Microbiology
09/05/2008
Transplantation Immunology
Transcriber: Laura Rayne
52:07 (Length of lecture)
Slide 1: Title Slide
I’m going to talk today about something that’s a little bit different than what you’ve heard up
until now. What I am more concerned with is really something a bit more practical in a sense of trying
to use the knowledge that we have about immunology to make things work. So this obviously is the
subject of transplantation. In a way it is sort of the biological version of engineering. It is like trying to
fix a broken car when you have a broken part. So the obvious solution to primates like us, who are tool
users, is to replace the broken part. However, like many engineering problems the laws of physics get in
your way. Here you shall see how the laws of transplantation, the rules of biology and immunology, can
get in your way. So we have to understand how these things work.
So when we think about transplantation in general: let’s take it from the perspective of the
ancients. Let’s go back to 1500 BC. You’re a slave working for some guy and you’ve done something
that has really irritated them so they’ve whacked your nose of. They actually used to do this. They
would try things like for example taking a nose from a slave or from another individual and replace it. It
never worked. There were other extremely unusual ideas that were tried later, such as in the Middle
Ages. For example, it was thought that having bull testicles grafted into you would increase your virility.
There were people that actually made money doing this kind of thing. The bottom line is none of this
worked. The thing that people found all the way through up until about the 1950s to the 1960s was that
if you tried to swap body parts between different individuals it never ever (almost) worked. So why is
that? The answer to that comes from our knowledge of immunology and of how immunology works.
I have a picture here of Edward Jenner. He came up with an excellent observation and that is
that the immune system can be manipulated. He made an observation that one can immunize an
individual, although he didn’t know that’s what it was. If you take small amounts of material from Cow
pox pustules from people and rubbed it into somebody else through a wound or whatever they became
immune to smallpox. So this is one of the first observations that immunity could be manipulated.
Slide 2
The story remained relatively unchanged or the next 100-150 years until this gentleman here Sir Peter
Medawar, who was a zoologist in 1945 when the Nazis were bombing London they were getting a lot of
injuries. So the British medical community became very interested in how to deal with burns and other
types of large injuries. Peter Medawar was assisting another surgeon by the name of Gibson. What
Medawar observed was one particular patient – a woman who was minding her own business and a V2
rocket comes by and drops an incendiary on her head. She ended up with very extensive burns down
her torso to the point where they could not do all of the grafting that they wanted to do. The way they
treated burns like this was they would take parts of your skin that were still good and just pinch it
between the forefinger and just whack the top off, and take that piece and put it in the wound, and an
island of skin would be there and it would slowly grow out and slowly cover the wound. She didn’t have
enough skin for that. She had to get some skin from her brother and they transplanted that skin on her
and of course that skin was eventually rejected. So they did it again from the brother and Medawar
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Laura Rayne
pg. 2
made this seemingly trivial observation. He saw that the skin that was transplanted on her a second
time from her brother was rejected more quickly.
Slide 3
Now he noticed that the ability to reject skin more quickly the second time you see the skin (or
an antigen) is a characteristic of immunity. So he theorized at that point that maybe the rejection of
tissues was an immunologically-mediated response because it happened more quickly. And he later
began to do these experiments in rabbits. He published this humongous paper in 1945 called “The
Behavior and Fate of Skin Autografts and Skin Homografts in Rabbits”, where he worked out the
phenomenology of this entire thing and noticed that there were three characteristics with graft
rejection. These three characteristics were inducibility, meaning the response didn’t appear to be there
until you put the antigen or the skin there. That it exhibited memory and specificity. These are the
cardinal hallmarks of an immune response, so from this observation he concluded that graft rejection
was an immunologically-mediated response. Now to us that’s trivial, but in the mid-twentieth century
that was huge because it opened up an entire new field. Hence we call him the father of
transplantation.
Slide 4
So here are the types of experiments he did. Let’s say you have a couple of rabbits. You have
two rabbits that are genetically dissimilar. So I colored one red and one blue – I’ll call it strain blue and
strain red. So you take a piece of skin from the blue rabbit and put it on the red rabbit, and what you’ll
see is that rejection will occur in 7-8 days, roughly. In a normal graft, like say if you were to take a piece
of skin from your leg and graft it onto your arm, or like that poor woman who was bombed in 1945 a
piece from your leg onto a burn or whatever, what you’ll see is that as you put the graft in place it will sit
there. Then the vessels in the graft will begin to reanastamose themselves so you get revascularization.
You get healing in 7-10 days. You get a few neutrophils infiltrating the graft and then you have complete
resolution. So the graft looks fine.
Slide 5
That’s not the case when you actually place a graft from a different individual. What you see is
histologically in the image I just showed you of rabbit red being transplanted with a piece of skin from
rabbit blue is that you see some revascularization at 3 days or so and there is some variability of when
this occurs. But instead of resolution or healing in you see this massive cellular infiltrate come into the
graft , and then the graft will actually become necrotic and will eventually slough off and I’ll show you
what this actually looks like. This is called First Set Rejection, where you put the graft on for the first
time and it is rejected in 7-8 days. This is the observation that Medawar made.
Slide 6
Now what happens if you take another piece of graft from the same rabbit that you’ve been
using before and you put it on rabbit red again? This was to replicate the observation he made in the
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Laura Rayne
pg. 3
clinic, which was that when the graft was placed on the woman a second time from her brother it was
rejected more quickly. Low and behold if you do it in rabbits you see the same thing. The first graft is
there. You put the second graft on and it is rejected in about half the time, in 3-4 days. This is a
phenomenon he called Second Set Rejection. This is evidence of immunologic memory, meaning that it
remembers its prior exposure to the antigen and in doing so reacts more quickly to that antigen or skin.
Slide 7
So just like I showed you a moment ago you see that the graft is rejected and you see a mass of
cellular infiltrate, and he described this histologically as well in the original article.
Slide 8
Now evidence of specificity comes from the fact that if you take a piece of skin from a third
rabbit known as a third party - so you have this same rabbit here and you’ve done teo grafts on him and
you see that the first graft is rejected in 7-8 days. You put a second graft on and it is rejected in 3-4
days. At the same as this one you put another graft on from a different genetically unrelated rabbit who
has no relationship to either one of these rabbits. This shows evidence of First Set Rejection. So it is
rejected in 7-8 days. So what this is telling you is that this rabbit here is able to distinguish between a
graft from this rabbit and a graft from this rabbit. So it’s exhibiting a response that we call specific. This
was the basic observation that Medawar made and that is that graft recognition is a) an
immunologically-mediated response and b) is related to the genetic constitution of the individual. That
is, it has to do with the genetic relatedness of individuals.
Slide 9
When you look at a Second Set Rejection here what you see is that instead of seeing a healing in
phase like you see with first set rejection what you see is this neutrophil and lymphocyte riot that occurs
almost immediately within 3-4 days. You don’t even get healing in and the graft is rejected and you get
thrombosis and necrosis very quickly. So it’s a fairly violent response.
Slide 10
We’ve developed a terminology for this that is essentially built around the genetic relatedness
between individuals. The first item on the list that you see up there are called autografts meaning self.
These grafts here are from one site to another on the same individual. An isograft is between two
genetically identical individuals so this would be between two identical twins or in my case I do grafts
between genetically identical mice of the same strain.
Slide 11
An allograft is between two genetically distinct individuals of the same species, so this is the
grafts between the rabbits. What Medawar did was allografts. What we do clinically when we swap
kidneys and hearts and livers and lungs between people are allografts. The Xenograft is between
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Laura Rayne
pg. 4
different species so if you were to have a duck-billed platypus and you wanted to do a graft from that
animal to a rabbit you could that, or from pig to man which people would love to do.
Slide 12
If you were to look at a mouse and see what this actually looks like. This is the back of a black
mouse. What you see is a skin graft that has been placed on a mouse. This is the very beginning of
rejection here. The key feature that you start seeing from a gross morphological point of view is you
start seeing these little scabby portions appearing on the graft.
Slide 13
Then after a little while longer you see that the graft completely scabs over and then falls off. In
this animal here you see a rejected graft. So from a gross morphological point of view that is what it
looks like.
Slide 14
This is the basic phenomenology of it all. It works this way if you transplant skin, hearts, lungs,
liver all those kind of things that are clinically important to us and clinically important to you if you have
end-stage organ disease with no other solution available. That same phenomenology applies. The only
thing that is really different in an untreated individual is the timing. If you have a skin graft and it gets
rejected, the skin falls off. If you have a heart graft and it gets rejected you drop dead, but the end
results are that the graft stops working.
The question is how does it work? Medawar made this observation that there are all these odd
looking cells in the graft called lymphocytes in huge numbers. In the mid-twentieth century they didn’t
know what those were for. So you design an experiment that asks the question “Is graft rejection
mediated by T cells?” In a lab like this you will go into your mouse room and grab a mouse, we’ll call
him strain B, and we’ll do the same type of experiment that Medawar did. We’ll take a graft from
another stain, strain A, and put it on there. We’ll look in 14 days and in this case we see First Set
Rejection. The graft is rejected and we see necrosis, just like I showed you in the picture there.
If you were to take a second graft on the same mouse and do a second skin graft with strain A
just like Medawar observed you’d see that it would reject in about 6 days. But we want to know if T
cells have anything to do with it. You take this mouse and you take the spleen out, and you purify the T
cells, and you inject it into another mouse that has never seen or been transplanted with a piece of skin
graft. Then you do a transplant on that animal. What you discover is that the graft is rejected with
Second Set kinetics. What that means is that you used the T cells to transfer the memory of the
exposure to the graft. Hence, T cells are involved. A very simple yet very nice experiment that shows
exactly how this works.
Slide 15
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pg. 5
You know that a T cell isn’t a T cell. We can sit there and subdivide cells and decide which T cells
are involved in graft rejection. We can do another experiment: what if you take a mouse and wipe out
all the CD4+ cells or CD8+ cells and then observe what happens to graft rejection? This is a classic
survival plot. What it shows is the % of surviving grafts vs. time. When an animal (or more than one)
rejects in your group in a given time the survival goes down in a stair-step like fashion. If you just take
some animals shown in black as the control and just do your transplant as you should, you’ll see that
they are all gone by 15 days. But this is sort of variable. You don’t do grafts and then on day 10
everybody just rejects. It works where you see a continuum.
Now you take some other animals and you do the grafts on them and you inject them with an
anti-CD8 monoclonal Ab. What the Ab does is wipe out all the CD8+ cells. Now you have an animal with
mostly only CD4+ cells. What you find is these animals reject at the same rate. There is really no
difference. That tells you that CD8+ cells are not absolutely required for rejection.
Now you do the opposite experiment where you treat them with anti-CD4. So this wipes out all
the CD4+ cells leaving only CD8+. In this case what you see is the graft survival is much longer. That
means that they will reject if only CD8+ cells are there, but they don’t reject as quickly. It is clear that
CD8+ cells aren’t required but they do help in rejection.
When you wipe them both out rejection is vastly extended. The fact that it occurs at all is
because when you inject a mouse with anti-CD4 and anti-CD8 monoclonal Abs you wipe them out but
the animal begins to repopulate after a while. New cells come out of the thymus, they repopulate the
external lymphoid organs and they come back. When they do eventually the animal will reject unless
you keep treating it continuously.
So we can see that CD8+ and CD4+ cells are important in rejection. It is a T cell mediated
response. CD4+ cells are absolutely required for this to occur. CD8+ cells are helpful in this response
but are not absolutely required. So we get an idea of the mechanisms involved here.
We also know that transplant rejection is also related to the genetic relatedness between donor
and recipient. Clearly it is a genetically-driven response.
Slide 16
There were a number of observations that were made during the 1920s and 1930s using inbred
strains of mice. They came up with these laws of transplantation. So if you’re working with inbred
strains of mice, which are mice that have been bred over the last hundred years or so and within a given
strain they are almost all genetically identical. You can swap transplants between those inbred strains
within those strains and those grafts will take.
So the law is that transplants between individuals of the same inbred strain will succeed.
Transplants between inbred strains are rejected. It is just like people. If you have a transplant between
identical twins it will succeed. If you have transplants between two different individuals who are not
twins it will be rejected. Here’s something that is less intuitively obvious: if you do a transplant from a
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pg. 6
parent to an F1 (F1 being the progeny of the two parents) that transplant will succeed but the reverse
will fail. I show you this because it illustrates how genetics drives graft rejection.
Slide17
If we look at two strains, how would this work exactly? Remember, MHC gene products are
involved in restriction of antigen recognition at the T cell receptor (not sure what he means by this).
Strain b mice have MHC complex haplotypes called b, one allele from each parent. The same for this
strain called k, again receiving one copy from each parent. Each of these parents has one of each allele.
They have F1 that inherits an allele from each parent so this mouse now has a b allele and a k allele. So
if we think about how the genetic relatedness influences this, we find that the more similar you are, the
less likely you are to reject. Over the course of the years we have discovered that the main driving
force behind graft rejection genetically is the MHC complex (repeats this). What we see here is this
inheritance pattern of receiving the MHC genes then will influence how this individual behaves to
transplants.
Let’s go back and look at the rules again (previous slide). Look at the third one. If we look at the
way that these genes are inherited we see that if there are transplants from the parent to the F1, the F1
has the same alleles that the parent does. This mouse which has b, sees b as self, so it doesn’t reject the
graft. What if we go in the opposite direction? We’d go from b/k to b/b. Well the b mouse has no
experience with k and so it sees k and therefore rejects the graft. Transplants from the parent to F1 will
succeed, but the reverse will fail. It is simple recognition.
Of course the other question that someone will think of eventually is why don’t we do this in
people? The reason is that people aren’t homozygous like inbred mice are and the MHC is a multi gene
locus. So an individual human will have so many combinatorial possibilities that these rules work at one
locus or in homozygous individuals, but if my child needs a kidney from me there is no guarantee I can
donate that kidney to them and have it work, although there is matching that can take place.
Slide 18
When we look at humans, we see how the locus looks. In humans there are a number of
different histocompatibility genes that control graft rejection – MHC Class II and MHC class I are the
most important of those. These molecules have their own characteristics and distribution throughout
the body. Remember that class II are primarily present on APCs and B cells, and Class I are ubiquitously
distributed throughout the body.
Slide 19
So if we’re going to do a transplant on somebody in the clinic , it is driven by the degree of
genetic relatedness between one individual and another. This is the key. When we do clinical
transplants in people we expend a great deal of effort trying to determine the genetic relatedness
between a potential donor and a recipient. Of course, you don’t want to transplant an organ that you
think will be rejected immediately. You’re just going to kill them. One of the oldest methods we have
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pg. 7
used to distinguish between two individuals is to do histocompatibility typing. What one will do is take
cells from the donor and the recipient (frequently lymph node cells). You grind them up and separate
the cells and seed them into different little wells. You place those cells in the plates here and you use
Abs that you have raised against various MHC antigens. You expose the cells to specific Abs and if the
cells have those antigens on their surface, the Abs will bind to it and you can determine whether that Ab
is there by adding complement. Complement in combination with Ab bound to the surface will begin to
blow holes in the cell membrane. You can then treat these cells with a vital dye (for example trypan
blue) that goes into the cell if the cell has holes in it. Dead cells become blue, live cells remain white. In
the case where the Ab does bind the cells become leaky.
Slide 20
What you can do then is run a panel. You’re using Ab to different HLA antigens. Here you have
your recipient. You know that this person is say A1 and A7 (2 alleles, one from each parent) and donor 1
has A1 and A7 – a perfect match. Donor 2 has A2 and A3. Which donor would work best? Obviously the
first one would be the one that you would choose, all other things being equal. This is how many of the
decisions are made, particularly for kidney transplantation. These assays are actually done using beads
and other types of reagents now.
Slide 21
You can also do another type of assay called a Mixed Leukocyte Reaction (MLR or MLC) where
one can take for example, a spleen from two different strains of mice called blue and red, and if you
simply mix them together in a culture disk these cells will proliferate wildly in response to each other.
You can prove that this response is related to MHC because if you take two spleens from two inbred
strains of mice, say red and red or blue and blue, nothing happens. But if you take red and blue and put
them together and look for proliferation, a common way to do that is to use a DNA precursor like
thymidine which is labeled with tridium. What happens is when cells multiply they make DNA of course
and they take up this DNA precursor and become radioactive. So you can tell if the cells are
proliferating. You can see that simply mixing leukocytes together can cause them to proliferate rapidly if
the two individuals are genetically unrelated. You can tell if one individual or another is proliferating by
simply irradiating one of these for example with 2000 RADS. It renders them unable to proliferate but
the can stimulate just fine.
Slide 22
So now you transplant an organ clinically and what happens? Acute rejection appears anywhere
from about 10 days on post-transplantation and can occur anytime after, even 20 years later. It is
primarily cellular and you see a massive infiltration of macrophages and lymphocytes and you obviously
see decreased graft function.
Slide 23
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pg. 8
I work in cardiothoracic surgery so I am a little bit biased toward heart. This is a histological
section of heart muscle stained with H&E. You can see the heart muscle is largely a pink material.
Nuclei show up as a blue color and what you see is that this graft has some infiltrate that you don’t
normally see in healthy heart muscle but there is not much. The cytoarchitecture is largely intact.
Slide 24
When we look at a graft that is rejecting however, what we see is a much more serious
infiltration of tissue. This is a little bit higher magnification. One even sees eosinophils in this particular
sample and you see a large infiltrate of lymphocytes, macrophages, neutrophils and a marked disruption
of the cytoarchitecture of the tissue. (As an aside, we diagnose heart transplant rejection in individuals
by biopsying the heart. If you wait until they begin to have compromised graft function they have this
tendency to drop dead on you before you know anything is happening. So what we do is take a biopsy
sample and do histological sectioning. That is where this is from.) The primary indicator is signs of this
type of infiltration. It is treatable. We have a number of immunosuppressant drugs to do that,
particularly steroids.
Slide 25
Another type of rejection that we call chronic rejection is very different. It is characterized by a
slow and steady decline in graft functions and what one typically will see is a progressive occlusion of
vessels and passageways within the graft. In the heart it would be the coronary arteries. In the lungs
you’ll see it along the trachea and the bronchi. In the lungs they call this bronchiolitis obliterans. It
seems to be the result of cellular and humoral mechanisms. That really means that we don’t know. We
know cellular responses and antibodies are involved but we don’t know exactly how. And in fact we
really have no satisfactory treatments for chronic rejection. Very often this will progress over time,
usually a period of years, and there isn’t a whole lot we can do about it.
Slide 26
So what does it look like exactly? This is in a mouse model of chronic rejection done in the
laboratory. We do heart transplants and kidney transplants in mice in an attempt to model what is
happening in humans. If we look at a normal coronary vessel, what we see here is the vessel wall is
composed of a series of layers consisting of the inner layer, the intima, and layers of elastin and
connective tissue. In a heart that is undergoing chronic rejection what you instead see is the intima is
right here and you see this hyperplasia that occurs inside the intima such that the vessel diameter is
now reduced. This occlusion will progress to the point where this vessel will no longer carry blood. The
heart will eventually stop working.
Slide 27
The hyperacute rejection is the atomic bomb of transplant rejection. It was first observed
clinically between individuals who were blood type mismatched or they had some other reason that
resulted in preexisting antibodies occurring in the recipient. Hyperacute rejection is caused by
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pg. 9
preexisting antibodies. What you see is when you’re the surgeon and you’ve transplanted this kidney
and you pull the cross clamp off to allow the blood flow to go to the kidney, you see the kidney get nice
and pink and it begins to function and produce urine after a while. Now it’s different. You pull the cross
clamp off and the thing doesn’t pink up. Instead what you see is hemorrhagic loci appearing on the
outside of the organ and the thing will actually turn black.
You can take a rat heart out, for example, and hang it on a stand and perfuse it with oxygenated
buffer and that thing will just sit there and beat merrily away as long as you want. Then you switch it to
a blood supply from a different species of animal that has preformed antibodies against that rat heart
and within 30 seconds it stops doing any useful work at all and in 20 minutes all you see is this barely
quivering hunk of black meat.
So you obviously don’t want this to happen clinically. How does it work? Well it is due to
preformed Abs existing in the recipient. You release the cross clamp, blood surges into the organ and
brings this preformed Ab into the organ. The Ab immediately begins binding to the endothelium on the
capillary walls, fixes complement and begins blowing holes in the endothelium. Of course, platelets and
neutrophils begin to adhere to the walls of the endothelium and the vessel gets blocked off. This can
occur within minutes, resulting in the total destruction of the organ. It is a very, very rare phenomenon.
We don’t really see it anymore unless you’ve screwed up in a really gross way.
Student question: “Is there a way to tell when that’s going to happen?”
Answer: There is. In fact, we expend a fair amount of effort making sure that won’t happen. There are
ways that you can test for preformed Abs. One way is called the cross-match. Not tested on.
The rest of the material will not be tested on because he did not have time to cover it.
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pg. 10