Normal Anatomy and Function of Lymph Nodes and Spleen

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NORMAL ANATOMY 1 AND FUNCTION OF LYMPH NODES AND SPLEEN

The immune system is dedicated to the rec- and functional unit of the lymph node paren- ognition of foreign antigens and defense from chyma, termed the “lymphoid lobule,” has been invading microorganisms. It consists of primary proposed as an addition, although its relevance has (central) and secondary (peripheral) lymphoid not been widely recognized (5,15,26,54). In this tissues. The primary lymphoid tissues are the model, each lymph node contains one or several bone marrow and thymus gland, which contain functional units, or lobules, dependent on the size the precursors of the lymphoid cells and support of the lymph node. A lobule represents a portion of initial antigen-independent differentiation from lymph node parenchyma between two transverse the immature to mature stage. The secondary lym- sinuses, centered under one afferent lymphatic phoid tissues include lymph nodes, spleen, and vessel and including the superficial cortex with sites of mucosa-associated lymphoid tissue (MALT) including tonsils, appendix, and Peyer patches of the gastrointestinal tract. Secondary lymphoid tissues are the sites of antigen-dependent proliferation and differentiation of lymphoid cells. LYMPH NODE

Gross Anatomy The lymph nodes are small, ovoid or bean- shaped encapsulated lymphoid tissues, generally less than 1 cm in greatest dimension. The human body contains about 450 lymph nodes. They are located adjacent to lymphatic vessels, particularly in areas where these vessels converge. Lymph nodes are abundant in neck, axilla, medi- astinum, retroperitoneum, and inguinal regions, and generally in those areas draining organs/tissues in contact to the external environment (fig. 1-1). They are connected to the blood circulation via an artery and vein, and to the lymphatic system through afferent and efferent lymphatic vessels. The lymph node is the site where circulating naïve lymphocytes encounter antigens and antigen-presenting cells (APC), which travel from peripheral tissues through the lymph. This system provides effective immune surveillance for the screening of foreign pathogens. Microscopic Anatomy Figure 1-1 Traditionally, lymph node anatomy is organized LYMPH NODES AND EXTRANODAL LYMPHOID TISSUES into discrete compartments: cortex, paracortex, Schematic showing sites of lymph node groups and medulla, and sinuses. More recently, an anatomic extranodal sites of lymphoid tissue throughout the body.

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Figure 1-2 LYMPH NODE Schematic diagram of a mid-sagittal section of a lymph node containing three lymphoid “lobules.” Each lobule is centered under its own afferent lymphatic vessel. The follicles and interfollicular cortex of these lobules constitute the superficial cortex, the deep cortex constitutes the paracortex, and the medullary cords and medullary sinuses constitute the medulla. Left lobule: Arterioles (red) and venules (blue) converge in the medullary cords. Arterioles arborize in the paracortical cords and interfollicular cortex and give rise to capillary beds (purple). Capillaries are present in the follicles but are less dense than in the other areas (omitted from the medullary cords shown here for clarity). Capillaries empty into high endothelial venules. Center lobule: This lobule is shown with the reticular meshwork superimposed on the vasculature. The center lobule is separated from the left lobule by a transverse sinus. Right lobule: The lobule is shown as it appears in histologic sections. Densely packed lymphoid cells fill the lobular reticular meshwork. Five cortical follicles are represented. The paracortex and medulla constitute the remainder of the lobule. (Fig. 2 from Willard-Mack C. Normal structure, function and histology of lymph nodes. Toxicol Pathol 2006;34:411.) lymphoid follicles, paracortex, and medulla (fig. Circulating naïve B cells enter the lymph 1-2). As each afferent lymphatic vessel collects node, are activated in the paracortex by CD4-pos- from a different drainage field, each lobule is itive T cells and dendritic cells, and move into potentially exposed to a different set of antigens, the primary follicle (34,40). Once in the primary an arrangement that may explain why areas follicle, a subset of these activated B cells begins within a lymph node may show different levels to rapidly proliferate to form a germinal center of immunologic activity (fig. 1-3). (GC). The GCs are surrounded by a darker corona (mantle zone) of displaced IgM- and IgD-posi- Lymph Node Cortex tive activated B cells, establishing the structure The major component of the lymph node cortex known as a secondary lymphoid follicle (21). is the B-cell compartment, basically represented by Secondary Follicles. Secondary follicles are primary and secondary lymphoid follicles. defined by the presence of GCs, the main sites Primary Follicles. Primary follicles are round of antibody affinity maturation and where aggregates of activated small B cells within plasma cells and a subset of memory B cells are a small network of follicular dendritic cells generated. The GC reaction results in selective (FDCs). These cells express immunoglobulin survival and expansion of B-cell clones with a (Ig)M, IgD, CD23, and CD38, and are negative high affinity for antigens, resulting in a substan- for CD10 and CD27. tially more effective immune response.

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Figure 1-3 LYMPH NODE Left: A section of a lymph node shows the prominent cortex and medulla. Follicular hyperplasia is seen. The cortex has numerous secondary lymphoid follicles with germinal centers of different sizes, including some forming geographic configurations (top of the lymph node). Right: Five secondary follicles are present in region A and mainly primary follicles in region B. These different regions of the same lymph node represent two different lobules collecting lymph from different drainage fields, and thus exposed to a different set of antigens.

Germinal Centers. GCs are dynamic struc- able regions of the Ig heavy and light chain tures. A few days after exposure to an antigen, genes, resulting in a diversity of B-cell receptors a GC develops in the center of a primary follicle, with varying affinities for antigen. The light reaches its maximum size within approximately zone is where the B cells are selected to generate 2 weeks, and thereafter slowly involutes within plasma cells and memory B cells based on the several weeks as antigen levels decrease. The affinity for antigen of their mutated surface Ig kinetics of GC formation and involution are (i.e., B-cell receptor). variable and depend on the nature of the antigen The dark and light zones have a unique dis- and the ability of the immune system to clear the tribution within the GC. The dark zone usually antigen. For some viral or other types of chronic lies nearest the paracortex and the light zone infection, GC reactions may persist longer, up is often polarized toward the site of antigen to several months. The GC reaction ends with entry, the subcapsular sinus (which receives involution of the GC and dissolution of the the afferent lymphatic drainage) in the case follicle. Follicular dissolution can adopt several of lymph nodes, or the mucosal surface in the histologic patterns (23). The histologic pattern case of tonsils, appendix, or Peyer patches of of follicular dissolution is, in part, related to the the ileum (37). The organization of the GC into nature and duration of antigenic stimulation. dark and light zones depends on the differen- The most conspicuous morphologic feature of tial expression of chemokines and chemokine the GC is the presence of two distinct compart- receptors. Expression of the chemokine receptor ments, the dark and light zones (fig. 1-4) (7). The CXCR4 by GC B cells is tightly controlled and is dark zone consists almost entirely of centroblasts a major factor controlling cell position within (mitotically active B cells) and few FDCs, thus the dark versus light zone (fig. 1-5). CXCL12 appearing “dark” by light microscopy. By con- (previously known as SDF1) is the ligand of trast, the light zone is occupied by large and small CXCR4 and is more abundant in the dark zone centrocytes (nondividing B cells) interspersed where CXCL12 is produced locally by stromal among a rich network of FDCs, which imparts cells (1). CXCL13, the ligand for CXCR5, is its “lighter” appearance by light microscopy. more abundant in the light zone, where it is The light zone also contains naïve IgD-positive produced by FDCs. Alternating upregulation B cells, in transit through the GC, and T cells, and downregulation of CXCR4 and CXCR5 most of which are CD4-positive. Tingible body expression, as well as MYC expression by GC macrophages, which phagocytose apoptotic B B cells, promotes the cycling of cells back and cells, are also found throughout the GC (31). forth between the dark and light zones (1,10). In the dark zone, B-cell proliferation occurs Recent live-imaging studies have shown that GC along with somatic hypermutation of the vari- B cells move bidirectionally between the two

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Figure 1-4 SECONDARY LYMPHOID FOLLICLE Upper left: A secondary lymphoid follicle with a reactive germinal center is surrounded by the mantle zone. The germinal center shows distinct light (top) and dark (bottom) zones. The mantle zone is thicker in the subcapsular aspect (top) of the lymphoid follicle and thinner in the opposite site. Lower left: Another secondary lymphoid follicle with a reactive germinal center has polarized light and dark zones. Upper right: the light zone is mainly composed of centrocytes (small cleaved cells) (yellow arrows). Lower right: the dark zone is mainly composed of centroblasts (large noncleaved cells) (white arrows).

GC zones; B cells also reenter the dark zone for imparting a monocytoid appearance, and are additional rounds of proliferation and somatic commonly admixed with neutrophils (fig. 1-6). hypermutation (10,18,45). Cellular Elements of Germinal Centers Marginal Zone. The lymph nodes occasionally exhibit a well-developed marginal zone. The B Cells. Most cells in the GC are activated B marginal zone is a distinct pale-appearing corona cells that consist of small and large centrocytes composed of small cells with slightly irregular (cleaved cells) and small and large centroblasts nuclei and clear cytoplasm surrounding mantle (noncleaved cells). A putative GC B-founder cell zones. In particular, lymph nodes located in the (known as a pro-GC cell) has been identified and mesenteric region often show a marginal zone. characterized in human tonsils (29). The marginal zone is usually more prominent in Centroblasts are medium to large B cells with the spleen (see below) and in MALT (e.g., Peyer an oval nucleus and open chromatin, contain- patches) than in lymph nodes. In some reactive ing one to three small nucleoli often opposed to and neoplastic conditions, marginal zones are the nuclear membrane and a rim of basophilic expanded. A related finding is monocytoid cytoplasm. Centrocytes are small to large B cells B-cell hyperplasia, which usually fills and dis- with irregular to cleaved nuclei, dense nuclear tends nearby sinuses but can spill into marginal chromatin, inconspicuous nucleoli, and scant zones. Monocytoid B cells are of intermediate cytoplasm (fig. 1-4). Large centrocytes are gen- to large size, have abundant pale cytoplasm erally smaller than centroblasts.

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The GC B cells have the following immuno- concentrated in the light zone. The cell of origin phenotype: CD38(+hi), CD20(+hi), CD27(+), of the FDC has not been established definitively, and IgD(-). GC B cells also express BCL6, CD10, but FDCs are not derived from hematopoietic LIM domain only 2 (LMO2), activation-induced cytidine deaminase (AID), and telomerase (20). CXCR4 expression delineates a homogeneous GC B-cell subpopulation that corresponds to centroblasts (8). GC B cells are characteristical- ly negative for BCL2 and other antiapoptotic family members. The characteristic immunophenotypic profile of reactive GCs as detected by immunohistochemical studies is illustrated in figures 1-7 and 1-8. Gene expression studies have shown differences between centrocytes and centroblasts (8,52). Centroblasts upregulate genes involved in cell proliferation and mitosis whereas centrocytes upregulate genes related to selection and affinity maturation events including genes related to the NF-kB, B-cell receptor, and CD40 signaling pathways. Centrocytes that fail to be selected are enriched in the GC light zone where they eventually undergo apoptosis, as suggested by enhanced expression of proapoptotic genes Figure 1-5 in this zone. Follicular Dendritic Cells. FDCs are stromal GERMINAL CENTER cells that form a network of processes in primary A simplified schematic diagram shows the cellular composition of the germinal center. The centroblasts (large follicles and GCs. FDCs have the ability to capture round blue cells) and centrocytes (small cleaved blue cells) large amounts of antigen in the form of immune are distributed in the dark and light zones, respectively. The complexes in highly ordered units designated as centroblasts express CXCR4, a receptor for CXCL12 that iccosomes (9). In GCs, FDCs are more densely is secreted by the local stromal cells of the dark zone. The centrocytes express CXCR5, a receptor for CXCL13 that is secreted by follicular dendritic cells of the light zone.

Figure 1-6 SECONDARY LYMPHOID FOLLICLE Left: Monocytoid B cells are close to a secondary lymphoid follicle. At low-power magnifi cation, the cluster of mono cytoid B cells is paler than the reactive germinal center. Right: High-power magnifica tion of monocytoid B cells. These cells are intermediate to large, with round to slightly irregular nuclei and a moderate amount of clear cytoplasm. Neutrophils are frequently admixed with the monocytoid B cells.

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Figure 1-7 TYPICAL IMMUNOPHENOTYPE OF SECONDARY LYMPHOID FOLLICLES The germinal centers of the B cells are positive for CD20 and negative for BCL2. CD3 highlights scattered germinal center T cells. The characteristic high proliferation rate of the germinal center cells is easily demonstrated using a marker of cell proliferation, such as Ki-67.

Figure 1-8 TYPICAL IMMUNOPHENOTYPE OF SECONDARY LYMPHOID FOLLICLES The germinal center B cells are positive for BCL6 and CD10. CD21 is a follicular dendritic cell-associated antigen and im munohistochemistry highlights meshworks of follicular dendritic cells inside the lymphoid follicles. The programmed cell death protein 1 (PD1[CD279]) is expressed by a subset of germinal center T cells (T-follicular helper cells).

precursors and most likely develop from local cell adhesion molecule 1 (VCAM-1), and inter- resident mesenchymal cells in the GC. cellular adhesion molecule 1 (ICAM-1) (30). FDCs are recognized morphologically by the The maintenance of the GC structure and GC presence of double oval to rectangular-shaped B cells is dependent on the presence of FDCs. nuclei with vesicular chromatin and small nu- FDCs support the migration and positioning of cleoli. FDCs express Fc receptors such as FcgRIIb GC cells by secreting chemokines and survival (CD32); they also express FcgRII (CD23) and factors. CXCL13 is perhaps the most important complement receptors such as CR1 (CD35), CR2 chemokine as it is the major chemoattractant (CD21) (fig. 1-8), CR3 (CD11b/CD18), vascular for B cells and follicular T cells. Survival factors

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Figure 1-9 TINGIBLE BODY MACROPHAGES Left: The presence of tingible body macrophages is a char- acter istic feature of reactive germinal centers. Right: High magnification showing macrophages with phago cytized lympho cytes and cellular debris (tingible bodies) inside their cytoplasm.

for GC cells include IL-6, BAFF (B-cell activating marrow-derived dendritic cells. Tingible body factor), and hedgehog ligands (44). macrophages eliminate apoptotic B cells inside T Cells. T cells represent a minor (5 to 20 GCs. Defects in the internalization of apoptotic percent) population in GCs but their function cells by macrophages have been reported in is essential for GC maintenance (19). GC T patients with systemic lupus erythematosus (4). cells are heterogeneous. One population is The clearance of apoptotic cells by macrophages positive for CD40L (CD154); has a T-helper may be important to avoid immune responses to (Th) immunophenotype; is positive for CD4, nuclear antigens exposed during the process of CXCR5 (required for follicular homing), ICOS, apoptosis. GC-resident dendritic cells were first PD1 (CD279) (fig.1-7), IL-21, and CD57; and is identified in human tonsils by immunohisto- characterized by the secretion of IL-4. These cells chemistry, however, the function of these cells are designated as follicular helper T cells (27,46). in the GC is not yet elucidated (16). IL-21 promotes T- and B-cell survival. Other T-cell Germinal Center-Associated Events populations residing in the GC include CD8-positive T cells, Th17 cells, and FOXP3-positive The GC is a highly specialized and regulated regulatory cells, all of which play functional compartment where many cellular and molec- roles in regulating the GC reaction. ular events occur that result in the production GC T cells express BCL6, which is essential for of high-affinity antibody-secreting plasma cells CXCR5 expression, T-cell follicular homing, and and memory B cells (26). These events include: GC formation. The importance of GC T cells is 1) B-cell clonal expansion, 2) somatic hypermu- highlighted by the fact that a loss of function tation, 3) class switch recombination, and 4) mutation in either CD40L (CD154) or CD40 affinity-based selection and differentiation of (constitutively expressed by B cells) results in B cells into plasma cells and memory B cells. the complete absence of GCs (13). Conversely, B-cell clonal expansion occurs mainly in defects in T-cell apoptosis can lead to an exag- centroblasts (53). Centroblasts are among the gerated GC reaction. fastest proliferating cells in the human body, Tingible Body Macrophages and Bone with a cell cycle estimated between 6 and 12 Marrow-Derived Dendritic Cells. The GC also hours. Centroblasts express genes involved in contains a population of tingible body macro- cell proliferation and DNA replication, high- phages (fig. 1-9) and a small population of bone lighting the fact that these cells are proliferating

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and undergoing somatic hypermutation of the ing. BCL6 acts as a transcriptional repressor (28) variable regions of the Ig heavy and light chain and is strongly upregulated by GC B cells (cen- genes. Centroblasts activate telomerase to prevent troblasts and most, but not all, centrocytes) and the shortening of telomeres in each cell cycle, GC T cells (fig. 1-8). It controls several important downregulate antiapoptotic genes, and upregulate functions in the biology of GC cells (reviewed proapoptotic genes. The result of this proapoptotic in references 28 and 52). BCL6 silences the default program is to facilitate the survival of only antiapoptotic molecule BCL2, ensuring that those GC cells that receive survival signals. GC B cells are eliminated by apoptosis if they Somatic hypermutation of the Ig variable are not selected and rescued by survival signals. region genes occurs in centroblasts and increases BCL6 also silences sensors of DNA damage, such the affinity of the Ig variable region (encoded by as the TP53 and ataxia-telangiectasia-mutated a VDJ rearrangement) for a particular antigen. (ATM) and Rad3-related (ATR) genes that help This process of mutation, mainly single-nucle- to suppress DNA-damage responses, allowing otide exchanges or small insertions or deletions GC B cells to sustain the genotoxic stress as- of DNA, occurs after exposure to an antigen. So- sociated with high proliferation. BCL6 also matic mutations arise via double-stranded DNA silences Blimp-1, a transcription factor, and thus breaks prior to the mutational event and depend controls the differentiation of GC B cells into on the activity of the enzyme activation-induced plasma cells. Lastly, BCL6 promotes the expres- cytidine deaminase (AID) (32,41). This process sion of T-follicular/helper cell-related genes, results in B-cell clones with increased affinity for such as CXCR5, PD1, and ICOS. T cells lacking antigen. Class switch recombination occurs in BCL6 fail to differentiate into GC Th cells and centroblasts and is a process by which the heavy fail to support GC responses (22). chain class of an antibody produced by a GC B-cell Lymph Node Paracortex clone changes from IgM (usually associated with IgD) to IgG, IgA, or IgE. Since the variable region The paracortex is located between the su- does not change, class switching does not alter perficial cortex, the area with the lymphoid antigen specificity. Instead, the antibody retains follicles, and the medulla. The paracortex is affinity for the same antigen, but can interact enriched with T cells, but also contains dendritic with different effector molecules. Class switch cells of the interdigitating dendritic subtype, recommendation is mediated also by AID. fibroblastic reticular cells (FRC), scattered large In the light zone, GC B cells with increased B cells, plasmacytoid dendritic cells, and high affinity for antigen are selected over those with endothelial venules (HEVs). The paracortex is lower affinity; the end result is a substantially the region where T cells encounter antigen- more effective immune response. The selection presenting cells (APC) such as interdigitating process involves the following steps: interaction dendritic cells. APC and dendritic cells migrate between centrocytes with the antigen retained into the lymph nodes via afferent lymphatics on FDCs, processing of antigen by B cells, and while most circulating naïve T lymphocytes presentation of antigen to follicular T-helper enter the lymph node from the blood via HEVs cells. FDC-activated T cells provide survival sig- (fig. 1-10). The homing of naïve T cells and mi- nals to the selected centrocytes. These selected gratory dendritic cells to the nodal paracortex centrocytes may return to the dark zone for is mediated by CCR7, a chemokine receptor further rounds of proliferation and selection, in dendritic cells and naïve T cells, and by the whereas apoptotic centrocytes are removed ligands CCL19 and CCL21 expressed by FRCs from GCs by tingible body macrophages. and HEVs (12). Subsequent downregulation of All of these GC events are tightly regulated CCR7 and upregulation of CXCR5 contribute by a complex network of transcription factors. to follicular homing of activated T cells. BCL6 is the master regulator of the GC reac- Fibroblastic Reticular Cell. The FRC is a dis- tion and is required for GC formation. BCL6 is tinct type of cell in the paracortex that has hybrid a 95-kD nuclear phosphoprotein that belongs features between epithelial and fibroblastic cells. to a large family of nuclear factors that contain These cells are difficult to recognize in routine zinc-finger motifs to mediate specific DNA bind- hematoxylin and eosin (H&E)-stained sections,

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Figure 1-10 T-CELL PARACORTEX Left: Silver stains (retic ulin shown) highlight the reticular fiber network and the “corridors” of the paracortex (upper left). The reticular network is produced and maintained by fibroblastic reticular cells. Fibroblastic reticular cells are variably positive for desmin (lower left) and cytokeratins 8 and 18. Right: A schematic repre sentation of the “corridors and conduits” of the para cortex.

but can be identified with immunohistochemical to the perivascular spaces (24). Cytokines and studies (fig. 1-10). FRCs have long slender cyto- other soluble substances entering lymph nodes plasmic processes; express cytokeratins 8 and 18, from sites of inflammation through the subcap- vimentin, smooth muscle actin, and desmin; and sular sinuses use these conduits to modulate the form tight junctions with each other. adhesive properties of the HEVs and thus allow The connective tissue skeletal network of the for entry of lymphocytes into the lymph node. lymph node is composed of a thin external fi- FRCs participate dynamically in the cytokine brous capsule with internal prolongations or tra- microenvironment of the paracortex as well as beculae, and by a complex meshwork of reticular in the regulation of cell trafficking and access fibers (fig. 1-10). FRCs produce, ensheathe, and of T cells into the paracortex (2,49,51). FRCs maintain this reticular fiber network (15). The secrete several homeostatic chemokines, such network has been conceptualized as a concentric as CCL19, CCL21, and CXCL12, and present arrangement of nested cylinders or “corridors” on their surface immobilized CCR7 ligands lined by FRCs that encircle the HEVs and radiate that provide signals to maintain the motility outwards to the sinuses (15). FRCs form an epi- of paracortical naïve T cells. thelium-like monolayer separating the corridors Plasmacytoid Dendritic Cells. Clusters of from the interstitial reticular matrix (fig. 1-10). plasmacytoid dendritic cells (previously known The corridors are filled with lymphocytes, and as plasmacytoid T cells or plasmacytoid mono- provide spaces for cell trafficking and for anti- cytes) are occasionally seen in lymph nodes in gen-presenting cells and lymphocytes to meet. H&E-stained sections. They are easily identi- Another space between FRCs and the basal fied by immunostains using antibodies against membrane, termed a “conduit,” has been pos- CD68, CD123, CD303, or TCL1 (figs. 1-11, tulated to mediate lymph flow from the sinuses 1-12). The number of plasmacytoid dendritic

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Figure 1-11 PLASMACYTOID DENDRITIC CELLS Cluster of plasmacytoid dendritic cells between two secondary lymphoid follicles. Scattered tingible body macrophages are admixed with the plasmacytoid dendritic cells.

cells increases in certain inflammatory and neoplastic conditions, for example, hyaline- vascular Castleman disease and Kikuchi-Fuji- moto lymphadenopathy (11,17). Plasmacytoid dendritic cells are bone marrow derived, enter lymph nodes through the HEVs, and are a major source of interferon-a. High Endothelial Venules. HEVs are postcapillary venules located in the paracortex of lymph nodes and other secondary lymphoid organs (except the spleen), specialized for the recruitment of B and T cells into the paracortex. HEVs are lined by unique endothelial cells with distinctive, almost cuboidal morphology, unlike the flat morphology of typical endothelial cells. Experimental data suggest that these morphologic features are a result of continuous but transient accumulation of lymphocytes between the endothelial cells and the underlying basal layer (38). Pockets of four to five lymphocytes are frequently observed underneath endothelial cells of the HEV. The endothelial cells express high levels of sulphated carbohydrate ligands for L-selectin, which can be recognized Figure 1-12 by the monoclonal antibody MECA-79. PLASMACYTOID DENDRITIC CELLS Lymphocyte adhesion to HEV endothe- High-power magnification of figure 1-10. The lial cells is mediated by vascular addressins plasmacytoid dendritic cells are intermediate to large, with expressed by the endothelial cells. These ad- round to oval and slightly eccentrically located nuclei with dressins bind to homing receptors, such as L- clumped nuclear chromatin (plasmacytoid appearance). Tingible body macrophages are present. selectin and a4b2 integrins, expressed on the

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Figure 1-13 MEDULLARY CORDS AND SINUSES This lymph node biopsy specimen (left) shows predomin antly medulla, focal adipose tissue metaplasia, and, in the right lower corner, a small portion of paracortex. The medulla is composed of sinuses and medullary cords (upper and lower right). The sinuses contain macrophages and small lymphocytes.

surface of circulating lymphocytes (6,36). Lym- and other signaling agents generated by local phocyte recruitment by HEVs also is modulated inflammatory conditions in tissues draining to remotely by chemokines produced by FRCs lymph nodes. The sinuses are the channels that and dendritic cells in the lymph node micro- carry lymph from the afferent lymphatics to the environment (39,43). Postcapillary venules in efferent lymph vessels at the hilum. There are other tissues do not express lymphocyte adhe- cortical and medullary sinuses. Most of the cortical sion molecules unless they are stimulated by sinuses seem to be blunt-ended and are in prox- inflammatory mediators (36). HEVs can develop imity to HEVs. There are also sinuses, frequently in nonlymphoid tissues involved by chronic located adjacent to lymphoid follicles, connecting inflammatory diseases or cancer and are associ- the subcapsular and medullary sinuses. ated with high levels of lymphocyte infiltration The sinuses are lined, at least in part, by en- into these affected tissue sites. dothelial cells. There are some differences in the composition and structure of the lining between Lymph Node Medulla the subcapsular and medullary sinuses. The floor The medulla is located in the inner or hilar of the subcapsular sinus is lined by lymphatic portion of the lymph node and is composed endothelial cells interspersed with subcapsular of cords and sinuses (fig. 1-13). The medullary sinus macrophages (CD169 positive). These cords are usually clearly delineated because their macrophages have a head that protrudes into dense cellularity contrasts with the sparse cellu- the sinus lumens and a long tail of processes larity of the surrounding medullary sinuses. The that extends into the underlying lymphoid fol- medullary cords contain B cells, T cells, plasma licles. Sinus macrophages capture large antigens cells, macrophages, mast cells, and dendritic that enter the lymph node through the lymph, cells. A paired arteriole and venule runs along and display them to B cells. the central axis of each medullary cord and is In addition to being sites of B-cell encounter surrounded by the FRC network. with large soluble antigens, the cortical and medullary sinuses are the major gateways for Lymph Node Sinuses lymphocytes exiting the lymph node. Exper- The afferent lymphatics contain lymph that imental data show that expansion of cortical drains into the subcapsular sinus. This lymph and medullary sinuses participates in regulating originates in the interstitial spaces of most of the lymphocyte egress from lymph nodes during tissues and contains antigens, APC, chemokines, prolonged inflammation, providing exit routes

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Figure 1-14 SPLEEN SHOWING WHITE AND RED PULP The white pulp consists of T-cell zones that surround the splenic arterioles (also known as periarteriolar T-cell–rich lymphoid sheets, or PALS) and B-cell follicles (primary or secondary), usually at the periphery of the sheets of T cells.

for lymphocytes (48). Spingosine 1-phosphate splenorenal ligaments. When these ligaments (S1P) receptors on endothelial cells lining the have not developed properly, the spleen can be sinuses and CD69 on lymphocytes control the aberrantly located in other sites of the abdomen passage of lymphocytes into the sinuses to alter or pelvis (ectopic spleen). Single or multiple ac- exit from the lymph node. The lymphatic sys- cessory spleens are found in about 10 percent of tem converges onto a single lymphatic vessel, the general population; usually accessory spleens the thoracic duct, which drains the lymph into are of small size and located near the hilum of the bloodstream. the spleen or the pancreatic tail. Splenosis is the implantation of splenic tissues into the peri- SPLEEN toneum (and often throughout the abdomen) following traumatic rupture of the spleen. The Gross Anatomy spleen has two major distinct compartments, The spleen is the largest secondary lymphoid the white pulp and the red pulp (fig. 1-14), with organ and is responsible for defense against distinctive morphologic features and function. blood-borne pathogens (the spleen filters the Microscopic Anatomy blood as lymph nodes filter lymph). In particular, the spleen has a key role in the removal White Pulp. The white pulp is a continuous of encapsulated bacteria. Patients with splenec- layer of lymphoid tissue that surrounds the tomy or with splenic dysfunction need routine branching splenic arterioles. The white pulp prophylaxis to prevent Streptococcus pneumoniae, consists of T-cell zones (also known as the periar- Haemophilus influenzae, and Neisseria meningit- teriolar lymphoid sheath, or PALS) and B-cell fol- ides infections. Other relevant functions of the licles (primary and secondary) (figs. 1-14, 1-15), spleen include filtering the blood to remove resembling the organization of the lymph node, old or damaged erythrocytes and recycling iron except for the apparent absence of HEVs. (35). The spleen lacks an afferent lymphatic The organization of the white pulp is con- system, but receives a fair amount of the blood trolled by specific chemokines that attract T and B supply, about 5 percent of cardiac output. cells to their specific compartments: B cells are at- The spleen is an elongated, dark red organ, sur- tracted by CXCL13 (produced by FDC and stromal rounded by a thin capsule of connective tissue. cells) whereas T cells are attracted by CCL19 and In younger adults, the spleen weights 150 to 250 CCL21 (mainly produced by stromal cells in the T g, but the size decreases with age. Normally, the zone). Lymphoid follicles are frequently observed spleen is located in the abdomen in the left hy- at the periphery of T-cell aggregates and have his- pochondrium, attached by the gastrosplenic and tologic and immunophenotypic features identical

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Figure 1-15 SPLEEN: WHITE PULP CD3 immunostain highlights the T-cell zone that surrounds the splenic arterioles and CD20 immunostain identifies the B-cell compartment, in this case represented mostly by primary fol licles. The white pulp contains also a network of fibroblastic reticular cells, highlighted by cyto keratins 8 and 18, which provide the corridors or physical roads for T cells in the white pulp.

Figure 1-16 SPLENIC SECONDARY LYMPHOID FOLLICLE Splenic secondary lymphoid follicle with a germinal center, mantle zone, and marginal zone. The mantle zone, represented by a thin rim of dark small lymphocytes, surrounds the germinal center. The marginal zone is the corona that surrounds the mantle zone. The marginal zone is composed of medium-sized lymphoid cells with abundant pale cytoplasm and slightly irregular nuclear contours (monocytoid appearance).

to those of primary and secondary follicles of the Localized between the white and red pulp is the lymph node. In splenic lymphoid follicles, the splenic marginal zone (fig. 1-16) (33). The spleen processes described in lymph node secondary lacks a marginal zone sinus and the arterioles open follicles also occur, including clonal expansion in a large perifollicular area, poor in reticulin fibers, of activated B cells, somatic hypermutation, and located at the outer aspect of the marginal zone. isotype switching. Similar to the lymph node This area is recognized in routine H&E-stained paracortex, the white pulp of the spleen contains a sections by the presence of pools of erythrocytes network of FRCs (positive for cytokeratins 8 and (fig. 1-17). It has been suggested that blood flow is 18) that provide physical corridors to the T cells retarded in this area. In humans, the splenic mar- and participate in T-cell distribution within the ginal zone is limited to the follicular compartment white pulp (fig. 1-15) (3). and does not extend along the T-cell zone (47).

13 Tumors of the Lymph Nodes and Spleen

Figure 1-17 Figure 1-18 SPLENIC ARTERIOLES SPLENIC RED PULP In the human spleen, the splenic arterioles open The red pulp is composed of a network of cords and in a perifollicular area, poor in reticulin fibers, located venous sinuses. The sinuses are lined by sinusoidal cells immediately peripheral to the marginal zones where the that express endothelial markers as well as CD8. blood flow seems to be retarded. This area is recognized in routine hematoxylin and eosin (H&E)-stained sections by the presence of a corona of erythrocytes. Red Pulp. The red pulp is the most abundant The splenic marginal zone is an important compartment of the spleen and is composed transit area between the bloodstream and white of a network of cords (about 70 percent) and pulp, and is involved in both innate and adap- venous sinuses (about 30 percent) (fig. 1-18) tive immunity. The splenic marginal zone has a (50). The capillaries open to the splenic cords, specific and distinct cell composition that con- which form an open blood system without an tains a population of resident macrophages and endothelial lining and contains reticular fibers, marginal zone B cells. The macrophages express myofibroblasts, plasma cells, plasmablasts, and unique specific receptors, such as the C-type numerous macrophages that remove old and lectin SIGNR1 and the type I scavenger receptor damaged erythrocytes. MARCO (macrophage receptor with collagenous From the cords, blood passes into venous si- structure) (14,25). SIGNR1 binds polysaccharide nuses lined by sinusoidal cells. These sinusoidal antigens and participates in the uptake and clear- cells are positive for endothelial markers such ance of Mycobacterium tuberculosis and Streptococcus as factor VIII, and are also strongly positive pneumoniae. MARCO recognizes many pathogens, for CD8. A set of stress fibers extending under- including Escherichia coli and Staphylococcus aureus. neath the basal plasma membrane of sinusoi- In addition to the clearance of encapsulated bac- dal cells forms slits. These slits are difficult for teria, marginal zone macrophages are important ageing erythrocytes with stiff membranes to for the clearance of viruses. Marginal zone B cells pass through. The blood cells that cannot pass are considered to represent a first line of defense through sinusoidal cells are destroyed by macro- against blood-borne pathogens, particularly phages residing in the cords. As a consequence encapsulated bacteria. Marginal zone B cells are of cell destruction in the red pulp, pigments IgM(+), IgD(+/-), CD21(+), CD23(+/-), CD1c(+), such as hemosiderin and lipofuscin (less abun- and CD27(+) and seem to represent recirculating dant), accumulate and are frequently observed IgM-positive memory cells (42). in the cytoplasm of macrophages.

14 Normal Anatomy and Function of Lymph Nodes and Spleen

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