Immunologic Aspects of Organ Transplantation

Susan Smith MN, PhD

Disclosures

June 17, 2002

The Acquired Immune System

Maturation of the lymphoid system occurs during the fetal and neonatal periods when lymphoid stem cells differentiate into B cells or T cells. At this time, the mechanisms for conferring genetic specificity to lymphocytes develop. This property of specificity is what differentiates the lymphoid cell from the myeloid cell, which can react with any antigen. The process of lymphopoiesis (lymphocyte origination and differentiation into functional effector cells) begins in the yolk sac and continues later in life in the thymus gland, liver, spleen, and finally the bone marrow, which is the primary site of lymphopoiesis in the full-term neonate.

Primary lymphoid tissue consists of "central" organs that serve as major sites of lymphopoiesis (Figure 10). Lymphoid stem cells, which originate in the bone marrow, give rise to the various components of the acquired immune system. Secondary lymphoid tissue is "peripheral" tissue that provides an environment for lymphocytes to encounter antigens and proliferate if necessary. Secondary lymphoid tissue consists of the spleen, lymph nodes, bone marrow, liver, and mucosa-associated lymphoid tissue (MALT) in the tonsils, respiratory tract, gut, and urogenital tract. Location of secondary lymphoid tissue is not coincidental; all of these structures provide major portals for the entry of foreign microorganisms into the body. Once in secondary lymphoid tissues, lymphocytes may migrate from one lymphoid structure to another by vascular and lymphatic channel systems.

Figure 10.

Primary and secondary lymphoid tissue.

Lymphocytes

Lymphocytes, the primary defenders of the acquired immune system, play a central role in regulating immune responses to all antigens; they are the only cells that have the intrinsic ability to recognize specific antigens. Lymphocytes are the major components of lymph nodes. Only about 5% of lymphocytes are blood borne; the other 95% reside in the lymph nodes and spleen. There are 2 major populations of lymphocytes: B cells and T cells. Lymphocytes have surface receptors that are specific for surface molecules, sometimes called epitopes, located on the surfaces of foreign proteins. T cells have receptors for class I and class II antigens, and B cells have receptors for immunoglobulins. B cells produce antibodies and mediate what is called the "humoral" immune response. T cells are involved in immunologic regulation and mediate what is called the "cellular" immune response. For the sake of discussion, the 2 types of lymphocytes will be described separately, but in reality there is much interaction between them, and effective host defense is dependent on this interaction.

B Cells

B cells mature in secondary lymphoid tissue and account for approximately 1% to 3% of the differential WBC count. The antigen-specific receptor on the B cell is an immunoglobulin. Once activated by the binding of antigen specific for only that receptor, the B cell produces a large quantity of antibody of the exact specificity as the original immunoglobulin receptor.

B cells are effector cells that mediate the humoral immune response (humoral immunity) through the production of antibodies, which is their major function. B cells are important in defense against pyrogenic bacterial infections, and can destroy transplanted organs by mediating hyperacute graft rejection. When a B cell is stimulated by a particular antigen, it differentiates into a lymphoblast. The lymphoblast differentiates into a plasmablast, which further differentiates into a plasma cell. Plasma cells release antibody until the antigen is destroyed (Figure 11). Following exposure of a B cell to a specific antigen, the antibody it produces may combine with a toxic site on the antigen molecule or cause its removal by phagocytes. In addition, memory of the offending antigen is retained for at least several months.

Figure 11.

Antigenic stimulation and subsequent antibody production.

Antibodies

Antibodies are also referred to as immunoglobulins. Immunoglobulins are specifically modified proteins present in serum and tissue fluids that are capable of selectively reacting with inciting antigens. All antibodies have the same basic structure, but each antibody varies from all others in 1 region so that each antibody is unique. Therefore, each antibody is specific and can recognize only 1 antigen. The body produces several million antibodies that are capable of reacting with just as many antigens. Antibodies are involved in a wide range of immune responses that lead to destruction and elimination of potentially harmful antigens. When viruses or bacteria, for instance, enter the body, their structural surface features are recognized by the body as not belonging to it. Antibodies are then formed and attracted to these foreign structures for which they have identical matching receptors. In this way, antibodies bind with antigens, which is called antigen-antibody complex formation.

Antibodies can be divided into 5 major classifications: IgM, IgG, IgA, IgD, and IgE. IgM is the principle mediator of the primary immune response. IgM is a "natural" antibody; there is no known contact with the antigen that stimulated its production. About 10% of all antibodies are of the IgM type. IgG is the principal mediator of the secondary immune response, which requires repeated exposure to the same antigen. IgG is the major antibody against bacteria and viruses. About 75% of all antibodies are of the IgG type. IgA is the secretory immunoglobulin present in body secretions and offers natural protection against nonspecific foreign antigens. About 15% of all antibodies are of the IgA type. The function of IgD is not known, but about 1% of antibodies are of this type. Although only about 0.002% of antibodies are of the IgE type, IgE antibodies present on basophils and mast cells play a significant role in inflammatory and immune reactions.

Structurally, antibodies consist of 4 polypeptide chains, 2 heavy and 2 light chains (Figure 12). The light chains have an adaptor component (Fab) that binds to antigen, and the heavy chains have an adaptor component (Fc) that activates complement. Using these adaptor components, the antibody forms a bridge between the antigen and a phagocyte, which facilitates destruction of the antigen. Mechanisms of antigen inactivation by antibody include neutralization, opsonization, and complement fixation (Figure 13).

Figure 12.

B cell antigen receptor.

Figure 13.

Elimination of antigen by antibody.

Neutralization. Imagine that a bacterial neurotoxin has entered the lymph node. If not cleared, the toxin will seek and destroy neural cells. However, because the acquired immune response is in a ready state, the toxin binds to the surface of the B cell with the correct specificity, which responds by making the appropriate antitoxin antibody. By binding to the remaining toxin, the antibodies form a basket around the antigen, sequestering it from neurons within the host. This antigen-antibody complex is now a target for phagocytosis and elimination via the kidneys.

Opsonization. Certain complement protein fragments behave as opsonins. Antibodies are also efficient opsonins. By coating the surface of the antigen for which they bear specificity, antibodies attract phagocytes that bear receptors for the stems of the antibody molecules. The phagocytes then chew up antigen-antibody complexes, effectively removing antigen from the host.

Complement fixation. As noted previously, the classical pathway of complement activation is triggered by antigen-antibody complex formation, a signal for the initial proteins in the complement cascade to attach to the stem of the antibody molecule. The cascade ultimately generates the MAC, which punches holes in the offending cellular antigen.

T Cells

Immature T cells develop in the thymus. During this immature phase of T-cell development, reactivity of these cells to self-antigens is eliminated and tolerance to self-antigens occurs. Under continued thymic hormonal influence, functionally active T cells develop. T cells account for approximately 15% of the differential WBC count.

T cells have molecules on their surfaces called TcRs, which recognize peptide fragments presented with human leukocyte antigen (HLA) class I and HLA class II molecules found on the surface of APCs. The structure of the TcR is similar to the structure of an antibody and also varies in one region so that each TcR is unique (Figure 14).

Figure 14.

T cell receptor (TcR)

T cells are divided into 4 functionally distinct, but interactive, cell populations or subsets: cytotoxic (CTL), memory (Tm), Th, and suppressor (Ts). Tc and Tm cells are referred to as effector cells because they are cytotoxic to antigen-bearing cells. Th and Ts cells are referred to as regulatory cells.

Effector cells. CTLs respond to processed peptide presented by nucleated cells of the body that have been infected (ie, virus). Like Th cells, CTLs clonally proliferate, but finish their response under the influence of Th cell-derived cytokines. CTLs bind to target cells and facilitate their destruction via substances known as lymphokines that stimulate inflammatory cells and via the production of cytolytic proteins. Tm cells are T cells that have been sensitized to a specific antigen and then cloned to remember the antigen. Memory cells remain present in the body for many years and are therefore available for defense on repeated exposure to an antigen. Repeated exposure to an antigen that the host has been previously sensitized to will result in a more rapid and accelerated immune response than on the first exposure.

Regulatory cells: Th cells and Ts cells . Th cells produce multiple lymphokines that promote the proliferation and activation of other lymphocytes and macrophages. There are 2 types of Th cells: Th1 cells and Th2 cells. Th1 cells produce cytokines (IL-2 and IFN-gamma) that activate macrophages to kill antigen-containing cells. Th2 cells produce enzymes that activate B cells to produce antibody. The cytokines produced by Th2 cells give chemical instructions to immune cells to: enter a site of antigen concentration, proliferate in response to antigen (IL-4 and IL-5 induce proliferation of B cells), and directly kill or phagocytose antigen (cytotoxic cytokines affecting T cells include lymphotoxin and TNF-beta, IFN-gamma activates macrophages). Thus, cytokine-producing Th cells effect antigen clearance by producing the molecules that activate immunocompetent cells in the presence of antigen (Figure 15). Cytotoxic T cells effect antigen clearance by directly killing cells whose surface tissue antigens become altered by exposure to infectious organisms or damaging toxins. Like phagocytes, CTLs depend on Th cell-produced cytokines to do their job.

Figure 15.

Elimination of antigen by T cells.

Ts cells produce a soluble factor that suppresses the cytotoxic response by effector cells and inhibits antibody production.[7] Helper and suppressor T cell activity is normally balanced to maintain immunologic homeostasis. Too much suppressor T-cell function, for instance, will inhibit Th cell function.

In addition to being functionally distinct, T cell subset populations have different surface antigens or "markers" called clusters of differentiation (CD) that can be detected by mAbs. CD4 and CD8 molecules are found on the surfaces of Th and Tc cells, respectively. CD4+ T cells recognize antigen combined with HLA class II molecules; CD8+ T cells recognize antigen combined with HLA class I molecules.

As mediators of the cellular immune response (cell-mediated immunity), T cells defend the body against viruses, fungi, and some neoplastic conditions, and destroy transplanted organs by mediating accelerated and acute rejection responses. T cell function is inhibited by viral and parasitic infections, malnutrition, prolonged general anesthesia, radiation therapy, uremia, Hodgkin's disease, and advanced age.

Lymphokines

Lymphokines are 1 of the 2 soluble products of lymphocytes, the other being antibodies. Lymphokines and monokines (released from monocytes) are inflammatory and regulatory hormones of the immune system that serve a variety of functions, such as the recruitment of macrophages to antigen sites (chemotaxis), augmentation of T cell function in general, and inhibition of viral replication. Lymphokines carry molecular signals between immunocompetent cells for amplification of the immune response. Their role in amplification of the T-cell response is crucial to cellular immunity. There are many lymphokines, including families of related cytokines, which are listed in Table 5 .

The lymphokines most relevant to transplantation are IL-2 and IFN-gamma. IL-2 is produced and released by activated CD4 T cells. IL-2 activates B cells, NK cells, and monocytes, and promotes B- and T-cell division (and T-cell proliferation and maturation into CTLs) and the release of other cytokines such as IFN-gamma from T cells. IFN-gamma is produced by activated T cells and NK cells. IFN-gamma induces the expression of HLA molecules on cells, increases activity of APCs, activates T cells and macrophages, and promotes differentiation of B cells into plasma cells.

Antigen Recognition by Receptors

All cells express foreign antigens. Foreign cells, of course, express antigens that are genetically different from those of the host. It is through specific "receptors" on the surfaces of lymphocytes that B cells and T cells can be differentiated, and it is also through these receptors that B cells and T cells are able to recognize foreign antigens. During lymphocyte maturation, each B cell and T cell acquires specific cell membrane surface receptors that allow them to "match up" with certain foreign antigens. On B-cell surfaces, immunoglobulins function as receptors; the nature of T-cell surface antigens is not entirely clear. This matching between host lymphocytes and foreign antigens is the recognition phase of the acquired immune response. When this occurs, lymphocytes are activated to differentiate, proliferate, and clone to mount an effective immune response against the offending antigen.

Specific genes are responsible for the receptors on lymphocytes and distinguish those that respond to particular antigens from those that do not. These genes are responsible for the property of uniqueness that allows each individual to recognize antigens that are genetically different from their own. No 2 persons are identical with respect to which antigens will incite an immune response; instead, the human population is extremely heterogenous in this respect.

Chromosomes and Genes

All somatic cells in the human body contain within their nuclei the genetic material (chromosomes) that provides the instructions for our life processes (Figure 16). The 23 chromosomes within each cell contain a large number of genes that encode proteins with specific functions. Each gene contains deoxyribonucleic acid that provides blueprints for proteins. Proteins can be expressed on the surface of the cells that make them (eg, receptors for growth factors), or they can be secreted by the cells that make them (eg, insulin and cytokines).

Figure 16.

Genes and proteins.

Each gene is located in a particular location (locus) on a particular chromosome, waiting to be activated by biochemical signals. While every cell in the body contains the exact same genetic material, cells receive signals to turn on only those genes that are appropriate to their function. For example, all cells in the body possess the gene that encodes insulin. However, only the beta cells of the pancreas receive the biochemical signals that turn on the insulin gene, resulting in the production of insulin.

Genes come in different "flavors" called alleles. Alleles are alternative forms of a gene that provide a variety of phenotypes or appearances. That is, different alleles of a gene recognize variants of the gene product.

The Major Histocompatibility Complex (MHC)

Each species expresses a set of proteins on their cell surfaces that identify their tissues as belonging to that species, and that species only. These proteins are encoded by a set of genes we call the MHC (Figure 17). In humans, the MHC is also known as the HLA complex. The HLA complex is a cluster of genes located on the short arm of chromosome 6 (Figure 18).

Figure 17.

The major histocompatibility complex.

Figure 18.

Association between HLA protein and self - non-self peptides.

HLA-encoded proteins are the most polymorphic proteins known. However, the diversity afforded by polymorphism of HLA alleles gives populations survival advantages. The consequences of inadequate diversity were illustrated by the smallpox epidemic among North American indigenous populations in the 17th-19th centuries. Seeking trade and the opportunity to convert indigenous peoples to Christianity, Europeans inadvertently carried smallpox to Indian villages. Relative homogeneity in the HLA allele pool among Indians and lack of previous exposure to smallpox resulted in decimation of the population. As much as 80% of village populations were lost to smallpox by the turn of the 20th century. These populations have yet to recover. The lesson here is that as much as we are drawn to the things we share, the differences among us may indeed be healthy!

In humans, the genetic factor that determines specific antigen recognition is called the MHC.[8] The MHC is the HLA genetic complex located on the short arm of the 6th chromosome. (Figure 17) The major function of the MHC is regulation of immune responsiveness. The HLA gene complex encodes cell surface molecules (antigens) that are highly immunogenic or antigenic against cells lacking the same genetic makeup. HLA molecules facilitate the distinction by the lymphoid system of self from nonself. HLA antigens are present on most cells in the body, including leukocytes and platelets, and are widely distributed in tissues and organs. They are not present on mature erythrocytes. These antigens are responsible for leukocyte reactions during blood transfusions and are important determinants of allograft rejection, hence the name "histocompatibility antigens." (Figure 19)

Figure 19.

Histocompatibility and transplantation immunology.

The gene products of the MHC (HLA molecules or antigens) are divided into 2 classes on the basis of structure, tissue distribution, function, and the specific types of antigen expressed on their surfaces. The 2 classes of HLA molecules are known as class I and class II antigens (Figure 20) that function as "billboards" to display what is going on inside the cell (class I) or outside the cell (class II).[9]

Figure 20.

Importance of the HLA complex and HLA-encoded proteins.

Class I antigens. Class I antigens (HLA A, B, and C antigens) are expressed on the plasma membranes of all nucleated cells.[10] They function as surface recognition molecules for CTLs. CTLs have receptors that recognize class I antigens and are activated against an antigen only if they share at least 1 class I HLA determinant with the antigen. Antiviral and antitumor activity and acute graft rejection are facilitated by the recognition of "nonself" class I antigens on foreign cell surfaces by host T cells. HLA types A, B, and C express class I antigens. Class I HLA molecules represent a problem for some patients awaiting transplantation because they greatly decrease the number of potentially compatible donors. Many individuals are exposed to class I HLA molecules through pregnancy, blood transfusions, or prior transplants. Those previously exposed develop antibodies to HLA class I molecules.

When considering class I HLA proteins and their relationship to foreign antigen, it is useful to think of the example of viral infection. In reading the viral genome, the host cell produces viral proteins that become complexed to HLA class I molecules in the course of normal turnover of the cell membrane proteins. In other words, when the nucleated cell changes its coat, this foreign viral protein appears on its insignia. This complexing of foreign peptide to the self class I protein is the first signal in the activation cascade for CD8+ CTLs (Figure 21).

Figure 21.

T cell-HLA protein interactions.

Class II antigens. Class II antigens (HLA-DR, HLA-DQ, and HLA-DP antigens) have limited tissue distribution. In contrast to class I antigens, class II antigens are expressed only on immune cells that have the ability to process and present antigens to T cells.[10] Regulatory T cells have receptors that recognize class II antigens. Th cells and Ts cells can recognize antigen only in the context of the class II molecules that they carry. If a macrophage or dendritic cell has internalized and processed antigen, a small peptide fragment of that antigen is complexed to the class II proteins normally expressed on the surface of the cell. This combination of self-tissue protein (MHC/HLA class II) and peptide fragment is the first signal in the activation cascade for CD4+ Th cells (Figure 21).

Any contact with foreign antigen leads to processing by host APCs, which leads to formation of a complex between a fragment of the antigen and the HLA self protein that is normally expressed on the surface of the APC (Figure 22). The T cell with specificity for that antigen fragment makes contact with this particular APC. This is the signal for the T cell to sit up and take notice that foreign peptide has complexed to self-HLA protein.

Figure 22.

Direct antigen presentation.

Inheritance of HLA Antigens

HLAs are inherited as a set of the 3 HLA groups: HLA-A, HLA-B, and HLA-DR. Each locus contains many individual alleles. The allelic specificities are designated by numbers following the locus symbol (eg, HLA-A1 or HLA-B5). Because human chromosomes exist in pairs, it is possible to identify a total of 6 HLA antigens in a given individual, 2 from each chromosome (HLA-A, HLA-B, and HLA -DR). The expression of HLA antigens on the individual's 6th chromosome is called a phenotype (Figure 23). One haplotype, or half of the total HLA identity, is inherited from each parent. Therefore, each individual has 2 haplotypes and there are a total of 4 different haplotype combinations from 2 parents. The 2 inherited haplotypes represent the individual's genotype. Each offspring of the same biologic parents has a 25% possibility of having the same HLA type as a sibling, a 25% possibility of having a totally dissimilar HLA type as a sibling, and a 50% possibility of sharing 1 haplotype with a sibling. If 2 children inherit the very same HLA from their parents, they are an HLA "identical match."

Figure 23.

Inheritance of HLA haplotypes.

The Cellular Immune Response: Th1 Cells

The cellular immune response is, again, mediated by T cells. Whereas B cells bind to soluble antigen on the surface of macrophages, T cells can recognize antigen only in association with a class I MHC antigen displayed on the surfaces of macrophages or APCs. T cells are triggered by stimulation of antigen receptors on their cell surfaces. Under the influence of alloantigen and Th1-derived IL-2, clones of alloantigen-responsive CTLs proliferate and seek direct contact with alloantigen-bearing cells (Figure 24). CTLs are programmed to produce molecules such as perforin (closely related to the C9 component of complement) and granzymes (serine esterases). These molecules pass from the T-cell cytoplasm into the donor cell, where they mediate the killing of the donor cell. The steps in the cellular immune response can be summarized in the following sequence:

  • The presence of a foreign antigen is necessary to initiate the response.

  • Initially, macrophages encounter the antigen and begin to phagocytize it. Antigenic fragments complexed with MHC antigens are released, which are carried to T cells in the lymph nodes by APCs.

  • Resting virgin or memory T cells are activated when the antigen-APC complex binds with the T-cell surface receptor.

  • APCs are stimulated to produce IL-1, which summons Th cells.

  • Th cells are then responsible for a number of actions, including the release of IL-2, which causes the differentiation and proliferation of T cells. The Th cells also stimulate antibody production by B cells.

  • Clonal expansion increases the sensitized T cell population approximately a 1000-fold.

  • Ultimately, the antigen-bearing cells are destroyed by the direct cytotoxic effect of effector T cells. Some sensitized T cells are returned to the lymphoid system with the memory of the antigen for future challenge.

Examples of cellular immune responses include tumor cell surveillance, destruction defense against viral and fungal infection, acute organ rejection (a type IV hypersensitivity reaction), graft-vs-host disease (GVHD, a type IV hypersensitivity reaction), and autoimmune diseases (type IV hypersensitivity reactions).

Figure 24.

The allogeneic response - Th1 cells.

The Humoral Immune Response: Th2 Cells (Figure 25)

Th2 cells drive the humoral arm of the allogeneic response by producing cytokines that stimulate B-cell proliferation and differentiation. In addition to being carried by APCs, alloantigen enters the lymph nodes directly, where it binds to specific immunoglobulin receptors on the surfaces of B cells. B-cell clonal proliferation occurs under the influence of the combination of alloantigen and Th2-derived IL-4 and IL-5. IL-2 produced by Th1 cells also stimulate the burst of proliferation experienced by B cells under the influence of alloantigen.

Figure 25.

The allogeneic response - Th2 cells and B cells

Following the proliferation of separate clones of B cells responding to a variety of alloantigens in this manner, there is a period of differentiation driven by the Th2-derived cytokine IL-6. At this time, the morphology of B cells changes drastically, surface immunoglobulin becomes practically undetectable, and the rough endoplasmic reticulum responsible for protein production predominates in the cytoplasm. B cells differentiate into plasma cells (the antibody factories of the immune system) under the influence of alloantigens and Th2-derived cytokines. The steps in the humoral immune response can be summarized in the following sequence:

  • As with the cellular immune response, the presence of a foreign antigen is necessary to initiate the process. Unlike T cells, B cells can recognize antigen in its native configuration.

  • Initially, macrophages encounter the antigen and begin to phagocytize it. Antigenic fragments are released that are carried to B cells in the lymph nodes and spleen by APCs. Resting virgin or memory B cells are activated when antigen binds to surface immunoglobulin.

  • IL-1 is released by APCs, and Th cells stimulate the sensitization and clonal proliferation of effector B cells. B cells are activated to differentiate and produce antibody when antigen binds to their receptors.

  • Antigen-antibody complexes form and ultimately the antigen-bearing cells are destroyed.

  • As in the cellular immune response, some of the plasma cells with specific memory of the antigen are cloned and returned to the lymphoid system.

In addition, B cells can process and present antigen to T cells. Examples of humoral immune responses include resistance to encapsulated pyrogenic bacteria such as pneumococci, streptococci, meningococci, and Haemophilus influenzae, and type II and III hypersensitivity reactions, including hyperacute rejection of a transplanted organ.

A first exposure of an antigen to an activated lymphocyte evokes a primary immune response. Repeated exposure of the identical antigen to activated lymphocytes evokes an accelerated secondary response. In the secondary immune response, the latent period is shorter and the amount of antigen required to initiate the response is less.

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