IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 2. Cells Executing the Immune Response

■ Many Cell types of diverse origins are designed to perform specialized functions in the Immune Response.

■ B AND T lymphocytes express antigen-binding receptors and other molecules (markers) on their surface that are necessary for carrying out a variety of functions.

■ The T-cell response requires the presentation of Antigens by antigen-presenting Cells. B cells are capable of recognizing native antigens that have not been processed or presented by other cells.

■ Various functional subpopulations of T lymphocytes exhibit helper, suppressor, or cytotoxic activity.

■ Phagocytic cells bearing specific surface markers circulate in the bloodstream (monocytes and granulocytes) and are present in Tissues (e.g., Kupffer cells in the Liver).

■ Eosinophils, basophils, mast cells, and platelets participate in the inflammatory response.

The precursors of the cells of The Immune System are pluripotent stem cells, which undergo two Main Pathways of differentiation (Fig. 2.1):

✵ lymphopoiesis — The formation of lymphocytes;

✵ myelopoiesis — the formation of phagocytes (monocytes, macrophages, and granulocytes), as well as other cells.

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Fig. 2.1. All cells of hematopoietic origin are formed from pluripotent stem cells, which give rise to cells of two main hematopoietic lineages: lymphoid and myeloid. Depending on the microenvironment, a lymphoid progenitor cell can differentiate into either a T cell or a B cell. In mammals, T cells mature in the Thymus, whereas B cells develop initially in the fetal liver and after birth in the Bone Marrow. The exact origin of individual types of antigen-presenting cells (APCs) remains unknown, although generally all of them are derived from hematopoietic stem cells. Natural killer (NK) cells likely originate from common lymphoid progenitors that mature in the fetal liver and subsequently in the bone marrow after birth. Myeloid progenitors differentiate into mature (committed) cells shown on the left. Eosinophils, neutrophils, and basophils are grouped together under the collective term granulocytes.

Lymphocytes can be classified as T, B, and NK cells. The two major populations of lymphocytes are named T cells and B cells. T cells develop from their precursors in the thymus, whereas in mammals B cells initially differentiate in the fetal liver and after birth in the Cytology/practical/86.html">Red bone marrow. In birds, B Cell Differentiation takes place in the bursa of Fabricius, an organ unique to this class of vertebrates. The Organs where lymphocyte differentiation occurs are referred to as central, or primary, Lymphoid organs. It is within these organs that precursors of B and T lymphocytes acquire The ability to recognize antigens through the expression of antigen-specific surface receptors.

Lymphocytes of the third population, which do not express antigen-binding receptors, are termed natural killer (NK) cells. They originate from lymphoid progenitor cells in the bone marrow and are functionally distinguished from T AND B cells by their ability to lyse certain tumor cell lines in vitro (rather than freshly excised tumors) without prior immunization. Morphologically, these are large granular lymphocytes (LGLs) (see below).

Monocytes/macrophages or polymorphonuclear granulocytes can function as phagocytes. Like lymphocytes, phagocytes are also represented by two populations: monocytes/macrophages and polymorphonuclear granulocytes. The latter have an irregularly shaped, segmented (polymorphic) nucleus. Depending on the staining characteristics of their cytoplasmic granules with acidic and basic Dyes, granulocytes are classified as neutrophils, basophils, or eosinophils. The EFFECTOR FUNCTIONS OF these three cell types differ. Neutrophils are the most abundant, also referred to as polymorphonuclear neutrophils (PMNs), and constitute the majority of white Blood Cells in circulating blood (approximately 60–70% in healthy adults).

Accessory cells (A cells). In addition to lymphocytes and phagocytes, the immune system includes A number of accessory cells.

✵ Antigen-presenting cells (APCs) — present antigens to T and B cells.

✵ Platelets — participate in blood clotting and inflammatory reactions.

✵ Mast cells, structurally and functionally similar to basophilic polymorphonuclear granulocytes — participate in certain types of inflammation (see Ch. 23).

✵ Endothelial cells — express molecules capable of recognizing circulating leukocytes, thereby mediating their adhesion and distribution within the vascular bed.

LYMPHOID CELLS

Every day, a significant number of lymphocytes are produced in the primary (central) lymphoid organs — the thymus and postnatal bone marrow. Some of these cells migrate from the bloodstream into secondary lymphoid tissues — the Spleen, Lymph Nodes, and mucosal-associated lymphoid tissues. The adult human body contains approximately 10¹² lymphoid cells, and lymphoid tissue as a whole accounts for roughly 2% of total body weight. Meanwhile, lymphoid cells make up about 20% of the leukocytes circulating in the blood. Many mature lymphoid cells are long-lived and can persist for years as immunological memory cells.

Lymphocytes are morphologically diverse. In a standard blood smear, lymphocytes vary in both size (6–10 µm in diameter) and Morphology. The Nucleus-to-Cytoplasm (N:C) ratio varies, as does the shape of the nucleus itself. The cytoplasm of some lymphocytes may contain azurophilic granules (Fig. 2.2).

Fig. 2.2. Morphological diversity of lymphocytes. 1. A small lymphocyte has a round nucleus, a high nuclear-cytoplasmic (N:C) ratio, and no granules in the cytoplasm. 2. A large granular lymphocyte has a lower N:C ratio, an indented nucleus, and azurophilic granules in the cytoplasm. Giemsa stain. (Photographs kindly provided by Dr. A. Stevens and Prof. J. Lowe.)

Light Microscopy of blood smears stained, for instance, with the Romanowsky-type Giemsa stain reveals two morphologically distinct types of circulating lymphocytes: the first consists of relatively small, typically agranular cells with a high N:C ratio, while the second comprises larger cells with a lower N:C ratio that contain cytoplasmic granules, commonly referred to as large granular lymphocytes. (LGLs should not be confused with granulocytes, monocytes, or their precursors, which also contain azurophilic granules.)

Resting T cells in blood. The majority of these cells express αβ T-cell receptors (αβ T cells; see below) and exhibit one of the two morphologies described above. Most helper T cells (Th; 95%) and a subset of cytotoxic T lymphocytes (Tc; 50%) are small, agranular lymphocytes with a high N:C ratio. Furthermore, their cytoplasm contains a specialized Structure known as the Ghالی body (or Ghall body), which is an aggregate of primary Lysosomes situated near a lipid droplet. The Ghall body is readily visualized by Electron microscopy (Fig. 2.3) or cytochemically via lysosomal enzyme detection Methods. Fewer than 5% of Th cells and approximately half of Tc cells display the alternative LGL-like morphology, characterized by scattered primary lysosomes in the cytoplasm and a well-developed Golgi apparatus (Fig. 2.4). Interestingly, mice lack cytotoxic T cells with an LGL-like morphology.

Fig. 2.3. Ultrastructure of non-granular T cells. This electron micrograph demonstrates the Ghall body (GB), a feature characteristic of the morphology of most resting T cells. It consists of primary lysosomes and a lipid droplet (indicated by the arrow); ×10,500. Inset: Under light microscopy, following non-specific esterase staining, the Ghall body appears as a homogeneous spot; ×400. From Zucker-Franklin D., Greaves M.F., Grossi C.E., et al., eds. 1988. Atlas of Blood Cells: Function and Pathology. Vol. II, 2nd ed. Milan: E.E. Ermes; Philadelphia: Lea & Febiger.

Fig. 2.4. Ultrastructure of granular T cells. These cells are characterized by electron-dense, peroxidase-negative granules (primary lysosomes, PL) scattered throughout the cytoplasm and partially concentrated near a well-developed Golgi apparatus (GA), alongside numerous Mitochondria (M); ×10,000. Inset: In light microscopy, T-cell granules can be demonstrated cytochemically using an acid phosphatase stain; ×400. From Zucker-Franklin D., Greaves M.F., Grossi C.E., et al.

1988. Atlas of Blood Cells: Function and Pathology. Vol. II, 2nd ed. Milan: E.E. Ermes; Philadelphia: Lea & Febiger.

The characteristics of large granular lymphocytes are also shared by another T-lymphocyte subpopulation, namely γδ T cells (γδ T cells). In lymphoid tissues, these

cells exhibit a dendritic morphology (Fig. 2.5); when cultured in vitro, they are capable of substrate adhesion, adopting a variety of shapes As a result (Fig. 2.6).

Fig. 2.5. Dendritic morphology of γδ T cells in tonsillar tissue. These γδ T cells are located predominantly in interfollicular, T-dependent areas, displaying a branching morphology. Stained with fluorochrome- and peroxidase-conjugated γδ-T-cell-specific Monoclonal Antibodies; ×900. (Eur. J. Immunol. 1991;21:173. Photograph kindly provided by Dr. A. Favre.)

Fig. 2.6. Morphological changes in cloned γδ T cells during in vitro culture. 1. Cells adhere to the substrate in a manner similar to macrophages; ×6000. 2. Cells elongate, forming uropods with prominent filopodia at their poles; ×2000. 3. Adhesive contact sites form at the tips of the filopodia; ×20,000. Eur. J. Immunol. 1991;21:173. Photographs kindly provided by Dr. G. Aranda and Dr. W. Maiolini.

Unstimulated B cells in blood. These cells lack Ghall bodies and do not morphologically resemble large granular lymphocytes; their cytoplasm is largely packed with dispersed monoribosomes (Fig. 2.7). Activated B cells with a well-developed rough Endoplasmic reticulum can occasionally be observed in the bloodstream (Fig. 2.8).

Fig. 2.7. Ultrastructure of resting B cells. The cytoplasm of these cells lacks Ghall bodies and granules, but contains scattered Ribosomes (R) and cisternae of the rough endoplasmic reticulum (ER). Upon activation, the Golgi apparatus associates with lysosomes in B cells; ×11,500.

Fig. 2.8. Ultrastructure of B lymphoblasts. The primary hallmark of B-cell activation is The Development of structures dedicated to immunoglobulin synthesis: the rough endoplasmic reticulum (ER), free polyribosomes, and the Golgi apparatus (GA), which is involved in immunoglobulin glycosylation; ×7500.

NK cells. Natural killer cells, much like γδ T cells and one of the Tc subpopulations, exhibit an LGL morphology. However, their cytoplasm typically contains more azurophilic granules than that of granular T cells.

Lymphocytes express unique surface markers specific to each subpopulation. The surface of lymphocytes (as well as other leukocytes) bears a multitude of diverse molecules that serve as markers for various subpopulations. A significant proportion of these cellular markers can now be readily identified using specific monoclonal antibodies. A standardized nomenclature for marker molecules has been established, wherein groups of monoclonal antibodies—each specifically binding to a distinct marker designated by the symbol CD (Cluster of Differentiation)—are classified accordingly. The CD nomenclature is primarily based on the Specificity of murine monoclonal antibodies directed against human leukocyte antigens. Numerous specialized laboratories worldwide contribute to the development of this Classification. To reach a consensus, a series of international workshops has been held to define characteristic panels of monoclonal antibodies that bind to different leukocyte populations, as well as the molecular weights of the recognized markers. Monoclonal antibodies sharing identical binding specificity are grouped together and assigned a CD number. Recently, however, this designation has come to refer not to the antibody groups themselves, but rather to the surface marker molecules recognized by these antibodies. (A comprehensive list of CD markers is provided in the Appendix.)

Subsequently, molecular markers began to be classified According to the information they provide about the cells expressing them, for example:

✵ Lineage markers, which serve as Characteristic Features of a given hematopoietic lineage; an example is the CD3 marker, found exclusively on T cells;

✵ differentiation markers, transiently expressed during maturation; an example is the CD1 marker, present on developing thymocytes but absent from mature T cells;

✵ activation markers, such as CD25—the low-affinity interleukin-2 (IL-2) receptor expressed solely on antigen-activated T cells.

While this approach to classifying markers is quite useful, it is not always feasible. In some cell populations, the activation marker and the differentiation marker are one and the same molecule. For example, CD10, which is present on immature B cells, disappears upon maturation but reappears upon activation (see Fig. 12.14). In addition, activation markers may be constitutively expressed on cells at low concentrations, but at much higher levels following activation. For instance, Treatment with IFNγ increases the expression of class II Major Histocompatibility Complex (MHC) molecules on monocytes.

Cellular markers form several families. Cell surface components belong to various families whose genes likely evolved from several ancestral ones. Marker molecules from different families differ in structure and form the following main groups:

✵ the immunoglobulin superfamily, which includes molecules structurally similar to antibodies; this group includes CD2, CD3, CD4, CD8, CD28, class I and II MHC molecules, and many others;

✵ the integrin family—heterodimeric molecules formed by $\alpha$- and $\beta$-chains; there are several subfamilies of Integrins; all members of a given subfamily share a common $\beta$-chain, but have distinct, uniquely determined $\alpha$-chains; in one of the subfamilies ($\beta_2$-integrins), the $\beta$-chain represents the CD18 marker. In combination with CD11a, CD11b, CD11c, or $\alpha$D, it forms lymphocyte function-associated antigens LFA-1, Mac-1 (CR3), and p150,95 (CR4), as well as the $\alpha$D$\beta_2$ cell surface molecule, frequently detected on leukocytes. In the second subfamily ($\beta_1$-integrins), the $\beta$-chain represents the CD29 marker; combined with various $\alpha$-chains, it forms markers of the late activation stage (VLAs, for very late antigens);

✵ selectins (E, L, and P), expressed on leukocytes (L) or on activated endothelial cells (E and P). They exhibit lectin-like specificity for sugars within highly glycosylated membrane Glycoproteins; Examples of selectins include CD43;

✵ Proteoglycans (a typical marker is CD44), possessing a number of glycosaminoglycan binding sites; an example is chondroitin sulfate.

Other families of cell markers include the tumor necrosis factor (TNF) and nerve growth factor (NGF) receptor superfamily, the C-type lectin superfamily, which includes CD23, and the multi-domain transmembrane receptor protein superfamily, which encompasses the IL-6 receptor.

It should be emphasized that markers expressed by lymphocytes can also be found on cells of other lineages. For example, CD44 is frequently detected on epithelial cells. Cell surface molecules can be identified using fluorescent antibodies as probes (Fig. 2.9). This approach forms The basis of flow immunofluorescence cytometry, a method that allows cells to be sorted and counted based on their size and fluorescence parameters (see Chapter 29). This technique enables the detailed sorting of lymphoid cell populations.

Fig. 2.9. Mouse monoclonal antibodies specific for a cytotoxic T lymphocyte (Tc) antigen (e.g., CD8) bind specifically to Tcs rather than helper T cells (Th). Bound antibodies are easily detected using fluorochrome-conjugated anti-mouse immunoglobulin antibodies. This method allows the identification and quantification of T cells belonging to different subpopulations.

T cells

T cells differ in their antigen-recognizing receptors. The hallmark marker of the T-cell lineage is the T-cell antigen receptor (TCR). There are two distinct types of TCR, both of which are heterodimers composed of two disulfide-linked polypeptide chains. The type I TCR is formed by $\alpha$- and $\beta$-chains, while the structurally similar type II TCR consists of $\gamma$- and $\delta$-chains. Both receptors are associated on The Cell surface with five Polypeptides of the CD3 complex, together forming the T-cell receptor complex (TCR–CD3 complex, see Chapter 7). Approximately 90–95% of blood T cells are $\alpha\beta$ T cells, with the remaining 5–10% being $\gamma\delta$ T cells.

$\alpha\beta$ T cells, in turn, differ in their expression of CD4 or CD8. $\alpha\beta$ T cells are subdivided into two distinct, non-overlapping subpopulations: cells of one subpopulation bear the CD4 marker and predominantly "help" or "induce" the immune response (Th), whereas Cells of the other bear the CD8 marker and exhibit primarily cytotoxic activity (Tc). CD4⁺ T cells recognize their specific antigens in association with class II MHC molecules, whereas CD8⁺ T cells recognize antigens in association with class I MHC molecules (see Chapter 7). Thus, the ability of a T cell to interact with another cell type depends on (is restricted by) the presence of the CD4 or CD8 marker on the former (Fig. 2.10). A small fraction of $\alpha\beta$ T cells expresses neither CD4 nor CD8. Similarly, most circulating $\gamma\delta$ T cells are "double negative," although some do bear CD8. Conversely, the majority of tissue-resident $\gamma\delta$ T cells express this marker.

Fig. 2.10. T cells express either $\gamma\delta$ or $\alpha\beta$ TCRs and are subdivided into CD4⁺ and CD8⁺ subpopulations that recognize peptide antigens in association with class I or class II MHC molecules, respectively. CD4⁺ T cells can be further divided into Th1 and Th2 subpopulations based on the set of cytokines they produce. Clear data regarding distinct cytokine profiles in $\gamma\delta$ T cells and CD8⁺ $\alpha\beta$ T cells are currently lacking.

CD4⁺ and CD8⁺ $\alpha\beta$ T cells are subdivided into functionally distinct subpopulations. As noted above, approximately 95% of CD4⁺ T cells and 50% of CD8⁺ T cells are morphologically small non-granular lymphocytes. These populations can be further differentiated based on the phenotypic expression of CD28 and CTLA-4 into functionally distinct subpopulations. The CD28 marker expressed by CD4⁺ T cells transmits a costimulatory activation signal upon antigen recognition. (In the absence of such a signal, TCR contact with antigen may induce anergy.) The ligands for CD28 are B7-1 (CD80) and B7-2 (CD86) molecules on APCs. CTLA-4, a molecule homologous to CD28, begins to be expressed by CD4⁺ T cells following activation. CTLA-4 binds to the same ligands as CD28, thereby limiting activation (see Chapter 11). In addition, $\alpha\beta$ T cells express various isoforms of the common leukocyte antigen, CD45. It is believed that CD45RO, rather than CD45RA, is associated with cellular activation. Other criteria are also used to isolate functionally distinct subpopulations of $\alpha\beta$ T cells, in particular the expression of natural killer cell markers (CD56, CD57, and CD11b/CD18), which are found on 5–10% of circulating T cells. These cells produce IL-4 but not IL-2, and show a weak proliferative response to antigens and mitogens.

$\alpha\beta$ T lymphocytes can also be classified by their cytokine profile. The functional diversity of T cells can be demonstrated by analyzing the cytokine secretion profiles of different T-helper clones. Two groups of CD4⁺ T-cell clones have been identified in both mice and humans. The Th1 subpopulation secretes IL-2 and IFN$\gamma$, whereas the Th2 subpopulation secretes IL-4, IL-5, IL-6, and IL-10 (Fig. 2.10; see also Chapter 11). Th1 cells participate in the activation of cytotoxic T cells and local inflammatory responses. Consequently, they are crucial for combating intracellular viral, bacterial, or parasitic infections. Th2 cells, on the other hand, are more effective in stimulating B-cell proliferation and antibody production, and their functions are therefore primarily associated with DEFENSE AGAINST EXTRACELLULAR microbes (humoral Immunity).

A list of the main markers of human and mouse $\alpha\beta$ T cells is presented in Fig. 2.11.

Fig. 2.11. To date, the molecular marker CD7 has been identified only in humans, whereas Thy-1 has been found only in mice. Other markers shown in square brackets are murine equivalents of human markers. Most of these markers belong to the immunoglobulin superfamily of Cell Adhesion molecules.

$\gamma\delta$ T cells are a common component of mucous membranes and the epidermis in mice. $\gamma\delta$ T cells are relatively frequent in mucosal epithelia but represent only a minor subpopulation among circulating T cells. In mice, almost all intraepithelial lymphocytes are $\gamma\delta$ T cells expressing CD8—a marker absent from most circulating $\gamma\delta$ T cells. CD8⁺ $\gamma\delta$ T cells have been shown to possess a distinct repertoire of T-cell receptors specific for certain bacterial and viral antigens (superantigens). Current consensus suggests that these cells may play an important role in protecting the body's mucous membranes against infection.

T cells share a number of common markers with cells of other lineages. So far, we have described cellular markers and antigen-specific receptors characteristic of specific T-lymphocyte subpopulations. However, a number of molecules are expressed On the surface of all T cells ("pan-T-cell markers") as well as on cells of other lineages. A good example is the sheep erythrocyte receptor (CD2). Normally, the CD2 molecule, by binding to its corresponding ligands, participates in T-cell activation alongside the TCR–CD3 complex and other membrane glycoproteins. At the same time, CD2 is also detected on 75% of CD3^- NK cells. Another molecule involved in T-cell activation is the CD5 marker, expressed on all T cells and on a subpopulation of B cells. The CD5 molecule can bind to CD72, but the question of its role as a physiological B-cell Ligand remains open. The CD7 marker is present on nearly all NK and T cells. A complete list of T-cell CD markers, some of which are also expressed on other hematopoietic cells, is provided in the appendix. Mouse T cells express markers similar to those found on human T cells.

Suppressor T cells. Clear functional evidence exists for the existence of antigen-specific suppressor T cells (Ts), yet these cells do not appear to constitute a distinct T-cell subpopulation dedicated exclusively to suppressive function. It has also been demonstrated that both CD4⁺ and CD8⁺ T cells are capable of suppressing immune responses either through direct cytotoxic action on antigen-presenting cells, by secreting "suppressive" cytokines (see Chapter 11), by delivering negative regulatory signals (via CTLA-4 binding to its ligands; see above), or through idiotype–anti-idiotype network interactions (see Chapter 13).

B cells

Between 5% and 15% of circulating blood lymphoid cells are B lymphocytes, identified by the presence of surface IMMUNOGLOBULINS (Ig). Ig molecules are constitutively synthesized; they are embedded in the cell's cytoplasmic membrane and function as antigen-specific receptors. Such receptors can be detected on the cell surface using fluorochrome-labeled anti-immunoglobulin antibodies. Immunofluorescent staining forms a ring around the B cell (Fig. 2.12). Bivalent anti-immunoglobulin antibodies, by interacting with these receptors, cause their cross-linking, leading to the formation of irregularly shaped immunoglobulin "patches" on the cell surface. Upon warming, most of these patches actively move across the cell surface and coalesce at one of its poles to form a "cap" (Fig. 2.12, inset). This process, termed capping, is followed by the internalization of the immunoglobulin molecules into the cell, where they undergo degradation. Capping can also be observed with other surface glycoproteins and is not restricted to B cells.

Fig. 2.12. B cells stained to detect surface immunoglobulin. Stained with fluorescent anti-IgM antibodies in the cold, B cells exhibit ring-like fluorescence under ultraviolet light. Incubation at 37 °C with these antibodies leads to the redistribution (capping) of the fluorescent label to one pole of the cell (see inset). x300. After Zucker-Franklin D., Greaves M.F., Grossi С E. et at. 1988. Atlas of Blood Cells: Function and Pathology. Vol. II. 2nd edn. Milan: E.E. Ermes, Philadelphia: Lea and Febiger.

The B-cell "receptor complex". Most human peripheral blood B cells express two immunoglobulin isotypes on their surface, IgM and IgD (see Ch. 6). On each individual B cell, the antigen-binding sites of these isotypes are identical. Less than 10% of circulating blood B cells express IgG, IgA, and IgE, but in certain body regions such cells are found with greater frequency; for example, IgA-bearing B cells are numerous in the intestinal mucosa. By associating with other molecules on the B-cell surface, immunoglobulin forms the antigen-recognition B-cell receptor complex. These other "accessory" molecules include disulfide-linked heterodimers consisting of Igα (CD79a) and Igβ (CD79b). These heterodimers, by interacting (similarly to the constituent PARTS OF THE TCR-CD3 complex of T cells) with the transmembrane segments of the immunoglobulin receptor, participate in The process of B-cell activation.

Other B-cell markers and subpopulations. A number of other molecular markers are expressed on both mouse and human B cells (Fig. 2.13). Most B cells carry class II MHC antigens on their surface, which are important for cooperative (contact) interactions with T cells. In the mouse, these are I-A or I-E antigens, and in humans, HLA-DP, HLA-DQ, and HLA-DR. Receptors for Complement components C3b (CR1, CD35) and C3d (CR2, CD21), detectable on almost all B cells, are involved in cell activation processes and likely in "homing". The interaction of CD19/CD21 with the complement + antigen complex plays a role in B-cell activation mediated by the antigen-binding antibody receptor. B cells also possess Fc receptors for exogenous IgG (FcγRII, CD32), which transmit negative regulatory signals to B cells.

The main markers currently used to identify human B cells are CD19, CD20, and CD22. Other human B-cell markers, CD72 and CD78, are also known. The CD72 marker has also been detected on mouse B cells (Lyb-2) along with B220, which is a high-molecular-weight (220 kDa) isoform of the CD45 marker (Lyb-5). A crucial role in contact interactions between T and B cells belongs to the CD40 marker (see Fig. 11.5).

Fig. 2.13. Many human and mouse B-cell markers are homologous; they are shown in the same color. Human markers equivalent to mouse markers are indicated in square brackets. The B220 marker was previously designated as Lyb-5. Igα and Igβ chains, by interacting with surface immunoglobulin (sIg), form the B-cell receptor complex. CD classification numbers are given in parentheses after the functional name.

B cells can be divided into two subpopulations: B-1 (Mac-1⁺, CD23^-) and B-2 (Mac-1^-, CD23⁺). The majority of B-1 cells express the CD5 (Ly-1) marker, originally discovered only on T cells. The function of CD5 in human B cells is currently unknown; this marker is associated with the B-cell receptor and may be involved in regulating the B-cell activation process. B-1 cells spontaneously synthesize so-called natural antibodies against certain bacterial antigens as well as autoantigens, such as DNA, the Fc fragment of IgG, Phospholipids, and cytoskeletal Proteins. In humans, B-1 cells are particularly frequent in newborn blood, whereas in mice they are predominantly found in the peritoneal cavity. According to some data, they undergo a special differentiation pathway that differs from the maturation of "conventional" B cells, which belong to the B-2 subpopulation.

In addition to the CD5 marker shared with T cells, B cells share markers with other cells, such as the CD40 marker, which is present on certain dendritic cells.

Natural Killer Cells

Cells designated as natural killer (NK) cells constitute up to 15% of blood lymphocytes; they express neither T- nor B-cell antigen-binding receptors.

Phenotypic markers of NK cells. Most antigens detected on the surface of NK cells using monoclonal antibodies are also present on T cells and monocytes/macrophages. The main markers of human NK cells, along with their cross-reactivity, are listed in Fig. 2.14. In purified lymphocyte populations, NK cells are most frequently identified using monoclonal antibodies against CD16 (FcγRIII). The CD16 marker participates in one of the NK activation mechanisms and is also expressed by neutrophils, certain types of macrophages, and γδ T cells.

Fig. 2.14. None of these markers is exclusively specific to NK cells. (KIR = killer cell inhibitory receptors; KAR = killer cell activation receptors.)

In granulocytes, the CD16 marker is linked to the cytoplasmic membrane via a phosphatidylinositol glycan anchor, whereas NK cells, macrophages, and γδ T cells express a transmembrane form of this marker molecule. Another important marker for NK identification is CD56, which is a homophilic neural cell adhesion molecule (NCAM) belonging to the immunoglobulin superfamily. In humans, lymphocytes lacking CD3 but expressing either or both CD56 and CD16 markers most likely belong to NK cells, although both markers are also present on a small fraction of T cells. Resting NK cells additionally express the IL-2 receptor β-chain (an intermediate-affinity receptor with a molecular mass of 70 kDa) and the signal-transducing γ-chain shared by receptors binding IL-2 and other cytokines. Naturally, direct stimulation with interleukin-2 triggers NK activation. It is of interest that the 70 kDa receptor is also expressed on all T cells with large granular lymphocyte (LGL) morphology, namely γδ T cells and a subset of CD8⁺ αβ T cells. Under METABOLISM/18.html">The Influence of IL-2, all these cells, including NK cells, acquire nonspecific cytotoxic activity, turning into cells commonly known as lymphokine-activated killer (LAK) cells. LAK cells exhibit a cytotoxic effect on freshly isolated tumor cells, and their target spectrum is much broader than that of resting NK cells.

Compared to human NK cells, mouse NK cells contain fewer azurophilic granules in their cytoplasm, but these granules are much larger. The list of phenotypic markers for mouse NK cells is presented in Fig. 2.15.

Fig. 2.15. Almost all molecular markers of mouse NK cells are also expressed on other cells, yet NK-specific markers have also been identified.

Functions of natural killer cells. The function of NK cells is the recognition and destruction of certain tumor cells (Fig. 2.16) as well as virus-infected cells. The recognition mechanism remains fully unclear. NK subpopulations express immunoglobulin superfamily molecules that regulate their cytotoxic activity. Products of certain HLA class I alleles can protect target cells from NK cells, whereas products of others, conversely, enhance their cytolytic action. For instance, certain HLA class I molecules (A, B, and C) expressed by a target cell, upon binding to "killer cell inhibitory receptors" (KIRs; e.g., p58 or p70) on NK cells, prevent cytolysis. "Killer cell activation receptors" (KARs; e.g., p50.1, 50.2, 50.3) expressed by NK cells can also bind to products of various HLA alleles on target cells. These interactions, either alone or together with others, such as CD2/LFA-1 binding, induce NK activation. For example, K562 cells, commonly used to study NK function, do not express HLA molecules and therefore lack ligands for KIRs. However, they possess LFA-1, which is sufficient for CD2-bearing NK cells to exert their action. Each natural killer cell carries at least one KIR specific for self MHC antigens, which is why autologous NK cells do not attack their own normal cells. Destruction of these cells by natural killer cells is possible upon modification of KIR-specific HLA ligands in cases of spontaneous malignant transformation or viral infection.

Fig. 2.16. A natural killer (NK) cell attacking a target cell (T). x 4500. (Photograph kindly provided by Dr. G. Aranda and Malorni, Rome.)

Natural killer cells are also capable of lysing antibody-coated target cells via their IgG receptors (FcγRIII, or CD16), a process known as antibody-dependent cell-mediated cytotoxicity (ADCC). Activated NK cells secrete interferon-gamma (IFN-γ) and other cytokines (such as IL-1 and GM-CSF), which may play an important role in regulating hemopoiesis and immune responses.

Activation of B and T cells

B and T cells are activated upon binding specific antigens. T cells require "seeing" the antigen presented in association with MHC molecules on antigen-presenting cells, whereas B cells can bind native antigens, but their activation requires help from T cells (in the case of certain polymeric antigens or mitogenic molecules, such T-cell help is not required).

In addition to specific antigen binding by receptors, effective activation of T and B cells requires intercellular interaction involving other surface components, such as CD28 in the case of T cells (see Ch. 11). Antigen-induced activation and differentiation of T and B cells typically occur in lymphoid tissues and can be reproduced in vitro by culturing lymphocytes in the presence of an activating agent. Such agents can include:

✵ an antigen recognized by the cell's surface antigen-binding receptor;

✵ monoclonal antibodies to the TCR–CD3 complex, and

✵ Lectins [e.g., phytohemagglutinin (PHA), concanavalin A (ConA), and pokeweed mitogen].

Lectins are carbohydrate-binding proteins of plant and bacterial origin. Some of them can activate lymphocytes by cross-linking BCRs or TCRs, thereby acting as mitogens (proliferation Inducers). Mitogenic stimulation of lymphocytes in vitro is thought to closely mimic activation by specific antigens. PHA and ConA stimulate mouse and human T lymphocytes. Bacterial lipopolysaccharide (LPS) stimulates mouse B cells, whereas pokeweed mitogen induces the proliferation of both human B and T cells (Fig. 2.17).

Fig. 2.17. Mitogen- or antigen-induced lymphocyte blastogenesis. The human T and B cells shown in the photograph were activated with pokeweed mitogen. 1. Increased cytoplasmic basophilia and cell enlargement. 2. Chromosome Condensation during Cell Division, making the Chromosomes clearly visible. Giemsa stain. ×1500. After Zucker-Franklin D., Greaves M.F., Grossi С.Е. et al. 1988. Atlas of Blood Cells: Function and Pathology. Vol. II. 2nd edn. Milan: E.E. Ermes, Philadelphia: Lea and Febiger.

In vitro studies using these agents have demonstrated that T- and B-cell activation triggers the synthesis of CYTOKINES AND THEIR receptors. Cytokine–receptor interaction drives cells into the division cycle (proliferation) and subsequent maturation into effector or memory cells. Under in vitro conditions, memory cells recirculate and ultimately home to the T- and B-dependent areas of lymphoid tissues, where they reside long-term, remaining poised to respond upon re-exposure to the same antigen.

Activation signals are mediated by "second messengers." The interaction of resting lymphocytes with an antigen sets off a cascade of biochemical processes leading to the generation of second messengers inside B or T cells. These messengers are responsible for subsequent Gene-level changes. Although several major mechanisms of lymphocyte activation exist, they are not yet fully understood. In both T and B cells, signal Transduction involves a guanosine triphosphate-binding (GTP-dependent) protein (G protein) that stimulates phosphatidylinositol metabolism. This generates two second messengers: Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers the release of Ca²⁺ ions from intracellular stores, whereas DAG activates protein kinase C, which, along with other Kinases, phosphorylates a number of Plasma Membrane components, leading to the appearance of Transcription factors and the subsequent expression of specific genes. Thus, immediately following contact with an antigen, T lymphocytes express several molecules on their surface, including gp39 and the IL-2 receptor. Subsequent cell–cell interactions mediated by these molecules drive lymphocyte proliferation and differentiation.

B-cell differentiation gives rise to plasma cells and memory cells. Following activation by a mitogen or antigen, T and B cells undergo characteristic ultrastructural changes, transforming into lymphoblasts (Figs. 2.8 and 2.18). Subsequently, many B lymphoblasts mature into antibody-forming cells (AFCs), which in vivo develop into terminally differentiated plasma cells. Some B lymphoblasts do not develop cisternae of the rough endoplasmic reticulum (ER). Such cells are found in germinal centers within lymphoid follicles and are referred to as follicular central cells or centrocytes (Fig. 2.19).

Fig. 2.18. Ultrastructure of T lymphoblasts. T lymphoblasts, emerging from T lymphocytes following antigen or mitogen stimulation, are large cells with well-developed cytoplasm containing diverse Organelles, including mitochondria (M) and free polyribosomes. Lymphoblasts may be "agranular" (1) or granular (2) depending on the presence or absence of electron-dense granules (G). Lipid droplets (LD) are also visible in the cytoplasm of the granular lymphoblast. ×3200.

Fig. 2.19. Follicular central cell. A well-developed cytoplasm containing polyribosomes (P) and several tubules of the rough endoplasmic reticulum (ER) is visible, whereas stacks of cisternae (parallel arrays of ER) are absent. A large, eccentrically positioned nucleolus (N) abutting the nuclear envelope is noticeable. These cells likely represent B memory cells. Certain lymphomas, known as centroblastic or centrocytic lymphomas, exhibit a similar morphology. ×8500.

Light microscopy reveals that the cytoplasm of plasma cells is basophilic, meaning it has an affinity for basic dyes. This cytoplasmic property is due to the presence of large amounts of RNA, which Supports antibody synthesis on the ribosomes of the rough ER (Fig. 2.20). Electron microscopy shows parallel arrays of rough ER within plasma cells (Fig. 2.21). These cells rarely appear in the bloodstream, accounting for no more than 0.1% of circulating lymphocytes. Normally, plasma cells are found exclusively in Secondary Lymphoid Organs and tissues, and they are also quite abundant in the red bone marrow. Antibodies produced by a single plasma cell share the same antigenic specificity and belong to a single immunoglobulin isotype. They can be detected in the cytoplasm of these cells using fluorochrome-labeled antiglobulin antibodies (Fig. 2.22). Plasma cells have a short lifespan; surviving for only a few days, they die via apoptosis (Fig. 2.23).

Fig. 2.20. Plasma cell morphology. A mature plasma cell is characterized by an eccentric nucleus and well-developed basophilic cytoplasm (basophilia is driven by the high RNA content required for Protein Synthesis). The Golgi apparatus is located in a slightly lighter perinuclear zone. May-Grünwald Giemsa stain. ×1000. After Zucker-Franklin D., Greaves M.F., Grossi С.Е. et al. 1988. Atlas of Blood Cells: Function and Pathology. Vol. II. 2nd edn. Milan: E.E. Ermes, Philadelphia: Lea and Febiger.

Fig. 2.22. Immunofluorescence staining of intracellular immunoglobulin in plasma cells. Fixed human plasma cells stained with fluorescein-labeled antibodies to human IgM (green fluorescence) and rhodamine-labeled antibodies to human IgG (red fluorescence). Intense intracytoplasmic fluorescence is visible. The distinct staining patterns of the two plasma cells indicate that, normally, each of them produces antibodies of only a single class or subclass (isotype). ×1500. After Zucker-Franklin D., Greaves M.F., Grossi С.Е. et al. 1988. Atlas of Blood Cells: Function and Pathology. Vol. II. 2nd edn. Milan: E.E. Ermes, Philadelphia: Lea and Febiger.

Fig. 2.21. Plasma cell ultrastructure. Characteristic parallel arrays of the rough endoplasmic reticulum (ER) are visible. In mature cells, the ER cisternae are packed with immunoglobulins. Mitochondria (M) are also visible. ×5000. After Zucker-Franklin D., Greaves M.F., Grossi С.Е. et al. 1988. Atlas of Blood Cells: Function and Pathology. Vol. II. 2nd edn. Milan:

E.E. Ermes, Philadelphia: Lea and Febiger.

Fig. 2.23. Apoptotic death of a plasma cell. Plasma cells are short-lived and die via apoptosis (programmed self-destruction). Changes in nuclear Chromatin typical of apoptosis are visible. ×5000.

Activation markers on lymphocytes. T- and B-cell activation induces the de novo synthesis of several surface markers and upregulates the expression of others.

These activation markers include cell adhesion molecules that ensure more efficient interactions of activated cells with others, as well as growth and differentiation factor receptors essential for sustained cell proliferation and maturation. One of these is the IL-2 receptor (IL-2R), expressed by T cells following activation; it consists of three subunits. Resting T cells constitutively express the γ chain (CD134) of this receptor, and some of them also express its β chain (CD122). Activation triggers the Synthesis of the IL-2R α subunit (CD25) and the Formation of the heterotrimeric high-affinity IL-2R. T-cell activation also transiently induces the expression of gp39 (CD40L), transferrin receptors (CD71, essential for proliferation), CD38, and CD69. These markers appear during early T-cell ontogeny but disappear during intrathymic development (see Ch. 12). MHC class II molecules serve as late activation markers on human T cells (they are not detected on mouse T cells). T cells, particularly memory T cells, express CD29 (the VLA β1 chain) as a late activation marker. Therefore, the "memory" function of the CD4⁺CD29⁺ T-cell subpopulation can be interpreted as an activation-induced increase in various cell adhesion molecules that facilitate interactions between these T cells and other cells upon re-encountering a given antigen.

B-cell activation markers include the high-affinity IL-2R and other receptors for growth and differentiation factors such as IL-3, IL-4, IL-5, and IL-6 (see Ch. 10). All of these receptors have been investigated using molecular cloning and sequencing techniques. In addition, activated B cells express transferrin receptors (CD71) and elevated levels of MHC class II membrane antigens. The CD23 marker (FcεRII, a low-affinity IgE receptor) expressed on activated human and mouse B cells is involved in the induction of cell division. The CD38 marker is absent on mature human B cells, but is detectable at the terminal stage of differentiation in plasma cells and germinal center cells, as well as on very early maturation-stage B cells. Specific plasmacytic antigen-1 molecules are found on human B cells exclusively at the plasmacytic stage of their differentiation. Immunological memory cells identified within germinal centers inside secondary lymphoid follicles (see Ch. 12) express neither IgD nor CD22.

NK-cell activation markers include MHC class II molecules.