Chapter 62: Major Histocompatibility Complex & Transplantation

The success of tissue and organ transplants depends on the donor’s and recipient’s major histocompatibility complex (MHC) proteins, which present antigens to T cells. The MHC proteins are alloantigens (i.e., they differ among members of the same species). In humans, these proteins are encoded by the human leukocyte antigen (HLA) genes, clustered on chromosome 6. (Note that we will use MHC and HLA interchangeably.) Three of these genes (HLA-A, HLA-B, and HLA-C) code for the class I MHC proteins. Several HLA-D loci determine the class II MHC proteins (i.e., DP, DQ, and DR) (Figure 62–1). The features of class I and class II MHC proteins are compared in Table 62–1. If the HLA proteins on the donor’s cells differ from those on the recipient’s cells, then an immune response occurs in the recipient.

Each person has two haplotypes (i.e., two sets of these genes—one on the paternal and the other on the maternal chromosome 6). These genes are very diverse (polymorphic) (i.e., there are many alleles of the class I and class II genes). For example, as of 2019, there are at least 4,300 HLA-A alleles, 5,200 HLA-B alleles, 3,900 HLA-C alleles, and more than 5,000 HLA-D alleles, and more are being discovered. However, an individual inherits only a single allele at each locus from each parent. Expression of these genes is codominant (i.e., the proteins encoded by both the paternal and maternal genes are produced).

The class I MHC proteins consist of one HLA-encoded polypeptide (an “alpha” chain), so each individual can have one set encoded by paternal genes and one set encoded by maternal genes. However, the class II MHC proteins consist of two HLA-encoded polypeptides (an “alpha” chain and a “beta” chain), so each individual can have as many as four of each class II MHC proteins because the maternal- and paternal-encoded peptides can mix and match.

HLA gene polymorphism causes us each to have somewhat different proteins on the surface of our cells. Each HLA protein can bind certain peptides better than others, which means that, if an infectious pandemic swept across our species, we are ensured to have some individuals who would be more likely to survive! But these protein differences can be recognized as non-self when introduced to another person’s immune system. The MHC is called major because these HLA differences are often responsible for acute rejection of a transplant, and these are the genes most crucial for matching donors and recipients.

In addition to the major antigens encoded by the HLA genes, there is a large number of minor antigens encoded by genes at sites other than the HLA locus. These minor antigens are various normal body proteins that have one or more amino acid differences from one person to another (i.e., they are “allelic variants”). Like the HLA proteins, these proteins therefore have amino acid differences and can be recognized as non-self by another person’s immune system. But minor antigens induce a weak immune response, resulting in slow rejection of a transplant, or the cumulative effect of several minor antigens can lead to a more rapid rejection. Predicting rejection on the basis of minor antigens is difficult, so donors and recipients are not routinely tested for specific minor histocompatibility antigens. In view of these differences in minor antigens, all recipients routinely receive immunosuppressive drugs, even if their major histocompatibility loci are well-matched.

Class I MHC Proteins

These are found on the surface of virtually all nucleated cells. The complete class I protein is composed of a 45,000-molecular-weight heavy chain (called the alpha chain) noncovalently bound to a β₂-microglobulin. The alpha heavy chain is highly polymorphic and is similar to an immunoglobulin molecule; it has hypervariable regions that bind and present short peptides to T cells. The polymorphism of these molecules is important in the recognition of self and nonself. Stated another way, if these molecules were more similar, our ability to accept foreign grafts would be correspondingly improved. The heavy chain also has a constant region where the CD8 protein of the cytotoxic T cell binds.

Class II MHC Proteins

These are glycoproteins found only on the surface of professional antigen-presenting cells (APCs), such as dendritic cells, macrophages, and B cells (see Chapter 60). They are highly polymorphic glycoproteins composed of two chains (called alpha and beta) that are noncovalently bound. Like class I proteins, the class II proteins have hypervariable regions that present the short peptides to T cells and provide much of the polymorphism. Unlike class I proteins, which have only one chain encoded by the MHC locus (pairing with β₂-microglobulin), both the alpha and beta chains of the class II proteins are encoded by the MHC locus. The two peptides also have a constant region where the CD4 proteins of the helper T cells bind.

The ability of T cells to recognize antigen is dependent on association of the antigen with either class I or class II proteins (see Chapter 60). For example, CD8-positive cytotoxic T cells only respond to antigen in association with class I MHC proteins. Thus, a cytotoxic T cell that is activated to kill a virus-infected cell will not kill a cell infected with the same virus if the cell does not also express the appropriate class I proteins. (This was determined by mixing cytotoxic T cells from individual “A,” bearing one set of class I MHC proteins, with virus-infected cells bearing a set of class I MHC proteins from individual “B.” Because of the class I MHC mismatch, no killing of the virus-infected “B” cells occurred.) This peptide–MHC requirement also holds for an individual’s CD4-positive T helper cell with respect to that individual’s class II proteins. The requirement that antigen recognition occurs in association with a “self” MHC protein is called MHC restriction and is a result of positive thymic selection (see Chapter 59).

As noted above, the success of organ transplants is, in large part, determined by the compatibility of the MHC genes of the donor and recipient (see later). Another context where MHC genes and proteins are important is in autoimmune diseases, many of which occur frequently in people who carry certain MHC genes (see Chapter 66).

The likelihood that a transplanted organ, or graft, is accepted by the recipient’s immune system depends on the genetic similarity between the recipient and the donor. On one end of the spectrum, an autograft (transfer of an individual’s own tissue to another site in the body) is always permanently accepted (i.e., it always “takes”). A syngeneic graft is a transfer of tissue between genetically identical individuals (i.e., identical twins) and almost always “takes.” On the other end of the spectrum, a xenograft, a transfer of tissue between different species, is the least likely to succeed except under certain unusual circumstances.

An allograft is a graft between genetically different members of the same species (e.g., from one human to another). Allografts are usually rejected unless the recipient is given immunosuppressive drugs. The severity and rapidity of the rejection will vary depending on the degree of difference between the donor and the recipient at the MHC loci.

Solid Organ Allograft Rejection

Even with perfect HLA matching, the presence of minor antigens means that immunosuppression is required after a transplant to prevent allograft rejection. As HLA mismatching increases, more immunosuppression is needed. In acute allograft rejection, vascularization of the graft is normal initially, but, in 11 to 14 days, marked reduction in circulation and mononuclear cell infiltration occurs, with eventual necrosis. This is also called a primary (first-set) reaction. A T-cell–mediated reaction is the main cause of acute rejection of many types of grafts, but antibodies contribute to the rejection of certain transplants. (In experimental animals, rejection of most types of grafts can be transferred by cells, not serum. In addition, T-cell–deficient animals do not reject grafts, but B-cell–deficient animals do.)

If a second allograft from the same donor is applied to a sensitized recipient, it is rejected in 5 to 6 days. This accelerated (second-set) reaction is caused primarily by presensitized cytotoxic T cells.

A graft that survives an acute allograft reaction can nevertheless undergo chronic rejection. This causes gradual loss of graft function and can occur months to years after engraftment. The main pathologic finding in grafts undergoing chronic rejection is atherosclerosis of the vascular endothelium. The immunologic stimulus that causes chronic rejection is complex and multifactorial and can occur even in HLA-matched donor–recipient pairs due to the presence of minor histocompatibility antigens. The adverse effects of long-term use of immunosuppressive drugs may also play a role in chronic rejection. Chronic rejection generally does not respond to treatment, and it carries a poor prognosis.

In addition to acute and chronic rejection, a third type called hyperacute rejection can occur. Hyperacute rejection typically occurs within minutes of a solid organ transplant graft and is due to the reaction of preformed anti-ABO antibodies in the recipient with ABO antigens on the surface of the endothelium of the graft. In this severe rejection reaction, the ABO blood group of donors and recipients must be matched, and a cross-matching test (see later) must be done. The laboratory tests used to determine ABO blood groups are described in Chapter 64.

Depending on the type of graft and the type of rejection, mismatching of the HLA-A, HLA-B, and HLA-DR alleles is the most predictive of solid organ transplant rejection. The donor alloantigens encoded by these alleles lead to activation of antigen-specific recipient helper and cytotoxic T cells. The strength of the response to foreign MHC proteins can be explained by the observation that there are two immune pathways by which the recipient’s immune response is stimulated (Figure 62–2).

These pathways are summarized as follows and are differentiated by whether the sensitizing APC is donor-derived or recipient-derived:

1. In the direct pathway of allograft recognition, there must be donor–recipient HLA mismatch. In this pathway, the donor’s APCs contained within the grafted organ migrate to a nearby secondary lymphoid tissue and present peptides in association with their class I and class II MHC proteins. The mere presence of the donor HLA protein that is presenting the peptide is enough to make the peptide–MHC complex appear to be nonself to the recipient’s T cells, regardless of the peptide. Unlike the conventional activation of T cells by cognate peptides complexed with MHC (see Chapter 60), “direct” recognition of these nonself HLA proteins triggers a polyclonal activation of a much larger percentage of recipient T-cell clones, by some estimates up to 10% of the recipient T cells. This is likely because the diversity of peptides complexed to the donor MHC proteins can trigger a similarly diverse array of T-cell clones.

   If the nonself HLA proteins are class I, they will activate CD8-positive T cells to become cytotoxic T lymphocytes (CTLs), which infiltrate the graft and kill the graft cells because they express the same class I proteins. “Direct” recognition of class II HLA proteins can also trigger activation of the recipient’s CD4-positive T cells, which can provide the cytokine “help” that enhances CD8-positive T-cell activation, as described in Chapter 60.

2. In the indirect pathway of allograft recognition, the recipient’s APCs present the donor’s proteins. The donor’s proteins that are shed by damaged cells of the graft are taken up by recipient dendritic cells, processed, and presented to T cells as “foreign” proteins in a draining lymph node. (If there is HLA mismatch, the donor HLA proteins are often the antigens responsible for this pathway because HLA proteins are highly polymorphic and immunogenic. But even without HLA mismatch, there are minor antigens that can bring about the indirect pathway of rejection.) This results in activation of CD4-positive helper T cells (see Chapter 60). The newly activated T helper cells can (a) migrate back to the graft and activate macrophages and (b) recruit neutrophils, or (c) migrate to the B-cell follicle and induce antibodies against the graft cells. Note that if the donor and recipient are not matched at the class I HLA loci, then the indirect pathway cannot involve cytotoxic T cells. Recipient cytotoxic T cells activated through the indirect pathway will only be able to recognize peptides presented on “self” HLA proteins, whereas those in the graft will be “nonself.”

Compared with the direct pathway, the indirect pathway takes longer because the recipient dendritic cells have to enter the graft, take up nonself proteins, and migrate to the draining lymph node to activate the adaptive immune response. Also, as time passes, the graft’s donor APCs are gradually replaced by recipient APCs, and the risk of direct recognition being a mechanism for rejection declines. Therefore, whereas both the direct and indirect pathways can contribute to acute rejection, the indirect pathway is primarily responsible for chronic rejection.

In all rejection scenarios, the activation of T cells must be accompanied by inflammatory stimuli, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These are necessary to induce the co-stimulatory signals, such as B7 molecules, on the APCs in order to fully activate the T cells (see Chapter 60). This is clinically relevant because limiting inflammation and tissue damage at the time of transplantation significantly limits the likelihood of graft rejection and improves outcomes. This explains why, given a choice, surgeons prefer to transplant organs from live donors whenever possible.

Hematopoietic Stem Cell Transplants

Malignancies of the hematologic system, particularly leukemia, are often treated with transplant of hematopoietic stem cells. The principle of this approach is to use aggressive chemotherapy to ablate all of the patient’s hematopoietic cells, which includes most of the malignant cells, and then replace them with healthy stem cells that can repopulate the hematopoietic system (see Figure 58–1). In the past, these transplants were called “bone marrow transplants” because the stem cells were isolated from bone marrow aspirates, but now the stem cells can also be derived from leukopheresis of peripheral blood or from a sample of banked umbilical cord blood.

Unlike most solid organ transplants, transplanted hematopoietic stem cells can be autologous (from the patient’s own stem cell pool) or allogeneic (from a donor). Autologous cell transplants are safer and avoid the need to find a matched donor, so you might think this would be the preferred approach in all cases. However, the main advantage to using allogeneic cells is that once these cells engraft, the T cells will actually attack any surviving malignant cells. This graft-versus-malignancy effect can occur with HLA-matched or -unmatched stem cells because of the minor antigens recognized by the donor cells. Without this effect, autologous transplants have higher relapse rates.

Graft-Versus-Host Reaction

As described earlier, one of the major advantages of allogeneic stem cell transplants is that the transplanted cells attack the malignant cells, even in well-matched donor–recipient pairs. An unfortunate adverse effect in these transplants is that transplanted cells may subsequently attack host cells. This graft-versus-host (GVH) reaction develops in 30% to 70% of recipients, depending on the type of donor cells and other factors.

This reaction occurs because grafted immunocompetent T cells proliferate in the immunocompromised host and reject host cells with “foreign” proteins, resulting in severe organ dysfunction. The donor’s cytotoxic T cells play a major role in destroying the recipient’s cells. These reactions occur primarily in “barrier tissues,” such as the skin and gastrointestinal system, causing severe rash, oral ulcers, diarrhea, and hepatitis. Many GVH reactions end in overwhelming infections and death.

There are three requirements for a GVH reaction to occur: (1) the graft must contain immunocompetent T cells, (2) the recipient must be immunocompromised, and (3) the recipient must express antigens (e.g., MHC proteins) foreign to the donor (i.e., the donor T cells recognize the recipient cells as foreign). Note that, like the graft-versus-malignancy effect, even when donor and recipient have identical class I and class II MHC proteins, a GVH reaction can occur because of differences in minor antigens. Risk of GVH reactions can be reduced by depleting the donor cell pool of T cells before the transplant, but this also reduces the graft-versus-malignancy effect and therefore increases relapse rates.

Once it occurs, GVH is treated with immunosuppressive agents (see below). Although these drugs suppress the allograft reaction, most patients must take these drugs for their entire lives. Immunosuppression increases the risk of disease relapse (by limiting graft-versus-malignancy effect) and also increases the risk of developing other malignancies as well as opportunistic infections.

HLA Typing in the Laboratory

Prior to transplantation, laboratory tests, commonly called HLA typing or tissue typing, are performed to determine the closest MHC match between the donor and the recipient. The most important alleles to match are HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ, and a donor–recipient pair in which all 10 of the maternal and paternal alleles of these five genes match is called a “10/10 match.” In the past, serologic assays were used to determine the Class 1 and Class II MHC proteins of the donor and recipient. However, serologic assays have now been largely replaced by DNA sequencing using polymerase chain reaction (PCR) amplification. DNA sequencing determines the different alleles carried by donors and recipients with great accuracy down to the molecular level.

In addition to the tests used for matching, preformed cytotoxic antibodies in the recipient’s serum reactive against the graft are detected by observing the lysis of donor lymphocytes by the recipient’s serum. This is called cross-matching and is done to prevent hyperacute rejections from occurring. In solid organ transplants, the donor and recipient are also matched for the compatibility of their ABO blood groups. The laboratory tests used to determine ABO blood groups are described in Chapter 64.

Among siblings in a single family, there is a 25% chance for both haplotypes to be shared, a 50% chance for one haplotype to be shared, and a 25% chance for no haplotypes to be shared. For example, if the father is haplotype A/B, the mother is CD, and the recipient child is AC, there is a 25% chance for a sibling to be AC (i.e., a two-haplotype match), a 50% chance for a sibling to be either BC or AD (i.e., a one-haplotype match), and a 25% chance for a sibling to be BD (i.e., a zero-haplotype match).

The Fetus Is an Allograft that Is Not Rejected

A fetus has MHC genes inherited from the father that are foreign to the mother, yet allograft rejection of the fetus does not occur. This is true despite many pregnancies from the same mother–father combination that produce offspring with the same MHC haplotypes. The reason that the mother fails to reject the fetus is unclear. The mother forms antibodies against the foreign paternal MHC proteins; therefore, the reason is not that the mother is not exposed to fetal antigens. Some possible explanations are (1) that the placenta does not allow maternal T cells to enter the fetus and (2) the maternal T cells within the placenta are biased toward a T-regulatory subset, which promotes tolerance of fetal antigens (see Chapter 60).

To reduce the rejection of transplanted cells or to treat GVH disease, immunosuppressive measures are generally required (Table 62–2 and Figure 62–3). These fall under the categories of corticosteroids (prednisone), DNA synthesis inhibitors (azathioprine, methotrexate, mycophenolate), calcineurin inhibitors (cyclosporine and tacrolimus), mammalian target of rapamycin (mTOR) inhibitors (sirolimus), signaling blockade (belatacept, basiliximab, etc.), and cell-depleting antibodies (antithymocyte globulin [ATG]).

Corticosteroids bind to glucocorticoid receptors that result in altered gene transcription in a variety of cell types. In immune cells, steroids act by inhibiting synthesis of leukotrienes, prostaglandins, and cytokines (e.g., IL-2) and by inducing apoptosis of rapidly dividing T cells. Corticosteroids inhibit cytokine production by blocking transcription factors, such as nuclear factor-κB and AP-1, which prevents the mRNA for these cytokines from being synthesized. Glucocorticoid receptors are found in nearly every cell in the body, causing widespread off-target changes in gene transcription. Therefore, the major disadvantage of corticosteroids that limits their chronic use is that they have numerous adverse endocrine, neuropsychiatric, metabolic, and cardiovascular side effects.

Azathioprine (which is converted to 6-mercaptopurine in the body), methotrexate, and mycophenolate mofetil inhibit different aspects of the nucleotide synthesis and metabolism, shutting down DNA synthesis and thereby blocking T-cell proliferation. Methotrexate may also have a variety of other effects on cell signaling through inhibition of signaling enzymes.

Cyclosporine prevents the activation of T lymphocytes by inhibiting the synthesis of interleukin (IL)-2 and IL-2 receptor. It does so by inhibiting calcineurin—a phosphatase enzyme that is activated by calcium flux following binding of the T-cell receptor, the first step in the cascade that ultimately leads to transcription of the genes encoding IL-2 and the IL-2 receptor. Tacrolimus binds a different protein (FKBP1A) but has a similar effect on calcineurin. Thus, cyclosporine and tacrolimus inhibit one of the earliest steps in the first phase of T-cell activation.

Sirolimus inhibits signal transduction through mTOR, which is primarily involved in signal transduction downstream of IL-2. Therefore, sirolimus inhibits later steps in the second phase of T-cell activation, in a pathway different from that of cyclosporine and tacrolimus. These drugs are more selective than steroids and therefore have fewer toxicities.

A variety of recombinant proteins, small molecules, and monoclonal antibodies can be used in immunosuppressive regimens, both to prevent rejection and to treat rejection episodes. Belatacept is a fusion protein consisting of cytotoxic T lymphocyte antigen-4 (CTLA-4) fused to the Fc fragment of human IgG. CTLA-4 competes with CD28 for binding to B7 proteins, but does so with higher affinity, thereby blocking co-stimulation of T cells and preventing graft rejection. Muromonab (OKT3) was the first approved monoclonal antibody. It a mouse antibody against CD3 that blocks signal transduction through the T-cell receptor. Basiliximab is a chimeric monoclonal antibody that blocks the IL-2 receptor, preventing T-cell proliferation.

Antithymocyte globulin (ATG) is a polyclonal cocktail of horse (Atgam) or rabbit (Thymoglobulin) antibodies against human thymocytes. ATG contains antibodies against many lymphocyte antigens (e.g., CD3, CD4, CD8, and others). After binding to their targets on the surfaces of T cells, these antibodies lead to cell death through complement-mediated lysis of the cell (among other potential mechanisms). As a consequence, ATG has a broader immunosuppressive effect than do the more targeted monoclonal antibodies described in the previous paragraph.

Unfortunately, immunosuppression greatly enhances the recipient’s susceptibility to opportunistic infections and neoplasms. For example, some patients undergoing treatment for multiple sclerosis with the monoclonal antibody natalizumab developed progressive multifocal leukoencephalopathy (see Chapter 44 for a description of this viral disease). The incidence of cancer is increased as much as 100-fold in transplant recipients who have been immunosuppressed for a long time. Common cancers in these patients include squamous cell carcinoma of the skin, adenocarcinoma of the colon and the lung, and lymphoma.