Session 3: Bridging Innate & Adaptive Immunity

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 naïve, 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. This is a video in which we are just able to visualize
00:25:31.24 the movement of some of these lysosome derived tubules from their site of
00:25:35.10 formation in lysosomes out to the periphery of cells and while I'm going
00:25:39.04 to show this to you, there are various biophysical techniques that you can use
00:25:42.05 to actually show that these tubules and vesicles derived from them will
00:25:45.22 physically fuse with the plasma membrane of the cell, delivering not only
00:25:50.15 the MHC class II molecule, but obviously also the antigen that has bound
00:25:55.28 to it as a consequence of the peptide loading event that took place
00:26:01.01 in the lysosome just at the moment of dendritic cell maturation.
00:26:05.08 All of these events take place as I said in just a couple of hours
00:26:09.07 if you wait overnight you can now see live dendritic cells that look ever much like
00:26:14.29 the static images I showed you earlier. Here a cell with all of its MHC class II molecules
00:26:21.24 on the surface, lysosomes are now stained red because none of the GFP
00:26:26.14 or green fluorescent protein coupled class II molecules are present within them anymore.
00:26:31.21 So what that means then is here one more unexpected feature of the system,
00:26:38.21 which is that MHC class II molecules can escape from lysosomes
00:26:43.21 to dendritic cell surface, by a pathway which we refer to as your retrograde transport pathway.
00:26:49.26 Anterograde transport refers to what happens when molecules
00:26:54.15 such as antigens come from the outside by endocytosis into the lysosomal compartment,
00:26:59.14 retrograde refers to what happens when they go out. Again, this is probably,
00:27:04.06 or almost certainly I should say, a microtubule driven process,
00:27:07.15 but what’s most remarkable is that it happens at all. Our conventional view of
00:27:12.00 lysosomes is really kind of summarized here in this early medieval representation,
00:27:16.19 even prior to Metchnikoff, showing that there is no escape.
00:27:20.25 This is at least the conventional view. Proteins, antigens, whatever microbes are collected,
00:27:28.03 delivered into lysosomes and simply degraded. So we had not really anticipated
00:27:34.12 that there was going to be a pathway anywhere in cell biology
00:27:39.12 that was going to allow us to recover or allow a cell such as a dendritic cell
00:27:45.04 to recover molecules in a very selective and very efficient fashion
00:27:49.19 so they can move from this degradative compartment out to the cell surface.
00:27:54.00 Now when they get out to the surface, why do they stay there?
00:27:58.03 Why don't they just come back into lysosomes by endocytosis.
00:28:01.05 Well you might say endocytosis is shut off and indeed, as I already mentioned,
00:28:05.22 it is as a consequence of down regulating active forms of Rho family GTPases,
00:28:11.29 such as Cdc42 and Rac, all of which are involved in actin assembly. So the normal
00:28:20.02 ability of immature dendritic cells to capture antigen by such processes
00:28:24.11 as macropinocytosis or phagocytosis, which are both strongly actin driven
00:28:30.18 processes as we've seen in the first video, indeed are turned off. But uptake
00:28:36.18 via endocytosis by clathrin coated pits, which are much smaller vesicular carriers
00:28:43.23 that can be formed about only 0.2 microns in diameter, as opposed to the
00:28:48.17 phagosome, which can be one or two or three or even five microns in diameter,
00:28:52.24 this pathway continues. And indeed we know that MHC class II molecules
00:28:57.19 can enter cells, and indeed can enter dendritic cells by clathrin mediated
00:29:03.00 endocytosis. So why is it that class II molecules do not enter in the mature cell?
00:29:11.27 That brings us to another emerging fundamental feature of membrane traffic,
00:29:17.11 and that is the role of protein based ubiquitination in controlling the movement
00:29:22.18 of membrane proteins from one compartment to the next. Over the last several
00:29:27.05 years, a large number of investigators have demonstrated that ubiquitin plays
00:29:32.21 a critical role in signaling, either the endocytosis of endocytic receptors into
00:29:38.09 clathrin coated pits and/or the ability of those receptors at the level of endosomes
00:29:44.08 to be sequestered into these rather oddly shaped structures
00:29:48.28 called multivesicular bodies. We know from the work of Scott Ember and others
00:29:54.09 for example that when ubiquitin is modified or a membrane protein is
00:29:59.12 modified by ubiquitin, after delivery or upon delivery to the endosomal compartment,
00:30:04.19 these ubiquitinated membrane proteins are further captured
00:30:08.25 by the endosome sequestered in these little internal vesicles that then
00:30:14.07 are delivered to late endosomes and eventually to lysosomes where they can finally
00:30:20.22 be degraded. Now this happens in dendritic cells because if you look actually
00:30:26.21 by electron microscopy in a picture taken by the late Marc Pypaert is shown here
00:30:35.11 that the class II molecules are not found on the limiting membrane of lysosomes,
00:30:40.11 but rather are found associated to a first instance with these internal vesicles.
00:30:46.09 Now this was a real surprise, a double surprise because we further thought
00:30:51.15 that of course not only everything that would go into a lysosome would be
00:30:56.06 degraded, but certainly everything that was associated with the multivesicular body
00:30:59.24 would be degraded somehow dendritic cells and class II molecules have figured this out.
00:31:05.20 Now this is how the ubiquitin system works, and I'll show you the solution
00:31:10.21 to this problem at least the first solution to the problem. Ubiquitin is a small
00:31:15.15 protein that is now well known as a consequence of a series or cascade of events
00:31:22.13 to be covalently linked to a variety of different acceptors both cytosolic proteins
00:31:29.02 as well as membrane proteins by virtue of the activity of at least two large
00:31:34.19 families of so called E3 ligases that fall either into the HECT family or
00:31:40.11 the RING family, the details of this at the moment are not important,
00:31:44.10 but what is important is that both of these E3 ligases can affix ubiquitin molecules
00:31:49.16 usually the lysine acceptors on a variety of different proteins.
00:31:54.04 Now it turns out that of all the MHC class II molecules that have been
00:32:00.26 sequenced so far there’s enormous diversity in terms of the sequences
00:32:04.28 that one finds in the cytoplasmic domains of these molecules
00:32:08.13 except for a single conserved lysine residue shown here at position 4 in the cytoplasmic domain
00:32:16.13 of the beta chain of the MHC class II molecule. When Jeoung-Sook Shin
00:32:21.06 who is now at UCSF as a faculty member was in my lab, she made this
00:32:27.02 realization and further surmised that gee if there is such an important
00:32:31.21 and well conserved lysine residue present in class II molecules,
00:32:35.15 perhaps class II molecules are subject to ubiquitination. And, indeed, not only
00:32:40.25 did she find that they are subject to ubiquitination, but that ubiquitination
00:32:45.01 is exquisitely well regulated. So here you are looking
00:32:50.09 at an SDS gel which was subjected to western blot antibody labeling procedure
00:32:57.16 to detect molecules of MHC class II that are either ubiquitinated or not ubiquitinated.
00:33:03.29 So here as you can see in immature dendritic cells class II molecules
00:33:07.26 on the beta chain are nicely ubiquitinated, but again, shortly after maturation
00:33:12.25 of these cells by stimulation of the Toll-like receptor system,
00:33:16.20 you now find that those ubiquitin molecules are no longer present on class II
00:33:22.18 we believe because they are no longer added but the most important
00:33:26.14 conclusion is that they are not there, and because they are not there,
00:33:30.03 they now lack the ability to enter the cell and become sequestered
00:33:34.06 in multivesicular bodies and in lysosomes. So the bottom line is that in
00:33:39.21 short dendritic cell because ubiquitination does not occur,
00:33:43.09 those MHC class II molecule peptide complexes that are recovered by retrograde transport
00:33:49.16 from the lysosomal compartment to the plasma membrane stay put
00:33:53.19 because they lack the information, namely the ubiquitin molecule
00:33:57.02 linked to the class II that happens in immature dendritic cells.
00:34:00.23 They lack this ubiquitin molecule and as a consequence these class II peptide complexes
00:34:06.08 stay where they can best serve the interest of the immune system
00:34:10.07 and be available for recognition by Cd4 positive T-cells.
00:34:14.09 And this is what that looks like. Dendritic cells shown here in red
00:34:19.19 can complex with an enormous number of T-cells because they express
00:34:24.29 at such high levels these MHC peptide complexes in the case of class II because
00:34:31.06 those complexes cannot be internalized after maturation by endocytosis.
00:34:37.12 Class I is a different story, and I'd like to turn to that now because it illustrates
00:34:43.07 another aspect of the system as why dendritic cells are indeed so special,
00:34:48.15 not only in terms of the role they play in the immune response,
00:34:52.17 but how that role is determined by alterations, in fact some rather
00:34:57.28 unexpected alterations in membrane traffic.
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.

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 naïve, 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. This is a video in which we are just able to visualize
00:25:31.24 the movement of some of these lysosome derived tubules from their site of
00:25:35.10 formation in lysosomes out to the periphery of cells and while I'm going
00:25:39.04 to show this to you, there are various biophysical techniques that you can use
00:25:42.05 to actually show that these tubules and vesicles derived from them will
00:25:45.22 physically fuse with the plasma membrane of the cell, delivering not only
00:25:50.15 the MHC class II molecule, but obviously also the antigen that has bound
00:25:55.28 to it as a consequence of the peptide loading event that took place
00:26:01.01 in the lysosome just at the moment of dendritic cell maturation.
00:26:05.08 All of these events take place as I said in just a couple of hours
00:26:09.07 if you wait overnight you can now see live dendritic cells that look ever much like
00:26:14.29 the static images I showed you earlier. Here a cell with all of its MHC class II molecules
00:26:21.24 on the surface, lysosomes are now stained red because none of the GFP
00:26:26.14 or green fluorescent protein coupled class II molecules are present within them anymore.
00:26:31.21 So what that means then is here one more unexpected feature of the system,
00:26:38.21 which is that MHC class II molecules can escape from lysosomes
00:26:43.21 to dendritic cell surface, by a pathway which we refer to as your retrograde transport pathway.
00:26:49.26 Anterograde transport refers to what happens when molecules
00:26:54.15 such as antigens come from the outside by endocytosis into the lysosomal compartment,
00:26:59.14 retrograde refers to what happens when they go out. Again, this is probably,
00:27:04.06 or almost certainly I should say, a microtubule driven process,
00:27:07.15 but what’s most remarkable is that it happens at all. Our conventional view of
00:27:12.00 lysosomes is really kind of summarized here in this early medieval representation,
00:27:16.19 even prior to Metchnikoff, showing that there is no escape.
00:27:20.25 This is at least the conventional view. Proteins, antigens, whatever microbes are collected,
00:27:28.03 delivered into lysosomes and simply degraded. So we had not really anticipated
00:27:34.12 that there was going to be a pathway anywhere in cell biology
00:27:39.12 that was going to allow us to recover or allow a cell such as a dendritic cell
00:27:45.04 to recover molecules in a very selective and very efficient fashion
00:27:49.19 so they can move from this degradative compartment out to the cell surface.
00:27:54.00 Now when they get out to the surface, why do they stay there?
00:27:58.03 Why don't they just come back into lysosomes by endocytosis.
00:28:01.05 Well you might say endocytosis is shut off and indeed, as I already mentioned,
00:28:05.22 it is as a consequence of down regulating active forms of Rho family GTPases,
00:28:11.29 such as Cdc42 and Rac, all of which are involved in actin assembly. So the normal
00:28:20.02 ability of immature dendritic cells to capture antigen by such processes
00:28:24.11 as macropinocytosis or phagocytosis, which are both strongly actin driven
00:28:30.18 processes as we've seen in the first video, indeed are turned off. But uptake
00:28:36.18 via endocytosis by clathrin coated pits, which are much smaller vesicular carriers
00:28:43.23 that can be formed about only 0.2 microns in diameter, as opposed to the
00:28:48.17 phagosome, which can be one or two or three or even five microns in diameter,
00:28:52.24 this pathway continues. And indeed we know that MHC class II molecules
00:28:57.19 can enter cells, and indeed can enter dendritic cells by clathrin mediated
00:29:03.00 endocytosis. So why is it that class II molecules do not enter in the mature cell?
00:29:11.27 That brings us to another emerging fundamental feature of membrane traffic,
00:29:17.11 and that is the role of protein based ubiquitination in controlling the movement
00:29:22.18 of membrane proteins from one compartment to the next. Over the last several
00:29:27.05 years, a large number of investigators have demonstrated that ubiquitin plays
00:29:32.21 a critical role in signaling, either the endocytosis of endocytic receptors into
00:29:38.09 clathrin coated pits and/or the ability of those receptors at the level of endosomes
00:29:44.08 to be sequestered into these rather oddly shaped structures
00:29:48.28 called multivesicular bodies. We know from the work of Scott Ember and others
00:29:54.09 for example that when ubiquitin is modified or a membrane protein is
00:29:59.12 modified by ubiquitin, after delivery or upon delivery to the endosomal compartment,
00:30:04.19 these ubiquitinated membrane proteins are further captured
00:30:08.25 by the endosome sequestered in these little internal vesicles that then
00:30:14.07 are delivered to late endosomes and eventually to lysosomes where they can finally
00:30:20.22 be degraded. Now this happens in dendritic cells because if you look actually
00:30:26.21 by electron microscopy in a picture taken by the late Marc Pypaert is shown here
00:30:35.11 that the class II molecules are not found on the limiting membrane of lysosomes,
00:30:40.11 but rather are found associated to a first instance with these internal vesicles.
00:30:46.09 Now this was a real surprise, a double surprise because we further thought
00:30:51.15 that of course not only everything that would go into a lysosome would be
00:30:56.06 degraded, but certainly everything that was associated with the multivesicular body
00:30:59.24 would be degraded somehow dendritic cells and class II molecules have figured this out.
00:31:05.20 Now this is how the ubiquitin system works, and I'll show you the solution
00:31:10.21 to this problem at least the first solution to the problem. Ubiquitin is a small
00:31:15.15 protein that is now well known as a consequence of a series or cascade of events
00:31:22.13 to be covalently linked to a variety of different acceptors both cytosolic proteins
00:31:29.02 as well as membrane proteins by virtue of the activity of at least two large
00:31:34.19 families of so called E3 ligases that fall either into the HECT family or
00:31:40.11 the RING family, the details of this at the moment are not important,
00:31:44.10 but what is important is that both of these E3 ligases can affix ubiquitin molecules
00:31:49.16 usually the lysine acceptors on a variety of different proteins.
00:31:54.04 Now it turns out that of all the MHC class II molecules that have been
00:32:00.26 sequenced so far there’s enormous diversity in terms of the sequences
00:32:04.28 that one finds in the cytoplasmic domains of these molecules
00:32:08.13 except for a single conserved lysine residue shown here at position 4 in the cytoplasmic domain
00:32:16.13 of the beta chain of the MHC class II molecule. When Jeoung-Sook Shin
00:32:21.06 who is now at UCSF as a faculty member was in my lab, she made this
00:32:27.02 realization and further surmised that gee if there is such an important
00:32:31.21 and well conserved lysine residue present in class II molecules,
00:32:35.15 perhaps class II molecules are subject to ubiquitination. And, indeed, not only
00:32:40.25 did she find that they are subject to ubiquitination, but that ubiquitination
00:32:45.01 is exquisitely well regulated. So here you are looking
00:32:50.09 at an SDS gel which was subjected to western blot antibody labeling procedure
00:32:57.16 to detect molecules of MHC class II that are either ubiquitinated or not ubiquitinated.
00:33:03.29 So here as you can see in immature dendritic cells class II molecules
00:33:07.26 on the beta chain are nicely ubiquitinated, but again, shortly after maturation
00:33:12.25 of these cells by stimulation of the Toll-like receptor system,
00:33:16.20 you now find that those ubiquitin molecules are no longer present on class II
00:33:22.18 we believe because they are no longer added but the most important
00:33:26.14 conclusion is that they are not there, and because they are not there,
00:33:30.03 they now lack the ability to enter the cell and become sequestered
00:33:34.06 in multivesicular bodies and in lysosomes. So the bottom line is that in
00:33:39.21 short dendritic cell because ubiquitination does not occur,
00:33:43.09 those MHC class II molecule peptide complexes that are recovered by retrograde transport
00:33:49.16 from the lysosomal compartment to the plasma membrane stay put
00:33:53.19 because they lack the information, namely the ubiquitin molecule
00:33:57.02 linked to the class II that happens in immature dendritic cells.
00:34:00.23 They lack this ubiquitin molecule and as a consequence these class II peptide complexes
00:34:06.08 stay where they can best serve the interest of the immune system
00:34:10.07 and be available for recognition by Cd4 positive T-cells.
00:34:14.09 And this is what that looks like. Dendritic cells shown here in red
00:34:19.19 can complex with an enormous number of T-cells because they express
00:34:24.29 at such high levels these MHC peptide complexes in the case of class II because
00:34:31.06 those complexes cannot be internalized after maturation by endocytosis.
00:34:37.12 Class I is a different story, and I'd like to turn to that now because it illustrates
00:34:43.07 another aspect of the system as why dendritic cells are indeed so special,
00:34:48.15 not only in terms of the role they play in the immune response,
00:34:52.17 but how that role is determined by alterations, in fact some rather
00:34:57.28 unexpected alterations in membrane traffic.
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.

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.