Allergy and autoimmunity occur when immune cells are activated by non-pathogenic antigens. In the case of allergies, the immune system recognizes innocuous non-self-antigens (e.g. proteins in peanuts), while in autoimmunity the immune system attacks cells expressing self-antigens. This session explores the molecular underpinnings of allergy and autoimmunity. Mast cells and IgE antibodies take center stage in the allergic response, while rogue T or B cells are the primary actors in autoimmunity.
00:00:09.17 My name is Avery August,
00:00:10.27 I'm at Cornell University,
00:00:12.02 and today I'm going to tell you a little bit about allergies
00:00:15.02 and the immune system.
00:00:16.20 So, this is an image of some peanuts,
00:00:19.12 and this simple nut
00:00:21.28 can have devastating consequences
00:00:23.21 in some individuals.
00:00:24.29 And the reason for this
00:00:26.20 is because they're allergic to proteins
00:00:28.09 that are found in peanuts.
00:00:30.11 So what I will be telling you about
00:00:32.29 is why they're allergic to proteins
00:00:34.23 found in peanuts.
00:00:37.05 So, the allergic response
00:00:39.19 is actually the combination
00:00:41.11 of the response of a number of different cells,
00:00:43.24 and these cells are shown over here
00:00:45.17 -- the innate lymphoid cell,
00:00:47.10 T helper 2 cell,
00:00:48.19 B cell,
00:00:50.07 and a mast cell or basophil --
00:00:51.28 and these cells interact
00:00:53.22 to drive the allergic response.
00:00:57.17 The other thing that's important for allergies
00:01:01.16 is the antibody IgE.
00:01:03.23 In the early to late 1960s,
00:01:06.07 Ishizaka and Lichtenstein discovered
00:01:09.23 that IgE was actually responsible for allergies.
00:01:13.15 Now, IgE is an isotype
00:01:15.26 of an antibody
00:01:17.23 and it looks just like an antibody.
00:01:19.08 It has two binding sites,
00:01:20.27 it has an Fc portion,
00:01:23.01 and it can interact with a receptor,
00:01:24.08 and so we'll come back to this structure later
00:01:26.25 when we look at the actual response to IgE.
00:01:30.17 Allergies are actually the result
00:01:32.18 of an immune response
00:01:34.00 and the immune response is divided
00:01:35.19 into two main types of responses.
00:01:37.15 An innate immune response,
00:01:38.29 on the right,
00:01:40.13 and an adaptive immune response,
00:01:41.26 on the left.
00:01:43.02 Now, the innate immune response
00:01:44.22 can respond to an allergen very quickly,
00:01:46.12 within minutes to hours,
00:01:47.28 but it does so in a non-specific way
00:01:50.18 because it only recognizes patterns
00:01:52.06 that are found on allergens.
00:01:54.15 But this response
00:01:56.17 can dictate the type of adaptive immune response
00:01:58.23 that we have.
00:02:01.00 By contrast, the adaptive immune response
00:02:03.21 is actually what is responsible for the allergy.
00:02:07.19 That adaptive immune response
00:02:09.15 takes some time to respond
00:02:11.01 -- it takes up to days to respond --
00:02:12.23 but its exquisitely specific,
00:02:15.17 and it results in the generation of antibodies,
00:02:18.07 and those antibodies can then determine
00:02:20.01 the clearance of pathogens
00:02:21.24 or, in the case of allergies,
00:02:23.06 whether the person is allergic or not.
00:02:26.25 So, what makes something allergenic?
00:02:29.09 Allergens have to be able to generate a B cell response
00:02:32.29 because B cells are what make antibodies.
00:02:35.21 Those allergens are generally proteins,
00:02:37.26 such as peanut allergens or egg white proteins.
00:02:40.23 They can be carbohydrates,
00:02:42.13 such as those found in meat.
00:02:44.03 Quite a few allergens are actually proteases
00:02:46.27 or are able to bind lipids.
00:02:48.29 Most of them are water soluble,
00:02:51.00 they're stable,
00:02:52.17 and they're small.
00:02:53.19 In some cases, they're resistant to heat.
00:02:55.15 And generally they can bind to pattern recognition receptors
00:02:58.02 on innate cells,
00:02:59.20 or protease activated receptors on epithelial cells,
00:03:02.16 then initiate the immune response to them.
00:03:07.12 So, how does an allergic response develop?
00:03:11.25 Well, first, you're exposed to an allergen
00:03:15.27 and generally that allergen will interact
00:03:17.24 with an epithelial cell.
00:03:19.06 That epithelial cell will produce cytokines,
00:03:22.00 such as IL-33 and interleukin-25 (IL-25),
00:03:25.13 and those two cytokines can drive the activation
00:03:28.08 of the innate lymphoid type 2.
00:03:31.03 That ILC2 cell
00:03:32.29 will produce a cytokine called interleukin-4,
00:03:36.03 and what interleukin-4 does...
00:03:37.23 it can condition dendritic cells
00:03:40.20 and T cells
00:03:42.27 to change the nature of that immune response.
00:03:45.05 Dendritic cells will pick up those allergens,
00:03:47.20 will process them into small pieces,
00:03:49.28 and present that allergen to T cells,
00:03:52.08 and when that T cell is interacting
00:03:54.07 with the dendritic cell,
00:03:55.19 under the influence of interleukin-4,
00:03:57.25 that T cell becomes a T helper 2 cell (Th2),
00:04:00.11 and that T helper 2 cell
00:04:02.09 is critical for the development of an allergy
00:04:04.07 because that T helper 2 cell
00:04:06.08 is what's responsible for
00:04:08.10 making interleukin-4 and interleukin-4
00:04:10.15 is really important for the production of IgE.
00:04:14.15 IgE is made by B cells.
00:04:17.17 And so, here,
00:04:19.08 during the development of that initial immune response
00:04:21.03 to the allergen,
00:04:22.29 there's no symptom,
00:04:24.07 the immune symptom is responding,
00:04:26.01 and it's responding
00:04:28.09 by picking up the allergen,
00:04:29.16 taking it to the lymph nodes,
00:04:32.11 and when that allergen reaches the lymph node,
00:04:34.07 it interacts with a B cell, and that B cell,
00:04:36.28 a very small number of B cells, one or two,
00:04:38.24 can interact with that allergen
00:04:40.09 and get activated.
00:04:41.13 And under the right conditions,
00:04:42.17 with the right help from T cells,
00:04:44.04 that B cell divides and multiplies
00:04:45.29 to become a larger number of B cells,
00:04:48.18 and so now we have a large number of B cells
00:04:50.27 that recognize that antigen.
00:04:53.05 Under the right conditions,
00:04:54.22 in the presence of help from T helper 2 cells,
00:04:58.23 particularly interleukin-4,
00:05:00.16 that B cell undergoes what's called class switch.
00:05:03.04 It changes the type of [antibody] that it makes
00:05:06.06 from IgM to IgE.
00:05:09.09 Once that B cell undergoes class switch,
00:05:11.20 now it can start to make IgE
00:05:14.02 by becoming a plasma cell
00:05:15.22 and secreting IgE.
00:05:17.22 Once that IgE is in circulation,
00:05:20.02 now the individual
00:05:21.28 can now respond to whatever that allergen is
00:05:24.08 the second time they get exposed.
00:05:27.06 So, the first time you're exposed to allergen,
00:05:29.12 you don't really notice it,
00:05:31.00 but your immune system is responding
00:05:32.22 and it's generating this whole process
00:05:34.27 that leads to the production of IgE.
00:05:37.07 So the second time you get exposed
00:05:39.03 to the allergen,
00:05:40.17 now you're more susceptible
00:05:42.17 to developing a allergic response,
00:05:45.03 and that occurs
00:05:47.01 because there are these other cell types in the body,
00:05:48.23 called mast cells or basophils,
00:05:50.17 that have receptors for IgE.
00:05:53.01 And those mast cells are found
00:05:55.01 in the mucosal areas of the tissues,
00:05:56.22 the respiratory, GI tract,
00:05:58.10 and the skin,
00:05:59.22 and there are basophils that are found in the blood,
00:06:01.18 and both cell types
00:06:02.24 have these receptors for IgE.
00:06:06.01 So here's what a basophil looks like in a blood smear.
00:06:08.26 You can see the staining
00:06:10.19 and you can see the granules
00:06:12.07 that these basophils have,
00:06:14.03 and red cells around them.
00:06:15.25 And here is a section of the skin
00:06:18.04 from a mouse
00:06:20.01 that we've stained with Toluidine blue
00:06:21.14 and you can see that the mast cells
00:06:22.28 are situated in the skin,
00:06:24.15 and you can see the granules, again,
00:06:26.00 in these mast cells.
00:06:30.07 Here is an electron micrograph
00:06:31.28 of a skin mast cell
00:06:33.09 and you can see, at very high resolution,
00:06:35.08 the structures of the granules,
00:06:37.23 including the one circled here in white,
00:06:40.11 that are filled with pharmacological agents
00:06:42.21 that these cells will release
00:06:44.15 when they get activated.
00:06:48.23 So, the contents of these granules
00:06:50.09 include histamine,
00:06:55.09 and cytokines,
00:06:56.15 and these pharmacological agents
00:06:58.07 have different physiological effects.
00:06:59.11 For example, histamine increases vascular permeability
00:07:01.22 and smooth muscle contraction,
00:07:03.24 whereas heparin induces swelling,
00:07:06.01 anaphylactic and inflammatory symptoms,
00:07:08.10 and proteases can remodel the extracellular matrix
00:07:11.10 and cause changes in the migration of cells.
00:07:15.27 And the mast cells can also produce cytokines
00:07:17.27 that can further promote inflammation
00:07:19.23 and other types of responses.
00:07:23.13 Mast cells also make other products:
00:07:25.12 other cytokines that are made later after activation,
00:07:29.02 chemokines that can attract other immune cells
00:07:30.28 to the site of activation,
00:07:32.13 and lipid mediators
00:07:34.18 that can also have effects on smooth muscle cells
00:07:36.17 and induce mucous secretion.
00:07:39.25 So what happens, then,
00:07:42.01 when this circulating IgE is made
00:07:44.28 the first time you got exposed to this allergen?
00:07:46.17 Well, that circulating IgE
00:07:48.00 can now interact with the receptors
00:07:50.03 on these mast cells and basophils
00:07:52.05 and we call that "arming"
00:07:54.04 of these mast cells and basophils with IgE,
00:07:56.14 because now the receptors are occupied with the IgE
00:08:00.04 and the mast cell or basophil
00:08:01.24 is now primed
00:08:03.19 to be able to respond a second time,
00:08:04.25 or subsequent times,
00:08:06.07 that you actually get exposed to that allergen.
00:08:09.23 So, here's the structure of IgE.
00:08:12.07 You can see the antigen binding site
00:08:14.07 bound to its receptor,
00:08:15.27 and this is the receptor that will be found
00:08:17.25 on these basophils and mast cells.
00:08:19.21 You can see, now, that
00:08:21.28 when this IgE binds to its receptor
00:08:23.07 it's now ready and it looks like a receptor
00:08:25.13 that can respond to antigen.
00:08:29.12 So, now, the second time
00:08:31.09 you get exposed to that peanut allergen,
00:08:33.16 the allergen interacts with the IgE
00:08:35.19 that's found on the mast cell and basophil
00:08:39.02 and it triggers the degranulation
00:08:41.19 of this mast cell or basophil,
00:08:43.00 where they release all their contents,
00:08:44.29 and those contents start to have physiological effects.
00:08:48.12 So, the first thing that happens
00:08:49.29 when that allergen binds to the IgE
00:08:52.28 found in the receptor
00:08:54.17 is the activation of calcium,
00:08:56.01 and so in this image
00:08:57.21 you will see that in the upper right-hand corner
00:08:58.24 antigen is added,
00:09:00.07 and then the cells start to respond
00:09:01.20 by increasing intracellular calcium,
00:09:03.14 shown in bright green,
00:09:05.05 as the cells get activated,
00:09:06.20 and you can see that, eventually,
00:09:08.03 the cells start getting activated in this field.
00:09:14.03 The next thing that happens after activation
00:09:16.19 is degranulation,
00:09:18.00 and in this particular video
00:09:19.23 what you'll see is that the mast cell is activated,
00:09:21.29 and as the granules are released
00:09:23.25 they pick up a dye
00:09:25.17 that allows you to visualize
00:09:27.09 the degranulated granules,
00:09:29.27 and that dye is shown in red.
00:09:42.02 Now you can start to see
00:09:43.25 the mast cell degranulate,
00:09:45.09 you can see the granules reach the cell surface,
00:09:46.21 they pick up the dye,
00:09:48.06 and now we can now see that the granules
00:09:49.24 have been released.
00:09:57.28 Once those granules have been released,
00:10:00.09 they now look empty.
00:10:01.14 So, here's an EM, an electron micrograph,
00:10:03.16 of a non-degranulated mast cell
00:10:06.22 on the [left]
00:10:08.08 and a degranulated mast cell on the [right],
00:10:09.24 and you can see that the granules are less dark,
00:10:12.14 indicating that they've released their contents.
00:10:18.08 So, what happens after these mast cells degranulate?
00:10:20.25 Well, blood vessels
00:10:23.01 start to increase in their size
00:10:25.19 because of the contents of the mast cells.
00:10:28.08 You get increased blood flow to the area
00:10:30.14 and this can cause reduced blood pressure,
00:10:32.23 irregular heart beat,
00:10:34.28 and, in some cases,
00:10:36.14 can result in systemic anaphylactic shock.
00:10:39.03 If this happens in the airways,
00:10:40.15 you get airway smooth muscle contraction,
00:10:42.20 you get an increase in mucus production,
00:10:44.24 and this can cause difficulty breathing,
00:10:46.21 swallowing, or wheezing
00:10:48.20 in those people who have allergic responses
00:10:50.23 in the airways.
00:10:51.24 If it happens in the GI tract,
00:10:53.13 if you get exposed to peanut allergen in the GI tract,
00:10:55.20 here the GI smooth muscle cells contract,
00:10:58.22 and that then leads to peristalsis
00:11:00.26 and fluid secretion,
00:11:02.13 it can cause stomach cramps,
00:11:03.22 vomiting, diarrhea...
00:11:05.03 classic symptoms of food allergy.
00:11:08.23 So, in the airways,
00:11:10.20 one can actually see mast cells
00:11:12.18 lining the trachea
00:11:14.04 in between the epithelial cells,
00:11:16.08 and those mast cells actually
00:11:18.16 reach in between the epithelial cells
00:11:19.23 and sample antigens and allergens
00:11:21.26 that are coming into the airways,
00:11:23.13 and you can see that here.
00:11:24.19 You can see that these are sections of trachea
00:11:27.12 that are stained with a mast cell protein,
00:11:32.04 and you can see it's stained in red,
00:11:33.16 and you can see an airway, here,
00:11:36.02 that is lined with mast cells.
00:11:39.17 And those mast cells actually
00:11:41.21 are continually sampling, in the video on the left,
00:11:43.22 continually sampling contents
00:11:46.00 that are passing through the airways
00:11:47.15 to determine whether there's anything
00:11:49.04 that they recognize and,
00:11:50.29 if there's something that they recognize,
00:11:52.11 then they will respond and degranulate,
00:11:54.15 and you can have some of these symptoms.
00:12:00.07 And so, in the end,
00:12:01.19 if this happens in the airway,
00:12:03.15 there's a cartoon, here,
00:12:04.25 of an open airway on the [left]
00:12:06.07 -- this is a normal airway
00:12:07.17 , the individual can breathe very clearly --
00:12:09.29 when the individual is exposed to allergens,
00:12:12.14 the airway smooth muscle cells expand,
00:12:14.20 the epithelial cells expand,
00:12:16.18 and you have mucus
00:12:18.06 that partially blocks the airway,
00:12:19.16 making it more difficult to breathe.
00:12:25.25 So, how do we actually block this response?
00:12:28.22 Well, you can prevent
00:12:31.16 the release of these granules
00:12:32.25 by blocking either the activation of the mast cell
00:12:36.02 or you can block the release of the contents,
00:12:37.19 and if you do that then
00:12:40.04 you can potentially reduce symptoms of allergies.
00:12:42.28 And so there are a number of agents that have been discovered
00:12:45.06 over the years
00:12:46.14 that can actually block
00:12:48.23 the release of mast cell contents,
00:12:51.07 including drugs that are based on Chromolyn,
00:12:53.12 such as Nasacrom,
00:12:55.07 and what that does,
00:12:56.18 in a way that we don't quite understand,
00:12:57.28 is it prevents the mast cell from releasing its granules
00:13:00.16 when it gets activated,
00:13:01.21 so by preventing the release of the granules,
00:13:04.02 you prevent the release of
00:13:06.02 histamine, heparin, TNF,
00:13:07.28 and all the other cytokines
00:13:10.06 that are responsible for the allergic response.
00:13:12.18 In other cases,
00:13:14.18 we don't actually block the release of the granules,
00:13:16.26 but we in some cases can block
00:13:18.28 the effects of the granule contents,
00:13:20.14 such as antihistamines,
00:13:22.21 which can reduce increased vascular permeability
00:13:25.11 and smooth muscle contraction
00:13:27.05 due to the histamine release.
00:13:28.15 So, when you get an allergic response,
00:13:30.03 you take an anti-histamine.
00:13:31.17 What you're actually doing
00:13:32.28 is preventing histamine
00:13:34.18 from having an effect on these smooth muscle cells.
00:13:38.26 If the histamine release has already occurred,
00:13:42.18 in the case of systemic anaphylactic shock,
00:13:44.24 we can actually counter the effects of histamine
00:13:47.14 by taking epinephrine,
00:13:48.23 which counteracts the effects of histamine,
00:13:51.23 and here's a picture of an EpiPen
00:13:54.02 that individuals who have severe allergies
00:13:56.27 will carry around with them
00:13:59.00 to use in case of an emergency,
00:14:00.18 so that they can counteract the effects of histamine.
00:14:04.24 In addition, there are other types of drugs,
00:14:07.10 such as the drugs that are based on the Singulair series,
00:14:10.12 and these drugs actually do
00:14:13.00 similar things to the anti-histamines.
00:14:14.12 They actually block the effects
00:14:16.12 of the leukotrienes that are produced by mast cells,
00:14:18.28 which have the same effects
00:14:20.14 -- smooth muscle contraction and edema.
00:14:22.13 And the way they do this is by competing
00:14:25.07 with the leukotrienes for the receptors
00:14:26.24 on these smooth muscle cells,
00:14:28.09 therefore preventing their action on these cells.
00:14:31.19 Another drug that was recently discovered
00:14:34.15 by a company called Genentech
00:14:36.14 is a drug that actually targets the IgE
00:14:40.24 and blocks it from interacting with its receptor,
00:14:43.19 and in that particular drug
00:14:45.00 -- it's a drug called Xolair --
00:14:46.08 what it actually does is...
00:14:48.01 actually it's an antibody
00:14:49.25 that binds to the Fc portion of IgE
00:14:51.25 and prevents it from interacting with its receptor.
00:14:54.16 And when it does so,
00:14:55.22 now the mast cell or basophil
00:14:57.07 can no longer respond,
00:14:58.13 because they don't have the IgE on their surface,
00:15:00.11 they cannot recognize the allergen,
00:15:01.27 and so you reduce the symptoms
00:15:03.17 of all allergic responses.
00:15:06.05 And so Xolair has actually worked very well
00:15:08.09 in reducing the symptoms of allergies
00:15:10.20 because it reduces the ability
00:15:12.21 of mast cells and basophils to respond.
00:15:14.28 So, in summary, then,
00:15:16.17 what we see is that
00:15:18.02 the first time you get exposed to an allergen,
00:15:19.27 you generate an immune response
00:15:21.26 that leads to the generation of IgE.
00:15:24.18 That IgE then coats
00:15:27.23 the mast cells and basophils,
00:15:29.06 and when those mast cells and basophils
00:15:30.22 actually come in contact with the allergen,
00:15:32.14 they respond to the allergen
00:15:34.08 and release their contents,
00:15:35.20 leading to the symptoms of allergies.
00:00:08.15 So, I'm Diane Mathis.
00:00:10.07 I'm a professor of immunology
00:00:11.29 at Harvard Medical School,
00:00:13.17 and this talk, my second one,
00:00:17.23 is focused on one particular mechanism
00:00:21.29 of enforcing T cell tolerance,
00:00:24.07 which depends on the transcription factor Aire.
00:00:29.11 Now, in my first talk,
00:00:31.09 I explained to you that
00:00:34.13 tolerance mechanisms are needed
00:00:37.25 because of the random manner in which
00:00:40.25 the repertoires of antigen-specific receptors
00:00:44.15 displayed on B cells and T cells
00:00:48.09 are generated during their differentiation
00:00:51.24 in the thymus and bone marrow.
00:00:56.10 And then I went on to
00:00:59.21 define and describe several of the mechanisms,
00:01:03.00 the major mechanisms of imposing tolerance.
00:01:07.13 And then, finally,
00:01:10.10 I explained that these tolerance mechanisms
00:01:15.01 break down, actually, relatively frequently,
00:01:17.20 and the result is the development of autoimmune disease,
00:01:21.15 diseases like type-1 diabetes, or myasthenia gravis,
00:01:25.26 or multiple sclerosis.
00:01:30.11 Now, this talk is focused on
00:01:36.00 one of the more recently discovered
00:01:40.06 means of enforcing tolerance,
00:01:42.17 that is, it was discovered about 15 years ago,
00:01:45.25 and it depends on a transcription factor called Aire.
00:01:51.25 It's an important mechanism
00:01:54.03 and a fascinating mechanism,
00:01:57.08 at least I think so and I hope you'll agree with me
00:01:59.29 by the time we get to the end of the talk.
00:02:03.25 this mechanism actually makes...
00:02:07.26 links together two of the important mechanisms,
00:02:12.14 one in central tolerance
00:02:14.11 and one in peripheral tolerance.
00:02:16.28 So, the Aire story starts with
00:02:20.17 a human disease called APS-1,
00:02:24.17 for autoimmune polyglandular syndrome type-1.
00:02:29.08 And these individuals have
00:02:31.15 severe Candidiasis infections
00:02:34.13 of mucosal surfaces.
00:02:36.02 They also have autoimmune attack
00:02:38.27 on the parathyroid glands
00:02:40.19 and autoimmune attack on the adrenal glands.
00:02:43.25 Now, these are the three most frequent symptoms
00:02:46.01 and they're the symptoms which are used
00:02:49.22 to diagnose the disease,
00:02:51.19 but all of the individuals with APS-1
00:02:55.10 have multiple other autoimmune manifestations.
00:02:58.17 They might have type-1 diabetes
00:03:01.00 or they have autoimmune attack on the ovaries or the liver,
00:03:03.18 and what these organ targets are
00:03:07.08 varies from individual to individual,
00:03:09.02 even for people that are in the same family
00:03:12.02 and have the same mutation.
00:03:16.10 So, in 1997,
00:03:19.21 two groups independently cloned
00:03:23.09 the gene underlying APS-1
00:03:25.08 and they called the protein that it encodes
00:03:29.01 Aire, for autoimmune regulator.
00:03:30.29 Aire was a quite large protein
00:03:33.21 of more than 500 amino acids,
00:03:35.27 and by now more than 50 mutations,
00:03:38.15 scattered through the Aire gene,
00:03:42.29 have been identified in different individuals with APS-1.
00:03:50.11 So, from the beginning,
00:03:52.07 Aire was thought to be some kind of transcriptional regulator,
00:03:57.00 and I'll show you later on that this, indeed,
00:03:59.16 turns out to be the case.
00:04:02.24 So, one important clue
00:04:05.04 to how Aire is working came
00:04:09.05 just from knowing where it's expressed.
00:04:10.28 So, it's expressed primarily in the thymus,
00:04:14.01 not by the differentiating T cells themselves,
00:04:17.11 but rather by the stromal cells,
00:04:19.16 the epithelial cells,
00:04:23.07 which nurture their differentiation and allows the different processes
00:04:27.24 that need to take place for T cell maturation.
00:04:31.16 They're localized...
00:04:34.13 Aire is localized in the medulla
00:04:36.10 and, within the medulla,
00:04:38.21 specifically in a very small subset of epithelial cells
00:04:42.27 which we call medullary epithelial cells,
00:04:45.14 and they make up only 0.5%
00:04:48.27 of the stromal cells in the thymus.
00:04:52.27 And the reason that this finding elicited interest
00:04:57.29 was that, at that time,
00:05:00.06 there was a body of data that was growing larger and larger
00:05:02.22 that these cells
00:05:06.08 actually express a large repertoire of RNA transcripts
00:05:10.04 encoding what we normally think of as
00:05:14.01 proteins particular for fully differentiated cells.
00:05:18.19 So, for example, one is insulin
00:05:21.09 or there might be myelin basic protein,
00:05:24.10 or a heart protein, or a liver protein.
00:05:28.04 In fact, when it was looked at very carefully,
00:05:32.13 it was found that many tissues in the body,
00:05:36.04 more than 30,
00:05:37.25 are represented by transcripts
00:05:41.13 in this very small population of cells.
00:05:46.17 And so the notion developed that these transcripts
00:05:50.11 would be translated into proteins
00:05:53.02 and, actually, if you have good antibodies
00:05:55.19 you can find these proteins
00:05:57.09 -- you can find insulin in these cells, for example --
00:06:00.29 and these proteins would be degraded
00:06:04.03 by normal mechanisms of antigen processing
00:06:07.08 and loaded onto MHC molecules
00:06:09.17 and be shuttled to the surface of the cell.
00:06:13.07 And then, as the self-reactive, differentiating T cell
00:06:17.23 is percolating through the thymus,
00:06:20.06 if its T cell receptor recognizes
00:06:22.23 this peptide-MHC complex
00:06:25.28 in a particular window of affinity or avidity,
00:06:30.04 T cell tolerance will take place.
00:06:34.03 And it was our hypothesis that,
00:06:36.12 actually, Aire is controlling the transcription
00:06:39.20 of this repertoire of transcripts
00:06:44.11 encoding peripheral tissue antigen...
00:06:48.20 proteins, or PTAs is what we call them.
00:06:51.23 And that was because of the overlap
00:06:55.25 in where Aire is expressed,
00:06:57.17 as well as the fact that individuals that have an Aire mutation
00:07:01.04 have a multi-organ autoimmune disease.
00:07:04.06 So, to evaluate our hypothesis, we
00:07:09.19 -- and, actually, other investigators, independently --
00:07:12.29 made mice which were lacking Aire
00:07:16.02 -- Aire knockout mice.
00:07:18.24 And when we looked at these mice, indeed,
00:07:21.00 they had inflammatory infiltrates in several organs.
00:07:27.03 I'm showing the salivary gland, here, and the thyroid gland,
00:07:29.20 where you can see these infiltrating leukocytes,
00:07:33.18 which you don't see in the wild type...
00:07:36.18 corresponding wild type tissues.
00:07:39.24 These mice also have auto-antibodies,
00:07:42.16 circulating auto-antibodies,
00:07:44.13 against many different organs.
00:07:46.18 So, basically, what you do here is
00:07:49.28 you just take serum from a wild type mouse
00:07:52.22 or a knockout mouse and use it...
00:07:56.04 incubate it with normal tissue
00:07:59.03 and then come in with a secondary step,
00:08:01.24 which allows you to light up
00:08:05.23 those regions where the antibody bound.
00:08:09.06 And so, the serum from the Aire knockout mice,
00:08:11.10 but not from the Aire wild type,
00:08:12.29 lit up a lot of different organs,
00:08:15.01 and actually some specific structures in different organs.
00:08:20.15 So, it was the parietal cells of the stomach
00:08:22.07 and the rods and cones region of the retina, for example.
00:08:26.01 Then a very critical experiment
00:08:30.12 was the one that's depicted here,
00:08:32.10 where we isolated RNA
00:08:35.22 from medullary epithelial cells
00:08:38.14 coming from an Aire wild type mouse,
00:08:40.18 on the x axis,
00:08:42.19 or an Aire knockout mouse, on the y axis,
00:08:44.27 and what I'm showing you is
00:08:47.16 whole-genome expression profiling,
00:08:52.13 where each dots represents expression of a particular gene
00:08:57.10 and how it's expressed in the wild type
00:08:59.24 is its point on the y axis... on the x axis,
00:09:03.09 and how it's expressed in the knockout is its point on the [y axis].
00:09:06.06 And where its induced by Aire
00:09:10.15 falls below the diagonal.
00:09:12.14 And when we looked at this,
00:09:14.28 what we found was that
00:09:17.11 Aire actually induced hundreds of transcripts
00:09:19.25 in medullary epithelial cells,
00:09:21.24 in particular, transcripts that were encoding these PTA proteins,
00:09:27.11 or peripheral tissue antigens.
00:09:31.05 Now, these data which I've shown you
00:09:33.13 were the original data that we got back in 2002,
00:09:37.19 and we could see that Aire was controlling hundreds of transcripts.
00:09:40.12 And more recently, there are more performant methods
00:09:43.24 to look at gene expression,
00:09:47.24 and, for example, what we call RNAseq,
00:09:51.08 which allows us to look at a lot more transcripts
00:09:53.12 and in a more quantitative way,
00:09:55.10 and what we found there surprised us in the fact that
00:09:59.10 Aire actually induces thousands of transcripts,
00:10:02.28 more than a quarter of the genome.
00:10:08.18 And then, the last experiment is
00:10:12.03 what I call closing the circle,
00:10:15.01 and what we did there was to take Aire knockout mice,
00:10:18.02 and in those mice there are circulating auto-antibodies,
00:10:22.15 and some of these are against stomach proteins,
00:10:26.02 which I showed you on the previous slide.
00:10:29.18 We then isolated...
00:10:31.04 we then identified the antigen
00:10:33.09 that these stomach antibodies saw
00:10:37.00 and found out that it's the mucin protein.
00:10:41.02 And then we went back
00:10:43.02 and looked into the medullary epithelial
00:10:47.26 gene expression profile and found that, indeed,
00:10:50.25 Aire was regulating mucin transcripts,
00:10:53.16 because when Aire wasn't there
00:10:56.13 mucin transcript levels in the thymus were reduced.
00:10:59.04 Other investigators did similar experiments,
00:11:02.24 one with an eye antigen
00:11:05.26 and another with salivary gland antigen,
00:11:08.29 and came out with the same conclusion.
00:11:11.21 So, I think that the model which we proposed
00:11:16.17 actually turned out to be correct,
00:11:18.23 according to a number of criteria,
00:11:20.19 and the model was close enough to the human disease
00:11:24.23 that it could be used to dissect specific mechanisms.
00:11:31.15 So, let's look a little bit at the cellular mechanisms involved.
00:11:36.07 First of all, you might have noticed that
00:11:40.04 I was very vague about what happened
00:11:42.18 once the differentiating thymocytes
00:11:44.23 saw the MHC-self-peptide complex.
00:11:46.19 I just said T cell tolerance takes place.
00:11:49.23 Now, there are several mechanisms
00:11:52.28 by which this might happen.
00:11:55.26 It could be that Aire promotes negative selection,
00:11:59.11 or clonal deletion,
00:12:01.14 of the self-reactive effector cells
00:12:03.25 that are going to get out into the periphery
00:12:07.08 and wreak havoc.
00:12:09.05 It could instead, or in addition,
00:12:11.15 be that Aire promotes positive selection
00:12:14.09 of regulatory T cells
00:12:16.25 that control the activities of these effector T cells.
00:12:21.13 Or Aire could do something completely different,
00:12:23.12 maybe something totally unexpected,
00:12:25.16 for example, it could control
00:12:28.15 antigen-presenting cells
00:12:30.20 or some other kind of innate cell
00:12:34.14 that starts off an autoimmune response.
00:12:40.00 Indeed, we found that Aire does promote negative selection,
00:12:43.23 or clonal deletion of self-reactive cells,
00:12:45.22 and I'll show you the original experiment
00:12:51.03 which demonstrated that.
00:12:53.11 So, this takes advantage of a trick
00:12:58.14 that immunologists use,
00:13:00.10 the creation of T cell receptor transgenic mice,
00:13:02.22 and this just tries to get around the fact that
00:13:08.06 any particular antigen specificity
00:13:10.09 is usually at a very low frequency,
00:13:12.17 so only 1 in 10^4 to 1 in 10^6 T cells
00:13:17.09 will be specific for a particular antigen.
00:13:19.19 And so, what one can do is
00:13:22.16 isolate T cell receptor transgenes
00:13:25.08 from a particular T cell clone
00:13:27.07 that are already rearranged,
00:13:29.10 introduce those into mice,
00:13:31.13 and that will shut off endogenous T cell receptor gene rearrangements,
00:13:38.16 and so the mouse will have a repertoire
00:13:40.14 that's highly skewed for that antigen specificity.
00:13:43.24 And in this case we add an additional twist onto that,
00:13:47.04 in that we create one mouse
00:13:50.18 that has a neo-self-antigen
00:13:53.19 expressed somewhere,
00:13:55.06 and then we create a T cell receptor transgenic mouse
00:13:57.20 that's capable of seeing this neo-self antigen.
00:14:02.08 So, an example is, for the neo-self antigen,
00:14:06.07 we make a mouse which is expressed ovalbumin,
00:14:10.12 membrane-bound ovalbumin,
00:14:13.04 under the dictates of the rat insulin promoter,
00:14:16.05 so it should be expressed in the pancreas
00:14:19.15 and, as I now should have convinced you,
00:14:21.24 is also expressed in the thymus.
00:14:25.26 And the second mouse, the reporter mouse,
00:14:28.05 is a T cell receptor transgenic mouse
00:14:33.17 which is capable of seeing a peptide
00:14:37.11 for membrane-ovalbumin.
00:14:39.25 So, you can see that when we cross these two mice together,
00:14:42.14 we create a potentially explosive situation
00:14:47.12 where there are many, many T cells
00:14:50.05 expressing this self-reactive specificity.
00:14:53.11 So, this slide shows you what happens
00:14:56.29 in the thymus of these mice.
00:14:59.17 We're looking at thymocytes
00:15:03.09 by staining for the CD4 and CD8 co-receptors,
00:15:07.21 and what you see is that,
00:15:09.15 in the absence of the neo-self antigen,
00:15:13.23 just the plain T cell receptor transgenic,
00:15:15.26 we find a lot of CD4...
00:15:18.15 fully mature CD4 single-positive T cells maturing.
00:15:24.15 And that's because the original clone
00:15:27.04 that we started with was a CD4+ T cell clone,
00:15:30.04 so that's expected.
00:15:31.28 And this is what happens
00:15:34.20 when you now cross the mice to the transgenic line
00:15:38.01 which is expressing the neo-self antigen.
00:15:41.03 In the presence of Aire, these CD45...
00:15:44.08 these CD4+ T cells are absent
00:15:48.24 -- they disappear, they're clonally deleted.
00:15:50.24 However, in the absence of Aire,
00:15:54.14 those T cells come back.
00:15:58.20 So, that clonal deletion does not take place
00:16:01.22 when Aire's not there.
00:16:03.16 And, actually, the animals develop
00:16:06.28 autoreactivity of the pancreas.
00:16:13.12 So, other labs did similar experiments
00:16:17.01 using other TCR transgenic
00:16:20.27 neo-self antigen models
00:16:23.01 and, in addition,
00:16:25.01 non-transgenic systems
00:16:28.04 could demonstrate that Aire was required
00:16:31.12 for clonal deletion.
00:16:33.04 So, I think we can be more specific here
00:16:35.06 and say that Aire is
00:16:39.05 controlling negative selection of self-reactive effector T cells.
00:16:43.16 Now, it turns out that Aire also controls
00:16:46.09 positive selection of regulatory T cells.
00:16:49.29 When we first started studying this,
00:16:51.29 we didn't think that that was the case
00:16:54.06 because we looked at a lot of mice
00:16:56.01 and we didn't see much difference
00:16:58.18 in the frequency or the type of Treg cell
00:17:03.17 that these mice had
00:17:06.13 -- so, CD4+/Foxp3+ regulatory T cells --
00:17:10.28 and that's because we were always looking at adult mice.
00:17:14.05 Later, we looked earlier
00:17:17.14 and did find that there was a deficit
00:17:19.26 in this regulatory T cell population
00:17:22.29 before 10 days of age.
00:17:25.14 Now, we wondered if this was very meaningful,
00:17:30.01 because it's not a huge difference
00:17:32.06 -- the cells aren't totally gone --
00:17:34.09 and it's only during this quite narrow time window
00:17:38.01 where one sees the difference.
00:17:40.00 So, we designed an experiment
00:17:42.05 which allowed us to show
00:17:45.03 whether regulatory T cells, during that time period,
00:17:47.20 are important.
00:17:49.07 So, we could do both a loss-of-function experiment
00:17:52.01 and a gain-of-function experiment.
00:17:54.22 So, in the loss-of-function experiment,
00:17:56.24 we took a mouse
00:18:00.19 where we could turn Foxp3 off
00:18:03.20 whenever we wanted to.
00:18:05.28 And so if we turn off Foxp3
00:18:08.14 then we won't get Tregs made during that time window.
00:18:12.14 So, we used that mouse to
00:18:15.03 deplete regulatory T cells
00:18:20.14 that are being generated during the first ten days of life,
00:18:23.16 and when we did that the mice developed multi-organ autoimmunity,
00:18:25.18 as indicated by the shaded blocks
00:18:29.28 that I show you, here.
00:18:32.03 If we did the same experiment,
00:18:34.10 however we took an adult mouse
00:18:37.16 where we depleted Tregs for 10 days,
00:18:39.06 we saw only rare and quite sporadic autoimmunity.
00:18:45.09 Now, for the gain-of-function experiment,
00:18:48.01 what we did was take Aire knockout mice,
00:18:50.15 which, as you saw before,
00:18:52.11 will develop multi-organ autoimmunity,
00:18:55.09 and then we took Tregs that were generated specifically
00:18:58.27 during that 10-day age window just after birth,
00:19:02.14 and we added those in,
00:19:05.20 and those mice were highly protected from autoimmunity.
00:19:08.27 However, if we took regulatory T cells
00:19:12.12 that were made during a 10-day window in an adult,
00:19:16.01 they were not protected.
00:19:17.18 So, by both the loss-of-function and gain-of-function experiments,
00:19:22.14 it was clear that Aire
00:19:27.16 was controlling an important population of regulatory T cells,
00:19:31.16 and this was specifically
00:19:34.29 during a very early time window.
00:19:38.05 Now, that made sense to us,
00:19:40.03 because we had an earlier finding
00:19:42.20 which we were not able to understand
00:19:45.11 until we got these more recent results,
00:19:47.21 and that is that we made a mouse
00:19:49.27 where we could turn Aire on and off at will.
00:19:53.28 So, if we had Aire on during the whole life of the mouse,
00:19:59.11 there was tolerance and the mouse didn't develop any autoimmunity.
00:20:03.10 And if we had Aire off during the whole life of the mouse,
00:20:06.24 it did develop autoimmunity.
00:20:09.23 That wasn't very surprising;
00:20:11.10 that's what we expected at that point.
00:20:13.14 However, what was surprising was that
00:20:17.00 if we had Aire on only for the first 7 or 10 days of life,
00:20:22.15 and then turned it off,
00:20:25.21 the mice were tolerant -- they didn't develop any signs of autoimmunity.
00:20:28.10 And then, conversely,
00:20:31.18 if we had Aire off during the first 7-10 days of life
00:20:35.10 and then turned it on for the rest of the life of the animal,
00:20:38.15 they did develop severe autoimmunity.
00:20:41.15 So, the expression of Aire during this very early,
00:20:47.06 this perinatal time window,
00:20:49.18 seemed to be necessary and sufficient to predict...
00:20:53.13 to protect from the autoimmune disease
00:20:56.02 that develops in the absence of Aire.
00:20:59.23 And so, we can also add that
00:21:03.07 Aire is also functioning
00:21:07.02 to promote positive selection of regulatory T cells.
00:21:10.05 I would be remiss if I didn't add that
00:21:13.13 other experiments in other laboratories
00:21:15.21 have suggested that Aire might have additional functions.
00:21:19.12 It might be involved in
00:21:22.01 differentiation of medullary epithelial cells,
00:21:24.11 differentiation of 𝛾:δ cells,
00:21:27.00 the turnover of medullary epithelial cells,
00:21:31.00 or peripheral tolerance.
00:21:35.15 Okay, ummm...
00:21:37.23 so, I think those are the major points
00:21:40.07 I'd like to make about the cellular mechanisms of Aire.
00:21:42.17 Now, I'd like to turn to the molecular mechanisms.
00:21:45.20 And I'll start out by saying this is an area
00:21:47.28 where it's been very dark for some time,
00:21:52.24 and light is just beginning to be shed.
00:21:57.29 People have been fascinated
00:22:00.04 by the molecular mechanism of Aire
00:22:02.02 since the beginning,
00:22:03.14 and the reason for that is that
00:22:05.22 here is a transcription factor
00:22:08.06 that's controlling thousands of genes
00:22:10.26 in a very small population of cells in the thymus.
00:22:16.11 And these genes, in the periphery,
00:22:20.00 are expressed very differently.
00:22:23.08 They're expressed in different cells,
00:22:25.11 they're expressed at different levels,
00:22:27.07 and they're expressed at different times
00:22:30.10 during the ontogeny of the individual,
00:22:33.22 so how can this one transcription factor do that?
00:22:38.22 So, from the beginning,
00:22:40.08 it was thought that Aire
00:22:43.19 was a transcriptional regulator,
00:22:45.09 and that's because it has
00:22:49.08 structural and functional features
00:22:52.05 of a transcriptional regulator.
00:22:53.27 So, structurally, it has a SAND domain
00:22:58.09 and, in other transcription factors,
00:23:00.28 the SAND domain is a DNA-binding domain.
00:23:03.12 However, I should mention that the important
00:23:09.24 amino acids in Aire...
00:23:11.26 the important amino acids for DNA binding in other proteins
00:23:15.25 are mutated in Aire
00:23:18.03 -- they're not the same as they are in these other proteins.
00:23:20.19 Aire has a nuclear localization signal.
00:23:23.10 It has a CARD domain,
00:23:25.11 which is important for homo-oligomerization.
00:23:29.10 It also has two PHD domains,
00:23:31.23 which are used in various types of protein-protein interactions.
00:23:41.02 As far as functional features,
00:23:42.27 Aire is localized in the nucleus,
00:23:45.01 as one would expect for a transcription factor.
00:23:47.06 It can induce transcription
00:23:50.02 -- so, if you make an expression vector where...
00:23:56.25 which will allow Aire expression if you transduce or transfect
00:24:01.17 the plasmid into a cultured cell,
00:24:08.19 and you also transfect a reporter gene
00:24:12.27 driven by the interferon promoter,
00:24:16.20 Aire will induce expression of that reporter.
00:24:20.06 So, it also binds to known transcription factors.
00:24:24.00 The first one identified was CBP,
00:24:27.10 or CREB-binding protein.
00:24:29.09 Now, Aire only binds very weakly
00:24:32.07 and non-specifically to DNA,
00:24:34.22 even though it has the SAND domain,
00:24:38.18 which I mentioned.
00:24:40.04 It seems to be more involved in binding
00:24:43.07 to different chromatin proteins
00:24:45.05 than directly to DNA itself.
00:24:50.17 So, even though it looked and smelled like a transcription factor,
00:24:52.29 there were always some odd things about Aire
00:24:55.18 which made one question
00:24:58.26 whether it was a classical transcription factor
00:25:01.29 that would bind to a promoter and induce or repress transcription
00:25:06.14 of a particular locus.
00:25:09.17 So, first of all, it's regulating thousands of genes,
00:25:14.01 so it seemed unlikely that they would
00:25:16.26 all have a specific binding site for Aire.
00:25:20.06 Secondly, it induces genes
00:25:25.26 which encode proteins that are found in many different cell types.
00:25:29.23 And, thirdly, it's possible to
00:25:34.04 introduce Aire artificially into different cell types,
00:25:38.04 either in culture or making transgenic mice,
00:25:41.21 for example, putting Aire behind the rat insulin promoter,
00:25:44.06 and in all these different cell types
00:25:46.27 Aire will induce batteries of transcripts.
00:25:49.14 However, the particular transcripts
00:25:52.17 that it does induce
00:25:54.27 differ from cell type to cell type,
00:25:56.23 and usually there's only about a 10-20% overlap
00:26:00.01 when you're comparing different cell types.
00:26:04.14 So, people have been working quite hard on this puzzle,
00:26:09.13 and I have to say that
00:26:13.16 we don't have a complete answer yet,
00:26:15.19 but we have learned some very important things
00:26:18.25 over the past couple of years.
00:26:20.21 So, one of them is that Aire
00:26:24.08 partners with many different proteins.
00:26:29.20 Now, the way that this experiment was done
00:26:33.23 was to take chromatin
00:26:37.09 from an Aire-expressing cell,
00:26:39.23 immunoprecipitate Aire
00:26:42.22 and then use mass spectromety...
00:26:49.17 to identify the proteins that are binding to Aire.
00:26:52.05 And of course you have to do
00:26:54.23 many different types of controls
00:26:57.09 to prove the validity of this assay,
00:27:01.06 which were done.
00:27:03.00 And, even after these controls,
00:27:05.19 it became clear that Aire binds to...
00:27:08.20 interacts with scores of proteins,
00:27:10.22 either directly or within the same complex.
00:27:15.10 Up here, I'm showing about 40,
00:27:17.13 but even since this figure was made
00:27:20.07 there are another 5-10 which have been identified.
00:27:23.21 Now, some of these... and these proteins fall into four different classes:
00:27:27.24 nuclear transport,
00:27:30.12 pre-messenger RNA processing,
00:27:32.11 chromatin function,
00:27:34.10 and the DMA-damage response
00:27:38.24 and some transcriptional activation function.
00:27:41.19 So, it's not surprising that Aire would bind to proteins
00:27:47.08 involved in nuclear transport,
00:27:48.23 because it is in the nucleus,
00:27:50.27 or chromatin function,
00:27:53.01 because we know it's a transcriptional regulator somehow,
00:27:55.12 but some of the other classes of proteins
00:27:57.26 were somewhat surprising,
00:27:59.21 like pre-mRNA processing.
00:28:01.16 And, after this was found, it...
00:28:05.10 experiments were done which showed that
00:28:08.12 Aire does actually control pre-mRNA processing.
00:28:10.26 And then, lastly,
00:28:14.17 these proteins that are involved in transcription
00:28:19.00 but are also part of the DNA-damage response.
00:28:24.01 So, that's one important thing.
00:28:25.29 The second important thing is that
00:28:28.08 Aire seems to be operating very early
00:28:30.28 in the process of gene transcription
00:28:33.25 by RNA polymerase-II.
00:28:37.05 So, when I was learning about transcription,
00:28:41.25 some years ago,
00:28:44.14 we used to think that there were two types of genes:
00:28:47.24 genes that had RNA polymerase on them,
00:28:50.02 and they were transcribed;
00:28:52.07 and genes that didn't have RNA polymerase on them,
00:28:55.15 and they weren't transcribed.
00:28:56.20 But, over the last, say, 5-8 years,
00:28:59.14 it's become clear that there's a third class of genes
00:29:03.13 which is very important,
00:29:06.09 and these genes are ones which RNA polymerase binds,
00:29:11.01 it moves a little bit into the gene,
00:29:13.09 and then just stops.
00:29:14.29 And these are called paused or stalled polymerase genes,
00:29:20.13 and it's clear that every cell type
00:29:23.11 that's been looked at so far
00:29:25.17 has a group of genes
00:29:27.27 which have paused polymerase on them.
00:29:29.29 So, to explain this a little better,
00:29:34.06 let me give you some detail.
00:29:36.27 So, what happens is that
00:29:40.09 RNA polymerase binds to the transcriptional start site
00:29:43.12 and it has at its C-terminal end
00:29:47.09 a set of repeats of a particular sequence
00:29:50.16 that has serines in it that become phosphorylated.
00:29:54.23 And so it binds at the transcriptional start site
00:29:57.28 and lets its C-terminal domain hang out,
00:30:02.18 and then it clears the promoter,
00:30:06.03 once there has been phosphorylation
00:30:11.06 of the serine residues at position 5 in this repeat
00:30:14.21 by the transcriptional factor TFIIH.
00:30:20.09 It proceeds a little further
00:30:23.22 and then just stops.
00:30:26.07 This is a paused polymerase
00:30:28.21 and it has been thought that one of the reasons that it pauses
00:30:32.11 is to allow time to allow capping of the RNA to take place.
00:30:36.22 Now, pausing is enforced by these two transcription factors,
00:30:40.28 NELF and DSIF,
00:30:44.04 and it's lifted, or released,
00:30:47.16 by the combined action of Brd4 and the heterodimer P-TEFb.
00:30:55.07 So, this set of proteins comes in,
00:30:57.28 causes additional phosphorylation of the C-terminal domain,
00:31:02.09 causes phosphorylation of NELF,
00:31:05.04 which releases it,
00:31:07.13 and turns DSIF into a positive transcriptional regulator.
00:31:13.13 And then elongation can proceed.
00:31:15.29 So, what Aire does is that
00:31:21.26 it releases RNA polymerase pausing
00:31:26.03 on paused genes.
00:31:27.20 And it's now become clear that
00:31:30.24 this site of RNA polymerase pausing
00:31:33.24 is a site of impact for many important types of
00:31:38.15 transcriptional regulation.
00:31:40.02 It's where c-Myc works,
00:31:41.27 it's where much of the regulation of LPS
00:31:44.28 -- lipopolysaccharide --
00:31:48.22 induced genes in macrophages is controlled,
00:31:53.07 and this is where Aire is operating.
00:31:56.15 So, we have a number of piece of evidence that point to that.
00:32:01.12 First of all, if we go back to
00:32:05.05 our genome-wide gene expression profiles
00:32:07.08 and, instead of looking at the whole gene...
00:32:10.14 the whole...
00:32:13.02 a group of Aire-induced genes and looking at the whole gene,
00:32:15.29 we look at different regions along the gene,
00:32:19.10 what we find is that Aire's impact
00:32:22.11 at the beginning of the gene is relatively low,
00:32:24.29 but it's impact later on is much higher,
00:32:27.26 which is indicative of an effect on elongation.
00:32:33.11 Secondly, Aire interacts directly with
00:32:38.29 Brd4 protein and also P-TEBb proteins.
00:32:43.03 And, thirdly, if we take inhibitors of
00:32:47.01 Brd4 or P-TEFb
00:32:49.28 or things which lift polymerase pausing,
00:32:54.05 those inhibitors also inhibit
00:32:57.15 Aire-induced gene transcription.
00:33:03.10 So, we're quite convinced that this is
00:33:06.27 a key element of Aire's molecular mechanism of action.
00:33:11.12 So, in fact, this mechanism goes quite far
00:33:14.15 in explaining many of these Aire oddities,
00:33:18.26 which I mentioned before.
00:33:21.08 It's clear, then, that it can
00:33:24.01 simultaneously control thousands of genes,
00:33:25.28 because thousands of genes
00:33:27.27 have polymerase paused on them.
00:33:29.15 It can induce
00:33:32.04 peripheral tissue antigen genes
00:33:34.23 associated with many cell types,
00:33:36.15 because, in fact, it's been found that,
00:33:38.22 preferentially, genes that are paused
00:33:42.01 are genes which are going to later be induced
00:33:44.20 or are later important for cell type differentiation.
00:33:49.26 And then, finally, it explains why,
00:33:52.19 if you put Aire into different cell types,
00:33:55.17 different sets of transcripts are induced,
00:33:57.16 and that's because different cell types
00:33:59.28 have different repertoires of paused genes.
00:34:04.18 And it also explains the large number of protein partners
00:34:09.07 that Aire has,
00:34:11.08 and that's because this scaffold,
00:34:14.21 this tail, this C-terminal domain tail,
00:34:18.10 which becomes phosphorylated
00:34:21.04 when polymerase pausing is lifted,
00:34:24.23 is an important scaffold for a number of cellular processes.
00:34:29.06 So, this... these phosphorylation events control RNA capping.
00:34:36.06 They'll also be very important in regulating splicing,
00:34:42.10 and even nuclear export.
00:34:45.15 And then, lastly, both DSIF
00:34:48.19 and the C-terminal domain
00:34:50.16 have been implicated in controlling different histone modifications.
00:34:56.26 So, that's where we stand today
00:35:01.24 with both the cellular and molecular mechanism of Aire,
00:35:05.12 and of course there are some very interesting questions
00:35:08.00 which remain to be answered.
00:35:10.15 If we look back at the cellular mechanisms,
00:35:13.00 I think the most pressing issue is to determine...
00:35:18.15 is to determine,
00:35:22.16 what's so special about this repertoire of regulatory T cells
00:35:25.04 that develops in the first 10 days of a mouse?
00:35:27.22 Do they have a particular repertoire?
00:35:29.29 What are the antigens that they are seeing?
00:35:32.25 And why are they so important
00:35:35.13 for protecting against autoimmunity,
00:35:37.28 when regulatory T cells are of course generated
00:35:40.17 throughout the life of the animal?
00:35:42.17 If we look at the molecular mechanism,
00:35:45.01 it's clear that we have some important bits and pieces
00:35:51.03 about how Aire operates at the molecular level,
00:35:55.19 but we need to learn, still, some important things,
00:35:58.29 like, how does Aire actually target the genes
00:36:01.22 that it is going to induce?
00:36:04.05 Is it sufficient that the gene just be paused
00:36:06.08 and there's something about that configuration
00:36:08.21 which Aire can recognize,
00:36:10.16 or is there some other element
00:36:14.03 which is used for recognition?
00:36:15.24 And then, finally, what I haven't told you yet
00:36:19.10 is that Aire does control
00:36:22.08 a lot of these peripheral tissue antigen transcripts
00:36:25.11 in medullary epithelial cells,
00:36:27.15 but there are also some that Aire doesn't control.
00:36:30.19 So, is there an Aire-2?
00:36:33.24 And, if that's the cause, what's its identity?
00:36:38.10 there's no homologous protein,
00:36:40.29 or no highly homologous protein, I should say.
00:36:43.16 So, what's its identity,
00:36:46.05 and does it operate in the same way that Aire does.
00:36:49.22 And, lastly, I'd like to thank or acknowledge
00:36:53.26 the people in the lab who have worked on Aire
00:36:56.26 over the past 15 years,
00:37:01.08 and I think found some very interesting things.
00:00:07.22 I'm Ted Yednock and I'm an immunologist trained at the University of California in San Francisco.
00:00:11.17 And for a number of years, I worked for a company called Elan pharmaceuticals.
00:00:15.09 At Elan, and we developed a drug called Tysabri for the treatment of multiple sclerosis.
00:00:20.09 So, I'm going to talk about the identification and development of Tysabri in two lectures.
00:00:26.04 Today, I'm going to talk about the scientific underpinnings of Tysabri and how we think
00:00:31.02 it works by inhibiting immune cell infiltration of the central nervous system,
00:00:35.15 preventing the damage associated with the autoimmunity in MS.
00:00:40.11 I'll also talk about the preclinical work and the clinical studies that we did in order
00:00:44.10 to demonstrate that the drug was safe and efficacious.
00:00:48.03 In the second lecture, I'll talk about how we believe the selective mechanism of this drug
00:00:52.20 allowed for the emergence of a very rare but serious brain infection called
00:00:57.27 progressive multifocal leukoencephalopathy, or PML.
00:01:01.19 And I'll also talk about the complexity that that caused for patients, and physicians,
00:01:06.25 and the FDA, as well as the companies, in trying to understand how PML was associated
00:01:12.07 with Tysabri.
00:01:13.22 So, at first, I...
00:01:15.05 I will say a few words about multiple sclerosis.
00:01:22.00 So, MS is an autoimmune disease, of the CNS, as I mentioned.
00:01:26.02 It's involved in the destruction of myelin.
00:01:27.25 So, there's a very strong T and B cell response against myelin.
00:01:31.27 And once myelin is lost, you have impaired nerve conduction and neuronal loss.
00:01:36.11 Now, MS is... can happen anywhere in the central nervous system, so it can affect any part
00:01:40.26 of your body.
00:01:41.27 A patient may feel tingling in an arm or hand, or they may have loss of vision, or issues
00:01:49.15 with control of bladder function, or... or not be able to walk.
00:01:54.06 It affects about 300,000 patients in the United States.
00:01:57.06 It's more common in women than men.
00:01:59.12 And, as I've alluded to, it's a devastating disease and it affects people in the
00:02:02.25 prime of life, between the ages of 20 and 40.
00:02:06.04 But it can be seen at any age.
00:02:08.23 Now, usually, MS is a relapsing... it begins as a relapsing-remitting disease.
00:02:14.05 So, the attacks can come along very quickly, you know, vision loss can happen very quickly,
00:02:19.17 and then, over a period of days to months, there will be a degree of recovery to varying levels.
00:02:25.08 But after 15 or 20 years, the disease often becomes progressive.
00:02:30.18 And this happens in about 65% of patients.
00:02:33.23 And now, it's more of a chronic neurodegenerative disease, without the acute inflammatory attacks.
00:02:40.12 So, MRI is actually a very useful diagnostic and monitoring tool for multiple sclerosis.
00:02:46.14 So, this is an MRI image.
00:02:48.14 And what... what is often done in MS is that patients are given a contrasting agent called
00:02:54.22 It's injected intravenously, and normally it's excluded from the brain.
00:02:58.27 However, in MS, when there's an active lesion, the blood-brain barrier is leaky and so gadolinium
00:03:04.03 will enter the brain and show up very prominently in an MRI.
00:03:08.03 Now, if you were to look at this lesion through a microscope, you would see this.
00:03:13.17 This is a... a blood vessel in multiple sclerosis.
00:03:17.09 And what's happened here is there are immune cells traveling throughout the body in the bloodstream
00:03:22.27 and... now, normally, they just go right through the brain.
00:03:25.10 But in the case of MS, there's something happening to the blood vessel wall, and the immune cells
00:03:30.12 will adhere to the vessel wall, migrate in, and accumulate in these large clusters around
00:03:36.15 the blood vessel.
00:03:37.15 And this basically is ground zero for demyelination and neuronal damage.
00:03:41.18 So, in 1990, we had a very simple hypothesis, and that was that if we could inhibit immune
00:03:48.00 cell attachment to the blood vessel wall we could prevent their migration into the brain
00:03:51.19 and all the damage associated with that.
00:03:53.26 And so... when I say adhesion molecule, picture Velcro.
00:03:57.22 So, we're trying to identify the little hooks on the... on the immune cell that allow it
00:04:01.22 to attach to the blood vessel wall, specifically at times of inflammation.
00:04:05.28 Now, since this is a brain disease, it's really important to be able to do these kind of studies
00:04:11.09 using an... an animal model.
00:04:12.23 So, the best animal model for multiple sclerosis is experimental autoimmune encephalomyelitis,
00:04:18.24 or EAE.
00:04:20.20 Now, in EAE... this is a... a... a model in the guinea pig, but we also use models in
00:04:25.19 mice and rats.
00:04:26.26 So, in this model, this is a brain... a slice through the brain of a guinea pig with EAE.
00:04:32.03 You can see a large blood vessel.
00:04:34.05 And in the blood vessel there are infiltrating immune cells, which are the blue cells, here.
00:04:39.23 And the section is stained with a red dye for myelin.
00:04:42.18 So, you can see that around the blood vessel there's a huge loss of myelin.
00:04:47.22 And then, also, the... the little green dots are axons, or nerve fibers, that are... are...
00:04:54.02 you're looking at them in cross-section.
00:04:56.07 And normally, over here, you can see that the nerve fibers are surrounded by red myelin,
00:05:01.12 so they're quite well insulated from each other.
00:05:03.27 But when the myelin is lost, the nerve cells or the nerve fibers are pushed around and clustered,
00:05:07.24 and the spacing between is occupied by immune cells and by edema.
00:05:11.15 And so you can see, with this kind of damage, how nerve conduction would be impaired.
00:05:18.18 This is the way the model progresses in animals.
00:05:21.03 So, in the guinea pig, we immunize the animals with spinal cord homogenate.
00:05:26.17 This causes a very strong response, again, of B cells and T cells to myelin.
00:05:31.19 And so that... by day 9 or 10, the immune cells will begin to infiltrate the
00:05:35.14 central nervous system, and you'll begin to see a degree of hind limb paralysis in the... in
00:05:40.01 the acute phase.
00:05:41.01 And then the animals begin to get a little bit better.
00:05:43.21 But then, by day 15 or so, the disease begins to, well, progress, and enters the chronic phase.
00:05:50.18 And the... the difference here is this is when demyelination begins.
00:05:53.20 So, once demyelination begins, the nerve fibers are pushed around, the immune cells are even
00:05:58.15 more activated, and the disease progresses with just more and more demyelination.
00:06:03.27 When we were first doing these experiments, we were a very small lab in a...
00:06:07.05 in a small company and we really didn't have a good model of EAE.
00:06:11.19 In fact, we weren't working in this area at all.
00:06:14.17 We... we were trying to develop therapeutics for Alzheimer's disease.
00:06:18.14 And we were trying to develop an animal model of Alzheimer's.
00:06:21.06 It would have been the world's first model, which... which we eventually did, but
00:06:25.15 our initial attempts weren't so good.
00:06:27.07 And in fact, we really were inducing a model in which there was a chronic inflammation.
00:06:33.21 And it's that kind of inflammation that initially got us interested in this area to begin with.
00:06:37.26 So, we took inflamed brains from these animals in this strange model of Alzheimer's disease,
00:06:43.24 and did a very simple experiment.
00:06:45.27 We took these sections and overlaid them or exposed them to a suspension of human lymphocytes.
00:06:51.20 And we found that the lymphocytes would adhere selectively to these inflamed vessels.
00:06:55.26 So, you can see these very dark blue, large circles are the human lymphocytes, and they're
00:07:02.06 adhering right in the center of these blood vessels, right where you would expect to see
00:07:06.06 blood cells in the circulation.
00:07:08.13 They don't bind elsewhere in the section and they didn't bind to non-inflamed vessels.
00:07:12.16 So, the fact that we were getting physical adhesion of these cells to inflamed vessels
00:07:18.21 suggests that this is probably a physiologically relevant... relevant interaction.
00:07:22.26 Because these ligands -- whatever these adhesive ligands... whatever they are -- were being
00:07:26.07 induced in the context of a whole brain.
00:07:29.04 We also isolated endothelial cells from rat brains and put them into culture,
00:07:34.25 stimulated them with TNF, and we found, there,
00:07:37.17 that lymphocytes would adhere here as well.
00:07:39.17 So, that was great.
00:07:41.00 We now had two different assays by which we could look at lymphocyte interaction with
00:07:44.21 inflamed endothelium, so we could screen for inhibitors of that interaction.
00:07:49.12 What we did is made thousands of antibodies against the lymphocyte surface, at random,
00:07:54.22 and screened for antibodies that would inhibit this.
00:07:56.25 So, in this assay, we could screen thousands of antibodies.
00:08:00.15 And in this one, we could screen umm... perhaps in dozens.
00:08:03.07 It was a much more cumbersome assay.
00:08:06.00 But, nonetheless, both assays gave us exactly the same answer.
00:08:09.22 And that is that alpha 4 beta 1 integrin is very important for lymphocytes and monocytes
00:08:15.28 to adhere to the... to the inflamed brain vessel.
00:08:18.16 So, in the presence of anti-alpha 4 or anti-beta 1, you can see that there was no longer any
00:08:24.00 human lymphocyte attachment to the vessels in this section.
00:08:29.09 So, this is what we thought was going on.
00:08:33.10 Lymphocytes and monocytes expressed alpha 4 beta 1 integrin and they were binding to
00:08:37.01 some ligand expressed by inflamed brain endothelial cells.
00:08:41.19 So, we also knew in 1990... and, in fact, I was...
00:08:45.01 I was kind of disappointed with this observation.
00:08:48.19 Because we knew that alpha 4 integrin was expressed by many cells in the circulation
00:08:53.13 -- lymphocytes, monocytes, eosinophils, basophils.
00:08:56.22 Unfortunately, it was low or negative on neutrophils, red blood cells, and platelets.
00:09:02.06 However, we had been hoping to find sort of a brain-specific adhesion molecule that was
00:09:06.15 expressed by a small subset of... of pathology-causing immune cells.
00:09:10.27 But, in fact, it was expressed very broadly.
00:09:13.20 Nonetheless, as you'll see, I believe that alpha 4 beta 1 integrin turned out to be
00:09:17.23 a pretty selective adhesion molecule for the CNS.
00:09:22.00 The molecule, alpha 4, had been cloned the year before by Yoshi Takada and Martin Hemler's group.
00:09:27.06 And a number of groups were also identifying the ligands for alpha 4.
00:09:32.13 For example, Elizabeth Wayner had found that an alternatively spliced form of fibronectin
00:09:36.20 was a ligand for alpha 4, and also Roy Lobb at Biogen had identified VCAM-1,
00:09:43.14 expressed by human umbilical vein endothelial cells in culture.
00:09:47.18 So... but no one really knew how alpha 4 beta 1 worked in vivo and how it contributed to
00:09:52.16 disease pathology.
00:09:54.00 And that's when we go back to umm... the need for a good animal model of EAE.
00:09:58.15 So, the model that we had with our Alzheimer's-inflamed brain wasn't really going to be good enough
00:10:03.21 for that.
00:10:04.21 So, we went down the street and visited Larry Steinman at Stanford.
00:10:09.19 And Larry Steinman is a world expert in multiple sclerosis and in EAE.
00:10:14.10 And we took... we went to him and showed him our results with alpha 4 beta 1 integrin,
00:10:18.17 and we had antibodies against alpha 4, and we asked him if he could help us test it in
00:10:23.05 a... in a really good model of EAE.
00:10:25.09 And that's exactly what he did.
00:10:28.03 Nathan Karin, who was a postdoc in his lab at the time, was working with a rat model
00:10:32.21 of EAE.
00:10:33.21 So, the first thing we did was confirm that using brain... sections of inflamed brain
00:10:38.11 from animals with... rats with EAE, that we had the same sort of lymphocyte adhesion,
00:10:43.04 and that it was completely dependent upon alpha 4 integrin.
00:10:46.14 Nathan then tested one of our antibodies against alpha 4 and found that it was very protective
00:10:52.00 in preventing disease in these... in this animal model.
00:10:54.21 So, that was great.
00:10:56.09 Based on that, we... we started bringing in a number of different EAE models into our...
00:11:01.07 our little lab.
00:11:02.26 This was one, again, in the guinea pig.
00:11:04.25 And here we wanted to characterize more thoroughly what... how alpha 4 integrin works, how quickly
00:11:10.12 it works, and the impact that it can have on the disease.
00:11:13.15 So, in this study, we labeled lymphocytes with indium-111, which is a very high gamma emitter.
00:11:20.19 And we found that if you inject the cells back into the bloodstream they will migrate
00:11:25.18 to the brain.
00:11:26.18 So, you can just take out the brain and do a gamma count and see how many cells there.
00:11:30.07 But, in the presence of anti-alpha 4, inhibition was 80 or 90%.
00:11:35.11 So, in this short-term migration assay, lasting about 18 hours, alpha 4 integrin was clearly
00:11:41.09 affecting the ability of cells to migrate into the brain.
00:11:45.17 And then we treated guinea pigs with the antibody, just at the onset of disease.
00:11:51.06 This was like day 10 or 11, just when paralysis was beginning but before demyelination had happened.
00:11:57.28 And what we found is that by day 20 we had a very strong impact in preventing demyelination.
00:12:02.28 So, in the control-treated animal, you can see that they're... we have stained these
00:12:07.19 sections of spinal cord with a blue dye for myelin, and there's an extensive loss of myelin
00:12:14.01 by day 20 in the control animals.
00:12:16.01 However, in the presence of anti-alpha 4, the myelin is almost completely intact.
00:12:20.05 So, there was very strong protection against the loss of myelin.
00:12:26.22 We next... we next treated animals at day 20.
00:12:30.15 So, this is after demyelination has already occurred.
00:12:34.02 And we treated them for a long time -- 56 days -- because you wanted to see if it would
00:12:38.06 impact the disease and if that impact would last.
00:12:42.04 And what we saw was this: a very strong reversal of the paralysis and... that lasted for
00:12:47.17 the entire time of the treatment.
00:12:50.19 Okay, so we did this experiment a number of times, looking at different time points to
00:12:54.19 see what impact that we would have on myelin.
00:12:58.07 So, in this study, we treated the animals after demyelination had already occurred,
00:13:05.03 and then we inhibited alpha 4 integrin for various periods of time.
00:13:09.17 We found that after 20 or 40 days of treatment that we actually would allow myelin to repair.
00:13:15.26 So, the way we knew this was that under control conditions myelin is either present or it's not.
00:13:22.21 So, over here, you can see that... here is myelin, this is an area where the myelin
00:13:27.08 has been eliminated, and the... the line between the two is very, very stark.
00:13:31.25 So, it's either there or it's not.
00:13:34.17 However, in animals that had been treated with an inhibitor of alpha 4 integrin,
00:13:37.23 we saw this very pale blue pallor, which we confirmed by electron microscopy to be regeneration
00:13:44.16 of myelin.
00:13:45.16 So, we believe that what was happening is that by inhibiting immune cell infiltration
00:13:49.14 into the CNS we were dampening down this inflammatory response and basically creating an environment
00:13:55.22 that was permissive for remyelination.
00:13:57.22 So, these are the studies that led to the development of Tysabri, which is a... we took
00:14:05.20 our best antibody against alpha 4 integrin and humanized it by CDR grafting.
00:14:10.16 And this was done in collaboration with the MRCC in London.
00:14:14.19 And fortunately, the antibody retained the full potency of its murine parent.
00:14:22.00 Just to give a perspective on scale, this is a model of an antibody next to a model
00:14:26.26 of an integrin.
00:14:28.10 Umm... the... the antibody actually binds to the head group, up here, which is the
00:14:33.02 business end of the integrin.
00:14:34.02 This is where it binds to ligands.
00:14:35.22 Now, recently, the crystal structure of natalizumab bound to the head groups of alpha 4 integrin
00:14:42.04 has actually been solved, by Yamel Yu in Timothy Spring... in Tim Springer's group.
00:14:48.11 So, this is looking at the crystal structure, just at the head groups of the integrin umm...
00:14:54.23 and the blue is beta 1 and the green is alpha 4.
00:14:58.18 And the ligand binding site -- this is for VCAM-1 -- is right in between the two.
00:15:03.03 So, there are critical contact residues on both sides of the line.
00:15:06.12 Now, natalizumab, he found, bounds right next to this.
00:15:10.11 So, the binding actually doesn't overlap.
00:15:12.26 And, in fact, if you take the business end of VCAM, the first domain, it will bind just
00:15:18.16 fine in the presence of... of Tysabri.
00:15:21.20 However, the second domain of VCAM causes steric hindrance, so that, when natalizumab...
00:15:29.10 when natalizumab is bound in the presence of the full-length ligand, its binding energy
00:15:34.10 is greatly decreased because of the steric hindrance.
00:15:37.06 What they also noticed in the lab was three critical contact residues within the natalizumab
00:15:43.14 binding site that help to explain observations that we had made a number of years earlier.
00:15:48.12 So, when you're developing a drug, it's important to have animal species for efficacy testing,
00:15:53.05 but you also need to have two different animal species for toxicology.
00:15:56.27 You need to be able to show that it's safe in at least two species.
00:16:00.28 So, we were disappointed because our... our best model for EAE at the time was in the rat.
00:16:06.22 However, the rat has a mutation in one of these critical residues, and we found that
00:16:10.16 the antibody didn't react.
00:16:12.28 There also was an EAE model in the marmoset monkey, which was a model that was set up
00:16:18.05 at UCSF.
00:16:19.05 And, again, we found that our antibody did not react with the monkey, which... we were
00:16:22.11 really surprised, because it's very closely related to the human.
00:16:26.12 But now it makes sense with... with the finding that there's a mutation in one of these critical
00:16:31.02 amino acids.
00:16:32.12 So, we did find, however, that the... the antibody reacted with cynomolgus monkey and
00:16:38.20 with guinea pig.
00:16:39.20 And, as you can see, that the sequence of these... of the three critical contact residues
00:16:44.03 in these species are identical to those in human.
00:16:46.27 And these are the species that we had chosen for our toxicology.
00:16:52.21 So, in order to develop a drug, you need to do a lot of toxicology studies.
00:16:58.21 This is just a subset of the things that we did.
00:17:00.21 And I'm not going to go through all these, other than to mention a couple findings.
00:17:05.18 First of all, in the first study up here, it was a six-month study in cynomolgus monkey
00:17:10.15 involving very high doses of... of natalizumab.
00:17:13.08 Umm... natalizumab, by the way, is the generic name for Tysabri.
00:17:17.12 So, we did 60 milligrams per kilogram dosing every week.
00:17:22.12 And, in patients, we find that 3 milligrams per kilogram dosed every month
00:17:27.06 is what it takes for efficacy.
00:17:29.15 So, in fact, in the monkeys, we were achieving blood levels more than a thousand-fold that
00:17:33.28 you would find in humans.
00:17:35.10 So, at the end of 6 months, we looked to see what impact it would have, particularly on
00:17:40.02 the immune system.
00:17:41.03 Remember, alpha 4 integrin is expressed by almost all circulating mononuclear cells.
00:17:46.00 And so we were quite interested to see what would happen in the immune system.
00:17:50.17 And much to our relief, we found that hematopoiesis... bone marrow was... was very much intact, so
00:17:57.10 lymphocytes and red blood cells were being made normally.
00:18:00.05 And this was important because alpha 4 integrin had been shown to be involved in hematopoiesis.
00:18:04.11 We also did immunotoxics... immunotox... immunotoxicity profiling and found that if you vaccinated
00:18:13.20 animals in the presence of Tysabri the... they responded just... just fine to the vaccine.
00:18:18.24 There was a slight delay in the initial antibody response, but the animals quickly caught up.
00:18:25.01 And other than that, in these studies we also looked at the impact of natalizumab on implantation,
00:18:31.03 in development, and... as well as on development of the fetus during the full pregnancy,
00:18:37.16 and found very little impact there.
00:18:39.25 So, to summarize our... our preclinical and our toxicology studies, functional screens
00:18:47.09 are... are really important when trying to understand a disease process.
00:18:50.10 And this may be an obvious thing to say, but I'm just so impressed with the simplicity
00:18:54.26 of the tissue assay that we had performed, looking at the endogenous induction of ligands
00:19:01.13 involved in this inflammatory process.
00:19:03.19 In fact, this tissue assay is something that was established by Stamper and Woodruff in the 70s,
00:19:08.22 and had... has been used successfully for identifying a number of immune adhesion receptors.
00:19:14.20 Our other findings were that anti-alpha 4 was effective in multiple models of CNS inflammation,
00:19:19.19 whether it was a very funny Alzheimer's model or EAE.
00:19:22.18 And, in fact, in different labs around the world, it's been shown to be effective in
00:19:27.10 rat, mouse, guinea pig, and primate EAE, as well as
00:19:31.03 two different models of CNS viral inflammation.
00:19:34.14 So, these are models in which there's a CNS infection by a virus, the immune system goes
00:19:39.26 in to fight the virus and causes damage, and we found that anti-alpha 4 was actually protective there.
00:19:46.04 It protected against the damage and the... the body was still able to effectively fight
00:19:51.00 the virus.
00:19:52.00 So, again, it was suggest... suggesting that... that natalizu... natalizumab would be safe.
00:19:57.20 There was a lot of criticism of EAE models, because it doesn't exactly mimic the processes
00:20:04.07 of multiple sclerosis.
00:20:05.26 But in a case like this, where we're focusing on the immune infiltration aspect, I think
00:20:10.25 it's actually a very good model.
00:20:12.20 Because immune cells need to enter the brain to effectively induce EAE, and they also need
00:20:17.05 to go into the brain to cause MS.
00:20:19.24 So, by studying mechanisms involved in this infiltration process, I think that EAE reflects
00:20:25.22 what's happening in humans.
00:20:28.08 And then, finally, natalizumab appears to be safe and well tolerated in preclinical safety studies.
00:20:33.17 Again, this was important because... because of alpha 4 integrin's broad distribution.
00:20:38.00 It just speaks to the fact that toxicology is an empirical science.
00:20:42.12 You really don't know until you test it.
00:20:45.15 And then, even then, it's important to remember that you always need to be vigilant.
00:20:50.19 So, these are the studies that led to the clinical program for natalizumab.
00:20:55.19 In the first case, we looked at a Phase I study in healthy volunteers and found that
00:21:00.14 the antibody was well tolerated with low immunogenicity.
00:21:03.07 And this is just with a single dose.
00:21:05.11 And we also were able to determine that it has a 12 day half-life, which is sufficient
00:21:10.02 for once a month dosing at 3 milligrams per kilogram.
00:21:13.26 So, reasonable dosing.
00:21:16.15 As we saw in the animal models, there was a two-fold increase in the number of circulating lymphocytes.
00:21:22.14 This is reflec... this is consistent with the drug's mechanism of action.
00:21:26.11 It's making it a bit more difficult for cells to get out of the blood stream into tissues.
00:21:30.02 And so you quickly establish a new equilibrium, which involves more lymphocytes in the blood.
00:21:36.13 But this two-fold increase really represents just the higher end of normal range.
00:21:41.24 Based on those studies, it went into a Phase II study.
00:21:44.02 And... and initially we... our idea was to treat acute flares in MS.
00:21:49.26 So, a patient would have a flare, they would come in to the doctor, get a dose of natalizumab,
00:21:54.21 and hopefully we would quickly dampen down that flare and prevent the damage associated
00:21:59.02 with it.
00:22:00.02 In fact, what we found... and this is with two doses covering two months... we found
00:22:05.18 that there was no impact at all on the time of resolution or the outcome of that relapse.
00:22:12.02 So apparently, by the time a patient is feeling the onset of a relapse, the immune cells are
00:22:17.05 already in the brain, and by inhibiting more it really does not have an impact.
00:22:22.00 However, what we did notice in the study was that by the end of the second month the new
00:22:28.12 lesion development in MRI... by MRI had basically stopped.
00:22:31.28 So, this is looking at the cumulative number of new gadolinium-enhancing lesions by MRI.
00:22:38.11 And you can see that in the placebo group, in red, that there's a pretty consistent accumulation
00:22:43.02 of new lesions.
00:22:44.05 But, by the fourth week on natalizumab, these lines are beginning to diverge.
00:22:49.02 And so that... by the eighth week, the second month, when the drug is still on board,
00:22:53.11 you can see that new lesion activity had... had almost stopped.
00:22:57.05 At the next time point, when the drug was gone, MRI activity began to return.
00:23:01.14 So, this result led us to the idea that, since the drug appeared to be safe, perhaps we could
00:23:06.08 take it into MS for chronic treatment -- prevent the development of new lesions and hopefully
00:23:11.07 have an impact on the progression of disease.
00:23:14.12 So, this led to the our Phase IIb study, and this was a six-month study involving
00:23:21.18 monthly dosing as well as MRIs, which are indicated by the Ms, and then we follow the patients
00:23:27.27 for six months after that.
00:23:29.24 210 patients, looking at placebo and two different doses of drug.
00:23:34.11 And the primary endpoint would be looking... was looking at lesion burden, as well as relapse rate.
00:23:40.23 This is what we found.
00:23:41.27 So, in the months before treatment began, and at the time of treatment, all three groups
00:23:46.17 had sim... very similar levels of MRI activity.
00:23:51.00 And then, looking at the placebo group, they maintain this level of activity pretty consistently
00:23:55.11 over the six-month period.
00:23:57.03 However, in the groups that were treated with natalizumab,
00:24:00.06 and this is both for the 3 and the 6 milligram group,
00:24:03.12 you can see that MRI activity almost completely stopped.
00:24:06.19 So, again, by the second month, activity was very, very low.
00:24:10.08 Now, I have to say I...
00:24:11.15 I was sitting in the audience of the company when we first saw these results and it...
00:24:19.07 it really took my breath away.
00:24:20.15 This is... this was a day that I will remember in my career.
00:24:23.07 And I think that it... it really changed the way I look at things from then on out,
00:24:28.07 because as a scientist working in the bench it's really hard to believe that you can have an impact
00:24:32.20 on human disease.
00:24:33.22 And so this...this really made me believe that and, as I said, has had a long-lasting impact.
00:24:41.08 The drug also affected the number of relapses.
00:24:43.28 So, there was about a 50% reduction in the number of relapses, as well as a number of
00:24:48.02 patients who had a relapse.
00:24:50.00 And also decreased the need for steroid rescue.
00:24:52.25 So, these patients, if they had a flare, could... had the option of having steroid and on natalizumab
00:24:59.11 they did not feel the need to do that.
00:25:02.25 This is looking at the same data, the MRI data.
00:25:07.06 So, the blue line is 6 milligrams per kilogram, the red line is 3 milligrams per kilogram.
00:25:13.23 And so... you can see that... you can see that the drug is... for 3 milligrams per kilogram,
00:25:20.26 when it begins to go away, MRI activity returns.
00:25:24.25 And with 6 milligrams per kilogram, it takes a little bit longer for... for activity to resume.
00:25:29.07 You can overlay lymphocyte counts on top of this.
00:25:33.06 So, this is looking at the number of lymphocytes in the bloodstream.
00:25:36.22 And at a very beginning, you can see that there's this... this increase to lymphocyte
00:25:39.22 counts that's maintained very stably throughout the treatment period.
00:25:43.01 But then, in the 3 milligram per kilogram due... dose group, you can see that as the
00:25:47.12 drug goes away the lymphocyte counts drop, and the MRI activity goes up.
00:25:52.07 And with the 6 milligram per kilogram group, you can see this is... this is basically shifted
00:25:56.11 by a month.
00:25:57.11 So, the cell counts drop and the MRI activity comes up a little bit later.
00:26:01.07 So, this is very consistent.
00:26:02.16 This finding is very consistent with the mechanism of action that we're thinking.
00:26:06.07 As blood cells leave the... as immune cells leave the bloodstream, the MRI activity would
00:26:13.14 Alright, so our... for our Phase III studies in relapsing-remitting multiple sclerosis,
00:26:19.08 in order to get approval for a drug you actually have to have two separate Phase III studies.
00:26:24.03 And so both of these were about a thousand patients.
00:26:26.15 Umm... the first one involved monotherapy, so it was just Tysabri versus placebo.
00:26:31.11 The second one was on... with patients who were already on interferon beta, which was
00:26:36.05 the standard of care for MS patients.
00:26:38.28 And in this case, these patients were having disease breakthrough even on interferon.
00:26:42.27 So, the trial was natalizumab... natalizumab plus interferon versus interferon by itself.
00:26:49.05 So, this was a randomized double-blind study, as were the Phase II, and involved patients
00:26:55.08 who had to have at least one relapse in the prior year to the study.
00:26:58.14 It was a two-year treatment with an early assessment at one year.
00:27:04.24 And the findings were very consistent with what we saw in Phase II.
00:27:07.13 The number of gadolinium-enhancing lesions, both in first year... in the first year,
00:27:12.28 as well as the second year, were inhibited by 92%.
00:27:15.17 So, very strong inhibition of... of this apparently acute inflammatory reaction.
00:27:21.15 And this translated to a 68% reduction in relapse rate, again, both in the first year
00:27:26.26 and the second year, as well as in the third year.
00:27:30.02 This is an open label extension study at this point, so there isn't a placebo control because
00:27:34.02 everyone's on drug.
00:27:35.13 But, in all three years, the disease activity or the relapse activity was reduced to the
00:27:40.22 same basal level.
00:27:45.21 And this is an interesting finding.
00:27:47.05 This is a subset analysis looking at how different levels of activity would impact the efficacy
00:27:54.03 of the drug.
00:27:55.03 So, this is comparing patients who had had one relapse at the onset of when they were
00:27:59.14 coming into the trial in the previous year versus three relapses in the previous year.
00:28:04.22 And so you can see that patients who had high levels of disease activity, relapse activity
00:28:09.11 was inhibited down to the same level as everyone else.
00:28:12.07 So, it appears as though natalizumab has a very strong effect on patients with
00:28:16.23 highly active disease, inhibiting the... the relapse rate down to the same level.
00:28:24.14 And then, finally, this is the critical primary endpoint, at the second year of analysis.
00:28:29.04 And this was, how does it affect the overall progression of the disease in patients,
00:28:33.04 as measured by the standard EDSS scale?
00:28:36.24 And you can see that there's about a 54% reduction when looking at a sustained response over
00:28:41.28 six months.
00:28:42.28 Again, another subset analysis, looking at patients with highly active disease.
00:28:48.05 Again, there's an even stronger reduction, a 64% reduction, in the progression of EDS scores
00:28:54.22 in patients with... with more than two relapses and gadolinium lesions before they
00:28:59.15 came into the study.
00:29:02.28 And then, finally, this was not a point... this was not a formal endpoint in the study,
00:29:07.07 but nonetheless was a measure that was done.
00:29:09.23 And this is the MSFC scale.
00:29:12.22 And here you can see that the placebo patients... well, actually, what's interesting about this
00:29:16.08 scale is that it involves ability to walk, ability to have dexterity in the hands,
00:29:22.09 as well as cognitive function, a measure of cognitive function.
00:29:26.21 You can see that the placebo group maintained fairly stably over the two-year period, whereas
00:29:31.21 patients on natalizumab actually showed a level of improvement.
00:29:36.18 Importantly, these findings have been consistent across a number of studies.
00:29:42.04 These are actually looking at registrations and observational studies done by MS investigators
00:29:47.09 across the world, and very similar levels of efficacy with respect to relapse rate.
00:29:52.15 So, in summary, two years of treatment with natalizumab decreases disease activity, reduces
00:29:58.24 the risk of relapse by 68%, and reduces the risk of sustained disability progression by
00:30:06.04 To summarize the drug's safety in these clinical trials, these two-year clinical trials,
00:30:13.06 common adverse events were headache, fatigue, and arthralgias,
00:30:15.25 which are commonly seen in clinical trials.
00:30:18.22 There also were a few more infusion site reactions in natalizumab versus placebo.
00:30:24.21 Natalizumab actually causes a persistent anti... anti-natalizumab response in about 6% of patients.
00:30:32.04 And so, in these patients, actually, the drug loses this efficacy because the antibody response
00:30:36.26 causes it to be cleared very, very quickly, which is consistent with the infusion site reactions.
00:30:43.19 Importantly, there was a similar incidence of infections and malignancies between the
00:30:48.13 two groups.
00:30:49.13 In fact, if you look at this more closely, this is looking at the risk of all infections,
00:30:54.17 and this is for both studies combined.
00:30:57.05 So, both the monotherapy as well as the interferon therapy trial.
00:31:03.10 And you can see that there is no measurable difference in the rate of overall infection
00:31:08.16 between natali... natalizumab and placebo.
00:31:14.05 So, this is just the timeline of development, looking back in the beginning at 1990,
00:31:20.27 when the target was... of alpha 4 integrin was first identified for multiple sclerosis.
00:31:25.13 The antibody was humanized.
00:31:27.28 About five years later we started the clinical trials.
00:31:31.14 Biogen came in with Elan in about the year 2000.
00:31:36.01 And when developing a drug like this for multiple sclerosis, it's very expensive, because the
00:31:41.01 trials are large, there's a lot of MRI measurements, and so it's really important to have a partner.
00:31:45.18 And, in fact, Biogen was a great partner, because they already had a highly effective
00:31:51.03 drug for multiple sclerosis in the clinic and they knew a lot about... about the disease.
00:31:55.25 Shortly after the partnership was when we had the readout of the Phase II data,
00:32:00.16 you know, the day that... that changed my career.
00:32:03.01 So, I think that Biogen was very happy about that as well.
00:32:06.24 The phase III study was then shortly initiated.
00:32:09.20 The drug was approved in... in November of 2004.
00:32:13.14 The Phase... the second year of the Phase III study completed and read out shortly thereafter.
00:32:19.01 So, all said, 15 years from conception, 10 years of clinical experience involving 4,000 patients.
00:32:28.02 Now, in the second part of my talk, I'm going to discuss what happens next.
00:32:32.13 And this was just three months after the drug was approved.
00:32:35.15 This was in February of 2005.
00:32:37.23 The entire world for Tysabri changed.
00:32:41.03 So, in February of that year, we found our first two cases of PML.
00:32:45.21 And PML is a viral infection in the central nervous system that usually results in death
00:32:50.04 or severe disability.
00:32:51.04 So, it's a very serious infection.
00:32:55.01 Two patients in the MS trial had developed PML.
00:32:58.22 And amazingly, they had developed PML almost within a week of each other.
00:33:02.12 So, after ten years of clinical experience, two patients developed PML within a week.
00:33:06.23 And so this was alarming to everyone, because it... it suggested, well, maybe this is
00:33:11.04 just the tip of the iceberg, and half the patients could have PML.
00:33:14.13 So, dosing of the drug was suspended.
00:33:17.21 And we undertook a comprehensive safety evaluation at that point.
00:33:21.11 This was extremely complicated, because there were 3,000 patients involved or already
00:33:26.05 are still... are still in clinical trials, as well a number of patients who were just beginning
00:33:30.24 to take the drug from its approval.
00:33:34.04 So, what I'll talk about in my second part, in the second lecture, is how the medical
00:33:39.15 and regulatory communities working together with the companies established a path to allow
00:33:44.04 natalizumab to return, as well as the work that we've done since then to better understand
00:33:48.17 the safety of natalizumab and how PML can be monitored.
00:33:51.25 So, at this point, I just want to end by thanking all of the collaborators in the... both the
00:33:58.17 preclinical and clinical studies.
00:34:01.05 I will at the end of my second lecture actually give a more detailed list of some of those contributors.
00:34:06.10 But needless to say, there were a ton of very talented people involved in the development
00:34:11.10 of this drug.
00:34:12.11 And also I want to thank the many patients who participated in the clinical trials.
00:34:17.22 Without their participation, it would be impossible to dev... to develop therapies for... for disease.
- Avery August iBioSeminar: Allergies and the Immune System
- Diane Mathis iBioSeminar: T Cell Tolerance
- Ted Yednock iBioSeminar: Discovery and Development of Tysabri (Natalizumab) for the Treatment of Multiple Sclerosis
When Avery August was a teenager, he moved with his family from Belize to Los Angeles. Unhappy with high school in Los Angeles, August quit and instead completed his GED. He spent two years at community college before transferring to California State University in Los Angeles. Supportive mentors at CSU gave August a chance to… Continue Reading
Diane Mathis is a Professor in the Division of Immunology and the Department of Microbiology and Immunobiology at Harvard Medical School. She is also a principal member of the Harvard Stem Cell Institute and an associate member of the Broad Institute. Mathis’ lab studies the genetic, cellular and molecular mechanisms that determine immunological tolerance, and… Continue Reading
Dr. Yednock recently joined the Drug Advisory Board of the Myelin Repair Foundation, a nonprofit organization focused on the discovery and development of therapeutics for multiple sclerosis (MS). Previously Dr. Yednock was the Executive Vice President and Head of Global Research for Elan Pharmaceuticals and a scientist at Athena Neurosciences. Through his scientific work at… Continue Reading