Every year a significant number of patients require organ transplantation for survival. In order to reduce the chances of organ rejection, the recipient and donor immune systems need to match. If they don’t match, the recipient immune system will recognize the donated organ as non-self. In this session, you will learn about the major types of transplantation, evaluate the immune cells involved in organ rejection, and understand the basics of transplantation tolerance (long-term graft acceptance without the long-term use of immunosuppressants). In order to achieve tolerance, a new transplantation protocol that induces mixed chimerism in patients has been developed. This protocol results in a patient’s immune system resembling both the donor and the recipient, and allows for transplanting organs between donors and recipients that aren’t an immunological “match”.
Introduction to Transplantation: The Immune Response to Allo- and Xenotransplantation
Concepts: Allo- and Xenotransplantation, immune cells involved in transplantation rejection, HLA matching, acute & chronic rejection, direct & indirect T Cell response, immunosuppressive drugs, and hematopoietic cell 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.
00:08:16.10 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.
00:09:23.24 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
00:11:16.25 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.
00:12:26.07 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.
00:14:36.09 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.
00:16:44.03 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.
00:24:18.20 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.
00:25:51.23 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.
00:28:04.24 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.
00:29:25.13 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.
00:30:46.00 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
00:31:57.07 So, that's the end of the first presentation.
00:31:59.11 Thank you for your attention.
Taming and Tracking the Human Alloresponse
Concepts: Transplantation tolerance, and mixed chimerism
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
00:02:50.00 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.
00:17:18.04 Well, there's... deletion is one possibility, that the donor-specific T cells actually got
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
00:25:17.07 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.
00:25:48.24 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.
Concepts: Xenotransplantation, 𝛼1,3-Gal, genetically engineered pigs for organ transplants, xenograft rejection, tolerance induction, mixed chimerism, CD47 signaling/macrophages in xenotransplantation
Note: This video is optional for this session.
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.
00:07:37.04 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.
00:14:40.23 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
00:17:12.03 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.
00:21:22.03 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.
Megan Sykes iBioSeminar: The Immune Response to Allo- and Xenotransplantation
Dr. Megan Sykes is the Friedlander Professor of Medicine, a Professor of Microbiology and Immunology and a Professor of Surgical Sciences at Columbia University Medical Center, and the director of the Columbia Center for Translational Immunology. In 1982, Sykes completed her medical degree at University of Toronto and continued her medical training in Montreal and… Continue Reading
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