Immunology sheet # 13 - Safa'a Makhool

TRANSPLANTATION

In this lecture we will continue talking about transplantation ..
As we said in the last lecture, the mechanisms of rejection are divided into four types: Hyper acute, accelerating, acute, and chronic rejection.

They also can be classified according to the cell types and processes involved:
Hyper acute preformed antibodies
Acute  also antibodies
While acute and chronic  involve T-lymphocytes.

Hyper acute rejection

It is mediated by preformed antibodies which target the vascularized organs. They usually target antigens expressed on the endothelial cells of the vascularized organs like the kidney, the heart, and the pancreas.

And this type of rejection does not affect non-vascularized organs like the Skin.
So skin is not affected by hyper acute rejection but it’s mediated by T-cells immunity.
However sometimes the kidney is damaged within minutes to hours because of these preformed antibodies.

Now how these antibodies could be preformed? Why?
Either because of the type of the antigens, like red blood cells antigens, these antigens are not only expressed on the RBCs but also they’re presented in the endothelial cells of other tissues which are ABO antigens.
Although they are weak and usually the response is mediated by IgM, they can be the cause of hyper acute rejection.

Or because of HLA antibodies which are produced in cases of pregnancy, abortion, blood transfusion, or transplantation.
HLA  Human Leukocyte Antigen

So there are ways to synthesize antibodies against HLA antigens…like in the pregnant women who develops maternal antibodies against the antigens presented in the father which may be presented in other individuals.. abortion is the same story.

-So these antibodies which are formed will attack vascularized organs like kidney which will turn blue and mottled shortly after vascularization is established.

-Extensive vascular thrombosis and hemorrhage with little evidence of a mononuclear cell infiltrate.
-The main manifestations that take place in hyper acute rejection are thrombosis and hemorrhage.

















Components of hyper acute rejection:
-Endothelial MHC antigens or carbohydrate determinants like those in blood groups.

-Preformed antibodies that can bind those antigens.

-The Complement regulatory proteins that can modify complement activation, and anticoagulants that can modify the coagulation pathway.
As you know the antigen-antibody complex will activate the compliment system by the classical pathway and this will activate the clotting system which activates kinin and fibronylitic system .
These are inter related system in which one activates the other.
So many systems are switched on as a consequence of formation of immune complex “antigen on the endothelial cells + preformed antibodies”.

-Again , The target is the donor vascular endothelium.



-The crucial event is the formation of the membrane attack complex (MAC).




If you remember binding of C5B to the cell will be followed by binding of C6, C7, C8, C9 which forms a giant molecule, one million Dalton molecular weight, on the surface of the target cell.



Complement activation is controlled to avoid the damage which is caused by several regulatory factors like:
1- Serum (Soluble) Complement Receptor 1  sCr1
2- Decay Accelerating Factor  DAF
3- Membrane Cofactor Protein or CD46
4- CD59
These act at different stages of the cascade to prevent compliment activation.

The Initial stimulus for activation must be strong enough to overcome these down regulating molecules. Which means that when the complement system switched on immediately other regulatory mechanisms start to operate and in most cases they will succeed in stopping the complement activation but if the reaction is very strong it may overcome these regulatory mechanism.
So in tissue transplantation one of the most important features is the strength of reaction and that’s why the in rejection reaction down regulatory mechanism FAIL to prevent complement activation then death of the organ will occur within minutes or hours.

So preformed antiMHC and antiRBC antibodies are responsible for hyper acute rejection but antibodies against RBC antigens “antiRBC antibodies” are responsible for only 25% of cases so they don’t always lead to hyper acute rejection.

But remember that we can’t transplant ignoring the ABO compatibility, the donor and the recipient must have the same blood group in addition to HLA matching (tissue matching).
So we can’t take a kidney from an A donor to B recipient this will potentially leads to hyper acute rejection.

Xenografting
is the transplantation of living cells, tissues or organs from one species to another, such as from pigs to humans.

Hyper acute rejection in xenografting is very severe and homologous restriction will occur why?
Because compliment regulatory factors present in the tissue of the donor will not down regulate complement component of recipient because they are different species.. so the regulatory factors FAIL to down regulate the complement activation.


Now the figure below shows antigen presented on an endothelial cell and antibody binds to it complement becomes activated and this will cause type 1 cell activation .
The regulatory factors will act against it, but if the reaction was very strong they will not act (like in case of Xenografting)  finally death of he organ will take place.









Type 1 Endothelial Activation:
Due to the effect of MAC on the donor vascular endothelium, even before cell lysis. Manifestations of this activation are:
a) cell retraction, leading to gaps between endothelial cells
b) loss of antithrombotic molecules from the endothelium.

- Thus, type 1 endothelial activation is responsible for the two principal pathologic findings in hyper acute rejection:
a) extra vascular hemorrhage and edema  blood and fluid ooze from
the endothelial gaps.

b) intravascular thrombosis  because of the loss of anti thrombotic
(anticoagulant) molecules and increase in vascular permeability 
the kidney becomes swollen and the thrombosis will lead to closure
(occlusion) and stop of perfusion …death of the transplanted organ is
the end result.

There is NO treatment for hyper acute rejection, when this reaction takes place all we can do is watching it so nothing can be done to prevent the reaction.

The only solution is preventing the reaction by matching donor and recipient HLA and blood groups.


Not all the organs are susceptible to the same extent , kidney and heart are very susceptible.
Skin and bone are not susceptible.
Liver although it’s a vascularized organ but it’s highly resistant so there is no risk of damaging the liver by hyper acute rejection.

So understanding the reaction will lead to prevent it, which is the only way to treat this kind of rejection.


Accelerated Rejection
Early rejection caused by induced antibodies.
Takes place within 5 days of transplantation.
Now because antibodies are unlikely to be formed by such a short period of time many authors believe that this reaction doesn’t occur unless it’s a different type of hyper acute rejection that’s produced against minor histocompatibility antigen.
An individual who is exposed to such antigens before and produced antibodies against them in the past as a consequence a second exposure will cause the formation of antibodies within 5 days only…
But naturally we transplant a tissue which contains antigens are not known to the recipient so we need 14 days at least to form antibodies.

And that’s why many authors believe that it’s not important.
The only explanation : these antibodies are formed against minor histocompatibility antigen which is very rare.


The most common type of rejection is the ACUTE rejection.
Why is it very common?
Because hyper acute rejection is very rare when we plan and prepare for the transplantation and we avoid it if there are preformed antibodies or different blood groups.
Accelerated type is not important as we said ….
that’s why Most rejections are of this type “acute” with decreasing frequency after the first three months.


Acute rejection is mediated by T-lymphocytes.
Individuals who lack t-lymphocytes may have one of these:
DiGeorge syndrome : which is caused by absence of gene that’s responsible about thymus formation so they are born without thymus.
OR nod mice syndrome: which is caused by thymectomy in neonatal period.

If a tissue or an organ is transplanted and there are no preformed antibodies, the rejection between allogenic donor and recipient takes place at day 11-15.

So 11-15 days are required for the evidence of rejection which will be in form of infiltration of the organ with mononuclear cells with an inflammatory response that will accompanish such reaction.
This is known as First set rejection.

If the same individual is transplanted with the same tissues “the same antigens” the rejection will takes place within 6-8 days and this is the Second set rejection.

If the same individual who mounted a second set rejection is injected with new antigens he will develop a rejection with in 11-15 days “first set rejection”.


So here exposure is he most important, recognition of the same antigen 2nd set rejection but if it’s different the same time of the first set is needed…

Strategies have been developed leading to improvement in graft survival (>80% for one year). And sometimes 90-95% because of matching and immuno suppression therapy .
Matching alone will decrease the chance of rejection but it does not prevent it.

Allogenic responses are very important and they are the result of direct and indirect recognition.
In terms of importance the most important one is CD4 direct recognition followed by CD4 indirect recognition and then CD8 direct recognition. Why?
Because CD4 recognize major histocompatibility antigens by either direct or indirect, they are also important in generation both humeral and cell mediated response against antigens.
While CD8 recognizes minor histocompatibility antigens.

Relative importance depends on:
type of graft vascularized or not
antigenic disparity how genetically different the donor and the recipient are
time of transplantation and the previous history of recipient.

Effector mechanisms “that are involved in the rejection” include DTH “delayed type hypersensitivity”, cytotoxic T cells, cytokines, toxic molecules (nitric oxide) as a consequence of activation of macrophages , and NK cells “natural killer cells”.

All of them are result of t-cells activation NOT antibody production.


Responses to minor histocompatibility antigens are much less potent than responses to MHC “major” the differences because the frequency of responding T cells is much lower. But they also mediate rejection.
Because every nucleotide cell express an MHC class I antigen so every cell in the transplanted organ is a target for rejection.
And since the whole organ has cells that express minor antigens then every single cell is a target so it’s impossible to prevent rejection.
Even if you have HLA identical saplings there is a chance of rejection because of the minor antigens.
Note: increase the number of family members will increase the chance to find identical HLA.

So that It is no wonder that successful transplantation requires the use of potent immunosuppressive drugs.

Now if the patient is subjected to immuno suppression therapy and the rejection is delayed by matching he will later develop a rejection reaction which is termed: Chronic rejection.

Which is a kind of rejection that’s delayed for years because of the use of immuno suppressive drugs..
It usually involves B and T cells

Even when 1 year graft survival has been achieved, the loss of transplanted organs continues to occur at a rate of about 3-5% per year so after 10 years only 50% of these transplants will still function due to chronic rejection.

Pathologic manifestations vary but always involve narrowing of the vascular bed.

Important observations made are;
-the presence of anti-donor antibody

-refractoriness to increases in immuno suppression means if you increase the immuno suppression therapy that will not improve the survival and that increase has its own risks like cancer or infection and usually these patients die because of sever infection or cancer

-high correlation between the onset of chronic rejection and history of early acute rejection episodes.













The figure in the previous page shows the difference between one year survival and long term survival.
As you can see over a period of 20 years (1975-1995) the one year survival improved from 40% to >85% with improvement of immuno suppression therapy .

Where as for a long term it goes from 20% to 22% , very minimal improvement due to chronic rejection.

Remember: the rejection in the first 3 months is acute, after that it’s chronic.

Study slide 28 which shows the 4 forms of rejection ..it’s very useful.

As you notice in the figure above in any transplants the T-lymphocytes are very important even for antibody production.. because they are the stimulants of DTH.
- CD4 which reacts with B cells to produce antibodies this will cause release of cytokines
- activation of macrophages which will release lytic enzymes and cause membrane destruction.
So once the organ is transplanted it usually stimulates variety of mechanisms starting with T-cells but at the end many specific and non specific mechanisms will act to destruct the membrane.

Manipulations to Prevent Graft Rejection
- Donor recipient matching and that involves MHC matching .
We should test the tissue type first “HLA” antigens. Which is known as HLA typing.
HLA typing can improve the success rate but it does not prevent the rejection, because it imprecise owing to the polymorphism and complexity of the human MHC, because we use methods that are used are not sensitive to detect minimal changes.

Example: if two individuals have the same HLA, they may have differences because of mutation or minor changes that can’t be detected by antibodies.

SO Grafts between HLA identical siblings are invariably rejected, albeit more slowly, unless donor and recipient are identical twins (minor H antigens).

As you can notice in the figure, there is no significant difference between full house (complete matching) and good or even with fair.
So minor improvement achieved by complete matching, because of the presence of other mechanisms. And this emphasis that the only way to achieve a successful transplantation is to use potent immuno suppression drugs.
So there is continuous improvement in immuno suppression therapy.

Prevention and Treatment of Rejection

-Transplantation almost invariably results in some form of rejection.

-Strategies used to avoid or delay rejection are general immunosuppression and minimizing the strength of the specific allogeneic reaction.

-Approaches for inducing donor-specific tolerance are also nearing clinical trials.

How can we induce tolerance?
As you know tolerance involves exposure but how?
This can be achieved by repeated blood transfusion, small amount 3-4 times.
Individuals will either develop anti HLA antibodies and exclude them, or they will not develop anti HLA antibodies and become a good recipient.

So blood transfusion detects and eliminates potential donors.

We also can induce tolerance by injection of CTLA14 which compute CD28 to bind to B71 or 2

Immunosuppressive Drugs

1- Inhibit or lyse T Lymphocytes:

- Cyclosporine A : blocks IL-2 dependent growth and differentiation of T
cells. (it’s the most potent one)

- Tacrolimus(FK 506): inhibits T cell activation.

- Rapamycin: inhibits T cell proliferation

Usually more than one drug is given , like when u give cyclosporine A with Rapamycin if u don’t block activation u kill the cell, so they act sequentially.
Better results achieved with using combinations.

2-Metabolic Toxins that kill proliferating T cells

• Inhibit maturation of lymphocytes and kill proliferating mature T cells that have been stimulated by alloantigens.

• Mycophenolate mofetil is the newest of these agents that is routinely used with Cyclosporine-A.

3-Antibodies reactive with T cell surface structures

Anti CD3 CD3 is essential for recognition of antigen by TCR this way we block this recognition.
anti-CD25 (α subunit of IL-2 receptor), anti-CD4, anti-CD8, and anti- ICAM

4-Anti Inflammatory agents
Corticosteroids: inhibit synthesis and secretion of cytokines including TNF and IL-1 by mononuclear phagocytes.

5-Tolerance Induction
As we said before , by blood transfusion, Soluble CTLA-4, Anti-CD40 ligand,
Anti-IL-2 receptor, and anti MHC donor peptide MHC peptide is presented to CD4 if u block it you will prevent that presentation of antigens.


At the level of transplantation:
Anti CD3 mono clone antibody  blocking of recognition by TCR
CTLA-4 blocks recognition by CD26 and B7.
Anti CD25 blocks recognition of IL2
Cyclosporine A + FK506  inhibit proliferation of T-lymphocyte
Rapamycin killing of the cell

Generally used drugs such as methotroxate, azathioprine, ….
















The improvement of immuno suppression therapy is to maintain the immunity of the host. And that can be achieved by using cyclosporine A or Tacrolimus.
Where as Methotroxate cause damaging of the immune system cells so it’s not recommended.

The organs that can be transplanted:
Cornea (from cadaver) , lung , kidney, heart, …..


Gene technology
Gene technology offers the possibility to breed the desired organs in animals: Lack of organs is no longer a problem

Gene technology makes it possible to humanize the bred organs; the immune system identifies the organ as its own tissue: Immune system rejection is prevented

From which animals are we able to transplant organs?
1. The Chimpanzee Its DNA sequence differs from ours by only 2%
2. The Baboon Its organs are too small for a large adult human
3. The Pig  Surprisingly similar to our anatomy and physiology

Organ breeding
-A transgenic animal carries a foreign gene inserted into its genome.
-The transgenic animal shows the specific characteristics which are coded on the inserted gene
-A gene which is responsible for the construction of a human organ makes the organism produce the organ additionally.










Done by : Safa’a Makhool
Immuno lec #5 of dr. Azmi
Date of the lec.:27-12-2010

a correction 4 this sheet was posted by safa2 on our fb group ...
here it is :
""‎9aba7 el5er.... I found some mistakes in my sheet and here is the correction.
in page #1: el sa6r el sades its not acute its accelerated
page #11: elsa6r 13 mktoob CTLA14 it's CTLA4
page #12 fo2 el rasmeh b km sa6er.. CD26"wrong" it's CD28
sooo sorry for these mistakes
...Good luck all :)""