Session 7: Autoimmunity and Allergy

00:00:08.03 Hello.
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,
00:06:52.01 heparin,
00:06:53.17 proteases,
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,
00:11:30.04 mMCP6,
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...
00:26:47.05 spectrometry...
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,
00:34:39.21 elongation,
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.

00:00:07.02 Hi.
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
00:02:53.22 gadolinium.
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.
00:10:27.03 Ni...
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
00:26:12.14 resume.
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
00:30:05.04 42-54%.
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.
00:34:00.05 I...
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.