Hi, I'm Megan Sykes.
I'm a professor at Columbia University, where I'm the Director of the Columbia Center
for Translational Immunology.
Today, I'm going to tell you about some of our research on taming and tracking the human alloresponse.
So, while we've made great advances in the field of transplantation, the success is
still limited by: one, the complications of the drugs that we use to avoid rejections;
and secondly, by chronic rejection, which remains a problem, even with the use of
optimal immunosuppression.
So, in our field of transplantation, the induction of immune tolerance has become a major goal,
because this state of tolerance would overcome both of these limitations.
What do I mean by tolerance?
Tolerance implies long-term graft acceptance without immunosuppressive therapy.
But importantly, with an otherwise intact immune system, that can recognize
foreign antigens and protect one from infections.
Successful tolerance induction, really, in my view, requires intentional, planned
immunosuppression withdrawal, achieving tolerance in a high proportion of individuals.
It's been known for some time that the rare person removing themselves from immunosuppression
may not reject.
The vast majority do.
But there are examples of tolerance induction through that... that rather dangerous and
inefficient process of immunosuppression withdrawal.
I'm referring, when I say tolerance induction is successful, to a process where it works
in most people, and it's intentional.
Okay, so how do we get to clinical trials of tolerance induction.
Of course, it begins with experimental models.
But if you look in the literature, you'll find actually hundreds or even thousands
of models of tolerance in rodents.
Unfortunately, almost none of have been successfully applied in humans.
And there are a number of steps that we need to take before we go to humans.
Because if... if we're going to... if we think we have tolerance, we're removing the standard of care.
We're removing the chronic immunosuppressive therapy that is otherwise used to prevent
rejection.
This is necessary if we want to have a normal... normally function immune... functioning immune system.
So, that's why one of the main reasons why we want tolerance, so that we don't have to have
that chronic immunosuppression.
But if we're going to remove that standard of care, we need to know that it works.
And this requires, first of all, in stringent rodent models, showing that it works.
And that usually involves extensively MHC-mismatched skin grafts, which are very difficult to
get acceptance of.
Secondly, we need to have other animal models.
It's very difficult to go from a mouse to a human in withdrawing immunosuppression.
We really need large animal models that are closer to humans.
And thirdly, it's desirable to have some experience with whatever drugs or agents you're
going to use in your tolerance protocol in other clinical settings.
As it happens, the approach that we've...
I and my colleagues have worked on for many years, involving hematopoietic cell transplantation
and induction of mixed hematopoietic chimerism, has come closest to meeting these criteria,
and has gotten into clinical trials.
So, if we're going to use hematopoietic cell transplantation to induce tolerance,
we can't use it in the usual way that it's used, for example, in a patient with leukemia or lymphoma.
In those settings, the transplant, of bone marrow or other hematopoietic cells, is done
after very heavy treatment of the recipient aimed at eradicating as many of the cancerous cells
as possible.
In a patient who doesn't have cancer, who needs an organ transplant, we can't justify
doing such toxic treatments.
Instead, we have to develop a way of preparing that recipient for their bone marrow transplant
-- that has the capacity to re-educate their immune system and induce tolerance --
that is far less toxic.
It has to not eliminate the recipient's bone marrow cells, so that if you failed to get
engraftment of your donor bone marrow you still have a functioning bone marrow and normal hematopoiesis.
And nevertheless, this treatment has to be strong enough to overcome the very strong
immune resistance in the recipient to the donor.
And we know we've succeeded in doing that if we achieve mixed chimerism.
And what I mean by mixed chimerism is coexistence of donor and recipient hematopoietic elements.
The donor ones aren't rejected, and so you see them in the circulation,
and the recipient ones have not been killed off by the conditioning,
and so you see them together with the donor cells.
Now, I mentioned in my introductory lecture that graft-versus-host disease is
a major complication of HLA-mismatched and even -matched hematopoietic cell transplantation.
It has some benefit in treating leukemia... in malignant diseases, but is absolutely
an unacceptable complication to introduce in somebody who doesn't have a malignant disease
but needs an organ transplant.
So, this is a big challenge: to cross HLA barriers, avoid GVHD, overcome the host resistance
of the donor, and do all of this with minimal toxicity.
It took many years for our groups at Mass General Hospital to reach a point
where we could actually try that.
And our pathway to clinical trials of tolerance induction using hematopoietic cell transplantation
actually involved rodent models, shown on the left side of this slide,
some involved in studies of treating leukemias and lymphomas
that ended up bringing us to a mixed chimerism approach
with non-myeloablative conditioning.
Meanwhile, our studies in rodents had shown that we could induce organ tolerance with
a non-myeloablative regimen for mixed chimerism induction.
And our studies in leukemias and lymphomas actually led us to an approach that
we could try in patients.
Because, in that setting, you can sometimes go directly from rodents to humans because
the patients who will try an experimental protocol have failed all other possibilities,
if they have a very advanced malignant disease.
And so we had the opportunity to try this mixed chimerism approach in some of
these patients who... in whom it was used as a platform for immunotherapy.
However, on the organ transplantation side, we didn't go straight from mice to humans.
We actually had a non-human primate model in the middle, which is very similar to humans
in how it responds to transplants, and was a very critical step in allowing us to do
bone marrow transplants for tolerance induction in patients with no malignant disease.
The protocols that we tried at Mass General for inducing tolerance in these patients underwent
several iterations.
A total of ten patients were transplanted under these three protocols.
The first one... and these were all supported by the immune tolerance network of the NIAID...
and the first one is shown here, and all of them have in common that they utilized non-myeloablative doses
of cyclophosphamide; local irradiation to the thymus;
a monoclonal antibody against CD2 that is given to deplete the recipient and donor T cells in vivo;
and then a combined kidney and bone marrow transplant on day 0.
And the post-transplant immunosuppression has involved a calcineurin inhibitor
for a period of 9-12 months.
The second iteration of the protocol brought in some steroids for a very short period
after the transplant to avoid an engraftment syndrome, as well as treatment with a B cell depleting agent,
rituximab, to avoid antibody-mediated rejection.
And the third iteration involved even more rituximab, several treatments with it,
and a slightly longer period of steroid treatment, but was otherwise similar.
This work has been published already, in these two papers shown here.
And I'll just very briefly summarize the clinical results.
Out of ten patients, seven were removed from immunosuppression successfully for periods
of years.
And their... their outcomes are shown here.
These first four patients had the first two regimens in the first ITN trial.
And that first patient is now more than 14 years off immunosuppression, doing very well.
This second patient is more than eight years off.
These other two patients had several years of no immunosuppression, but eventually returned
to immunosuppression, unfortunately, due to a low-grade chronic rejection.
In the second trial, where we added more B cell depletion to avoid this low...
very low-level antibody-mediated rejection that led to these patients returning to immunosuppression...
in the second trial, we added more rituximab, and these patients actually are now more than
seven years post-transplant and doing very well without any antibody-mediated rejection.
Now, I mentioned this term, mixed chimerism, where the donor and recipient cells coexist
in the patient.
And this is illustrated here for... for these four patients in the second trial.
And what you see is, in multiple bone marrow lineages -- lymphocytes, granulocytes, monocytes, etc --
we see a contribution of the donor in the circulating population, but only
for a very short period of time, for a period of 1-2 weeks.
So, this is very transient chimerism, and it's very different than what we can achieve,
for example, in rodents, where the chimerism persists forever.
But we knew from our non-human primate studies, and from some other studies in patients
with malignancies, that transient chimerism, when achieved with a kidney transplant at the same time,
could lead to tolerance.
So, in the lab, we've been studying what the mechanisms of this tolerance are.
And one of the things that we've observed in the patients who got this treatment is
that there's a marked enrichment for what we call regulatory T cells among the T cells
that initially come back after the transplant.
So, this slide here shows you the percentage of T cells in the CD4 lineage that have
this regulatory cell phenotype over time post-transplant.
And each type of symbol represents an individual patient.
And what you see is that these percentages of regulatory cells in that first year post-transplant
are very, very high compared to the pre-transplant level, which you see here is very, very low.
It's normally a very small percentage of CD4 T cells, but it goes way up after the transplant.
It eventually comes down to normal over a period of about a year.
Now, in... if you look at the actual absolute numbers of those regulatory T cells,
you can see, over here, that they are in fact depleted at one week post-transplant, but that
within a couple of weeks they come pretty close to the pre-transplant baseline level.
In contrast, the non-regulatory T cells, the conventional CD4 T cells
-- here, you see their pre-transplant values, and here you see post-transplant --
they remain low for a very, very long time.
And that explains why we see such a marked enrichment of the regulatory cell population
among those CD4 T cells.
And these are bona fide regulatory T cells, because FOXP3 is the transcription factor
that is a master regulator of the Treg program.
And demethylation of the... of the FOXP3 region in the genome is a hallmark of a...
of a bona fide Treg.
And we can see here, in this bottom plot, that the level of TSDR methylation
actually correlates very well with the percentage of regulatory T cells we detect.
So, these are really Tregs.
Why are they so enriched?
Well, it looks like there's a few things going on.
But the main one is probably that they are expanding in the periphery after we
deplete the T cells, the vast majority of the T cells.
So, it seems that they... some of them are spared from the depleting T cell antibody,
and the ones that remain undergo a lot of proliferation.
And we can see this here in this upper right plot, where we're looking at the percentage
of these regulatory T cells that express Ki67, which is a marker of proliferating cells.
And you can see that it's way up at 1 and 2 weeks post-transplant compared to baseline levels.
So that... and this is something that happens when you deplete lymphocytes,
that the ones that remain undergo proliferation.
So, that's one of the major mechanisms of this enrichment.
Well, what role do these Tregs play in the tolerance that we see in these patients?
Well, we can look at this by looking at alloresponses in in vitro mixed lymphocyte reactions and
cytotoxic T lymphocyte assays.
And what we have found in all of our tolerant patients is that they become very hyporesponsive
toward their donor.
Here, we're looking at their proliferative response, in red, to the donor,
and in blue, to a third party individual to... that they're not tolerant to.
You see that the response to the donor is markedly reduced.
And this this particular sample was taken one year after a transplant.
But what you see over here is that when you remove the regulatory T cells, the Tregs,
from that patient's cell population, and now measure the response to the donor,
a response is revealed.
So, this indicates that those regulatory T cells were suppressing the anti-donor response.
However, in this same patient, when we went back and did similar studies at 8 and 18 months post-transplant,
the result was a bit different.
Now, you still see that patient is hyporesponsive to the donor -- you can't even see a red symbol here,
because there's no response to the donor -- but now depleting the regulatory T cells
doesn't really reveal any anti-donor reactivity.
There's still very little response there, even though we've enhanced the response
to third party by depleting Tregs.
So, this suggested to us that we no longer depended on regulatory T cells to see
this donor-specific hyporesponsiveness at 18 months post-transplant, and that something else
might be going on.
And we hypothesized that maybe that large number of alloreactive T cells present
prior to the transplant had been deleted of those that recognized the donor.
So, to summarize these functional assays, tolerance in seven of seven cases in this study
was associated with the development of donor-specific unresponsiveness in
these in vitro assays.
We saw a regulatory T cell enrichment in all of these patients after the transplant.
And in some assays, we could see that those regulatory T cells were playing a role in
suppressing the anti-donor response.
But when we looked late, more than a year post-transplant, we did not see a role
for regulatory cells in suppressing that response in the longer term.
So, this made us think that perhaps the long-term tolerance might be deletional.
Okay.
Well, there's... deletion is one possibility, that the donor-specific T cells actually got
eliminated.
So, here, if we think of the red cells as the ones that recognize the donor,
if they're deleted they're actually gone from the immune repertoire after the transplant.
But another possibility is that they're anergic, meaning that they persist but they
simply don't respond when stimulated through their T cell receptor.
They're in the state of anergy.
And functionally, with the kinds of assays that I've just told you about,
there was no way to distinguish those two possibilities.
So, we really wanted to find a way of distinguishing them, and actually seeing what happens to
alloreactive T cells.
And this is a big challenge, because T cells recognizing a given set of alloantigens
on an MHC-mismatched donor represent a very large number of cells and specificities.
It's thought that 1-10% of T cells respond to a given donor.
And this is thought to be due to recognition of thousands of different peptide/MHC specificities
on an allogeneic MHC.
And all the studies that had been done show that there's no particular predictable
dominant immune response.
And so there's really no way to pick out clones that you can track over time with tetramers,
for example.
So, the approach that we used was really facilitated by the development of a technology for
high-throughput sequencing of the hypervariable region of the T cell receptor beta chain,
known as the CDR3.
And this is the... the part of the T cell receptor that is most specific for the peptide
that is seen by that T cell receptor.
And this hypervariable region is formed by the rearrangement of the V, the D, and the J segments
of the T cell receptor, along with N insertions that give it additional diversity.
And a commercially available platform was developed for actually sequencing up to
millions of these unique sequences simultaneously.
And this led us to hypothesize that high-throughput CDR3 sequencing of transplant recipient's
donor-reactive T cells prior to a transplant would allow us to identify
the repertoire of TCRs... of clones that recognize the donor's alloantigens.
And that we could then carry out such sequencing after the transplant to track the fate
of those T cells.
Using this approach, we actually succeeded in developing a method for tracking
a patient anti-donor T cell response and obtaining evidence for clonal deletion
as a mechanism of tolerance in the patients that I've been speaking about.
And what this assay involves is taking patient lymphocytes, whole PBMCs;
labeling them with a CFSE dye, which is a fluorescent dye that dilutes each time the cell divides,
so the level of CFSE staining is a marker of how much a given cell has divided;
and stimulating those in a co-culture for six days with donor PBMCs that have been irradiated,
so they can't divide, and also labeled with a different dye, a violet dye;
co-culturing for six days, collecting the cells, and specifically sorting the recipient, the responder cells,
that have divided -- those that have diluted their CFSE dye -- and separately sorting, on a FACS sorter,
CD4 and CD8 cells of that recipient that have divided in response to donor antigens.
And what we did is then subjected each of these populations, these divided cell populations,
to high-throughput sequencing of the T cell receptor CDR beta...
CDR three region.
And also did the same thing on CD4 and CD8 cells from the unstimulated T cell population
of that patient.
And this is all prior to the transplant.
And then we can actually define a sequence as alloreactive... donor-reactive if there...
if it's expanded more than fivefold in this mixed lymphocyte reaction compared to
its frequency in the unstimulated population, shown over here.
So, this... this is a way of identifying a repertoire, a set, of T cells that we call
a fingerprint of the anti-donor alloresponse.
And this is what... when we developed this assay, we tested it on our tolerant patients.
And what we found was that in all three of the tolerant patients who we studied
there was a significant... a statistically significant decline in the frequency of donor-reactive
CD4 and CD8 cells in the circulation, over time, after the transplant.
And we saw this in all three patients compared to the pre-transplant level.
We also had one patient in this trial who failed to achieve tolerance, who got the same treatment
but rejected the kidney after the immunosuppression was withdrawn.
And what you see here is that this patient did not show any significant reduction
in the frequency of anti-donor clones in the circulation.
So, it suggests that this method actually distinguishes the tolerant from the non-tolerant patients.
We've also tested this method on patients who don't get a tolerance-inducing regimen
but who just get a kidney transplant with conventional immunosuppression.
And some of our typical results are shown here.
Interestingly, we don't see any reduction... these are two different individuals, two different recipients,
looking at the frequency of donor-reactive clones over time.
Here's pre-transplant, and here's post-transplant.
You can see that in both of these patients there's a statistically significant increase
in the number of circulating donor-reactive CD4 clones after the transplant, showing you
the stimulation of the immune response by the transplant.
So, that helps to validate this assay as showing us something very biologically meaningful.
And what we were able to conclude from this study is that high-throughput deep CDR sequencing
of recipient's donor-reactive T cells pre-transplant enables identification of a specific set of
donor-reactive T cells.
And these donor-reactive clones can then be tracked in the post-transplant period to
tell us something about what's going on immunologically.
And our studies indicate that we are identifying biologically relevant T cells with this pre-transplant MLR,
because their frequency goes up in a conventional transplant recipient
after the transplant.
And our data suggests that in the tolerant patients who get
combined kidney and bone marrow transplantation,
deletion of donor-reactive T cells is a long-term mechanism of tolerance.
And in studies I didn't have time to go through, this deletion seems to be the result
both of global T cell depletion with the conditioning and specific exposure to the donor antigens.
In contrast, expansion of circulating donor-reactive clones is detected in conventional transplant
recipients.
And so far, this deletion analysis has outperformed the functional assays that I referred to earlier
because functional studies actually showed donor-unresponsiveness in the patient
who failed tolerance in addition to those who succeeded, suggesting that that patient
was demonstrating anergy, at least under the conditions of our in vitro assay, whereas this
deletional assay actually distinguished the tolerant from the non-tolerant patient.
Okay.
I'm just going to spend a few minutes talking about how this TCR tracking method can also
be used to better understand what's going on within an allograft.
And this study actually involves patients who receive intestinal transplants.
And at our center, at Columbia, our patients are actually followed by surveillance biopsies
of the intestinal allograft through a stoma that is created at the time of transplant,
because the symptoms of rejection can be quite nonspecific.
And doing these surveillance biopsies is a way of making sure that we're on top of
a rejection if it does occur.
So, it's looked at histologically.
So, this approach has actually given us an opportunity to look not only in the circulating
cell populations of these patients but also at what's going on in the graft biopsy specimens
in real time.
And we've taken advantage of this to look at... within the graft at the
alloreactive T cells that we've identified with the method I just spoke about.
So, just to give you a bit of background, intestinal transplant outcomes are...
are not as good as we would like them at this point.
And there's a lot of rejection that occurs.
And particularly in patients who get intestinal transplants alone.
Some patients don't just get an intestinal transplant; they get a liver transplant with it,
because their liver has failed for a variety of reasons, often due to chronic TPN
used to treat the intestinal failure.
But what you see in this slide is that the patients who get multivisceral transplants
-- liver, pancreas, stomach, and everything along with the intestine --
actually have lower rejection rates than patients who get intestines alone.
So, we hypothesized that this might have to do with the interplay of graft-versus-host
and host-versus-graft reactivity in these patients.
Now, I should say that the intestine comes with a very big load of lymphocytes.
And it's known that intestinal transplantation can cause graft-versus-host disease.
So, we've looked at this in our patients, and hypothesized that, in fact,
lymphocytes from that graft may go into the circulation, and that may be a marker of patients who
won't have rejection.
And this can occur without graft-versus-host disease.
And in fact, when we investigated this hypothesis, it turned out to be the case.
What we found in a lot of these patients, and particularly those who got the multivisceral transplants,
shown with the circles... we saw very high levels of donor chimerism
in the circulation, spontaneously, without any bone marrow transplant.
And most of these patients did not have graft-versus-host disease.
In 14 patients shown here, 8 showed this mixed chimerism in the circulation, but only one
had graft-versus-host disease, and it was very mild and self-limited.
Interestingly, many patients did not have this macrochimerism.
And we define macrochimerism as more than 4% donor T cells in the circulation at its peak.
And it's most commonly the patients getting intestinal transplants, the ones here
with triangles, did not get macrochimerism.
But what was striking is this association down here.
We observed that the patients who have this macrochimerism, as we've defined it,
have much lower graft rejection rates than those who don't have macrochimerism,
consistent with the hypothesis that we started out with, that this macrochimerism may protect the patients
from rejection, and can occur without graft-versus-host disease, as we've seen.
Now, as I mentioned, we also study the grafts, and we can do flow cytometry with
multiple parameters to actually look at the replacement of donor cells by the recipient within the graft.
And we can look at all sorts of different subsets of cells within those mucosal biopsies
over time.
And this is just an example of one such biopsy, where we're looking at different lymphocyte subsets,
and we have specific markers that... this antibody that goes up in the y-axis distinguishes
recipient cells, whereas those that are negative for that antibody are donor-derived.
So already, you're seeing mixed chimerism in this intestinal graft.
And what we noticed is that there was a highly variable rate of replacement of
the donor T lymphocytes that come in that graft by recipient T cells from patient to patient.
And that there was an association with rejection, and development of donor-specific antibodies,
with more rapid replacement by the recipient.
So, this part of the slide is showing you the rate at which... the percentage of recipient cells
in these different T cell subsets.
And you can see that it's quite high quite early on in these patients who undergo rejection.
Each line is a different patient.
In contrast, patients who don't have rejection, or have a DSA-negative rejection,
the rate of replacement of donor T cells by the recipient within the graft is very slow.
Very interesting.
And this part of the slide on the right just represents this in a different way,
making the same point.
So, what... what's going on here?
It looks like donor cells appearing in the blood and recipient cell... recipient T cells
not replacing the donor cells in the graft is associated with less rejection.
Well, our original hypothesis was that, in fact, all of this is reflecting a balance
between the graft-versus-host response, caused by T cells in the graft,
and the host-versus-graft response, which is a systemic immune response.
And using this T cell receptor tracking method that I just spoke about, we could actually
look at this in both directions: in the graft-versus-host and host-versus-graft directions.
So, this is the same assay that I mentioned earlier, and now we're doing it in both directions
on pre-transplant donor and recipient cells to identify the GvH and the host-versus-graft
T cell repertoires.
And now we can interrogate these biopsy specimens for these clones.
And what we found was quite striking.
In the early period post-transplant, particularly in those patients who have slow replacement
of their graft T cells by the recipient, there's a marked expansion of graft-versus-host reactive
T cells within the graft.
It's... they're much more frequent than what we see in the lymphoid tissue prior to transplant,
for example, shown in the black bars.
So, there's a huge expansion of GvH-reactive CD4 and, over here, CD8 cells
in the graft compared to what was in the donor lymphoid system.
And this was interesting.
And we wondered why that was.
Our analyses, our flow cytometric analyses, included analyses of the antigen-presenting cell
populations in the graft.
And what we found was, in contrast to T cell replacement rates, which were extremely variable,
as I showed you...
patients with rejection tended to have rapid replacement of T cells by the recipient,
whereas those without rejection had slower replacement... in contrast to all that, all of the patients
showed quite rapid replacement of antigen-presenting cells, of myeloid cells,
with a dendritic cell phenotype by the recipient.
This one is at day 16.
Almost all the APCs are recipient-derived, whereas the T cells are still mostly donor.
There's very few recipient ones.
So, that was quite a uniform finding: early replacement of donor APCs by the recipient.
And that could explain this expansion of graft-versus-host-reactive cells in the graft.
They're having recipient antigens presented to them on these recipient APCs that enter the graft,
expanding this GvH response.
And we think this is very protective.
We have additional studies that I don't have time to take you through, but that GvH response
also goes into the circulation, contributing to the macrochimerism.
The other thing that we can see with this technique... we can interrogate the biopsies
for host-versus-graft clones, and what we see is something that hasn't been shown before.
During a rejection of a human allograft, there's a huge enrichment of host-versus-graft alloreactive
T cells within those grafts.
And that may be obvious, but there's some dogma from the literature that most T cells
infiltrating a graft during a rejection are bystanders; they're nonspecific.
That is clearly not the case here.
A very high percentage of these T cells during a rejection are host-versus-graft reactive,
as defined by our TCR tracking method.
This goes down as the rejection resolves, but it's still an enrichment for...
those host-versus-graft cells persist long-term in these patients, and we think they may pose
a constant risk for rejection.
So, I'll end here with a summary.
We have found that there's a direct correlation between early region and accelerated replacement
of donor T cell populations in the graft by recipient T cells that look like those
in the circulation.
They have a blood-like phenotype.
Host-versus-graft clones predominate among those host T cells within rejecting grafts.
They persist at lower levels long-term.
And what I didn't show you is that they changed their phenotype long-term;
they look more like tissue resident lymphocytes, and they seem to seed the entire gut.
And we think these pose a constant threat of rejection.
Thirdly, in contrast to the highly variable replacement rate of donor T cells by the recipient
in the gut, antigen-presenting cell replacement is uniformly rapid.
And finally, we think these rapidly immigrating recipient APCs are driving the local expansion
and activation of GvH-reactive T cells coming with the graft, and that these
may actually control the host-versus-graft-reactive clones, curbing rejection and replacement of
donor T cells by the recipient within the graft.
So, I'm going to end there.
Obviously, I've talked about a lot of different studies, and that's involved a huge number
of people, both at Columbia and originally at Mass General,
where we did the clinical trials of tolerance induction.
And the intestinal transplant studies have involved many people in the lab,
but also in... on the clinical side as well.
So, thank you very much for your attention, and I'll stop there.
