Chapter 73: The Structure of Lymphocytes and Plasma Cells

Lymphocytes and plasma cells first were described in 1774 and 1875, respectively.¹ Studies during the subsequent 75 years with improved histologic techniques and light microscope optics furthered understanding of the lymphoid organs and the distribution of lymphocytes.^2,3,4,5,6 By the mid-20th century, awareness that the immune system had at least two components—one governing humoral immunity and one governing cellular immunity—led to early concepts of different lymphocyte subsets. Also, at the same time came the discovery that the thymus and bursa of Fabricius in birds were the source of what came to be known as T (thymic-derived) and B (bursa-derived) lymphocytes, respectively, and that the marrow was the bursa equivalent in humans (human B cells therefore could represent marrow-derived cells). This discovery coupled with descriptions of inherited absence of the thymus leading to loss of cellular immunity but retention of humoral immunity and cases of retention of cellular immunity in children who were deficient in antibody production, eventually led to our current understanding of the division of labor among what originally appeared to be a common lymphocyte pool, morphologically. The later advent of monoclonal antibodies against numerous surface antigens coupled with flow cytometry, in vitro functional assays, molecular techniques to distinguish between B cells and T cells, and experiments using inbred strains of mice brought us to our current state of knowledge of the immune response and its abnormalities.

Flow cytometry identifies a multitude of lymphocyte subsets based on antigen expression patterns. These immunophenotypic subsets correlate closely with function as determined by in vitro and in vivo testing. T lymphocytes, B lymphocytes, and natural killer (NK) cells represent three major blood lymphocyte functional subsets. The marrow and thymus contain precursor cells that resemble lymphocytes but lack function without differentiation and maturation into various lymphocyte subsets. Plasma cells are terminally differentiated B lymphocytes that produce immunoglobulin and mostly reside in marrow, lymph nodes, and other lymphoid tissues. (Chap. 6).

LIGHT MICROSCOPY

Classic studies of blood and tissues defined lymphocytes as spherical and/or ovoid cells that have diameters from 6 to 15 μm when flattened on glass slides.⁴ Some of these studies described two separate broad types of lymphocytes based on size: small lymphocytes with diameters of 6 to 9 μm and large lymphocytes with diameters of 9 to 15 μm. Patients with acute viral illnesses have increased numbers of circulating large, “reactive,” lymphocytes. Other illnesses, such as infection with Bordetella pertussis and autoimmune disorders, can cause blood to have increased small lymphocytes or lymphocytes with plasma cell-like morphology (Chap. 78). The mean absolute number of circulating small lymphocytes in normal adults is 2.5 × 10⁹/L (Chap. 2).⁷ Children have higher lymphocyte counts that trend downward until they reach adult levels at approximately 8 to 10 years of age (Chap. 7).⁸

Most lymphocytes in normal blood are small with an ovoid or kidney-shaped nucleus that stains purple, has densely packed nuclear chromatin, and occupies approximately 90 percent of the cell area (Fig. 73–1A and B) by Romanowsky polychromatic stains (e.g., Giemsa or Wright) of air-dried films. A small rim of cytoplasm stains light blue. Nucleoli rarely are observed in Wright-stained films, but nucleoli in these cells may become visible in certain preparations, such as cytospin slides, or after prolonged storage in anti-coagulated blood collection tubes.

A minority of lymphocytes in normal blood have morphology that defines them as large granular lymphocytes (LGLs).⁹ These LGLs are slightly larger than most lymphocytes, having an increased area of light blue or clear cytoplasm. LGL cytoplasm contains a number of coarse pink granules, usually 5 to 15 per cell, and occasional clear vacuoles. In a normal adult, approximately 5 percent but up to 10 to 15 percent of blood lymphocytes are LGLs (see Fig. 73–1E).⁹ The LGLs in blood are composed of NK cells and a subset of cluster of differentiation (CD) 8+ T lymphocytes, indistinguishable by their morphology.

PHASE-CONTRAST MICROSCOPY

Active movement of lymphocytes is studied by phase-contrast, or interference-contrast, microscopy. Lymphocytes move slowly with a “hand mirror” appearance. Cytoplasmic spreading does not occur. However, during cell movement, a thickening occurs in the cytoplasmic rim, which houses most of the cell’s organelles, including the Golgi apparatus.

TRANSMISSION ELECTRON MICROSCOPY AND CYTOCHEMISTRY

The blood lymphocyte measures approximately 5 μm in spherical diameter as visualized by transmission electron microscopy.¹⁰ The nucleus has an abundance of electron-dense, condensed heterochromatin, a feature characteristic of nonproliferating cells. The nucleoli are round in section, approximately 0.5 to 1.4 μm in diameter. They are composed of three distinct and concentrically arranged structural units: the central region or agranular zone; the middle, fibrillar region; and the granular zone, which contains intranucleolar chromatin. The lymphocyte’s nuclear membrane contains nuclear pores and a perinuclear space.

The cytoplasmic organelles of the lymphocytes are characteristic of eukaryotic cells. Some organelles, such as the Golgi zone, are poorly developed. The cytoplasm contains free ribosomes, occasional ribosome clusters, and strands of rough-surfaced endoplasmic reticulum (Fig. 73–2). Centrioles, mitochondria, microtubules (diameter approximately 0.25 μm), and microfilaments (diameter approximately 0.07 μm) are present in the cytoplasm adjacent to the cell membrane. The cytoplasm also contains lysosomes, which are approximately 0.4 μm in diameter, are electron opaque, and contain classic lysosomal enzymes (e.g., acid phosphatase, β-glucuronidase, and acid ribonuclease).¹¹ The lymphocyte plasma membrane stains with colloidal iron, a marker for membrane sialic acid. Lymphocyte cell membranes and cell coat glycoproteins are shown with other electron-dense markers, including phosphotungstic acid, lanthanum colloid, and ruthenium red.

Most T lymphocytes have a localized “dot” pattern when stained for acid phosphatase, acid and neutral nonspecific esterases, β-glucuronidase, and N-acetyl-β-glucosaminidase.¹² LGLs stain for acid hydrolases with a dispersed, granular reaction pattern.¹³ B lymphocytes either lack esterase and acid phosphatase or show minimal scattered granular staining.

SCANNING ELECTRON MICROSCOPY

Scanning electron microscopy provides three-dimensional information.¹⁴ However, the resolution achieved with scanning electron microscopy, approximately 0.1 μm, is considerably less than that possible with transmission electron microscopy, generally 0.002 to 0.0039 μm. Normal blood lymphocytes, washed and collected on silver membranes and fixed in glutaraldehyde, have a spherical topography with varying numbers of stubby or finger-like microvilli (Fig. 73–3).¹⁵ In contrast, monocytes are much larger, have few microvilli, and display ruffled membranes and ridge-like profiles (Chap. 67).

Lymphocyte microvilli contain parallel bundles of actin filaments that undergo continuous assembly and disassembly.¹⁶ The function of lymphocyte microvilli probably includes segregating surface receptors involved in extravasation. Two receptors involved in the initial rolling phase of extravasation, L-selectin and α₄β₇ integrin,¹⁷ localize to microvillar tips. In contrast, the β₂ integrins that mediate stable adhesion and diapedesis localize to nonprotrusive regions of the cell surface. This spatial separation of surface receptors might enable a temporal segregation of adhesive function during extravasation. Lymphocytes expressing chimeric L-selectin constructs that no longer localize to microvilli do not roll on L-selectin ligands, supporting this hypothesis.¹⁸

Lymphocyte stimulation is associated with a complex sequence of morphologic and biochemical events. Activation of B and T lymphocytes results in the transformation of the small, resting lymphocyte into proliferating large cells with abundant highly basophilic cytoplasm, irregularly condensed or smudgy chromatin, and round to slightly irregular nuclear outlines (see Fig. 73–1D). Nucleoli may be evident by light microscopy in these cells, but they usually are not prominent. Infection with B. pertussis causes an increase in blood lymphocytes with a particular activated morphology characterized by small size, scant cytoplasm, and cleaved nuclei with mature chromatin (see Fig. 73–1C).

Activated lymphocytes proliferate and mature into effector lymphocytes and memory cells. Effector cells include helper T cells, cytolytic T cells, and plasma cells (see Figs. 73-1F and G, 73–4, and 73–5). In vitro, plant lectins, bacterial products, polymeric substances, and enzymes activate lymphocytes and cause mitosis. Such agents are called mitogens. Some mitogens are specific for either B or T lymphocytes, whereas other mitogens stimulate both.¹⁹

Approximately 4 hours after mitogen stimulation, lymphocytes show increased nucleolar size and an increase in the number and concentration of granules in the granular zone. These changes are followed by an increase in fibrillar zones and increased intranucleolar chromatin. Nucleolar chromatin becomes more electron lucent or dispersed. From 48 to 72 hours following the addition of phytohemagglutinin, the volume of the cytoplasm increases. In addition, the cytoplasm contains an increased number of ribosomal clusters and more rough-surfaced endoplasmic reticulum. The activated cell has increased numbers of lysosomes and a larger Golgi complex with more components.²⁰ Under some circumstances (e.g., cultures of human lymphocytes stimulated for 7 to 10 days with pokeweed mitogen), some B cells form well-developed Golgi and plasmacytoid features.²¹ Similar plasmacytoid cells are observed in antigen-stimulated lymph nodes, during graft rejection in vivo, and in some in vitro systems, including the mixed lymphocyte culture. In lymph nodes, the stimulated lymphoid cells may be referred to by various pathologists as lymphoblasts, immunoblasts, centroblasts, or large lymphoid cells. Morphologic criteria for these cells overlap.

Following stimulation with antigen or mitogens, the lymphocyte enters the cell cycle. The fate and function of lymphocytes that traverse the cell cycle can be divided into two pathways. Some lymphocytes undergo several mitotic cycles and then return to the G₀ phase, indistinguishable in morphology from the original nonactivated cells. Some of them then become memory cells, programmed to remember the stimulating antigen and thus respond more rapidly to reexposure to the original antigen. Alternatively, they become terminally differentiated effector lymphocytes, such as plasma cells or cytotoxic T cells (Chaps. 75 and 76).

MORPHOLOGIC STUDIES

Plasma cells derive from small B lymphocytes after activation in the correct environment. The characteristic feature of plasma cells is abundant cytoplasmic and secretory immunoglobulin (Ig). A fully mature plasma cell lacks surface Ig expression. Each plasma cell has the same clonal rearrangement of its V(D)J (variable diversity joining) Ig genes as its predecessor B lymphocyte (Chap. 75). Several mitotic divisions may occur during cellular differentiation from the resting lymphocyte to the plasmablast to the immature plasma cell. Immature plasma cells can undergo successive waves of mitosis in the medullary cords of lymph nodes in response to antigen.²² Cell transfer experiments demonstrated that these transformed cells later mature into antibody-producing plasma cells.²³

Pokeweed mitogen induces B lymphocytes to transform into plasma cells after 7 to 10 days of culture.²⁴ These plasma cells infrequently contain large electron-dense inclusions (Russell bodies), which may measure 2 to 3 μm in diameter (see Fig. 73–1H).²⁵ Russell bodies, cytoplasmic Ig in the endoplasmic reticulum, sometimes are dissolved during the staining procedure. They usually occur in pathologic states but may be found in plasma cells from normal lymph nodes or marrow.

LIGHT MICROSCOPY, HISTOCHEMISTRY, AND ELECTRON MICROSCOPY

The mature plasma cell has a characteristic basophilic cytoplasm and an eccentric nucleus when treated with a polychrome stain. The nuclear polarity is attributable to a large paranuclear zone, which corresponds to the Golgi apparatus. The typical mature plasma cell spread on a slide usually is round or oval and has a diameter of 9 to 20 μm, with a mean cell diameter of 14 μm and a mean nuclear diameter of 8.5 μm (see Fig. 73–1F and G).²⁶ The nuclear heterochromatin is coarse and distributed in a pattern that sometimes resembles the spokes of a wheel (cartwheel nucleus) on paraffin sections. Normal plasma cells may occasionally have two or more nuclei. Cytochemical features of plasma cells include positive staining for β-glucuronidase and mitochondrial enzyme markers. They do not stain for peroxidase or nonspecific esterase.²⁷

Plasma cells in patients with certain diseases may have different histochemical properties. Plasma cell size and morphology may be altered substantially in myeloma and macroglobulinemia (Chaps. 107 and 109, respectively). Plasma cells with two or three nuclei are more frequent in marrows from patients with plasma cell dyscrasias. Periodic acid-Schiff stains may reveal cytoplasmic or nuclear inclusions in clonal plasma cells.²⁸ Under some circumstances, amyloid inclusions in plasma cells have been detected by electron microscopy.²⁹ In hemochromatosis and hemosiderosis, plasma cells may contain hemosiderin when examined by electron microscopy.³⁰

The plasma cell is packed with a rough-surfaced endoplasmic reticulum having numerous attached ribosomes as seen by electron microscopy. A large, circumscribed Golgi zone forms a paranuclear halo when observed by light microscopy. The nucleus has dense areas of heterochromatin. The Golgi zone contains lamellae, vesicles, vacuoles, and a number of granules. Mitochondria are located between the strands of endoplasmic reticulum.³¹

B LYMPHOCYTE ANTIGENS

Figure 73–6 summarizes the expression of antigens on cells of the B-lymphocyte lineage, including committed progenitor B cells and pre-B cells. Chapter 74 discusses these cells and the maturation stages they represent. Figure 73–6 also lists antigens that are expressed or increased upon B-cell activation. Of the B-cell–associated antigens that are commonly used, only a few are restricted to cells of the B lineage. Of these antigens, only CD20, CD22, and Pax 5 are not found on other cell types. Pax 5, a transcription factor, is a “master regulator” of B-cell development^32,33 that is expressed from the precursor stage through all B-cell maturation until it is lost at the plasma cell stage. Demonstration of monoclonal surface Ig allows diagnosis of clonal, neoplastic B cells. CD20 is the target of rituximab, a monoclonal antibody commonly used for treatment of B-cell neoplasms. CD19 is restricted mostly to B cells, but may be expressed weakly by follicular dendritic cells. CD19 is expressed by B cells at all stages of maturation, including the committed B-cell progenitor and most normal plasma cells. As such, it is the best-defined pan–B-cell surface antigen.

In addition to the CD antigens and Igs, B cells express the three major histocompatibility complex (MHC) class II antigens: DR, DP, DQ. These antigens are heterodimers of heavy chains and light chains that are encoded by genes within the D complex of the human leukocyte antigen (HLA) complex (Chap. 137). MHC class I antigens are expressed on all nucleated cells.

B-1 B Cells and CD5+ B Cells

B-1 B cells have distinctive activation requirements and high levels of CD44 and interleukin (IL)-5 receptor α (IL-5Rα). They proliferate more rapidly than other B cells to stimuli such as IgM crosslinking, possibly because of having constitutively activated nuclear signal transducer and activator of transcription 3 (STAT3).³⁴ Many, but not all, B-1 cells express CD5, a 67-kDa transmembrane glycoprotein that is more brightly expressed by T cells. These cells are designated CD5 B cells.³⁵ B-1 B cells do not express other T-cell markers but do express all other pan–B-cell surface antigens. Various agents modulate B-cell expression of CD5.³⁶ B-1 B cells are found in umbilical cord blood,³⁷ adult blood, the pleura and peritoneum, and all major secondary lymphoid organs; they are rare in the marrow.³⁸ These cells apparently are enriched for cells that spontaneously produce polyreactive autoantibodies.^39,40,41

Plasma Cells

Many B cell differentiation antigens are not expressed by the mature plasma cell, including CD20, Pax-5, surface Ig, and HLA class II antigens (see Fig. 73–6). Of the cells of the B lineage, plasma cells are distinctive in that they express CD138 and bright CD38.⁴² Clonal plasma cell neoplasms usually have antigen expression distinct from normal plasma cells including aberrant expression of CD20, CD28, CD56, and CD117. Clonal plasma cells usually aberrantly lack expression of CD19 and CD27.⁴³

T-LYMPHOCYTE AND NATURAL KILLER CELL ANTIGENS

Figure 73–7 and Table 73–1 summarize the expression of antigens on cells of the T-lymphocyte and NK lineages. All lymphocyte progenitors originate in the marrow, but T lymphocytes have their own special organ for maturation—the thymus—whereas NK cells appear to differentiate in secondary lymphoid tissue (Chap. 6).

Thymocyte

The thymus promotes the development of antigen-specific T lymphocytes and eliminates self-reactive T lymphocytes. There are three general stages of thymocyte maturation based on the surface CD4 and CD8 expression: double negative, double positive, and single positive. These stages have corresponding anatomic localization within the thymus with the least-mature double-negative cells located in the subcapsular area and the most-mature single-positive cells located in the medulla (Chap. 6). The most immature T lymphocytes in the thymus populate the subcapsular areas and express CD2, CD5, and CD7, antigens present on T lymphocytes of all stages. Capsular, “double-negative” thymocytes also express CD1a, cytoplasmic CD3, and terminal deoxynucleotidyl transferase (TdT). The majority of thymocytes are at the “double-positive” stage within the cortical area. These are the cells undergoing positive selection. Once the thymocytes achieve their “education” without dying, they mature to the single-positive stage in the medulla. These varying stages of immature T-cell maturation in the thymus have corresponding cell phenotypes in T-lymphoblastic leukemia/lymphoma.

Mature T Lymphocytes

Small, mature T lymphocytes are the most common lymphocytes in blood. T lymphocytes recognize antigen in the context of the MHC through binding with the T-cell receptor (TCR). Signaling from the TCR involves many membrane proteins, including CD3, a three-subunit complex expressed by early thymocytes and mature T cells.⁴⁴ It is tightly linked to the T-cell antigen receptor (Chap. 76). Most T lymphocytes have the α/β TCR on their surface, but a few percent of blood lymphocytes have the γ/δ TCR.

CD4 and CD8 Lymphocyte Subsets

Mature T cells express either CD4 or CD8, but not both. CD4, a member of the Ig supergene family, is a single-chain transmembrane glycoprotein.⁴⁵ CD8 is a 34-kDa dimeric transmembrane glycoprotein.⁴⁶ Most T cells express the α and β subunits of CD8. CD4 and CD8 act as coreceptors during T-cell activation by antigen. CD4 recognizes MHC II and CD8 recognizes MHC class I (Chap. 76). CD4 also is a coreceptor for the human immunodeficiency virus⁴⁷ (Chap. 81), as are CCR5 (chemokine receptor 5) and CXCR4 (chemokine-related receptor). The majority of CD8 cells are cytolytic when appropriately activated.

Subsets of CD4+ T cells have helper function for activation and maturation of cytolytic cells or B cells. Other CD4 subsets have regulatory activity including the T-regulatory (T_REG) cells that induce immune tolerance, and follicular T-helper (T_FH) cells that promote B cell maturation and differentiation in germinal centers. T_REG and T_FH cells have unique phenotypes. T_REG cells express the low affinity receptor for IL-2 (CD25); and the transcription factor forkhead box P3 (FoxP3).⁴⁸ Follicular helper T-T_FH cells express CD10 and CD57. Malignant counterparts to both subsets occur. Adult T-cell leukemia/lymphoma expresses both CD25 and FoxP3 and is associated with marked immunosuppression.⁴⁹ Angioimmunoblastic T-cell lymphoma has characteristic clonal T cells that express CD10 and CD57 just like T_FH cells, and this lymphoma is associated with polyclonal hypergammaglobulinemia and the expansion and proliferation of both B cells and CD21+ follicular dendritic cells.⁵⁰ T-helper-17 (Th17) cells that secrete IL-17 exhibit critical roles in mucosal defense and in autoimmune disease pathogenesis.⁵¹

Natural Killer Cells

The NK cell is defined as an effector cell that is not MHC restricted and has the capacity for spontaneous cytotoxicity toward various target cells (Chap. 77). Most NK cells have LGL morphology (see Fig. 73–1E).⁵² However, not all NK cells have LGL morphology, and not all LGL cells are NK cells. Many are cytolytic T lymphocytes. Cytolytic T lymphocytes and NK cells share many granule contents that can be detected by immunohistochemistry or flow cytometry. These include TIA-1, an RNA binding protein, and several granzymes, which are granule enzymes with serine protease activity.

Despite their relative morphologic homogeneity, NK cells comprise several subpopulations with distinct phenotypes. Human NK cells characteristically express CD16 (FcγRIII) and CD56 but not TCRα/β or TCRγ/δ, CD3, or CD4.^53,54 CD8 is found on approximately 30 to 50 percent of NK cells. CD8 on NK cells is dim by flow cytometry and is of the β-homodimer form. CD16 (FcγRIII) is a low-affinity receptor that binds to IgG, which is bound specifically to antigens present on cells targeted for destruction in antibody-dependent cell-mediated cytotoxicity.⁵⁵ CD16 is expressed on all NK cells, neutrophils, and tissue macrophages. CD56 is the neural cell adhesion molecule and is seen on most NK cells in either low (“dim”) or high (“bright”) density.^53,56 This 200-kDa protein is expressed at higher levels following activation.

LYMPHOCYTE SURFACE ANTIGENS

Lymphocyte subsets generally cannot be distinguished from one another by morphology. Most resting lymphocytes appear as small round cells with a dense nucleus and little cytoplasm. However, this homogeneous appearance is deceptive, as these cells comprise many functionally distinct subpopulations.

These subsets can be distinguished through the differential expression of cell-surface proteins, each of which can be recognized by a specific monoclonal antibody. Coupled with the biochemical analyses of the surface molecules that are recognized by each of these antibodies, many lymphocyte surface antigens have been defined.

Typically, it is necessary to monitor for coexpression of two or more cell-surface proteins to define a functional subset of lymphocytes. The same cell-surface protein is often expressed by more than one cell subset. For example, both helper and cytotoxic T cells express CD3, the proteins associated with the TCR for antigen (Chap. 76). Expression of both CD3 and CD4 helps to distinguish mature Th cells from cytotoxic T cells that express CD3 and CD8, and from other cells, such as dendritic cells, that express CD4 but lack expression of CD3 (Chap. 76). As noted above (see “CD4 and CD8 Lymphocyte Subsets”), T_REG cells are defined by the coexpression of CD3, CD4, CD25, and cytoplasmic FoxP3.⁴⁸ For these and other types of lymphocytes, it is the expression of a characteristic constellation of surface and cytoplasmic molecules, rather than the expression of any one particular marker, that generally helps to distinguish one subset of lymphocytes from another.

Fluorescent probes also can be used to identify antigen-specific lymphocytes.⁵⁷ Each clone of B lymphocytes expresses Ig capable of binding a particular antigen (Chap. 75). The frequencies of B cells specific for one antigen are estimated to range from 1 in 100,000 to 1 in 1,000,000 cells or less. Populations of lymphocytes enriched for B cells binding to a specific antigen can be stained using antigen coupled to probes, allowing for the detection and isolation of antigen-specific B cells using flow cytometry.⁵⁸ Alternatively, flow-based techniques can be used to monitor for antigen-specific B cells that are activated by contact with antigen.⁵⁹ T lymphocytes, however, generally recognize antigen in the form of peptides nestled into molecules of the MHC (Chap. 76). Thus identification and isolation of antigen-specific T cells require more complex probes using multimeric complexes comprised of specific peptide antigen complexed with the relevant MHC molecule.⁵⁷

Unfortunately, few studies of the composition and biochemistry of lymphocytes have used purified lymphocyte subpopulations. Because mature helper T cells are the predominant blood lymphocyte of normal adults, many reported biochemical parameters are most relevant to this subpopulation.

ION AND WATER CONTENT

The resting blood lymphocyte has a mean cell volume of 200 μm³ and contains 71 ± 1.2 percent by weight of water.⁶⁰ The total lymphocyte cation content is 35 femtomoles (fmol) per cell, of which 22 to 28 fmol per cell is potassium, and 7.9 ± 3.2 fmol per cell is sodium.⁶⁰ Lymphocyte membranes have both voltage-gated and calcium-activated potassium channels that regulate cell volume. Pharmacologic inhibition of these channels blocks T-cell activation. The calcium content of resting lymphocytes has been estimated at 580 to 800 pmol/10⁶ cells.⁶¹ Cytosolic free calcium concentrations are relatively low in resting lymphocytes (approximately 0.1 μM) but increase severalfold after activation.⁶²

LYMPHOCYTE MEMBRANE

The lymphocyte plasma membrane is composed of equal parts of weight of protein and glycosphingolipids and 6 percent by weight of carbohydrate.⁶³ The molar ratio of cholesterol to phospholipid is approximately 0.5.^64,65 Phosphatidylcholine is the predominant phospholipid in the lymphocyte plasma membrane, but phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin are also present. Approximately half the membrane fatty acids are saturated. The membrane proteins are usually glycosylated.

The glycosphingolipids and protein receptors of lymphocytes often are organized in glycolipoprotein microdomains termed lipid rafts.^66,67 Such lipid rafts sequester various protein receptors, coreceptors, and accessory molecules that together are involved in lymphocyte cell signaling, cytoskeletal reorganization, and/or membrane trafficking.⁶⁸ As such, the surface molecules on lymphocytes are not randomly distributed.

Extracellular Membrane-Associated Enzymes (Ectoenzymes)

Exposed on the exterior surface of lymphocytes are several enzymes called ectoenzymes (Table 73–2). Generally, the number of surface enzyme molecules is low compared with that of other surface molecules, such as those involved in lymphocyte adhesion. This probably reflects the fact that these molecules are catalytic and have a higher functional specific activity than do molecules involved in adhesion events, where multiple interactions over large surface areas are required. As such, it is possible that many more enzymes are present than the ones currently recognized because they are expressed at levels that are not detectable by conventional methods using monoclonal antibodies and flow cytometry.

Some of the surface enzymes are involved in nucleotide metabolism (see Table 73–2). For example, CD73 is an ecto-5′-nucleotidase that catalyzes the 5′ dephosphorylation of purine and pyrimidine ribo- and deoxyribonucleoside monophosphates to nucleosides that can be taken up by transport systems.⁶⁹ This ecto-5′-nucleotidase is attached to the plasma membrane by a glycerol phosphatidylinositol anchor. In addition, lymphocytes express CD26,⁷⁰ which is a membrane protein that can associate with adenosine deaminase, the levels of which are increased after activation.⁷¹ The shedding of adenosine deaminase by stimulated cells may explain why plasma levels of this enzyme are increased in early HIV infection and in other diseases associated with immune activation.⁷²

The ectoenzymes of nucleotide metabolism may regulate lymphocyte and granulocyte function at sites of inflammation. Activated T lymphocytes can release ATP, which, in turn, can bind to specific plasma membrane ATP receptors.⁷³ In addition, CD38 can catalyze the transient formation of cyclic adenosine 5′-diphosphate-ribose, a new second-messenger molecule directly involved in the control of calcium homeostasis by means of receptor-mediated release of calcium from ryanodine-sensitive intracellular stores.⁷⁴ The consequent increase in calcium mobilization and phospholipid breakdown can provoke activation or death, depending on the target cell. Subsequently, the dephosphorylation of ATP generates adenosine, which can interact with A2 receptors on the plasma membranes of neutrophils, monocytes, and lymphocytes.⁷⁵ The engagement of A2 receptors elevates adenosine 3′,5′-cyclic phosphate levels, counteracting the effects of ATP on cell activation. The deamination of adenosine permits the cycle to begin anew.

The ectodomains of several other surface antigens can possess proteolytic activity. For example, CD10 (or CALLA) also has neutral endopeptidase activity,⁷⁶ and CD26 has dipeptidyl peptidase IV activity.⁷⁷ These enzymes may play a role in modulating the binding of lymphocytes to other cells and to the extracellular matrix. In addition, inhibition of the catalytic activity of CD26 can provoke many cellular effects, including induction of tyrosine phosphorylation and p38 mitogen-activated protein kinase activation, as well as suppression of DNA synthesis and reduced production of various cytokines. As such, these ectoenzymes could play an important role in lymphocyte activation.

Some membrane-bound proteases have a disintegrin and a metalloprotease domain, termed ADAM (a disintegrin and a metalloprotease).⁷⁸ One such member of this family of proteins is the tumor necrosis factor-α converting enzyme, otherwise known as ADAM17 (CD156b).⁷⁹ These enzymes cleave other surface molecules, such as tumor necrosis factor, thereby releasing the soluble active cytokine. In addition, they may play an important role in modifying the activity of cytokines or other cell-surface molecules that are present in the vicinity of the plasma membrane.

Intracellular Membrane-Associated Enzymes

Transmembrane proteins that have cytoplasmic regions with kinase or phosphatase activities are common in biology although relatively few of these are restricted to lymphocytes. Nevertheless, many cytoplasmic domains of transmembrane proteins interact directly with enzymes that are restricted or preferentially expressed by lymphocytes or lymphocyte subsets (Chaps. 75 and 76). B lymphocytes, for example, selectively express Bruton tyrosine kinase (BTK), a tyrosine kinase that plays a critical role in signal transduction via surface Ig receptors.⁸⁰ Moreover, mutations that disrupt the function of such kinases can impair B-cell development, leading to dysregulated B-cell function or immune deficiency.⁸¹ On the other hand, T-cell development and function rely heavily on cytoplasmic receptor-associated tyrosine kinases, such as the zeta-associated protein of 70 kDa (ZAP-70), leukocyte tyrosine kinase (lck), or fyn. ZAP-70 interacts with the ζ-chain (CD247) of the TCR for antigen,⁸² whereas the latter enzymes, lck and fyn, are Src family tyrosine kinases that interact with cytoplasmic domains of various accessory molecules, including CD2, CD4, CD8, CD44, CD50, and/or CD137.⁸³ Through such interactions, these receptor protein tyrosine kinases play important roles in signal transduction following immune recognition and/or cognate intercellular immune interactions.

In addition, lymphocytes possess an important class of intracellular molecules, known collectively as adapter proteins, that have no intrinsic enzymatic activity.⁸⁴ These adaptor proteins can serve as a scaffolding for the assembly of kinases and other signaling molecules following antigen-receptor ligation. One important adaptor protein expressed in B lymphocytes is B-cell linker protein (BLNK; Chap. 75).⁸⁵ On the other hand, T cells use a distinct adaptor protein called linker for activation of T cells (LAT).⁸⁶ These molecules couple proximal biochemical events initiated by surface-receptor ligation with more distal signaling pathways by recruiting other cytosolic proteins (Chaps. 75 and 76).

CYTOMATRIX

Beneath the lymphocyte’s plasma membrane is a fully developed cytomatrix with several different structural and mechanical proteins, including tubulin, actin, myosin, tropomyosin, α-actinin, filamin, and a spectrin-like molecule, which are important in the formation of the immunologic synapse that forms during cognate intercellular interactions.⁸⁷ These are arranged into typical microfilaments, microtubules, and intermediate filaments. Lymphocyte activation by antigens or mitogens can lead to changes in the interaction of membrane components with the cytoskeleton, allowing for antigen processing, Ig secretion, or cell-mediated cytotoxic reactions.⁸⁸

ORGANELLES

In large part the composition and metabolism of long-lived blood T lymphocytes reflect their resting state. The T cells have a high nuclear-to-cytoplasmic ratio, few ribosomes or mitochondria, and scant endoplasmic reticulum. Glycogen stores are meager. The DNA content of the resting small lymphocyte, 8 pg per cell, is the same amount in other diploid cells. In contrast, the RNA content averages 2.5 pg per cell, yielding an RNA-to-DNA ratio of approximately 0.32.⁸⁹ This value is less than in most other human cells, as a result of the small amount of ribosomal RNA in most lymphocytes.

In contrast to most lymphocytes, however, plasma cells have a high RNA-to-DNA ratio. These cells are the end products of B-cell differentiation and are committed to the synthesis, assembly, and secretion of Ig. Accordingly, these cells have a well-developed rough endoplasmic reticulum and Golgi apparatus, but lack many of the surface receptors found on most lymphocytes. Mature plasma cells are probably terminally differentiated and have a low rate of DNA synthesis and abundant RNA, reflecting the plasma cell’s high-level synthesis of Ig.

Lysosomes

The few lysosomes in blood lymphocytes contain several different acid hydrolases, including acid phosphatase, β-glucuronidase, β-galactosidase, β-hexosaminidase, α-arabinosidase, α-galactosidase, α-mannosidase, α-glucosidase, and β-glucosidase.^90,91,92 Acid hydrolase activities are generally higher in T cells than in non-T lymphocytes. Lysosomal acid esterase, assayed histochemically with α-naphthyl acetate as substrate, has a characteristic punctate appearance in mature T lymphocytes.⁹³ Secretory lysosomes are specialized organelles that combine catabolic functions of conventional lysosomes with the capacity to be secreted upon induction.⁹⁴ An example of such secretory lysosomes are the specialized cytoplasmic granules of T cells and NK cells that are responsible for the cytotoxic effector function of these cells.

Cytoplasmic Granules

In contrast to other lymphocytes, cytotoxic T lymphocytes and NK cells possess abundant cytoplasmic granules. These contain a pore-forming proteolytic enzyme, termed perforin, and a series of serine proteinases with specific proapoptotic activity, called granzymes.⁹⁵ To protect against possible autolysis by granule contents, cytotoxic lymphocytes possess serine-proteinase inhibitors, termed serpins.⁹⁶ As an additional safeguard, the granzymes of resting lymphocytes are stored as inactive proenzymes.

Cytotoxic lymphocytes rely primarily on the perforin/granzyme system to kill their targets.⁹⁷ Upon contact with its target cell, the cytotoxic lymphocyte converts the granzymes into active forms by a lysosomal cysteine protease called dipeptidyl peptidase I.⁹⁸ Then perforin introduces a pore in the membrane, allowing the activated granzymes and other granule contents to pass into the cytoplasm and then the nucleus of the cell targeted for destruction.⁹⁵ In vitro studies indicate that granzyme nuclear import is independent of ATP, cannot be inhibited by nonhydrolyzable guanosine triphosphate analogues, and involves binding within the nucleus, unlike conventional signal-dependent nuclear protein import. The perforin-dependent nuclear entry of granzymes precedes the nuclear events of apoptosis, such as DNA fragmentation and breakdown of the nuclear envelope (Chap. 15).

ACKNOWLEDGMENTS

This chapter was adapted from “Morphology of Lymphocytes and Plasma Cells” by H. Elizabeth Broome and “Composition and Biochemistry of Lymphocytes and Plasma Cells” by Thomas J. Kipps in the earlier edition.