Session 9: The Immunology of Organ Transplantation

Transcript of Part 3: Xenotransplantation

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