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Transplantation

This module will help you

• understand the mechanisms by which the immune system recognizes and rejects transplants.
• see some of the techniques used to suppress unwanted immune responses.
• test your knowledge and immunology problem-solving skills.

Transplantation
Clinical Immunosuppression

Transplantation

The cause of transplant rejection is recognition of foreign MHC antigens by T cells and activation of those T cells to become effector cytotoxic or helper T cells. T cell activation occurs in the case of vascularized grafts of nucleated cells expressing MHC. Corneas are not vascularized and can be successfully transplanted even in unmatched individuals. RBC express no MHC; recipients must be matched for ABO and Rh blood types to prevent damage from preexisting (natural) antibodies to these antigens.

Autografts (such as skin transplanted from one location to cover burns on another) and grafts from an identical twin do not have foreign MHC antigens and are usually accepted without medication to prevent rejection. Allografts come from members of the same species who may have identical or non-identical alleles at MHC loci. Siblings have a 1 in 4 chance of sharing all MHC antigens and being ideal donors. Close relatives usually share more MHC antigens than unrelated people, while populations from different parts of the world are less likely to share MHC antigens than people from similar origins. Xenografts come from members of different species; their MHC may be so foreign that it is not recognized by T cells and does not activate them, but other antigens (including adhesion molecules and other cell surface carbohydrates to which humans already have antibodies) can cause very rapid graft rejection.

*average values; closer matching generally increases survival time.

Tissue matching involves identifying MHC antigens on both donor and recipient cells and using donor cells with as many MHC alleles identical to those of the recipient as possible. Matching MHC Class I (especially HLA-B) and Class II HLA-DR alleles is more important for successful transplantation than matching other MHC antigens; and matching MHC is more important than matching minor histocompatibility antigens. Transplants must always be matched for blood type antigens, which are found on other body tissues.

HLA matching is done in two ways. Serological assays use antibodies to HLA alleles to type donor and recipient cells. Binding can either be detected by flow cytometry or by adding complement and assaying for cell viability. The Mixed Lymphocyte Reaction (MLR) is used to measure CD4 cell division in response to foreign Class II MHC or CD8 T cell division in response to foreign Class I MHC. Stimulator cells from the donor, usually B cells activated to express MHC and treated with mitomycin C so they cannot divide, are mixed with responder T cells from the recipient (the process is reversed for bone marrow transplants). Several days later, the culture is pulsed with ³H-thymidine and uptake by the responder cells is measured. The higher the counts, the stronger is the response to the foreign cells. Limit dilution assays (see ToolBox) are even better than MLR at quantifying the frequency of responding cells.

HLA matching improves graft survival but does not prevent rejection, even in MHC-identical siblings (except for identical twins). One reason is that MHC typing using anti-HLA antibodies is imprecise; available antibodies do not detect all MHC alleles and the same antibody may bind two similar but non-identical alleles. Another is the presence of minor histocompatibility (minor H) antigens; these antigens stimulate slow but eventual graft rejection. Finally, time for full typing is limited for cadaver grafts because the organs can survive for only a limited time; heart transplants are matched only for ABO and Rh blood types (blood type antigens are also present on vascular epithelium).

Allogeneic MHC is recognized by either CD8 T cells (Class I) or CD4 T cells (Class II); up to 10% of T cells can recognize a given allogeneic MHC because it resembles self MHC + foreign peptide (see MHC: Antigen Processing and Presentation). Minor H antigens are usually recognized by CD8 T cells and the number of responding T cells more closely resembles that for a foreign antigen (0.01-0.001% of T cells). Minor H differences are due to polymorphism in proteins between members of the same species. An example is proteins encoded only on the Y chromosome (H-Y antigens). Since females do not make these proteins, Y antigen peptides are foreign antigens and elicit immune (anti-male) responses. Some minor H antigens may actually be foreign peptides presented on self MHC, so that two people with the same MHC alleles could differ because the donor cells were presenting foreign peptide that could be recognized by the recipient and induce an immune response that would kill the donor cells. Most minor H antigens have not been identified. All cells on the graft express minor H antigens and can be recognized and destroyed by recipient T cells.

Host T cells are activated in the draining lymph nodes to graft antigens by two mechanisms. Grafts contain passenger leukocytes, APC bearing both MHC and co-stimulatory molecules. Passenger leukocytes travel to the draining lymph nodes and activate recipient T cells (direct alloreactivity). Direct activation of recipient T cells is responsible for acute graft rejection that occurs in the first weeks following transplantation; effectors are primarily CTL. Symptoms of acute rejection include fever, a skin rash, impaired organ function (such as decreased urine output from a transplanted kidney), and a mononuclear (T cell) infiltrate into the graft visible on biopsy. Indirect alloreactivity comes from uptake of graft antigens by recipient APC and presentation on self MHC. Peptides from both MHC and minor H antigens are presented by recipient APC. Effectors are usually Th1 cells that activate macrophages to cause tissue injury and scarring that can cause chronic rejection or organ failure.

Hyperacute graft rejection occurs immediately upon transplantation. It is due to preformed antibodies, either natural antibodies to blood type antigens or anti-MHC antibodies formed in response to blood transfusions or previous transplants, or developed during pregnancy to the baby's paternal MHC antigens. Antibodies react with antigens on vascular endothelial cells and activate complement. Resulting damage blocks blood vessels and starves the organ for oxygen. Hyperacute rejection fatally damages the organ and cannot be reversed; the only treatment is immediate removal of the graft. It can be prevented by careful cross matching of donor and recipient blood.

Because of the shortage of human organs for transplantation, there has been an increasing interest in using animal organs for that purpose . Pigs are inexpensive, easy to breed, and already raised to serve as human food. Their organs are similar in size to human organs. Heart valves and skin are already transplanted from pigs to people. Skin serves as a temporary covering for severely burned patients but is eventually rejected because of its foreign antigens; heart valves are not rejected because living pig cells are removed before transplantation.

Xenograft rejection usually occurs by hyperacute mechanisms: preformed human antibodies to pig carbohydrates and adhesion molecules bind the pig organs and activate complement-mediated inflammation and tissue destruction. Pig complement-inhibiting molecules like DAF do not block human complement function. Transgenic pigs have been created which have five human genes: CD46, CD55, CD59, DAF, and H-transferase. The first four of these encode human complement-inhibitory proteins that block human complement from damaging the pig organs. H-transferase changes a pig surface carbohydrate residue to a human one, so that antibodies will not react to the pig epitope and activate complement. These transgenic pig organs survive for 30 hours in baboons instead of one hour, but much more work needs to be done before xenografting from pigs to humans is practical. One potential problem that must be avoided, even if the transplantation rejection can be dealt with, is transfer of potentially lethal viruses from animals to humans via xenografts.

Chronic (long term) rejection usually results in arteriosclerosis of graft vessels; in kidney grafts fibrosis and atrophy of the glomerulus and tubules occurs. Chronic rejection and organ failure are usually due to alloreactivity, ischemic-reperfusion injury during transplantation, chronic toxicity of anti-rejection drugs, and infection with CMV. T cells infiltrate the graft and produce cytokines that upregulate CAM expression on vascular endothelium and attract macrophages. Macrophages secrete IL-1, TNFa, and the chemokine MCP to cause chronic inflammation.

Bone marrow transplantation is used to treat inherited immune deficiencies, other deficiencies in the hematopoietic system, and leukemia. In bone marrow transplantation, the hematopoietic system of the recipient is completely destroyed by irradiation or cytotoxic drugs. Such preparation is required to make room for the transplanted marrow and may also be used to kill cancer cells if the transplant is being used as cancer therapy. Rejection in bone marrow recipients is called graft-versus-host disease (GVHD), since mature T cells present in donor marrow may be activated to reject host antigens. GVHD responses occur to both MHC and minor H antigens. Symptoms of GVHD include rashes, diarrhea, and pneumonitis. Donor marrow can be treated before transplantation with antibodies to markers on mature T cells (anti-CD3, anti-CD4, and CD8) to reduce the possibility of GVHD. Cord blood from the placentas of newborns contains high frequencies of stem cells and low frequencies of mature T cells, and can replace bone marrow as a source of hematopoietic cell transplants. Interestingly, GVHD responses are beneficial in patients with leukemia. The minor H antigen HB-1 is expressed on acute lymphoblastic leukemia cells and on B cells transformed by EBV (which can cause B cell tumors in bone marrow recipients). Eliminating all mature T cells before transplanting the marrow increases the likelihood of recurrent leukemia or EBV-mediated B cell tumors.

Improved success in transplantation is due to increasing technical expertise, the availability of transplant centers to do HLA matching and minimize organ delivery time, and the availability of immunosuppressive drugs (cyclosporin and tacrolimus) that block T cell activation to alloantigens. Still problematic are shortages of organs, the ability of existing disease to destroy the transplanted organ (diabetes and HBV infection are two examples), side effects of immunosuppressive drugs (see below), and high cost.

The fetus is an almost perfect allograft. We do not understand why most pregnancies are not rejected even though half of the baby's antigens are foreign to the mother. Cells from the fetus come in contact with the mother's cells in the placenta and may even enter the mother's circulation during pregnancy. The mother does make antibodies to the father's HLA antigens; women who have had several pregnancies are the best source of anti-HLA antibodies for serological typing.

The placenta, particularly the trophoblast (fetal tissue in closest contact with maternal tissue), plays a major role in preventing rejection. Trophoblast cells do not express classical Class I or Class II MHC that would activate maternal CD8 and CD4 T cells. Instead, trophoblast cells express HLA-G which binds KIR on NK cells and blocks their recognition (NK cells kill cells not expressing MHC in the absence of KIR binding). Cells in the placenta also express an enzyme IDO that rapidly catabolizes tryptophan, which starves maternal T cells for this amino acid and reduces their ability to respond. Pregnant mice given inhibitors of IDO reject allogeneic but not syngeneic fetuses. Evidence also exists for specific T cell tolerance to paternal MHC during the pregnancy. Finally, trophoblast cells secrete TGFb, IL-1, and IL-10 that suppress Th1 responses.

Clinical Immunosuppression

Unwanted immune responses are seen in allergy, in autoimmunity, and in transplant rejection. Suppression of allergic responses is generally aimed at the IgE effector mechanisms. Treating autoimmunity is more difficult than suppressing transplant rejection because autoimmunity involves established responses while transplantation rejection can be anticipated and to a certain extent prevented. Development of immune suppressing drugs has been empirical (try it and see if it works). Classes of drugs that are used include anti-inflammatory agents, cytotoxic agents that kill effector cells, and drugs that block T cell activation. Most immune suppression is not antigen-specific and increases the risk of infection.

*Adapted from Janeway et al. Immunobiology, 5th edition, Garland Publishing Company, New York, 2001.

Corticosteroids block inflammation. The most commonly used is prednisone, a synthetic analogue of cortisol. Steroids are lipid soluble and pass through the plasma membrane to bind cytosolic steroid receptors that then move to the nucleus and bind DNA. Steroid receptors regulate expression of about 1% of human genes. Prednisone affects the activation of many genes, especially when given at high doses. Side effects include fluid retention and weight gain, diabetes, skin thinning and bone mineral loss. For long-term use, steroids are given at lower doses with cytotoxic drugs to minimize side effects.

Cytotoxic drugs block DNA synthesis and affect rapidly dividing cells. Azathioprine and cyclophosphamide are the most commonly used cytotoxic drugs for immune suppression. Side effects include nausea and vomiting, hair loss, low numbers of erythrocytes, platelets, and leukocytes, bladder hemorrhage or tumors, and fetal damage or death. Cytotoxic drugs are usually given at high doses with a transplant to block acute rejection and then at lower doses with corticosteroids for maintenance.

The drugs which really made transplantation possible are cyclosporin A and tacrolimus. Cyclosporin A is derived from the Norwegian soil fungus Tolypodcladium inflatum. Tacrolimus (FK 506) comes from the Japanese filamentous bacterium Streptomyces tsukabaensis; rapamycin, a closely related drug also from Streptomyces is currently in clinical trials as an immunosuppressant. Cyclosporin A and tacrolimus cause non antigen-specific immunosuppression and are expensive and toxic to the kidneys and other organs. They are usually administered in high doses with a transplant to block acute rejection and then at lower doses for maintenance.

*Adapted from Janeway et al. Immunobiology, 5th edition, Garland Publishing Company, New York, 2001.

Anti-Lymphocyte Globulin (ALG) is one of the original immune suppressants and is still used in episodes of acute rejection. ALG is serum from a horse immunized with human lymphocytes; when injected into humans it kills lymphocytes. It must be given in large doses injected intramuscularly (which is very painful), it kills all lymphocytes (not just those rejecting the graft), and it causes serum sickness in recipients who make antibodies to the horse IgG. Nevertheless, it is very effective at blocking acute rejection.

Immunosuppressive monoclonal antibodies are being developed (see Designer Antibodies for more information). Monoclonal anti-CD3 is used now in humans to block graft rejection. Other antibodies being tested include anti-CD4, anti CD40L, and a fusion protein of CTLA-4 and the Fc region of human antibody.

Autoimmune disease treatment involves the same groups of drugs that are used for transplant patients, along with disease-specific remedial drugs such as insulin for diabetes. Antibodies to TNFa have induced remission in some people with rheumatoid arthritis. Antibodies to CD18 which block leukocyte binding to vascular endothelium have reduced inflammation in some animal models of autoimmune disease. Experimental approaches to developing monoclonal antibodies for treatment of autoimmunity have been targeted to making anti-idiotype against clonally restricted TCR or BCR of autoimmune effector cells or blocking MHC presentation of key peptides with antibodies or antagonist peptides.

Another strategy under investigation for treating autoimmunity is immune modulation, inducing changes in T cell cytokine secretion. It has already been demonstrated in EAE that induction of TGFb-producing cells blocks disease symptoms, presumably by interfering with effector Th1 cell functions. A more complete understanding of regulatory T cells is needed before these strategies are practical; however, they do avoid the requirement for identifying the specific antigen to which the response is being made.

Finally, the use of peptides to regulate T cell-mediated autoimmunity is being investigated. Oral administration of some peptides has been shown to prime Th2 cells that make IL-4 or TGFb to block Th1 responses or reduce the severity of established disease. Top date this treatment has had minimal success in humans with rheumatoid arthritis.

Practice Quiz

Pick the one BEST answer for each question by clicking on the letter of the correct choice.

1. John Doe needs a kidney transplant. The best MHC match for John would probably be

a. an unrelated cadaver.
b. his brother
c. his cousin.
d . his mother.
e. his wife.

2. The serological tissue typing test is

a. an ELISA for donor antibodies to recipient MHC antigens.
b. the best test to predict if the recipient will reject the donor tissue.
c. used to match Class I MHC for a heart transplant.
d. used to match Class I MHC for a kidney transplant.
e. used to match Minor H antigens between donor and recipient.

3. One of the ways by which allograft rejection is prevented is through administration of

a. antibodies to CD3.
b. antibody to the foreign MHC.
c. IL-2.
d. IgE.
e. Rhogam.

4. Hyperacute graft rejection

a. always occurs if HLA-B or HLA-DR alleles are mismatched.
b. can be controlled with Cyclosporin A and anti-CD3.
c. causes most transplantation failures.
d. is due to the presence of antibodies against graft surface antigens.
e. is due to T cell recognition of more than three differences in MHC alleles.

5. The best test to determine whether a kidney graft from a relative would be rejected would be

a. agglutination of donor kidney cells by anti-MHC antibodies from the recipient.
b. ⁵¹Cr release assay using donor target cells and recipient T cells.
c. ELISA of donor kidney molecules with enzyme-labeled recipient anti-kidney antibodies.
d. flow cytometry of donor cells using antibodies to recipient MHC alleles.
e. limit dilution assays of the frequency of recipient cells which will recognize the donor HLA.

6. Acute rejection occurs due to

a. activation of recipient B cells by donor MHC on recipient Th2 cells.
b. activation of recipient Tc cells by donor MHC on donor APC.
c. activation of recipient Tc cells by donor MHC on recipient APC.
d. activation of recipient Th1 cells by donor MHC on donor APC.
e. activation of recipient Th1 cells by donor minor H antigens on recipient APC.

7. All of the following statements about tissue matching are true EXCEPT

a. Many HLA antigens have not yet been identified.
b. Minor H antigens are often not encoded in the MHC.
c. The serological typing test can report a match when HLA antigens are different.
d. Tissue between HLA-identical siblings can sometimes be rejected.
e. Time for bone marrow typing is limited by the ability of the marrow cells to survive outside the donor.

8. Xenografts of pig organs into people has so far failed because

a. high numbers of human T cells recognize pig MHC as foreign.
b. humans are allergic to pig tissue.
c. humans have preexisting antibodies to pig carbohydrates.
d. humans have preexisting antibodies to pig MHC.
e. pig organs are too small for most humans.

9. Long-term allograft survival is affected by all of the following EXCEPT

a. cause of the original organ failure.
b. CMV infections.
c. number of HLA antigens that are matched.
d. pre-formed antibodies to alloantigens.
e. toxicity of the anti-rejection drugs.

10. Possible immunotherapeutic strategy for treating autoimmunity would NOT include administration of

a. adjuvants to tolerize APC.
b. antibodies to CD3.
c. azathioprine to block T cell division.
d. Cyclosporin A to block IL-2 production during T cell activation.
e. prednisone to reduce inflammation.

11. Bone marrow transplantation differs from organ transplantation because

a. activation of Tc cells by foreign HLA is the principal mechanism of acute rejection.
b. minor H antigens do not occur on marrow cells and do not contribute to rejection.
c. mature Tc cells in the graft can recognize and reject recipient tissue.
d. the donor is alive and can give permission for the transplant.
e. the recipient has no immune system and cannot reject the donor marrow cells..

12. The developing fetus is not rejected by the pregnant mother because

a. the fetus has no antigens that are foreign to the mother's immune system.
b. the mother cannot make antibodies to paternal HLA antigens.
c. the mother's T cells are never exposed to fetal antigens.
d. trophoblast cells express MHC identical to that of the mother.
e. trophoblast cells secrete immunosuppressive cytokines.

13. Side effects of immune suppression include all of the following EXCEPT

a. cancer.
b. hair loss.
c. HAMA response.
d. infections.
e. weight gain.

14. Prednisone is a(n)

a. anti-inflammatory drug.
b. cholesterol drug.
c. cytotoxic drug.
d. drug that blocks T cell activation.
e. steroid inhibitor.

15. It is easier to treat transplant rejection than to treat autoimmunity because

a. HLA antigens are better characterized than autoantigens.
b. mechanisms for graft rejection are better understood than those for autoimmunity.
c. more drugs are available for controlling rejection than for controlling autoimmunity.
d. once an immune response is established it is harder to block.
e. the foreign antigens can be removed with a transplant but not with a self antigen.

Problem

How would monoclonal anti-CD4, anti CD40L, and a fusion protein of CTLA-4 and the Fc region of human antibody inhibit graft rejection? What specific effector cells would they target? Would the inhibition be less likely to increase infection risk than current therapy?

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