The introduction of monoclonal antibody (MAb) testing (see Chapter 2) led to the present identification of surface membrane markers on lymphocytes and other cells. In practical terms, surface markers are used to identify and enumerate various lymphocyte subsets, establish lymphocyte maturity, classify leukemias, and monitor patients on immunosuppressive therapy.

Cell surface molecules recognized by MAbs are called antigens, because antibodies can be produced against them, or markers, because they identify and discriminate between, or “mark,” different cell populations. Originally, surface markers were named according to the antibodies that reacted with them, but a uniform nomenclature system has now been adopted.

In this system, a surface marker that identifies a particular lineage or differentiation stage with a defined structure, and that can be identified with a group or cluster of MAbs, is called a member of a cluster of differentiation (CD; Fig. 4-2). Markers can be categorized as follows:

Figure 4-2 Lymphocyte membrane marker development—lymphoid CD antigen expression in T lymphocytes. *TdT,* Terminal deoxynucleotidyl transferase. *CD,* cluster of differentiation; *HLA,* human leukocyte antigen; *Ig,* immunoglobulin.

• Some markers are specific for cells of a particular lineage or maturational pathway.

• Some markers vary in expression, depending on the state of activation or differentiation of the same cells—for example, when CD antigen identification is used to classify lymphocyte subsets (e.g., CD4 and CD8).

In addition to using CD classification for the identification and separation of lymphocytes, CD antigens are involved in various lymphocyte functions, usually the following:

• Promotion of cell to cell interactions and adhesion

• Transduction of signals that lead to lymphocyte activation

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.

Figure 4-3 Development of immunologic organs.
The anatomy of the human fetus illustrates the development of the mammalian immune system. Cells of the pharyngeal pouches migrate into the chest and form the thymus. Precursors of lymphocytes originate early in embryonic life in the yolk sac and eventually migrate to the bone marrow via the spleen and liver.

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).

Figure 4-4 Human primary and secondary tissues. (Adapted from Turgeon ML: Clinical hematology: theory and procedures, ed 4, Philadelphia, 2005, Lippincott Williams & Wilkins.)

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.

Figure 4-5 Thymic development.
Histology of the thymus changes with age. The main feature of these changes is a loss of cellularity with increasing age.

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.

Figure 4-6 A, Lymph node (×90). B, Enlargement of cortical nodule seen in A (×450). (From Anthony CP, Thibodeau GA: Textbook of anatomy and physiology, ed 12, St Louis, 1987, Mosby.)

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

Virgin or Naïve Lymphocytes

Virgin or naïve lymphocytes are cells that have not encountered their specific antigen. These cells do express high-molecular-weight variants of leukocyte common antigen.

Memory cells are populations of long-lived T or B cells that have been stimulated by antigen. They can make a quick response to a previously encountered antigen. Memory B cells carry surface IgG as their antigen receptor; memory T cells express the CD45RO variant of the leukocyte common antigen and increased levels of cell-adhesion molecules (CAMs), chemical mediators involved in inflammatory processes throughout the body (Fig. 4-8).

Figure 4-8 Phases of an adaptive immune response.
An adaptive immune response consists of distinct phases; the first three are the recognition of antigen, activation of lymphocytes, and elimination of antigen (effector phase). The response declines as antigen-stimulated lymphocytes die by apoptosis, restoring homeostasis, and the antigen-specific cells that survive are responsible for memory. The duration of each phase may vary in different immune responses. The y-axis represents an arbitrary measure of the magnitude of the response. These principles apply to humoral immunity (measured by B lymphocytes) and cell-mediated immunity (mediated by T lymphocytes). (From Abbas AK, Lichtman AH: Basic immunology: functions and disorders of the immune system, updated edition, ed 3, Philadelphia, 2011, Saunders.)

Development of T Lymphocytes

Most lymphocytes (see Color Plate 8) found in the circulating blood are T cells derived from bone marrow progenitor cells that mature in the thymus gland (Table 4-2). These cells are responsible for cellular immune responses and are involved in the regulation of antibody reactions in conjunction with B lymphocytes.

Table 4-2

Lymphocyte Characteristics

Type	Function(s)	Phenotypic Marker	Peripheral Blood (% of Total)
Helper T (Th) cells	Stimulate B cell growth and differentiation (humoral immunity); macrophage activation by secreted cytokines (cell-mediated immunity)	CD3+, CD4+, CD8−	50-60
Cytotoxic T (Tc) cells	Lysis of virus-infected cells, tumor cells, and allografts (cell-mediated immunity); macrophage activation by secreted cytokines (cell-mediated immunity)	CD3+, CD4−, CD8+	20-25
Natural killer (NK) cells	Lysis of virus-infected cells, (antibody-dependent cellular cytotoxicity)	Fc receptor for IgG or cells CD16	∼10
B cells	Antibody production (humoral immunity)	Fc receptors, MHC class II, CD19, CD21	10-15

During cellular development, T lymphocyte function–associated antigens vary in expression. Some antigens appear early in cellular development and remain on mature T cells. Others appear at an early or intermediate stage of cellular maturation and are lost before maturity.

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.

Double-Negative Thymocytes

Early thymocytes lacking CD4 and CD8 surface membrane markers are referred to as double-negative thymocytes. These cells proliferate in the outer cortex of the thymus under the influence of interleukin-7 (IL7). IL-7 is critical for this growth and differentiation.

Rearrangement of the genes that code for the antigen receptor, the T cell receptor (TCR), begin at this developmental stage. CD3 constitutes the main part of the T cell antigen receptor. The configuration of two of the eight chains of the receptor have variable regions that recognize specific antigens. These are coded for by selecting gene segments and deleting others.

Rearrangement of the beta (β) chain occurs first; the appearance of a functional β chain on the cell surface sends a signal to suppress any further β chain gene rearrangements. The combination of the β chain with the CD3 forms the pre– T-cell antigen receptor (TRC). Signaling by the β chain promotes the development of a CD4+ and CD8+ thymocyte.

Thymocytes that express gamma (γ ) and delta (δ) chains follow a different developmental pathway. Cells expressing gamma-delta (γδ) chains typically remain both CD4− and CD8−. These double-negative cells represent most of the population of T lymphocytes in the skin and intestinal and pulmonary epithelium.

Circulating CD3+ double-negative lymphocytes are phenotypically and functionally distinct from single-positive CD3+CD4+ and CD3+CD8+ lymphocytes and are thought to represent a distinct T cell lineage. The presence of low numbers of double-negative T cells in healthy individuals and the increase observed in association with lymphoproliferative disorders, graft-versus-host disease, and autoimmune diseases suggest a pathogenic or immunoregulatory role for this population of T lymphocytes.

Double-Positive Thymocytes

Cells with both CD4+ and CD8+, or double-positive, surface markers represent the second stage of thymocyte development. These thymocytes begin to demonstrate rearranged genes coding for the alpha (α) chain. When the CD3-αβ receptor complex (TCR) is expressed on the cell surface, a process known as positive selection permits only double-positive cells with functional TCR receptors to survive. T cells must recognize foreign antigen in association with class I or II major histocompatibility complex (MHC) molecules. Any thymocyte that is unable to recognize self-MHC dies without ever leaving the thymus gland. Functioning T lymphocytes must be able to recognize a foreign antigen along with MHC molecules. A second selection process, negative selection, takes place among the surviving double-positive T cells. Only 1% to 3% of double-positive thymocytes survive in the cortex.

Double-positive (DP) CD4CD8 Tαβ cells have been reported in normal individuals as well as in different pathologic conditions, including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated. Double-negative cells may act like natural killer (NK) cells because they are capable of binding to many natural, unprocessed cell surface molecules. In addition, these cells are capable of recognizing antigens without being presented by MHC proteins. Consequently, NK cells may represent an important bridge between natural and adaptive immunity.

Later Cellular Differentiation and Development of T Lymphocytes

When mature T cells leave the thymus, their T cell receptors (TCRs) are CD4+ or CD8+. Survivors of selection exhibit only one type of marker, CD4+ or CD8+ and migrate to the medulla. These cells gain functional maturity with their entry into the peripheral blood circulation.

T cells develop into a variety of clones. Each lymphocyte displays a single type of structurally unique receptor. The repertoire of antigen receptors in the entire population of lymphocytes is extremely large and diverse. This increases the probability that an individual lymphocyte will encounter an antigen that binds to its receptor, thereby triggering activation and proliferation of the cell. This process, clonal selection, accounts for most of the basic properties of the adaptive immune system.

Antigen receptors for common pathogens need to be reinvented by every generation of cells. Because the binding sites of antigen receptors arise from random genetic mechanisms, the receptor repertoire contains binding sites that can react not only with infectious microorganisms, but also with innocuous environmental antigens and self antigens.

T-Lymphocyte Subsets

The CD4+ subset was initially described as representing the helper-inducer T cell; the CD8+ subset was initially described as representing the suppressor-cytotoxic T cell. Lymphocytes can be subdivided into several populations using various operational and phenotypic parameters. For example, CD4 lymphocytes express both CD3 and CD4. The surface marker CD45RA subset delineates a naïve helper T cell population. The CD45RO subset delineates a memory helper T cell population. CD8+ lymphocytes express both CD3 and CD8 surface membrane markers.

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).

Figure 4-9 Differentiation of naïve CD4+ helper T (Th) cells into Th1 and Th2 effector cells.
After their activation by antigen and costimulators, naïve helper T cells may differentiate into Th1 and Th2 cells under the influence of cytokines. IL-12 produced by microbe-activated macrophages and dendritic cells stimulates differentiation of CD4+ T cells into Th1 effectors. In the absence of IL-12, the T cells themselves (and perhaps other cells) produce IL-4, which stimulates their differentiation into Th2 effectors. (Adapted from Abbas AK, Lichtman AH: Basic immunology: functions and disorders of the immune system, ed 3, Philadelphia, 2008, Saunders.)

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.