Ethical issues are an important factor in living donation

The shortage of organs from cadaveric donors has increased the use of living donors. Clearly this is only possible in situations where donation of an organ poses little risk to the living donor. Kidney transplantation is routinely performed from living donors, as it is quite possible to live with just one healthy kidney. It is also possible to donate a lobe of a liver, as it can grow back again. Donation of a lung lobe is also possible, though this is a much riskier procedure. Hematopoietic stem cell transplants also involve living donors, though the issues with this form of transplant are somewhat different than for solid organ transplants as the stem cells are rapidly replaced.

Before any donation from a living donor is carried out, it is important to ensure that the potential donor is healthy, that the donation is compatible (of the appropriate blood group), and that the donor is aware of the risks and has not been coerced into giving an organ. It would be unethical for a donor to be put under extreme pressure to donate an organ, and the transplant teams must take great care to ensure that this is not happening. In the majority of cases donation is carried out between close relatives (including spouses) or sometimes friends. However, there are a few ‘altruistic donors’ that give their organs to be used by whoever needs them. In most countries it is not allowed to sell organs for transplantation, and the majority of those involved in transplantation would see that as unethical. However, there is a real problem with ‘transplant tourism’ where people from rich, developed, countries travel to buy organs from poor, marginalized, donors.

Organ exchange programs have been set up to maximize the number of transplants performed. In the simplest cases these are paired donations. Jane Smith may need a kidney and have a relative, Steve Smith, who is willing to donate it. However, if Steve Smith’s kidney is incompatible with Jane then he cannot donate it to her. However, if there is another couple in a similar predicament, so that Jim Jones needs a kidney and Stella Jones is willing to donate one but they are incompatible, then it might be possible for Stella Jones to donate her kidney to Jane Smith and for Steve Smith to donate to Jim Jones. More complex arrangements are also possible, with three or more pairs donating in such a way as to maximize the number of transplants. Indeed it is possible to use an altruistic donor to initiate a chain of donation, such that they donate to recipient A, recipient A’s potential donor gives to recipient B, their donor gives to C and so on, until the final recipient is an individual who does not have a suitable donor.

In addition to transplantation of organs, there is a large program of transplanting hematopoietic stem cells (cells capable of regenerating blood cells), for example in patients with leukemia or with primary immune deficiencies. These stem cells were previously normally harvested from the bone marrow of (living) donors, though increasingly peripheral stem cells obtained from the blood are used – stem cell transplantation has its own particular problems.

Clinical trials of stem cell therapy, using mesenchymal stem cells or embryonic stem cells are now underway. The aim of these is that the stem cells can differentiate and repair damaged organs. In some cases the stem cells are taken from the patient, in which case there is no immunological issues. However, if they are taken from another individual then rejection is possible.

Host versus graft responses cause transplant rejection

Immune recognition of the antigenic differences between the donor organ and the recipient will, unless treated, lead to an immune response in which the host immune system responds to, and attacks, the donor tissue.

The nature of the host versus graft response is discussed in more detail below.

Similar to any other adaptive immune response, the immune response against a graft shows memory of previous encounters with an antigen. Therefore, once an animal has rejected an organ graft for the first time, if a second graft is performed from the same species or donor then it is rejected more rapidly (second set rejection).

There is a high frequency of T cells recognizing the graft

One of the main features of the immune response against a transplanted organ is that it is much more vigorous and strong than the response against a pathogen, such as a virus. This is largely reflected by the frequency of T cells that recognize the graft as foreign and react against it.

Thus, in a naive or unimmunized individual fewer than 1/100 000 T cells respond upon exposure to a virus or a protein immunization; however, 1/100–1/1000 T cells respond to allogeneic antigen-presenting cells (APCs). This is reflected in the strong T cell response (proliferation) seen when naive T cells are stimulated with allogeneic dendritic cells (Fig. 21.4)

Fig. 21.4 Measuring the strength of the alloresponse

The strength of the alloresponse can be measured in a mixed lymphocyte reaction. In this assay T cells from an individual were mixed with varying numbers of dendritic cells (DCs) from either the same (autologous DC) or a different (allogeneic DC) donor. The dendritic cells were irradiated to prevent their proliferation. As a control cultures were included that contained just the dendritic cells or just the T cells. Five days later, T cell proliferation was measured by incorporation of ³ H-thymidine, which is incorporated into the DNA of dividing cells. There is strong proliferation of T cells exposed to allogeneic dendritic cells, even though these T cells have not been exposed to the allogeneic cells, i.e. it represents a primary immune response.

Histocompatibility antigens are the targets for rejection

Early experiments showed that the bulk of the allospecific response is against molecules of the MHC. We now know that these molecules are the MHC class I or class II molecules (see Chapter 5), which are responsible for presenting antigen (in the form of peptides) to either:

As discussed in Chapter 8, MHC molecules are highly polymorphic, and it is these polymorphic differences that are seen by alloreactive T cells.

Why is there such a high frequency of allospecific T cells?

Several models seek to explain why there is such a high frequency of allospecific cells in the T cell repertoire.

The first model (high determinant density model) (Fig. 21.w1) suggests that allospecific T cells recognize the foreign MHC molecules directly, with a low affinity, in a peptide-independent manner. The affinity of the interaction would normally be too low to activate the T cells; however, because the T cells see the MHC molecules directly, and are not recognizing the peptide, this low affinity is compensated for by the high concentration of MHC molecules on allogeneic cells.

Fig. 21.w1 Why is there a high frequency of allospecific cells?

The high precursor frequency of alloreactive T cells can be explained by one of two models. In the first (high determinant density model) the T cell receptor recognizes the MHC molecules on the surface of the APC with a moderate to low affinity. However, this recognition is largely independent of the type of peptide present in the groove of the MHC molecule. The low affinity of the interaction is compensated for by the multiple interactions that form, which provide an avidity advantage. In the multiple determinant model the T cells recognize, with a high affinity, peptide and MHC molecule complexes. The peptides are derived from normal self proteins. As the T cells have not been tolerated in the thymus to those antigens in the context of the donor MHC (though any T cells reactive with the same antigens in the context of self MHC would have been deleted), there is a high frequency of alloreactivity. Which form of allorecognition is most important will depend on the MHC combination of the donor and recipient.

The second model (multiple determinant model) states that what the allospecific T cells are recognizing are peptides derived from normal, non-polymorphic, host proteins that bind to and are presented by the foreign MHC molecules, but are not presented by self MHC. Due to lack of presentation by self MHC, the T cell repertoire is not tolerant to such peptides. The high frequency of the response is due to the large number of such antigens that can be presented by the graft.

Indirect recognition is important in chronic rejection

In a primary alloresponse (see Fig. 21.4), most of the alloreactive CD4 or CD8 T cells directly recognize the donor MHC molecules.

However, there are other forms of alloresponse, including:

Fig. 21.5 Direct and indirect allorecognition

(1) In direct allorecognition the CD4 or CD8 T cell directly recognizes the donor APC, with the T cell receptor (TCR) binding donor MHC molecules bearing donor peptide. (2) Indirect allorecognition is more similar to conventional T cell recognition, in that the recipient CD4 T cells recognize foreign (donor derived) antigen that has been taken up and processed by recipient APCs. The TCR therefore recognizes recipient MHC bearing donor peptide. CD8 cells only see alloantigen by the direct pathway.

The indirect response is very similar to conventional T cell recognition of normal antigens, such as those from a pathogen, which are processed by host APCs and presented in the context of host MHC molecules.

Nevertheless, the indirect pathway of recognition is important during chronic rejection, when the number of donor-derived professional APCs is no longer high enough to stimulate a direct immune response. It is also important in the rejection of corneal grafts because the cornea lacks large numbers of APCs.

Minor antigens can be targets of rejection even when donor and recipient MHC are identical

Although the MHC is the major target of the alloimmune response, there are also minor histocompatibility antigens. These can serve as targets of rejection even when the MHC is identical between donor and recipient.

The nature of most minor histocompatibility antigens is unknown, though they are assumed to be normal polymorphic molecules, peptides from which bind to host MHC and induce an immune response. In some cases they are expressed in a tissue-specific manner.

Perhaps the best studied minor histocompatibility antigen system is the H-Y system. These are antigens encoded by the Y chromosome, and so are expressed only on male cells. Thus, following immunization, it is possible to demonstrate immune responses and rejection of male organs or skin following transplantation mediated by female animals (2X chromosomes) against male cells (X and Y chromosome). It is not possible to show responses against female antigens by male animals because the male animals have one X chromosome, and so are tolerant to all antigens encoded on it (Fig. 21.6).

Fig. 21.6 H-Y minor histocompatibility antigens

The H-Y antigens are minor histocompatibility antigens expressed on male animals only. Their existence can be demonstrated by skin grafting between animals of the same strain. As would be expected grafts from female mice are always accepted, while those from male mice are accepted on male recipients, but not female recipients.

Graft versus host reactions result when donor lymphocytes attack the graft recipient

Although it is usual to think of the immune response recognizing and destroying the transplanted organ, the situation is different when competent immune cells are transplanted into a recipient. This can happen during bone marrow transplantation, when normal donor T cells may be infused into the recipient. In such circumstances the T cells can recognize the MHC molecules and/or minor histocompatibility antigens of the recipient as foreign, and produce an immune response against the recipient. This is known as graft versus host disease (GvHD).

GvHD can be lethal, causing damage in particular to the skin and gut. It can be demonstrated in animal models by transfer of bone marrow to irradiated recipient animals (Fig. 21.7). It can be avoided by:

Fig. 21.7 Graft versus host disease

Graft versus host disease is caused by immunocompetent cells reacting against their host animal. Immunocompetent cells from a donor of type A are injected into an immunosuppressed (X-irradiated) host of type C, or a normal (A × B) F₁ recipient. The immunosuppressed individual is unable to reject the cells and the F₁ animal is fully tolerant to parental type A cells. In both cases the donor cells recognize the foreign tissue types B or C of the recipient. They divide and react against the recipient tissue cells and recruit large numbers of host cells to inflammatory sites. Very often the process leads to the death of the recipient.

Hyperacute rejection is immediate and mediated by antibody

Hyperacute rejection is seen when the recipient animal has pre-existing antibodies that are reactive with the donor tissue. This may be because:

A special case is seen in xenotransplantation, where humans and Old World monkeys and apes all have pre-existing antibodies to a carbohydrate antigen α-galactosyl. This carbohydrate is expressed on cell surface proteins of all other donors. Therefore, a xenotransplant of a cellular organ from a pig (or most other species) into a primate is at risk of hyperacute rejection.

Hyperacute rejection is seen within minutes of connecting the circulation into the transplanted organ. It is caused by the pre-existing antibodies binding to the endothelial cells lining the blood vessels and by initiating immune effector functions.

Complement activation can lead to death of the endothelium, or, when the damage is sub-lethal, activation of the endothelial cells. This not only causes an inflammatory response, increasing vascular leakage, but can also cause blood coagulation The result is rapid destruction of the graft (Fig. 21.9).

Fig. 21.9 Renal histology showing hyperacute graft rejection

Hyperacute rejection is caused by pre-exisiting antibody at the time of transplantation. There is extensive necrosis of the glomerular capillary associated with massive interstitial hemorrhage. This extensive necrosis is preceded by an intense polymorphonuclear infiltration, which occurs within the first hour of the graft’s revascularization. The changes shown here occurred 24–48 hours after this. H&E stain. × 200.

Prevention of hyperacute rejection is performed by carefully avoiding transplanting an organ into an individual with pre-existing antibodies to that tissue. This is done by:

This involves incubating donor leukocytes with recipient serum in the presence of complement; cell death indicates the presence of anti-donor antibody and is a contraindication to proceeding with transplantation. Such cross-matching is normally performed immediately before surgery. There is increasing success in transplanting across ABO incompatible barriers, though this requires very careful preparation of the recipient.

ABO incompatible transplantation

As discussed above, transplantation across an ABO incompatibility barrier results in hyperacute rejection of the organ. However, there is increasing experience in such transplants. To give a practical example, let us take a patient Isaac Bloggs who has blood group B and requires a kidney. He therefore has anti-A antibodies. His brother, Mohammed Bloggs, is willing to donate a kidney and is a perfect HLA match. However, he is blood group A, and so antibodies in Isaac’s blood will bind to Mohammed’s cells and destroy them though hyperacute rejection. However, the transplant team decide to go ahead with the transplant.

The first stage is to deplete Isaac’s blood of anti-A antibodies. This can be done by plasmapheresing him (taking his blood out, replacing the plasma that contains the antibodies and then returning it to him). Alternatively his blood can be passed down an affinity column that removes the antibodies. This process will need to be repeated multiple times in order to get the levels of anti-A antibody low. In some hospitals Isaac will also be given antibodies to deplete B cells or have his spleen removed, to reduce the immune response following transplantation.

Once the antibody levels are low enough, then Mohammed’s kidney can be transplanted into Isaac. There is a need for careful monitoring in the early days, and it may be necessary to continue plasmapheresis. Isaac will also receive the conventional immunosuppressive drugs.

In most cases these transplants do very well. Interestingly the levels of anti-A antibody in Isaac are likely to return to close to the levels seen before the procedures over a few months. However, these antibodies do not destroy the donated graft, even though they would have resulted in hyperacute rejection if they had been there at the time of transplantation. The reason is that the cells in the graft, most probably the endothelial cells, have undergone a process termed accommodation, in which they survive even though there are high levels of antibody capable of binding to them. The process of accommodation involves upregulation of a number of protective genes in the cells of the donated kidney.

In the case of xenotransplantation, one solution being developed is the generation of pigs that lack the α-galactosyl carbohydrate epitopes. This is done by knocking out the gene encoding the enzyme (α-galactosyl transferase) that generates this carbohydrate. In preclinical models in which pig organs are transplanted into primates, this approach has led to prolonged graft survival with no evidence of hyperacute rejection.

Acute rejection occurs days to weeks after transplantation

Acute rejection is normally seen days to weeks after transplantation, and is caused by activation of allospecific T cells capable of damaging the graft.

Donor dendritic cells (sometimes called passenger leukocytes) play an important role in triggering acute rejection. Dendritic cells that are present in the organ, following transplantation into the recipient, migrate to the lymph nodes draining the organ and stimulate a primary alloimmune response.

The importance of these dendritic cells can be shown by ‘parking experiments’ in which:

Fig. 21.10 Importance of passenger leukocytes in graft sensitization

The role of passenger leukocytes (dendritic cells) can be shown by ‘parking’ experiments in which kidneys are grafted from a rat of strain A into a recipient of strain B. Immunosuppression is used to prevent the animal from rejecting the graft. After a period the grafts are then retransplanted into a fresh strain B rat. There is very slow rejection of the graft (when compared to the rapid rejection seen when the kidney is transferred first into a strain A rat), which is thought to be due to the inability of the kidney from the strain A rat to immunize the strain B recipient due to the loss of dendritic cells during the period when the graft was ‘parked’ in the first strain A animal. The slow rejection probably occurs via the indirect pathway. The rejection occurs at the normal rapid tempo if strain A dendritic cells are injected into the recipient animal at the same time as the graft, suggesting that dendritic cells are capable of sensitizing the animal to the graft.

However, if the third animal is injected with donor-derived dendritic cells there is rapid graft rejection. These data highlight the contribution of donor dendritic cells in initiating the alloresponse.

Although the direct pathway is thought to predominate in acute rejection, the indirect alloresponse, though significantly weaker, can also cause acute rejection in some animal models.

Once activated the T cells migrate to the organ and lead to tissue damage by standard immunological effector mechanisms (Fig. 21.11). These include:

Fig. 21.11 Renal histology showing acute graft rejection

(1) Small lymphocytes and other cells are accumulating in the interstitium of the graft. Such infiltration (I) is characteristic of acute rejection and occurs before the appearance of any clinical signs. H&E stain. (2) H&E stain of acutely rejecting kidney showing vascular obstruction. (3) van Gieson’s stain of acutely rejecting kidney showing the end stage of this process. (G, glomerulus.)

If the animal or patient has already been exposed to the alloantigens expressed by the graft, and as a consequence has been immunized, there will be alloreactive memory cells. This will lead to a much more rapid (accelerated) rejection of the graft (Fig. 21.12).

Fig. 21.12 Graft rejection displays immunological memory

A human skin allograft at day 5 (1) is fully vascularized and the cells are dividing, but by day 12 (2) is totally destroyed. A second graft (‘second-set’ graft) from the same donor shown here on day 7 (3) does not become vascularized and is destroyed rapidly. This indicates that sensitization to the first graft produces immunological memory.

Chronic rejection is seen months or years after transplantation

In vascularized organs chronic rejection presents as occlusion of blood vessels, which on histological analysis show a thickening of the intima, similar in some respects to the thickening seen as a result of atherosclerosis (Fig. 21.13). Smooth muscle cell proliferation is often seen, together with a macrophage infiltrate (together with some lymphocytes). This eventually leads to blockage of the blood vessels and subsequent ischemia of the organ.

Fig. 21.13 Chronic rejection

Grafts that survive acute rejection are still capable of undergoing chronic rejection. (1) Section taken from a patient with chronic rejection of their heart graft. The lumen of the blood vessel in the heart has been narrowed as a result of thickening of the wall of the vessel, limiting the blood supply to the heart. (2) Section taken from a patient with chronic rejection of the lung, showing obliterative bronchiolitis (arrow) blocking the airways.

(Kindly supplied by Professor Marlene Rose, Imperial College London, Harefield Hospital, and Dr Margaret Burke, Pathology Department, Royal Brompton Hospital and Harefield Hospital.)

A number of mechanisms can lead to chronic rejection. They include:

Non-immunological processes are also important, such as:

In some cases, initiation of chronic rejection may be immunological in nature, but its progression is due to non-immunological mechanisms.

Chronic rejection responds poorly to current immunosuppressive therapy. Therefore, although there has been considerable improvement in overall graft survival over the past decades, this improvement is mostly seen in the first year following transplantation – the subsequent survival of grafts has hardly altered over the past 20–30 years (Fig. 21.14). This indicates the need to improve the treatment of chronic rejection.

Fig. 21.14 Long-term survival of kidney grafts

The graph shows the survival of kidney grafts in patients transplanted from heart-beating donors in the UK 1995–2007. For comparison survival in the UK transplant program 1975–1979 is also included. There has been a considerable improvement in graft survival since 1975, but most of the improvement is seen in the first year. After that time the half-life of graft survival is similar for all patient groups.

(Data from Statistics and Audit Directorate, NHS Blood and Transplant, 2009.)

The better the HLA matching of donor and recipient, the less the strength of rejection

The major antigenic differences recognized by the alloimmune response are found on the MHC molecules (HLA in humans). These highly polymorphic molecules have a vital role in presenting antigens to T cells.

There are many different alleles of the MHC molecules, and one way to reduce the strength of a rejection response is to match the donor and recipient so that they share as many alleles as possible. In general matching is now performed using molecular techniques, with polymerase chain reactions (PCR) that are specific for the different alleles.

In humans HLA matching is rarely perfect between unrelated donors because of the difficulty in matching all MHC class I and class II gene loci and the high level of polymorphism at each locus.

The extensive polymorphism means that there can hundreds of variants of each antigen. For example the number of (protein) variants of the HLA molecules identified up to April 2011 was: HLA-A 1176; HLA-B 1641; HLA-C 808; HLA-DRA 2, -DRB 774; HLA-DQA 27, -DQB 106; HLA-DPA 16, -DPB 129.

Therefore, even before one considers other polymorphic molecules associated with the HLA locus, there is considerable complexity, which makes it highly unlikely for there to be a complete match.

In cases where the transplant is between living related donors (such as brothers and sisters) there is a greater opportunity for a match because in general the HLA locus is inherited en bloc as a single set (or haplotype) from each parent.

Loci outside the MHC can also lead to rejection (the minor histocompatibility antigens). However, there is no attempt to match for these antigens because there is little possibility of getting a good match and the effect of matching any single minor antigen is too small to be significant.

It should be noted that even when siblings are perfectly matched at the MHC locus they will not be matched (unless they are identical twins) for the minor histocompatibility antigens.

The importance of HLA matching is not always crucial

HLA matching is very important in bone marrow transplantation (where a large potential donor pool can reduce the risk of GvHD) and has a significant influence on the outcome in kidney transplantation. For other organs the importance is less clear, for example:

6-MP, azathioprine, and MPA are antiproliferative drugs

The first antiproliferative drug to be used in patients was 6-mercaptopurine (6-MP). This was followed by the introduction of azathioprine, the parent compound that is converted in vivo to 6-MP. Azathioprine was given to all patients until a more potent alternative arrived on the scene, mycophenolic acid (MPA).

These antiproliferative drugs have related modes of action, namely inhibition of the synthesis of purines that are required for cell division:

Clearly purine synthesis is needed in all cell types. Consequently, the risk of such agents is inhibition of other populations of rapidly dividing cell systems, such as the bone marrow. Indeed, close monitoring of white cell and platelet numbers is necessary in patients on antiproliferative drugs.

T cells are particularly sensitive to antiproliferative agents, partly because they are largely dependent on the de-novo synthesis of purines, whereas other cell types have a more efficient salvage pathway. Consequently, at conventional doses there is selective inhibition of T cell-dependent immunity.

Ciclosporin, tacrolimus, and sirolimus are inhibitors of T cell activation

Ciclosporin inhibits the production of IL-2

Drugs that inhibit T cell activation are the mainstay of immunosuppressive regimens. The first such drug to be discovered was ciclosporin, a fungal metabolite (Fig. 21.w2).

Fig. 21.w2 Structure of immunosuppressive fungal macrolides

The immunosuppressive fungal macrolides, ciclosporin, FK506 (tacrolimus), and rapamycin (sirolimus), have quite different structures. They act on lymphocytes in different ways – ciclosporin and FK506 affect cytokine production and rapamycin interferes with signaling through the IL-2 receptor (IL-2R).

The introduction of ciclosporin revolutionized clinical transplantation in that it led to a marked improvement in early success rates, which rose from approximately 70% to over 90%. Furthermore, the doses of other drugs that were required were substantially lower, so drug side effects were less troublesome.

Because of the much greater potency of ciclosporin, and its ability to prevent early acute rejection, transplantation of organs other than kidney became routine. Programs of heart, liver, and more recently lung transplantation were established in major transplant centers around the world.

The key effect of ciclosporin is to inhibit the production of the major growth factor for T cells, interleukin-2 (IL-2).

The intracellular mechanism of action of ciclosporin involves binding to cyclophilin, and the consequent inhibition of the calcium-dependent phosphatase, calcineurin, which would otherwise activate the NFAT complex, and lead to IL-2 gene transcription.

Corticosteroids are anti-inflammatory drugs used for transplant immunosuppression

Corticosteroids, given in combination with azathioprine, were the mainstay of immunosuppression for several decades. Only now is the possibility of rapid corticosteroid withdrawal, after 1 or 2 weeks, being explored.

Corticosteroids are pharmacological derivatives of the glucocorticoid family of steroid hormones, and act through intracellular receptors that are almost ubiquitously expressed.

The anti-inflammatory effects of steroids are highly complex, reflecting the fact that as many as 1% of genes may be regulated by glucocorticoids. Some of the most important effects are:

The problem of side effects is a major issue with the use of corticosteroids, particularly at high doses. Central weight gain, fluid retention, diabetes mellitus, bone mineral loss, and thinning of the skin, all result from protracted corticosteroid use. It is for this reason that much attention is being devoted to protocols that allow steroid minimization. However, for corneal transplants, where topical (local) administration of the drug is possible (thus minimizing side effects), corticosteroids are the main form of immunosuppression used.

Antibody therapy

Antibodies have long been used for preventing graft rejection. One of the earliest forms of immunosuppression was anti-lymphocyte globulin or anti-thymocyte globulin; polyclonal antibodies raised by immunizing rabbits or horses with lymphocytes or thymocytes. These antibodies deplete circulating T cells. These are still used, though they are increasingly being replaced with monoclonal antibodies. The first monoclonal antibody licensed for any purpose was Muromonab-CD3 which was approved by the FDA in 1986 for the treatment of rejection episodes following renal transplantation. This binds to CD3 on T cells, and suppresses T cell responses. The action of Muromonab binding CD3 can result in a cytokine release syndrome on infusion, caused by the secretion of inflammatory cytokines by the T cells following engagment of CD3. Two anti IL2 receptors (daclizumab and basiliximab) are now widely used in transplantation, as part of standard immunosuppressive agents. These antibodies operate by blocking the IL2 receptor, rather than depleting T cells. Alemtuzumab (commonly known as CAMPATH-1 H), which depletes CD52 expressing leukocytes, has been widely used for transplantation though it is not yet licensed for that application. Other antibodies are being investigated for particular applications in transplantation (for example the depletion of B cells). Antibody like molecules (e.g. abatacept and belatacept which are two forms of a fusion protein of CTLA4 with the Fc portion of an antibody (see Chapter 8)) are also being evaluated for clinical use.

There is evidence for the induction of tolerance in humans

There are two sources of evidence for the induction of tolerance in humans.

First there are patients who have received grafts, but are no longer on immunosuppressive regimens because they cannot tolerate the drugs. They can show long-term graft survival. This is not formal evidence of tolerance, but it is highly suggestive that some people can have an operational tolerance whereby they fail to destroy their organ graft.

Second, it is possible to look at the frequency of alloreactive T cells in patients with grafts. In some groups there is a reduced frequency of these cells, but the response to other antigens remains normal (Fig. 21.15). Again, this is not a formal proof of tolerance because it is not yet known how the in-vitro assays relate to the response in patients. However, it does indicate that it might be possible to develop tests that will allow us to monitor the development of tolerance in patients, and so know how to tailor treatment to the individual (e.g. removing them from immunosuppression when indicated).

Fig. 21.15 Reduction in allospecific T cells in patients with kidney grafts

The graph shows the frequency of T cells capable of producing IL-2 from patients with long-term kidney grafts that react with cells bearing the donor alloantigen or control (third party) alloantigens. The data show two groups of patients – those with evidence of chronic rejection and those without. Both groups of patient show a reduced frequency of T cells capable of recognizing alloantigen from the donor of the organ when compared to the frequency against third party alloantigen. This indicates that the patients show a degree of reduced reactivity to alloantigens.

Third, there are now studies that do demonstrate long term graft survival (and so apparent tolerance) in patients that have received joint renal and hematopoetic stem cell transplants. These patients had a short course of immunosupression, and have since seen long term graft survival with no rejection. While the number of patients treated is limited, these data are very encouraging.

Novel methods for inducing tolerance are being developed

There are various ways in which tolerance (or the appearance of tolerance) can occur. Most of these are discussed in more detail in Chapter 19. An understanding of the mechanisms by which tolerance is induced and maintained allows the development of novel methods for inducing tolerance.

Central tolerance results from deletion of T cells in the thymus and is the most important form of tolerance induction for preventing autoimmunity. It has been harnessed for the induction of tolerance in experimental systems by transplanting the thymus from the donor into the recipient. This approach may be particularly useful in the context of xenotransplantation, where there is an opportunity to manipulate the donor and/or recipient before grafting of the organ.

Alloreactive cells can be made anergic

In the peripheral organs tolerance induction can result from deletion. However, it is also possible for alloreactive cells to be anergized. Anergy describes a state in which the cell is not deleted, but has been rendered unresponsive to further stimulation by the same antigen.

Blockade of co-stimulatory molecules such as CD80 and CD86 with agents like CTLA-4–Ig (a fusion protein between CTLA-4, a ligand for CD80 and CD86, and the Fc part of an antibody molecule) can be used to induce anergy in alloreactive cells (Fig. 21.16). However, it should be noted that the situation can be more complex than this. In many APCs cross-linking of CD80 and CD86 with CTLA-4–Ig results in upregulation of an immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). This enzyme catabolizes tryptophan, and as a result prevents T cell activation both as a result of:

Fig. 21.16 Co-stimulation blockade

For full T cell activation to occur the T cell needs to receive two signals – signal 1 through the T cell receptor binding the appropriate peptide–MHC complex (which provides antigenic specificity) and signal 2 through co-stimulatory molecules (the most important of which for naive T cells is CD28 on the T cell binding CD80, or CD86 on the APC). Interaction of a T cell with an APC that lacks CD80 or CD86, so that signal 1 only is received, fails to activate the T cell and can result in apoptosis or anergy of the lymphocyte. In experimental or clinical settings it is possible to block the CD28 interaction with CD80/86 by addition of a soluble molecule, CTLA-4–Ig, which consists of the extracellular part of CTLA-4 (an alternative ligand for CD80/86) fused to the Fc of immunoglobulin. This molecule prevents the interaction of CD28 with CD80/86. It can also result in upregulation of immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO), as described in the text.

The role of IDO in immune regulation was first recognized in the placenta, where it protects the fetus from immunological rejection – inhibition of IDO causes rejection of histoincompatible fetuses.

Another alternative is to induce a regulatory response to the alloantigen. The phenomenon of T cell regulation has long been recognized (Fig. 21.17), and can be shown in experimental models by transferring T cells from a tolerant animal to a naive recipient, and showing that this results in a transfer of the tolerance. Several types of T cell are capable of regulating the immune response, and strategies that seek to expand these cells may be one method to induce tolerance in vivo.

Fig. 21.17 Tolerance to grafts can be transferred by regulatory cells

An animal of strain A that has been made tolerant to a graft B can contain regulatory cells. If these cells are removed from the animal and transferred to a lightly irradiated strain A recipient, and this animal is then given a skin graft from strain B, then that graft may be accepted. In most cases this tolerance is specific, so if the second mouse received a graft of strain C it would be rejected.

Immune privilege can be a property of the tissue or site of transplant

Although a failure to reject a graft could be the result of tolerance (or immunosuppression), an alternative is that the graft is immune privileged and protected from the immune response against it (see Chapter 12):

It is important to note that immune privilege is not an absolute term. Corneal grafts are rejected, albeit less vigorously than other grafts. It is probably best to think of immune privilege as a spectrum ranging from grafts that show a high degree of privilege (cornea) to those where the immune response against them is very strong (skin), with other grafts somewhere in between.

Several mechanisms can be responsible for immune privilege. These include ‘ignorance’ – the immune system does not see the graft. The cornea is not vascularized, and has a poor lymphatic drainage. It also has a very low concentration of dendritic cells in the center. These features reduce the ability of the graft to stimulate an immune response against it. If this ignorance is disrupted (e.g. by inducing vascularization in the graft bed) then the cornea is more rapidly rejected.

In addition the tissue can deviate the immune response. In the anterior chamber of the eye there is a cytokine environment (high in TGFβ and α-melanocyte stimulating hormone) that deviates the immune response away from a tissue destructive to a non-inflammatory response.

Finally, the tissue being transplanted can itself be privileged. This is also seen in the cornea, which expresses high levels of Fas ligand.

Alternative approaches to overcoming the shortage of donor organs are being investigated

Although it is very important to increase the donor pool, the approaches discussed above will never provide all the organs needed. Alternatives include:

Xenotransplantation

The most favored animal for developing xenotransplantation is the pig, for reasons that include the physiological and anatomical compatibility between pigs and humans and the ability to breed large numbers of animals rapidly.

There are several barriers to xenotransplantation, including public acceptability, safety, and scientific issues.

The main safety concern is the risk of transmission of viruses from the pig to the human, though this is a diminishing concern. It should be noted that acellular xenografts have been performed for many years and are highly successful (e.g. in the case of pig heart valves, which can be used to replace diseased valves). These are not subject to immunological rejection, nor are they potential sources of viral infection.

The scientific issues revolve around preventing graft rejection. As indicated above, a pig organ transplanted into a human would undergo hyperacute rejection as a result of preformed anti-α-galactosyl antibodies in the recipient’s circulation. One solution has been to engineer pigs (by nuclear cloning from cell lines) that lack the galactosyl transferase enzyme responsible for creating α-galactosyl residues. Organs from these pigs show reasonably long-term survival in primates.

As well as offering scientific challenges, there are also opportunities for using pig organs. The ability to genetically engineer the pig should make it easier to develop tolerance to the organ. This, as well as the ability to carry out transplants in a pre-planned manner, offers considerable advantages over conventional transplantation.