Transplantation Immunobiology and Immunosuppression


Results of the World’s First Successful Hand Transplant.

Only a few short decades ago, there were no options for patients dying of end-stage organ failure. The concept of transplanting an organ from one individual to another was thought to be impossible. The evolution of clinical transplantation and transplant immunology is one of the bright success stories of modern medicine. It was through understanding of the immune response to the transplanted tissue that pioneers in the field were able to develop therapies to manipulate the immune response and to prevent rejection of the transplanted organ. Today, there are more than 25,000 transplants performed annually, and over 100,000 patients are currently listed and awaiting an organ.

The concept of transplantation is certainly not new. History is replete with legends and myths recounting the replacement of limbs and organs. An oft-repeated myth of early transplantation is derived from the miracle of Saints Cosmas and Damian (brothers and subsequently patron saints of physicians and surgeons) in which they successfully replaced the gangrenous leg of the Roman deacon Justinian with a leg from a recently deceased Ethiopian ( Fig. 25.1 ). It was not, however, until the French surgeon Alexis Carrel developed a method for joining blood vessels in the late nineteenth century that the transplantation of organs became technically feasible and verifiable accounts of transplantation began ( Fig. 25.2 ). He was awarded the Nobel Prize (Medicine) in 1912 “in recognition of his work on vascular suture and the transplantation of blood vessels and organs.” Having established the technical component, Carrel himself noted that there were two issues to be resolved regarding “the transplantation of tissues and organs…the surgical and the biological.” He had solved one aspect, the surgical, but he also understood that “it will only be through a more fundamental study of the biological relationships existing between living tissues” that the more difficult problem of the biology would come to be solved.

Fig. 25.1
A fifteenth century painting of Cosmas and Damian, patron saints of physicians and surgeons. The legend of the Miracle of the Black Leg depicts the removal of the diseased leg of Roman Justinian and replacement with the leg of a recently deceased Ethiopian man.

Fig. 25.2
Triangulation technique of vascular anastomosis by Alexis Carrel.

Reprinted from Edwards WS, Edwards PD. Alexis Carrel: Visionary Surgeon . Springfield, IL: Charles C Thomas; 1974.

Forty years would pass before another set of eventual Nobel Prize winners, Peter Medawar and Frank Macfarlane Burnet, would begin to define the process by which one individual rejects another’s tissue ( Fig. 25.3 ). Medawar and Burnet had developed an overall theory on the immunologic nature of self and the concept of immunologic tolerance. Burnet hypothesized that the definition of “self” was not preprogrammed but rather actively defined during embryonic development through the interaction of the host’s immune cells with its own tissue. This hypothesis implied that tolerance could be induced if donor cells were introduced to the embryo within this developmental time period. Burnet was proven correct when Medawar showed that mouse embryos receiving cells from a different mouse strain accepted grafts from the strain later in life while rejecting grafts from other strains. These seminal studies were the first reports to demonstrate that it was possible to manipulate the immune system to accept allografts.

Fig. 25.3
(A) Sir Peter Medawar. (Courtesy Bern Schwartz Collection, National Portrait Gallery, London.) (B) Sir Frank Macfarlane Burnet.

Courtesy Walter and Eliza Hall Institute of Medical Research.

Shortly thereafter, Joseph Murray, Nobel Laureate 1990, performed the first successful renal transplant between identical twins in 1954. At the same time, Gertrude Elion, who worked as an assistant to George Hitchings at Wellcome Research Laboratories, developed several new immunosuppressive compounds including 6-mercaptopurine and azathioprine. Roy Calne, a budding surgeon-scientist who came from the United Kingdom to study with Murray, subsequently tested these reagents in animals and then introduced them into clinical practice, permitting nonidentical transplantation to be successful. Elion and Hitchings later shared the Nobel Prize in 1988 for their work on “the important principles of drug development.” Subsequent discovery of increasingly effective agents to suppress the rejection response has led to the success in allograft survival that we enjoy today. It is this collaboration between scientists and surgeons that has driven our understanding of the immune system as it relates to transplantation. In this chapter, we provide an overview of the immune response with specific attention to transplant immunity and the rejection process, review the specific immunosuppressive agents that are employed to prevent rejection, and provide a glimpse into the future of the field.

The Immune Response

The immune system, of course, did not evolve to prevent the transplantation of another individual’s tissue or organs; rejection, rather, is a consequence of a system that has developed over thousands of years to protect against invasion by pathogens and to prevent subsequent disease. To understand the rejection process and in particular to appreciate the consequences of pharmacologic suppression of rejection, a general understanding of immune response as it functions in a physiologic setting is required.

The immune system has evolved to include two complementary divisions to respond to disease: the innate and acquired immune systems. Broadly speaking, the innate immune system recognizes general characteristics that have, through selective pressure, come to represent universal pathologic challenges to our species (ischemia, necrosis, trauma, and certain nonhuman cell surfaces). The acquired arm, on the other hand, recognizes specific structural aspects of foreign substances, usually peptide or carbohydrate moieties, recognized by receptors generated randomly and selected to avoid self-recognition. Although the two systems differ in their specific responsibilities, they act in concert to influence each other to achieve an optimal overall response.

Innate Immunity

The innate immune system is thought to be a holdover from an evolutionarily distant response to foreign pathogens. In contrast to the acquired immune system, which employs an innumerable host of specificities to identify any possible antigen, the innate system uses a select number of protein receptors to identify specific motifs consistent with foreign or altered and damaged tissues. These receptors can exist on cells, such as macrophages, neutrophils, and natural killer (NK) cells, or free in the circulation, as is the case for complement. Whereas they fail to exhibit the specificity of the T-cell receptor (TCR) or antibody, they are broadly reactive against common components of pathogenic organisms, for example, lipopolysaccharides on gram-negative organisms or other glycoconjugates. Thus, the receptors of innate immunity are the same from one individual to another within a species and, in general, do not play a role in the direct recognition of a transplanted organ. They do, however, exert their effects indirectly through the identification of “injured tissue” (e.g., as is the case when an ischemic, damaged organ is moved from one individual to another).

Once activated, the innate system performs two vital functions. It initiates cytolytic pathways for the destruction of the offending organism, primarily through the complement cascade ( Fig. 25.4 ). In addition, the innate system can convey the encounter to the acquired immune system for a more specific response through byproducts of complement activation by activation of antigen-presenting cells (APCs). Macrophages and dendritic cells not only engulf foreign organisms that have been bound by complement, but they can also distinguish pathogens as they can be identified through receptors for foreign carbohydrates (e.g., mannose receptors). Recently, a highly evolutionarily conserved family of proteins known as Toll-like receptors (TLRs) has been described to play an important role as activation molecules for innate APCs. They bind to pathogen-associated molecular patterns, motifs common to pathogenic organisms. Some examples of TLR ligands include lipopolysaccharide, flagellin (from bacterial flagella), double-stranded viral RNA, unmethylated CpG islands of bacterial and viral DNA, zymosan (β-glucan found in fungi), and numerous heat shock proteins. In contrast to pathogen-associated molecular patterns, which initiate a response to an infectious challenge, danger-associated molecular pattern molecules (DAMPs), also called alarmins, trigger the innate inflammatory response to noninfectious cell death and injury. Many DAMPs are nuclear or cytosolic proteins or even DNA that is released or exposed in the setting of cell injury. These signals alert the innate immune system that injury has occurred and a response is required. DAMP receptors include some of the TLRs, including TLR2 and TLR4, but also a variety of other proteins, such as receptor for advanced glycosylation end-products (RAGE) and triggering receptor expressed on myeloid cells 1 (TREM-1). In the setting of a transplant surgery where an organ is cut out of one individual with a period of obligatory ischemia, cooled to near freezing, and then replaced in another individual, DAMPs play an active role in stimulating the innate inflammatory response. Once an injury or infectious insult has been identified, the cellular components of the innate system begin to initiate a response.

Fig. 25.4, Complement activation. There are three distinct pathways that lead to complement activation. All three pathways lead to production of C3b , which initiates the late steps of complement activation. C3b binds to the microbe and promotes opsonization and phagocytosis. C5a stimulates the local inflammatory response and catalyzes formation of the membrane attack complex, which results in microbial cell membrane disruption and death by lysis.

Monocytes

Mononuclear phagocytes are bone marrow–derived cells that initially emerge as monocytes within peripheral blood. In the setting of certain inflammatory signals, they are home to sites of injury or inflammation, where they mature and become macrophages. Their function is to acquire, process, and present antigen as well as to serve as effector cells in certain situations. Once activated, they elaborate various cytokines that regulate the local immune response. They play a significant role in facilitating the acquired T-cell response through antigen presentation, and their cytokines induce substantial tissue dysfunction in sites of inflammation. Thus, their recruitment to sites of injury and cell death can subsequently provoke T-cell activation and rejection.

Dendritic Cells

Dendritic cells are specialized macrophages that are regarded as professional APCs. They are the most potent cells that present antigen and are distributed throughout the lymphoid and nonlymphoid tissues of the body. Immature dendritic cells can be found along the gut mucosa, within the skin, and in other sites of antigen entry. Once they have encountered antigen in sites of injury, they undergo a process of maturation, including the upregulation of both major histocompatibility complex (MHC) molecules, class I and class II, as well as various costimulatory molecules. They also begin to migrate toward peripheral lymphoid tissue (i.e., lymph nodes), where they can interact with antigen-specific T cells and potentiate their activation. The dendritic cell is involved in the licensing of CD8+ T cells for cytotoxic function, stimulates T-cell clonal expansion, and provides signals for helper T cell (Th) differentiation. There are also subsets of dendritic cells that serve distinct functions in inducing and regulating the cellular response. For example, myeloid dendritic cells are more immunogenic, whereas plasmacytoid dendritic cells are more tolerogenic and may work to suppress the immune response.

Natural Killer Cells

NK cells are large granular lymphocytes with potent cytolytic function that constitute a critical component of innate immunity. They were initially discovered during studies focused on tumor immunology. There was a small subset of lymphocytes that exhibited the ability to lyse tumor cells in the absence of prior sensitization, described as “naturally” reactive. These “natural killer” cells exhibited rapid cytolytic activity and existed in a relatively mature state (i.e., morphology characteristic of activated cytotoxic lymphocytes—large size, high protein synthesis activity with abundant endoplasmic reticulum, and rapid killing activity). Further studies have indicated that NK cells lyse cell targets that lack expression of self class I MHC, termed the missing self hypothesis, a situation that could arise as a result of viral infection with suppression of self class I molecules or in tumors under strong selection pressure of killer T cells. Since those initial studies, NK cells have been found to express cell surface inhibitory receptors, which include killer inhibitory receptors. These molecules function to deliver inhibitory signals when they bind class I MHC molecules, thus preventing NK-mediated cytolysis on otherwise healthy host cells. NK cells produce various cytokines, including interferon-γ (IFN-γ), which may function to activate macrophages, which can in turn eliminate host cells infected by intracellular microbes. Similar to macrophages, NK cells express cell surface Fc receptors, which bind antibody and participate in antibody-dependent cellular cytotoxicity. NK cells also play an important role in the immune response after bone marrow transplantation and xenotransplantation. Their role in solid organ transplantation is less well defined.

Acquired Immunity

The distinguishing feature of the acquired immune system is specific recognition and disposition of foreign elements as well as the ability to recall prior challenges and to respond appropriately. Highly specific receptors, discussed later, have evolved to distinguish foreign from normal tissue through antigen binding. The term antigen is used to describe a molecule that can be recognized by the acquired immune system. An epitope is the portion of the antigen, generally a carbohydrate or peptide moiety, that actually serves as the binding site for the immune system receptor and is the base unit of antigen recognition. Thus, there may be one or many epitopes on any given antigen. The acquired response is divided into two distinct arms: cellular and humoral. The predominant effector cell in each arm is the T cell and B cell, respectively. Accordingly, the two main types of receptors that the immune system employs to recognize any given epitope are the TCR and B-cell receptor or antibody. In general, individual T or B lymphocytes express identical receptors, each of which binds only to a single epitope. This mechanism establishes the specificity of the acquired immune response. The antigenic encounter alters the immune system such that future challenges with the same antigen provoke a more rapid and vigorous response, a phenomenon known as immunologic memory. There are vast differences in the way each division of the acquired immune response identifies an antigen. The B-cell receptor or antibody can identify its epitope directly without preparation of the antigen, either on an invading pathogen itself or as a free-floating molecule in the extracellular fluid. T cells, however, recognize only their specific epitope after it has been processed and bound to a set of proteins, unique to the individual, which are responsible for presentation of the antigen. This set of proteins, crucial to antigen presentation, are termed histocompatibility proteins and, as their name suggests, were defined through studies examining tissue transplantation. The case of the immune response in tissue transplantation is unique and is discussed in its own section.

Major Histocompatibility Locus: Transplant Antigens

The MHC refers to a cluster of highly conserved polymorphic genes on the sixth human chromosome. Much of what we know about the details of the immune response grew from initial studies defining the immunogenetics of the MHC. Studies began in mice in which the MHC gene complex, termed H-2 , was described by Gorer and Snell as a genetic locus that segregated with transplanted tumor survival. Subsequent serologic studies identified a similar genetic locus in humans called the human leukocyte antigen (HLA) locus. The products of these genes are expressed on a wide variety of cell types and play a pivotal role in the immune response. They are also the antigens primarily responsible for human transplant rejection, and their clinical implications are discussed later.

MHC molecules play a role in both the innate and acquired immune systems. Their predominant role, however, lies in antigen presentation within the acquired response. As mentioned earlier, the TCR does not recognize its specific antigen directly; rather, it binds to the processed antigen that is bound to cell surface proteins. It is the MHC molecule that binds the peptide antigen and interacts with the TCR, a process called antigen presentation. Thus, all T cells are restricted to an MHC for their response. There are two classes of MHC molecules, class I and class II. In general, CD8+ T cells bind to antigen within class I MHC, and CD4+ T cells bind to antigen within class II MHC.

Human Histocompatibility Complex

The antigens primarily responsible for human allograft rejection are those encoded by the HLA region of chromosome 6 ( Fig. 25.5 ). The polymorphic proteins encoded by this locus include class I molecules (HLA-A, B, and C) and class II molecules (HLA-DP, DQ, and DR). There are additional class I genes with limited polymorphism (E, F, G, H, and J), but they are not currently used in tissue typing for transplantation and are not considered here. There are class III genes as well, but they are not cell surface proteins involved in antigen presentation directly but rather include molecules that are pertinent to the immune response by various mechanisms: tumor necrosis factor-α (TNF-α), lymphotoxin β, components of the complement cascade, nuclear transcription factor-β, and heat shock protein 70. Other conserved genes within the HLA include genes necessary for class I and class II presentation of peptides, such as the peptide transporter proteins TAP1 and TAP2 and proteasome proteases LMP2 and LMP7. Although other polymorphic genes, referred to as minor histocompatibility antigens, exist in the genome outside of the HLA locus, they play a less significant role in transplant rejection and are not covered here. It is, however, important to point out that even HLA-identical individuals are subject to rejection on the basis of these minor differences. The blood group antigens of the ABO system must also be considered transplant antigens, and their biology is critical to humoral rejection.

Fig. 25.5, Location and organization of the HLA complex on human chromosome 6 and H-2 complex on murine chromosome 17. The complex is conventionally divided into regions I and II. (Adapted from Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology. 9th ed. Philadelphia: Saunders Elsevier; 2018.) TAP , Transporter associated with antigen processing; TNF-α , tumor necrosis factor-α.

Although initially identified as transplant antigens, class I and class II MHC molecules actually play vital roles in all immune responses, not just those to transplanted tissue. HLA class I molecules are present on all nucleated cells. In contrast, class II molecules are found almost exclusively on cells associated with the immune system (macrophages, dendritic cells, B cells, and activated T cells) but can be upregulated and appear on other parenchymal cells in the setting of cytokine release due to an immune response or injury.

The importance to transplantation of MHC gene products stems from their polymorphism. Unlike most genes, which are identical within a given species, polymorphic gene products differ in detail while still conforming to the same basic structure. Thus, polymorphic MHC proteins from one individual are foreign alloantigens to another individual. Recombination within the HLA locus is uncommon, occurring in approximately 1% of molecules. Consequently, the HLA type of the offspring is predictable. The unit of inheritance is the haplotype, which consists of one chromosome 6 and therefore one copy of each class I and class II locus (HLA-A, B, C, DP, DQ, and DR). Thus, donor-recipient pairings that are matched at these HLA loci are referred to as HLA-identical allografts, and those matched at half of the HLA loci are termed haploidentical . Note that HLA-identical allografts still differ genetically at other genetic loci and are distinct from isografts. Isografts are organs transplanted between identical twins and are immunologically indistinguishable and thus are not naturally rejected. The genetics of HLA is particularly important in understanding clinical living related donor transplantation. Each child inherits one haplotype from each parent; therefore, the chance of siblings being HLA identical is 25%. Haploidentical siblings occur 50% of the time, and completely nonidentical or HLA-distinct siblings occur 25% of the time. Biologic parents are haploidentical with their children unless there has been a rare recombination event. The degree of HLA match can also improve if the parents are homozygous for a given allele, thus giving the same allele to all children. Likewise, if the parents share the same allele, the likelihood of that allele being inherited improves to 50%. This is even more important in the field of bone marrow transplantation in which the risks of donor-mediated cytotoxicity and resultant graft-versus-host disease become a more relevant issue.

Each class I molecule is encoded by a single polymorphic gene that is combined with the nonpolymorphic protein β2-microglobulin (chromosome 15) for expression. The polymorphism of each class I molecule is extreme, with 30 to 50 alleles per locus. Class II molecules are made up of two chains, α and β, and individuals differ not only in the alleles represented at each locus but also in the number of loci present in the HLA class II region. The polymorphism of class II is thus increased by combinations of α and β chains as well as by hybrid assembly of chains from one class II locus to another. As the HLA sequence varies, the ability of various peptides to bind to the molecule and to be presented for T-cell recognition changes. Teleologically, this extreme diversity is thought to improve the likelihood that a given pathogenic peptide will fit into the binding site of these antigen-presenting molecules, thus preventing a single viral agent from evading detection by T cells of an entire population.

Class I Major Histocompatibility Complex

The three-dimensional structure of class I molecules (HLA-A, B, and C) was first elucidated in 1987. The class I molecule is composed of a 44-kDa transmembrane glycoprotein (α chain) in a noncovalent complex with a nonpolymorphic 12-kDa polypeptide called β 2 -microglobulin. The α chain has three domains, α 1 , α 2 , and α 3 . The critical structural feature of class I molecules is the presence of a groove formed by two α helices mounted on a β pleated sheet in the α 1 and α 2 domains ( Fig. 25.6 ). Within this groove, a 9–amino acid peptide, formed from fragments of proteins being synthesized in the cell’s endoplasmic reticulum, is mounted for presentation to T cells. Almost all the significant sequence polymorphism of class I is located in the region of the peptide-binding groove and in areas of direct T-cell contact ( Fig. 25.7 ). The assembly of class I is dependent on association of the α chain with β2-microglobulin and native peptide within the groove. Incomplete molecules are not expressed. In general, all peptides made by a cell are candidates for presentation, although sequence alterations in this region favor certain sequences over others. The α 3 immunoglobulin (Ig)-like domain, which is the domain closest to the membrane and interacts with the CD8 molecule on the T cell, demonstrates limited polymorphism and is conserved to preserve interactions with CD8+ T cells.

Fig. 25.6, Structure of the major histocompatibility complex class I molecule. Class I molecules are composed of polymorphic α chain noncovalently attached to the nonpolymorphic β 2 -microglobulin (β 2 m). (A) Schematic diagram. (B) The ribbon diagram shows the extracellular structure of a class I molecule with a bound peptide.

Fig. 25.7, Polymorphic residues of major histocompatibility complex (MHC) molecules. The polymorphic residues of class I and class II MHC molecules are located in the peptide-binding grooves and the α helices around the grooves. The regions of greatest variability among different human leukocyte antigen (HLA) alleles are indicated in red , of intermediate variability in green , and of the lowest variability in blue .

Human class I presentation occurs on all nucleated cells, and expression can be increased by certain cytokines, thus allowing the immune system to inspect and to approve of ongoing protein synthesis. IFNs (IFN-α, IFN-β, and IFN-γ) induce an increase in the expression of class I molecules on a given cell by increasing levels of gene expression. T-cell activation occurs when a given T cell encounters a class I MHC molecule carrying a peptide from a nonself protein presented in the proper context (e.g., viral protein is processed in an infected cell and the peptide fragments are presented on class I molecules for T-cell recognition). So-called cross-presentation may also occur in which certain APCs, namely, a subset of dendritic cells, have the ability to take up and process exogenous antigen and present it on class I molecules to CD8+ T cells. In the case of transplantation, this activation is not only possible when foreign peptide is identified after the donor MHC has been processed and presented on recipient APCs but is thought to more commonly occur when a T cell interacts directly with the donor nonself class I MHC, the so-called direct alloresponse.

Class II Major Histocompatibility Complex

The class II molecules are products of the HLA-DP, HLA-DQ, and HLA-DR genes. The structural features of class II molecules are strikingly similar to those of class I molecules. The three-dimensional structure of class II molecules was inferred by sequence homology to class I in 1988 and eventually proven by x-ray crystallography in 1993 ( Fig. 25.8 ). The class II molecules contain two polymorphic chains, one approximately 32 kDa and the other approximately 30 kDa. The peptide-binding region is composed of the α1 and β1 domains. As with the class I molecule, significant polymorphic residues of class II are located in the peptide-binding clefts and in the alpha helices around these clefts ( Fig. 25.7 ). The Ig-like domain is composed of the α2 and β2 segments. Similar to the class I Ig-like α3 domain, there is limited polymorphism in these segments, and the β2 domain, in particular, is involved in the binding of the CD4 molecule, helping to restrict class II interactions to CD4+ T cells. Class II molecule assembly requires association of both the α chain and β chain in combination with a temporary protein called the invariant chain. This third protein covers the peptide-binding groove until the class II molecule is out of the endoplasmic reticulum and is sequestered in an endosome. Proteins that are engulfed by a phagocytic cell are degraded at the same time as the invariant chain is removed, allowing peptides of external sources to be associated with and presented by class II. In this way, the acquired immune system can inspect and approve of proteins that are present in circulation or that have been liberated from foreign cells or pathogens through the phagocytic process. Accordingly, class II molecules, in contrast to class I molecules, are confined to cells related to the immune response, particularly APCs (macrophages, dendritic cells, B cells, and monocytes). Class II expression can also be induced on other cells, including endothelial cells, under the appropriate conditions. After binding class II molecules, CD4+ T cells participate in APC-mediated activation of CD8+ T cells and antibody-producing B cells. In the case of transplanted organs, ischemic injury at the time of transplantation accentuates the potential for T-cell activation by upregulation of both class I and class II molecules locally on the recipient. The trauma of surgery and ischemia also upregulate class II on all cells of the allograft, making nonself MHC more abundant. Host CD4+ T cells may then recognize donor MHC directly (direct alloresponse) or after antigen processing on the recipient’s own MHC (indirect alloresponse) and then proceed to participate in rejection.

Fig. 25.8, Structure of the major histocompatibility complex class II molecule. Class II molecules are composed of a polymorphic α chain noncovalently attached to a polymorphic β chain. (A) Schematic diagram. (B) The ribbon diagram shows the extracellular structure of a class II molecule with a bound peptide.

Human Leukocyte Antigen Typing: Implications for Transplantation

For the reasons already discussed, closely matched or less mismatched transplants are less likely to be recognized and rejected than are similar grafts differing by multiple alleles at the MHC. HLA matching has clear influence on the prolongation of graft survival. Humans potentially have two different HLA-A, B, and DR alleles (one from each parent, six in total). Although clearly biologically important, the HLA-C, DP, and DQ loci have historically been administratively dismissed in general organ allocation. However, reporting requirements on the genetic typing of donors have recently been expanded to include HLA-C, DP, and DQ so that these HLA molecules can also be considered for the purpose of organ allocation. Whereas current immunosuppressive regimens negate much of the impact of matching, several studies have demonstrated better renal allograft survival when the six primary alleles (A, B, and DR) are matched between donor and recipient, a so-called six-antigen match or zero-antigen mismatch ( Fig. 25.9 ). Historically, MHC compatibility had been defined using two cellular assays: the lymphocytotoxicity assay and the mixed lymphocyte reaction. Both assays identify MHC epitopes but do not comprehensively define the entire antigen or the exact HLA genetic disparity involved. More precise molecular techniques now exist for genotyping that distinguish the nucleotide sequence of an individual’s MHC.

Fig. 25.9, Influence of human leukocyte antigen (HLA) matching on renal allograft survival. Matching of HLA alleles between donor and recipient significantly improves renal allograft survival. The data are shown for deceased donor renal allografts stratified by number of matched HLA alleles.

The mixed lymphocyte reaction is performed by incubating recipient T cells with irradiated donor cells in the presence of 3 H-thymidine (the irradiation treatment ensures that the assay measures only proliferation of recipient T cells). If the cells differ at the class II MHC locus, recipient CD4+ T cells produce interleukin-2 (IL-2), which stimulates proliferation. Proliferating cells incorporate the labeled nucleotide into their newly manufactured DNA, which can be detected and quantified. Whereas class II polymorphism is detected by this assay, it takes several days to complete one assay. Thus, use of the mixed lymphocyte reaction as a prospective typing assay is limited to living related donors. The specific MHC alleles are not identified with this assay; instead, they are inferred from a series of reactions. Although this assay has been extremely valuable historically, it has now been largely supplanted by more modern molecular techniques. The lymphocytotoxicity assay involves taking serum from individuals with anti-MHC antibodies of known specificity and mixing it with lymphocytes from the individual in question. Exogenous complement is added, as is a vital dye, which is not taken up by intact cells. If the antibody binds to MHC, it activates the complement and leads to cell membrane disruption, and the cell takes up the vital stain. Microscopic examination of the cells can then determine if the MHC antigen was present on the cells. This, too, has been supplanted by more modern methods of MHC-specific antibody detection.

The sequencing of the HLA class I and class II loci has allowed several genetic-based techniques to be used for histocompatibility testing. These methods include restriction fragment length polymorphism, oligonucleotide hybridization, and polymorphism-specific amplification using the polymerase chain reaction and sequence-specific primers. Of these methods, the polymerase chain reaction with sequence-specific primers technique is most commonly employed for class II typing. Serologic techniques are still the predominant method for class I typing because of the complexity of class I sequence polymorphism. Sequence polymorphisms that do not alter the TCR-MHC interface are unlikely to affect allograft survival; thus, the enhanced precision of molecular typing may provide more information than is actually clinically relevant.

Cellular Components of the Acquired Immune System

The key cellular components of the immune system, T cells, B cells, and APCs, are hematopoietically derived and arise from a common progenitor stem cell. The development of the lymphoid system begins with pluripotent stem cells in the liver and bone marrow of the fetus. As the fetus matures, the bone marrow becomes the primary site of lymphopoiesis. B cells were named after the primary lymphoid organ that produces B cells in birds, the bursa of Fabricius. In humans and most other mammals, precursor B cells remain within the bone marrow as they mature and fully develop. Although precursor T cells also originate in the bone marrow, they soon migrate to the thymus, the primary site of T-cell maturation, where they become “educated” to self and acquire their specific cell surface receptors and the ability to generate effector function. Mature lymphocytes are then released from the primary lymphoid organs, the bone marrow and thymus, to populate the secondary lymphoid organs including lymph nodes, spleen, and gut, as well as peripheral tissues. Each of these cells has a unique role in establishing the immune response. The highly coordinated network is regulated in part through the use of cytokines ( Table 25.1 ).

Table 25.1
Summary of cytokines.
Adapted from Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology . 9th ed. Philadelphia: Saunders Elsevier; 2018.
Cytokine Source Principal Cellular Targets and Biologic Effects
Interleukin-1 Macrophages, endothelial cells, some epithelial cells Endothelial cell: activation (inflammation, coagulation)
Hypothalamus: fever
Liver: synthesis of acute-phase proteins
Interleukin-2 T cells T cells: proliferation, ↑ cytokine synthesis, survival, potentiates Fas-mediated apoptosis, promotes regulatory T-cell development
NK cells: proliferation, activation
B cells: proliferation, antibody synthesis (in vitro)
Interleukin-3 T cells Immature hematopoietic progenitor cells: stimulates differentiation into myeloid lineage, proliferation of myeloid lineage cells
Interleukin-4 CD4+ T cells (Th2), mast cells B cells: isotype switching to IgE
T cells: Th2 differentiation, proliferation
Macrophages: inhibition of IFN-γ–mediated activation
Mast cells: stimulates proliferation
Interleukin-5 CD4+ T cells (Th2) Eosinophils: activation, ↑ production
B cells: proliferation, IgA production
Interleukin-6 Macrophages, endothelial cells, T cells Liver: ↑ synthesis of acute-phase proteins
B cells: proliferation of antibody-producing cells
Interleukin-7 Fibroblasts, bone marrow stromal cells Immature hematopoietic progenitor cells: stimulates differentiation into lymphoid lineage
T and B cells: important for survival during development as well as for T-cell memory
Tumor necrosis factor Macrophages, T cells Endothelial cells: activation (inflammation, coagulation)
Neutrophils: activation
Hypothalamus: fever
Liver: ↑ synthesis of acute-phase proteins
Muscle, fat: catabolism (cachexia)
Many cell types: apoptosis
Interferon-γ T cells (Th1, CD8+ T cells), NK cells Macrophages: activation (increased microbicidal functions)
B cells: isotype switching to IgG subclasses that facilitate complement fixation and opsonization
T cells: Th1 differentiation
Various cells: ↑ expression of class I and class II MHC, ↑ antigen processing and presentation to T cells
Type I interferons (IFN-α, IFN-β) Macrophages: IFN-α
Fibroblasts: IFN-β
All cells: stimulates antiviral activity including ↑ class I MHC expression
NK cells: activation
Transforming growth factor-β T cells, macrophages, other cell types T cells: inhibition of proliferation and effector functions
B cells: inhibition of proliferation, ↑ IgA production
Macrophages: inhibits activation, stimulates angiogenic factors
Fibroblasts: increased collagen synthesis
Lymphotoxin T cells Lymphoid organogenesis
Neutrophils: increased recruitment and activation
BAFF (CD257) Follicular dendritic cells, monocytes, B cells B cells: survival and proliferation
APRIL (CD256) T cells, follicular dendritic cells, monocytes B cells: survival and proliferation
Interleukin-8 Lymphocytes, monocytes Stimulates granulocyte activity
Chemotactic activity
Interleukin-9 Activated Th2 lymphocytes Enhances proliferation of T cells, mast cells
Interleukin-10 Macrophages, T cells (mainly regulatory T cells) Macrophages and dendritic cells: inhibition of IL-12 production, stimulates expression of costimulatory molecules and class II MHC
Interleukin-11 Bone marrow stromal cells Megakaryocytes: thrombopoiesis
Liver: induces acute-phase proteins
B cells: stimulates T-dependent antibody production
Interleukin-12 Macrophages, dendritic cells T cells: Th1 differentiation
NK and T cells: IFN-γ synthesis, increased cytotoxic activity
Interleukin-13 CD4+ T cells (Th2), NKT cells, mast cells B cells: isotype switching to IgE
Epithelial cells: increased mucus production
Fibroblasts and macrophages: increased collagen synthesis
Interleukin-14 T cells, some B-cell tumors B cells: enhances proliferation of activated B cells, stimulates Ig production
Interleukin-15 Macrophages, others NK cells: proliferation
T cells: proliferation (memory CD8+ T cells)
Interleukin-17 T cells Endothelial cells: increased chemokine production
Macrophages: increased chemokine/cytokine production
Epithelial cells: GM-CSF and G-CSF production
Interleukin-18 Macrophages NK and T cells: IFN-γ synthesis
Interleukin-21 Th2, Th17, Tfh Drives development of Th17 and Tfh
B cells: activation, proliferation, differentiation
NK cells: functional maturation
Interleukin-22 Th17 Epithelial cells: production of defensins, increased barrier functions
Promotes hepatocyte survival
Interleukin-23 Macrophages, dendritic cells T cells: maintenance of IL-17–producing T cells
Interleukin-27 Macrophages, dendritic cells T cells: inhibits production of IL-17/Th17 cells, promotes Th1 differentiation
NK cells: IFN-γ synthesis
Interleukin-33 Endothelial cells, smooth muscle cells, keratinocytes, fibroblasts Th2 development and cytokine production
APRIL , A proliferation-inducing ligand; BAFF , B cell–activating factor; G-CSF , granulocyte-colony stimulating factor; GM-CSF , granulocyte-macrophage colony-stimulating factor; IFN , interferon; Ig , immunoglobulin; MHC , major histocompatibility complex; NK , natural killer; NKT , natural killer T cell; Tfh , T follicular helper.

Both B and T cells are integral components of a highly specific response that must be prepared to recognize a seemingly endless array of pathogens. This is accomplished through a unique method that allows random generation of almost unlimited receptor specificity yet controls the ultimate product by eliminating or suppressing those that might react against self and perpetuate an autoimmune response. There are fundamental differences in the manner in which T and B cells recognize antigen. B cells are structured to respond to whole antigen and in response synthesize and secrete antibody that can interact with antigen at distant sites. T cells, on the other hand, are responsible for cell-mediated immunity and of necessity must interact with cells in the periphery to neutralize and to eliminate foreign antigens. From the peripheral blood, T cells enter the lymph nodes or spleen through highly specialized regions in the postcapillary venules. Within the secondary lymphoid organ, T cells interact with specific APCs, where they receive the appropriate signals that in effect license them for effector function. They then exit the lymphoid tissues through the efferent lymph, eventually percolating through the thoracic duct and returning to the bloodstream. From there, they can return to the site of the immune response, where they encounter their specific antigen and carry out their predefined functions.

T-Cell Receptor

Considerable progress has been made in defining the mechanisms of T-cell maturation and the development of a functional TCR. The formation of the TCR is fundamental to the understanding of its function. When precursor T cells migrate from the fetal liver and bone marrow to the thymus, they have yet to obtain their specialized TCR or accessory molecules. On arrival to the thymus, T cells undergo a remarkable rearrangement of the DNA that encodes the various chains of the TCR (α, β, γ, and δ) ( Fig. 25.10 ). The order of genetic rearrangement recapitulates the evolution of the TCR. T cells first attempt to recombine the γ and δ TCR genes and then, if recombination fails to yield a properly formed receptor, resort to the more diverse α and β TCR genes. The γδ configuration is typically not successful, and thus, most T cells are αβ T cells. T cells expressing the γδ TCR have more primitive functions, including recognition of heat shock proteins and activity similar to NK cells as well as MHC recognition, whereas αβ T cells are more typically limited to recognition of MHC complexed with processed peptide.

Fig. 25.10, T-cell receptor (TCR) recombination and expression (α and β loci shown here). There is an elaborate genetic rearrangement that leads to the formation of a diverse repertoire of TCRs. Genomic DNA is spliced under the direction of specific enzymes active during T-cell development within the thymus. Random segments from regions termed variable (V) , joining (J) , diversity (D) , and constant (C) are brought together to form a unique gene responsible for a unique TCR chain. The γ and δ loci recombine first, and if successful, a γδ TCR is formed. If unsuccessful, then α and β regions recombine to form an αβ TCR. Approximately 95% of T cells progress to express an αβ TCR.

Regardless of the genes used, individual cells recombine to express a TCR with only a single specificity. These rearrangements occur randomly and can theoretically produce 10 15 different TCRs; however, 10 15 T cells would weigh 500 kg and cannot all be contained in the human body. Based on computational models of homeostasis of multiclonal populations of T cells, it is estimated that approximately 10 9 naïve T-cell clonotypes (i.e., T cells with the same TCR specificity) are present at any point in time. As a result, the frequency of naïve T cells available to respond to any given pathogen is relatively small, estimated to be between 1 in 200,000 and 1 in 500,000. These developing T cells also express both CD4 and CD8, accessory molecules that strengthen TCR binding to MHC. These accessory molecules further increase the binding repertoire of the population to include either class I or class II MHC molecules. If the process of T-cell maturation ended at this stage, there would be a host of T cells that could recognize self MHC–peptide complexes, resulting in an uncontrolled, global autoimmune response. To avoid the release of autoreactive T cells, developing cells undergo a process following recombination known as thymic selection ( Fig. 25.11 ). Cells initially interact with the MHC-expressing cortical thymic epithelium, which produces hormones (thymopoietin and thymosin) as well as cytokines (e.g., IL-7) that are critical to T-cell development. If binding does not occur to self MHC, those cells are useless to the individual (e.g., they cannot bind self cells to assess for infection), and they are permitted to die by neglect through apoptosis, a process called positive selection. Thus, positive selection ensures that T cells are restricted to self MHC. Cells surviving positive selection then move to the thymic medulla and normally eventually lose either CD4 or CD8. If binding to self MHC in the medulla occurs with an unacceptably high affinity, there is an active process whereby death-promoting signals are delivered and programmed cell death is initiated, a process termed negative selection. Negative selection stands in contrast to the death that occurs by neglect when immature lymphocytes are not positively selected. Another possible, although less common, outcome of a high-affinity interaction with self peptide–MHC is the development of a regulatory T-cell (Treg) phenotype. The precise nature of this affinity threshold remains a matter of intense investigation and involves interaction with hematopoietic cells that reside in the thymus as well as medullary thymic epithelial cells. These thymically derived “natural” Tregs emerge from the thymus and are involved in the suppression of autoreactive T cells in the periphery, which is discussed later.

Fig. 25.11, T-cell maturation. Initially, bone marrow-derived T-cell precursors arrive in the thymic cortex lacking CD4, CD8, or a T-cell receptor (TCR) and are referred to as double negative. The genes responsible for expression of the TCR chains subsequently undergo a series of recombination events resulting in expression of either a γδ TCR or, more commonly (>90%), an αβ TCR on the cell surface. The γδ T cells proceed through a distinct selection process that is independent of major histocompatibility complex (MHC) restriction. The αβ T cells acquire expression of both CD4 and CD8 and are then referred to as double positive. They then proceed to undergo the process of positive and negative selection and ultimately express only CD4 or CD8, depending on which class of MHC they restrict to.

The only cells released into the periphery are those that can both bind self MHC and avoid activation. Whereas T cells are restricted to bind self MHC–peptide complexes without activation, the selection process does not consider foreign MHC. Thus, by random chance, some cells with appropriate affinity for self MHC survive and have inappropriately high affinity for the MHC molecules of other individuals. In the setting of transplantation, these recipient T cells are able to recognize donor MHC–peptide complexes because there are sufficient conserved motifs shared between donor and self MHC molecules. However, because donor MHC was not present during the thymic education process, the binding of donor MHC by an “alloreactive” T cell leads to activation, and rejection ensues. The precursor frequency or the number of alloreactive T cells is much higher than the 1 in 200,000 or 1 in 500,000 T cells available to react toward any given antigen. Because T cells are selected to bind self MHC, the frequency specific for a similar, nonself MHC (i.e., alloreactive) is estimated to be between 1% and 10% of all T cells.

In addition to thymic selection, it is now clear that mechanisms exist for peripheral modification of the T-cell repertoire. Many of these mechanisms are in place for removal of T cells after an immune response and downregulation of activated clones. CD95, a molecule known as Fas, is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on activated T cells. Under appropriate conditions, binding of this molecule to its ligand, CD178, promotes programmed cell death of a cohort of activated T cells. This method is dependent on TCR binding and the activation state of the T cell. Complementing this deletional method to TCR repertoire control are nondeletional mechanisms that selectively anergize (make unreactive) specific T-cell clones. In addition to signaling through the TCR complex, T cells require additional costimulatory signals (described in detail later). TCR binding leads to T-cell activation only if the costimulatory signals are present, generally delivered by APCs. In the absence of costimulation, the cell remains unable to proceed toward activation and in some circumstances becomes refractory to activation even with the appropriate signals. Thus, TCR binding that occurs to self in the absence of appropriate antigen presentation or active inflammation results in an aborted activation and prevents self-reactivity.

T-Cell Activation

T-cell activation is a sophisticated series of events that has only recently been more fully described. The TCR, unlike antibody, recognizes its ligand only in the context of MHC. By requiring that T cells respond only to antigen encountered when it is physically embedded on self cells, the system avoids constant activation by soluble molecules.

T cells can then specifically recognize and destroy cells that make peptide products of mutation or viral infection. Because the number of potential antigens is high and the likelihood is that self-antigens vary minimally from foreign antigens, the nature of the TCR-binding event has evolved such that a single interaction with an MHC molecule is not sufficient to cause activation. In fact, a T cell must register a signal from approximately 8000 TCR-ligand interactions with the same antigen before a threshold of activation is reached. Each event results in the internalization of the TCR. Because resting T cells have low TCR density, sequential binding and internalization during several hours are required. Transient encounters are not sufficient. This threshold is reduced considerably by appropriate costimulation signals (detailed later).

Most TCRs are heterodimers composed of two transmembrane polypeptide chains, α and β. The αβ-TCR is noncovalently associated with several other transmembrane signaling proteins, including CD3 (composed of three separate chains, γ, δ, and ε) and ζ chain molecules, as well as the appropriate accessory molecule from the T cell, either CD4 or CD8, which associates with its respective MHC molecule. Together, these proteins are known as the TCR complex. When the TCR is bound to an MHC molecule and the proper configuration of accessory molecules stabilizes its binding, a signal is initiated by intracytoplasmic protein tyrosine kinases ( Fig. 25.12 ). These protein tyrosine kinases include p56lck (on CD4 or CD8), p59Fyn, and ZAP-70, the last two of which are associated with CD3. Repetitive binding signals combined with the appropriate secondary costimulation eventually activate phospholipase-γ1, which in turn hydrolyzes the membrane lipid phosphatidylinositol bisphosphate, thereby releasing inositol trisphosphate and diacylglycerol. Inositol trisphosphate binds to the endoplasmic reticulum, causing a release of calcium that induces calmodulin to bind to and activate calcineurin. Calcineurin dephosphorylates the critical cytokine transcription factor nuclear factor of activated T cells (NFAT), prompting it, with the transcription factor nuclear factor κB (NF-κB), to initiate transcription of cytokines including IL-2 and its receptor. Resting T cells express only low levels of the IL-2 receptor (CD25), but with activation, IL-2R expression is increased. As the activated T cell begins to produce IL-2 secondary to events initiated by TCR activation, the cytokine begins to work in both autocrine and paracrine fashions, potentiating diacylglycerol activation of protein kinase C. Protein kinase C is important in activating many gene regulatory steps critical for cell division. This effect, however, is restricted only to T cells that have undergone activation after encountering their specific antigen leading to IL-2R expression. Thus, the process limits proliferation and expansion to only those clones specific for the offending antigen. As the antigenic stimulus is removed, IL-2R density decreases and the TCR complex is reexpressed on the cell surface. There is a negative feedback system between the TCR and the IL-2R, resulting in a highly regulated and efficient system that is reactive only in the presence of antigen and ceases to function once antigen is removed. Not surprisingly, many of these steps in T-cell activation have been targeted in the development of immunosuppressive agents. These are discussed in detail in a subsequent section of this chapter.

Fig. 25.12, T-cell activation. On antigen recognition, there is a clustering of T-cell receptor (TCR) complexes and coreceptors that initiates a cascade of signaling events within the T cell. Tyrosine kinases associated with the coreceptors (e.g., Lck) phosphorylate CD3 and the ζ chain (A). The ζ chain association protein kinase (ZAP-70) subsequently associates with these regions and becomes activated. ZAP-70 phosphorylates various adaptor molecules, such as LAT (B). These adaptors become docking sites for other enzymes such as PLCγ1 that ultimately lead to the activation of upstream cellular enzymes (e.g., Ras and MAPK pathways) (C). These enzymes then activate transcription factors that promote expression of various genes involved in proliferation and T-cell responses.

Costimulation

Recognition of the antigenic peptide–MHC complex through TCR binding is usually not sufficient alone to generate a response in a naïve T cell. Additional signals through so-called costimulatory pathways are required for optimal T-cell activation. , In fact, receipt of TCR complex signaling, often referred to as signal 1, in the absence of costimulation or signal 2 not only fails to achieve activation but also can lead to a state of inaction or anergy ( Fig. 25.13 ). An anergic T cell is rendered unable to respond even if given both of the appropriate stimuli. This characteristic of the immune system is thought to be one of the major mechanisms in tolerance to self-antigens in the periphery, crucial in the prevention of autoimmunity. Researchers have exploited this discovery using antibodies or receptor fusion proteins designed to block interactions between key costimulatory molecules at the time of antigen exposure. Much of the research to date has focused on the interactions of two costimulatory pathways, the CD28/B7 pathway (Ig-like superfamily members) and CD40/CD154 pathway (TNF/TNFR superfamily members). There have been, however, many additional pairings within these same families and others that have been found to have distinct roles in costimulatory function ( Table 25.2 ).

Fig. 25.13, T-cell costimulation. Naïve T cells require multiple signals for efficient activation. (A) Signal 1 occurs when the T-cell receptor (TCR) recognizes its putative major histocompatibility complex–peptide combination. In the absence of any additional signals, there is an aborted response or anergy, a state in which the cell is no longer available for stimulation. (B) TCR signaling in conjunction with signals received through costimulatory molecules (e.g., B7 molecules), signal 2, promotes effective T-cell activation and function.

Table 25.2
Costimulatory molecules.
Receptor Distribution Ligand Distribution Principal Effects and Functions
CD28 T cells CD80/CD86 Activated APCs Lowers the threshold for T-cell activation
Promotes survival, ↑ antiapoptotic factors
Promotes Th1 phenotype
CD40 Dendritic cells, B cells, macrophages, endothelial cells CD154 T cells, soluble platelets Induces CD80/CD86 expression on APCs
CD27 T cells, NK cells, B cells CD70 Thymic epithelium, activated T cells, activated B cells, mature dendritic cells Enhances T-cell proliferation and survival
Acts after CD28 to sustain effector T-cell survival
Influences secondary responses more than primary
Promotes B-cell differentiation and memory formation
CD30 Activated T cells, activated B cells CD153 B cells, activated T cells Maintains survival of primed and memory T cells
Promotes Th2 > Th1
CD95 (Fas) T cells, B cells, APCs, stromal cells CD178 (FasL) T cells, APCs, stromal cells Involved in peripheral T-cell homeostasis through “fratricide,” may deliver costimulatory signal
CD134 (OX40) Activated T cells
CD4+ > CD8+
CD252 (OX40L) Activated T cells, mature dendritic cells, activated B cells Important for CD4+ T-cell expansion and survival
↑ Antiapoptotic factors
Functions after CD28 to sustain CD4+ T-cell survival
Enhances cytokine production
Augments effector and memory CD4+ T-cell function
Promotes Th2 > Th1
CD137 (4-1BB) Activated T cells
CD8+ > CD4+
Monocytes, follicular dendritic cells, NK cells
4-1BBL Mature dendritic cells, activated B cells, activated macrophages Sustains rather than initiates CD8+ T-cell responses
Functions after CD28 to sustain T-cell survival
Important in antiviral immunity
Promotes CD8+ effector function and cell survival
CD152 (CTLA-4) Activated T cells CD80/CD86 Activated APCs Higher affinity for CD80/CD86 than CD28, inhibits T-cell response
HVEM T cells, monocytes, immature dendritic cells CD258 (LIGHT) Activated lymphocytes, immature dendritic cells, NK cells Augments T-cell responses, CD8+ > CD4+
Promotes dendritic cell maturation
CD272 (BTLA) Activated T cells, B cells, dendritic cells Negative costimulator, inhibits IL-2 production
BTLA remains expressed on Th1 but not Th2
CD160 NK cells, cytolytic CD8+ T cells, γδ T cells Negative regulator of CD4+ T-cell activation
Inhibits proliferation and cytokine production
CD265 (RANK) Dendritic cells CD254 (TRANCE) Activated T cells
CD4+ > CD8+
Enhances dendritic cell survival, upregulates Bcl-xl, possibly enhances IFN-γ production
CD279 (PD-1) T cells CD274 (PD-L1) T cells, B cells, APCs, some parenchymal cells Inhibits activation, proliferation, and acquisition of effector cell function
Th1 > Th2
CD273 (PD-L2) Dendritic cells, macrophages Inhibits activation, proliferation, and acquisition of effector cell function
Th2 > Th1
CD278 (ICOS) Activated T cells, memory T cells CD275 (ICOSL) Dendritic cells, B cells, macrophages Promotes survival and expansion of effector T cells, possibly promotes Th2 responses
GITR Tregs, CD8+ T cells, B cells, macrophages GITRL B cells, dendritic cells, macrophages, endothelial cells Marker for Tregs, allows proliferation of Tregs
Promotes T-cell proliferation and cytokine production
Negative regulator for NK function
APC , Antigen-presenting cell; BTLA , B and T lymphocyte–associated; CTLA , cytotoxic T lymphocyte–associated; GITR, glucocorticoid-induced tumor necrosis factor receptor; GITRL, glucocorticoid-inducted tumor necrosis factor receptor ligand; HVEM , herpes virus entry mediator; ICOS , inducible costimulator; ICOSL, inducible costimulator ligand; NK, natural killer; PD , programmed death; RANK, receptor activator of NFκB; Treg , regulatory T cell.

CD28, present on T cells, and the B7 molecules CD80 and CD86 on APCs were among the first costimulatory molecules to be described. Ligation of CD28 is necessary for optimal IL-2 production and can lead to the production of additional cytokines, such as IL-4 and IL-8, and chemokines, such as Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES), as well as protect T cells from activation-induced apoptosis through the upregulation of antiapoptotic factors such as Bcl-X L and Bcl-2. CD28 is expressed constitutively on most T cells, whereas the expression of CD80 and CD86 is largely restricted to professional APCs, such as dendritic cells, monocytes, and macrophages. The kinetics of CD80/CD86 expression is complex, but they are typically increased with the induction of the immune response. Another ligand for CD80 and CD86 is CTLA-4 (CD152). This molecule is upregulated and expressed on the surface of T cells after activation, and it binds CD80/CD86 with 10 to 20 times greater affinity than CD28. CTLA-4 has been shown to have a negative regulatory effect on T-cell activation and proliferation, an observation supported by the fact that CTLA-4–deficient mice develop a lethal lymphoproliferative disorder. The negative regulatory effect of CTLA-4 is mediated through both cell intrinsic activation of intracellular phosphatases and cell extrinsic mechanism in which CTLA-4 binding actually removes CD80/CD86 from the surface of the APC, thereby limiting the availability of ligands for CD28 costimulation. The therapeutic potential of costimulation blockade was first made apparent through the development of CTLA-4–Ig, an engineered fusion protein composed of the extracellular portion of the CTLA-4 molecule and a portion of the human Ig molecule. This compound binds CD80 and CD86 and prevents costimulation through CD28. Several clinical trials in autoimmunity have demonstrated the efficacy of CTLA-4–Ig (abatacept). More recently, a higher-affinity, second-generation version, belatacept, has been tested with success as a replacement for calcineurin inhibitors and was approved in 2011 for kidney transplant recipients. ,

Closely related to the CD28/B7 pathway is the CD40/CD154 (CD40L) pathway. Evidence for the crucial role of the CD40/CD154 pathway in the immune response came to light after the observation that hyper-IgM syndrome results from a mutational defect in the gene encoding CD154. In addition to defects in the generation of T cell–dependent antibody responses, patients with hyper-IgM syndrome also have defects in T cell–mediated immune responses. CD40 is a cell surface molecule expressed on endothelium, B cells, dendritic cells, and other APCs. Its ligand, CD154, is primarily found on activated T cells. Upregulation of CD154 after TCR signaling allows signals to be sent to the APC through CD40; in particular, it is a critical signal for B-cell activation and proliferation. CD40 binding is required for APCs to stimulate a cytotoxic T-cell response. It leads to the release of activating cytokines, particularly IL-12, and the upregulation of B7 molecules. It also initiates innate functions of APCs, including nitric oxide synthesis and phagocytosis. Interestingly, CD154 is also released in soluble form by activated platelets. Thus, sites of trauma that attract activated platelets simultaneously recruit the ligand required to activate tissue-based APCs, providing a link between innate and acquired immunity. Antibody preparations against CD154 have shown great promise in experimental models, but initial clinical trials were halted because of concern for unexpected thrombotic complications. There continues to be hope that anti-CD154 antibodies that bind distinct epitopes, Fc-silent domain antibodies devoid of cross-linking abilities, or antibodies directed toward CD40 may circumvent this issue (See “Immunosuppression” section).

Since earlier investigations, multiple other pairings of molecules have been characterized and shown to demonstrate costimulatory or coinhibitory activity. It is the sum of these positive costimulatory and negative coinhibitory signals that shapes the character and magnitude of the T-cell response. CD278 (inducible costimulator, or ICOS) is a CD28 superfamily expressed on activated T cells, and its ligand, CD275 (ICOSL or B7-H2), is expressed on APCs. Unlike CD28, ICOS is not present on naïve T cells, but instead expression is upregulated after T-cell activation and persists on memory T cells. ICOS can function to boost activation of effector T cells in general but in particular plays a critical role in the function of T follicular helper (Tfh) cells, a specialized CD4+ T-cell subset involved in the germinal center reaction and generation of class-switched antibody. Another member of the CD28 superfamily, programmed death (PD-1) (CD279), and its ligands PD-L1 (CD274) and PD-L2 (CD273), both B7 family members, have been shown to be involved in negative regulation of cellular immunity. More recently, coinhibitory molecules PD-1H (also known as VISTA for V domain Ig suppressor of T cell activation) and B and T lymphocyte–associated (BTLA) have joined this list. Several members of the TNF/TNFR superfamily have been shown to play important roles in T-cell costimulation. These include CD134/CD252 (OX40/OX40L), CD137/CD137L (4-1BB/4-1BBL), CD27/CD70, CD95/CD178 (Fas/FasL), CD30/CD153, receptor activator of NFκB/TNF-related activation-induced cytokines (RANK/TRANCE), and others. Furthermore, members of the CD2 family function in both costimulatory (i.e., CD2) and coinhibitory (i.e., 2B4) roles during the execution of an alloimmune response. Finally, the T cell–Ig mucin-like family of molecules has been shown to play important coinhibitory roles during alloimmunity, both on effector cells and on Tregs.

In addition to the multitude of costimulatory molecules, many other adhesion molecules expressed on the cell surface (intercellular adhesion molecule, selectins, integrins) control the movement of immune cells through the body, regulate their trafficking to specific areas of inflammation, and nonspecifically strengthen the TCR-MHC binding interaction. They differ from costimulation molecules in that they enhance the interaction of the T cell with other cell types and antigen without directly influencing the quality of the TCR response. There are two main families of cellular adhesion molecules within the immune system: the selectins and the integrins. The selectin family of adhesion molecules is responsible for “rolling,” the initial attachment of leukocytes to vascular endothelial cells at sites of tissue injury and inflammation before their firm adhesion (mediated by integrin binding). The selectin family of proteins is composed of three closely related molecules, each having differential expression on immune cells: L-selectin is expressed on leukocytes, P-selectin is expressed on platelets, and E-selectin is expressed on endothelium. Structurally, all selectins share an amino-terminal lectin domain that interacts with a carbohydrate ligand, an epidermal growth factor–like domain, and two to nine short repeating units that share homology with sequences found in some complement-binding proteins. In contrast to most other adhesion molecules that also possess some signaling or costimulatory functionality, selectins function solely to facilitate leukocyte binding to vascular endothelium. This selectin-mediated loose binding is converted into tight adhesion after activation of leukocyte integrins. Integrins are transmembrane receptors that serve as bridges for cell-cell as well as for cell–extracellular matrix interactions. Many are expressed constitutively on cells of the immune system (i.e., leukocyte function antigen 1) but on sensing inflammatory cytokine or chemokine signals, such as IL-8, are induced to change conformation that results in higher avidity interaction with integrin ligands, resulting in leukocyte extravasation into inflamed tissue. Both selectins and integrins are potential therapeutic targets to inhibit access of donor-reactive T cells into the allograft and weaken proimmune interactions.

T-Cell Effector Functions

During thymic education, most T cells initially express both CD4 and CD8 molecules, but subsequently, T cells become either CD4+ or CD8+, depending on which MHC class they restrict to. Thus, these accessory molecules govern which type of MHC and by extension which types of cells a given T cell can interact with and evaluate. Because there is nearly ubiquitous expression of class I MHC, all cell types are surveyed. These class I molecules display peptides that are generated within the cell (e.g., peptides from normal cellular processes or from internal viral replication). T cells responsible for inspecting all cells express the accessory molecule CD8, which in turn binds to class I and specifically stabilizes a TCR interaction with a class I–presented antigen. Thus, CD8+ T cells evaluate most cell types and mediate destruction of altered cells. Appropriately, they have been termed cytotoxic T cells.

APCs are the predominant cell type that expresses class II MHC molecules in addition to class I. Class II molecules display peptides that have been sampled from surrounding extracellular spaces through phagocytosis and thus usually represent the presentation of newly acquired antigen. Cells initiating an immune response need to have access to this newly processed antigen. CD4 binds class II MHC and stabilizes the interaction of the TCR with the class II–peptide complex. Thus, under physiologic conditions, CD4+ T cells are first alerted to an invasion of the body by hematopoietically derived APCs that present their newly acquired antigen in the form of processed peptide in a class II molecule. As a consequence of their MHC restriction, these subpopulations of T cells have several different functions. CD4+ T cells typically contribute to the response in a helper or regulatory role, whereas CD8+ T cells are much more likely to play a part in cell elimination through cytotoxic functions.

After activation, CD4+ T cells initially play a critical role in the expansion of the immune response. After encountering an APC that expresses the specific antigenic peptide–MHC class II pairing, the CD4+ T cell can then signal back to the APC to promote factors that allow CD8+ T-cell activation. This process is accomplished by expression of specific costimulatory molecules and the release of certain cytokines. This licensing of CD8+ T cells for cytotoxic function is a key step within the immune response. This describes in part how CD4+ T cells become helper cells. More recently, there has been further elucidation of their cellular differentiation into several well-defined Th subsets, including Th1, Th2, Th17, and Tfh cells, which are largely defined on the basis of the distinct transcription factors they express and the cytokines they elaborate ( Fig. 25.14 ). The main cytokine driving the differentiation of Th1 cells is IL-12, and mature Th1 cells mediate effector function through the release of IFN-γ and TNF. The predominant role of IFN-γ is to enhance macrophage function and activity as well as to promote cell-mediated immunity. Activated macrophages then proceed to ingest and to kill invading microbes, and at the same time, the acquired immune system is directed to produce antibodies that promote opsonization, thereby enhancing the overall process. Th2 cell differentiation, in contrast, is driven by the presence of IL-4 and results in release of IL-4, IL-5, IL-10, and IL-13, which ultimately inhibit macrophage activation and promote IgE production and eosinophil activation. Th17 cells are an inflammatory CD4+ subset that plays a major role in the protective immune response against fungal pathogens and extracellular bacteria. Th17 cells are generated in the presence of transforming growth factor-β (TGF-β) and IL-6 and are potent secretors of the inflammatory cytokines IL-17 and IL-23. Interestingly, in addition to their role in protective immunity, Th17 cells have been associated with several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and psoriasis, and several immunomodulatory therapies are being developed to impair their activity in these patients. Finally, Tfh cells are ICOS+ PD-1+ cells that home to lymphoid germinal centers by virtue of their expression of the chemokine receptor CXCR5, where they provide help for the generation of class-switched, high-affinity IgG responses. Tfh cells provide this help in the form of CD154 expression and the secretion of IL-21.

Fig. 25.14, T-cell subsets. Naïve CD4+ T cells may differentiate into distinct subsets of effector cells in response to antigen, costimulatory, or coinhibitory signals and cytokines. Th1 cells produce interferon-γ (IFN-γ) , which activates macrophages to kill intracellular microbes. Th2 cells produce cytokines (interleukin [IL] -4, IL-5, and others) that stimulate immunoglobulin E production and activate eosinophils in response to parasitic infection. Th17 cells secrete IL-17 and IL-22; they play an important role in responses to fungi and contribute to several autoimmune inflammatory diseases. Tfh cells produce IL-21 and provide help to B cells for antibody production.

An important feature of these CD4+ Th cells is the ability of one subset to regulate the activity of the other. For example, IL-10 produced by Th2 cells and Tregs negatively regulates transcription of IFN-γ mRNA. Thus, the initial steps in differentiation depend greatly on the surrounding immunologic milieu, which ultimately influences the character of the immune response. Furthermore, more recent fate mapping studies have revealed a high degree of plasticity between Th subsets, demonstrating that cells of one Th subset can under certain conditions transdifferentiate into another Th subset.

Another subset of CD4+ T cells that has been described to play a critical role in the ability of the immune system to temper its response is the Treg population. Tregs suppress immune responses either through direct cell-cell contact with effector cells or indirectly through their interaction with APCs. These cells not only have the ability to suppress cytokines, adhesion molecules, and costimulatory signals but are also able to focus this response by expression of integrins, which allow Tregs to home to the location of immune engagement. The most extensively studied population of Tregs are those CD4+ T cells that express CD25 (the high-affinity α chain of the IL-2 receptor). CD4+CD25+ cells express the transcription factor Foxp3, a protein that has been shown to be both necessary and sufficient for the differentiation of CD4+ T cells into Tregs. Indeed, both mice and humans that lack functional Foxp3 molecule develop severe systemic autoimmunity. Thus, CD4+CD25+ Foxp3+ T cells have been the target of numerous attempts to alter immune function and are being tested in clinical trials of cellular immunotherapy to control graft rejection after transplantation and to mitigate autoimmunity. Foxp3+ Tregs develop during T-cell thymic development after recognition of self-antigen in the thymus (with signal strength that is not sufficient to induce negative selection). These so-called natural Tregs (also termed thymic Tregs) express a TCR repertoire distinct from that of conventional T cells and are important for maintaining immune homeostasis and preventing autoimmunity. However, Foxp3+ Tregs can also develop extrathymically during the course of an immune response, and studies have shown that these cells are elicited by stimulation with low-dose antigen or under conditions of limited CD154 costimulation. These so-called induced Tregs (also termed peripheral Tregs) are highly specific for the antigen by which they were elicited and thus may be more potent suppressors of autoimmunity and transplant rejection when used as cellular immunotherapy.

Unlike CD4+ T cells, CD8+ T cells function primarily to eliminate infected or defective cells. As mentioned before, licensing occurs through APC interactions, and subsequent cell killing occurs by either a calcium-dependent secretory mechanism or a calcium-independent mechanism that requires direct cell contact. In the calcium-dependent mechanism, the rise in intracellular calcium after activation triggers exocytosis of cytolytic granules. These granules contain a lytic protein called perforin and serine proteases called granzymes. Perforin polymerization creates defects in the target cell’s membrane, allowing granzyme activity to lyse the cell. In the absence of calcium, T cells can induce apoptosis of a target cell through a Fas-dependent mechanism. It occurs when surface CD95 (Fas) is bound by its ligand CD178 (FasL). Cytotoxic T cells upregulate CD178 on activation. This, in turn, binds CD95 on target cells, resulting in programmed cell death.

Cytokines

Cell surface receptors provide an interface through which adjacent cells can transfer signals vital to the immune response. Whereas this cell-to-cell contact is a critical component of cellular communication, soluble mediators are also used extensively to accomplish similar tasks. These polypeptides, termed cytokines, are critical to the development and function of both the innate and acquired immune processes. The action of cytokines, also known as interleukins (see Table 25.1 ), may be autocrine (on the same cell) or paracrine (on adjacent cells), but it is usually not endocrine. They are released by multiple cell types and may function to activate, to suppress, or even to amplify the response of adjacent cells. The prototypical cytokine of T-cell activation is IL-2. Once a given T cell encounters its specific antigen in the setting of appropriate costimulation, it will subsequently produce and release IL-2 as well as other cytokines that will influence any cell within its vicinity. As mentioned before, Th cellular subsets are differentiated on the basis of the pattern of cytokine expression. Th1 cells, which mediate cytotoxic responses such as delayed-type hypersensitivity, express IL-2, IL-12, IL-15, and IFN-γ. Th2 cells support the development of humoral or eosinophilic responses and consequently express IL-4, IL-5, IL-10, and IL-13. Th17 cells, a more recently described subset, are distinguished by their production of IL-17, IL-21, and IL-22.

Cytokine receptors are now known to function through Janus kinase (JAK) signal transduction proteins. They convey signals to signal transducers and activators of transcriptions (STATs), DNA-binding proteins that translocate to the nucleus to influence gene transcription. As is the case with most of the immune response, this pathway is tightly regulated. For example, suppressors of cytokine signaling proteins act in a negative feedback loop to inhibit STAT phosphorylation by binding and inhibiting JAKs or competing with STATs for phosphotyrosine-binding sites on cytokine receptors. There is evidence emerging for the involvement of suppressors of cytokine signaling proteins in human disease, which raises the possibility that therapeutic strategies based on the manipulation of suppressors of cytokine signaling activity might be of clinical benefit.

One particular subset of cytokines is termed chemokines for their ability to influence the movement of leukocytes and to regulate their migration to and from secondary lymphoid organs, blood, and tissues. Chemokines, or chemotactic cytokine chemokines, are a unique set of cytokines that are structurally homologous, 8- to 10-kDa polypeptides that have a varying number of cysteine residues in conserved locations that are key to forming their three-dimensional shape. The two major families are CC chemokines (also called β), in which the two defining cysteine residues are adjacent, and the CXC (or α) chemokine family, in which these residues are separated by one amino acid. There are numerous CC (1–28) and CXC (1–16) chemokines with various targets and functions. The CC and CXC chemokines are produced not only by leukocytes but also by several other cell types, such as endothelial and epithelial cells as well as fibroblasts. In many circumstances, these cell types are stimulated to produce and to release the chemokines after recognition of microbes or other tissue injury signals detected by the various cellular receptors of the innate immune system discussed earlier. Although there are exceptions, recruitment of neutrophils is mainly mediated by CXC chemokines, monocyte recruitment is more dependent on CC chemokines, and lymphocyte homing is modulated by both CXC and CC chemokines. Chemokine receptors are G protein–coupled receptors containing seven transmembrane domains. These receptors initiate intracellular responses that stimulate cytoskeletal changes and polymerization of actin and myosin filaments, resulting in increased cell motility. These signals may also change the conformation of cell surface integrins, increasing their affinity for their ligands, thus affecting migration, rolling, and diapedesis. Thus, chemokines work in concert with adhesion molecules, such as integrins and selectins, and their ligands to regulate the migration of leukocytes into tissues. Distinct combinations of chemokine receptors are expressed on various types of leukocytes, resulting in the differential patterns of migrations of those leukocytes. Chemokines or their receptors have been exploited by viruses such as human immunodeficiency virus (HIV) (CCR5 and CXCR4 expressed on CD4 T cells are used as entry coreceptors) or used as therapeutic targets, such as CCR7 (FTY720 or fingolimod, an S1PR1 modulator, promotes sequestration of T cells in the lymph node through a CCR7-dependent mechanism; see later). In addition to cytokines, there are a host of other soluble, small-molecule mediators that are released during an immune response or with other types of inflammation. These function to increase blood flow to the area and to improve the exposure of the area to lymphocytes and the innate immune system.

B Cells and Antibody Production

The primary lymphoid organ responsible for B-cell differentiation is the bone marrow. Similar to all other cells in the immune system, B cells are derived from pluripotent bone marrow stem cells. IL-7, produced by bone marrow stromal cells, is a growth factor for pre-B cells. IL-4, IL-5, and IL-6 are cytokines that stimulate the maturation and proliferation of mature primed B cells. The principal function of B cells is to produce antibodies against foreign antigens (i.e., the humoral immune response) as well as to be involved in antigen presentation. B-cell development occurs through several stages, each stage representing a change in the genomic content at the antibody loci. During the differentiation process, there is an elegant series of nucleotide rearrangements that results in a nearly unlimited array of specificities, allowing for a diverse recognition repertoire.

Similar to the T cell and its receptor, each B cell has a unique membrane-bound receptor through which it recognizes specific antigen. In the case of the B cell, this Ig molecule may also be produced in a secreted form that can interact with the extracellular environment far from its cellular origin. Each mature B cell produces antibody of a single specificity.

Each antibody is composed of two heavy chains and two light chains. Five different heavy chain loci (μ, γ, α, ε, and δ) are found on chromosome 14 and two light chain loci (κ and λ) are located on chromosome 2. Each chain is composed of V, D, J, and C regions, which are brought together randomly by the RAG1 and RAG2 complex to form a functional antigen receptor. Ig has a basic structure of four chains, two of which are identical heavy chains and two of which are identical light chains ( Fig. 25.15 ). Both heavy and light chains have a constant region as well as a variable, antigen-binding region. The antigen-binding site is composed of both the heavy and light chain variable regions. The ability of antibody to neutralize microbes is entirely a function of this antigen-binding region.

Fig. 25.15, Structure of immunoglobulin (Ig) . (A) Representation of secreted immunoglobulin G (IgG) molecule. The antigen-binding regions are formed by the variable regions of both light (V L) and heavy (V H ) chains. The constant region of the heavy chain (C H) is responsible for the Fc receptor and complement-binding sites. (B) Schematic diagram of membrane-bound immunoglobulin G (IgM) . The membrane form of the antibody has C-terminal transmembrane and cytoplasmic portions that anchor the molecule in the plasma membrane. (C) X-ray crystallography representation of IgG molecule. Heavy chains are colored blue and red , light chains are colored green , and carbohydrates are shown in gray .

In humans, there are nine different Ig subclasses or isotypes: IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgE. Heavy chain use defines the subtype of any given antibody. Whereas the variable regions are involved in antigen binding, the constant regions have functionality as well. The fragment crystallizable region or Fc region is in the tail portion composed of the two heavy chain constant regions. It interacts with Fc receptors on phagocytic cells of the innate immune system to facilitate opsonization and subsequent destruction of the antigen to which the antibody is bound, as well as facilitating antigenic peptide processing. The Fc portion of IgM and some classes of IgG also serves to activate complement. Distinct immune effector functions are assigned to each isotype. IgM and IgG antibodies provide a pivotal role in the endogenous or intravascular immune response. IgA is primarily responsible for mucosal immunity and is largely confined to the gastrointestinal and respiratory tracts. Resting B cells that have not yet been exposed to antigen express IgD and IgM on their cell surface. After interaction with antigen, the first isotype produced is IgM, which is efficient at binding complement to facilitate phagocytosis or cell lysis. Further activation and differentiation of the B cell occur after interactions with CD4+ Tfh cells. B cells undergo isotype switching, which results in a decrease in IgM titer with a concomitant rise in IgG titer. Unlike the TCR, the Ig loci undergo continued alteration after B-cell stimulation to improve the affinity and functionality of the secreted antibody. A primed B cell may undergo further mutation within the variable regions that leads to increased affinity of antibody, termed somatic hypermutation. Such B cells are retained to provide the ability to generate a more vigorous response if the antigen happens to be re-encountered ( Fig. 25.16 ).

Fig. 25.16, B-cell differentiation. Naïve B cells recognize their specific antigen as it binds to surface-bound antibody. Under the influence of helper T cells, costimulatory signals, and other stimuli, B cells become activated and clonally expand, producing many B cells of the same specificity. They also differentiate into antibody-secreting cells, plasma cells. Some of the activated B cells undergo heavy chain class switching and affinity maturation. Ultimately, a small subset become long-lived memory cells, primed for future responses.

B-cell activation occurs when antigen is bound by two surface antibodies (or a multimeric form of antibody) and the antibodies are brought together on the cell surface in a process known as cross-linking. This event stimulates B-cell activation, proliferation, and differentiation into various B-cell subsets and antibody-secreting plasma cells. As for T cell, the threshold for B-cell activation is high. This can be lowered 100-fold by costimulatory signals received by the transmembrane complex CD19-CD21. B cells can also internalize antigens bound to surface antibodies and process them for presentation to T cells, thus participating in antigen presentation. As discussed earlier, B cells may provide and receive certain costimulatory signals. For example, B cells express CD40, and when bound by CD154 expressed on activated T cells, the result is upregulation of B7 molecules on the B cell and delivery of important costimulatory signals to T cells as well.

Plasma cells reside in the bone marrow and are distinguished histologically by their hypertrophied Golgi apparatus resulting from their high degree of protein synthesis. They secrete large amounts of monoclonal (single specificity) antibody and exhibit phenotypic and functional characteristics distinct from other B cells that are the focus of therapeutic strategies to target plasma cells either for oncologic purposes or for the control of alloantibodies in transplantation.

In addition to being secreted in an adaptive manner after exposure to antigen, antibody can also exist as part of the innate or natural immune repertoire in the circulation for initial response to common pathogens. Antigen exposure generally leads to B-cell affinity maturation and isotype switching and produces high-affinity IgG antibodies. Naturally occurring antibodies, however, are generally IgM antibodies with low affinity and are generally thought to bind to a broad array of carbohydrate epitopes found on many common bacterial pathogens. Natural antibody is responsible for ABO blood group antigen responses and discordant xenograft rejection (see “Xenotransplantation”).

This portion of the chapter has reviewed the various components of the immune system and their function in the context of conventional, infectious immune challenges. The next sections address the unique nature of the immune response to transplanted tissue and organs.

Transplant Immunity

The study of modern transplant immunology is traditionally attributed to the experiments of Sir Peter Medawar fueled by attempts to use skin transplantation as a treatment for burned aviators during World War II. While monitoring the victims with autologous (syngeneic) and homologous (allogeneic) skin grafts, he noted that not only did all allogeneic grafts universally fail promptly, but also secondary grafts from the same donor were rejected even more vigorously, suggesting immune involvement. He pursued this hypothesis with extensive experiments in rabbits, wherein he confirmed his previous observation and noted the presence of a heavy lymphocyte infiltrate in the rejecting graft. It was N.A. Mitchison, working in the early 1950s, who definitively identified a role for lymphocytes in the rejection of foreign tissue. Subsequent studies in tumor immunology as well as work by Snell using strains of genetically identical mice identified the genetic basis for graft rejection as the MHC, known in humans as HLA and in mice as the H-2 locus. These series of experiments during a short period of several years demonstrated that rejection of transplanted tissue was an immunologic process, implicated lymphocytes as the principal effector cells, and identified the MHC as the primary source of antigen in the rejection response. These pivotal studies laid the groundwork for the transition of transplantation from the experimental to the clinical realm.

Whereas the technical skill for the transplantation of skin and other organs had been available for some time, the vigorous rejection of allografts had prevented its widespread use for many years. It was not until 1954, after Medawar’s critical studies had been published, that the first successful organ transplantation was performed. Despite Medawar’s claim that the “biological force” responsible for rejection would “forever inhibit transplantation from one individual to another,” Joseph Murray, a surgeon-scientist, persevered in his pursuit of making clinical transplantation a reality. At the time, there was evidence to suggest that the overall immunologic barrier was lacking between identical twins, and coincidentally, Murray was busily perfecting a surgical technique for kidney transplantation in dogs. In 1954, the opportunity presented itself to test the hypothesis. Richard Herrick, who had incurable kidney damage, was the first candidate, and his identical sibling, Ronald, was willing to donate a kidney for transplantation to his brother. Murray confirmed the lack of immunologic reactivity between the two brothers by first placing skin grafts from each twin onto the other. Once he confirmed the lack of a response, he used the technique that he had perfected in the canine model, performing the first successful kidney transplant between identical twins in December 1954. The operation proceeded without complication, and the kidney functioned well without the need for immunosuppression. Despite this landmark advance in transplantation, the majority of individuals in need of a transplant did not have an identical twin to donate an organ. Thereafter, the focus of the field was appropriately directed toward the development of methods to control the rejection response.

During the 1950s and 1960s, several discoveries were made that were of the utmost importance for future successes in transplantation. Following Gorer and Snell’s description of the murine MHC system, Jean Dausset described the equivalent in humans using antibodies developed against HLA. This led to the first serologically based typing system for human transplant antigens. Snell and Dausset shared the Nobel Prize in 1980 for their observations.

In the late 1960s, Paul Terasaki reported on the significance of preformed antibody directed against donor MHC molecules and its impact on kidney graft survival. He developed the microlymphocyte cytotoxicity test, allowing pretransplantation detection of recipient-derived antidonor antibody. This formed the basis for the physical crossmatch assay that is used today to screen potential donor-recipient pairings. These techniques, along with the development of new immunosuppressive compounds, including 6-mercaptopurine and azathioprine, led to the first successful kidney transplantation between relatives who were not identical twins and also to the first successful transplant using a kidney from a deceased donor.

Although early attempts at immunosuppression permitted extended allograft survival in selected patients, both the reproducibility and durability of results were far from adequate. In the 1970s, investigators sought novel treatments to improve the success rate for transplantation; these modalities included thoracic duct drainage and the use of antilymphocyte serum. Despite these efforts, the results for kidney transplantation remained poor, with the best centers achieving 1-year survival rates of 70% for living related kidney grafts and 50% for deceased donor kidney transplants. Then a chance discovery of a promising agent from a fungal isolate dramatically changed the outlook for kidney and other types of transplantation. Jean-François Borel identified an active metabolite, cyclosporine A (CsA), that showed selective in vitro inhibition of lymphocyte cultures but no significant myelotoxic effects. Promising results in dogs eventually led to clinical trials in humans, and the modern era of transplantation had begun.

The introduction of CsA ushered in the most dramatic improvement in the field of transplantation. Liver and heart transplant survival rates doubled, and the improved immunosuppression encouraged transplant teams around the world to begin broader investigational use, transplanting lung, small bowel, and pancreas. Now, with the use of CsA and newer agents such as tacrolimus, 1-year graft survival has exceeded 90% for virtually all organs except the small intestine. Despite the discovery and clinical introduction of ever increasingly potent immunosuppressants, the field of transplantation has many areas in need of improvement. Drug-related side effects and the intractable problem of chronic rejection still plague practitioners. One area of focus of current research is the development of a clinically applicable strategy to promote “transplantation tolerance,” thereby eliminating the pitfalls and shortcomings of current immunosuppressive therapy.

Rejection

There are three classic histopathologic definitions of allograft rejection that are based on not only the predominant mediator but also the timing of the process ( Fig. 25.17 ).

  • 1.

    Hyperacute rejection occurs within minutes to days after transplantation and is primarily mediated by preformed antibody.

  • 2.

    Acute rejection is a process mediated most commonly by T cells but is often accompanied by an acquired antibody response and generally occurs within the first few weeks to months of transplantation but can occur at any time.

  • 3.

    Chronic rejection is a common contributing cause of long-term allograft loss and is an indolent fibrotic process that occurs over months to years. It is thought to be secondary to chronic immunologic injury from both T and B cell–mediated processes (including antidonor antibodies) but is difficult to completely separate from nonimmune mechanisms of chronic organ damage (e.g., drug toxicity and cardiovascular comorbid diseases).

Fig. 25.17, Mechanisms of rejection. (A) Hyperacute rejection occurs when preformed antibodies react with donor antigens on the vascular endothelium of the graft. Subsequent complement activation triggers rapid intravascular thrombosis and graft necrosis. (B) Acute cellular rejection is predominantly mediated by a cellular infiltrate of alloreactive T cells that attack donor cells both in the endothelium and in the parenchyma. Alloreactive antibodies can also develop after engraftment and lead to antibody-mediated rejection that contributes to parenchymal and vascular injury. (C) Chronic rejection is characterized by graft arteriosclerosis and fibrosis. Immune- and non–immune-mediated mechanisms are responsible for abnormal proliferation of cells within the intima and media of the vessels of the graft, eventually leading to luminal occlusion. (Adapted from Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology . 9th ed. Philadelphia: Saunders Elsevier; 2018.) APC , Antigen-presenting cell.

Hyperacute Rejection

Although essentially untreatable, hyperacute rejection is nearly universally avoidable with the proper use of the lymphocytotoxic crossmatch or other means of detecting antidonor antibodies before transplantation. This form of rejection occurs when preformed antibodies against the donor, commonly referred to as donor-specific antibodies, are present in the recipient’s system before transplantation. These antibodies may be the result of “natural processes,” such as the formation of antibody to blood group antigens, or the product of prior exposure to antigens with similar enough specificities as those expressed by the donor that cross-reactivity can occur. In the latter, sensitization is usually the result of prior transplantation, transfusion, or pregnancy but may also result from prior environmental antigen exposure. As expected, hyperacute rejection can occur within the first minutes to hours after graft reperfusion. Antibodies bind to the donor tissue or endothelium and initiate complement-mediated lysis and endothelial cell activation, resulting in a procoagulant state and immediate graft thrombosis. On histologic evaluation, there may be platelet and fibrin thrombi, early neutrophil infiltration, and positive staining for the complement product C4d on the endothelial lining of small blood vessels ( Fig. 25.18 ). Thankfully, this type of rejection is avoidable with pretransplantation testing by current crossmatch assays.

Fig. 25.18, Histology of rejection. (A) Hyperacute rejection of a kidney allograft with characteristic endothelial damage, thrombus, and early neutrophil infiltrates. (B) Acute cellular rejection of kidney with inflammatory cells within the connective tissue around the tubules and between tubular epithelial cells. (C) Acute antibody-mediated rejection of kidney allograft with inflammatory reaction within a graft vessel resulting in endothelial disruption (arrow) . (D) C4d deposition in the small vessels of the transplanted kidney. (E) Chronic rejection in a transplanted kidney with graft arteriosclerosis. The vascular lumen has been replaced with smooth muscle cells and fibrotic response.

Similar to the lymphocytotoxicity assay described previously that is used for MHC class I typing, the physical crossmatch is performed by mixing cells from the donor with serum from the recipient and the addition of complement if needed. Lysis of the donor cells indicates that antibodies directed against the donor are present in the recipient’s serum; this is called a positive crossmatch. Thus, a negative crossmatch assay coupled with proper ABO matching will effectively prevent hyperacute rejection in 99.5% of transplants. Newer crossmatch techniques have become increasingly sophisticated, including those directed at both class I and class II antibodies, flow cytometric techniques, and bead-based screening assays to exclude non-HLA antibodies. As a given patient’s sensitivity status may change over time, a more common technique for screening a patient’s sensitization status is to screen a potential recipient’s serum against a panel of random donor cells representing the anticipated regional donor pool. Known as the panel reactive antibody (PRA) assay, the results are expressed as a percentage of the panel within the randomly selected cell set that lyses when recipient serum is added. Thus, a nonsensitized patient would be given a score of 0%, and a highly sensitized patient might have a PRA score up to 100%. These screens can now be performed without the need for cells by using polystyrene beads coated with HLA antigens. In this situation, the laboratory detects all anti-HLA antibodies and calculates a PRA score on the basis of the expected frequency of the HLA types in the donor pool. In the event a compatible donor is not available for a highly sensitized recipient, clinical protocols exist to attempt desensitization that uses plasmapheresis or intravenous immune globulin (IVIG) to reduce circulating antibody and prevent hyperacute rejection. However, the need for desensitization is decreasing with advancements in deceased donor allocation algorithms and living donor paired donor exchange programs aimed at avoiding crossmatch-positive donor-recipient pairs.

Acute Rejection

Of the three types of rejection, only acute rejection can be successfully reversed once it is established. T cells constitute the core element responsible for acute rejection, often termed T cell–mediated rejection . There is also a form of acute rejection that is particularly aggressive and involves vascular invasion by T cells known as acute vascular rejection. Finally, a more recently recognized form of acute rejection mediated by the humoral immune system, known as antibody-mediated rejection (AMR), is discussed briefly later. With the advent of increasingly effective immunosuppression, allograft loss from acute cellular rejection has become increasingly rare. Acute rejection can occur at any time after the first few postoperative days, the time needed to mount an acquired immune response; it most commonly occurs within the first 6 months after transplantation. Without adequate immunosuppression, the cellular response will progress during the course of days to a few weeks, ultimately destroying the allograft. As described earlier, there are two main pathways through which rejection can proceed, the direct and indirect alloresponses ( Fig. 25.19 ). In either case, alloreactive T cells encounter their specific antigen (either processed donor MHC peptides indirectly presented on self MHC or directly recognized donor MHC), undergo activation, and promote similar rejection responses. The precursor frequency of T cells specific for either direct allorecognition or indirect allorecognition differs. Indirect allorecognition is theoretically similar to that of any given pathogen. Donor MHC protein is processed into peptides and presented on self MHC. The number of T cells specific for this antigen is approximately 1 in 200,000 to 1 in 500,000. Direct allorecognition, however, has a much higher precursor frequency. These T cells recognize donor MHC directly without processing ( Fig. 25.20 ). Given that T cells are selected to recognize self MHC molecules and that there are similarities between donor and recipient MHC, it is no surprise that a substantial number of T cells are alloreactive. Some estimates suggest that somewhere between 1% and 10% of all T cells are directly alloreactive. This high precursor frequency likely overwhelms many of the regulatory processes in place to control the much lower cell frequencies involved in physiologic immune responses. These alloreactive T cells, once activated, move to attack the graft. Subsequently, there is massive infiltration of T cells and monocytes into the allograft, resulting in organ injury through direct cytolysis and a general inflammatory milieu that leads to generalized parenchymal dysfunction and endothelial injury resulting in thrombosis (see Fig. 25.18 ).

Fig. 25.19, Direct versus indirect allorecognition. (A) Direct allorecognition occurs when recipient T cells bind directly to donor MHC molecules on graft cells. (B) Indirect allorecognition results when recipient antigen-presenting cells (APCs) take up donor MHC and process the alloantigen. Allopeptides are then presented on recipient (self) MHC molecules in standard fashion to alloreactive T cells. (Adapted from Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology. 9th ed. Philadelphia: Saunders Elsevier; 2018.) CTL, Cytotoxic lymphocyte; DC , dendritic cell; MHC , major histocompatibility complex.

Fig. 25.20, Molecular basis for direct allorecognition. Recipient T cells may recognize donor major histocompatibility complex (MHC) molecules directly because of the similarities between MHC alleles but become activated because only T cells strongly reactive to self MHC were deleted in the thymus through negative selection. (A) Normally, T cells encounter self MHC complexed with foreign peptide and become activated in the appropriate context. (B) T cells may encounter allogeneic MHC complexed with endogenous peptide that together resemble self MHC bound with foreign peptide. (C) Alternatively, allogeneic MHC alone may contribute to allorecognition and T-cell activation independent of self peptide.

The bulk of current immunosuppressive agents are directed toward the T cells themselves or interruption of pathways essential to their activation or effector functions. In an effort to prevent acute cellular rejection, induction therapy is generally used during the initial stages after transplantation. These agents are discussed in the subsequent section but are most often antibody therapies that serve to globally deplete or inactivate T cells during the immediate postoperative period of engraftment when ischemia-reperfusion injury is most likely to promote immune recognition. Immunosuppressive regimens are frequently designed to favor more intensive initial immunosuppression in the immediate postoperative period and are then tapered to lower, less toxic levels over time.

T cell–specific treatments lead to the prevention of acute rejection in approximately 70% of transplants, and when it does occur, it can be reversed in most cases. Similar to hyperacute rejection resulting from preformed antibody responses, T-cell presensitization will result in an accelerated form of cellular rejection mediated by memory T cells. It generally occurs within the first 2 or 3 days after transplantation and can be accompanied by a significant humoral response.

The humoral equivalent to acute cellular rejection is AMR. This occurs when offending antibodies specific for alloantigen exist in the circulation at levels undetectable by crossmatch assays or, alternatively, B-cell clones capable of producing donor-specific antibody are activated and stimulated to produce de novo alloantibodies. These antibodies are thought to bind HLA antigens within the graft, recruit innate and adaptive immune mechanisms, and acutely injure the transplanted organ ( Fig. 25.18 ). The former scenario is often seen in patients with a high PRA score that has decreased over time. Transplantation presumably leads to restimulation of memory B cells responsible for the donor-specific antibodies. The result is initial graft function, followed by rapid deterioration within the first few postoperative days. Implementation of a more aggressive immunosuppressive regimen, including higher doses of steroids combined with nonspecific antibody reduction by plasmapheresis or IVIG (nonspecific Ig), is occasionally successful in acutely reversing AMR.

Prompt recognition of acute rejection is essential to ensure optimal graft survival. Untreated rejection leads to expansion of the immune response to involve multiple pathways, some of which are less sensitive to T cell–specific therapies. In addition, damage to the allograft, particularly for kidney, pancreas, and heart, is generally accompanied by a permanent loss of function that is proportional to the magnitude of involvement. Most acute rejection episodes are initially asymptomatic until the secondary effects of organ dysfunction occur. By this point, the rejection process has proceeded to a point that it is often more difficult to reverse. Accordingly, monitoring for acute rejection is usually intense initially, particularly during the first year after transplantation. In general, any unexplained graft dysfunction should prompt biopsy and evaluation for the lymphocytic infiltration, antibody deposition, and parenchymal necrosis characteristic of acute rejection.

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