Session 9: The Immunology of Organ Transplantation

Transcript of Part 2: Taming and Tracking the Human Alloresponse

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