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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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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