Immune responses that mediate allograft rejection are funda mentally similar to responses that fight microbes. The main difference is the nature of what is recognized as foreign. Adaptive immune responses play the major role in graft rejection, but innate responses may contribute in some situations. In this section, we focus on adaptive immune responses to alloantigens and mention the possible role of innate immunity later.
Alloantigens elicit both cellular and humoral immune responses. The mechanisms of allorecognition are best under stood by considering the graft antigens that stimulate allogeneic responses and the properties of the responding lymphocytes.
The Nature of Alloantigens
Most of the antigens that stimulate adaptive immune responses against allografts are proteins encoded by polymorphic genes that differ among individuals. These proteins are called histocompatibility molecules because they determine if the grafted tissue (histo, tissue) is compatible or incompatible with the host’s immune system. The most important of these molecules are the major histocompatibility complex (MHC) proteins. All the individuals of an inbred strain of animals, such as mice, are genetically identical, and they are homozygous for all genes, including MHC genes (except genes on the sex chromosomes in males). In contrast, inbred animals of different strains, and individuals in an outbred species (except identical twins), differ in many of the genes they inherit, including MHC genes. The basic rules of transplantation immunology were first established from experiments done with genetically defined inbred mice and from observations made during early attempts at clinical transplantation. These studies showed the following (Fig. 1):
• Cells or organs transplanted between genetically identical individuals (identical twins or members of the same inbred strain of animals) are not rejected.
• Cells or organs transplanted between genetically nonidentical people or members of two different inbred strains of a species are almost always rejected.
• The offspring of a mating between two different inbred strains of animal will not reject solid tissue or organ grafts from either parent. In other words, an (A × B) F1 animal will not reject grafts from an A or B strain animal.
• A graft derived from the offspring of a mating between two different inbred strains of animal will be rejected by either parent. In other words, a graft from an (A × B) F1 animal will be rejected by either an A or a B strain animal.
Fig1. The genetics of graft rejection. In the illustration, the two different mouse colors represent inbred strains with different sets of genes, referred to here as A or B, encoding molecules important for graft rejection, called major histocompatibility complex (MHC) molecules. Inherited MHC alleles from both parents are codominantly expressed in the skin of an A × B offspring, and therefore these mice are represented by both colors. Syngeneic grafts are not rejected (A). Allografts are always rejected (B). Grafts from an A or a B parent will not be rejected by an (A × B) F1 offspring (C), but grafts from the F1 will be rejected by either parent (D). These phenomena are due to the fact that MHC gene products are responsible for graft rejection; grafts are rejected only if they express an MHC type (represented by green or orange) that is not expressed by the recipient mouse.
Such results indicated that the molecules in the grafts that are responsible for eliciting rejection must be polymorphic and their expression is codominant. Polymorphic refers to the property that these graft antigens differ among the individuals of a species (other than identical twins) or between different inbred strains of animals. Codominant expression means that every individual inherits genes encoding these molecules from both parents, and both parental alleles are expressed. Therefore, (A × B) F1 animals express both A and B alleles, see both A and B tissues as self, and are tolerant to both A and B proteins. By contrast, inbred A or B animals express only the A or B allele, are not tolerant to the proteins they do not express and see (A × B) F1 tissues as partly foreign. Thus, an (A × B) F1 animal does not reject either A or B strain grafts, whereas both A and B strain recipients reject an (A × B) F1 graft.
The genetics of graft rejection provided one of the earliest lines of evidence that the adaptive immune system distinguishes self from foreign antigens. All individuals are normally tolerant to self antigens and do not react against these, but they do react against foreign antigens. This is now known to be a funda mental property of the normal immune system, where foreign most often refers to microbial antigens. In the context of organ transplantation, foreign usually means products of polymorphic MHC genes that differ from the host's genes.
The molecules responsible for strong and rapid rejection reactions are MHC molecules whose physiologic function is to bind and present peptides to T cells. MHC molecules, described in Chapter 6, were named before their normal function was understood. George Snell and colleagues produced pairs of congenic strains of inbred mice that were bred to be genetically identical to each other, except for genes needed for graft rejection. They used these mice to identify the polymorphic genes, which were called MHC genes, that encode the molecular targets of allograft rejection. Transplants of most tis sues between any pair of unrelated individuals will be rejected because the genes encoding MHC molecules are so polymorphic that two unrelated individuals are extremely unlikely to inherit the same alleles. The role of MHC molecules as the antigens that cause graft rejection is a consequence of the nature of T-cell antigen recognition, as we will discuss later. Recall that human MHC molecules are called human leukocyte antigens (HLAs), and in the context of human transplantation, the terms MHC and HLA are used interchangeably.
In any transplant between genetically nonidentical donor and recipient, there will be polymorphic antigens other than MHC molecules against which the recipient may mount an immune response. These antigens typically induce weak or slower (more gradual) rejection reactions than do MHC molecules and are therefore called minor histocompatibility antigens. The relevance of minor histocompatibility antigens in clinical solid-organ transplantation is uncertain, mainly because there has been little success in identifying the relevant antigens. In mice, the male H-Y antigen appears to be a target of immune recognition by female recipients of grafts from male donors. Although in humans there is a slightly higher risk for rejection of heart transplants from male donor to female recipient, compared with gender-matched transplants, given the scarcity of donor hearts, gender matching is not practical. Minor histocompatibility antigens play a more significant role in stimulating graft-versus-host responses after HSC transplantation, discussed later, but the nature of the relevant antigens in that setting is also not defined.
Recognition of Alloantigens by T Cells
Allogeneic MHC molecules of a graft can be presented for rec ognition by the recipient’s T cells in two different ways, called direct and indirect (Fig. 2). Initial studies showed that the T cells of a graft recipient recognize intact, unprocessed MHC molecules in the graft, and this is called direct presentation (or direct recognition) of alloantigens. Subsequent studies showed that sometimes the recipient T cells recognize graft (donor) MHC molecules only in the context of the recipient’s MHC molecules, implying that the recipient’s MHC molecules must be presenting peptides derived from allogeneic donor MHC proteins to recipient T cells. This process is called indirect presentation (or indirect recognition), and it is essentially the same as the recognition of any foreign (e.g., microbial) protein anti gen. The initial T-cell response to MHC alloantigens, whether it results from direct or indirect recognition, most likely occurs in lymph nodes draining the graft, as we will discuss later.
Fig2. Direct and indirect alloantigen recognition. (A) Direct alloantigen recognition occurs when alloreactive T cells bind directly to an intact allogeneic major histocompatibility complex (MHC) molecule with bound peptide on a graft (donor) dendritic cell (DC) or other antigen-presenting cell (APC) within the lymph nodes. Recipient CD4+ or CD8+ T cells can directly recognize donor MHC-II or MHC-I molecules, respectively, and will differentiate into helper T cells or cytotoxic T lymphocytes (CTLs). The CTLs will directly recognize the same donor MHC-peptide complex displayed on graft tissue cells and kill these cells. (B) Indirect alloantigen recognition occurs when allogeneic MHC molecules from graft cells are taken up and processed by recipient APCs and peptide fragments of the allogeneic MHC molecules containing polymorphic amino acid residues are bound and presented by recipient (self) MHC molecules. Donor MHC–specific helper T cells that are generated in this way can help B cells to produce donor MHC–specific antibodies that can damage graft cells. The helper T cells also can be activated in the graft by recipient macrophages presenting the same donor MHC–derived peptides, leading to inflammatory damage to the graft.
Direct Recognition of Major Histocompatibility Complex Alloantigens on Donor Cells
In the case of direct recognition, intact MHC molecules dis played by cells in the graft are recognized by recipient T cells without a need for processing by host antigen-presenting cells (APCs) (see Fig. 2A). It may seem puzzling that T cells, which are normally selected during their maturation to be self MHC–restricted, are capable of recognizing foreign (allogeneic or xenogeneic) MHC molecules. A likely explanation is that T-cell receptors (TCRs) have some intrinsic affinity for MHC molecules, regardless of whether they are self or foreign. Furthermore, during T-cell development in the thymus, positive selection promotes survival of T cells with weak self MHC reactivity, and among these T cells, there may be many with strong reactivity to allogeneic MHC molecules. Negative selection in the thymus efficiently eliminates T cells with high affinity for self MHC, but it will not necessarily eliminate T cells that bind strongly to allogeneic MHC molecules, simply because allogenic MHC molecules are not present in the thymus. The result is that the mature repertoire includes many T cells that bind allogeneic MHC molecules with high affinity. One can think of direct allorecognition as an example of an immunologic cross-reaction in which a T cell that was selected to be self MHC–restricted is able to bind structurally similar allogeneic MHC molecules with sufficiently high affinity to permit activation of the T cell (Fig. 3).
Fig3. Molecular basis of direct recognition of allogeneic major histocompatibility complex (MHC) molecules. Direct recognition of allogeneic MHC molecules may be thought of as a cross-reaction in which a T cell specific for a self MHC molecule–foreign peptide complex (A) also recognizes an allogeneic MHC molecule (B and C). Donor or self (recipient) peptides that bind to MHC molecules in the graft may contribute to allorecognition (B) or they may not (C).
MHC molecules that are expressed on cell surfaces normally contain bound peptides, and in some cases the peptide contributes to the structure recognized by the alloreactive T cell, exactly like the role of peptides in the normal recognition of foreign anti gens by self MHC–restricted T cells (see Fig. 3B). Even though these peptides may be derived from proteins that are present in both donor and recipient, on the graft cells they are displayed by allogeneic MHC molecules. Therefore, the complexes of peptides (self or foreign) with allogeneic MHC molecules will appear different from self peptide–self MHC complexes. In other cases, direct recognition and activation of an alloreactive T cell may occur regardless of which peptide is carried by the allogeneic MHC molecule, because the polymorphic amino acid residues of the allogeneic MHC molecule alone form a structure that resembles self MHC plus peptide (see Fig. 3C).
T-cell responses to directly presented allogeneic MHC molecules are very strong because there is a high frequency of T cells that can directly recognize any single allogeneic MHC protein. It is estimated that as many as 1% to 10% of all T cells in an individual can directly recognize and react against an allogeneic MHC molecule on a donor cell. In striking contrast, in an infection, the frequency of naive T cells that react against any microbial peptide displayed by self MHC molecules is approximately 1 in 105 or 106 T cells. There are several explanations for the high frequency of alloreactive T cells.
• Many different peptides derived from donor cellular proteins may combine with a single allogeneic MHC molecule, and each of these peptide-MHC combinations can theoretically activate a different clone of recipient T cells. In contrast, most microbes or protein antigens contain relatively few immunodominant peptides that can be displayed by the self MHC molecules of an individual at any time, so few T-cell clones are activated.
• It is estimated that each APC displays thousands of MHC molecules on its surface. On an allogenic APC, most of these can be recognized by alloreactive T cells at one time. However, in the case of an infection, less than 1% (and perhaps as few as 0.1%) of the self MHC molecules on an APC normally present peptides from that microbe at one time, and only these can be recognized by T cells specific for the microbial antigen.
• Allogeneic MHC molecules can display not only foreign peptides from donor cells but also self peptides, and these self peptide–foreign MHC complexes can activate T cells. Because these complexes are not normally expressed in the thymus or peripheral tissues, they have not participated in negative selection of T cells that could recognize allogeneic graft cells and potentially cause graft damage. In contrast, T cells specific for self peptides displayed by self MHC molecules are eliminated by negative selection in the thymus and by peripheral tolerance mechanisms.
• Many of the T cells that respond to an allogeneic MHC molecule, even on first exposure, are memory T cells. It is likely that these memory cells were generated during previous exposure to other foreign (e.g., microbial) anti gens and cross-react with allogeneic MHC molecules. These memory cells not only are expanded populations of antigen-specific cells but also are more rapid and powerful responders than are naive lymphocytes. Thus, these alloreactive memory T cells contribute to the greater strength of the initial T-cell response to a new allograft, compared to the response to the first encounter with a microbial antigen.
Direct allorecognition can generate both CD4+ and CD8+ T cells that recognize graft antigens and contribute to rejection. The role of the alloreactive T-cell response in rejection is described later.
Indirect Recognition of Alloantigens
In the indirect pathway, donor (allogeneic) MHC molecules are captured and processed by recipient APCs, and peptides derived from the allogeneic MHC molecules are presented in association with self MHC molecules (see Fig. 1B). Thus, peptides from the allogeneic MHC molecules are displayed by host APCs and recognized by T cells like conventional foreign (e.g., microbial) protein antigens. Because allogeneic MHC molecules have amino acid sequences different from those of the host, they themselves can serve as foreign antigens and generate foreign peptides associated with self MHC molecules on the surface of host APCs. Each allogeneic MHC molecule may give rise to multiple peptides that are foreign for the host, each recognized by a different clone of T cells. Indirect presentation may result in allorecognition by CD4+ T cells because alloantigens are acquired by host APCs primarily through the endosomal vesicular pathway (i.e., as a consequence of phagocytosis) and are therefore presented by MHC class II (MHC-II) molecules. Some antigens of phagocytosed graft cells do enter the MHC class I (MHC-I) pathway of antigen presentation and are indirectly recognized by CD8+ T cells. This phenomenon is an example of cross-presentation or cross-priming (see Fig. 6.14), in which dendritic cells (DCs) ingest proteins of another cell such as from the graft; the proteins are delivered to the cytosol, where they are processed into peptides by proteasomes; and the peptides are presented on MHC-I molecules to activate (prime) CD8+ T lymphocytes. However, as discussed later, CD8+ T cells activated by the indirect pathway do not recognize intact MHC molecules on graft cells and cannot directly kill these cells.
Evidence that indirect recognition of allogeneic MHC molecules plays a significant role in graft rejection comes from studies with knockout mice lacking MHC-II expression. For example, skin grafts from donor mice lacking MHC-II are able to induce recipient CD4+ (i.e., MHC-II–restricted) T-cell responses to peptides derived from donor MHC-I molecules. In these experiments, the donor MHC-I molecules are processed and presented by self MHC-II molecules on the recipient’s APCs and stimulate the recipient’s helper T cells. Evidence has also been obtained that indirect antigen presentation may contribute to chronic rejection of human allografts. CD4+ T cells from heart and liver allograft recipients recognize and are activated by peptides derived from donor MHC when presented by the patient’s own APCs. The indirect pathway may become more important over time after transplantation, as donor DCs in the graft are replaced by host DCs. Additional evidence for a role of indirect presentation of MHC molecules in graft rejection is the fact that T-dependent high-affinity anti body responses specific for donor MHC-II molecules contribute to many cases of renal allograft rejection. As discussed later, this indicates that recipient B cells specific for unprocessed epi topes on donor MHC-II molecules indirectly present processed peptides from the same donor MHC-II molecules to recipient helper T cells specific for those peptides. This would be necessary for the germinal center reaction and affinity maturation of the alloantibody response.
Activation and Effector Functions of Alloreactive T Lymphocytes
When lymphocytes recognize alloantigens, they become activated to proliferate, differentiate, and perform effector functions that can damage grafts. The activation steps are similar to those we have described for lymphocytes reacting to microbial antigens.
Activation of Alloreactive T Lymphocytes
The T-cell response to an organ graft may be initiated in the lymph nodes that drain the graft (Fig. 4). Most organs contain resident APCs, such as DCs, and therefore transplanted organs carry with them APCs that express donor MHC molecules. These donor APCs can migrate to regional lymph nodes and present unprocessed allogeneic MHC-I or MHC-II molecules to the recipient’s CD8+ or CD4+ T cells, respectively (direct MHC allorecognition). Host DCs from the recipient may also migrate into the graft, pick up graft alloantigens, transport these back to the draining lymph nodes, and process them into peptides that are displayed bound to recipient MHC molecules (the indirect pathway). The lymphatic vessels in allografts are surgically disrupted during the process of transplantation, and it is likely that lymphatic drainage to recipient lymph nodes is reestablished by growth of new lymphatic channels in response to inflammatory stimuli generated during grafting. Naive CD4+ and CD8+ lymphocytes that normally traffic through the lymph node encounter these alloantigens and are induced to proliferate and differentiate into effector helper T cells and cytotoxic T lymphocytes (CTLs). This process is sometimes called sensitization to alloantigens. The effector cells migrate back into the graft and mediate rejection by mechanisms that are discussed later.
Fig4. Activation of alloreactive T cells. (A) In the case of direct allorecognition, donor dendritic cells in the allograft migrate to secondary lymphoid tissues, where they directly present allogeneic major histocompatibility complex (MHC) molecules to host T cells. Only CD8+ T cells recognizing donor MHC-I is shown, but CD4+ T cells can also directly recognize donor MHC-II. In the case of indirect allorecognition, recipient dendritic cells that have entered the allograft transport donor MHC proteins to secondary lymphoid tissues and present peptides derived from these MHC proteins to alloreactive host T cells. This is shown for CD4+ T cells, and indirect recognition of allogeneic MHC by CD8+ T cells is likely less important. After both indirect and direct allorecognition, the T cells become activated and differentiate into effector CD4+ helper T cells and CD8+ cytotoxic T lymphocytes (CTLs). (B) The alloreactive effector T cells migrate into the allograft, become reactivated by alloantigen, and mediate damage. In the graft, direct recognition of allogeneic MHC-I by CD8+ CTLs is required for killing of graft parenchymal cells because these cells express only allogeneic MHC. In contrast, CD4+ helper T cells that can directly or indirectly recognize allogeneic MHC-II can be activated by donor or host antigen-presenting cells (APCs), respectively, and both can promote inflammation that damages the graft.
As mentioned earlier, many of the T cells that respond to the allogeneic MHC antigens in a new graft are cross-reactive memory T cells previously generated against environmental antigens before transplantation. Unlike naive T cells, memory T cells may not need to see antigens presented by DCs in lymph nodes to be activated, and they may migrate directly into grafts, where they can be activated by APCs or tissue cells displaying alloantigen.
The response of alloreactive T cells to foreign MHC molecules can be analyzed in vitro by the mixed lymphocyte reaction (MLR), in which lymphocytes from two genetically distinct individuals are mixed together in cell culture. The T cells from one individual become activated by recognition of allogeneic MHC molecules on the cells of the other. The MLR was used clinically in the past as a predictive test of T-cell–mediated graft rejection and as an in vitro model to study the mechanisms of alloreactivity, but now it is mainly of historical significance.
Role of Costimulation in T-Cell Responses to Alloantigens
In addition to recognition of alloantigen, costimulation of T cells primarily by B7 molecules on APCs is important for activating alloreactive T cells. Costimulation is likely most important to activate naive alloreactive T cells, but even alloreactive memory T-cell responses can be enhanced by costimulation. Rejection of allografts and stimulation of alloreactive T cells in an MLR can be inhibited by agents that block B7 molecules. Mouse allografts survive for longer periods when they are transplanted into knockout animals lacking B7-1 (CD80) and B7-2 (CD86) compared with transplants into normal mice. As we will discuss later, blocking B7 costimulators is a therapeutic strategy to inhibit graft rejection in humans as well.
The requirement for costimulation leads to the interesting question of why these costimulators are expressed by graft APCs in the absence of infection, which we have previously discussed as the physiologic stimulus for the expression of costimulators. One possibility is that the innate immune response to ischemic damage of some cells in the graft, discussed later, results in increased expression of costimulators on APCs.
Effector Functions of Alloreactive T Cells
Alloreactive CD4+ and CD8+ T cells that are activated by graft alloantigens cause rejection by distinct mechanisms (see Fig. 4). The CD4+ helper T cells differentiate into cytokine producing effector cells that damage grafts by cytokine-mediated inflammation, similar to a delayed-type hypersensitivity (DTH) reaction. CD8+ T cells differentiate into CTLs, which directly kill graft cells.
Only CTLs that are generated by direct allorecognition can kill graft cells, whereas both CTLs and helper T cells generated by either direct or indirect alloantigen recognition can cause cytokine-mediated damage to grafts. CD8+ CTLs that are generated by direct allorecognition of donor MHC molecules on donor APCs can recognize the same MHC molecules on parenchymal cells in the graft and kill those cells. In contrast, any CD8+ CTLs that are generated in response to indirect recognition of allogeneic MHC are restricted to recognition of peptides from these allogeneic MHC molecules bound to recipient (self) MHC molecules, and therefore the T cells will not be able to kill the foreign graft cells because the graft cells do not express recipient MHC molecules. When CD4+ effector T cells are generated by direct or indirect recognition of allogeneic MHC, the principal mechanism of rejection is inflammation caused by the cytokines produced by the effector T cells. CD8+ T cells that may be activated by the indirect pathway may also contribute to rejection by producing inflammatory cytokines. Presumably, effector cells activated by the indirect pathway infiltrate the graft and recognize peptides from graft MHC molecules being dis played by host APCs that have also entered the graft.
Activation of Alloreactive B Cells and Production and Functions of Alloantibodies
Antibodies against graft antigens, called donor-specific anti bodies, also contribute to rejection. High-affinity alloantibodies are mostly produced by helper T-cell–dependent activation of alloreactive B cells, much like antibodies against other protein antigens (see Chapter 12). The antigens most frequently recognized by alloantibodies are donor MHC molecules, including both MHC-I and MHC-II proteins. The likely sequence of events leading to the generation of these alloantibody-producing cells is that naive B lymphocytes recognize the allogenic MHC molecules, internalize and process these proteins, and present peptides derived from them to helper T cells that were previously activated by the same peptides presented by DCs (see Fig. 2). Thus, activation of alloreactive B cells is an example of indirect presentation of alloantigens. In addition, donor- specific antibodies against non-HLA alloantigens also contribute to rejection.
The alloreactive antibodies produced in graft recipients engage the same effector mechanisms that antibodies use to combat infections, including complement activation and Fc receptor–mediated binding and activation of neutrophils, macrophages, and NK cells. Because MHC antigens are expressed on endothelial cells, much of the alloantibody-mediated dam age is targeted at the graft vasculature, as discussed later.
Innate Immune Responses to Allografts
In addition to the adaptive immune responses specific for alloantigens that differ between donor and host, innate immunity plays a role in the outcome of transplantation. The interruption of blood supply to tissues and organs during the time between removal from a donor and placement in a host usually causes some ischemic damage. This can result in the expression of dam age-associated molecular patterns in the graft, which simulate innate responses mediated by both innate cells within the graft and the recipient’s innate immune system. These innate responses can directly cause graft injury, but they are also thought to enhance adaptive responses by increasing migration of alloreactive circulating memory T cells into the graft and by activating APCs, as is the case in immune responses to microbes. APC activation increases the expression of costimulators and production of cytokines and thus the priming of naive alloreactive T lymphocytes. In addition, host NK cells can respond to the absence of self MHC molecules on donor graft cells and therefore contribute to graft rejection.
