,
00:46:55.08 a critical process called negative selection occurs. Now during negative selection,
00:47:00.05 either dendritic cells in the thymus or a related but nevertheless different
00:47:05.03 type of cell which has the same function or is presumed to have the same function
00:47:09.27 in the thymus called thymic epithelial cells have the remarkable function
00:47:15.06 of being able to turn on at the level of transcription, the expression of a wide array
00:47:22.00 of almost all of the proteins that we know that will be expressed in differentiated cells
00:47:27.03 later in life in the pancreas, in the liver, in the kidney, all of these are
00:47:32.22 expressed by these cells early on in the thymus, creating peptide MHC complexes
00:47:38.02 that are recognized by these thymocytes or forming T-cells that are being born
00:47:45.11 and developing in the thymus at this very very early stage of life. Now what
00:47:50.21 happens at this stage though is really quite interesting
00:47:53.21 and also quite important, and indeed quite profound.
00:47:56.14 Rather than the recognition event of the cognate peptide MHC by the T-cell receptors
00:48:04.11 on these developing T-cells, rather than causing an immune response or
00:48:09.28 an unrestricted proliferation of the T-cells, instead the T-cells
00:48:14.02 are induced to undergo apoptosis and die. In immunological parlance,
00:48:20.03 these cells are referred to as deleted. So any T-cell that recognizes
00:48:24.18 its antigen in the environment of the thymus early in development
00:48:29.06 is negatively selected and removed from the repertoire of
00:48:36.28 all of the antigens that could possibly be seen by the T-cell receptor later in life.
00:48:43.06 So this is a terrifically important first pass, whereby the immune system,
00:48:48.07 thymic and epithelial cells and dendritic cells due to the special properties of the thymus,
00:48:53.17 which are still not quite understood, are capable of removing a wide array
00:49:00.08 of T-cell specificities that would otherwise recognize host or self proteins
00:49:07.03 causing auto-reactivity and auto-immunity. As powerful and as important
00:49:13.07 as this process is, its not 100% efficient. Some self antigens are missed,
00:49:18.20 but another critically important class of antigen that's missed
00:49:23.11 is environmental antigens, since after birth we are all bombarded
00:49:28.13 and in fact bathed in a wide number of environmental allergens such as pollen in the air,
00:49:35.07 food allergens of various types, things that may penetrate through the skin,
00:49:40.17 and if we were to amount an immune response to each one of these foreign
00:49:44.08 antigens we would be hyper-allergic and not be in a very good state.
00:49:50.14 Now there is no way that the thymus can educate our T-cell responses or
00:49:54.29 the dendritic cells can educate our T-cell responses in the thymus
00:49:57.27 to delete T-cells that might be specific for a pollen or environmental antigens.
00:50:04.25 That has to occur after birth because obviously as a fetus we are not generally
00:50:09.16 speaking exposed to too many environmental antigens that are allergic in this sense.
00:50:14.05 So this is a process that's been left to the formation of this final and critically
00:50:21.21 important and yet incredibly poorly understood form of T-lymphocyte
00:50:26.10 called the T reg, or regulatory T-cell. Now a lot of these are in fact formed
00:50:31.12 in the thymus, so in addition to deletion of T-cell reactivities, one also finds
00:50:36.24 that the thymus will produce an array of regulatory T-cells that recognize
00:50:42.18 a variety of self antigens that will then have a tendency to help turn off
00:50:48.01 T-cell responses that occur inappropriately later. Okay, but many regulatory T-cells,
00:50:54.26 or T regs, are not produced in the thymus but rather are produced in the periphery
00:50:59.26 as a consequence of not so much a negative selection process but a tolerogenic
00:51:04.25 process which in this case is mediated almost exclusively by the dendritic cell
00:51:09.11 not by the thymus. Now this happens under steady-state condition.
00:51:13.27 So what I mean by that is if an antigen is encountered by dendritic cells that have not
00:51:21.09 received a stimulus via a microbial type Toll-like receptor ligand to mature,
00:51:29.22 what one finds then is that all of the same processes of antigen processing
00:51:34.12 and presentation and transport of the peptide of MHC complexes
00:51:38.10 to the surface take place, dendritic cell develops to a form that is capable of efficiently
00:51:44.09 doing this and efficiently generating T-cell recognition, but nevertheless
00:51:49.14 under these conditions in the absence of a Toll-like receptor stimulus
00:51:53.18 or another inflammatory stimulus, the type of T-cell that emerges is a
00:51:59.03 regulatory T-cell, or an induced regulatory T-cell, totally as a consequence
00:52:03.06 of peripheral recognition events. So these T regs instructed again and formed
00:52:09.26 almost uniquely by peripheral dendritic cells found in our lymph nodes,
00:52:15.05 lymphoid organs and indeed all of our peripheral tissues are our last line
00:52:21.12 and in many cases our most important line of defense against mistakes
00:52:26.21 that can be made by the immune system between self and non-self,
00:52:31.11 between foreign and endogenous antigens. Again, serving to control
00:52:36.09 this balance between tolerance and immunity. Again, it is the T-cell that does it,
00:52:42.13 although to be fair we don't really understand very much about how T regs work,
00:52:47.10 this is an emerging field, an emerging problem at the moment.
00:52:51.11 But what it does seem to be quite clear based on genetic deletion results
00:52:56.04 and antibody blocking results that have been done quite recently, it is these
00:53:01.04 dendritic cells that exist under steady state non-inflammatory conditions
00:53:05.10 that really are responsible for generating these T regs.
00:53:09.21 Now this is critically important for a variety of reasons because every time a
00:53:14.19 dendritic cell matures as a consequence of being stimulated by Toll-like receptor
00:53:19.24 ligand, those dendritic cells present not only the foreign antigen
00:53:23.18 but also present every self antigen in the body that they can come in contact
00:53:27.26 with over that period of time. So every time we respond to a foreign stimulus,
00:53:33.23 we run the risk of responding to one of our own self antigens
00:53:38.21 and run the risk of developing auto-immunity. So in order to maintain
00:53:45.06 this very very careful relationship, this very very careful balance
00:53:50.07 that must occur lest auto-immune pathologies set in between immunity and tolerance,
00:53:57.13 there is this continuing production of T regs that occurs that provide with us
00:54:03.20 this important break, this important line of defense to maintain equilibrium,
00:54:09.07 maintain homeostasis, and maintain health. So the way I like to frame this
00:54:15.16 is as a hypothesis, which I stress by saying is really no more than a hypothesis
00:54:22.06 at this point, but nevertheless I think summarizes quite well the process as many
00:54:27.20 of us really believe it occurs at this point. So as I was saying,
00:54:32.00 under conditions of no infection, under the steady-state, one finds
00:54:35.28 immature dendritic cells in peripheral tissues, as shown here in the skin,
00:54:40.02 again using the same diagram we've been looking at during the course of these two lectures.
00:54:45.11 No infection, immature in the periphery, and at some point these cells
00:54:51.09 either just due to stochastic processes or due to some inductive process
00:54:56.11 migrate from the skin via the lymphatics into the lymphoid tissues.
00:55:00.22 Along the way, they undergo a type of maturation, because now in lymphoid
00:55:06.11 organs they are capable of presenting antigens, all self-antigens in this case,
00:55:10.17 or all environmental antigens, but nevertheless the type of maturation
00:55:14.20 that they undergo is one that leads to tolerance. So they are not antigen
00:55:19.08 presenting, they are not very effective antigen presenting cells as immature cells
00:55:23.21 in the periphery, they are much better at it when they are in lymphoid organs
00:55:27.18 but they are nevertheless still tolerogenic. Again, these are conditions under
00:55:31.11 steady state, meaning no infection, no inflammation. Everything changes though
00:55:36.04 when we go to the condition of infection, or inflammation. Here now
00:55:41.10 what you find is that again the dendritic cells are still immature in the periphery,
00:55:45.23 but now they encounter a Toll-like receptor stimulus as a consequence
00:55:50.16 of the advent of one or more microbes as we've been discussing. The migration
00:55:56.29 process is the same, the delivery to lymphoid organs is more or less the same,
00:56:01.14 but now the maturation that takes place is one which is not tolerogenic but rather immunogenetic.
00:56:08.19 Okay, so the T-cells that are produced by this same progenitor population of
00:56:14.22 dendritic cells, possibly, possibly there are subsets, but possibly the same
00:56:19.09 progenitor population of dendritic cells under conditions of infection,
00:56:23.11 under conditions of inflammation, yields T-cells that undergo development
00:56:27.05 not to produce T regs, but rather to produce one of the many types of
00:56:30.28 inflammatory or immunogenetic T-cells that I listed for you just a moment ago.
00:56:36.12 Now this to me comprises probably one of the most profound of all problems that
00:56:44.16 remain in the immune system. I've stated it in what may appear to some of you
00:56:49.23 at least to be relatively reasonable terms, but the fact of the matter
00:56:53.04 is we have almost no idea how these events are interconnected.
00:56:57.18 We know small details, all of which are indeed enticing, beginning with why
00:57:03.14 is it that transcriptionally one can find so many different differentiated gene
00:57:07.28 products expressed early on in the thymus? We know something about what
00:57:12.18 the transcription factors are that do this, but how all of this is regulated,
00:57:15.26 how it actually works, very very few of the details are really known,
00:57:20.03 and it really represents a terrific area for research of basic biology and also to
00:57:25.26 come up with still solutions to one of the great problems left in immunology.
00:57:31.19 There are many, but this is certainly one that tops my list. Now another reason
00:57:37.16 why this is so important is not just because of the basic biological aspect,
00:57:42.03 but also because of the disease aspect. I think increasingly as size progresses
00:57:47.21 in our understanding and our ability to do more and more complex experiments,
00:57:52.23 particularly at the systems level, an equally valid path to take in studying basic
00:58:00.10 biology is to understand disease processes. This of course has been done
00:58:05.03 by many in the past, but I think increasingly so at earlier and earlier stages
00:58:09.00 in ones scientific career and ones scientific interests, its possible to begin
00:58:14.03 to do real basic solid experiments where your question is what happens during
00:58:20.02 a particular disease process. So how does tolerance and immunity
00:58:23.18 fit into this? I've already hinted at it several times, but if you have a situation
00:58:28.06 where there is too little tolerance in other words the dendritic cells
00:58:32.10 or the thymus were not optimally efficient at deleting auto-reactive T-cells
00:58:37.24 or turning auto-reactive T-cells into T regs, or regulatory T-cells,
00:58:43.00 one can find a wide variety of diseases that fall into the broad class of auto-immune disorders,
00:58:49.24 such as auto-immune diabetes, lupus, or Myasthenia gravis. These are mediated
00:58:55.04 either by the production of pathogenic antibodies to self proteins,
00:58:59.08 or T-cells that exert direct cytotoxic effects on normal host tissues.
00:59:05.24 Another possibility is chronic inflammation, so diseases such as arthritis,
00:59:11.19 or asthma, Crohn's disease, ulcerative colitis, Multiple Sclerosis, possibly
00:59:16.22 have to do with the fact that an inflammation starts and then can't be turned off.
00:59:20.28 These may not be strictly auto-immune in many cases because for a lot of these
00:59:26.00 diseases there may not be a single antigen against which T-cells continuously
00:59:30.21 are producing new antibody via B-cell production or new cytotoxic secretions
00:59:39.03 as a consequence of the T-cells own activities, but nevertheless these
00:59:43.17 are processes that keep going because of disregulation of the balance
00:59:48.27 between tolerance and immunity. And again, in many ways one can attribute
01:00:00.00 the brute cause of all of this to dendritic cells misbehaving,
01:00:04.25 presenting antigens in the wrong context, producing the wrong type
01:00:08.26 of T-cells under a condition that doesn't call for that type of T-cell response,
01:00:13.24 and then the do-loops that emerge simply don't get turned off.
01:00:17.27 So how do we intervene in all of these things and how can we do this not only to
01:00:24.09 understand the biology, which is of course paramount, but also to understand
01:00:28.00 how therapeutically we can begin to intervene in these diseases processes
01:00:32.16 with ever higher degrees of specificity and exactness so that we can turn off
01:00:38.04 just the disease process and not interfere with normal ongoing processes
01:00:43.04 or actually do more harm than good. So now that I've moved myself from
01:00:48.23 academia to a biotech company, these are problems that are coming to the fore
01:00:54.23 on a daily basis, and its of no small matter to try and understand and grapple
01:01:01.28 with these problems, not only as a basic scientist but also as someone
01:01:06.06 who is now committed to understand how it is that you can turn that
01:01:10.07 basic science knowledge into dealing with major major health problems such as these.
01:01:15.09 It's also possible that you have too much tolerance and two sets
01:01:21.17 of very bad things can occur under these circumstances. This is different from diseases
01:01:27.05 that lead to immunodeficiency. Here you have diseases where the immune system
01:01:32.16 is often intact, but has been educated by the pathogenic organism
01:01:38.21 or as shown here by cancer cells, to evade the immune response. Cancer is I think
01:01:47.01 a particularly challenging example. Immunotherapy in cancer is something
01:01:51.16 that is now just starting to gain steam now with the first immunotherapeutic
01:01:56.28 to prostate cancer just having been approved this year, but what we
01:02:02.00 understand about cancer and the immune system tells us, at least to a first approximation,
01:02:07.25 that many cancers in fact are capable of generating immune responses,
01:02:11.14 either due to mutation or to ectopic expression of proteins that are not normally produced by a given cell.
01:02:20.03 Cancer cells in fact can elicit T-cell responses, but they've figured out,
01:02:25.12 or if they haven't figured out at least they've been selected for cells that are
01:02:29.25 capable of subverting those T-cell responses, either by turning them off,
01:02:34.02 such that when the T-cell penetrates into a tumor bed and tries to kill its target,
01:02:39.00 the target protects itself by secreting or placing on its surface molecules that in
01:02:45.05 fact will abrogate T-cell responses rendering them as immunologically, I'll just say, anergic.
01:02:53.21 Another possibility though is that cancer cells in fact by much the same way that
01:02:59.26 dendritic cells seem to do it, will seem to generate a T-regulatory or T reg responses,
01:03:05.15 again having the same effect subverting T-cell responses to cancerous cells
01:03:12.10 that would otherwise be amenable to be controlled by those T-cells, at least in theory.
01:03:18.10 Another, and I think in many ways more clear example, occurs in the case of many
01:03:25.01 chronic viral infections, such as CMV or HIV. CMV is a particularly good example,
01:03:31.25 but many other chronic viruses are as well. What happens in these cases is that
01:03:37.00 viruses have figured out how to down regulate proteins on the surface of
01:03:43.10 the infected cell, or in some cases even on the surface of dendritic cells
01:03:47.06 in such a way as to prevent, again, T-cell recognition or in some cases even T-cell responses.
01:03:54.15 The immune system in a sense gets educated to understand that the viral proteins
01:03:59.21 are not actually foreign, but indeed are part of the own host protein repertoire,
01:04:09.04 and as a consequence by tricking the immune system in this way,
01:04:12.12 the virus can replicate and can maintain its infection with impunity without
01:04:19.19 risk of detection by the immune system. So in the case of diseases where
01:04:24.29 there is too much tolerance, therapeutic intervention that one can imagine,
01:04:30.04 is how is it that you reactivate T-cell responses by convincing the dendritic cells
01:04:36.24 to break tolerance as we say and re-introduce antigens either derived from cancer cells
01:04:43.21 or from viruses under conditions that now can generate positive
01:04:48.15 immunogenic immune responses rather than just tolerogenic immune responses.
01:04:54.05 So both of these types of disease states, again, embody I think some of the most exciting
01:05:00.02 biology, both immunology and cell biology that one can think of
01:05:05.00 in the immune system, plus also offer the opportunity to the lucky and interested
01:05:10.09 scientists, which hopefully includes me, to understand how it is one can
01:05:17.00 actually make either biological agents or even small molecule drugs
01:05:21.13 to either induce tolerance under conditions where you would like to turn off
01:05:26.20 chronic inflammation or turn off auto-immunity or overcome tolerance
01:05:31.17 under conditions where you would like to re-activate the immune system,
01:05:35.08 re-educating it to go about its job and combating what are effectively
01:05:41.24 foreign agents such as cancer or chronic viruses both for therapeutic benefit. Thank you.
- Duration: 00:20:58
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.
Medzhitov R. (2001) Toll-like receptors and innate immunity. Nat Rev Immunol. 1(2):135-45
- Ira Mellman iBioSeminar: Cell Biology of the Immune Response
- Ruslan Medzhitov Discovery Talk: The Role of Toll-Like Receptors in the Control of Adaptive Immunity
Dr. Ruslan Medzhitov is a Sterling Professor of immunology at Yale School of Medicine and a Howard Hughes Medical Institute investigator. His laboratory studies the signals that initiate and control the process of inflammation, allergic reaction, and immune response. His laboratory also studies tissue biology, and the communication circuits that help to establish stable cellular… Continue Reading
Ira Mellman is Vice President of Research Oncology at Genentech. Ira is a cell biologist with a long standing interest in membrane traffic. His lab is reponsible for key observations leading to the initial discovery of endosomes, the mechanisms of epithelial cell polarity, and the cellular basis of dendritic cell function. Until 2007, Ira was… Continue Reading