Sites of Lymphocyte Development

In mammalian immunologic development, the precursors of lymphocytes arise from progenitor cells of the yolk sac and liver (Fig. 4-3). Later in fetal development, and throughout the life cycle, the bone marrow becomes the sole provider of undifferentiated progenitor cells, which can further develop into lymphoblasts. Continued cellular development and proliferation of lymphoid precursors occur as the cells travel to the primary and secondary lymphoid tissues.

Primary Lymphoid Tissue

In mammals, both the bone marrow (and/or fetal liver) and thymus are classified as primary or central lymphoid organs (Fig. 4-4).

Thymus

Early in embryonic development, the stroma and nonlymphoid epithelium of the thymus are derived from the third and fourth pharyngeal pouches. The characteristics of the thymus gland change with aging. Older persons are immunologically challenged because aging causes a reduction in the production of naïve T cells by the thymus. Intrinsic defects in mature T cell function, alterations in the life span of naïve T cells and in naïve or memory T cell ratios in the peripheral lymphoid tissues, occur as the result of the decline of the T cell response in older persons.

The thymus, located in the mediastinum, exercises control over the entire immune system. It is believed that the development of diversity occurs mainly in the thymus and bone marrow, although clonal expansion can occur anywhere in the peripheral lymphoid tissue.

Progenitor cells that migrate to the thymus proliferate and differentiate under the influence of the humoral factor, thymosin. These lymphocyte precursors with acquired surface membrane antigens are referred to as thymocytes.

The reticular structure of the thymus allows a significant number of lymphocytes to pass through it to become fully immunocompetent (able to function in the immune response), thymus-derived T cells. The thymus also regulates immune function by the secretion of multiple soluble hormones.

Many cells die in the thymus and apparently are phagocytized, a mechanism to eliminate lymphocyte clones reactive against self. It is estimated that approximately 97% of the cortical cells die in the thymus before becoming mature T cells. Viable cells migrate to the secondary tissues. The absence or abnormal development of the thymus results in a T lymphocyte deficiency.

Involution of the thymus is the first age-related change occurring in the human immune system. In postnatal life, the thymus is the primary organ that produces naïve T cells for the peripheral T cell pool but production of cells declines as early as 3 months of age. The thymus gradually loses up to 95% of its mass during the first 50 years of life (Fig. 4-5). The accompanying functional changes of decreased synthesis of thymic hormones and the loss of ability to differentiate immature lymphocytes are reflected in an increased number of immature lymphocytes within the thymus and as circulating peripheral blood T cells. Most changes in immune function, such as dysfunction of T and B lymphocytes, elevated levels of circulating immune complexes, increases in autoantibodies, and monoclonal gammopathies are correlated with involution of the thymus (see Chapter 27). Immune senescence may account for the increased susceptibility of older adults to infections, autoimmune disease, and neoplasms.

Bone Marrow

The bone marrow is the source of progenitor cells. These cells can differentiate into lymphocytes and other hematopoietic cells (e.g., granulocytes, erythrocytes, megakaryocyte populations). In mammals, the bone marrow also supports eventual differentiation of mature T and B lymphocytes, probably from a common lymphoid cell progenitor. It is believed that the bone marrow and gut-associated lymphoid tissue (GALT) may also play a role in the differentiation of progenitor cells into B lymphocytes.

Secondary Lymphoid Organs

Secondary lymphoid organs provide a unique microenvironment for the initiation and development of immune responses. The secondary lymphoid tissues include lymph nodes, spleen, GALT, thoracic duct, bronchus-associated lymphoid tissue (BALT), skin-associated lymphoid tissue, and blood. Mature lymphocytes and accessory cells (e.g., antigen-presenting cells) are found throughout the body, although the relative percentages of T and B cells vary in different locations (Table 4-1).

Table 4-1

Approximate Percentage of Lymphocytes in Lymphoid Organs

Lymphoid Organ	T Lymphocytes (%)	B Lymphocytes (%)
Thymus	100	0
Blood	80	20
Lymph nodes	60	40
Spleen	45	55
Bone marrow	10	90

Adapted from Claman HN: The biology of the immune response, JAMA 268:2790–2796, 1992.

The highly sophisticated structure of secondary lymphoid organs allows migration and interactions between antigen-presenting cells, T and B lymphocytes, and follicular dendritic cells (FDCs) and other stromal cells. The cooperative activities of lymphoid cells within secondary organs dramatically increase the probability of interactions of rare B, T, and APCs that results in effective generation of humoral immune responses.

Tumor necrosis factor (TNF) and lymphotoxin are essential to the formation and maintenance of secondary organs. These cytokines are produced by B and T lymphocytes. Proliferation of the T and B lymphocytes in the secondary or peripheral lymphoid tissues (Fig. 4-6) is primarily dependent on antigenic stimulation.

The T lymphocytes or T cells populate the following:

1. Perifollicular and paracortical regions of the lymph nodes

2. Medullary cords of the lymph nodes

3. Periarteriolar regions of the spleen

4. Thoracic duct of the circulatory system

The B lymphocytes or B cells multiply and populate the following:

1. Follicular and medullary (germinal centers) of the lymph nodes

2. Primary follicles and red pulp of the spleen

3. Follicular regions of GALT

4. Medullary cords of the lymph nodes

Lymph Nodes

Lymph nodes act as lymphoid filters in the lymphatic system. Lymph nodes respond to antigens introduced distally and routed to them by afferent lymphatics (Fig. 4-7). Generalized lymph node reactivity can occur after systemic antigen challenge (e.g., serum sickness).

Spleen

The spleen acts as a lymphatic filter within the blood vascular tree. It is an important site of antibody production in response to IV particulate antigens (e.g., bacteria). The spleen is also a major organ for the clearance particles.

Gut-Associated Lymphoid Tissue

GALT includes lymphoid tissue in the intestines (Peyer’s patches) and the liver. GALT features immunoglobulin A (IgA) production and involves a unique pattern of lymphocyte recirculation. Pre–B cells develop in Peyer’s patches and, after meeting antigen from the gut, many enter the general circulation and then return back to the gut. GALT is also important for the development of tolerance to ingested antigens.

Thoracic Duct

The thoracic duct lymph is a rich source of mature T cells. Chronic thoracic duct drainage can cause T cell depletion and has been used as a method of immunosuppression.

Bronchus-Associated Lymphoid Tissue

BALT includes lymphoid tissue in the lower respiratory tract and hilar lymph nodes. It is mainly associated with IgA production in response to inhaled antigens.

Skin-Associated Lymphoid Tissue

Antigens introduced through the skin are presented by epidermal Langerhans cells, which are bone marrow–derived accessory cells. These epidermal cells then interact with lymphocytes in the skin and in draining lymph nodes.

Blood

The blood is an important lymphoid organ and immunologic effector tissue. Circulating blood has enough mature T cells to produce a graft-versus-host reaction. In addition, blood transfusions have been responsible for inducing acquired immunologic tolerance in kidney allograft patients.

Blood is the most frequently sampled lymphoid organ. It is assumed that what is found in blood samples represents what is present in other lymphoid tissues. Although this may be a true representation, it is not always accurate.

Circulation of Lymphocytes

Mature T lymphocytes survive for several months or years, whereas the average life span of B lymphocytes is only a few days. Lymphocytes move freely between the blood and lymphoid tissues. This activity, termed lymphocyte recirculation, enables lymphocytes to come into contact with processed foreign antigens and disseminate antigen-sensitized memory cells throughout the lymphoid system. Clonal expansion may occur regionally, as in lymph nodes draining a contact allergic reaction, and then the whole body becomes susceptible to rechallenge because T cells recirculate, but generally are excluded from returning to the thymus. Research has shown that a pool of T cell clonal elements is developed by a combination of positive selection of clones able to recognize and react to foreign antigens, and negative selection (purging) of clones able to interact with self-antigens in a damaging way.

Recirculation of lymphocytes back to the blood is through the major lymphatic ducts. Lymphocytes enter the lymph node from the blood circulation via arterioles and capillaries to reach the specialized postcapillary venules. From the venule, the lymphocytes enter the node and remain in the node or pass through the node and return to the circulating blood. Lymphatic fluid, lymphocytes, and antigens from certain body sites enter the lymph node through the afferent lymphatic duct and exit the lymph node through the efferent lymphatic duct (see Fig. 4-7).

Early Cellular Differentiation and Development

Differentiation of a lymphocyte begins in the thymus as a thymocyte. Early surface markers on thymocytes that are committed to becoming T cells include CD44 and CD25. As thymocytes develop, there is an orderly rearrangement of the genes coding for an antigen receptor.

Maturation is a complicated process that lasts for a period of 3 weeks. During this period, cells filter through the cortex to the medulla of the thymus. Thymic stromal cells include fibroblasts, macrophages, epithelial cells, and dendritic cells; all these cell types play a role in T cell development.

Helper T Lymphocytes

Helper T lymphocytes, or T-helper (Th) cells, can be assigned to one of several subsets, including the following:

• Helper T type 1 (Th1) cells are responsible for cell-mediated effector mechanisms.

• Helper T type 2 (Th2) cells play a greater role in the regulation of antibody production.

• Regulatory T (Treg) cells are an immunoregulatory type of Th cells.

These divisions are not absolute, with considerable overlap or redundancy in function among the different subsets. This classification is based on the in vitro blends of cytokines that they produce. Th1 and Th2 cells can promote the development of cytotoxic cells and are believed to develop from Th0 cells. Th1 cells interact most effectively with mononuclear phagocytes; Th2 cells release cytokines that are required for B cell differentiation (Fig. 4-9).

Characterized by high interferon-gamma (IFN-γ) production, Th1 responses promote the elimination of intracellular pathogens (Fig. 4-10, A). Characterized by IL-4 and IL-5, Th2 responses promote a different type of effector response that involves immunoglobulin E (IgE) production and eosinophils capable of eliminating larger extracellular pathogens, such as helminths (see Fig. 4-10, B). In situations of repeated pathogen exposure or persistent infection, the polarization of T cell responses serves to focus the antigen-specific response on a specific effector pathway.

The following factors can influence the terminal differentiation of lymphocytes:

• Type of antigen-presenting cell (APC)

• Affinity of the specific antigenic peptide

• Types of costimulatory molecules expressed by APCs

• Cytokines acting on T cells during primary activation through TCRs

A hierarchy is apparent among these factors and is determined by how they influence T cell differentiation. Certain cytokines acting directly on T cells during primary activation appear to be the most proximal or direct mediators of CD4+ T cell differentiation. The presence of IL-12 during primary T cell activation leads to strong development of Th1 responses, and IL-4 promotes Th2 development. Activation through the TCR is a requirement for initiating terminal differentiation, but the signals from the TCR appear to be phenotype-neutral.

Certain T cells carry out delayed hypersensitivity reactions. These T cells react with antigen MHC class II on APCs and create their effects mainly through cytokine production. These cells generally are of the CD4+ phenotype.

T cells can also be differentiated into two populations depending on whether they use an αβ (TCR2) or γδ (TCR1) antigen receptor. The TCR consists of a heterodimer and a number of associated polypeptides that form the CD3 complex. The dimer recognizes processed antigen associated with an MHC molecule. The CD3 complex is required for receptor expression and is involved in signal transduction. TCR1 cells constitute less than 5% of total lymphocytes but appear in greater proportions in some sites (e.g., skin, vagina). TCR1 cells appear to recognize different antigens than TCR2 cells, including carbohydrate and intact protein antigens. In addition, some TCR1 cells do not require antigen to be processed or presented by MHC molecules.

Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes, or T cytotoxic (Tc) cells, are effector cells found in the peripheral blood that are capable of directly destroying virally infected target cells. Most Tc cells are CD8+ and recognize antigen on the target cell surface associated with MHC class I molecules (e.g., human leukocyte antigen [HLA] types A, B, and C) or MHC class I alone. This process is demonstrated by the immune response to virus-infected cells or tumor cells (Fig. 4-11).

In a primary viral infection, naïve CD8+ T cells are primed in secondary lymph nodes and consequently proliferate and differentiate into effector CD8+ T cells to eliminate virus-infected cells. After clearance of the virus, most effector CD8+ T cells contract because of apoptosis but a small number of these CD8+ T cells form a memory T cell pool.

Studies have demonstrated that human CD8+ T cells undergo a change in the expression of costimulatory molecules (e.g., CD27, CD28, and CD45RA) on their surface, according to their differentiation and maturation. Cytolytic effector molecules, perforin, and granzyme A-B, are considered to be markers for effector CD8+ T cells because they are the actual functional molecules for killing target cells.

Naïve and central memory CD8+ T cells express the membrane marker, CCR7, for homing to secondary lymph nodes, but effector memory and effector CD8+ T cells express the chemokine receptors for inflammatory cytokines, which enable the cells to migrate toward infected and inflamed sites. A unique subset of the effector CD8+ T cell population expresses CXCR1. These CXCR1 CD8+ T cells possess chemotactic activity toward the CDCR1 ligand IL-8, a potent inflammatory cytokine produced in inflamed tissues and in tissues infected with some viruses, such as human cytomegalovirus (HCMV) or influenza A. This suggests that these CXCR1+ effector CD8+ T cells immediately migrate to inflamed and infected sites to exert their effector function in the initial stage of an immune response. It is possible that effector CD8+ T cell subsets are functionally distinct populations of T lymphocytes.

In addition to destruction of virally infected, MHC class I–bearing targets, Tc cells are major effectors in allograft organ rejection. Tc cells express CD4 or CD8, depending on the MHC antigen restriction that governs their antigen recognition (i.e., class I or II antigens; Fig. 4-12).

Suppressor T lymphocytes, or T suppressor (Ts) cells, are functionally defined T cells that downregulate the actions of other T and B cells. Ts cells have no unique markers. Although antigen-specific suppression was described in 1970, and many investigators believe that Ts cells are critical in various phases of immunoregulation, peripheral tolerance, and autoimmunity, their mode of action is unclear. Many Ts cells are CD8+ and may operate through secretion of free TCRs.

Antigen Processing and Antigen Presentation to T Cells

Antigen-presenting cells (APCs) are a group of functionally defined cells capable of taking up antigens and presenting them to lymphocytes in a form that they can recognize. APCs take up antigens (e.g., dendritic cells, macrophages, B cells, even tissue cells) in various ways. Some are collected in the periphery and transported to the secondary lymphoid tissues; other APCs normally reside in lymphoid tissues and intercept antigen as it arrives. B cells recognize antigen in a native form.

There are two major pathways of antigen processing for the APC and target cell, endogenous and exogenous. The endogenous pathway processes proteins that have been internalized, processed into fragments, and reexpressed at the cell surface membrane in association with MHC molecules. In this pathway, proteins in the cytoplasm are cleaved into peptide fragments about 20 amino acids in length. These fragments are then transported into the lumen of the endoplasmic reticulum by the transporter associated with the antigen-processing complex, where the fragments encounter newly formed, heavy-chain molecules of MHC class I and their associated beta₂-microglobulin (β₂m) light chains. The heavy chain, light chain, and peptide form a trimeric complex, which is then transported to and expressed on the cell surface.

T cells that express the CD8+ cell surface marker recognize antigens presented by MHC class I molecules. CD8+ functions as a coreceptor in this process, binding to an invariant region of the MHC class I molecule. Pathogen clearance requires that CD8+ effector cells produce inflammatory cytokines and develop cytolytic activity against infected target cells, after which a small number of memory cells survive that rapidly regain effector function in the event of rechallenge. During this process, a relatively homogeneous pool of naïve CD8+ T cells differentiates into heterogeneous pools of effector and memory CD8+ T cells.

In the exogenous pathway, soluble proteins are taken up from the extracellular environment, generally by specialized or so-called professional APCs. The antigens are then processed in a series of intracellular acidic vesicles called endosomes. During this process, the endosomes intersect with vesicles that are transporting MHC class II molecules to the cell surface. CD4+ T cells recognize antigens that are presented by MHC class II molecules. As with CD8, the CD4 molecule functions as a coreceptor, increasing the strength of the interaction between the T cell and APC.

For both systems of antigen presentation, recognition of the antigen by the T cells is described as being MHC-restricted, a process whereby T cells recognize only antigen presented by self MHC molecules.

Antigen Recognition by T Cells

T cells are clonally restricted, so that each T cell expresses a receptor that can interact with a given peptide. Each lymphocyte makes only one type of antigen receptor and can recognize only a very limited number of antigens. Because receptors differ on each clone of cells, the entire lymphocyte population has an enormous number of different, specific antigen receptors.

The TCR of most T lymphocytes is composed of an alpha and beta polypeptide chain, with constant regions located close to the cell surface and the part that binds to the antigenic peptide of appropriate fit located away from the cell surface. The difference in structure of the distal regions of the alpha and beta chains allows the development of different clones of T cells. The TCR reacts with antigen in the context of MHC class I or II molecules on an APC (Fig. 4-13).

T cells recognize protein antigens in the form of peptide fragments presented at the cell surface by MHC I or II molecules. When the antigen-specific TCR on the T cell surface (specifically the zeta-beta chains) of the CD3 complex interacts with the appropriate peptide-MHC complex, it triggers phosphorylation of the intracellular domains of the CD3 zeta chains. Subsequently, the zeta-associated protein 70 (ZAP-70) binds to the phosphorylated zeta chains and is activated.

Simultaneous colligation of the cell marker CD4 (or CD8) with the MHC class II (or I) molecule results in the phosphorylation of particular kinases. These events stimulate the activation of at least three intracellular signaling cascades. T cell activation also requires a second costimulatory signal (e.g., interaction between marker CD28 on T cells and marker CD80 on APCs). This interaction also triggers several intracellular signaling pathways.

T-Independent Antigen Triggering

Some antigens, particularly polysaccharide polymers (e.g., dextran), can trigger B cells without help from T cells. These T-independent antigens generally are not strong, provoke mainly IgM responses, and induce minimal immunologic memory.

Natural Killer Cells

Natural killer (NK) cells are essential mediators of virus immunity. Their deficiency in humans lead to uncontrolled viral replication and poor clinical outcome. MHC class I (MCH I) is essential to NK and T cell effector and surveillance functions. A total of 70% to 80% of NK cells have the appearance of large granular lymphocytes (LGLs). Up to about 75% of LGLs function as NK cells and LGLs appear to account fully for the NK activity in mixed cell populations.

NK cells destroy target cells through an extracellular nonphagocytic mechanism referred to as a cytotoxic reaction, MHC-unrestricted cytolysis. Target cells include tumor cells, some cells of the embryo, cells of the normal bone marrow and thymus, and microbial agents. Studies have suggested that a considerable number of NK cells may be present in other tissues, particularly in the lungs and liver, where they may play important roles in inflammatory reactions and in host defense, including defense against certain viruses (e.g., cytomegalovirus, hepatitis). NK cells will actively kill virally infected target cells and, if this activity is completed before the virus has time to replicate, a viral infection may be stopped.

Several cytokines affect NK cell activation and proliferation. NK cells are highly responsive to IL-2, IL-7, and IL-12. These cytokines generate high cytokine-activated killer activity in these cells. In addition, NK cells synthesize a number of cytokines involved in the modulation of hematopoiesis and immune responses and in the regulation of their own activities.

Target cell recognition and the molecular identification and analysis of the involved NK cell receptors are undergoing intensive research. These molecules are mainly classified under the family of cell adhesion molecules (CAMs). The main class of effector CAMs shown to mediate NK cell functions is the leukocyte integrins—more specifically, the β₂ class of integrins.

Several NK cell surface molecules involved in target cell recognition and binding have been identified. NK cells recognize targets using several cell surface molecular receptors (e.g., CD2, CD69, NKR-P1) and a high density of the Fc receptor CD16 of IgG (FC-R III). They also receive inhibitory signals from MHC class I on potential target cells, transduced by a killer inhibitory receptor on the NK cell. CD56 may mediate interactions between effector and target cells. NK cells are able to bind and lyse antibody-coated nucleated cells through a membrane Fc receptor that can recognize part of the heavy chain of immunoglobulins. This enables NK cells to mediate antibody-dependent, cell-mediated cytotoxic (ADCC) activities. Some, if not all, of the activation of NK cells is mediated by CD16, which exerts a regulatory role in their cytolytic function. NK cells respond to cross-linking of CD16 and CD69 as follows:

K-Type Lymphocytes

K-type killer cells are mononuclear cells that can kill target cells sensitized with antibody, which they engage through their Fc receptors. Most K-type cells are non-T, non-B lymphocytes, but macrophages and eosinophils can also have K cell activity.

K-type cells exhibit a different cytotoxic mechanism than NK cells. The target cell must be coated with low concentrations of IgG antibody, referred to as an ADCC reaction. An ADCC reaction may be exhibited by both K cells and phagocytic and nonphagocytic myelogenous-type leukocytes. K cells are capable of lysing tumor cells. Although morphologically similar to a small lymphocyte, the precise lineage of the K cell is uncertain.

Cell Surface Markers

B lymphocytes are best known to express CD19 but not CD3 surface membrane markers. During B-cell differentiation in the bone marrow, the surface molecule CD19 appears early and remains on the B cell unit until it differentiates into a plasma cell. Four proteins on the surface of mature B cells-CD19, CD21, CD81, and CD225—from the CD19 complex.

Primitive B cell precursors have δ chains in their cytoplasm and no Ig on their surface. More differentiated (but still immature) B cells have intact cytoplasmic IgM and surface IgM. Mature B cells lose their cytoplasmic IgM and add surface IgD to the surface IgM. These changes appear to occur in the absence of antigen and depend on cytokines.

In humans, there is evidence of four types of B cell surface markers:

After binding and cooperative interaction with T cells, B cells undergo transformation into plasma cells. The secreted antibody, in turn, has the same specificity as the Ig receptor on the B cell. Almost all the antibody produced by plasma cells is secreted (plasma cells have few Ig receptors), but 90% of the antibody produced by B cells is expressed as surface Ig receptors. Some antigens (e.g., lipopolysaccharides from some gram-negative organisms) can bind to the Ig receptor and also stimulate an antibody response independent of T cell cooperation (T-independent antigens). This type of response is generally of low intensity and is class-restricted to the production of IgM antibody.

B cells have surface immunoglobulin (sIg), except for very immature lymphocytes and mature plasma cells, that are normally polyclonal (i.e., kappa and lambda light chains are present on the cytoplasmic membrane of B cells). Mu and delta heavy chains are usually found with kappa or lambda chains on any one cell surface. Gamma and alpha chains are rarely found on the surface of properly prepared, normal lymphocytes.

B Cell Activation

B cells can be stimulated in their resting state to enlarge, develop synthetic machinery, divide, mature, and secrete antibody. The proper signals for this sequence depend on the type of triggers, which can be specific or nonspecific and polyclonal. Specific activation involves the antigen that is complementary to the particular Ig on the surface. Nonspecific activation occurs with B cell mitogens.

Efficient antibody production to complex protein antigens requires T cell help, which in turn develops from APCs presenting antigen to the T cell. Activated T cells secrete a variety of cytokines that together with the specific antigen, trigger the B cell to develop into an antibody-secreting cell. This process also involves class switching.

In the immune response to a foreign protein, the first antibodies to appear are of the IgM class (or isotype). As the response proceeds, other isotypes (IgG, IgA, and IgE) emerge from Ig class switching. The isotype switch has considerable clinical importance because each of the four major isotypes has specialized biologic properties. IgG is the principal class of antibody in interstitial fluids and IgA is the protective antibody of mucosal surfaces. Isotype switching requires collaboration between antibody-synthesizing B cells and helper CD4+ T cells. The B cell uses IgM molecules on its surface to capture the antigen and present the antigen to the T cell. Contact between the collaborating lymphocytes is enhanced by complementary pairs of CAMs. Some CAMs (e.g., CD4, MHC class II antigens) are constitutively expressed on the surface of T and B cells, whereas others are induced. For example, contact between B and T cells induces the T cell to express a ligand for the B cell surface molecule CD40. In turn, CD40 interacts with the newly expressed CD40 ligand on the T cell, which leads to the expression of another B cell surface molecule, B7. The latter’s partner on the surface of the T lymphocyte is CD28. These cooperative and synergistic interactions between T and B cells induce the secretion of cytokines such as IL-2 and IL-4.

Isotype switching requires two signals. The first is delivered by an interleukin and the second by the binding of CD40 to its ligand on the T cell. In the process of switching from IgM synthesis to IgE synthesis, IL-4 makes the IgE gene in the B cell accessible to the switch machinery initiated when CD40 binds to its ligand. In this process, the gene that encodes the variable region (the part of the antibody molecule that contains the antigen-binding site) moves from its position near the gene that encodes for IgM to a position near the gene that encodes for IgE.

Changes With Aging

Except for inconsistent values seen in extremely old adults, the total number of T cells in the peripheral blood is relatively stable throughout adult life. However, there is a change in the distribution of T cell subpopulations. A decrease in the number of suppressor cells and an increase in the helper cell population are demonstrated in older adults.

The effect of aging on the immune response is highly variable, but the ability to respond immunologically to disease is age-related. Faulty immunologic reactions (e.g., aberrant functioning of immunoregulatory cells, effector T cells, and antibody-producing B cells) may contribute to poor immunity in older adults. Functional deficits of T lymphocytes have been identified with aging, causing impairment of cell-mediated immunity. In addition, skin testing reveals decreases in the intensity of delayed hypersensitivity in older adults. The proliferative response of T lymphocytes to mitogens or antigens such as Mycobacterium tuberculosis or varicella-zoster virus is impaired.

A decrease in Th cells is the primary cause of the impaired humoral response in older adults. Although the total number of B cells and total Ig concentration remain unchanged, the serum concentration of IgM is decreased and IgA and IgG concentrations are increased.

Cell-Mediated Immune System

Deficiencies of cell-mediated immunity are often suspected in individuals with recurrent viral, fungal, parasitic, and protozoal infections. Patients with AIDS exhibit some of the most severe manifestations of cell-mediated immunity (see Chapter 25).

One avenue of testing involves delayed hypersensitivity skin testing to determine the integrity of the patient’s cell-mediated immune response. More than 90% of normal adults will react to one of the following antigens within 48 hours after antigen exposure: Candida albicans, Trichophyton, tetanus toxoid, mumps, and streptokinase-streptodornase. Reactivity to histoplasmin or purified protein derivative (PPD) is positive in patients with active infection or previous exposure to histoplasmosis or tuberculosis, respectively; therefore these tests are not useful for the assessment of anergy.

The number of T lymphocytes, the primary effector cells in cell-mediated reactions, can be determined by several techniques. Previously, the gold standard was the E rosette technique (erythrocyte rosette formation), but the development of flow cytometry with MAbs has replaced this technique. Testing for the functionality of lymphocytes is just as important as a quantitative count of CD4+ and CD8+ cells.

The in vitro diagnostic test (IVD; see later, “Assessment of Cellular Immune Status”) is the newest approach to testing the functionality of T lymphocytes. It is important to recognize that CD4 counts do not always reflect the actual status of the patient’s immune system. ImmuKnow (Cylex, Columbia, Md) is the first U.S. Food and Drug Administration (FDA)–approved immune function test. It is widely considered to be the gold standard of immune function testing.

The QuantiFERON-CMI kit (Cellestis, Valencia, Calif) is an in vitro assay for measuring cell-mediated immune functionality. The procedure is a single-step enzyme-linked immunosorbent assay (ELISA) to determine T cell responses by measuring IFN-γ levels in plasma. This is a specific marker cytokine for a cell-mediated or inflammatory immune response (e.g., bacterial, parasitic, or viral).

Humoral System

The humoral system can be screened for abnormalities by quantitating the concentrations of IgM, IgG, and IgA. An initial simple screening can be determined by the presence and titer of antibodies to type A and B red blood cell (RBC) antigens.

Primary Immunodeficiency Disorders

Primary immunodeficiencies (PID) are rare genetic disorders of the innate and adaptive immune system. Classic primary immunodeficiency disorders (PIDs) are usually monogenic (mendelian) disorders affecting host defenses (Box 4-3). More than 200 clinical phenotypes of PID have been described. Over 120 different gene mutations have been identified which cause impairment in the differentiation and/or function of immune cells with different degrees of severity. Diseases associated with a primary defect in the immune response are comprised of about 40% T cell disorders, 50% B cell disorders, 6% phagocytic abnormalities, and 4% complement alterations (Fig. 4-14). The most common T cell deficiency states are those associated with a concurrent B cell abnormality. Primary immunodeficiency disorders are predominantly seen (75%) in children younger than 5 years.

Gene therapy with hematopoietic stem cells (HSC) is a therapeutic strategy for the treatment of several forms of primary immunodeficiency. Current approaches use gene transfer of the therapeutic gene into autologus HSC by retroviral vector-mediated gene transfer. This method has been successful in severe combined immunodeficiencies (SCID-1) and chronic granulomatous disease (CGD). Wiskott-Aldrich syndrome is another good candidate for gene therapy treatment.

Secondary Immunodeficiency Disorders

A secondary immunodeficiency can result from a disease process that causes a defect in normal immune function, which leads to a temporary or permanent impairment of one or multiple components of immunity in the host (Box 4-4). Patients with secondary immunodeficiencies, which are much more common than primary deficiencies, have an increased susceptibility to infections, as seen in the PIDs.

Immunosuppressive agents and burns are major causes of secondary immunodeficiencies. In varying degrees, immunosuppressive agents have been demonstrated to affect every component of the immune response. In burn patients, septicemia is a common complication in those who survive the initial period of hemodynamic shock. The mechanism that seems most critical in thermal injury is disruption of the skin; however, interference with phagocytosis and deficiencies of serum Ig and complement levels have also been observed.

Immune-Mediated Disease

The immune system is normally efficient in eliminating foreign antigens. The nature of the antigen or the genetic makeup of the host, however, can cause alterations of the immune response that can be injurious and lead to immune-mediated disease (Table 4-8). In these disorders, the immune response is normal but the reactivity is heightened, prolonged, or inappropriate.

A major concern is allergic reactions, characterized by an immediate response on exposure to an offending antigen and the release of mediators (e.g., histamine, leukotrienes, prostaglandins) capable of initiating signs and symptoms (see Chapter 26). Although allergic reactions are associated with IgE, not all allergic reactions are IgE-mediated. Complement activation by immune complexes or through the alternative complement pathway has been shown to release complement C3a and C5a anaphylatoxins, which are capable of producing similar reactions.

Autoimmune disease is thought to be caused by antibody or T cell sensitization with autologous self-antigens (see Chapter 28). Postulated mechanisms of this process include the following:

Autoimmune Lymphoproliferative Syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is a disease in which a genetic defect in programmed cell death, or apoptosis, leads to breakdown of lymphocyte homeostasis and normal immunologic tolerance. ALPS is the first pediatric syndrome described in which the primary defect is in apoptosis.

Defective apoptosis in lymphocytes (and, in ALPS type II, dendritic cells) leads to accumulation of these cells in the lymphoid organs after they would normally be eliminated. As a result, cells with autoimmune potential are unchecked and can induce a variety of autoimmune diseases; the risk for malignant transformation to lymphoma is increased.

Patients with ALPS have chronic enlargement of the spleen and lymph nodes, various manifestations of autoimmunity, and elevation of a normally rare population of double-negative T cells (DNTs). When lymphocytes from ALPS patients are cultured in vitro, they are resistant to apoptosis, as compared with cells from healthy controls.

Most ALPS patients have mutations in a TNF receptor gene that is a member of a superfamily (TNFRSF6). This gene, previously known as APT1, encodes the cell surface receptor for the major apoptosis pathway in mature lymphocytes. This receptor has many names, including Fas. The Fas apoptotic pathway is important for eliminating excess T cells after they have been activated and also eliminating antigen-driven and autoreactive T cell clones. Fas is a functional trimer residing at the cell membrane that when engaged by trimeric Fas ligand (FasL), initiates a proteolytic cascade leading to chromosomal DNA degradation and cell death.

Principle

Phytohemagglutinin (PHA) is a nonspecific mitogen that can be used to stimulate cell division in CD4 T lymphocytes, regardless of their antigenic specificity or memory status. Therefore, PHA is considered to be a global stimulator of the immune system. The production of intracellular adenosine triphosphate (ATP) is one of the first steps in cellular activation following stimulation with mitogens such as PHA. ATP is a multifunctional nucleotide that plays an indispensable role in the transfer of intracellular chemical energy.

When a sample of patient blood is incubated with PHA, increased ATP production (see figure below) occurs within PHA-activated CD4 T cells. These cells are then isolated by the addition of magnetic beads coated with anti-CD4 monoclonal antibody. The isolated CD4 cells are washed on a magnetic tray and lysed to release intracellular ATP. The amount of measured light emitted following the addition of a luminescence reagent is proportional to the amount of ATP present. An established calibration curve is used to characterize the cellular immune function of the sample.

Procedural Protocol

∗See for a complete description of the method.