Transplantation Immunologypg. 1

Transplantation Immunologypg. 1

Laura Rayne

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 thebiological 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 getin 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 transplantationin 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 Cowpox 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 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 rejectskin 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 thisentire 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 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 calledSecond 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 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

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.

Whenyou 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 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.