Session 9: The Immunology of Organ Transplantation

00:00:14.19 Hi, I'm Megan Sykes.
00:00:16.03 I'm a professor at Columbia University, and I'm the Director of the Columbia Center
00:00:21.10 for Translational Immunology.
00:00:23.17 Today's lecture is an introduction to the field of transplantation, particularly transplantation immunology.
00:00:34.16 Transplantation is inseparable from immunology, because organ transplant rejection
00:00:41.02 and graft-versus-host disease are the result of immune responses caused by genetic differences from...
00:00:48.18 between the transplant donor and the recipient.
00:00:53.14 There are a few definitions I'd like you to understand before I go through my lecture,
00:00:58.20 because I will be using some of these words as I go... go forward.
00:01:02.28 Autologous means an organ or tissue from the self.
00:01:07.19 There are some autologous transplants that are done sometimes,
00:01:11.20 for example pancreatic islets in a person whose pancreas needs to be removed due to pancreatitis.
00:01:17.17 The person will get their islets back so they don't get diabetes.
00:01:23.03 Syngeneic is not from the self, but it's from the next best thing, a genetically identical individual
00:01:29.02 -- an identical twin.
00:01:31.20 In mice, we use syngeneic donors all the time because we have inbred mice --
00:01:39.04 hundreds and thousands of identical twins.
00:01:42.00 In humans, we may have one identical twin in the rare case... in the rare person.
00:01:50.02 Allogeneic is the commonest kind of transplant, that means from another individual,
00:01:54.17 a genetically different individual, of the same species.
00:01:58.13 And that includes anybody who isn't an identical twin: a brother, a sister, a parent.
00:02:04.06 Those are all allogeneic to us.
00:02:06.28 And then finally, xenogeneic.
00:02:08.12 That'll be the topic of my third lecture.
00:02:10.26 A xenogeneic transplant is something that we don't do right now, but we hope to do
00:02:16.10 in the near future.
00:02:17.13 And that involves transplantation from a different species.
00:02:21.21 Okay, well, let's focus for a while on allogeneic organs and tissues.
00:02:27.14 Now, the immune responses that we have to think about when we put an organ
00:02:32.09 from one genetically different individual into another is what we call the host-versus-graft response,
00:02:39.11 the response of the recipient's immune system against the donor, foreign antigens.
00:02:45.25 And T lymphocytes are the main players in this response.
00:02:50.14 Antibodies also happen, and there are specific conditions when antibodies are a concern
00:02:56.00 that I'll talk about later, but many antibody responses are dependent on T cells.
00:03:01.20 And so the T cell can be thought of as a central player in rejection in most instances.
00:03:07.27 Now, in the second part of the slide, I mentioned allogeneic hematopoietic cells.
00:03:15.22 That's what many of you may have heard of as a bone marrow transplant in somebody
00:03:19.22 who has leukemia or lymphoma.
00:03:22.19 That's a commonest reason for doing a hematopoietic cell transplant.
00:03:27.13 It's not always bone marrow.
00:03:28.26 Often, we use mobilized peripheral blood stem cells for a hematopoietic cell transplant.
00:03:35.22 And there, we have to think about immune responses in both directions, because the donor is,
00:03:41.10 again, foreign to the recipient.
00:03:43.16 And again, we have to think about the recipient rejecting the donor.
00:03:47.15 And the main players there are T cells and natural killer cells, and also sometimes antibodies
00:03:53.18 can play a role.
00:03:55.25 But there's another direction we have to think about, because when we do a hematopoietic cell transplant
00:04:01.12 we do things to compromise the recipient's immunity very, very extensively.
00:04:06.10 And then the immune cells that come with the graft can actually attack the recipient.
00:04:12.14 So, it's a donor anti-recipient attack that we call graft-versus-host responses.
00:04:19.03 And T cells and, to some degree, natural killer cells play major roles there.
00:04:25.07 Then, we can think about xenogeneic organs, cells, and tissues.
00:04:30.02 And there, it's mainly the host barrier to the donor that we have to think about,
00:04:35.06 the host-versus-graft response.
00:04:36.09 And it's a very, very strong response involving T cells, natural killer cells, macrophages,
00:04:42.07 and antibodies, even antibodies that aren't T cell dependent.
00:04:45.24 A very strong immune response.
00:04:48.08 Now, there are many ways that we can try to avoid rejection.
00:04:54.02 And I'm speaking now, again, about allogeneic organs, which is the type of organ that
00:04:59.13 we currently transplant.
00:05:00.26 Rarely, we may have an identical twin as a donor.
00:05:04.03 And the very first transplant in humans was done with an identical twin who was able
00:05:09.19 to donate a kidney to his twin brother.
00:05:12.08 Secondly, most... since most people don't have identical twins, we use immunosuppressive drugs.
00:05:19.02 And that really is the standard of care for allowing a graft to be taken without rejection.
00:05:25.14 And finally, I'm going to speak in my second talk about what's sort of
00:05:30.02 the holy grail of transplantation, which is to induce tolerance, immune tolerance.
00:05:34.20 And there have been some recent trials that have actually achieved this in small groups
00:05:39.10 of patients.
00:05:41.05 Now, there's a particular genetic locus that we have to think about when we consider
00:05:47.01 any kind of a transplant.
00:05:48.09 And that's called the HLA, the human leukocyte antigens.
00:05:53.09 Because HLA antigens are extremely polymorphic -- meaning any two individuals who are unrelated
00:06:00.22 are very likely to have completely different HLA genes... and these HLA genes elicit
00:06:08.13 the strongest immune responses by T cells and antibodies.
00:06:13.20 And this slide, here, shows you the importance of HLA matching, that when you have
00:06:23.12 a closely HLA-matched organ -- this is kidney transplantation in a large series --
00:06:31.01 you can see that the rate of graft loss... now, this is following these grafts over 20 years, that there are...
00:06:39.06 is a constant rate of graft loss over time.
00:06:41.20 Grafts... many grafts eventually get rejected.
00:06:44.21 That's called chronic rejection, which I'll come back to.
00:06:47.24 But these lines are a little bit divergent.
00:06:50.07 The ones that are most closely HLA-matched have the longest half-life, the slowest rate of chronic rejection
00:06:57.17 whereas those that are HLA-mismatched more extensively have
00:07:02.24 poorer outcomes over time.
00:07:04.07 So, HLA is important in this regard.
00:07:08.11 Now, what is the cause of rejection.
00:07:11.00 As I mentioned, T cells are central players in the process.
00:07:15.10 Now, acute rejection is... is something that we avoid.
00:07:20.03 We don't like to lose the organ -- ever -- completely.
00:07:23.24 And that's why we use these immunosuppressive drugs.
00:07:26.12 And if we have an episode of rejection, we can usually control it by
00:07:30.17 adding more immunosuppression temporarily.
00:07:33.16 But as I mentioned, these drugs must be taken chronically.
00:07:38.16 And... otherwise, the graft will almost certainly be rejected.
00:07:43.00 Now, I mentioned chronic rejection as a cause of late graft loss in the slide that
00:07:49.05 I just showed you.
00:07:50.14 Unfortunately, that is an ongoing problem in the field.
00:07:53.25 It hasn't been improved by the improvements that we've made in immunosuppressive therapies
00:08:00.14 in the last few decades.
00:08:02.04 So, that is one reason why tolerance is sort of the holy grail in transplantation,
00:08:07.27 because tolerance would mean the immune system treats the donor as self, and chronic rejection
00:08:13.10 wouldn't happen either.
00:08:15.10 Alright.
00:08:16.10 Now, I've talked about rejection as if it's one thing.
00:08:21.10 But in fact there are several different types of graft rejection.
00:08:25.17 One is caused by pre-existing antibodies.
00:08:30.14 And we call that hyperacute rejection.
00:08:32.15 I'll say more about that.
00:08:34.15 Another kind of antibody-mediated rejection is called acute vascular rejection.
00:08:39.23 And that's when antibodies come... get formed after the transplant, but can cause
00:08:45.00 a fairly rapid rejection once they get formed, whereas hyperacute rejection is caused by antibodies
00:08:51.06 that are there before the transplant.
00:08:53.23 Now, the cellular rejection is one that is mediated, caused, by T cells alone,
00:09:00.24 with no antibody role.
00:09:02.12 But as I mentioned, antibodies also depend on T cells for their formation,
00:09:07.05 so some of this acute vascular rejection is antibody... is T cell-dependent.
00:09:13.06 And then chronic rejection, also, is thought to be dependent on T cells, even though
00:09:18.19 there's fairly good evidence that antibodies also play a role.
00:09:22.24 Okay.
00:09:23.24 Now, the first and second types of rejection -- hyperacute and acute vascular --
00:09:28.10 we really try very hard to avoid by selecting our donors and recipients, and in some cases doing
00:09:34.15 special preparation of our recipient, if we think they're at high risk of this.
00:09:39.00 So, let's talk a little bit about hyperacute rejection.
00:09:42.09 Hyperacute rejection can occur in a couple of circumstances.
00:09:46.26 One is if we have blood group-mismatched donors, for example if the recipient is blood group B
00:09:52.27 and the donor is blood group A.
00:09:56.01 The recipient has anti-A antibodies in the... in their serum.
00:09:59.28 And that antibody will immediately bind to the blood vessels of the kidney.
00:10:06.08 As soon as the graft is hooked up, it'll bind to the endothelial cells lining the blood vessels.
00:10:13.07 And what that does is it fixes complement, initiates a whole cascade of inflammatory events
00:10:19.22 that ultimately will activate the coagulation cascade and occlude the blood vessels.
00:10:26.08 So, that graft is lost very, very quickly.
00:10:29.19 And the same thing can happen in... even in a blood group-matched situation, but in which
00:10:35.22 the recipient has made antibodies against donor HLA antigens prior to the transplant.
00:10:43.04 And this can happen because of a prior pregnancy, a prior transplant, or due to blood transfusions
00:10:49.22 that the recipient may have had.
00:10:51.18 And it's exactly the same process.
00:10:53.24 The antibodies in the patient circulation bind to the endothelial cells and fix complement
00:11:01.09 and initiate that same coagulation cascade and occlude the blood vessels of the graft.
00:11:07.04 So, as I just said, it can... hyperacute rejection can occur in blood group-mismatched and presensitized
00:11:14.18 recipients.
00:11:16.25 And on the bottom, it says that hyperacute rejection has presented a barrier to xenotransplantation.
00:11:24.27 As I'll discuss in the xenotransplantation lecture, we've found a way of avoiding that.
00:11:31.09 But it was a major limitation to the field of xenotransplantation over the years.
00:11:36.15 Now, just to show you what hyperacute rejection looks like, this is an experimental study
00:11:42.02 where a rat heart was put into a mouse that had lots of natural antibody against that rat.
00:11:51.20 That's the one on the right, here.
00:11:53.07 And you can see that that heart looks black.
00:11:56.04 And that's because that occlusion of the blood vessels has happened in that very short period of time
00:12:01.22 since the graft was put in.
00:12:03.21 This is half an hour after the transplant.
00:12:06.10 Over here, you see a pink looking graft, and that's a much more normal looking graft.
00:12:12.09 And that's because this mouse that got the rat heart didn't have those high levels of
00:12:16.21 preformed antibody against the rat xenograft.
00:12:20.11 It's a xenograft because it's from one species to another.
00:12:25.07 Okay.
00:12:26.07 Now, acute cellular rejection is a major problem.
00:12:33.00 And it's a common cause of what we call rejection episodes.
00:12:37.25 And we think that this whole process, which... in which, again, the T cell is the central player,
00:12:44.05 gets initiated in part because of procedures that involve... are involved in
00:12:51.02 obtaining an organ from a donor, transplanting it to the recipient.
00:12:55.22 And there's a period where there's no blood flow to that graft, called ischemia.
00:13:01.00 And that ischemia can activate all sorts of components of the innate immune system.
00:13:07.13 And the innate immune system is very effective at activating T cells.
00:13:12.16 And it's actually T cells, as I mentioned, that are the real players in this graft rejection.
00:13:19.03 And the strongest rejections are directed against those HLA differences that I mentioned,
00:13:25.10 but also what's called minor histocompatibility antigens, which are basically peptides
00:13:31.03 that are presented by an HLA antigen, but they're peptides of proteins that have some polymorphism,
00:13:37.28 and may differ between the donor and recipient.
00:13:40.21 We have many such polymorphisms.
00:13:43.26 And so, even in the setting of HLA matching, there are many minor histocompatibility antigens
00:13:49.17 that can be presented and cause a T cell-mediated rejection.
00:13:54.15 And this is if you had a really bad T cell-mediated rejection that destroyed the graft,
00:13:59.27 this is what it would look like.
00:14:00.27 It's... it's quite swollen, and it's white.
00:14:03.04 And it's white because of all the leukocytes, the white cells, the lymphocytes, etc,
00:14:08.19 that are infiltrating the graft.
00:14:12.01 As I mentioned, it's a T cell-mediated process.
00:14:15.04 This stain here is an immunostain for... with anti-CD3.
00:14:18.19 And you can see there's a lot of those T cells that express CD3 in the graft.
00:14:25.03 And histologically, you can just see this gross infiltrate of cells with blue nuclei
00:14:30.28 and very little cytoplasm.
00:14:32.11 Those are largely lymphocytes.
00:14:35.02 Okay.
00:14:36.09 Well, what causes this T cell response?
00:14:41.00 There are two types of T cell responses.
00:14:44.10 One is called direct allorecognition, and the other is called indirect allorecognition,
00:14:50.23 in transplantation.
00:14:52.19 And indirect allorecognition is actually like... a lot like any other type of immune response.
00:14:58.04 And I'll tell you why in a minute.
00:15:00.25 Direct allorecognition is quite unique -- it's different from most immune responses --
00:15:06.19 because it actually is seeing the allogeneic HLA presenting a peptide.
00:15:14.17 And there are many, many such possible allogeneic HLA/peptide complexes on a donor graft.
00:15:23.23 And this elicits a very strong response.
00:15:25.26 And we have both CD4 and CD8 T cells that can recognize this donor.
00:15:32.13 And the essential point of direct allorecognition is that it's directly recognizing antigen
00:15:38.26 presented by a donor cell.
00:15:40.22 So, this is a donor dendritic cell, an antigen-presenting cell that is presenting these donor HLA molecules.
00:15:49.24 And that... that's the direct response.
00:15:52.24 Indirect allorecognition is a little bit different, because it in fact involves a recipient antigen-presenting...
00:16:01.11 presenting cell, here, a recipient dendritic cell.
00:16:04.06 And what happens is that recipient antigen-presenting cell picks up donor antigens,
00:16:09.28 such as these HLA molecules, internalizes them,
00:16:14.27 processes them through the antigen processing pathway,
00:16:18.04 and re-presents them on a recipient MHC molecule, a class II molecule, that is on the surface
00:16:25.13 of the recipient dendritic cell.
00:16:27.12 So, this recipient CD4 T cell, in this slide, is actually recognizing a donor HLA-derived peptide
00:16:36.13 presented by a recipient HLA molecule on a recipient antigen-presenting cell.
00:16:43.02 Alright.
00:16:44.03 Well, HLA differences between donors and recipients activate a very large number of T cells
00:16:51.11 of different specificities.
00:16:53.10 And this results in the response to HLA
00:16:56.23 -- which is the human form of the major histocompatibility complex, the MHC --
00:17:02.22 being stronger than any ordinary immune response.
00:17:07.08 So, this is an extraordinary response.
00:17:09.17 Now, the reason for this is that T cell receptors are... have evolved to recognize MHC/peptide.
00:17:18.05 And so there's this inherent fit between T cell receptors and MHC/peptide complexes.
00:17:25.01 And T cells get positively selected in the thymus to weakly recognize a self MHC/peptide complex.
00:17:32.15 But what happens, in order to avoid autoimmunity, is that they undergo this other process
00:17:38.02 called negative selection, in the thymus, that weeds out the T cells that strongly recognize
00:17:43.15 self MHC/peptide complexes.
00:17:46.09 But there's no donor cells in that... in your thymus that contribute to that process,
00:17:51.13 so you don't weed out T cells that recognize alloantigens.
00:17:55.16 So, this combination of inherent MHC recognition, and not being weeded out for the donor,
00:18:03.01 results in a lot of T cells that see the donor.
00:18:06.06 And one of the results of this is that we can actually measure responses
00:18:10.26 to an allogeneic HLA-mismatched donor just in cells that we take right out of a person.
00:18:17.03 We don't have to give any immunization to see this.
00:18:20.23 And these two responses, called the mixed lymphocyte reaction, in which we measure
00:18:27.17 either T cell proliferation or cytotoxic T cell activity against the donor cells,
00:18:35.13 are our measures of this very strong response.
00:18:39.15 You don't see that for other types of immune responses.
00:18:42.27 You have to immunize.
00:18:44.05 And in fact, it's been estimated that anywhere from about 1-10% of T cells will recognize
00:18:52.16 a given MHC-mismatched allogeneic donor, which is a huge, huge proportion of this
00:19:00.19 very diverse T cell repertoire that we have.
00:19:03.25 I've been talking mainly about acute rejection up until now.
00:19:07.15 And chronic rejection is another type of rejection that is much more slow than acute rejection.
00:19:15.24 And we've made pretty good inroads into reducing acute rejection and graft loss rates
00:19:21.07 with modern immunosuppression.
00:19:23.10 But we haven't done much to eliminate this slower form of rejection, that we call chronic rejection.
00:19:30.12 And the mechanisms of chronic rejection are not fully understood.
00:19:34.20 They may involve, probably, some of the same processes that I've referred to.
00:19:39.17 But it's thought that indirect recognition plays a major role, mainly because the antigen-presenting cells,
00:19:45.20 the professional APCs that come with the graft,
00:19:49.09 do get replaced over time by the recipient's.
00:19:53.00 So, you have more recipient APCs around to present antigen.
00:19:56.10 And indirect recognition is very effective at inducing antibody responses.
00:20:01.14 And antibodies may play a very important role in many instances of chronic rejection.
00:20:09.09 Now, the reason for that is that the B cells are particularly focused on alloantigens.
00:20:20.23 Because... here's an example of a B cell that has a receptor, a surface immunoglobulin receptor,
00:20:29.03 that recognizes an allogeneic donor HLA class I molecule.
00:20:34.08 And what happens is that B cell, by recognizing that molecule, will selectively pick it up
00:20:41.12 and process it, and present it through its class II molecule to a CD4 cell, shown here.
00:20:50.09 So, that CD4 cell helps that B cell, which has focused its antigen... its peptide antigen that
00:20:57.04 that CD4 cell recognizes, and helps the B cell.
00:21:01.26 So, it's a T cell/B cell interaction.
00:21:04.25 Over here, you have the dendritic cell that initially primed that CD4 cell, but that CD4
00:21:11.02 now can very effectively induce antibody responses by that particular B cell.
00:21:17.00 So, that's the indirect pathway of inducing alloantibody responses.
00:21:22.10 Now, there's a third pathway of allorecognition that's only been recognized in the...
00:21:27.26 in the last few years, and that's called semidirect allorecognition.
00:21:32.12 And what that means is... it... it's still a recipient dendritic cell, a recipient antigen-presenting cell
00:21:41.06 that is presenting antigen to the T cell.
00:21:44.16 But instead of picking up and processing antigen, as we saw for the indirect pathway, over here,
00:21:53.22 what happens is that recipient antigen-presenting cell actually steals entire MHC/peptide complexes
00:22:04.02 away from the donor cell.
00:22:05.19 It picks it up in intact form from the cell membrane, and presents it on its own cell membrane.
00:22:13.15 Some people refer to it as crossdressing.
00:22:15.27 It's the recipient dendritic cell looking like the donor in terms of the HLA/peptide complexes
00:22:23.02 that it presents.
00:22:24.26 And that's important because we know we have donor HLA around as long as we have
00:22:29.20 a donor graft around.
00:22:31.23 And it means that... since the T cells that recognize these donor HLA/peptide complexes
00:22:38.22 are the ones that we originally described as directly alloreactive, it means those cells,
00:22:44.01 which are so abundant, have a constant trigger through this pathway.
00:22:49.19 Okay, how do we prevent all this?
00:22:52.16 I mentioned that lifelong immunosuppressive drugs are the standard of care.
00:22:57.00 That's how we get organs to be accepted for many years in many people.
00:23:02.20 And there are several types of drugs that we use in combination.
00:23:07.27 Steroids are not on this list, but corticosteroids that have very broad effects on
00:23:14.10 the immune system are commonly used as one of three drugs.
00:23:18.25 The second drug is often a cytotoxic drug, such as azathioprine or, more recently, mycophenolate mofetil.
00:23:27.11 And what these [drugs] do is they specifically target proliferating T cells...
00:23:33.02 proliferating cells, like T cells and B cells.
00:23:37.09 And then finally, the third drug is often a calcineurin inhibitor, like cyclosporine
00:23:42.07 or tacrolimus or, instead, rapamycin,
00:23:47.00 all of which are very selective for targeting T cell activation.
00:23:54.08 And rapamycin is a little bit different in its mechanism of action compared to
00:23:59.06 the calcineurin inhibitors.
00:24:02.11 Rapamycin actually inhibits the mammalian target of rapamycin, which is a different pathway,
00:24:08.26 pand may have advantages in sparing a regulatory subset of T cells when it's blocked.
00:24:17.20 Okay.
00:24:18.20 So, that's organ transplantation, rejection, how we prevent rejection.
00:24:23.02 I'm now going to end with just a few slides about hematopoietic cell transplantation,
00:24:28.24 which I mentioned early on.
00:24:30.28 Hematopoietic cell transplantation can involve bone marrow, which used to be the usual type
00:24:37.16 of transplant.
00:24:39.06 But more recently, a lot of people get mobilized peripheral blood instead.
00:24:44.10 And what that involves is treating the donor with a drug, GCSF, or there are some
00:24:50.28 more recent drugs, that actually cause the bone marrow stem cells to leave their home
00:24:55.09 in the bone marrow and go into the circulation.
00:24:58.06 And so now you can just, using a leukapheresis catheter, collect peripheral blood that is
00:25:04.04 very enriched for the bone marrow stem cells that we need for our transplant.
00:25:10.10 Cord blood is another source of hematopoietic cell transplants.
00:25:14.21 And finally, we can actually enrich the hematopoietic stem cells from any of these tissues.
00:25:20.22 CD34 is a marker that is on stem cells, as well as a variety of other cells,
00:25:26.24 and often CD34 selection is used to enrich the stem cells.
00:25:30.25 And the main application of bone marrow... of hematopoietic cell transplantation is
00:25:36.22 in the treatment of hematologic malignancies, like leukemias and lymphomas,
00:25:43.02 but also in treating genetic diseases of blood forming cells, immunodeficiency disease and
00:25:48.15 sickle cell disease, etc.
00:25:50.23 Okay.
00:25:51.23 Well, as I mentioned at the beginning, we have an issue.
00:25:56.00 In addition to the recipient rejecting the donor, we have the issue of graft-versus-host disease
00:26:01.17 when we do hematopoietic cell transplants.
00:26:04.17 And that's in a sense the donor rejecting the recipient, the donor attacking the recipient.
00:26:10.11 And that is also -- just like rejection -- dependent on T cells.
00:26:14.03 They're the key players.
00:26:15.24 And what happens is donor T cells seeing foreign antigens in the heavily compromised recipient
00:26:22.13 get activated, and they attack cells of the... of the epithelium... of the epithelial tissues,
00:26:29.00 skin, anywhere in the intestine... in the intestinal system, and liver are
00:26:34.01 the main targets.
00:26:35.11 And there are acute and chronic forms, just like rejection.
00:26:39.28 And through most of the clinical field of hematopoietic cell transplantation,
00:26:45.20 graft-versus-host disease has been such a big problem when HLA barriers are crossed
00:26:51.07 that it has been necessary to find an HLA-matched donor.
00:26:56.10 And of course that could be a sibling, because we all have two different HLA loci.
00:27:03.03 With any given sibling who's not an identical twin, there's a 1-in-4 chance that there will be
00:27:08.28 a complete HLA match at both loci, at both alleles.
00:27:17.04 Now, that obviously is only 25% of people who are lucky enough to have an HLA-identical sibling,
00:27:23.02 and so this has led to the development of large bone marrow transplant registries,
00:27:28.01 where people have volunteered to be HLA-typed, and then to give their hematopoietic cells
00:27:32.21 to somebody who may need a transplant who they don't know and are unrelated to.
00:27:37.14 But that takes millions and millions of people, because of the extensive polymorphism of HLA.
00:27:43.28 Finding a matched donor is like finding a needle in the haystack, but it's done.
00:27:49.02 Now, recently, the field has advanced so that we are able to perform
00:27:54.11 more extensively mismatched transplants, HLA-mismatched transplants.
00:27:58.16 And this is getting more and more common at multiple centers.
00:28:03.24 Okay.
00:28:04.24 So, what is this whole graft-versus-host disease?
00:28:07.06 How does this happen?
00:28:09.00 So, what happens is we give our allogeneic bone marrow transplant, for example,
00:28:16.13 and that contains mature T cells, okay?
00:28:20.00 Those T cells get infused with the graft, you know, through the vein.
00:28:24.12 And they come in, and they go into the recipient lymphoid structures, like the lymph node
00:28:29.12 and the spleen.
00:28:30.12 And they actually get trapped there, because those structures are so full of recipient
00:28:35.07 antigen-presenting cells.
00:28:36.07 So, over a period of several days, those donor T cells recognizing recipient antigens
00:28:42.07 on these recipient APCs will get activated and expand.
00:28:46.16 And then they will... they will leave the lymph node and go back into the circulation.
00:28:52.11 And if the recipient has been treated with a conditioning that causes inflammation
00:29:00.10 in those epithelial target tissues -- and conditioning being irradiation, chemotherapy, etc --
00:29:07.19 those tissues themselves are inflamed.
00:29:10.25 And those tissues will provide signals that allow those activated T cells that get in
00:29:17.03 the circulation to enter those epithelial tissues and cause this disease called
00:29:22.19 graft-versus-host disease.
00:29:24.13 Okay.
00:29:25.13 Well, how can we prevent it?
00:29:26.25 The obvious one is to deplete the T cells from the donor graft.
00:29:31.28 And that works.
00:29:33.02 But it has some caveats.
00:29:35.17 First of all, it turns out that those donor T cells help the bone marrow to engraft,
00:29:41.14 to overcome the recipient rejection of the donor.
00:29:43.17 And there is an increase in rejection if we T cell deplete our donors.
00:29:48.09 Secondly, most of these... many of these transplants are done to treat malignant diseases.
00:29:55.00 And it turns out that the T cells in the donor graft are a kind of immunotherapy.
00:30:00.11 They actually kill off residual leukemia or lymphoma cells in the recipient that
00:30:07.20 didn't get killed by the chemo or radiotherapy.
00:30:10.26 And so there are higher relapse rates when we T cell deplete our donor grafts;
00:30:15.11 we take away that immunotherapy.
00:30:18.15 And thirdly, adults have a very hard time reconstituting their immune systems
00:30:24.10 after they've had all this treatment.
00:30:26.04 And if you T cell deplete your donor graft, you are more... the recipient is more prone
00:30:32.05 to serious infections.
00:30:33.23 There's quite a long period of immunodeficiency before the immune system recovers.
00:30:38.09 So, donor T cells also have the risk of anti-infectious immunity.
00:30:45.00 Okay.
00:30:46.00 So, I mentioned that relapse rates are higher when we T cell deplete the donor.
00:30:50.18 And that's because that alloreactivity against the recipient, while it's harmful and
00:30:56.04 causes graft-versus-host disease, it also attacks residual malignant cells in the recipient.
00:31:01.00 And natural killer cells, at least with certain types of malignancies, also have the ability
00:31:06.24 to attack malignant cells.
00:31:09.07 And so they can also be beneficial.
00:31:11.22 And then there are a variety of other strategies people are looking at.
00:31:17.11 Differences... changing immunosuppression, and many, many forms of immunomodulation
00:31:24.25 that are being explored to reduce graft-versus-host disease.
00:31:28.09 And the holy grail is to try and preserve antitumor reactivity in this case.
00:31:35.17 And this can be done in ways that... for example, controlling the trafficking of T cells
00:31:41.13 so you can still use the alloreactivity within the lymphohematopoietic system to give you
00:31:46.23 an anti-leukemia effect.
00:31:49.10 Or many groups are working on specifically targeting tumor antigens with a variety of
00:31:56.07 approaches.
00:31:57.07 So, that's the end of the first presentation.
00:31:59.11 Thank you for your attention.

00:00:14.12 Hi, I'm Megan Sykes.
00:00:15.23 I'm a professor at Columbia University, where I'm the Director of the Columbia Center
00:00:20.28 for Translational Immunology.
00:00:22.11 Today, I'm going to tell you about some of our research on taming and tracking the human alloresponse.
00:00:30.11 So, while we've made great advances in the field of transplantation, the success is
00:00:38.06 still limited by: one, the complications of the drugs that we use to avoid rejections;
00:00:44.18 and secondly, by chronic rejection, which remains a problem, even with the use of
00:00:50.04 optimal immunosuppression.
00:00:52.07 So, in our field of transplantation, the induction of immune tolerance has become a major goal,
00:01:00.00 because this state of tolerance would overcome both of these limitations.
00:01:06.03 What do I mean by tolerance?
00:01:08.12 Tolerance implies long-term graft acceptance without immunosuppressive therapy.
00:01:14.21 But importantly, with an otherwise intact immune system, that can recognize
00:01:20.13 foreign antigens and protect one from infections.
00:01:25.21 Successful tolerance induction, really, in my view, requires intentional, planned
00:01:33.02 immunosuppression withdrawal, achieving tolerance in a high proportion of individuals.
00:01:37.28 It's been known for some time that the rare person removing themselves from immunosuppression
00:01:44.03 may not reject.
00:01:45.14 The vast majority do.
00:01:47.07 But there are examples of tolerance induction through that... that rather dangerous and
00:01:55.06 inefficient process of immunosuppression withdrawal.
00:01:59.18 I'm referring, when I say tolerance induction is successful, to a process where it works
00:02:05.23 in most people, and it's intentional.
00:02:09.06 Okay, so how do we get to clinical trials of tolerance induction.
00:02:14.03 Of course, it begins with experimental models.
00:02:16.20 But if you look in the literature, you'll find actually hundreds or even thousands
00:02:22.05 of models of tolerance in rodents.
00:02:25.11 Unfortunately, almost none of have been successfully applied in humans.
00:02:30.18 And there are a number of steps that we need to take before we go to humans.
00:02:34.20 Because if... if we're going to... if we think we have tolerance, we're removing the standard of care.
00:02:41.26 We're removing the chronic immunosuppressive therapy that is otherwise used to prevent
00:02:47.13 rejection.
00:02:50.00 This is necessary if we want to have a normal... normally function immune... functioning immune system.
00:02:56.24 So, that's why one of the main reasons why we want tolerance, so that we don't have to have
00:03:00.25 that chronic immunosuppression.
00:03:03.13 But if we're going to remove that standard of care, we need to know that it works.
00:03:07.18 And this requires, first of all, in stringent rodent models, showing that it works.
00:03:13.09 And that usually involves extensively MHC-mismatched skin grafts, which are very difficult to
00:03:19.25 get acceptance of.
00:03:21.10 Secondly, we need to have other animal models.
00:03:24.27 It's very difficult to go from a mouse to a human in withdrawing immunosuppression.
00:03:31.00 We really need large animal models that are closer to humans.
00:03:34.14 And thirdly, it's desirable to have some experience with whatever drugs or agents you're
00:03:41.06 going to use in your tolerance protocol in other clinical settings.
00:03:45.23 As it happens, the approach that we've...
00:03:48.07 I and my colleagues have worked on for many years, involving hematopoietic cell transplantation
00:03:53.19 and induction of mixed hematopoietic chimerism, has come closest to meeting these criteria,
00:03:59.26 and has gotten into clinical trials.
00:04:03.19 So, if we're going to use hematopoietic cell transplantation to induce tolerance,
00:04:11.00 we can't use it in the usual way that it's used, for example, in a patient with leukemia or lymphoma.
00:04:17.12 In those settings, the transplant, of bone marrow or other hematopoietic cells, is done
00:04:23.15 after very heavy treatment of the recipient aimed at eradicating as many of the cancerous cells
00:04:29.28 as possible.
00:04:31.25 In a patient who doesn't have cancer, who needs an organ transplant, we can't justify
00:04:37.22 doing such toxic treatments.
00:04:39.16 Instead, we have to develop a way of preparing that recipient for their bone marrow transplant
00:04:45.23 -- that has the capacity to re-educate their immune system and induce tolerance --
00:04:51.16 that is far less toxic.
00:04:53.05 It has to not eliminate the recipient's bone marrow cells, so that if you failed to get
00:05:00.14 engraftment of your donor bone marrow you still have a functioning bone marrow and normal hematopoiesis.
00:05:08.02 And nevertheless, this treatment has to be strong enough to overcome the very strong
00:05:13.14 immune resistance in the recipient to the donor.
00:05:17.14 And we know we've succeeded in doing that if we achieve mixed chimerism.
00:05:21.00 And what I mean by mixed chimerism is coexistence of donor and recipient hematopoietic elements.
00:05:29.18 The donor ones aren't rejected, and so you see them in the circulation,
00:05:34.10 and the recipient ones have not been killed off by the conditioning,
00:05:38.24 and so you see them together with the donor cells.
00:05:42.02 Now, I mentioned in my introductory lecture that graft-versus-host disease is
00:05:47.11 a major complication of HLA-mismatched and even -matched hematopoietic cell transplantation.
00:05:53.26 It has some benefit in treating leukemia... in malignant diseases, but is absolutely
00:05:59.19 an unacceptable complication to introduce in somebody who doesn't have a malignant disease
00:06:05.23 but needs an organ transplant.
00:06:07.26 So, this is a big challenge: to cross HLA barriers, avoid GVHD, overcome the host resistance
00:06:16.07 of the donor, and do all of this with minimal toxicity.
00:06:21.17 It took many years for our groups at Mass General Hospital to reach a point
00:06:27.07 where we could actually try that.
00:06:28.22 And our pathway to clinical trials of tolerance induction using hematopoietic cell transplantation
00:06:37.11 actually involved rodent models, shown on the left side of this slide,
00:06:44.28 some involved in studies of treating leukemias and lymphomas
00:06:48.24 that ended up bringing us to a mixed chimerism approach
00:06:51.09 with non-myeloablative conditioning.
00:06:53.23 Meanwhile, our studies in rodents had shown that we could induce organ tolerance with
00:06:58.18 a non-myeloablative regimen for mixed chimerism induction.
00:07:03.04 And our studies in leukemias and lymphomas actually led us to an approach that
00:07:07.26 we could try in patients.
00:07:09.12 Because, in that setting, you can sometimes go directly from rodents to humans because
00:07:17.11 the patients who will try an experimental protocol have failed all other possibilities,
00:07:22.08 if they have a very advanced malignant disease.
00:07:26.11 And so we had the opportunity to try this mixed chimerism approach in some of
00:07:31.18 these patients who... in whom it was used as a platform for immunotherapy.
00:07:36.19 However, on the organ transplantation side, we didn't go straight from mice to humans.
00:07:41.21 We actually had a non-human primate model in the middle, which is very similar to humans
00:07:47.12 in how it responds to transplants, and was a very critical step in allowing us to do
00:07:53.17 bone marrow transplants for tolerance induction in patients with no malignant disease.
00:07:59.24 The protocols that we tried at Mass General for inducing tolerance in these patients underwent
00:08:06.22 several iterations.
00:08:07.25 A total of ten patients were transplanted under these three protocols.
00:08:14.15 The first one... and these were all supported by the immune tolerance network of the NIAID...
00:08:21.04 and the first one is shown here, and all of them have in common that they utilized non-myeloablative doses
00:08:28.00 of cyclophosphamide; local irradiation to the thymus;
00:08:32.19 a monoclonal antibody against CD2 that is given to deplete the recipient and donor T cells in vivo;
00:08:39.06 and then a combined kidney and bone marrow transplant on day 0.
00:08:44.12 And the post-transplant immunosuppression has involved a calcineurin inhibitor
00:08:48.25 for a period of 9-12 months.
00:08:52.02 The second iteration of the protocol brought in some steroids for a very short period
00:08:58.20 after the transplant to avoid an engraftment syndrome, as well as treatment with a B cell depleting agent,
00:09:06.16 rituximab, to avoid antibody-mediated rejection.
00:09:11.20 And the third iteration involved even more rituximab, several treatments with it,
00:09:17.11 and a slightly longer period of steroid treatment, but was otherwise similar.
00:09:23.05 This work has been published already, in these two papers shown here.
00:09:27.16 And I'll just very briefly summarize the clinical results.
00:09:31.14 Out of ten patients, seven were removed from immunosuppression successfully for periods
00:09:37.28 of years.
00:09:39.07 And their... their outcomes are shown here.
00:09:41.28 These first four patients had the first two regimens in the first ITN trial.
00:09:49.07 And that first patient is now more than 14 years off immunosuppression, doing very well.
00:09:55.27 This second patient is more than eight years off.
00:09:59.04 These other two patients had several years of no immunosuppression, but eventually returned
00:10:04.08 to immunosuppression, unfortunately, due to a low-grade chronic rejection.
00:10:12.13 In the second trial, where we added more B cell depletion to avoid this low...
00:10:19.03 very low-level antibody-mediated rejection that led to these patients returning to immunosuppression...
00:10:25.20 in the second trial, we added more rituximab, and these patients actually are now more than
00:10:30.12 seven years post-transplant and doing very well without any antibody-mediated rejection.
00:10:36.17 Now, I mentioned this term, mixed chimerism, where the donor and recipient cells coexist
00:10:42.14 in the patient.
00:10:43.14 And this is illustrated here for... for these four patients in the second trial.
00:10:50.21 And what you see is, in multiple bone marrow lineages -- lymphocytes, granulocytes, monocytes, etc --
00:10:57.09 we see a contribution of the donor in the circulating population, but only
00:11:05.02 for a very short period of time, for a period of 1-2 weeks.
00:11:10.06 So, this is very transient chimerism, and it's very different than what we can achieve,
00:11:16.06 for example, in rodents, where the chimerism persists forever.
00:11:19.16 But we knew from our non-human primate studies, and from some other studies in patients
00:11:25.10 with malignancies, that transient chimerism, when achieved with a kidney transplant at the same time,
00:11:30.18 could lead to tolerance.
00:11:32.24 So, in the lab, we've been studying what the mechanisms of this tolerance are.
00:11:38.13 And one of the things that we've observed in the patients who got this treatment is
00:11:42.24 that there's a marked enrichment for what we call regulatory T cells among the T cells
00:11:49.15 that initially come back after the transplant.
00:11:52.22 So, this slide here shows you the percentage of T cells in the CD4 lineage that have
00:12:00.26 this regulatory cell phenotype over time post-transplant.
00:12:04.04 And each type of symbol represents an individual patient.
00:12:09.08 And what you see is that these percentages of regulatory cells in that first year post-transplant
00:12:16.17 are very, very high compared to the pre-transplant level, which you see here is very, very low.
00:12:24.06 It's normally a very small percentage of CD4 T cells, but it goes way up after the transplant.
00:12:28.28 It eventually comes down to normal over a period of about a year.
00:12:33.18 Now, in... if you look at the actual absolute numbers of those regulatory T cells,
00:12:39.17 you can see, over here, that they are in fact depleted at one week post-transplant, but that
00:12:45.27 within a couple of weeks they come pretty close to the pre-transplant baseline level.
00:12:51.06 In contrast, the non-regulatory T cells, the conventional CD4 T cells
00:12:55.27 -- here, you see their pre-transplant values, and here you see post-transplant --
00:13:00.12 they remain low for a very, very long time.
00:13:03.06 And that explains why we see such a marked enrichment of the regulatory cell population
00:13:08.28 among those CD4 T cells.
00:13:11.17 And these are bona fide regulatory T cells, because FOXP3 is the transcription factor
00:13:18.16 that is a master regulator of the Treg program.
00:13:22.20 And demethylation of the... of the FOXP3 region in the genome is a hallmark of a...
00:13:29.12 of a bona fide Treg.
00:13:30.24 And we can see here, in this bottom plot, that the level of TSDR methylation
00:13:36.27 actually correlates very well with the percentage of regulatory T cells we detect.
00:13:40.28 So, these are really Tregs.
00:13:42.10 Why are they so enriched?
00:13:45.02 Well, it looks like there's a few things going on.
00:13:48.24 But the main one is probably that they are expanding in the periphery after we
00:13:56.14 deplete the T cells, the vast majority of the T cells.
00:13:59.24 So, it seems that they... some of them are spared from the depleting T cell antibody,
00:14:06.09 and the ones that remain undergo a lot of proliferation.
00:14:09.12 And we can see this here in this upper right plot, where we're looking at the percentage
00:14:14.27 of these regulatory T cells that express Ki67, which is a marker of proliferating cells.
00:14:21.00 And you can see that it's way up at 1 and 2 weeks post-transplant compared to baseline levels.
00:14:28.13 So that... and this is something that happens when you deplete lymphocytes,
00:14:32.12 that the ones that remain undergo proliferation.
00:14:35.01 So, that's one of the major mechanisms of this enrichment.
00:14:40.02 Well, what role do these Tregs play in the tolerance that we see in these patients?
00:14:44.10 Well, we can look at this by looking at alloresponses in in vitro mixed lymphocyte reactions and
00:14:51.23 cytotoxic T lymphocyte assays.
00:14:54.18 And what we have found in all of our tolerant patients is that they become very hyporesponsive
00:15:01.11 toward their donor.
00:15:02.15 Here, we're looking at their proliferative response, in red, to the donor,
00:15:07.04 and in blue, to a third party individual to... that they're not tolerant to.
00:15:12.27 You see that the response to the donor is markedly reduced.
00:15:16.26 And this this particular sample was taken one year after a transplant.
00:15:21.10 But what you see over here is that when you remove the regulatory T cells, the Tregs,
00:15:26.15 from that patient's cell population, and now measure the response to the donor,
00:15:31.08 a response is revealed.
00:15:32.27 So, this indicates that those regulatory T cells were suppressing the anti-donor response.
00:15:38.22 However, in this same patient, when we went back and did similar studies at 8 and 18 months post-transplant,
00:15:45.08 the result was a bit different.
00:15:47.09 Now, you still see that patient is hyporesponsive to the donor -- you can't even see a red symbol here,
00:15:54.01 because there's no response to the donor -- but now depleting the regulatory T cells
00:15:59.23 doesn't really reveal any anti-donor reactivity.
00:16:02.17 There's still very little response there, even though we've enhanced the response
00:16:06.27 to third party by depleting Tregs.
00:16:09.07 So, this suggested to us that we no longer depended on regulatory T cells to see
00:16:15.15 this donor-specific hyporesponsiveness at 18 months post-transplant, and that something else
00:16:21.13 might be going on.
00:16:22.23 And we hypothesized that maybe that large number of alloreactive T cells present
00:16:27.18 prior to the transplant had been deleted of those that recognized the donor.
00:16:33.14 So, to summarize these functional assays, tolerance in seven of seven cases in this study
00:16:40.22 was associated with the development of donor-specific unresponsiveness in
00:16:45.20 these in vitro assays.
00:16:47.16 We saw a regulatory T cell enrichment in all of these patients after the transplant.
00:16:52.26 And in some assays, we could see that those regulatory T cells were playing a role in
00:16:57.05 suppressing the anti-donor response.
00:16:59.20 But when we looked late, more than a year post-transplant, we did not see a role
00:17:04.18 for regulatory cells in suppressing that response in the longer term.
00:17:10.13 So, this made us think that perhaps the long-term tolerance might be deletional.
00:17:16.27 Okay.
00:17:18.04 Well, there's... deletion is one possibility, that the donor-specific T cells actually got
00:17:23.13 eliminated.
00:17:24.13 So, here, if we think of the red cells as the ones that recognize the donor,
00:17:29.21 if they're deleted they're actually gone from the immune repertoire after the transplant.
00:17:33.16 But another possibility is that they're anergic, meaning that they persist but they
00:17:39.23 simply don't respond when stimulated through their T cell receptor.
00:17:43.01 They're in the state of anergy.
00:17:45.03 And functionally, with the kinds of assays that I've just told you about,
00:17:48.09 there was no way to distinguish those two possibilities.
00:17:51.25 So, we really wanted to find a way of distinguishing them, and actually seeing what happens to
00:17:58.07 alloreactive T cells.
00:18:00.08 And this is a big challenge, because T cells recognizing a given set of alloantigens
00:18:06.15 on an MHC-mismatched donor represent a very large number of cells and specificities.
00:18:14.10 It's thought that 1-10% of T cells respond to a given donor.
00:18:19.09 And this is thought to be due to recognition of thousands of different peptide/MHC specificities
00:18:25.13 on an allogeneic MHC.
00:18:28.23 And all the studies that had been done show that there's no particular predictable
00:18:36.01 dominant immune response.
00:18:37.10 And so there's really no way to pick out clones that you can track over time with tetramers,
00:18:43.11 for example.
00:18:44.13 So, the approach that we used was really facilitated by the development of a technology for
00:18:50.10 high-throughput sequencing of the hypervariable region of the T cell receptor beta chain,
00:18:56.22 known as the CDR3.
00:18:58.23 And this is the... the part of the T cell receptor that is most specific for the peptide
00:19:05.27 that is seen by that T cell receptor.
00:19:09.06 And this hypervariable region is formed by the rearrangement of the V, the D, and the J segments
00:19:16.08 of the T cell receptor, along with N insertions that give it additional diversity.
00:19:22.17 And a commercially available platform was developed for actually sequencing up to
00:19:30.18 millions of these unique sequences simultaneously.
00:19:35.15 And this led us to hypothesize that high-throughput CDR3 sequencing of transplant recipient's
00:19:43.13 donor-reactive T cells prior to a transplant would allow us to identify
00:19:49.13 the repertoire of TCRs... of clones that recognize the donor's alloantigens.
00:19:55.11 And that we could then carry out such sequencing after the transplant to track the fate
00:20:00.17 of those T cells.
00:20:02.23 Using this approach, we actually succeeded in developing a method for tracking
00:20:09.20 a patient anti-donor T cell response and obtaining evidence for clonal deletion
00:20:16.02 as a mechanism of tolerance in the patients that I've been speaking about.
00:20:20.10 And what this assay involves is taking patient lymphocytes, whole PBMCs;
00:20:27.14 labeling them with a CFSE dye, which is a fluorescent dye that dilutes each time the cell divides,
00:20:34.21 so the level of CFSE staining is a marker of how much a given cell has divided;
00:20:40.22 and stimulating those in a co-culture for six days with donor PBMCs that have been irradiated,
00:20:47.02 so they can't divide, and also labeled with a different dye, a violet dye;
00:20:53.16 co-culturing for six days, collecting the cells, and specifically sorting the recipient, the responder cells,
00:21:02.00 that have divided -- those that have diluted their CFSE dye -- and separately sorting, on a FACS sorter,
00:21:11.24 CD4 and CD8 cells of that recipient that have divided in response to donor antigens.
00:21:20.09 And what we did is then subjected each of these populations, these divided cell populations,
00:21:25.18 to high-throughput sequencing of the T cell receptor CDR beta...
00:21:30.15 CDR three region.
00:21:32.01 And also did the same thing on CD4 and CD8 cells from the unstimulated T cell population
00:21:37.24 of that patient.
00:21:38.24 And this is all prior to the transplant.
00:21:41.22 And then we can actually define a sequence as alloreactive... donor-reactive if there...
00:21:49.03 if it's expanded more than fivefold in this mixed lymphocyte reaction compared to
00:21:55.05 its frequency in the unstimulated population, shown over here.
00:22:01.09 So, this... this is a way of identifying a repertoire, a set, of T cells that we call
00:22:08.00 a fingerprint of the anti-donor alloresponse.
00:22:13.24 And this is what... when we developed this assay, we tested it on our tolerant patients.
00:22:19.14 And what we found was that in all three of the tolerant patients who we studied
00:22:25.16 there was a significant... a statistically significant decline in the frequency of donor-reactive
00:22:32.18 CD4 and CD8 cells in the circulation, over time, after the transplant.
00:22:37.22 And we saw this in all three patients compared to the pre-transplant level.
00:22:44.03 We also had one patient in this trial who failed to achieve tolerance, who got the same treatment
00:22:49.22 but rejected the kidney after the immunosuppression was withdrawn.
00:22:56.06 And what you see here is that this patient did not show any significant reduction
00:23:01.24 in the frequency of anti-donor clones in the circulation.
00:23:06.06 So, it suggests that this method actually distinguishes the tolerant from the non-tolerant patients.
00:23:14.13 We've also tested this method on patients who don't get a tolerance-inducing regimen
00:23:19.15 but who just get a kidney transplant with conventional immunosuppression.
00:23:24.25 And some of our typical results are shown here.
00:23:28.22 Interestingly, we don't see any reduction... these are two different individuals, two different recipients,
00:23:35.04 looking at the frequency of donor-reactive clones over time.
00:23:41.02 Here's pre-transplant, and here's post-transplant.
00:23:43.17 You can see that in both of these patients there's a statistically significant increase
00:23:48.11 in the number of circulating donor-reactive CD4 clones after the transplant, showing you
00:23:54.05 the stimulation of the immune response by the transplant.
00:23:58.09 So, that helps to validate this assay as showing us something very biologically meaningful.
00:24:06.01 And what we were able to conclude from this study is that high-throughput deep CDR sequencing
00:24:11.24 of recipient's donor-reactive T cells pre-transplant enables identification of a specific set of
00:24:19.23 donor-reactive T cells.
00:24:22.01 And these donor-reactive clones can then be tracked in the post-transplant period to
00:24:27.19 tell us something about what's going on immunologically.
00:24:32.14 And our studies indicate that we are identifying biologically relevant T cells with this pre-transplant MLR,
00:24:40.12 because their frequency goes up in a conventional transplant recipient
00:24:44.06 after the transplant.
00:24:46.11 And our data suggests that in the tolerant patients who get
00:24:49.05 combined kidney and bone marrow transplantation,
00:24:52.11 deletion of donor-reactive T cells is a long-term mechanism of tolerance.
00:24:56.28 And in studies I didn't have time to go through, this deletion seems to be the result
00:25:02.25 both of global T cell depletion with the conditioning and specific exposure to the donor antigens.
00:25:09.20 In contrast, expansion of circulating donor-reactive clones is detected in conventional transplant
00:25:15.22 recipients.
00:25:17.07 And so far, this deletion analysis has outperformed the functional assays that I referred to earlier
00:25:25.14 because functional studies actually showed donor-unresponsiveness in the patient
00:25:29.28 who failed tolerance in addition to those who succeeded, suggesting that that patient
00:25:35.16 was demonstrating anergy, at least under the conditions of our in vitro assay, whereas this
00:25:41.19 deletional assay actually distinguished the tolerant from the non-tolerant patient.
00:25:47.06 Okay.
00:25:48.24 I'm just going to spend a few minutes talking about how this TCR tracking method can also
00:25:54.26 be used to better understand what's going on within an allograft.
00:26:00.20 And this study actually involves patients who receive intestinal transplants.
00:26:06.26 And at our center, at Columbia, our patients are actually followed by surveillance biopsies
00:26:13.28 of the intestinal allograft through a stoma that is created at the time of transplant,
00:26:19.28 because the symptoms of rejection can be quite nonspecific.
00:26:23.15 And doing these surveillance biopsies is a way of making sure that we're on top of
00:26:31.08 a rejection if it does occur.
00:26:32.25 So, it's looked at histologically.
00:26:35.03 So, this approach has actually given us an opportunity to look not only in the circulating
00:26:42.04 cell populations of these patients but also at what's going on in the graft biopsy specimens
00:26:49.00 in real time.
00:26:50.00 And we've taken advantage of this to look at... within the graft at the
00:26:57.01 alloreactive T cells that we've identified with the method I just spoke about.
00:27:01.13 So, just to give you a bit of background, intestinal transplant outcomes are...
00:27:06.28 are not as good as we would like them at this point.
00:27:11.07 And there's a lot of rejection that occurs.
00:27:13.25 And particularly in patients who get intestinal transplants alone.
00:27:18.13 Some patients don't just get an intestinal transplant; they get a liver transplant with it,
00:27:22.19 because their liver has failed for a variety of reasons, often due to chronic TPN
00:27:29.06 used to treat the intestinal failure.
00:27:32.10 But what you see in this slide is that the patients who get multivisceral transplants
00:27:36.11 -- liver, pancreas, stomach, and everything along with the intestine --
00:27:40.07 actually have lower rejection rates than patients who get intestines alone.
00:27:44.16 So, we hypothesized that this might have to do with the interplay of graft-versus-host
00:27:52.04 and host-versus-graft reactivity in these patients.
00:27:56.20 Now, I should say that the intestine comes with a very big load of lymphocytes.
00:28:01.25 And it's known that intestinal transplantation can cause graft-versus-host disease.
00:28:07.13 So, we've looked at this in our patients, and hypothesized that, in fact,
00:28:14.26 lymphocytes from that graft may go into the circulation, and that may be a marker of patients who
00:28:22.11 won't have rejection.
00:28:24.15 And this can occur without graft-versus-host disease.
00:28:27.16 And in fact, when we investigated this hypothesis, it turned out to be the case.
00:28:32.01 What we found in a lot of these patients, and particularly those who got the multivisceral transplants,
00:28:38.03 shown with the circles... we saw very high levels of donor chimerism
00:28:44.00 in the circulation, spontaneously, without any bone marrow transplant.
00:28:48.24 And most of these patients did not have graft-versus-host disease.
00:28:52.13 In 14 patients shown here, 8 showed this mixed chimerism in the circulation, but only one
00:28:59.05 had graft-versus-host disease, and it was very mild and self-limited.
00:29:03.05 Interestingly, many patients did not have this macrochimerism.
00:29:08.20 And we define macrochimerism as more than 4% donor T cells in the circulation at its peak.
00:29:15.20 And it's most commonly the patients getting intestinal transplants, the ones here
00:29:20.00 with triangles, did not get macrochimerism.
00:29:23.03 But what was striking is this association down here.
00:29:26.16 We observed that the patients who have this macrochimerism, as we've defined it,
00:29:31.14 have much lower graft rejection rates than those who don't have macrochimerism,
00:29:37.00 consistent with the hypothesis that we started out with, that this macrochimerism may protect the patients
00:29:44.19 from rejection, and can occur without graft-versus-host disease, as we've seen.
00:29:50.06 Now, as I mentioned, we also study the grafts, and we can do flow cytometry with
00:29:55.08 multiple parameters to actually look at the replacement of donor cells by the recipient within the graft.
00:30:02.20 And we can look at all sorts of different subsets of cells within those mucosal biopsies
00:30:08.07 over time.
00:30:09.22 And this is just an example of one such biopsy, where we're looking at different lymphocyte subsets,
00:30:14.13 and we have specific markers that... this antibody that goes up in the y-axis distinguishes
00:30:22.10 recipient cells, whereas those that are negative for that antibody are donor-derived.
00:30:26.23 So already, you're seeing mixed chimerism in this intestinal graft.
00:30:32.20 And what we noticed is that there was a highly variable rate of replacement of
00:30:39.09 the donor T lymphocytes that come in that graft by recipient T cells from patient to patient.
00:30:47.19 And that there was an association with rejection, and development of donor-specific antibodies,
00:30:54.28 with more rapid replacement by the recipient.
00:30:58.21 So, this part of the slide is showing you the rate at which... the percentage of recipient cells
00:31:06.15 in these different T cell subsets.
00:31:08.16 And you can see that it's quite high quite early on in these patients who undergo rejection.
00:31:15.02 Each line is a different patient.
00:31:17.09 In contrast, patients who don't have rejection, or have a DSA-negative rejection,
00:31:22.24 the rate of replacement of donor T cells by the recipient within the graft is very slow.
00:31:28.20 Very interesting.
00:31:29.20 And this part of the slide on the right just represents this in a different way,
00:31:35.04 making the same point.
00:31:36.16 So, what... what's going on here?
00:31:39.21 It looks like donor cells appearing in the blood and recipient cell... recipient T cells
00:31:46.00 not replacing the donor cells in the graft is associated with less rejection.
00:31:51.20 Well, our original hypothesis was that, in fact, all of this is reflecting a balance
00:31:57.17 between the graft-versus-host response, caused by T cells in the graft,
00:32:02.18 and the host-versus-graft response, which is a systemic immune response.
00:32:08.02 And using this T cell receptor tracking method that I just spoke about, we could actually
00:32:13.08 look at this in both directions: in the graft-versus-host and host-versus-graft directions.
00:32:18.08 So, this is the same assay that I mentioned earlier, and now we're doing it in both directions
00:32:23.28 on pre-transplant donor and recipient cells to identify the GvH and the host-versus-graft
00:32:31.23 T cell repertoires.
00:32:33.24 And now we can interrogate these biopsy specimens for these clones.
00:32:39.13 And what we found was quite striking.
00:32:42.11 In the early period post-transplant, particularly in those patients who have slow replacement
00:32:48.17 of their graft T cells by the recipient, there's a marked expansion of graft-versus-host reactive
00:32:55.13 T cells within the graft.
00:32:57.20 It's... they're much more frequent than what we see in the lymphoid tissue prior to transplant,
00:33:04.06 for example, shown in the black bars.
00:33:06.09 So, there's a huge expansion of GvH-reactive CD4 and, over here, CD8 cells
00:33:12.08 in the graft compared to what was in the donor lymphoid system.
00:33:18.14 And this was interesting.
00:33:19.23 And we wondered why that was.
00:33:21.27 Our analyses, our flow cytometric analyses, included analyses of the antigen-presenting cell
00:33:29.20 populations in the graft.
00:33:32.23 And what we found was, in contrast to T cell replacement rates, which were extremely variable,
00:33:38.24 as I showed you...
00:33:40.11 patients with rejection tended to have rapid replacement of T cells by the recipient,
00:33:46.01 whereas those without rejection had slower replacement... in contrast to all that, all of the patients
00:33:51.21 showed quite rapid replacement of antigen-presenting cells, of myeloid cells,
00:33:57.03 with a dendritic cell phenotype by the recipient.
00:34:01.05 This one is at day 16.
00:34:03.11 Almost all the APCs are recipient-derived, whereas the T cells are still mostly donor.
00:34:08.25 There's very few recipient ones.
00:34:11.00 So, that was quite a uniform finding: early replacement of donor APCs by the recipient.
00:34:17.26 And that could explain this expansion of graft-versus-host-reactive cells in the graft.
00:34:23.07 They're having recipient antigens presented to them on these recipient APCs that enter the graft,
00:34:29.11 expanding this GvH response.
00:34:32.04 And we think this is very protective.
00:34:34.20 We have additional studies that I don't have time to take you through, but that GvH response
00:34:40.26 also goes into the circulation, contributing to the macrochimerism.
00:34:45.18 The other thing that we can see with this technique... we can interrogate the biopsies
00:34:50.06 for host-versus-graft clones, and what we see is something that hasn't been shown before.
00:34:56.07 During a rejection of a human allograft, there's a huge enrichment of host-versus-graft alloreactive
00:35:02.19 T cells within those grafts.
00:35:05.26 And that may be obvious, but there's some dogma from the literature that most T cells
00:35:11.02 infiltrating a graft during a rejection are bystanders; they're nonspecific.
00:35:16.02 That is clearly not the case here.
00:35:18.04 A very high percentage of these T cells during a rejection are host-versus-graft reactive,
00:35:24.16 as defined by our TCR tracking method.
00:35:27.13 This goes down as the rejection resolves, but it's still an enrichment for...
00:35:33.02 those host-versus-graft cells persist long-term in these patients, and we think they may pose
00:35:38.27 a constant risk for rejection.
00:35:41.10 So, I'll end here with a summary.
00:35:44.25 We have found that there's a direct correlation between early region and accelerated replacement
00:35:50.09 of donor T cell populations in the graft by recipient T cells that look like those
00:35:56.19 in the circulation.
00:35:57.25 They have a blood-like phenotype.
00:36:00.27 Host-versus-graft clones predominate among those host T cells within rejecting grafts.
00:36:05.12 They persist at lower levels long-term.
00:36:08.07 And what I didn't show you is that they changed their phenotype long-term;
00:36:11.28 they look more like tissue resident lymphocytes, and they seem to seed the entire gut.
00:36:16.20 And we think these pose a constant threat of rejection.
00:36:20.27 Thirdly, in contrast to the highly variable replacement rate of donor T cells by the recipient
00:36:26.06 in the gut, antigen-presenting cell replacement is uniformly rapid.
00:36:30.25 And finally, we think these rapidly immigrating recipient APCs are driving the local expansion
00:36:37.12 and activation of GvH-reactive T cells coming with the graft, and that these
00:36:42.15 may actually control the host-versus-graft-reactive clones, curbing rejection and replacement of
00:36:49.00 donor T cells by the recipient within the graft.
00:36:52.12 So, I'm going to end there.
00:36:54.13 Obviously, I've talked about a lot of different studies, and that's involved a huge number
00:36:59.23 of people, both at Columbia and originally at Mass General,
00:37:05.09 where we did the clinical trials of tolerance induction.
00:37:09.15 And the intestinal transplant studies have involved many people in the lab,
00:37:15.23 but also in... on the clinical side as well.
00:37:18.15 So, thank you very much for your attention, and I'll stop there.

00:00:14.27 Hi, I'm Megan Sykes.
00:00:16.04 I'm a professor at Columbia University and Director of the Columbia Center
00:00:20.16 for Translational Immunology.
00:00:22.13 I'm going to give you an overview of xenotransplantation in this lecture.
00:00:27.07 So, the field of transplantation is limited by drug treatment-related complications,
00:00:33.20 chronic rejection, and the availability of organs.
00:00:37.15 So, one solution that would overcome all of these problems would be xenotransplantation,
00:00:43.22 meaning transplantation of organs from another species, with induction of tolerance to avoid
00:00:51.11 the drug treatment-related complications and chronic rejection.
00:00:56.15 Currently, many more people need organ transplants than get them.
00:01:02.11 These... these ovals represent the people who need organ transplants,
00:01:08.01 who have end-stage organ failure,
00:01:11.19 versus those... only a fraction of those make it to the waiting list,
00:01:15.15 and only a smaller fraction yet of those actually come to transplant.
00:01:20.00 The unfortunate result of this is that many people actually die waiting for an organ.
00:01:26.19 120,000 people currently are on a waiting list in the United States.
00:01:31.18 And less than a third of these receive transplants, and many die waiting for an organ.
00:01:37.26 So, it would really be nice to have an unlimited supply of organs.
00:01:43.13 And xenotransplantation potentially could fill this need.
00:01:46.28 Most of the field feels that pigs would be the most desirable organ source for human transplantation
00:01:55.23 for a number of reasons, which I'll come back to.
00:01:59.26 However, pigs, and most mammalian species, and also non-mammalian species, in fact,
00:02:07.05 express an antigen that is quite ubiquitous and that causes... has posed a major barrier
00:02:12.19 to xenotransplantation for many years.
00:02:15.21 And that is an epitope called alpha1,3Gal, which is a terminal carbohydrate modification
00:02:23.04 of many glycoproteins and glycolipids, that is present in most species, and it's made
00:02:29.10 by an enzyme called alpha1,3Gal transferase.
00:02:33.01 As it happens, old-world monkeys, and subsequently humans, have a mutation in this enzyme gene
00:02:41.10 and therefore don't make this epitope.
00:02:44.02 And because many species like bacteria and other microbes do have the alpha1,3Gal epitope,
00:02:50.18 we all get exposed to it.
00:02:52.13 And therefore we have antibodies in our circulation that recognize alphaGal.
00:02:57.26 These are called natural antibodies because they're there without any known exposure
00:03:04.02 to a pig or any anything else, but nevertheless they're present in all human sera.
00:03:11.16 And what those can do, if you do a transplant from a pig to an old-world primate,
00:03:18.28 is they can immediately bind to the endothelial cells at the graft, fix complement, and cause
00:03:25.15 hyperacute rejection, or a more delayed form of rejection called delayed xenograft rejection.
00:03:30.15 So, this has been a major obstacle to the field that actually was overcome by
00:03:36.13 a technological advancement, which is the ability to genetically engineer pigs.
00:03:42.11 And in the early 2000s, this gene... this enzyme gene, alpha1,3-galactosyl transferase,
00:03:49.27 was actually knocked out of a pig.
00:03:52.06 And that has really helped to transform the field.
00:03:55.17 Now, these pigs that we use in our studies at Columbia are actually a special line of pigs
00:04:02.07 that have been generated for over 40 years through inbreeding by David Sachs,
00:04:09.13 who was part of our Columbia team, and that are miniature swine.
00:04:13.00 And so they're actually a good size for organ transplantation to human,
00:04:19.26 because they're closer to our size, rather than the thousand pounds that a regular pig can grow to.
00:04:26.00 So, the alpha1,3Gal gene was knocked out of these miniature swine in the early 2000s.
00:04:32.26 And this is a picture of the first such animal, and it was perfectly healthy.
00:04:37.18 And these animals, now, are available for research on a regular basis.
00:04:44.26 Now, other groups also have knocked out alphaGal from more conventional-sized pigs,
00:04:51.27 and this really led to a transformation in the field overall.
00:04:55.15 This sort of shows you the progress of the field in terms of pig organ survival in xenotransplantation
00:05:04.13 to primates.
00:05:05.26 Before 1980, it was minutes.
00:05:08.18 In the 1980s, immunoabsorption procedures were developed to get rid of natural antibodies,
00:05:15.16 and that prolonged graft survivals to hours.
00:05:18.07 In the 1990s, the first transgenic pigs were made that express human complement regulatory proteins,
00:05:24.22 and that, combined with immunosuppress... advances in immunosuppression, permitted xenograft
00:05:31.13 survivals of days to weeks.
00:05:34.04 And then in the 2000s, with the development of these Gal knockout pigs, survivals improved
00:05:39.15 to months.
00:05:40.22 And as I'll show you, in the 2010s, we've come even further than that.
00:05:45.13 Now, there are three approaches, major approaches, to overcome... overcoming xenograft barriers.
00:05:52.11 And these can really be used in combination.
00:05:55.12 One is immunosuppressive therapy.
00:05:57.03 The second is genetic engineering, as I've already mentioned.
00:06:00.24 And the third is tolerance induction.
00:06:02.18 And we think that tolerance is going to be an important component of successful
00:06:08.15 clinical xenotransplantation, because of the very high level of immunity that we have to
00:06:15.10 these highly disparate donors.
00:06:18.15 This is a slide showing a paper from Mohiuddin et al that was published a couple of years ago,
00:06:25.03 that shows how far we've come in getting graft survival from pigs into non-human primates
00:06:32.16 using immunosuppression and genetic engineering.
00:06:36.27 So, this study involved the use of pigs that had been engineered to... they were Gal...
00:06:46.23 alphaGal transferase knockout pigs that had this human complement regulatory protein,
00:06:52.19 CD46, and human thrombomodulin, which inhibits coagulation.
00:06:57.08 And these hearts were transplanted heterotopically, so they're not functioning hearts,
00:07:01.14 but they were put in the abdomen as a sort of accessory heart in these baboons.
00:07:06.11 And what you see is very, very long survival of the animals that got the full immunosuppressive regimen.
00:07:15.10 And unfortunately, the survival was very dependent on high doses of that immunosuppression.
00:07:20.23 As soon as it was reduced, the grafts were rejected.
00:07:24.02 But you can see that the number of days these grafts survived is close to a thousand.
00:07:29.20 So, we now have survival of these heart grafts for several years.
00:07:36.03 Okay.
00:07:37.04 Well, there's other things that can be done to pigs in terms of genetic engineering
00:07:42.20 to help facilitate xenotransplantation.
00:07:46.09 Some people are thinking about removing the major histocompatibility complex antigens,
00:07:52.14 the MHC of the pig, which is referred to as the SLA, so that they can't be seen
00:07:57.18 by the immune system.
00:07:59.03 This is an interesting approach, but it has some limitations.
00:08:02.00 First of all, indirect recognition of processed and presented antigen from the pig could lead
00:08:10.20 to destruction by other mechanisms involving cytokines, etc.
00:08:17.04 Secondly, a lack of MHC will make a cell more prone to be attacked by natural killer cells.
00:08:26.11 This can be overcome with some further genetic engineering, potentially.
00:08:29.10 But thirdly, if a graft doesn't have any SLA molecules, any MHC, it can't present antigen
00:08:37.10 to T cells at all, and so T cells can't protect the graft from infections,
00:08:41.19 so that's a potential problem.
00:08:44.09 So, our approach is not to get rid of the MHC on the... on the donor, but instead
00:08:53.03 to try and re-educate the recipient's immune system, to regard the donor itself, by inducing tolerance.
00:09:00.00 And there's two approaches to tolerance that I'm going to speak about.
00:09:02.28 One is mixed chimerism.
00:09:04.07 And as you'll see, this tolerizes T cells and B cells, and also even natural killer cells.
00:09:10.24 And the second is thymic transplantation, which tolerizes T cells.
00:09:15.22 So, many years ago, we actually tried to develop a non-myeloablative, low toxicity,
00:09:24.15 potentially clinically relevant method of inducing mixed chimerism in the closely related species rat to mice.
00:09:31.05 And this shows you the regimen that involved monoclonal antibodies against T cells
00:09:37.05 and natural killer cells, local radiation to the thymus, and a very low non-myeloablative dose
00:09:43.28 of total body irradiation.
00:09:46.14 And these animals did develop mixed chimerism and were tolerant of their donors.
00:09:51.00 And it showed... this slide shows you that these chimeric animals actually accepted
00:09:57.14 skin grafts from those rat donors without any immunosuppression,
00:10:01.17 whereas they were still competent to reject skin grafts from a third party rat.
00:10:06.11 So, the tolerance was quite specific for that rat bone marrow donor.
00:10:12.22 And interestingly, this chimerism was sort of... it lasted a long time, but only at
00:10:18.17 very, very low levels over time.
00:10:20.22 And yet we could see that the T cell tolerance was mediated by the presence of
00:10:25.13 donor antigen-presenting cell in the recipient thymus that led to central deletion of donor-reactive T cells.
00:10:34.16 Using this model, we were also able to look at how what happens to an innate immune response,
00:10:40.21 particularly natural antibodies.
00:10:44.11 Mice do have natural antibodies against the rat, and that's how we did our first studies.
00:10:48.14 But later, when this alphaGal enzyme was identified... mice, like most species, do have alphaGal,
00:10:55.26 so they don't have anti-Gal natural antibodies, but by knocking out the alphaGal transferase from mice,
00:11:03.04 one could now produce a mouse that resembled a human in having natural antibodies
00:11:07.21 against Gal.
00:11:09.03 And so now we could ask, what happens when we do a rat bone marrow transplant to these mice?
00:11:15.22 The rats do express alphaGal.
00:11:17.24 Will we tolerize the B cells specific for Gal in addition to everything else.
00:11:24.17 And what we found was, indeed, that we could tolerize anti-Gal natural antibody-forming B cells
00:11:29.16 by induction of mixed chimerism.
00:11:33.22 And that we could thereby prevent both T cell-mediated and antibody-mediated rejection.
00:11:39.15 That's illustrated here, where we have several groups of mice, both wild type and
00:11:46.23 alphaGal knockout mice -- so, GalT +/+ and -/- -- that either received the conditioning
00:11:54.00 but no rat bone marrow transplant,
00:11:56.12 or that received conditioning with a rat bone marrow transplant,
00:12:00.05 so that they developed mixed chimerism.
00:12:02.19 And what happens is this conditioning actually really bumps up the level of anti-Gal antibodies
00:12:07.15 in these mice.
00:12:08.15 So, if you just put in a rat heart to one of these mice, it's actually very quickly rejected.
00:12:14.24 Some of them are hyperacutely rejected, others with a more delayed vascular rejection
00:12:20.10 type of pattern.
00:12:21.18 In wild type mice that just get the conditioning, so they don't have the anti-Gal,
00:12:26.17 they still have lots of T cell immunity to the rat, and they reject, by a cellular rejection mechanism,
00:12:32.00 within a week or so.
00:12:33.22 But our mixed chimeras, whether they're wild type or Gal-knockout recipients,
00:12:39.08 uniformly accept those rat heart grafts, showing that both the antibody-mediated
00:12:44.06 and the T cell-mediated rejection processes are avoided by induction of mixed chimerism.
00:12:51.00 This slide illustrates one of those hyperacutely rejected in a conditioned Gal-knockout mouse.
00:12:57.01 You can see that within 30 minutes that graft has turned black.
00:13:00.16 It's completely rejected due to this antibody-mediated mechanism, whereas if you look
00:13:06.14 at the wild type animal here, that doesn't have anti-Gal antibody, the heart is still pink and beating
00:13:13.01 at that 30-minute time point.
00:13:15.25 So, to summarize, mixed chimerism can be induced in Gal transferase-knockout mice
00:13:20.28 using non-myeloablative conditioning and high doses of rat bone marrow.
00:13:25.20 Mixed chimerism in this model leads to rapid tolerization of anti-Gal-secreting B cells.
00:13:31.00 And through a number of studies, we showed that this was true tolerance of the B cells.
00:13:38.14 And in the grafting studies you saw, we were able to prevent hyperacute rejection,
00:13:45.02 delayed xenograft rejection, cellular rejection, and chronic rejection
00:13:49.06 of these primarily vascularized cardiac xenografts.
00:13:54.07 Another innate component of the innate immune system is natural killer cells.
00:13:59.06 And what we observed early on in these rat-to-mouse studies was they actually pose a very strong
00:14:04.21 barrier to engraftment of rat bone marrow, much more so than to allogeneic bone marrow,
00:14:10.27 for example.
00:14:12.25 And so, in this rat-to-mouse mixed chimerism model, we included antibodies to deplete
00:14:18.01 natural killer cells, as well as another sort of innate subset of T cells, the gamma delta T cells,
00:14:24.04 we found also had to be depleted to get mixed chimerism.
00:14:28.11 So, there's more barriers to xenograft... in xenograft... xenogeneic hematopoietic cells
00:14:34.20 than to allogeneic hematopoietic cells from the innate immune system.
00:14:39.23 Okay.
00:14:40.23 Well, what about natural killer cells?
00:14:42.20 So, we deplete them, but they come back after we've induced mixed chimerism with this regimen.
00:14:49.17 We wanted to know whether those NK cells that come back are tolerant to the rat, or whether
00:14:54.09 they still have anti-rat reactivity.
00:14:57.15 Well, this is a complicated slide showing an old-fashioned type of assay for measuring
00:15:03.00 NK cell activity in vitro... in vivo.
00:15:05.11 It's called the IuDR uptake assay.
00:15:08.05 And with this assay, we discovered that induction of mixed chimerism from the rat led to tolerance
00:15:15.19 to the rat, but that it was associated with a global hyporesponsiveness of NK cells.
00:15:21.07 In contrast, conditioning alone did not lead to this effect, so it clearly had to do
00:15:27.11 with the presence of rat chimerism.
00:15:29.27 So, from this, we concluded that mixed xenogeneic chimerism leads to tolerance of T cells,
00:15:36.20 B cells making natural antibodies of all specificities, and natural killer cells.
00:15:41.17 So, in more recent years, we have actually been able to test these ideas and see whether
00:15:47.10 it holds up in the pig/human combination, not by doing human transplants to...
00:15:52.04 pig transplants to humans, but instead by creating human immune systems in immunodeficient mice.
00:15:59.02 And these are what we call humanized mice.
00:16:01.18 And this is a model developed in the laboratory of Yong-Guang Yang several years ago,
00:16:08.24 where he takes immunodeficient mice, gives them a little bit of whole-body irradiation,
00:16:14.11 and then transplants a fetal thymic tissue under the kidney capsule, and gives human
00:16:22.09 bone marrow stem cells intravenously.
00:16:24.24 And he also constructed immunodeficient mice to express porcine cytokine genes that are
00:16:32.15 important for porcine hematopoiesis.
00:16:35.06 And in doing that, could then get pig bone marrow to engraft as well, when given to
00:16:41.07 these mice that get human hematopoietic stem cell grafts.
00:16:45.04 And used this model to ask whether or not tolerance could be achieved by induction of
00:16:52.07 mixed chimerism in the human immune system.
00:16:54.18 And this slide shows you the coexistence of pig and human cells in one immunodeficient mouse,
00:17:01.08 now 25 weeks post-transplant.
00:17:05.00 You can see that there are human cells and pig cells by flow cytometry with specific
00:17:11.03 antibodies.
00:17:12.03 This is a pig antibody, and this is a human CD45 antibody.
00:17:17.02 They're coexisting, lifelong, in these animals.
00:17:21.16 And most importantly, when pig skin grafts were put onto these... these mixed chimeras,
00:17:30.12 it was found that the animals rejected third-party pig skin grafts, but accepted the donor skin grafts.
00:17:40.00 This one animal died at 40 days, but the others showed this pattern of long-term acceptance
00:17:45.23 of the donor skin, and rejection of the third-party skin from the pig.
00:17:53.06 In contrast, animals that are not humanized, they're not reconstituted, they're not able
00:17:57.15 to reject any skin grafts.
00:17:59.21 Whereas those that are reconstituted with the human immune systems but
00:18:04.19 don't get mixed chimerism, they for the most part reject both types of pig skin grafts.
00:18:09.25 So, this shows you that human-specific tolerance to the pig can be induced by induction of
00:18:16.22 this mixed xenogeneic chimerism.
00:18:19.04 So... and with other studies, these results proved that central T cell tolerance of
00:18:26.04 human T cells can be achieved to porcine xenografts through induction of mixed hematopoietic chimerism.
00:18:32.12 More recently in our laboratory, we have asked whether human natural killer cells
00:18:38.00 can also be tolerized to pig by induction of mixed chimerism,
00:18:41.06 as we saw occurred in the rat-to-mouse model.
00:18:45.01 To do this, we've had to take humanized mice and induce mixed chimerism in the way
00:18:50.09 that I just showed you, with pig bone marrow and pig cytokine transgenic recipients.
00:18:56.15 And now do some maneuvers to induce production of human natural killer cells,
00:19:04.24 because they need human IL-15 to be produced.
00:19:06.15 But in doing this, we were able to show that some of these mixed chimeras do indeed
00:19:13.20 show specific tolerance to the pig donor.
00:19:17.06 And that's illustrated here.
00:19:19.15 If you look at the open symbols...
00:19:24.01 I hope you can see them... that both the open and closed symbols represent... the open symbols
00:19:31.06 represent mixed chimeras; the closed symbols represent controls that are not porcine mixed chimeras,
00:19:37.02 but are just humanized; and then we have normal human PBMC with a square
00:19:44.02 and the dashed line.
00:19:45.19 And you can see that the normal human PBMC, and that all these mixed chimeras,
00:19:50.14 have similar killing of this class I-deficient target human cell line, called K562.
00:19:56.05 So, they have function.
00:19:58.15 But if you look at their killing of pig targets, you see that only the non-chimeras
00:20:04.24 kill the human... the pig targets, whereas the mixed chimeras are specifically unresponsive
00:20:11.17 to the pig targets.
00:20:13.22 Now, that's one pattern that we saw.
00:20:16.16 In other animals, we saw this other pattern, called global hyporesponsiveness,
00:20:23.06 where the mixed chimeras, shown with the open symbols, now didn't respond to the common target,
00:20:31.10 K562, and also didn't respond to the pig.
00:20:33.01 So, there were two types of tolerance: one that was desirable,
00:20:37.10 where it was specific for the pig;
00:20:40.06 another where there was a more global dysfunction of the natural killer cells,
00:20:44.24 which is not the ultimate endpoint that we would like.
00:20:49.20 So now we're working on engineering pigs in a way that will make them resistant to
00:20:55.14 human natural killer cells.
00:20:57.26 But to summarize what I've said so far, mixed xenogeneic chimerism leads to tolerance of
00:21:02.22 T cells, B cells, natural killer cells.
00:21:06.28 And so far, these conclusions seem to apply in the human... pig-to-human combination
00:21:11.22 in humanized mice as well as the rat-to-mouse.
00:21:14.13 I haven't shown you human B cell tolerance, but in work that's ongoing, that seems
00:21:19.18 to occur as well.
00:21:21.03 Okay.
00:21:22.03 Well, there's another innate barrier that gets in the way of mixed chimerism,
00:21:27.25 and that is mediated by macrophages.
00:21:31.18 Studies in baboons and also in murine models have shown that mixed chimerism from
00:21:41.13 a highly disparate xenogeneic donor can be extremely short-lived due to destruction by macrophages
00:21:50.21 of those xenogeneic cells as soon as they come in.
00:21:53.05 Now, why are they so quickly eaten by macrophages?
00:21:56.09 Well, it turns out that there is a major molecular interaction that tells macrophages
00:22:04.00 not to eat circulating cells.
00:22:06.13 This is the CD47-SRIPalpha pathway, and this is what prevents our macrophages from normally
00:22:14.04 eating up our erythrocytes.
00:22:16.19 Only when an erythrocyte gets old and loses CD47 expression does it get taken up
00:22:23.06 by a macrophage and destroyed.
00:22:25.28 So, CD47 is a ligand for SIRPalpha, which is an inhibitory receptor expressed on macrophages
00:22:34.20 that gives the macrophage a "don't eat me" signal.
00:22:37.28 As it turns out, porcine CD47 does not transmit that inhibitory signal to human or baboon SIRPalpha,
00:22:48.05 resulting in just unopposed activation of those macrophages, and rapid destruction
00:22:54.12 of the porcine... porcine cells.
00:22:57.02 So, based on this, we hypothesized that another genetic modification of the pig,
00:23:04.26 namely putting in the human CD47 gene into these miniature swine, would make them better
00:23:12.03 hematopoietic cell donors and give us more lasting mixed chimerism.
00:23:15.04 And indeed, that seems to be the case.
00:23:17.16 So, this is a study from David Sachs' lab, showing that peripheral blood stem cells,
00:23:25.11 mobilized stem cells, from the CD47-high transgenic pig led to quite prolonged chimerism,
00:23:33.13 shown in the lower-right quadrant of each of these plots, compared to peripheral blood stem cells
00:23:40.27 from a CD47-low pig, that really didn't express detectable CD... human CD47.
00:23:47.11 So, this CD47 seems to be markedly prolonging the survival of pig hematopoietic cells
00:23:55.27 in the circulat... in the baboon recipient.
00:23:58.21 And this is associated with prolongation of pig skin grafts.
00:24:03.25 So, what you see on this slide is the survival... what these pig skin grafts on these baboons
00:24:11.05 looked like at 14 days, in the control animal that very quickly rejected this skin graft.
00:24:19.26 This animal got pig PBSCs, but without high CD47.
00:24:24.12 And you can see it's rejecting already at 14 days.
00:24:27.17 And histologically there's a lot of rejection there.
00:24:30.22 In contrast, the grafts were much prolonged in the CD47-high pig PBSC recipients.
00:24:40.00 And this graft at 42 days still looks beautiful with, really, very little infiltration of
00:24:46.07 lymphocytes at that point.
00:24:48.12 So, this is a very important aspect of the approach that we're using to induce
00:24:56.05 mixed chimerism and tolerance, now, in non-human primate recipients.
00:25:01.15 The second approach to xenograft tolerance is thymic transplantation.
00:25:06.13 And ultimately, we think this one is going to be useful in conjunction with the hematopoietic chimerism.
00:25:14.11 And this work actually builds on studies that we did back in the 1990s, where we showed that
00:25:20.21 if we took normal immunocompetent mice, removed their thymus, and T cell depleted them
00:25:26.26 temporarily... gave monoclonal antibodies to T cells to the mice, and then put in
00:25:33.21 a pig thymus graft, the pig thymus would replace the mouse thymus that we'd removed,
00:25:39.21 generate a whole new repertoire of T cells.
00:25:41.24 And those T cells were tolerant of the pigs, so that you could put this pig skin graft
00:25:46.04 on to an immunocompetent mouse, and it wasn't rejected.
00:25:50.23 So, we've shown that this same approach works in a human immune system, in human humanized mice.
00:25:58.11 Now, constructing these humanized mice instead of with a human... excuse me...
00:26:03.15 fetal thymus graft, putting in porcine fetal thymus tissue, and then giving the same human stem cells
00:26:11.05 to both groups.
00:26:13.04 And what we find... the graft that we put under the kidney capsule of these mice,
00:26:18.07 the fetal thymus graft, is very, very small: it's one cubic millimeter.
00:26:22.21 And you can see now these are... these are animals that are euthanized after the graft
00:26:27.18 has had a chance to grow.
00:26:29.03 And it was put under the kidney capsule.
00:26:31.26 And this reddish thing is the kidney, and the white thing is the graft that has grown
00:26:37.14 to be just as big as the kidney.
00:26:39.19 And the same occurs if it's a fetal pig thymus tissue, as we see with human thymus tissue.
00:26:50.18 And both of them really look the same in terms of the profile of cells in the thymus graft.
00:26:57.11 So, these are human thymocytes in the human thymus graft showing a very normal CD4/CD8 profile
00:27:02.24 showing regulatory T cells in normal percentages.
00:27:07.05 And this is the same stem cells developing in the pig thymus graft.
00:27:12.01 They're really quite indistinguishable, phenotypically, from what we see in a human thymus graft.
00:27:18.14 And most importantly, once again, putting in this pig thymus, allowing human T cells
00:27:24.04 to develop in a pig thymus, leads to specific tolerance to the pig.
00:27:28.24 So, what we're looking at here is survival of pig skin grafts from the pig...
00:27:35.20 the same genetically similar donor to the pig that gave the bone marrow cells versus
00:27:42.22 a third-party pig that is SLA-mismatched to the donor pig.
00:27:47.06 And in mice with a pig thymus, you can see that the donor pig skin graft is markedly
00:27:54.10 survived... skin grafts are markedly prolonged, whereas the third-party grafts are rejected
00:28:00.15 in most cases.
00:28:02.01 Whereas, when those human T cells developed in a human thymus, they uniformly reject
00:28:09.14 the pig skin grafts.
00:28:10.15 So, we've induced specific skin graft tolerance with this thymus transplant.
00:28:17.16 Applying this in a large animal model, Kaz Yamada and colleagues in our center have developed
00:28:23.28 a regimen for immunosuppression that all... and also have adapted the thymus transplant approach
00:28:30.13 to be primarily vascularized, so it functions more quickly.
00:28:35.20 Rather than just being put as a piece under the kidney capsule... he actually does this
00:28:40.07 in several ways, but one way is to take the donor pig, take out its thymus,
00:28:45.02 cut it into pieces, and put it under the kidney capsule -- multiple pieces of the donor pig --
00:28:51.12 and then sew that animal back up, and allow that thymus graft to coalesce and grow
00:28:56.22 and become functional.
00:28:59.14 And then, transplanting the thymal kidney en bloc, as a single graft, to a non-human primate.
00:29:05.22 The other approach is to just take out the thymus graft... thymus from the pig and
00:29:10.22 vascularize it primarily in the primate recipient.
00:29:15.06 And the results that we've achieved with this approach are very, very exciting.
00:29:20.10 We... this is an example of an animal that got... a baboon that got
00:29:27.06 a life-supporting pig kidney that has normal kidney function at six months post-transplant,
00:29:35.06 that's sustaining that baboon's life.
00:29:38.06 Normal creatinine.
00:29:39.21 And the kidney looks perfect.
00:29:42.01 There's really no histologic abnormality, and no antibody binding to that kidney.
00:29:49.15 This animal unfortunately had to be euthanized for other reasons.
00:29:54.09 And in vitro studies on this animal, that aren't on this slide, actually showed donor-specific
00:30:00.11 unresponsiveness to that pig.
00:30:03.25 So, we think this is really applicable and very exciting as an approach to tolerance induction.
00:30:10.09 So, to summarize, we can achieve human T cells and NK cell tolerance to porcine xenoantigens
00:30:16.18 via mixed chimerism induction in humanized mice.
00:30:20.24 Mixed xenogeneic chimerism can be enhanced in primates by using human CD47 transgenic pigs
00:30:26.15 as source animals.
00:30:29.08 Porcine thymic transplantation, as a regulatory mechanism to the tolerance,
00:30:34.01 which I didn't go into,
00:30:36.04 but that seems to be very important in suppressing T cells that you can't get rid of prior to the pig transplant.
00:30:43.17 And ultimately, we think a combination of mixed chimerism and thymic transplantation,
00:30:48.16 and some further genetic modifications of the pigs, will make xenotransplantation and
00:30:53.23 tolerance clinically achievable and safe.
00:30:57.16 So, many, many people have contributed to this work.
00:31:02.05 This is a list of some of the key players in the large animal studies.
00:31:06.24 And many people also in the humanized mouse work that I spoke about.
00:31:11.22 So, I'll thank you for your attention and stop there.