Microbial adherence occurs through pattern recognition receptors (see Chapters 6 and 7), followed by phagocytosis. Coating microbes with complement components and/or antibodies (opsonization) enhances phagocytosis by monocytes/macrophages and is mediated by specialized complement receptors and antibody receptors expressed by the phagocytic cells (see Chapters 3 and 4).

Q. How do macrophages recognize microbes that have been coated with antibody?

A. Macrophages have Fc receptors that recognize the constant domains of the heavy chains of an antibody molecule (see Figs 1.10 and 1.11).

There are three different types of polymorphonuclear granulocyte

The polymorphonuclear granulocytes (often referred to as polymorphs or granulocytes) consist mainly of neutrophils (PMNs). They:

• are released from the bone marrow at a rate of around 7 million per minute;

• are short-lived (2–3 days) relative to monocytes/macrophages, which may live for months or years.

Like monocytes, PMNs marginate (adhere to endothelial cells lining the blood vessels) and extravasate by squeezing between the endothelial cells to leave the circulation (see Fig. 1.17) to reach the site of infection in tissues. This process is known as diapedesis. Adhesion is mediated by receptors on the granulocytes and ligands on the endothelial cells, and is promoted by chemo-attractants (chemokines) such as interleukin-8 (IL-8) (see Chapter 6).

Like monocytes/macrophages, granulocytes also have pattern recognition receptors, and PMNs play an important role in acute inflammation (usually synergizing with antibodies and complement) in providing protection against microorganisms. Their predominant role is phagocytosis and destruction of pathogens.

The importance of granulocytes is evident from the observation of individuals who have a reduced number of white cells or who have rare genetic defects that prevent polymorph extravasation in response to chemotactic stimuli (see Chapter 16). These individuals have a markedly increased susceptibility to bacterial and fungal infection.

Neutrophils comprise over 95% of the circulating granulocytes

Neutrophils have a characteristic multilobed nucleus and are 10–20 μm in diameter (Fig. 2.5). Chemotactic agents attracting neutrophils to the site of infection include:

• protein fragments released when complement is activated (e.g. C5a);

• factors derived from the fibrinolytic and kinin systems;

• the products of other leukocytes and platelets; and

• the products of certain bacteria (see Chapter 6).

Fig. 2.5 Morphology of the neutrophil

Morphology of the neutrophil. At the ultrastructural level, the azurophilic (primary) granules are larger than the secondary (specific) granules with a strongly electron-dense matrix; the majority of granules are specific granules and contain a variety of toxic materials to kill microbes. A pseudopod (to the right) is devoid of granules. Arrows indicate nuclear pores. Go: Golgi region. Inset: A mature neutrophil in a blood smear showing a multilobed nucleus. × 1500.

(From Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

Neutrophils have a large arsenal of enzymes and antimicrobial proteins stored in two main types of granule:

• the primary (azurophilic) granules are lysosomes containing acid hydrolases, myeloperoxidase, and muramidase (lysozyme); they also contain the antimicrobial proteins including defensins, seprocidins, cathelicidins, and bacterial permeability inducing (BPI) protein; and

• the secondary granules (specific to neutrophils) contain lactoferrin and lysozyme (see Fig. 2.5).

During phagocytosis the lysosomes containing the antimicrobial proteins fuse with vacuoles containing ingested microbes (termed phagosomes) to become phagolysosomes where the killing takes place.

Neutrophils can also release granules and cytotoxic substances extracellularly when they are activated by immune complexes (antibodies bound to their specific antigen molecules) through their Fc receptors. This is an important example of collaboration between the innate and adaptive immune systems, and may be an important pathogenetic mechanism in immune complex diseases (type III hypersensitivity, see Chapter 25).

Granulocytes and mononuclear phagocytes develop from a common precursor

Studies in which colonies have been grown in vitro from individual stems cells have shown that the progenitor of the myeloid lineage (CFU-GEMM) can give rise to granulocytes, monocytes and megakaryocytes (Fig. 2.6). Monocytes and neutrophils develop from a common precursor cell, the CFU-granulocyte macrophage cells (CFU-GMs) (see Fig. 2.6). Myelopoiesis (the development of myeloid cells) commences in the liver of the human fetus at about 6 weeks of gestation.

Fig. 2.6 Development of granulocytes and monocytes

Pluripotent hematopoietic stem cells generate colony-forming units (CFUs) that can give rise to granulocytes, erythrocytes, monocytes, and megakaryocytes (CFU-GEMMs). CFU-GEMMs therefore have the potential to give rise to all blood cells except lymphocytes. IL-3 and granulocyte–macrophage colony-stimulating factor (GM-CSF) are required to induce the CFU-GEMM stem cell to enter one of five pathways (i.e. to give rise to megakaryocytes, erythrocytes via burst-forming units, basophils, neutrophils, or eosinophils). IL-3 and GM-CSF are also required during further differentiation of the granulocytes and monocytes. Eosinophil (Eo) differentiation from CFU-Eo is promoted by IL-5. Neutrophils and monocytes are derived from the CFU-GM through the effects of G-CSF and M-CSF, respectively. Both GM-CSF and M-CSF, and other cytokines (including IL-1, IL-4, and IL-6), promote the differentiation of monocytes into macrophages. Thrombopoietin (TP) promotes the growth of megakaryocytes. (B, basophil; BFU-E, erythrocytic burst-forming unit; DC, dendritic cell; Epo, erythropoietin; G, granulocyte; M, monocyte)

CFU-GEMMs mature under the influence of colony-stimulating factors (CSFs) and several interleukins (see Fig. 2.6). These factors, which are relevant for the positive regulation of hemopoiesis, are:

• derived mainly from stromal cells (connective tissue cells) in the bone marrow;

• also produced by mature forms of differentiated myeloid and lymphoid cells.

Bone marrow stromal cells, stromal cell matrix, and cytokines form the microenvironment to support stem cell differentiation into individual cell lineages. Stromal cells produce an extracellular matrix which is very important in establishing cell–cell interactions and enhancing stem cell differentiation. The major components of the matrix are proteoglycans, fibronectin, collagen, laminin, haemonectin and thrombospondin.

Other cytokines, such as transforming growth factor-β (TGFβ) may downregulate hemopoiesis. CFU-GMs taking the monocyte pathway give rise initially to proliferating monoblasts. Proliferating monoblasts differentiate into pro-monocytes and finally into mature circulating monocytes which serve as a replacement pool for the tissue-resident macrophages (e.g. lung macrophages).

Monocytes express CD14 and significant levels of MHC class II molecules

The non-differentiated hemopoietic stem cell marker CD34, like other early markers in this lineage, is lost in mature neutrophils and mononuclear phagocytes. Other markers may be lost as differentiation occurs along one pathway, but retained in the other. For example, the common precursor of monocytes and neutrophils, the CFU-GM cell, expresses major histocompatibility complex (MHC) class II molecules, but only monocytes continue to express significant levels of this marker.

Q. What is the functional significance of the expression of MHC molecules on monocytes?

A. Monocytes can present antigens to helper T cells, but neutrophils generally cannot.

Mononuclear phagocytes and granulocytes display different functional molecules. Mononuclear phagocytes express CD14 which is part of the receptor complex for the lipopolysaccharide of Gram-negative bacteria). In addition, they acquire many of the same surface molecules as mature or activated neutrophils (e.g. the adhesion molecules CD11a and b and Fc receptors which recognize the constant regions of antibodies e.g. CD64 and CD32 – FcγRI and FcγRII respectively).

Neutrophils express adhesion molecules and receptors involved in phagocytosis

CFU-GMs go through several differentiation stages to become neutrophils. As the CFU-GM cell differentiates along the neutrophil pathway, several distinct morphological stages are distinguished. Myeloblasts develop into promyelocytes and myelocytes, which mature and are released into the circulation as neutrophils.

The one-way differentiation of the CFU-GM into mature neutrophils is the result of acquiring specific receptors for growth and differentiation factors at progressive stages of development. Surface differentiation markers disappear or are expressed on the cells as they develop into granulocytes. For example, MHC class II molecules are expressed on the CFU-GM, but not on mature neutrophils.

Other surface molecules acquired during the differentiation process include:

• adhesion molecules (e.g. the leukocyte integrins CD11a, b, and c, associated with CD18 β₂ chains); and

• receptors involved in phagocytosis including complement and antibody Fc receptors.

Neutrophils constitutively express FcγRIII and FcγRII, and FcγRI is induced on activation.

It is difficult to assess the functional activity of different developmental stages of granulocytes, but it seems likely that the full functional potential is realized only when the cells are mature.

There is some evidence that neutrophil activity, as measured by phagocytosis or chemotaxis, is lower in fetal than in adult life. However, this may be due, in part, to the lower levels of opsonins (e.g. complement components and antibodies) in the fetal serum, rather than to a characteristic of the cells themselves.

To become active in the presence of opsonins, neutrophils must interact directly with microorganisms and/or with cytokines generated by a response to antigen. This limitation could reduce neutrophil activity in early life.

Activation of neutrophils by cytokines and chemokines is also a prerequisite for their migration into tissues (see Chapter 9).

Eosinophils, basophils, mast cells and platelets in inflammation

Eosinophils are thought to play a role in immunity to parasitic worms

Eosinophils comprise 2–5% of blood leukocytes in healthy, non-allergic individuals. Human blood eosinophils usually have a bilobed nucleus and many cytoplasmic granules, which stain with acidic dyes such as eosin (Fig. 2.7). Although not their primary function, eosinophils appear to be capable of phagocytosing and killing ingested microorganisms.

Fig. 2.7 Morphology of the eosinophil

The ultrastructure of a mature eosinophil shows granules (G) with central crystalloids. × 17 500. (ER, endoplasmic reticulum; Nu, nucleus; P, nuclear pores) Inset: A mature eosinophil in a blood smear is shown with a bilobed nucleus and eosinophilic granules. × 1000.

(From Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

The granules in mature eosinophils are membrane-bound organelles with crystalloid cores that differ in electron density from the surrounding matrix (see Fig. 2.7). The crystalloid core contains the major basic protein (MBP), which:

• is a potent toxin for helminth worms;

• induces histamine release from mast cells;

• activates neutrophils and platelets; and

• of relevance to allergy, provokes bronchospasm.

Other proteins with similar effects are found in the granule matrix, for example:

• eosinophil cationic protein (ECP); and

• eosinophil-derived neurotoxin (EDN).

Release of the granules on eosinophil activation is the only way in which eosinophils can kill large pathogens (e.g. schistosomula), which cannot be phagocytosed. Eosinophils are therefore thought to play a specialized role in immunity to parasitic worms using this mechanism (see Fig. 15.13).

Basophils and mast cells play a role in immunity against parasites

Basophils are found in very small numbers in the circulation and account for less than 0.2% of leukocytes (Fig. 2.8).

Fig. 2.8 Morphology of the basophil

Morphology of the basophil: ultrastructural analysis shows a segmented nucleus (N) and the large cytoplasmic granules (G). Arrows indicate nuclear pores. × 11 000. Inset: This blood smear shows a typical basophil with its deep violet-blue granules. × 1000.

(Adapted from Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

The mast cell (Fig. 2.9), which is present in tissues and not in the circulation, is indistinguishable from the basophil in a number of its characteristics, but displays some distinctive morphological features (Fig. 2.10). Their shared functions may indicate a convergent differentiation pathway.

Fig. 2.9 Histological appearance of human connective tissue in mast cells

This micrograph of a mast cell shows dark blue cytoplasm with purple granules. Alcian blue and safranin stain. × 600.

(Courtesy of Dr TS Orr.)

Fig. 2.10 Some distinctive and common characteristics of basophils and mast cells

Some distinctive and common characteristics of basophils and mast cells.

The stimulus for mast cell or basophil degranulation is often an allergen (i.e. an antigen causing an allergic reaction). To be effective, an allergen must cross-link IgE molecules bound to the surface of the mast cell or basophil via its high-affinity Fc receptors for IgE (FcεRI). Degranulation of a basophil or mast cell results in all contents of the granules being released very rapidly. This occurs by intracytoplasmic fusion of the granules, followed by discharge of their contents (Fig. 2.11).

Fig. 2.11 Electron micrograph study of rat mast cells

Rat peritoneal mast cells show electron-dense granules (1). Vacuolation with exocytosis of the granule contents has occurred after incubation with anti-IgE (2). Transmission electron micrographs. × 2700.

(Courtesy of Dr D Lawson.)

Mediators such as histamine, released by degranulation, cause the adverse symptoms of allergy, but, on the positive side, also play a role in immunity against parasites by enhancing acute inflammation.

Platelets have a role in clotting and inflammation

Blood platelets (Fig. 2.12) are not cells, but cell fragments derived from megakaryocytes in the bone marrow. They contain granules, microtubules, and actin/myosin filaments, which are involved in clot contraction. Platelets also participate in immune responses, especially in inflammation.

Fig. 2.12 Ultrastructure of a platelet

Cross-section of a platelet showing two types of granule (G) and bundles of microtubules (MT) at either end. × 42 000.

(Adapted from Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

The adult human produces 10¹¹ platelets each day. About 30% of platelets are stored in the spleen, but may be released if required.

Q. What circumstance might require the release of additional platelets into the circulation?

A. Severe blood loss.

Platelets express class I MHC products and receptors for IgG (CD32; FcγRII), which are important in platelet activation via IgG immune complexes. In addition, megakaryocytes and platelets carry:

• receptors for clotting factors (e.g. factor VIII); and

• other molecules important for their function, such as the GpIIb/IIIa complex (CD41) responsible for binding to fibrinogen, fibronectin, vitronectin (tissue matrix), and von Willebrand factor (another clotting factor).

Both receptors and adhesion molecules are important in the activation of platelets.

Following injury to endothelial cells, platelets adhere to and aggregate at the damaged endothelial surface. Release of platelet granule contents, which include de novo synthesized serotonin and endocytosed fibrinogen, results in:

• increased capillary permeability, a feature of inflammation;

• activation of complement (and hence attraction of leukocytes); and

• clotting.

NK cells

NK cells account for up to 15% of blood lymphocytes and express neither T cell nor B cell antigen receptors. They are derived from the bone marrow and morphologically have the appearance of large granular lymphocytes (see Fig. 2.19).

Fig. 2.19 Morphological heterogeneity of lymphocytes

Lymphocyte morphology. (1) The small lymphocyte has no granules, a round nucleus, and a high N:C ratio. (2) The large granular lymphocyte (LGL) has a lower N:C ratio, indented nucleus, and azurophilic granules in the cytoplasm. Giemsa stain. (3) Ultrastructure of the LGL shows characteristic electron-dense peroxidase-negative granules (primary lysosomes, PL), scattered throughout the cytoplasm, with some close to the Golgi apparatus (GA) and many mitochondria (M). × 10 000.

((1) Adapted from Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003. (2) Courtesy of Dr A Stevens and Professor J Lowe.)

Functional NK cells are found in the spleen, and cells found in lymph nodes that express CD56 but not CD16 (see below) might represent immature NK cells.

Nevertheless many surface markers are shared with T cells, monocytes/macrophages or neutrophils.

CD16 and CD56 are important markers of NK cells

The presence of CD16 (FcγRIII) is commonly used to identify NK cells in purified lymphocyte populations. CD16 is involved in one of the activation pathways of NK cells and is also expressed by neutrophils, some macrophages and γδ T cells (see below). However on neutrophils, CD16 is linked to the surface membrane by a glycoinositol phospholipid (GPI) linkage, whereas NK cells and γδ T cells express the transmembrane form of the molecule. The CD56 molecule, a homophilic adhesion molecule of the immunoglobulin superfamily (NCAM), is another important marker of NK cells. Combined with the absence of the T cell receptor (CD3), CD56 and CD16 are currently the most reliable markers for NK cells in humans.

Resting NK cells also express the β chain of the IL-2 receptor, and the signal transducing common γ chain of IL-2 and other cytokine receptors (see Fig. 8.18). Therefore, direct stimulation with IL-2 activates NK cells.

The function of NK cells is to recognize and kill virus-infected cells (Fig. 2.13) and certain tumor cells by mechanisms described in chapter 10.

Fig. 2.13 An NK cell attached to a target cell

An NK cell (NK) attached to a target cell (TC). × 4500.

(Courtesy of Dr G Arancia and W Malorni, Rome.)

Classical and non-classical MHC class I molecules (see Fig. 5.15) are ligands for inhibitory receptors on the NK cells which prevent killing and this explains why normal body cells (all of which normally express MHC class I molecules) are not targeted by NK cells.

Downregulation or modification of MHC molecules in virus-infected cells and some tumors makes them susceptible to NK cell-mediated killing.

Q. What advantage is there for a virus in causing the loss of MHC class I molecules in the cell it has infected?

A. The infected cell can no longer be recognized by cytotoxic T cells (see Fig. 1.12).

NK cells are also able to kill targets coated with IgG antibodies via their receptor for IgG (FcγRIII, CD16). This property is referred to as antibody-dependent cellular cytotoxicity (ADCC).

NK cells release interferon-γ (IFNγ) and other cytokines (e.g. IL-1 and GM-CSF) when activated, which might be important in the regulation of hemopoiesis and immune responses.

Antigen presenting cells

APCs are a heterogeneous population of leukocytes that are important in innate immunity (see Fig. 2.2) and play a pivotal role in the induction of functional activity of T helper (TH) cells.

In this regard, APCs are seen as a critical interface between the innate and adaptive immune systems. There are professional APCs (dendritic cells, macrophages and B cells) constitutively expressing MHC class II and co-stimulatory molecules, and non-professional APCs which express MHC class II and co-stimulatory molecules for short periods of time throughout sustained inflammatory responses. This group is comprised of fibroblasts, glial cells, pancreatic β cells, thymic epithelial cells, thyroid epithelial cells and vascular endothelial cells.

Both macrophages and B cells are rich in membrane MHC class II molecules, especially after activation, and are thus able to process and present specific antigens to (activated) T cells (see Chapter 8).

Somatic cells other than immune cells do not normally express class II MHC molecules, but cytokines such as IFNγ and tumor necrosis factor-α (TNFα) can induce the expression of class II molecules on some cell types, and thus allow them to present antigen (non-professional APCs). This induction of ‘inappropriate’ class II expression might contribute to the pathogenesis of autoimmune diseases and to prolonged inflammation (see Chapter 20).

Dendritic cells are derived from several different lineages

Functionally, dendritic cells (DC) are divided into those that both process and present foreign protein antigens to T cells – ‘classical’ dendritic cells (DCs) – and a separate type that passively presents foreign antigen in the form of immune complexes to B cells in lymphoid follicles – follicular dendritic cells (FDCs; Fig. 2.14).

Fig. 2.14 Different kinds of antigen-presenting cells (APCs)

There are two main types of dendritic cells – classical DC and follicular dendritic cells (FDCs). (1) Immature DCs are derived from bone marrow and interact mainly with T cells. They are highly phagocytic, take up microbes, process the foreign microbial antigens into small peptides, and become mature APCs carrying the processed antigen (a peptide) on their surface with specialized MHC molecules. Specific T cells recognize the displayed peptide in a complex with MHC and, in the presence of cytokines produced by the mature DC, proliferate and also produce cytokines. (2) FDCs are not bone marrow derived and interact with B cells. In the B cell follicles of lymphoid organs and tissues they bind small immune complexes (IC, called iccosomes). Antigen contained within the IC is presented to specific B cells in the lymphoid follicles. This protects the B cell from cell death. The B cell then proliferates and with T cell help can leave the follicle and become a plasma cell or a memory cell (see Fig. 2.48).

Most DCs derive from one of two precursors:

• a myeloid progenitor (DC1) that gives rise to myeloid DCs, otherwise called bone-marrow derived or bm-DCs; and

• a lymphoid progenitor (DC2) that develops into plasmacytoid DCs (pDCs).

A summary of the main properties of myeloid and plasmacytoid dendritic cells is shown in Figure 2.15.

Fig. 2.15 Myeloid and plasmacytoid dendritic cells (DCs)

There are two main types of dendritic cells defined by their origin. They have differences in their localization markers and cytokine.

Myeloid DCs can also be divided into at least three types: Langerhans’ cells (LCs), dermal or interstitial DCs (DDC-IDCs) and blood monocyte-derived DCs (moDCs).

Different populations of DCs can be identified by their surface markers. Myeloid DCs, but not pDCs express CD1a and CD208, whilst DDC-IDC and moDC express also CD11b. Langerhans’ cells have so called Birbeck granules containing Langerin. It appears that various populations of myeloid DCs may represent different stages in their maturation and migration in the body (see below).

BM-DCs express various receptors that are involved in antigen uptake:

• C-type lectin receptors – for glycosyl groups, e.g. macrophage mannose receptor (MMR) family;

• Fc receptors for IgG, IgE;

• receptors for heat shock protein–peptide complexes;

• receptors for apoptotic corpses;

• ‘scavenger’ receptors – for sugars, lipid etc;

• toll-like receptors (TLRs).

Before DCs take up antigen (become loaded) they are called immature DCs and express various markers characteristic for this, resting, stage, the most important being chemokine receptors CCR1, CCR5 and CCR6. DCs are attracted to the infection site by chemokines through these receptors (see Chapter 6).

Mature DCs loaded with antigen down-regulate expression of CCR1, 5, 6 and up-regulate CCR7. This encourages their migration from various tissues into peripheral lymphatics, where CCR7 interacts with secondary lymphoid tissue chemokine SLC (CCL21) expressed on vascular endothelium (see Fig. 6.15).

DCs are found primarily in the skin, lymph nodes, and spleen, and within or underneath most mucosal epithelia. They are also present in the thymus, where they present self antigens to developing T cells.

Langerhans’ cells and interdigitating dendritic cells are rich in MHC class II molecules

Langerhans’ cells in the epidermis and in other squamous epithelia migrate via the afferent lymphatics into the paracortex of the draining lymph nodes (Fig. 2.16). Here, they interact with T cells and are termed interdigitating cells (IDCs, Fig. 2.17). These DCs are rich in class II MHC molecules, which are important for presenting antigen to helper T cells.

Fig. 2.16 Migration of antigen-presenting cells (APCs) into lymphoid tissues

Bone marrow-derived dendritic cells (DCs) are found especially in lymphoid tissues, in the skin, and in mucosa. DCs in the form of Langerhans’ cells are found in the epidermis and in mucosa and are characterized by special granules (the tennis racquet-shaped Birbeck granules; not shown here). Langerhans’ cells are rich in MHC class II molecules, and carry processed antigens. They migrate via the afferent lymphatics (where they appear as ‘veiled’ cells) into the paracortex of the draining lymph nodes. Here they make contact with T cells. These ‘interdigitating dendritic cells (IDCs)’, localized in the T cell areas of the lymph node, present antigen to T helper cells. Antigen is exposed to B cells on the follicular dendritic cells (FDCs) in the germinal centers of B cell follicles. Some macrophages located in the outer cortex and marginal sinus may also act as APCs. In the thymus, APCs occur as IDCs in the medulla. (HEV, high endothelial venule)

Fig. 2.17 Ultrastructure of an interdigitating dendritic cell (IDC) in the T cell area of a rat lymph node

Intimate contacts are made by APCs with the membranes of the surrounding T cells. The cytoplasm contains a well-developed endosomal system and does not show the Birbeck granules characteristic of skin Langerhans’ cells. × 2000. (I, IDC nucleus; Mb, IDC membrane; T, T cell nucleus)

(Courtesy of Dr BH Balfour.)

Q. What function could the migration of Langerhans’ cells to the lymph nodes from the mucosa or skin serve?

A. The migration of Langerhans’ cells provides an efficient mechanism for carrying antigen from the skin and mucosa to the TH cells in the lymph nodes, and are rich in class II MHC molecules, which are important for presenting antigen to TH cells. Lymph nodes provide the appropriate environment for lymphocyte proliferation.

BM-DCs are also present within the germinal centers (GCs) of secondary lymphoid follicles (i.e. they are the MHC class II molecule-positive germinal center DCs [GCDCs]). In contrast to FDCs, they are migrating cells, which on arrival in the GC interact with germinal center T cells and are probably involved in antibody class switching (see Chapter 9).

The thymus is of crucial importance in the development and maturation of T cells. In thymus there are cortical DCs and IDCs which are especially abundant in the medulla (see Fig. 2.16). They participate in two important stages in T cell maturation/differentiation in thymus positive and negative selection respectively (see below).

FDCs lack class II MHC molecules and are found in B cell areas

Unlike the APCs that actively process and present protein antigens to T cells, FDCs have a passive role in presenting antigen in the form of immune complexes to B cells. They are therefore found in the primary and secondary follicles of the B cell areas of the lymph nodes, spleen, and MALT (see Fig. 2.16). They are a non-migratory population of cells and form a stable network (a kind of web) by establishing strong intercellular connections via desmosomes.

FDCs lack class II MHC molecules, but bind antigen via complement receptors (CD21 and CD35), which attach to complement associated with immune complexes (iccosomes; Fig. 2.18). They also express Fc receptors. The FDCs produce chemokines that are important in homing of B cells to the follicular areas in lymphoid tissues. They are not bone marrow derived, but are of mesenchymal origin.

Fig. 2.18 Follicular dendritic cell

An isolated follicular dendritic cell (FDC) from the lymph node of an immunized mouse 24 hours after injection of antigen. The FDC is of intermediate maturity with smooth filiform dendrites typical of young FDCs, and beaded dendrites, which participate in the formation of iccosomes (immune complexes) in mature FDCs. The adjacent small white cells are lymphocytes.

(Electron micrograph kindly provided by Dr Andras Szakal; reproduced by permission of the Journal of Immunology.)

Lymphocytes

Lymphocytes are phenotypically and functionally heterogeneous

Large numbers of lymphocytes are produced daily in the primary or central lymphoid organs (i.e. thymus and postnatal bone marrow). Some migrate via the circulation into the secondary lymphoid tissues (i.e. spleen, lymph nodes, and MALT).

The average human adult has about 2 × 10¹² lymphoid cells and lymphoid tissue as a whole represents about 2% of total body weight. Lymphoid cells account for about 20% of the leukocytes in the adult circulation.

Many mature lymphoid cells are long-lived, and persist as memory cells for many years.

Q. Given that there are roughly 10⁹ lymphocytes/liter of blood and an average of 5 liters of blood in an individual, and that roughly 2 × 10⁹ new cells are produced each day, what can you infer about the location and life span of lymphocytes within an individual?

A. This implies that less than 1% of an individual’s lymphocytes are in the circulation. This highly selective population of lymphocytes is mostly en route between tissues. The data also imply that many lymphocytes must die each day to maintain the overall balance of the lymphoid system, and that the average life span of a lymphocyte will be months to years. The actual values are, however, enormously variable, depending on the type of lymphocyte.

Lymphocytes are morphologically heterogeneous

In a conventional blood smear, lymphocytes vary in both size (from 6–10 μm in diameter) and morphology.

Differences are seen in:

• nuclear to cytoplasmic (N:C) ratio;

• nuclear shape; and

• the presence or absence of azurophilic granules.

Two distinct morphological types of lymphocyte are seen in the circulation as determined by light microscopy and a hematological stain such as Giemsa (Fig. 2.19):

• the first type is relatively small, is typically agranular and has a high nuclear to cytoplasmic (N:C) ratio (Fig. 2.19[1]);

• the second type is larger, has a lower N:C ratio, contains cytoplasmic azurophilic granules, and is known as the large granular lymphocyte (LGL).

LGLs should not be confused with granulocytes, monocytes, or their precursors, which also contain azurophilic granules.

Most T cells express the αβ T cell receptor (see below) and, when resting, can show either of the above morphological patterns.

Most T helper (TH) cells (approximately 95%) and a proportion (approximately 50%) of cytotoxic T cells (TC or CTL) have the morphology shown in Figure 2.19(1).

The LGL morphological pattern displayed in Figure 2.19(2) is shown by less than 5% of TH cells and by about 30–50% of TC cells. These cells display LGL morphology with primary lysosomes dispersed in the cytoplasm and a well-developed Golgi apparatus, as shown in Figure 2.19(3).

Most B cells, when resting, have a morphology similar to that seen in Figure 2.19(1) under light microscopy.

Lymphocytes express characteristic surface and cytoplasmic markers

Lymphocytes (and other leukocytes) express a large number of different functionally important molecules mostly on their surfaces but also in their cytoplasm, which can be used to distinguish (‘mark’) cell subsets. Many of these cell markers can be identified by specific monoclonal antibodies (mAb) and can be used to distinguish T cells from B cells (Fig. 2.20).

Fig. 2.20 Main distinguishing markers of T and B cells

Main distinguishing markers of T and B cells.

Lymphocytes express a variety of cell surface molecules that belong to different families, which have probably evolved from a few ancestral genes. These families of molecules are shared with other leukocytes and are distinguished by their structure. The major families include:

• the immunoglobulin superfamily;

• the integrin family;

• selectins;

• proteoglycans.

The immunoglobulin superfamily comprises molecules with structural characteristics similar to those of the immunoglobulins and includes CD2, CD3, CD4, CD8, CD28, MHC class I and II molecules, and many more.

The integrin family consists of heterodimeric molecules with α and β chains. There are several integrin subfamilies and all members of a particular subfamily share a common β chain, but each has a unique α chain:

• one integrin subfamily (the β₂-integrins) uses CD18 as the β chain, which can be associated with CD11a, CD11b, CD11c, or αd – these combinations make up the lymphocyte function antigens LFA-1, Mac-1 (CR3), p150, 95, and αdβ₂ surface molecules respectively – and are commonly found on leukocytes;

• a second subfamily (the β₁-integrins) has CD29 as the β chain, which again is associated with various other peptides and includes the VLA (very late activation) markers.

The selectins (CD62, E, L, and P) are expressed on leukocytes (L) or activated endothelial cells and platelets (E and P). They have lectin-like specificity for a variety of sugars expressed on heavily glycosylated membrane glycoproteins (e.g. CD43).

The proteoglycans, typically CD44, have a number of glycosaminoglycan (GAG) binding sites (e.g. for chondroitin sulfate), and bind to extracellular matrix components (typically, hyaluronic acid).

Other families include:

• the tumor necrosis factor (TNF) and nerve growth factor (NGF) receptor superfamily;

• the C-type lectin superfamily;

• the family of receptors with seven transmembrane segments (tm7); and

• the tetraspanins, a superfamily with four membrane-spanning segments (tm4), for example CD20.

Marker molecules allow lymphocytes to communicate with their environment

The major function of the families of marker molecules described above is to allow lymphocytes to communicate with their environment. They are extremely important in cell trafficking, adhesion, and activation.

Markers expressed by lymphocytes can often be detected on cells of other lineages (e.g. CD44 is commonly expressed by epithelial cells).

Identification of lymphocyte subsets

Human lymphocytes can be identified in tissues and their function can be measured as separated populations. Immunofluorescence techniques to identify leukocytes are shown in Method box 2.1.

Method box 2.1 Identification of cell populations

Molecules on or in cells can be identified using fluorescent antibodies as probes. The antibodies can be applied to tissue sections or used in flow cytometry.

Immunofluorescence

Immunofluorescence detects antigen in situ. A section is cut on a cryostat from a deep-frozen tissue block. This ensures that labile antigens are not damaged by fixatives.

Fig. MB2.1.1 Immunofluorescence.

Direct immunofluorescence

The test solution of antibody conjugated with a fluorescein, e.g. to CD3, is applied to the section in a drop, incubated, and washed off. Any bound antibody is then revealed under the microscope. Ultraviolet light is directed onto the section through the objective and as a result the field is dark and areas with bound fluorescent antibody on and in the T cells fluoresce.

Indirect immunofluorescence

Antibody applied to the section as a solution is unconjugated (fluorescence free) and is called a primary antibody. It is visualized using fluoresceinated secondary anti-immunoglobulin antibody, e.g. to the mouse anti-human CD3 antibodies in this case.

Indirect complement amplified – an elaboration of indirect immunofluorescence

This is an elaboration of the indirect immunofluorescence for the detection of complement-fixing primary antibody. In the second step fresh complement is added, which becomes fixed around the site of antibody binding. Due to the amplification steps in the classical complement pathway (see Chapter 4) one antibody molecule can cause many C3b molecules to bind to the section – these are then visualized with fluoresceinated anti-C3 secondary antibody.

Flow cytometry and cell sorting

Cells are stained with specific fluorescent reagents to detect surface molecules and are then introduced into the flow chamber of the flow cytometer. The cell stream passing out of the chamber is encased in a sheath of buffer fluid. The stream is illuminated by laser light and each cell is measured for size (forward light scatter) and granularity (90° side light scatter), as well as for fluorescence (green, orange, red, purple, blue – depending on the fluorescein conjugated to the antibody) to detect two different surface markers. In a cell sorter the flow chamber vibrates the cell stream causing it to break into droplets, which are then charged and may be steered by deflection plates under computer control to collect different cell populations according to the parameters measured. Plot (2.1) shows peripheral blood mononuclear cells double stained with FITC-conjugated (green) anti-CD3 antibody (x axis) and PE-labeled (orange) anti-CD8 antibody (y axis). Four populations can be seen, and the CD8 cells appear in the right upper quadrant. The CD8 cells are then selected (gated) and the isolated CD8 population is shown in plot (2.2).

Fig. MB2.1.2 Fluorescence-activated cell sorter (FACS).

ELISPOT assays

Individual B cells producing specific antibody or individual T cells secreting particular cytokines may be detected by enzyme linked immuno SPOT (ELISPOT) assay.

Fig. MB2.1.3 ELISPOT assays.

For detection of antibody-producing cells

The lymphocytes are plated onto a plate coated with antigen (antigen-sensitized). Secreted antibody binds antigen in the immediate vicinity of cells producing the specific antibody. The spots of bound antibody are then detected chromatographically using enzyme coupled to anti-immunoglobulin and a chromogen.

For detection of cytokine-producing cells

The plates are coated with anti-cytokine antibody and the captured cytokine is detected with a second, enzyme-coupled antibody, to a different epitope on the cytokine.

Marker molecules allow lymphocytes to be isolated from each other

The presence of characteristic surface molecules expressed by cell populations allows them to be identified using fluorescent antibodies as probes. These can be applied to tissue sections to identify cell populations or be used in flow cytometry to enumerate and separate cells in suspension on the basis of their size and fluorescent staining (see Method box 2.1). These techniques together with the expression of surface molecules allowing the cell populations to be isolated from each other using cell panning and immunomagnetic beads (Method box 2.2) have allowed a detailed dissection of lymphoid cell populations.

Method box 2.2 Isolating cell populations

Density-gradient separation of lymphocytes on Ficoll Isopaque

Lymphocytes can be separated from whole blood using a density gradient. Whole blood is defibrinated by shaking with glass beads and the resulting clot removed. The blood is then diluted in tissue culture medium and layered on top of a tube half full of Ficoll. Ficoll has a density greater than that of lymphocytes, but less than that of red cells and granulocytes (e.g. neutrophils).

After centrifugation the red cells and polymorphonuclear neutrophils (PMNs) pass down through the Ficoll to form a pellet at the bottom of the tube while lymphocytes settle at the interface of the medium and Ficoll.

The lymphocyte preparation can be further depleted of macrophages and residual PMNs by the addition of iron filings. These are taken up by phagocytes, which can then be drawn away with a strong magnet. Macrophages can also be removed by leaving the cell suspension to settle on a plastic dish. Macrophages adhere to plastic, whereas the lymphocytes can be washed off.

Fig. MB2.2.1 Density-gradient separation of lymphocytes on Ficoll Isopaque.

Isolating cell populations using their characteristic surface molecules

The presence of characteristic surface molecules expressed by cell populations allows the cell populations to be isolated from each other using cell panning and immunomagnetic beads.

Isolation of lymphocyte subpopulations – panning

Cell populations can be separated on antibody-sensitized plates. Antibody binds non-covalently to the plastic plate (as for ELISPOT immunoassay) and the cell mixture is applied to the plate. Antigen-positive cells (Ag⁺) bind to the antibody and the antigen-negative cells (Ag^–) can be carefully washed off.

By changing the culture conditions or by enzyme digestion of the cells on the plate, it is sometimes possible to recover the cells bound to the plate.

Often the cells that have bound to the plate are altered by their binding (e.g. binding to the plate cross-links the antigen, which can cause cell activation). The method is therefore most satisfactory for removing a subpopulation from the population, rather than isolating it.

Examples of the application of this method include:

• separating TH and TC cell populations using antibodies to CD4 or CD8; and

• separating T cells from B cells using anti-Ig (which binds to the surface antibody of the B cell).

In reverse, by sensitizing the plate with antigen, antigen-binding cells can be separated from non-binding cells.

Fig. MB2.2.2 Isolation of lymphocyte subpopulations – panning.

Cell separation by immunomagnetic beads

Direct method (shown in Fig. MB2.2.3)

The beads are coated with a monoclonal antibody to the cellular antigen of interest, by either:

• direct binding to the bead; or

• binding the primary antibody to secondary antibody-coated beads.

The coated beads are then incubated with the cell suspension (or even whole blood) and the cells bound by the antibody on the beads (positively selected cells) are immobilized by applying a magnetic field to the tube (concentrating on the test-tube wall around the magnet). The non-immobilized cells (negatively selected) are removed from the tube and the positively selected cells are recovered following washing and dissociation from the antibody-coated beads. This method can be also used to remove an unwanted population of the cells from a mixture. In this case the non-immobilized cells will be collected for further experiments.

Indirect method

The monoclonal antibody to the target cellular antigen is first added to the cell suspension. Following incubation with the antibody, the cells are washed and mixed with beads coated with the appropriate secondary anti-Ig antibody. Cells bound to the magnetic beads (positively selected) are then immobilized with a magnetic field and the non-immobilized cells (negatively selected) removed. The positively selected cells are then washed and dissociated from the beads.

The microbeads available these days allow us to abandon the dissociation procedure. They are so small that they can be left attached to the cell surface, and will not interfere with their function.

Cell sorting

Cells can be isolated by their surface markers using a flow cytometer with a sorting function (cell sorter). The sorter will direct the cells fluorescing due to a specific fluoresceinated antibody attached to them into a collection vessel. Using the example discussed in Method box 2.1, cells with attached FITC-conjugated antibody would be directed to one collection vessel for green cells and cells with attached PE-labeled (orange) antibody – to separate collection vessel for orange cells.

Fig. MB2.2.3 Cell separation by immunomagnetic beads.

T cells can be distinguished by their different antigen receptors

The definitive T cell lineage marker is the T cell antigen receptor (TCR). The two defined types of TCR are:

• a heterodimer of two disulfide-linked polypeptides (α and β);

• a structurally similar heterodimer consisting of γ and δ polypeptides.

Both receptors are associated with a set of five polypeptides (the CD3 complex) and together form the TCR complex (TCR–CD3 complex; see Chapter 5).

Approximately 90–95% of blood T cells in humans are αβ T cells and the remaining 5–10% are γδ T cells.

There are three major subpopulations of αβ T cells

• Helper T cells (TH) that express the CD4 marker (CD4⁺ T cells), and mainly ‘helps’ or ‘induces’ immune responses, divided into two main subsets (TH1 and TH2).

• Regulatory T cells (Tregs) that express the CD4 marker (CD4⁺T cells) and regulate immune responses.

• Cytotoxic T cells (TC) that express the CD8 marker (CD8⁺ T cells) – also called cytotoxic T lymphocytes (CTLs).

CD4⁺ T cells recognize their specific antigens in association with MHC class II molecules, whereas CD8⁺ T cells recognize antigens in association with MHC class I molecules (see Chapter 7). Thus, the presence of CD4 or CD8 limits (restricts) the type of cell with which the T cell can interact (Fig. 2.21).

Fig. 2.21 Functional T cell subsets

T cells express either γδ or αβ T cell receptors (TCR). T cells are divided into CD4⁺ and CD8⁺ subsets and these subsets determine whether they see antigen (peptides) with MHC class II or I molecules, respectively. CD4⁺ T cells can be further subdivided into TH1 and TH2 on the basis of their cytokine profiles.

A small proportion of αβ T cells express neither CD4 nor CD8; these ‘double negative’ T cells might have a regulatory function.

In contrast, while most circulating γδ cells are ‘double negative’, most γδ T cells in the tissues express CD8.

T helper subsets are distinguished by their cytokine profiles

CD4⁺ T helper cells can be further divided into functional subsets on the basis of the spectrum of the cytokines they produce:

• TH1 cells secrete IL-2 and IFNγ;

• TH2 cells produce IL-4, IL-5, IL-6, and IL-10 (see Fig. 11.4).

TH1 cells mediate several functions associated with cytotoxicity and local inflammatory reactions. They help cytotoxic T cell precursors develop into effector cells to kill virally-infected target cells and activate macrophages infected with intracellular pathogens (e.g. Mycobacterium, Chlamydia) enhancing intracellular killing of the pathogens by the production of IFNγ. Consequently, they are important for combating intracellular pathogens including viruses, bacteria, and parasites. Some TH1 cells also help B cells to produce different classes of antibodies.

Recently a TH17 cell has been described that is similar to the TH1 subset but its induction from TH0 cells is dependent on TGFβ and IL-21 and not IL-12 and IFNα. Their induction by TGFβ suggests that they are related to the regulatory T cell subsets. They produce both IL-17 and IL-22 and appear to play an important role in maintaining the integrity of mucosal surfaces and thus in protection against microbial entry into the body.

Q. Which cell type do TH1 cells interact with to help combat intracellular pathogens?

A. Mononuclear phagocytes.

TH2 cells are effective at stimulating B cells to proliferate and produce antibodies of some IgG subclasses and especially IgE and therefore function primarily to protect against free-living, extracellular, microorganisms (humoral immunity).

The number of cells producing a given cytokine can be measured using flow cytometry and antibodies that are allowed to penetrate the cells following permeabilization (see Method box 2.1). The same technique can be used to determine the number of B cells producing a particular antibody.

The measurement of single cells secreting a particular cytokine or antibody can be achieved using an enzyme-linked method, namely ELISPOT (see Method box 2.1).

Several CD4⁺ regulatory T cell populations have been described as being capable of suppressing T cell responses (see below).

Other T cell subsets include γδ T cells and NKT cells

γδ T cells may protect the mucosal surfaces of the body

γδ T cells are relatively frequent in mucosal epithelia, but form only a minor subpopulation of circulating T cells (around 5%). Most intraepithelial lymphocytes (IELs) are γδ T cells and express CD8, a marker not found on most circulating γδ T cells.

γδ T cells have a specific repertoire of TCRs biased towards certain bacterial/viral antigens (superantigens, see Fig. 14.16). Human blood γδ T cells have specificity for low molecular mass mycobacterial products (e.g. ethylamine and isopentenyl pyrophosphate).

Current opinion is that γδ T cells may play an important role in protecting the mucosal surfaces of the body. Some γδ T cells may recognize antigens directly (i.e. with no need for MHC molecule-mediated presentation).

γδ T cells display LGL characteristics (see Fig. 2.19) and some have a dendritic morphology in lymphoid tissues (Fig. 2.22). They appear to have a broader specificity for recognition of unconventional antigens such as heat shock proteins, phospholipids and phosphoproteins. Unlike αβ T cells, they do not generally recognize antigens in association with classical MHC class I and II molecules. There is evidence that γδ T cells show cytotoxicity and regulatory functions and subsets of them appear to have specific tissue locations.

Fig. 2.22 Dendritic morphology of γδ T cells in the tonsil

The γδ T cell population is predominantly localized in the interfollicular T cell-dependent zones. Note the dendritic morphology of the cells. Anti-γδ T cell monoclonal antibody and immunoperoxidase. × 900.

(Courtesy of Dr A Favre, from Eur J Immunol 1991;21:173, with permission.)

NKT cells may initiate T cell responses

NKT cells have T markers and also some NK cell markers: they express CD3 and have a unique αβ TCR (expressing an invariant Vα and Vβ11, see Chapter 5).

Q. What markers would one normally use to distinguish T cells from NK cells?

A. CD16 and CD56 are used to distinguish NK cells. CD3 is characteristic of T cells.

NKT cells are thought to recognize glycolipid antigens presented by CD1d molecules (see Chapter 5), but not conventional MHC molecules. In response to antigen they are capable of producing large amounts of IFNγ and IL-4.

NKT cells are therefore thought to act as an interface between the innate and adaptive systems by initiating T cell responses to non-peptide antigens.

NKT cells are also thought to regulate immune responses (especially dendritic cell function) through the production of cytokines (e.g. IL-10).

B cells recognize antigen using the B cell receptor complex

About 5–15% of the circulating lymphoid pool are B cells, which are defined by the presence of surface immunoglobulin, transmembrane molecules, which are constitutively produced and inserted into the B cell membrane, where they act as specific antigen receptors.

Most human B cells in peripheral blood express two immunoglobulin isotypes on their surface:

• IgM; and

• IgD (see Chapter 3).

On any B cell, the antigen-binding sites of these IgM and IgD isotypes are identical.

Fewer than 10% of the B cells in the circulation express IgG, IgA, or IgE, but B cells expressing IgG, IgA, or IgE are present in larger numbers in specific locations of the body (e.g. IgA-bearing cells in the intestinal mucosa).

Immunoglobulin associated with other ‘accessory’ molecules on the B cell surface forms the ‘B cell antigen receptor complex’ (BCR). These ‘accessory’ molecules consist of disulfide-bonded heterodimers of:

• Igα (CD79a); and

• Igβ (CD79b).

The heterodimers interact with the transmembrane segments of the immunoglobulin receptor (see Fig. 3.1), and, like the separate molecular components of the TCR/CD3 complex (see Fig. 5.2), are involved in cellular activation. Intracellular domains of CD79a/b have immunoreceptor tyrosine-based activation motifs (ITAMs). BCR interaction with specific antigen triggers ITAM phosphorylation and this initiates a downstream cascade of intracellular events leading to the activation-related changes in gene expression.

Other B cell markers include MHC class II antigens and complement and Fc receptors

Most B cells carry MHC class II antigens, which are important for cooperative (cognate) interactions with T cells (see Fig. 5.18).

Complement receptors for C3b (CD35) and C3d (CD21) are commonly found on B cells and are associated with activation and, together with chemokine receptors, possibly ‘homing’ of the cells in the peripheral lymphoid organs and tissues. CD19/CD21 interactions with complement, associated with antigen, play a role in antigen-induced B cell activation via the antigen-binding antibody receptor.

Fc receptors for exogenous IgG (FcγRII, CD32) are also present on B cells and play a role in negative signaling to the B cell (see Chapter 11).

CD19 and CD20 are the main markers currently used to identify human B cells. Other human B cell markers are CD22 and CD72 to CD78.

Murine B cells also express CD72 (Lyb-2) together with B220, a high molecular weight (220 kDa) isoform of CD45 (Lyb-5).

CD40 is an important molecule on B cells and is involved in cognate interactions between T and B cells (see Fig. 9.6) with T cells expressing CD40 ligand (CD40L).

Activated B cells upregulate expression of B7.1 (CD80) and B7.2 (CD86) molecules that interact with their CD28 expressed by T cells. This provides a co-stimulatory signal for T/B cognate interactions.

CD5⁺ B-1 cells and marginal zone B cells produce natural antibodies

CD5⁺ B-1 cells have a variety of roles

Many of the first B cells that appear during ontogeny express CD5, a marker originally found on T cells. These cells (termed B-1 cells) are found predominantly in the peritoneal cavity in mice, and there is some evidence for a separate differentiation pathway from ‘conventional’ B cells (termed B-2 cells).

CD5⁺ B-1 cells express their immunoglobulins from unmutated or minimally mutated germline genes (see Chapter 3) and produce mostly IgM, but also some IgG and IgA. These so-called natural antibodies are of low avidity, but, unusually, they are polyreactive and are found at high concentration in the adult serum. CD5⁺ B-1 cells:

• respond well to TI (T-independent) antigens (i.e. antigens that can directly stimulate B cells without T cell help);

• may be involved in antigen processing and antigen presentation to T cells; and

• probably play a role in both tolerance and antibody responses.

Functions proposed for natural antibodies include:

• the first line of defense against microorganisms;

• clearance of damaged self components; and

• regulatory ‘idiotype network’ interactions within the immune system.

Characteristically, natural antibodies react against autoantigens including:

• DNA;

• Fc of IgG;

• phospholipids; and

• cytoskeletal components.

CD5 has been shown to be expressed by B-2 cells when they are activated appropriately, so there is some controversy about whether CD5 represents an activation antigen on B cells. Current theories therefore support the notion for two different kinds of CD5⁺ B cells.

Although the function of CD5 on human B cells is unknown, it is associated with the BCR and may be involved in the regulation of B cell activation.

Marginal zone B cells are thought to protect against polysaccharide antigens

Much has been learned about marginal zone B cells over the past few years. These cells accumulate slowly in the marginal zone of the spleen – a process that takes between 1 and 2 years in humans.

Like B-1 cells, marginal zone B cells respond to thymus-independent antigens, and they are thought to be our main protection against polysaccharide antigens. They also produce natural antibodies, and together with B-1 cells have recently been termed ‘innate-like B cells’.

B cells can differentiate into antibody-secreting plasma cells

Following B cell activation, many B cell blasts mature into antibody-forming cells (AFCs), which progress in vivo to terminally differentiated plasma cells, whilst a subset of B cells remains in the periphery as long-lived memory B cells.

Some B cell blasts do not develop rough endoplasmic reticulum cisternae. These cells are found in germinal centers and are named follicle center cells or centrocytes.

Under light microscopy, the cytoplasm of the plasma cells is basophilic due to the large amount of RNA being used for antibody synthesis in the rough endoplasmic reticulum. At the ultrastructural level, the rough endoplasmic reticulum can often be seen in parallel arrays (Fig. 2.23).

Fig. 2.23 Ultrastructure of the plasma cell

The plasma cell is characterized by parallel arrays of rough endoplasmic reticulum (E). In mature cells, these cisternae become dilated with immunoglobulins. Mitochondria (M) are also seen. × 5000.

(Adapted from Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

Plasma cells are infrequent in the blood, comprising less than 0.1% of circulating lymphocytes. They are normally restricted to the secondary lymphoid organs and tissues, but are also abundant in the bone marrow. Since their sole function is to produce immunoglobulins, plasma cells have few surface receptors and do not respond to antigens. Unlike resting B cells or memory B cells, plasma cells do not express surface BCR or MHC class II.

Antibodies produced by a single plasma cell are of one specificity (idiotype) and immunoglobulin class (isotype and allotype; see Chapter 3).

Immunoglobulins can be visualized in the plasma cell cytoplasm by staining with fluorochrome-labeled specific antibodies (Fig. 2.24).

Fig. 2.24 Immunofluorescent staining of intracytoplasmic immunoglobulin in plasma cells

Fixed human plasma cells, treated with fluoresceinated anti-human-IgM (green) antibody and rhodaminated anti-human-IgG (red) antibody, show extensive intracytoplasmic staining. As the distinct staining of the two cells shows, plasma cells normally produce only one class or subclass (isotype) of antibody. × 1500.

(Adapted from Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

Many plasma cells have a short life span, surviving for a few days and dying by apoptosis (Fig. 2.25). However, a subset of plasma cells with a long life span (months) has recently been described in the bone marrow that might be important in giving rise to sustained antibody responses.

Fig. 2.25 Plasma cell death by apoptosis

Plasma cells are short-lived and die by apoptosis (cell suicide). Note the nuclear chromatin changes, which are characteristic of apoptosis. × 5000.

Lymphocyte development

Lymphocytes, the effector cells of the adaptive immune response, are the major component of organs and tissues that collectively form the lymphoid system.

Within the lymphoid organs, lymphocytes interact with other cell types of both hematopoietic and non-hematopoietic origin that are important for lymphocyte maturation, selection, function and disposal of terminally differentiated cells.

These other cell types are termed accessory cells and include:

• antigen presenting cells;

• macrophages;

• reticular cells; and

• epithelial cells.

The lymphoid system is arranged into either discrete encapsulated organs or accumulations of diffuse lymphoid tissue, which are classified into primary (central) and secondary (peripheral) organs or tissues (Fig. 2.26).

Fig. 2.26 Major lymphoid organs and tissues

Thymus and bone marrow are the primary (central) lymphoid organs. They are the sites of maturation for T and B cells, respectively. Cellular and humoral immune responses occur in the secondary (peripheral) lymphoid organs and tissues. Secondary lymphoid organs can be classified according to the body regions they defend. The spleen responds predominantly to blood-borne antigens. Lymph nodes mount immune responses to antigens circulating in the lymph, entering through the skin (subcutaneous lymph nodes) or through mucosal surfaces (visceral lymph nodes). Tonsils, Peyer’s patches, and other mucosa-associated lymphoid tissues (MALT) (blue boxes) react to antigens that have entered via the surface mucosal barriers. Note that the bone marrow is both a primary and a secondary lymphoid organ because it gives rise to B and NK cells, but is also the site of B cell terminal differentiation (long-lived plasma cells).

In essence, lymphocytes:

• are produced, mature, and are selected in primary lymphoid organs; and

• exert their effector functions in the secondary lymphoid organs and tissues.

Tertiary lymphoid tissues are anatomical sites that under normal conditions contain sparse lymphocytes, if any, but may be selectively populated by these cells in pathological conditions (e.g. skin, synovium, lungs).

Lymphoid stem cells develop and mature within primary lymphoid organs

In the primary lymphoid organs, lymphocytes (B and T cells):

• differentiate from lymphoid stem cells;

• proliferate;

• are selected; and

• mature into functional cells.

In mammals, T cells mature in the thymus and B cells mature in the fetal liver and postnatal bone marrow (see Chapter 9). Birds have a specialized site of B cell generation, the bursa of Fabricius.

In the primary lymphoid organs:

• lymphocytes acquire their repertoire of specific antigen receptors to cope with the antigenic challenges that individuals encounter during their lifetime;

• cells with receptors for autoantigens are mostly eliminated; and

• in the thymus, T cells also ‘learn’ to recognize appropriate self MHC molecules.

There is evidence that some lymphocyte development might occur outside primary lymphoid organs.

Q. Why do lymphocytes need to ‘learn’ what constitutes self MHC and self antigens?

A. Each individual is different and has a particular set of MHC molecules and particular variants of the many other molecules present in the body. The process of what constitutes immunological ‘self’ is different in each individual and therefore learning to recognize ‘self’ is a dialogue between T cells and APCs that takes place in each individual.

T cells develop in the thymus

The thymus in mammals is a bilobed organ in the thoracic cavity overlying the heart and major blood vessels. Each lobe is organized into lobules separated from each other by connective tissue trabeculae.

Within each lobule, the lymphoid cells (thymocytes) are arranged into:

• an outer tightly packed cortex, which contains the majority of relatively immature proliferating thymocytes; and

• an inner medulla containing more mature cells, implying a differentiation gradient from cortex to medulla (Fig. 2.27).

Fig. 2.27 Thymus section showing the lobular organization

This section shows the two main areas of the thymus lobule – an outer cortex of immature cells (C) and an inner medulla of more mature cells (M). Hassall’s corpuscles (H) are found in the medulla. H&E stain. × 25.

(Courtesy of Dr A Stevens and Professor J Lowe.)

The main blood vessels that regulate cell traffic in the thymus are high endothelial venules (HEVs; see Fig. 2.29) at the corticomedullary junction of thymic lobules. It is through these veins that T cell progenitors formed in the fetal liver and bone marrow enter the epithelial anlage and migrate towards the cortex.

Fig. 2.29 Schematic structure of the thymus

A schematic representation of the cell types found in a fully developed thymic lobule. Subcapsular epithelial cells that produce IL-7 (nurse cells) sustain T lymphoblast proliferation in the outer cortex. Developing T cells interact with the cortical epithelial network where they are positively selected. Apoptotic cells are phagocytosed by macrophages present in the deep cortex and in the medulla. TCR⁺ thymocytes co-expressing CD4 and CD8 undergo the process of negative selection by interacting with a variety of antigen-presenting cells (APCs), such as dendritic cells, interdigitating cells, macrophages, and epithelial cells. T cells that have survived the selection processes are exported from the thymus via high endothelial venules (HEVs) and lymphatic vessels.

(From Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

In the cortex of the thymus the T cell progenitors undergo proliferation and differentiation processes that lead to the generation of mature T cells through a corticomedullary gradient of migration.

A network of epithelial cells throughout the lobules plays a role in the differentiation and selection processes from fetal liver and bone marrow-derived prethymic cells to mature T cells.

The mature T cells probably leave the thymus through the same PCVs, at the corticomedullary junction from which the T cell progenitors entered (Fig. 2.28).

Fig. 2.28 Cell migration to and within the thymus

T cell progenitors enter the thymic lobule through postcapillary venules (PCVs) at the corticomedullary junction. These cells are double negative 1 (DN1) for CD4 and CD8 expression but are also CD25^–, but CD44⁺. They move progressively towards the outer cortex and differentiate into DN2 (CD25⁺, CD44⁺) and DN3 cells (CD25⁺, CD44^lo). Thymocytes accumulate in the subcapsular region where they actively proliferate and differentiate into double positive (DP; CD4⁺, CD8⁺) cells. DP thymocytes reverse their polarity and move towards the medulla. In the course of this migration, thymocytes are selected and as single positive (SP; CD4⁺ or CD8⁺) cells ultimately leave the thymus, presumably via HEVs at the corticomedullary junction.

Three types of thymic epithelial cell have important roles in T cell production

At least three types of epithelial cell can be distinguished in the thymic lobules according to distribution, structure, function, and phenotype:

• the epithelial nurse cells are in the outer cortex;

• the cortical thymic epithelial cells (TECs) form an epithelial network; and

• the medullary TECs are mostly organized into clusters (Fig. 2.29).

These three types of epithelial cell have different roles for thymocyte proliferation, maturation, and selection:

• nurse cells in the outer cortex sustain the proliferation of progenitor T cells, mainly through cytokine production (e.g. IL-7);

• cortical TECs are responsible for the positive selection of maturing thymocytes, allowing survival of cells that recognize MHC class I and II molecules with associated peptides via TCRs of intermediate affinity; and

• medullary TECs display a large variety of organ-specific self peptides through transcription factors such as AIRE (autoimmune regulator).

Q. What is the significance of the presence of organ-specific self peptides in the thymus?

A. An individual needs to be tolerant of antigens that are expressed in other tissues, not just the thymus. By presenting a library of self molecules, the thymus can delete or tolerize lymphocytes that might otherwise react against self molecules once they had migrated into other tissues. Thus TECs, together with other APCs (interdigitating cells and macrophages), play a role in negative selection (i.e. the deletion of self-reactive T cells).

Hassall’s corpuscles (see Fig. 2.27) are found in the thymic medulla. Their function is unknown, but they appear to contain degenerating epithelial cells rich in high molecular weight cytokeratins.

The mammalian thymus involutes with age (Fig. 2.30). In humans, atrophy begins at puberty and continues throughout life. Thymic involution begins within the cortex and this region may disappear completely, whereas medullary remnants persist.

Fig. 2.30 Atrophic adult thymus

There is an involution of the thymus with replacement by adipose tissue (AdT). The cortex (C) is largely reduced and the less cellular medulla (M) is still apparent.

(Courtesy of Dr A Stevens and Professor J Lowe.)

Cortical atrophy is related to a sensitivity of the cortical thymocytes to corticosteroid, and all conditions associated with an acute increase in corticosteroids (e.g. pregnancy and stress) promote thymic atrophy.

It is conceivable that T cell generation within the thymus continues into adult life, albeit at a low rate. Evidence for de novo T cell production in the thymus (recent thymic emigrants) has been shown in humans over the age of 76 years.

Stem cell migration to the thymus initiates T cell development

The thymus develops from the endoderm of the third pharyngeal pouch as an epithelial rudiment that becomes seeded with blood-borne stem cells. Relatively few stem cells appear to be needed to give rise to the enormous repertoire of mature T cells with diverse antigen receptor specificities.

From experimental studies, migration of stem cells into the thymus is not a random process, but results from chemotactic signals periodically emitted from the thymic rudiment. β₂-microglobulin, a component of the MHC class I molecule, is one such putative chemoattractant.

In birds, stem cells enter the thymus in two or possibly three waves, but it is not clear that there are such waves in mammals.

Once in the thymus, the stem cells begin to differentiate into thymic lymphocytes (called thymocytes), under the influence of the epithelial microenvironment.

Whether or not the stem cells are ‘pre-T cells’ (i.e. are committed to becoming T cells before they arrive in the thymus) is controversial. Although the stem cells express CD7, substantial evidence exists that they are in fact multipotent. Granulocytes, APCs, NK cells, B cells, and myeloid cells have all been generated in vitro from hematopoietic precursors isolated from the thymus. This suggests that the prethymic bone marrow-derived cell entering the thymic rudiment is multipotent.

Notch1 receptor has proved to be essential for T cell development, and is involved in T versus B cell fate determination through interaction with thymic epithelial cells expressing Notch ligands. At this particular level Notch1 acts as a lineage specifier. Notch1 deficient bone marrow progenitors migrate from the bone marrow to the thymus but can no longer develop towards the T cell lineage. Since these progenitors are still at least bi-potential they develop into B cells instead.

Epithelial cells, macrophages, and bone marrow-derived IDCs, molecules rich in MHC class II, are important for the differentiation of T cells from this multipotent stem cell. For example, specialized epithelial cells in the peripheral areas of the cortex (the thymic nurse cells, see above) contain thymocytes within pockets in their cytoplasm. The nurse cells support lymphocyte proliferation by producing the cytokine IL-7.

The subcapsular region of the thymus is the only site where thymocyte proliferation occurs. Thymocytes develop into large, actively proliferating, self-renewing lymphoblasts, which generate the thymocyte population.

There are many more developing lymphocytes (85–90%) in the thymic cortex than in the medulla, and studies of function and cell surface markers have indicated that cortical thymocytes are less mature than medullary thymocytes. This reflects the fact that cortical cells migrate to, and mature in, the medulla.

Most mature T cells leave the thymus via HEVs at the corticomedullary junction, though other routes of exit may exist, including lymphatic vessels.

T cells change their phenotype during maturation

As with the development of granulocytes and monocytes, ‘differentiation’ markers of functional significance appear or are lost during the progression from stem cell to mature T cell.

Analyses of genes encoding αβ and γδ TCRs and other studies examining changes in surface membrane antigens suggest that there are multiple pathways of T cell differentiation in the thymus. It is not known whether these pathways are distinct, but it seems more likely that they diverge from a common pathway.

Only a small proportion (< 1%) of mature T lymphocytes express the γδ TCR. Most thymocytes differentiate into αβ TCR cells, which account for the majority (> 95%) of T lymphocytes in secondary lymphoid tissues and in the circulation.

Phenotypic analyses have shown sequential changes in surface membrane antigens during T cell maturation (Fig. 2.w1). The phenotypic variations can be simplified into a three-stage model.

Fig. 2.w1 Expression of human T cell markers during development

Terminal deoxynucleotidyl transferase (TdT) is an enzyme in thymic stem cells. It decreases in stage II and is lost altogether in the medulla. Several surface glycoproteins appear during differentiation. CD1 is present on stage II cortical thymocytes and is lost in the medulla. CD2 and CD7 (the pan-T marker) appear very early in differentiation and are maintained through to the mature T cell stage. CD5 appears at an early stage and persists on mature T cells. CD3 is expressed first in the cytoplasm in stage I cells (cyto), and then on the surface simultaneously with the T cell receptor (TCR). In most stage II cells, both surface CD3 and the αβ TCR are expressed at low density, but these markers are present at high density on stage III cells. CD4 and CD8 are co-expressed on stage II cells (double positives). One of these molecules is lost during differentiation into mature stage III cells (single positives).

Positive and negative selection of developing T cells takes place in the thymus

The processes involved in the education of T cells are shown in Figure 2.31, and self tolerance is discussed fully in Chapter 11. Positive selection ensures only TCRs with an intermediate affinity for self MHC develop further.

Fig. 2.31 T cell differentiation within the thymus

In this model, pre-thymic T cells are attracted to and enter the thymic rudiment at the corticomedullary junction. They reach the subcapsular region where they proliferate as large lymphoblasts, which give rise to a pool of cells entering the differentiation pathway. Many of these cells are associated with epithelial thymic nurse cells. Cells in this region first acquire CD8 and then CD4 at low density. They also rearrange their T cell receptor (TCR) genes and may express the products of these genes at low density on the cell surface. Maturing cells move deeper into the cortex and adhere to cortical epithelial cells. These epithelial cells are elongated and branched, and thus provide a large surface area for contact with thymocytes. The TCRs on the thymocytes are exposed to epithelial MHC molecules through these contacts. This leads to positive selection. Those cells that are not selected undergo apoptosis and are phagocytosed by macrophages. There is an increased expression of CD3, TCR, CD4, and CD8 during thymocyte migration from the subcapsular region to the deeper cortex. Those TCRs with self reactivity are now deleted through contact with autoantigens presented by medullary thymic epithelial cells, interdigitating cells, and macrophages at the corticomedullary junction – a process called negative selection. Following this stage, cells expressing either CD4 or CD8 appear and exit to the periphery via specialized vessels at the corticomedullary junction.

(Adapted from D Zucker-Franklin, CE Grossi, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

T cells:

• recognize antigenic peptides only when presented by self MHC molecules on APCs; and

• show ‘dual recognition’ of both the antigenic peptides and the polymorphic part of the MHC molecules.

Positive selection (the first stage of thymic education) ensures that only those TCRs with an intermediate affinity for self MHC are allowed to develop further. There is evidence that positive selection is mediated by TECs acting as APCs.

T cells displaying very high or very low receptor affinities for self MHC undergo apoptosis and die in the cortex. Apoptosis is a pre-programmed ‘suicide’, achieved by activating endogenous nucleases that cause DNA fragmentation.

T cells with TCRs that have intermediate affinities are rescued from apoptosis, survive, and continue along their pathway of maturation. A possible exception is provided by some T cells equipped with γδ receptors, which (like B cells) recognize native antigenic conformations with no need for APCs.

Negative selection ensures that only T cells that fail to recognize self antigen proceed in their development. Some of the positively selected T cells may have TCRs that recognize self components other than self MHC. These cells are deleted by a ‘negative selection’ process, which occurs:

• in the deeper cortex;

• at the corticomedullary junction; and

• in the medulla.

T cells interact with antigen presented by interdigitating cells, macrophages, and medullary TECs. The role of medullary TECs for negative selection has been emphasized recently by the finding that these cells express genes for virtually all tissue antigens in the body, and that these genes are activated by certain transcription factors (TF) to express these antigens (e.g. AIRE).

Only T cells that fail to recognize self antigen are allowed to proceed in their development. The rest undergo apoptosis and are destroyed. These, and all the other apoptotic cells generated in the thymus, are phagocytosed by (tingible body) macrophages (see Fig. 2.45) in the deep cortex.

Fig. 2.45 Germinal center macrophages

Immunostaining for cathepsin D shows several macrophages localized in the germinal center (GC) of a secondary follicle. These macrophages, which phagocytose apoptotic B cells, are called tingible body macrophages (TBM).

(Courtesy of Dr A Stevens and Professor J Lowe.)

T cells at this stage of maturation (CD4⁺ CD8⁺ TCR^lo) go on to express TCR at high density and lose either CD4 or CD8 to become ‘single positive’ mature T cells.

The separate subsets of CD4⁺ and CD8⁺ cells possess specialized homing receptors (e.g. CD44), and exit to the T cell areas of the peripheral (secondary) lymphoid tissues where they function as mature ‘helper’ and ‘cytotoxic’ T cells, respectively.

Q. Which subset of T cells functions as TH and which as TC cells?

A. CD4⁺ T cells function mainly as TH cells whereas CD8⁺ T cells are predominantly TC cells.

Less than 5% of thymocytes leave the thymus as mature T cells. The rest die as the result of:

• selection processes; or

• failure to undergo productive rearrangements of antigen receptor genes.

Adhesion of maturing thymocytes to epithelial and accessory cells is crucial for T cell development

Adhesion of maturing thymocytes to epithelial and other accessory cells is mediated by the interaction of complementary adhesion molecules, such as:

• CD2 with LFA-3 (CD58); and

• LFA-1 (CD11a, CD18) with ICAM-1 (CD54).

These interactions induce the production of the cytokines IL-1, IL-3, IL-6, IL-7, and GM-CSF, which are required for T cell proliferation and maturation in the thymus.

Early thymocytes also express receptors for IL-2, which together with IL-7 sustains cell proliferation.

Negative selection may also occur outside the thymus in peripheral lymphoid tissues

Not all self-reactive T cells are eliminated during intrathymic development, probably because not all self antigens can be presented in the thymus. The thymic epithelial barrier that surrounds blood vessels may also limit access of some circulating antigens.

Given the survival of some self-reacting T cells, a separate mechanism is required to prevent them attacking the body. Experiments with transgenic mice have suggested that peripheral inactivation of self-reactive T cells (peripheral tolerance, see Chapter 19) could occur via several mechanisms as follows:

• downregulation of the TCR and CD8 (in cytotoxic cells) so that the cells are unable to interact with target autoantigens;

• anergy, due to the lack of crucial co-stimulatory signals provided by the target cells, followed by induction of apoptosis after interaction with autoantigen;

• regulatory T cells (Tregs).

Regulatory T cells are involved in peripheral tolerance

Tregs have been the subject of intensive research over the past few years, especially in the areas of autoimmunity and vaccine development.

In addition to NKT cells and γδ T cells regulating immune responses, there is now substantial evidence that separate CD4⁺ subsets also have this function. The general consensus is that there are two main types of Treg – naturally occurring, and inducible following activation by specific antigen.

Naturally occurring Tregs:

• constitutively express CD25 (the α chain of the receptor for IL-2);

• constitute about 5–10% of the peripheral CD4⁺ T cells;

• express the unique transcription factor FoxP3;

• constitutively express the marker CTLA4;

• do not proliferate in response to antigenic challenge;

• are thought to produce their suppressive effects through cell contact (e.g. with APCs, TH1 or TH2 cells).

Antigen-induced Tregs:

• also express CD25;

• can develop from CD25^–, CD4⁺ T cells;

• are believed to exert their suppressive effects through IL-10.

There is some evidence for extrathymic development of T cells

The vast majority of T cells require a functioning thymus for differentiation, but small numbers of cells carrying T cell markers that are often oligoclonal in nature have been found in athymic (‘nude’) mice. Although the possibility that these mice possess thymic remnants cannot be ruled out, there is accumulating evidence to suggest that bone marrow precursors can home to mucosal epithelia and mature without the need for a thymus to form functional T cells with γδ TCRs and probably also T cells with αβ TCRs.

The importance of extrathymic development in animals that are euthymic (i.e. that have a normal thymus) is at present unclear.

B cells develop mainly in the fetal liver and bone marrow

Unlike birds, which have a discrete organ for the generation of B cells (the bursa of Fabricius), in mammals B cells develop directly from lymphoid stem cells in the hematopoietic tissue of the fetal liver (Fig. 2.32). This occurs at 8–9 weeks of gestation in humans, and by about 14 days in the mouse. Later, the site of B cell production moves from the liver to the bone marrow, where it continues through adult life. This fetal liver-bone marrow migration of stem cells is also true for cells of other hematopoietic lineages such as erythrocytes, granulocytes, monocytes, and platelets. The B cell lineage specifier is the transcription factor PAX5 which is expressed initially in B cell precursors but then at all stages of B cell development

Fig. 2.32 Hemopoiesis in fetal liver

Section of human fetal liver showing islands of hemopoiesis (H). Hematopoietic stem cells are found in the sinusoidal spaces (S) between plates of liver cells (L).

(Courtesy of Dr A Stevens and Professor J Lowe.)

B cell progenitors are also present in the omental tissue of murine and human fetuses and are the precursors of a self-replicating B cell subset, the B-1 cells (see above).

B cell production in the bone marrow does not occur in distinct domains

B cell progenitors in the bone marrow are seen adjacent to the endosteum of the bone lamellae (Fig. 2.33). Each B cell progenitor, at the stage of immunoglobulin gene rearrangement may produce up to 64 progeny. The progeny migrate towards the center of each cavity of the spongy bone and reach the lumen of a venous sinusoid (Fig. 2.34).

Fig. 2.33 Schematic organization of B cell development in the bone marrow

The earliest B cell progenitors are found close to the endosteum (1) where they interact with stromal reticular cells (2). The stromal reticular cells prompt precursor B cell proliferation and maturation (3 and 4). During these processes, selection occurs, which implies B cell apoptosis and phagocytosis of apoptotic cells by macrophages (5). B cells that have survived selection mature further and interact with adventitial reticular cells (6), which may facilitate their ingress (7) into bone marrow sinusoids (8) and finally the central venous sinuses, from which they enter the general circulation. In this model, maturation and selection events follow a gradient from the periphery of the bone marrow tissue contained in the bony spaces towards the center.

Fig. 2.34 Bone marrow

(1) Low-power scanning electron micrograph showing the architecture of bone and its relationship to bone marrow. Within the cavities of spongy bone between the bony trabeculae, B cell lymphopoiesis takes place, with maturation occurring in a radial direction towards the center (from the endosteum to the central venous sinus). (2) The biopsy below shows hematopoietic bone marrow (HM) in the spaces between the bony trabeculae (lamellae) (T). Some of the space is also occupied by adipocytes (AdC).

(Courtesy of Dr A Stevens and Professor J Lowe.)

In the bone marrow, B cells mature in close association with stromal reticular cells, which are found both adjacent to the endosteum and in close association with the central sinus, where they are termed adventitial reticular cells.

Where the B cells differentiate, the reticular cells have mixed phenotypic features with some similarities to fibroblasts, endothelial cells, and myofibroblasts. The reticular cells produce type IV collagen, laminin and the smooth muscle form of actin. Experiments in vitro have shown that reticular cells sustain B cell differentiation, possibly by producing the cytokine IL-7.

Adventitial reticular cells may be important for the release of mature B cells into the central sinus.

B cells are subject to selection processes

Most B cells (> 75%) maturing in the bone marrow do not reach the circulation, but (like thymocytes) undergo a process of programmed cell death (apoptosis) and are phagocytosed by bone marrow macrophages.

Q. By analogy with T cell development, infer what determines whether a B cell will die during development in the bone marrow

A. B cells with a non-productive rearrangement of the immunoglobulin genes do not survive and many self-reactive B cells are also eliminated through negative selection.

B cell–stromal cell interactions enhance the survival of developing B cells and mediate a form of selection that rescues a minority of B cells with productive rearrangements of their immunoglobulin genes from programmed cell death.

Many self-reactive B cells are also eliminated through negative selection in the bone marrow.

From kinetic data, it is estimated that about 5 × 10⁷ murine B cells are produced each day. As the mouse spleen contains approximately 7.5 × 10⁷ B cells, a large proportion of B cells must die, probably at the pre-B cell stage, because of non-productive rearrangements of receptor genes or if they express self-reactive antibodies and are not rescued.

Immunoglobulins are the definitive B cell lineage markers

Lymphoid stem cells expressing terminal deoxynucleotidyl transferase (TdT) proliferate, differentiate, and undergo immunoglobulin gene rearrangements to emerge as pre-B cells (see Chapters 3 and 9). A sequence of immunoglobulin gene rearrangements and phenotypic changes takes place during B cell ontogeny (see Chapter 8), similar to that described above for T cells.

Heavy-chain gene rearrangements occur in B cell progenitors and represent the earliest indication of B lineage commitment. This is followed by light-chain gene rearrangements, which occur at later pre-B cell stages.

Once a B cell has synthesized light chains, it becomes committed to the antigen-binding specificity of its surface IgM (sIgM) antigen receptor.

One B cell can therefore make only one specific antibody – a central tenet of the clonal selection theory for antibody production.

Surface immunoglobulin-associated molecules Igα and Igβ (CD79a and b) are present by the pre-B cell stage of development.

B cells migrate to and function in the secondary lymphoid tissues

Early B cell immigrants into fetal lymph nodes (17 weeks in humans) are surface IgM⁺ and are B-1 cells. CD5⁺ B cell precursors are found in the fetal omentum.

Some CD5⁺ B cells are also found in the marginal zone of the spleen and mantle zone of secondary follicles in adult lymph nodes (see Fig. 2.42).

Fig. 2.42 Structure of the secondary follicle

A large germinal center (GC) is surrounded by the mantle zone (Mn).

Following antigenic stimulation, mature B cells can develop into memory cells or antibody-forming cells (AFCs).

Surface immunoglobulins (sIg) are usually lost from plasma cells (the terminally differentiated form of an AFC) because their function as a receptor is no longer required. Like any other terminally differentiated hematopoietic cell, the plasma cell has a limited life span, and eventually undergoes apoptosis.

Lymphoid organs

The generation of lymphocytes in primary lymphoid organs is followed by their migration into peripheral secondary tissues, which comprise:

• well-organized encapsulated organs, the spleen and lymph nodes (systemic lymphoid organs); and

• non-encapsulated accumulations of lymphoid tissue.

Lymphoid tissue found in association with mucosal surfaces is called mucosa-associated lymphoid tissue (MALT).

Lymphoid organs and tissues protect different body sites

The systemic lymphoid organs and the mucosal system have different functions in immunity:

• the spleen is responsive to blood-borne antigens, and patients who have had their spleen removed are much more susceptible to pathogens that reach the blood stream;

• the lymph nodes protect the body from antigens that come from skin or from internal surfaces and are transported via the lymphatic vessels;

• the MALT protects mucosal surfaces.

Responses to antigens encountered via the spleen and lymph nodes result in the secretion of antibodies into the circulation and local cell-mediated responses.

Being the major port of entry into the body for pathogens, the mucosa-associated lymphoid tissue is the site of first encounter (priming) of immune cells with antigens entering via mucosal surfaces, and lymphoid tissues are associated with surfaces lining:

• the intestinal tract – gut-associated lymphoid tissue (GALT);

• the respiratory tract – bronchus-associated lymphoid tissue (BALT); and

• the genitourinary tract.

The major effector mechanism at mucosal surfaces is secretory IgA antibody (sIgA), which is actively transported via the mucosal epithelial cells to the lumen of the tracts.

Q. More than 50% of the body’s lymphoid tissue is in the MALT and IgA is the most abundant immunoglobulin in the body. What reason could explain this preponderance of immune defenses in mucosal tissues?

A. The mucosal surfaces present a large surface area, vulnerable to infectious agents. Most infectious agents enter the body by infecting and/or crossing mucosal surfaces.

The spleen is made up of white pulp, red pulp, and a marginal zone

The spleen lies at the upper left quadrant of the abdomen, behind the stomach and close to the diaphragm. The adult spleen is around 13 × 8 cm in size and weighs approximately 180–250 g.

The outer layer of the spleen consists of a capsule of collagenous bundles of fibers, which enter the parenchyma of the organ as short trabeculae. These, together with a reticular framework, support two main types of splenic tissue:

• the white pulp; and

• the red pulp.

A third compartment, the marginal zone, is located at the outer limit of the white pulp.

The white pulp consists of lymphoid tissue

The white pulp of the spleen consists of lymphoid tissue, the bulk of which is arranged around a central arteriole to form the periarteriolar lymphoid sheaths (PALS; Fig. 2.35). PALS are composed of T and B cell areas:

• the T cells are found around the central arteriole;

• the B cells may be organized into either primary ‘unstimulated’ follicles (aggregates of virgin B cells) or secondary ‘stimulated’ follicles (which possess a germinal center with memory cells).

Fig. 2.35 White pulp of the spleen

Spleen section showing a white pulp lymphoid aggregate. A secondary lymphoid follicle, with germinal center (GC) and mantle (Mn), is surrounded by the marginal zone (MZ) and red pulp (RP). Adjacent to the follicle, an arteriole (A) is surrounded by the periarteriolar lymphoid sheath (PALS) consisting mainly of T cells. Note that the marginal zone is present only at one side of the secondary follicle.

(Courtesy of Professor I Maclennan.)

The germinal centers also contain follicular dendritic cells (FDCs) and phagocytic macrophages. Macrophages and the FDCs present antigen to B cells in the spleen.

B cells and other lymphocytes are free to leave and enter the PALS via branches of the central arterioles, which enter a system of blood vessels in the marginal zone (see below). Some lymphocytes, especially maturing plasmablasts, can pass across the marginal zone via bridges into the red pulp.

The red pulp consists of venous sinuses and cellular cords

The venous sinuses and cellular cords of the red pulp contain:

• resident macrophages;

• numerous plasma cells.

In addition to immunological functions, the spleen serves as a reservoir for platelets, erythrocytes, and granulocytes. Aged platelets and erythrocytes are destroyed in the red pulp in a process referred to as ‘hemocatheresis’.

The functions of the spleen are made possible by its vascular organization (Fig. 2.36). Central arteries surrounded by PALS end with arterial capillaries, which open freely into the red pulp cords. Circulating cells can therefore reach these cords and become trapped. Aged platelets and erythrocytes are recognized and phagocytosed by macrophages.

Fig. 2.36 Vascular organization of the spleen

The splenic artery branches to form trabecular arteries, which give rise to central arteries surrounded by periarteriolar lymphoid sheaths (PALS), which are the T cell areas of the white pulp. Leaving the PALS, the central arteries continue as penicillary arteries and sheathed capillaries, which open in the splenic cords of the red pulp. From the red pulp (where hemocatheresis takes place) the blood percolates through the wall of the venous sinuses. The central arterioles surrounded by PALS give collateral branches that reach a series of sinuses in the marginal zone. Most of the blood from the marginal sinuses enters the red pulp cords and then drains into the venous sinuses, but a proportion passes directly into the sinuses to form a closed circulation.

(Courtesy of Dr A Stevens and Professor J Lowe.)

Blood cells that are not ingested and destroyed can re-enter the blood circulation by squeezing through holes in the discontinuous endothelial wall of the venous sinuses, through which plasma flows freely.

The marginal zone contains B cells, macrophages, and dendritic cells

The marginal zone surrounds the white pulp and exhibits two major features, namely:

• a characteristic vascular organization; and

• unique subsets of resident cells (B cells, macrophages, and dendritic cells).

The blood vessels of the marginal zone form a system of communicating sinuses, which receive blood from branches of the central artery (see Fig. 2.36).

Most of the blood from the marginal sinuses enters the red pulp cords and then drains into the venous sinuses, but a small proportion passes directly into the venous sinuses to form a closed circulation.

Cells residing in the marginal zone comprise:

• various types of APC – metallophilic macrophages, marginal zone macrophages, dendritic cells;

• a subset of B cells with distinctive phenotype and function – they express high levels of IgM and low or absent IgD and in humans are long-lived recirculating cells;

• some B-1 cells.

Q. In humans, the marginal zone does not develop fully until 2 years of age. What is the functional consequence of this delay in development of the marginal zone?

A. Marginal zone B cells and B-1 cells respond strongly to thymus-independent antigens, including capsular polysaccharides of bacteria, and the main function of the marginal zone is to mount immune responses to bacteria that have reached the circulation (e.g. streptococci). Infants therefore have a reduced ability to respond to blood-borne infections with certain (encapsulated) bacteria.

Lymph nodes filter antigens from the interstitial tissue fluid and lymph

The lymph nodes form part of a network that filters antigens from the interstitial tissue fluid and lymph during its passage from the periphery to the thoracic duct and the other major collecting ducts (Fig. 2.37).

Fig. 2.37 The lymphatic system

Lymph nodes are found at junctions of lymphatic vessels and form a network that drains and filters interstitial fluid from the tissue spaces. They are either subcutaneous or visceral, the latter draining the deep tissues and internal organs of the body. The lymph eventually reaches the thoracic duct, which opens into the left subclavian vein and thus back into the circulation.

Lymph nodes frequently occur at the branches of the lymphatic vessels. Clusters of lymph nodes are strategically placed in areas that drain various superficial and deep regions of the body, such as the:

Lymph nodes protect the skin (superficial subcutaneous nodes) and mucosal surfaces of the respiratory, digestive, and genitourinary tracts (visceral or deep nodes).

Human lymph nodes are 2–10 mm in diameter, are round or kidney shaped, and have an indentation called the hilus where blood vessels enter and leave the node.

Lymph arrives at the lymph node via several afferent lymphatic vessels, and leaves the node through a single efferent lymphatic vessel at the hilus.

Lymph nodes consist of B and T cell areas and a medulla

A typical lymph node is surrounded by a collagenous capsule. Radial trabeculae, together with reticular fibers, support the various cellular components. The lymph node consists of:

• a B cell area (cortex);

• a T cell area (paracortex); and

• a central medulla, consisting of cellular cords containing T cells, B cells, abundant plasma cells, and macrophages (Figs 2.38–2.40).

Fig. 2.38 Lymph node section

The lymph node is surrounded by a connective tissue capsule and is organized into three main areas: the cortex (C), which is the B cell area; the paracortex (P), which is the T cell area; and the medulla (M), which contains cords of lymphoid tissue (T and B cell areas rich in plasma cells and macrophages). H&E stain. × 10.

(Adapted from Zucker-Franklin D, Grossi CE, eds. Atlas of blood cells: function and pathology, 3rd edn. Milan: Edi Ermes; 2003.)

Fig. 2.39 Histological structure of the lymph node

Cortex (C), paracortex (P), and medulla (M) are shown. The section has been stained to show the localization of T cells. They are most abundant in the paracortex, but a few are found in the germinal center (GC) of the secondary lymphoid follicle, in the cortex, and in the medullary cords (MC).

(Courtesy of Dr A Stevens and Professor J Lowe.)

Fig. 2.40 Schematic structure of the lymph node

Beneath the collagenous capsule is the subcapsular sinus, which is lined by endothelial and phagocytic cells. Lymphocytes and antigens from surrounding tissue spaces or adjacent nodes pass into the sinus via the afferent lymphatics. The cortex is mainly a B cell area. B cells are organized into primary or, more commonly, secondary follicles – that is, with a germinal center. The paracortex contains mainly T cells. Each lymph node has its own arterial and venous supply. Lymphocytes enter the node from the circulation through the highly specialized high endothelial venules (HEVs) in the paracortex. The medulla contains both T and B cells in addition to most of the lymph node plasma cells organized into cords of lymphoid tissue. Lymphocytes leave the node through the efferent lymphatic vessel.

The paracortex contains many APCs (interdigitating cells), which express high levels of MHC class II surface molecules. These are cells migrating from the skin (Langerhans’ cells) or from mucosae (dendritic cells), which transport processed antigens into the lymph nodes from the external and internal surfaces of the body (Fig. 2.41). The bulk of the lymphoid tissue is found in the cortex and paracortex.

Fig. 2.41 Intedigitating cells in the lymph node paracortex

Interdigitating dendritic cells (IDC; stained dark brown) form contacts with each other and with paracortical T cells (see also Fig. 2.16).

(Courtesy of Dr A Stevens and Professor J Lowe.)

The paracortex contains specialized postcapillary vessels – high endothelial venules (HEVs) – which allow the traffic of lymphocytes out of the circulation into the lymph node (see ‘Lymphocyte traffic’ below).

The medulla is organized into cords separated by lymph (medullary) sinuses, which drain into a terminal sinus – the origin of the efferent lymphatic vessel (see Fig. 2.40).

Scavenger phagocytic cells are arranged along the lymph sinuses, especially in the medulla. As the lymph passes across the nodes from the afferent to the efferent lymphatic vessels, particulate antigens are removed by the phagocytic cells and transported into the lymphoid tissue of the lymph node.

The cortex contains aggregates of B cells in the form of primary or secondary follicles.

B cells are also found in the subcapsular region, adjacent to the marginal sinus. It is possible that these cells are similar to the splenic marginal zone B cells that intercept incoming pathogens primarily by mounting a rapid, IgM-based, T-independent response.

T cells are found mainly in the paracortex. Therefore, if an area of skin or mucosa is challenged by a T-dependent antigen, the lymph nodes draining that particular area show active T cell proliferation in the paracortex.

Q. How is antigen transported from the skin to the paracortex of the regional lymph nodes?

A. On Langerhans’ cells/veiled cells via the afferent lymph.

Further evidence for this localization of T cells in the paracortex comes from patients with congenital thymic aplasia (DiGeorge syndrome), who have fewer T cells in the paracortex than normal. A similar feature is found in neonatally thymectomized or congenitally athymic (‘nude’) mice or rats.

Secondary follicles are made up of a germinal center and a mantle zone

Germinal centers in secondary follicles are seen in antigen-stimulated lymph nodes. These are similar to the germinal centers seen in the B cell areas of the splenic white pulp and of MALT.

Germinal centers are surrounded by a mantle zone of lymphocytes (Fig. 2.42). Mantle zone B cells (Fig. 2.43) co-express surface IgM, IgD, and CD44. This is taken as evidence that they are naive, actively recirculating B cells.