Chapter 26 Transplantation Immunobiology and Immunosuppression

Andrew B. Adams, Allan D. Kirk, Christian P. Larsen

Transplantation has revolutionized the treatment of end-stage organ failure. Today, there are over 25,000 transplantations performed annually and more than 100,000 patients are currently listed and awaiting an organ. The concept of tissue transplantation is certainly not new. As early as 800 BC, skin grafts were performed in India to conceal amputation of the nose, a punishment for adultery. History also is replete with legends and myths recounting the replacement of limbs and organs. One of the first references specifically to solid organ replacement as a therapeutic solution occurred when Tua-Ho, of China, reportedly replaced diseased organs with healthy ones approximately 200 AD. A more well-known myth of early transplantation 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. 26-1). However, it was not until the French surgeon Alexis Carrel developed a method for joining blood vessels in the late 19th century that the transplantation of organs became technically feasible and verifiable accounts of transplantation began (Fig. 26-2). He was awarded the Nobel Prize in 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”¹ that the more difficult problem of the biology would come to be solved. Forty years would pass before another set of eventual Nobel Prize winners, including Peter Medawar, would begin to define the process whereby one individual rejects another’s tissue (Fig. 26-3).² Shortly thereafter, Joseph Murray, Nobel Laureate 1990, performed the first successful renal transplant between identical twins in 1954 (Fig. 26-4).³ 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-MP and Aza. Murray and Roy Calne subsequently introduced these agents 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.” The subsequent discovery of increasingly potent 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 will provide a primer on rejection in the context of the broader immune response, review the specific agents that are used to suppress the rejection response, and provide a glimpse into the future of the field of immunology and the immune response.

FIGURE 26-1 Painting of Cosmas and Damian (15th C.), patron saints of physicians and surgeons. The legend of the Miracle of the Black Leg depicts the removal of the diseased leg of the Roman, Justinian, and replacement with the leg of a recently deceased Ethiopian man.

(Courtesy Wellcome Library, London.)

FIGURE 26-2 Triangulation technique of vascular anastomosis by Alexis Carrel.

(From Edwards WS, Edwards PD: Alexis Carrel: Visionary surgeon, Springfield, Ill, 1974, Charles C Thomas, pp 64–83.)

FIGURE 26-3 Sir Peter Medawar.

(Courtesy Bern Schwartz Collection, National Portrait Gallery, London.)

FIGURE 26-4 A, The first identical twin kidney transplantation, performed on December 23, 1954. B, Herrick twins (seated) with transplantation team.

(Courtesy Harvard Medical Library, Francis A. Countway Library of Medicine, Center for the History of Medicine, Boston.)

The Immune Response

The process of rejection did not evolve as a response to prevent the relatively recent developments in transplantation, but is part of a system that has developed over thousands of years to protect against invasion by pathogens and prevent subsequent disease. To understand the rejection process and, in particular, to appreciate the consequences of pharmacologic suppression of rejection, a general understanding of the 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 (e.g., ischemia, necrosis, trauma, certain nonhuman cell surfaces).⁴ Conversely, the acquired arm 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.

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 respond appropriately. Highly specific receptors (see 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 uses to recognize any given epitope are the T cell receptor (TCR) and B cell receptor, or antibody. In general, individual T or B lymphocytes express identical receptors, each of which only binds to a single epitope. This mechanism establishes the specificity of the acquired immune response. The antigenic encounter alters the immune system so 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, only recognize 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, is 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 will be discussed in its own section.

Major Histocompatibility Locus: Transplantation Antigens

The major histocompatibility complex (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 HLA (human leukocyte antigen) 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 will be discussed later.

MHC molecules play a role in the innate and acquired immune systems. Their predominant role, however, lies in antigen presentation within the acquired response. As noted, 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 in class I MHC and CD4⁺ T cells bind to antigen in 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. 26-5). The polymorphic proteins encoded by this locus include class I molecules (HLA-A, -B, and -C) and class II molecules (HLA-DR, -DP, and -DQ). 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 include molecules pertinent to the immune response by various mechanisms—tumor necrosis factors-α (TNF-α) and TNF-β, components of the complement cascade, nuclear transcription factor β, and HSP 70. Other conserved genes in HLA include genes necessary for class I and class II presentation of peptides, such as the peptide transporter proteins TAP-1 and TAP-2 and proteosome proteases LMP-2 and LMP-7.⁵ Although other polymorphic genes, referred to as minor histocompatibility antigens, exist in the genome outside of the HLA locus, they play a more limited role in transplant rejection and will not be 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 transplantation antigens and their biology is critical to humoral rejection.

FIGURE 26-5 Location and organization of the HLA complex on human chromosome 6. The complex is conventionally divided into three regions, I, II, and III. Class III genes are not related to class I and class II genes, structurally or functionally.

(Adapted from Klein J, Sato A: The HLA system—first of two parts. N Engl J Med 343:702–709, 2000.)

Although initially identified as transplantation 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 (e.g., macrophages, dendritic cells, B cells, activated T cells) but can be upregulated and appear on other parenchymal cells in the setting of cytokine release caused by an immune response or injury.

The importance of MHC gene products to transplantation stems from their polymorphism. Unlike most genes, which are identical in 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, -DR, -DP, and -DQ). Thus, donor-recipient pairings that are matched at all HLA loci are referred to as HLA-identical allografts and those matched at half of the HLA loci are referred to as 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, are immunologically indistinguishable, and thus do not reject. The genetics of HLA is particularly important in understanding clinical living-related donor (LRD) 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 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. Similarly, 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 (β₂M; chromosome 15) for expression. The polymorphism of each class I molecule is extreme, with 30 to 50 alleles/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 of 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 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.⁶

Class I Major Histocompatibility Complex

The three-dimensional structure of class I molecules (HLA-A, -B, and -C) was first elucidated in 1987.⁷ 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 β₂-M. 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. 26-6). Within this groove, a nine-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. The assembly of class I is dependent on association of the α chain with β₂-M 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-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.

FIGURE 26-6 Structure of the MHC class I molecule. Class I molecules are composed of a polymorphic α chain noncovalently attached to the nonpolymorphic β₂-microglobulin (β₂M). A, Schematic diagram. B, Ribbon diagram shows the extracellular structure of a class I molecule with a bound peptide.

(Adapted from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6, Philadelphia, 2010, Saunders-Elsevier.)

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 approve of ongoing protein synthesis. Interferons (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 antigen-presenting cells (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.⁸ 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 more commonly occurs when a T cell interacts directly with the nonself class I MHC, the so-called direct alloresponse.

Class II MHC

The class II molecules are products of the HLA-DR, HLA-DQ, and HLA-DP 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. 26-7).⁹ 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 α-1 and β-1 domains. The immunoglobulin-like domain is composed of the α-2 and β-2 segments. Similar to the class I immunoglobulin-like α-3 domain, there is limited polymorphism in these segments and the β-2 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 chains in combination with a temporary protein called the invariant chain.¹⁰ 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 (e.g., 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 the upregulation of class I and class II molecules locally on the recipient. The trauma of surgery and ischemia also upregulate class II molecules on all cells of an allograft, making nonself-MHC more abundant. Host CD4⁺ T cells may then recognize donor MHC directly (direct alloresponse) or after antigen processing (indirect alloresponse) and then proceed to participate in rejection.

FIGURE 26-7 Structure of the MHC class II molecule. Class II molecules are composed of a polymorphic α chain noncovalently attached to a polymorphic β chain. A, Schematic diagram. B, Ribbon diagram shows the extracellular structure of a class II molecule with a bound peptide.

(Adapted from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6, Philadelphia, 2010, Saunders-Elsevier.)

HLA Typing: Implications for Transplantation

For the reasons already discussed, closely matched transplants are less likely to be recognized and rejected than are similar grafts differing by multiple alleles at the MHC. HLA matching has a clear influence on the prolongation of graft survival. Humans have two different HLA-A, -B, and -DR alleles—one from each parent, six in total. Although clearly important, the HLA-C, -DP, and -DQ loci are administratively dismissed in general organ allocation. Although current immunosuppressive regimens negate much of the impact of matching, there have been several studies that have demonstrated improvements in renal allograft survival when the six primary alleles are matched between donor and recipient, a so-called six-antigen match (Fig. 26-8). Historically, MHC match has been defined using two cellular assays, the lymphocytotoxicity assay and the mixed lymphocyte reaction (MLC). Both assays define MHC epitopes but do not comprehensively define the entire antigen or the exact genetic disparity involved. Techniques now exist for precise genotyping via molecular techniques that distinguish the nucleotide sequence of an individual’s MHC.

FIGURE 26-8 Influence of HLA matching on renal allograft survival. Matching HLA alleles between donor and recipient significantly improves renal allograft survival. The data shown are for deceased donor renal allografts stratified by number of matched HLA alleles.

(Data from U.S. Organ Procurement and Transplant Network and the Scientific Registry of Transplant Patients: 2008 OPTN/SRTP annual report: Transplant data 1998–2007 [http://optn. transplant.hrsa.gov/ar2008].)

The MLC is performed by incubating recipient T cells with irradiated donor cells in the presence of ³H-thymidine; the irradiation treatment ensures that the assay only measures the 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. Class II polymorphism can be detected by this assay, but it takes several days to complete one assay. Thus, the use of MLC as a prospective typing assay is limited to LRDs. The specific MHC alleles are not identified with this assay per se; 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 well as a vital dye that 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 whether 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 class I and class II HLA loci has allowed several genetic-based techniques to be used for histocompatibility testing. These methods include restriction fragment length polymorphism (RFLP), oligonucleotide hybridization, and polymorphism-specific amplification using the polymerase chain reaction and sequence-specific primers (PCR-SSP). Of these methods, the PCR-SSP technique is most commonly used for class II typing. Serologic techniques are still the predominant method for class I typing because of the complexity of class I sequence polymorphism. It is important to note that 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 antigen-presenting cells, 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 26-1).

Table 26-1 Summary of Cytokines

CYTOKINE	SOURCE	PRINCIPAL CELLULAR TARGETS AND BIOLOGIC EFFECTS
IL-1	Macrophages, endothelial cells, some epithelial cells	Endothelial cell: Activation (inflammation, coagulation) Hypothalamus; Fever Liver: Synthesis of acute-phase proteins
IL-2	T cells	T cells: Proliferation, ↑ cytokine synthesis, survival, potentiate Fas-mediated apoptosis, promote regulatory T cell development NK cells: Proliferation, activation B cells: Proliferation, antibody synthesis (in vitro)
IL-3	T cells	Immature hematopoietic progenitor cells: Stimulate differentiation into myeloid lineage, proliferation of myeloid lineage cells
IL-4	CD4⁺ T cells (Th2), mast cells	B cells: Isotype switching to IgE T cells: Th2 differentiation, proliferation Macrophages: Inhibit IFN-γ mediated activation Mast cells: Stimulate proliferation
IL-5	CD4⁺ T cells (Th2)	Eosinophils: Activation, ↑ production B cells: Proliferation, IgA production
IL-6	Macrophages, endothelial cells, T cells	Liver: ↑ synthesis of acute-phase proteins B cells: Proliferation of antibody-producing cells
IL-7	Fibroblasts, bone marrow stromal cells	Immature hematopoietic progenitor cells: Stimulate differentiation into lymphoid lineage T and B cells: Important for survival during development as well as for T cell memory
TNF	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
IFN-γ	T cells (Th1, CD8⁺ T cells), NK cells	Macrophages: Activation (increased microbicidal functions) B cells: Isotype switching to IgG subclasses, which 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 IFNs (IFN-α, IFN-β)	Macrophages, IFN-α; fibroblasts, IFN-β	All cells: Stimulate antiviral activity, including ↑ class I MHC expression NK cells: Activation
TGF-β	T cells, macrophages, other cell types	T cells: Inhibit proliferation and effector functions B cells: Inhibit proliferation, ↑ IgA production Macrophages: Inhibit activation, stimulates angiogenic factors Fibroblasts: Increased collagen synthesis
Lymphotoxin (LT)	T cells	Lymphoid organogenesis Neutrophils: Increased recruitment and activation
IL-8	Lymphocytes, monocytes	Stimulate granulocyte activity, chemotactic activity
IL-9	Activated Th2 cells, lymphocytes	Enhance proliferation of T cells, mast cells
IL-10	Macrophages, T cells (mainly regulatory T cells)	Macrophages, dendritic cells: Inhibit IL-12 production, stimulate expression of costimulatory molecules and class II MHC
IL-11	Bone marrow stromal cells	Megakaryocytes: Thrombopoiesis Liver: Induces acute-phase proteins B cells: Stimulate T-dependent antibody production T cells: May skew toward Th2 phenotype
IL-12	Macrophages, dendritic cells	T cells: Th1 differentiation NK and T cells: IFN-γ synthesis, increased cytotoxic activity
IL-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
IL-14	T cells, some B cell tumors	B cells: Enhance proliferation of activated B cells, stimulate immunoglobulin production
IL-15	Macrophages, others	NK cells: Proliferation T cells: Proliferation (memory CD8⁺ T cells)
IL-17	T cells	Endothelial cells: Increased chemokine production Macrophages: Increased chemokine, cytokine production Epithelial cells: GM-CSF and G-CSF production
IL-18	Macrophages	NK and T cells: IFN-γ synthesis
IL-23	Macrophages, dendritic cells	T cells: Maintenance of IL-17 producing T cells
IL-27	Macrophages, dendritic cells	T cells: Inhibit production of IL-17/Th17 cells, promote Th1 differentiation NK cells: IFN-γ synthesis

G-CSF, Granulocyte-colony stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Adapted from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6, Philadelphia, 2010, Saunders-Elsevier.

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 for random generation of almost unlimited receptor specificity, yet controls the ultimate product by eliminating or suppressing those that might ultimately react against self and enable 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 eliminate foreign antigens. From the peripheral blood, T cells enter the lymph nodes or spleen through highly specialized regions in the postcapillary venules. In 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 Cells

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 understanding its function.¹¹ 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 their DNA, which encodes the various chains of the TCR (α, β, γ, and δ; Fig. 26-9). 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 natural killer (NK) cells, as well as MHC recognition, whereas αβ T cells are more typically limited to recognition of MHC complexed with processed peptide.

FIGURE 26-9 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 T cell receptors. Genomic DNA is spliced under the direction of specific enzymes active during T cell development in the thymus. Random segments from regions known as 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.

(Adapted from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6, Philadelphia, 2010, Saunders-Elsevier.)

Regardless of the genes used, individual cells recombine to express a TCR with only a single specificity. The rearrangements occur randomly, resulting in a population of T cells capable of binding 10⁹ different specificities, essentially all combinations of MHC and peptide. As a result, the frequency of naïve T cells available to respond to any given pathogen is relatively small, between 1 in 200,000 to 500,000. These developing T cells also express both CD4 and CD8, accessory molecules, which strengthen the TCR binding to MHC. These accessory molecules further increase the binding repertoire of the population to include 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. 26-10).¹² Cells initially interact with the MHC-expressing cortical thymic epithelium, which produces hormones (thymopoietin and thymosin) as well as cytokines (e.g., IL-7), which are critical to T cell development. If binding does not occur to self-MHC, those cells are useless to the individual—because they cannot bind self cells to assess for viral infection—and they undergo programmed self-destruction via apoptosis, a process called positive selection (Fig. 26-11). Cells surviving positive selection then move to the thymic medulla and, normally, eventually lose CD4 or CD8. If binding to self-MHC in the medulla occurs with an unacceptably high affinity, programmed death again results; this process is called negative selection. The precise nature of this affinity threshold remains a matter of intense investigation and involves interaction with hematopoietic cells that reside in the thymus. The only cells released into the periphery are those that can 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 an 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 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 between 1% and 10% of all T cells.

FIGURE 26-10 T cell maturation. Initially, T cell precursors arrive in the thymic cortex lacking CD4, CD8, or a TCR and are referred to as double-negative. The genes responsible for the expression of the TCR chains subsequently undergo a series recombination events, resulting in expression of a γδ TCR or, more commonly (>90%), an αβ TCR on the cell surface. γδ T cells proceed through a distinct selection process that is independent of MHC restriction. 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.

(Adapted from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6, Philadelphia, 2010, Saunders-Elsevier.)

FIGURE 26-11 Thymic selection. αβ T cells must go through positive and negative selection within the thymus to become a functional T cell. A, B, Positive selection. A, Double-positive thymocytes are required to interact with self MHC–endogenous peptide complexes expressed on mainly thymic epithelial cells to receive survival signals. B, Thymocytes whose TCR does not recognize self-MHC fail to receive the essential survival signals and go on to be deleted from the repertoire via programmed cell death. This process ensures that all mature T cells are restricted to self-MHC. C, Negative selection. T cells that are positively selected migrate through the thymic medulla to undergo negative selection. Negative selection is the process whereby self-reactive T cells are eliminated. If thymocytes bind with high avidity (i.e., strongly) to self-MHC–peptide complexes on bone marrow–derived thymic dendritic cells, they receive signals that promote apoptosis.

(Adapted from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6, Philadelphia, 2010, Saunders-Elsevier.)

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 the removal of T cells following an immune response and downregulation of activated clones. CD95, a molecule known as Fas, is a member of the 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 (see later). TCR binding only leads to T cell activation if the costimulatory signals are present, generally delivered via APCs. In the absence of costimulation, the cell remains unable to proceed toward activation and, in some cases, 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. As noted, the TCR, unlike antibody, only recognizes its ligand in the context of MHC. By requiring that T cells only respond 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 so 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.¹³ Each event results in the internalization of the TCR. Because resting T cells have low TCR density, sequential binding and internalization over several hours is required. Transient encounters are not sufficient. This threshold is reduced considerably by appropriate costimulation signals (see later).

As discussed in the previous section, 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. This is 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 stabilize its binding, a signal is initiated by intracytoplasmic protein tyrosine kinases (PTKs). These PTKs include p56lck (on CD4 or CD8), p59Fyn, and ZAP70; the latter two are associated with CD3. Repetitive binding signals combined with the appropriate costimulation eventually activate phosphokinase C-gamma (PLC-γ1), which in turn hydrolyzes the membrane lipid phosphatidyl inositol biphosphate (PIP2), thereby releasing inositol triphosphate (IP3) and diacyl glycerol (DAG). IP3 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 the transcription of cytokines, including IL-2 and its receptor (Fig. 26-12). Resting T cells express only low levels of the IL-2 receptor (IL-2R; 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 autocrine and paracrine fashions, potentiating DAG activation of protein kinase C (PKC). PKC 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 only reactive in the presence of antigen and ceases to function once antigen is removed. Many of these steps in T cell activation have been targeted in the development of immunosuppressive agents. These will be discussed in detail later in this chapter.

FIGURE 26-12 T cell activation. On antigen recognition, there is a clustering of TCR complexes and coreceptors, which 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. ζ-chain association protein kinase (Zap-70) subsequently associates with these regions and becomes activated. Zap-70 phosphorylates various adaptor and coreceptor proteins, ultimately activating numerous cellular enzymes, including calcineurin, PKC, and several MAP kinases. These enzymes then activate transcription factors that promote the expression of various genes involved in proliferation and T cell responses.

(Adapted from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6, Philadelphia, 2010, Saunders-Elsevier.)

Costimulation

As noted, recognition of the antigenic peptide–MHC complex via 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.^14,¹⁵ 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 can lead to a state of inaction, or anergy (Fig. 26-13). An anergic T cell is now unable to respond, even if given both appropriate stimuli.¹⁶ 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. Most research to date has focused on the interactions of two costimulatory pathways, the CD28-B7 pathway (immunoglobulin-like superfamily members) and CD40-CD154 pathway (tumor necrosis factor [TNF]–TNFR superfamily members). There have been, however, many additional pairings in these same families and others that have been found to have distinct roles in costimulatory function (Table 26-2).