Download Chapter 5 Normal Histology of the Lymphoid Tissues

Chapter 5
Normal Histology of the Lymphoid Tissues
J. Han van Krieken
Lymphoid tissue is found all over the body. It occurs in wellorganized lymphoid organs, such as the lymph nodes and the
spleen, or as extranodal lymphoid tissue as part of the gut,
skin, and lung. It may develop in any part of the body under
specific conditions.
The lymphoid organs are divided into central and peripheral organs. The bone marrow and the thymus constitute the
central lymphoid organs, and the lymph nodes, the spleen,
and extranodal lymphoid tissues constitute the peripheral lymphoid organs. The bone marrow is the site of lymphopoiesis.
Some lymphocytic precursors develop into B cells, but most
become T cells. The generation of a diversity of effector B cells
requires a two-step maturation process. The first step occurs
in the bone marrow and results in the development of virgin
or naive B cells. The second step takes place in the peripheral
lymphoid organs, where affinity maturation of the naive B cells
takes place on encounter with a specific antigen, resulting in
the formation of plasma cells and memory B cells. The generation of mature T cells occurs exclusively in the thymus, where
T-cell precursors develop into a variety of mature CD4-positive
(CD4+) and CD8+ T cells through a process of positive and negative selection. T cells do not require further affinity maturation
in secondary lymphoid tissues. In contrast to B cells, T cells
are activated only in secondary lymphoid organs and start to
­proliferate on appropriate antigen stimulation.
The architecture of the thymus and of the peripheral lymphoid organs offers the ideal microenvironment for these
processes. Knowledge of these structures and the immunophenotype of their cellular components within their natural
microanatomic environment has improved significantly by the
addition of immunohistochemistry to the morphologic studies
on lymphoid tissues. Immunophenotyping allows identification
of cell lineage and provides information on the stage of maturation, activation, and differentiation of lymphocytes. Immunohistochemistry has offered the opportunity to identify and
distinguish more precisely the nonlymphoid components of
the lymphoid tissues as well, including the monocyte-derived
antigen-presenting cells and macrophages. The use of antibodies against adhesion molecules expressed by endothelial lining
cells and by lymphocytes has led to a better understanding of
lymphocyte trafficking and homing.
Molecular techniques performed on tissue fragments and
particularly experiments carried out on selected and defined
areas, cell clusters, or on single cells dissected from tissue
sections, together with in vitro experiments, and the development of knockout and transgenic animal’s experiments have
profoundly changed our understanding of the structure of lymphoid tissues and have resulted in new views and concepts
of the functional microanatomy of these tissues as the morphologic substrate of the immune response. This knowledge
is essential in the analysis of lymphoid tissues in pathologic
conditions, because it is helpful to recognize underlying pathogenic mechanisms.
This chapter discusses the histology and the ­immunohistology
of the thymus, lymph node, and spleen and finally factors
governing lymphocyte trafficking. The historical description
of the microanatomy of these organs, as reported by classic
histology, is presented first. This information is complemented
by the results obtained by immunohistochemical analysis. The
functional significance of the various compartments identified
by morphology is discussed.
The bone marrow, which is the site of lymphopoiesis in postnatal life and represents an additional microenvironment for
B-cell maturation, is discussed in Chapter 1. Particular f­ eatures
of extranodal lymphoid tissues are the subject of Chapter 28.
THE THYMUS
The thymus is a lobulated, primary lymphoid organ and has
as main function the maturation of T lymphocytes. Its dominant cellular components are epithelial cells and T lymphocytes
that mature from the cortex, the peripheral part of the lobules,
to the medulla, the central part. The thymus is a completely
encapsulated, pyramid-shaped organ located in the anterosuperior mediastinum. It is composed of two lobes that join at
their lower poles, which may reach the level of the fourth costal
cartilage. The upper poles extend into the neck. The gray color
of the thymus during infancy turns yellow with increasing age
because of accumulation of fat tissue. Because this fat merely
takes the place of normal thymus parenchyma, the organ’s
shape and volume remain unchanged (1).
Atrophy
In relation to body weight, thymic weight is maximal at birth,
and its absolute weight peaks at puberty. Even after excluding age-related differences in the weight of this organ, large
interindividual variations exist. These concepts are based on
autopsy findings showing a thymus weighing 12 to 15 g at birth
and 30 to 40 g at puberty. This prominent increase in absolute mass is followed by a gradual decrease, or “age-related
thymic involution,” leaving a thymus weighing no more than
10 to 15 g at the age of 60 years. Involution is accompanied
by a gradual replacement of the thymic parenchyma by fat
tissue until 40 to 50 years of age, after which little changes
(1,2). Despite of its notable decrease in size, the thymus never
disappears completely, and it remains functionally active even
after puberty (1). Nevertheless, the impaired T-cell function
that accompanies ageing is attributed to thymic involution
­resulting in a smaller number of naïve T cells (3). Remnants of
the thymus with residual epithelium and cortical thymocytes
are preserved, permitting the thymus to act as a site of T-cell
differentiation and maturation throughout the entire life (4).
Microscopically, the involution is the result of atrophy of
the parenchyma and concomitant accumulation of fat cells
(Fig. 5.1A). This gradual decrease of the parenchyma is accompanied by an accentuation of so-called Hassall corpuscles (see
Fig. 5.1B). Some of the corpuscles calcify, but others transform
into thymic cysts (2). The morphology of the residual epithelial
1
Orazi9781609136826-ch05.indd 1
6/11/2013 3:50:25 AM
2
Knowles Neoplastic Hematopathology
FIGURE 5.1. Hematoxylin and eosin–stained, paraffin-embedded thymic tissue of an adolescent. A: Lobulation of the thymic parenchyma and clear delineation between the dark-stained cortex and the lighter medulla are shown (H&E stain, original magnification:
60× magnification). There is fatty replacement, but the morphology of the thymus tissue is retained. B: Detail of the cortex and medulla in
which a Hassall corpuscle is present (H&E stain, original magnification: 250× magnification). The cortex contains far more lymphocytes
than the medulla. In the medulla, the light-stained large nuclei of the thymic epithelium can be recognized.
cells does not show any significant changes, which probably
implies that they remain biologically active. Using immunohistochemistry it became clear that this persistent decrease in the
number of epithelial cells and thymocytes is not the only event
responsible for age-related thymic involution: there is an even
more pronounced decrease in medullary interdigitating dendritic cells (5).
Histology and Immunohistology
Because the fibrous capsule that surrounds the thymus extends
into the thymic parenchyma as loose septa, the organ is incompletely subdivided into various lobules measuring 0.5 to 2 mm.
These lobules represent the basic structural units of the thymus, which comprises two morphologically distinct areas,
a cortex and a medulla (Fig. 5.1). Epithelial cells and T lymphocytes or thymocytes constitute the major components of
both regions. The subcapsular area of the cortex is occupied
by somewhat larger thymocytes with a blastlike nucleus and a
high number of mitotic figures. The distinction between cortex
and medulla at the light microscopic level is evident because
the amount of lymphocytes in the cortex far outnumbers those
in the medulla. In a hematoxylin and eosin (H&E) stain, the
medulla appears less intensely stained than the cortex (Fig.
5.1B). The subcapsular and perivascular areas are considered
to be a separate compartment of the thymus, referred to as the
perivascular space (6).
Thymic Epithelium
The epithelial cells, which especially in the cortex are difficult
to recognize by routine light microscopy, are a heterogeneous
population of round to spindle-shaped cells. Ultrastructurally,
six different subtypes have been identified and described, of
which four variants are localized to the cortex and two are confined to the medulla (7). As a whole, epithelial cells provide the
appropriate microenvironment for T-cell maturation. However,
because the development of T lymphocytes is an extremely
complex process, each of the epithelial cell variants exercises
its own specific function in the establishment of an effective
T-cell compartment.
The epithelial network of the middle and deep cortex consists of cells characterized by their long cytoplasmic processes
embracing thymocytes. These cortical epithelial cells represent
Orazi9781609136826-ch05.indd 2
the in vivo equivalent of thymic nurse cells, a cell population that has been extensively studied in vitro (8). The results
of these in vitro experiments, using cell lines derived from
murine thymic nurse cells, suggest that they could be involved
in the negative selection process of thymocytes by inducing thymocytic apoptosis (9).
Despite the marked differences in the submicroscopic level
appearance of the various epithelial cell types, striking similarities in their morphology, accentuating their common origin,
are evident. First, every thymic epithelial cell displays slender
cytoplasmic processes, which explains the term dendritic cell.
These dendrites exhibit well-developed desmosomes at their
ends through which the epithelial cells are connected with one
another. The whole of thymic epithelial cells creates a firm
meshwork throughout the entire parenchyma in which the
other cell types are embedded. The epithelial origin of these
cells is further confirmed by the presence of a supporting
basement membrane. The basal lamina surrounding medullary epithelial cells shows focal gaps, and only cortical epithelial cells lining mesenchymal spaces have a continuous basal
membrane to separate them from the neighboring fibrous
tissue (1). Immunohistochemistry and electron microscopy of
thymic epithelial cells have shown the presence of intermediate and thin filaments within the cytoplasm of these cells. The
intermediate filaments, which correspond to tonofilaments, are
clustered in thick bundles attached to the desmosomes. They
form an extensive filamentous network within the cell body
and the cytoplasmic processes of the cortical and the medullary epithelial cells. Bundles of thin actin-like filaments, located
immediately underneath the plasma membrane, complete the
filamentous cytoskeleton of the epithelial cells. Subcortical epithelial cells typically show an abundance of thin filaments, but
this portion of the cytoskeleton is nearly undetectable in medullary epithelial cells (10).
Hassall corpuscles (Fig. 5.1B) are clusters of concentrically
arranged epithelial cells located in the medulla, but in addition
to their thymic epithelial cell features, they acquire characteristics of squamous epithelium by exhibiting a variable degree
of keratinization and staining with antibodies against terminally differentiated epithelium. Their precise function or significance is unresolved. Although interpreted in the past as a
terminal phase of a degenerative process, they are considered
a dynamic structure that is involved in the intrathymic maturation of T cells (11). The epithelial cells that compose the Hassall
6/11/2013 3:50:28 AM
corpuscles contain a particularly well-developed framework of
tonofilaments (10). These thymic corpuscles do not constitute
isolated epithelial islets within the thymic medulla but form,
together with the other epithelial ­
components of the inner
part of the thymic parenchyma, an uninterrupted epithelial
­structure.
Subcapsular epithelial cells, a minority of cortical epithelial
cells, and almost all medullary epithelial cells can be considered as a functionally distinct group of neuroendocrine cells.
These cells strongly express oxytocin, vasopressin, and neurophysin-like peptides (12). These peptides are synthesized
within these cells, where the proteins can be demonstrated
and the corresponding mRNA is found (13). It has been suggested that, analogous to neurosecretory cells found elsewhere,
neuroendocrine cells of the thymus convert neuronal signals
into neuropeptide secretion. Accepting this hypothesis, thymic
oxytocin and vasopressin, secreted as a result of yet undefined
neuronal influences, are expected to exert a direct immunomodulation on T-cell maturation (12).
Antibodies against other substances involved in intercellular signaling—the thymic hormone thymuline, members of
the thymosin family, and thymopoietin—immunoreact with a
­subpopulation of these neuroendocrine cells, particularly subcapsular and medullary epithelial cells (14). These hormones
also affect the maturation of T-cell precursors and the expression of T-cell antigens.
The entire cortical epithelium, including the nurse cells,
reacts with Mab-MR6, recognizing a component of the interleukin-4 (IL-4) receptor complex. Consequently, these cells may
act as an IL-4 reservoir for the surrounding immature cortical thymocytes (15). Antibodies against major histocompatibilty complex (MHC) class II molecules (e.g., HLA-DR) stain a
fine meshwork of cytoplasmic processes originating from the
epithelial cells in the outer cortex that embrace nonreactive
thymocytes, arranged singly or in small clusters. Considerable parts of the inner cortex and the medulla are negative for
HLA-DR (16).
T Lymphocytes
T lymphocytes (i.e., thymocytes) displaying heterogeneous
features corresponding to the various stages of thymocyte
maturation predominate in the thymic cortex. The immature
lymphoblasts are found in the subcapsular region. Maturation
occurs toward the medulla, resulting in medium-sized thymocytes throughout the cortex, and at the corticomedullary
Chapter 5 Normal Histology of the Lymphoid Tissues3
junction, mature small lymphocytes are scattered among the
epithelial cells in the medulla. This is a complex process involving many transcription factors with the Notch pathway as a key
player (17,18).
The most immature thymic T cells (i.e., T-cell precursors)
are identified by the expression of TdT, CD34, CD33, CD45RA,
and CD38low, typically lacking surface CD2, CD5, CD4, CD8,
CD1, and CD3 (15). They express the integrins very late activation (VLA) antigen-4 (VLA-4, a4b1, CD49d/CD29), VLA-5 (a5b1,
CD49e/CD29), and PGP-1 (CD44), which potentially mediate
homing of the precursors to the thymus (19,20). T-cell receptor (TCR) genes are still in the germline configuration. These
multipotent progenitor cells have the capacity to develop into
T cells and NK cells (21).
Unlike their predecessors, the earliest committed T-cell
progenitors have acquired surface CD1 (Fig. 5.2A), CD2, CD5,
CD7, and cytoplasmic CD3, but they are still devoid of surface
CD3, CD8, and CD4. These triple-negative thymocytes show an
intense proliferative activity (Fig. 5.2B), which depends on IL-7
(22–24) and stem cell factor (25,26). BCL-2, an anti-apoptosis
protein, may add to the prolonged cell survival of these early
thymocytes (27). During this stage of thymocyte development,
the TCR b chain is rearranged. After this pivotal event in T-cell
maturation, TCR b is expressed on the cell surface in a complex
with gp33 and the pre-TCR a chain. The resultant primitive
TCR complex occurs in association with CD3 (28–30). Signaling through this pre-TCR complex is crucial for the next step
in the generation of mature T lymphocytes, which comprises
three molecular biologic events: the concomitant ­up-regulation
of CD4 and CD8 resulting in double-positive thymocytes, rearrangement of the TCR a locus, and allelic exclusion of the
TCR b locus (28–30). As a consequence of this complex event,
CD4+CD8+TCRlow cortical thymocytes are brought about, representing the first thymic T-cell population to express the definitive TCR ab chain (19).
Subsequently, these CD3low, double-positive thymocytes are
positively or negatively selected by thymic stromal cells. Many
factors, such as the density of the MHC molecules and coreceptors expressed on the auxiliary thymic cells and the nature and
concentration of peptides presented on their surface determine
the ultimate fate of the T cells subject to this selection process
(31–33). However, the level of avidity between TCR and MHCpeptide complexes is the main factor mediating survival signals or deletion by apoptosis. High-affinity binding of the TCR
to peptides, derived from autoantigen, superantigens, or both,
presented in the context of self-MHC results in clonal deletion
FIGURE 5.2. A: Immunohistochemistry shows expression of CD1a in the cortical thymocytes associated with a high proliferation (B, mib1;
original magnifications: 250× magnification).
Orazi9781609136826-ch05.indd 3
6/11/2013 3:50:30 AM
4
Knowles Neoplastic Hematopathology
of autoreactive T cells (34,35). In contrast, low or intermediate
avidity confers to positive selection, which is indispensable for
the final maturation of double-positive thymocytes to CD4 or
CD8 single-positive T cells.
Positive selection in vivo is mediated predominantly by cortical epithelial cells that express MHC class I or class II molecules (36), whereas in vitro experiments suggest that negative
selection comes about most efficiently when the antigen is
presented by medullary dendritic cells (37). Most thymocytes,
unable to pass through the selection process because of defective TCR/MHC interaction, undergo apoptosis. All thymocytes
triggered to die, whether apoptosis is a result of negative selection or caused by lack of stimulation, are thoroughly eliminated
by the numerous phagocytes in the cortex and medulla, among
which are the aforementioned cortical epithelial nurse cells.
B Lymphocytes
The presence of B cells as a consistent component of the human
thymus is well established (38–41). There are several subsets of
B lymphocytes with distinct B-cell subsets in the perivascular
space and in the parenchyma, preferentially in the neighborhood of Hassall corpuscles. This latter subset is morphologically and phenotypically distinct from the B cells composing
the B follicle of the lymph node, but they do express most pan-Bcell markers, including CD19, CD20, CD22, CD37, CD72, CD76,
and weakly express IgM and IgD (38). An important subset is
CD2+ and CD40+, markers that are invariably present on T lymphocytes but only occasionally found on B cells (41). CD2 may
mediate the interaction of these B cells with the surrounding
thymocytes and epithelial cells through their ligand LFA-3. B
and T cells would benefit from this CD2 interaction through
the acquisition of improved self-antigen recognition capacity
(30,31). Another subpopulation of somewhat larger B cells displaying dendritic cytoplasmic extensions is seen near Hassall
corpuscles. These peculiar cells, also designated asteroid cells,
consistently lack IgD but do express an additional marker,
CD23, pointing out their activated status (38).
As a whole, the B cells of the thymic parenchyma belong
to the microenvironment of the medulla and do not represent
mere passengers derived from the perivascular space, which
also comprises a B-lymphocytic population, including B-cell
follicles. During fetal development, B lymphocytes initially are
restricted to the perivascular compartment, while progressively
increasing numbers of these cells are observed within the thymic parenchyma, and it seems likely that thymic medullary
B lymphocytes are acquired by migration from the extraparenchymal area. The number of B cells in the thymic medulla is
related to the number of individual B lymphocytes and B follicles
present in the extraparenchymal compartment, which supports
the hypothesis that the intramedullary and extramedullary
B-cell compartments do not constitute entirely separate regions
but that they are at least subject to similar influences (38).
Based on topographic, morphologic, and immunohistochemical similarities, it has been suggested that intrathymic B
cells represent a specific type of marginal zone B cell intrinsic
to the thymic parenchyma. Nevertheless, important immunophenotypic differences exist between B lymphocytes of the thymic parenchyma and marginal zone cells as they are observed
in the spleen, peripheral lymph, nodes, and mucosa-associated
lymphoid tissues (38). This particular subset of B cells awaits
further examination to elucidate its precise stage in B-lymphocytic differentiation.
Other Cell Types
Besides thymic epithelial cells and lymphocytes, several other
cells have been identified in the thymus. This minor population is composed of various cell types, including macrophages,
Orazi9781609136826-ch05.indd 4
interdigitating dendritic cells, and myoid cells. Macrophages
are mainly found in the cortex, but they also occur in the
medulla. As regular phagocytic cells, they are characterized
by their a-naphthol esterase and acid phosphatase activity.
Being devoid of HLA-DR antigens, these macrophages are
not expected to function as genuine antigen-presenting cells.
Together with the thymic nurse cells, they eliminate dying thymocytes that have been negatively selected during maturation
processes (4).
Interdigitating dendritic cells are exclusively localized in the
medulla. These cells stand out by their irregularly shaped and
folded nucleus and by their long cytoplasmic processes that
embrace the surrounding T cells. Because of this intimate contact and the expression of HLA-DR antigens by interdigitating
dendritic cells, investigators have speculated that these cells
contribute to the final maturation of medullary T lymphocytes.
Although Langerhans cells with characteristic Birbeck granules
do occur in the thymus of animals, these cells are consistently
lacking in human thymuses (4).
Myoid cells have been identified in adult and fetal thymuses.
These cells are unevenly distributed throughout the thymus,
with a preferential occurrence in small clusters, predominantly
located in the medullary parenchyma. These cells display the
ultrastructural features of degenerating striated muscle cells,
typically containing myosin and actin filaments in their cytoplasm (10). The presence of acetylcholine receptor–like material (11) has been demonstrated on the surface of thymic myoid
cells, a finding that might explain the link between myasthenia
gravis and the thymus (38).
Blood vessels and the associated perivascular spaces belong
to the extraparenchymal compartment of the thymus. The perivascular space, its macrophages, vascular endothelium, and
type 1 thymic epithelium represent the blood-thymus barrier,
which was thought to guarantee an antigen-free environment
in the thymic cortex, protecting thymocytes from inappropriate stimulation. Nieuwenhuis and associates (42) demonstrated
the existence of a transcapsular pathway by which antigens
may bypass the thymic-blood barrier. Since these results were
published, the functional significance of the blood-thymic barrier was seriously questioned. Nevertheless, cortical thymocytes are undoubtedly efficiently protected against blood-borne
antigens, whether this shelter is entirely provided for by the
described structures or not. Morphologically, the perivascular
space is based on an extensive reticulin meshwork that surrounds the complete vascular system of the thymus. On either
side, this specialized region is bordered by a basement membrane, with the one produced by the endothelium on the vascular side and the one lining the type 1 epithelial cells on the
other. The overall appearance and the cellular composition
of the perivascular area show considerable variation among
the two components of the thymic parenchyma. Whereas the
medulla generally is poorer in lymphocytes, its wide perivascular spaces contain many of these cells. The cortex is provided
with narrow perivascular areas, mostly devoid of lymphocytes.
The vascular network embedded in this fibrous tissue is
derived from interlobular arteries, in particular the arterioles at
the corticomedullary junction and a capillary network located in
the cortex. In the subcapsular area, they unite in an anastomosing arcade that drains in postcapillary venules. The extraparenchymal compartment contains lymphatics and nerves. Whereas
afferent lymphatic vessels are consistently absent, efferent ones,
arising from the medulla and the ­
corticomedullary junction,
run along with arteries and veins. Eventually, they leave the
organ also by perforating the c­ apsule, particularly in the clefts
formed by the interlobular septa, meanwhile having drained
the perivascular spaces (43). The thymus is innervated by autonomic nerves that are derived from the sympathetic chain and
the vagal nerve and are mainly restricted to the capsule and
its septa. A neural plexus is formed along the corticomedullary
6/11/2013 3:50:30 AM
Chapter 5 Normal Histology of the Lymphoid Tissues5
junction by closely interwoven sympathetic and vagal fibers.
This autonomic innervation is crucial to vasomotor control but
probably also serves other purposes (43).
THE LYMPH NODE
Lymph nodes are bean-shaped encapsulated lymphoid organs,
generally measuring only a few millimeters in the longest
dimension. In a stimulated state they enlarge to reach a size
of more than 1 cm. These organs occur throughout the entire
body, invariably intercalated in the lymph stream. They are
most frequent in the axillary, cervical, and inguinal regions,
in the mediastinum, and in the retroperitoneum. They serve
innate and specific immunity. Their macrophages ingest the
bulk of invading, lymph-borne microorganisms, reducing the
load of foreign antigens that is carried along with the lymphatics. Lymphocytes may continuously enter the lymph node
parenchyma through the highly specialized postcapillary
venules, allowing a recruitment of specific lymphocytes from a
large circulating pool. In this way, a system capable of generating an adequate immune response to nearly all lymph-borne
antigens is created (44).
Histology and Immunohistology
The lymph node has a fibrous capsule from which septa derive,
resulting in an incomplete subdivision of the parenchyma into
segments. Several afferent lymphatics reach the lymph node
at its convex margin to end into the subcapsular or marginal
sinus, which can be regarded as a lymph reservoir (Fig. 5.3).
Subsequently, the lymph percolates through the cortical sinuses
that communicate with the medullary sinuses and eventually
converge to give rise to only one efferent lymph vessel that
leaves the lymph node at its hilus. The sinus network does not
randomly drain the lymph node. Instead, it constitutes an ingenious irrigation system, relating each afferent lymphatic to a
well-defined functional compartment (45).
The sinuses form a labyrinth of wide, irregular spaces that
resemble thin-walled blood vessels (46). The sinus lacework
is bordered by a discontinuous monolayer of sinus lining cells
to which delicate collagen fibers are attached. This s­ upportive
fibrous skeleton stretches out in the lumen, preventing the
sinus walls from collapsing. Broad intercellular gaps in the
sinus lining allow unimpeded contact between the luminal
contents and the surrounding tissue. The absence of a basal
FIGURE 5.3. Lymph node capsule and septum derived from it with entering lymph vessels.
The subcapsular sinus is filled with lymphocytes and not well visible (125× magnification).
Orazi9781609136826-ch05.indd 5
membrane promotes direct interaction between the circulating
lymph and the lymph node parenchyma. In contrast, a basal
membrane is found underlying the cellular monolayer, lining
the capsular side of the marginal sinus (47).
In normal lymph nodes, sinus lining cells are inconspicuous and can hardly be distinguished from the macrophages
and other mononuclear cells abundantly present in the sinus
lumen. Sinus lining cells have long dendrites that connect the
cell with its neighbors through well-developed desmosomes.
Another feature of sinus lining cells is their intimate association with reticulin fibers from the sinus cavity. These components of the fibrous network supporting the sinuses, typically
composed of type IV collagen, are engulfed by slender protrusions extending from the cell’s body (47,48).
Based on marked morphologic similarities, it has been
suggested that the sinus lining cells originate from dendritic
cells. However, unlike follicular dendritic cells and interdigitating dendritic cells, they do not display any phagocytic ­activity.
Moreover, they consistently lack S-100 protein and CD1a, molecules frequently present on interdigitating dendritic cells. In
contrast, they do react with antibodies directed against the
highly restricted antigens Ki-M9 and Ki-M4, which have only
been identified on the surface of follicular dendritic cells. The
latter finding, together with the demonstration of IL-6 production by sinus lining cells (49), strongly suggests that these cells
really function as genuine antigen-binding and -presenting
cells (48).
The nodal arteries enter the lymph node through the hilus
and give rise to arterioles that follow the fibrous trabeculae.
From these small vessels, extensive capillary networks branch
off that are connected with postcapillary venules. Most of
these highly specialized vessels, also called high endothelial
venules because of their unusual morphology, are situated in
the paracortex. High endothelial venules, also designated as
epithelioid venules, are easily distinguished by their plump,
cuboidal to cylindrical endothelial cell lining and typically display a large, round nucleus and abundant cytoplasm. Scanning electron microscopy has shown that the tridimensional
structure of these vessels is unique. Extensive portions of the
high endothelial venules show a cobblestone surface with lymphocytes located in the crevices separating adjacent endothelial cells. Because of these peculiar features, turbulent blood
flow is brought about along the high endothelial venules, which
may account for an important improvement of the interactions
between circulating lymphocytes and the endothelial surface.
These specialized postcapillary vessels play a crucial part in
the recruitment of circulating lymphocytes into the lymph node
parenchyma, for this process is essentially based on cell-cell
interactions between endothelial cells and lymphocytes. Important molecules in this process are nepmucin and autotaxin,
both expressed by high endothelial venules (50). The trafficking is steered by several chemokines and chemoattractants (51)
with a key role for chemokine receptor 7 (CCR7) (52).
After its passage through the high endothelial venules,
the blood is drained by the nodal veins, which leave the node
together with the efferent lymphatic. In humans, no communications exist between the sinuses and the vascular system. A
detailed description of the microvascular structures supporting
the lymphocyte trafficking is beyond the scope of this book (53).
The lymph node parenchyma is subdivided in the cortex,
comprised of B-cell follicles, the paracortex, consisting predominantly of T cells, and the medulla, the innermost region
(Fig. 5.4). The B follicle is responsible for humoral immunity;
the T-cell area accounts for cellular immunity. Immunoglobulin-­
secreting plasma cells, together with long-lived, antigen-­
specific memory B cells, are generated in the former area, and
in the latter region, antigen-specific T lymphocytes become
activated, which results in an impressive clonal expansion of
these cells.
6/11/2013 3:50:31 AM
6
Knowles Neoplastic Hematopathology
FIGURE 5.4. Hematoxylin and eosin–stained, paraffin-embedded sections of reactive
lymph node, both with paracortical hyperplasia and secondary follicles.
The impressive variation in the appearance of a lymph node
as a reflection of different pathologic conditions represents
adaptation to the specific type of antigen. In follicular hyperplasia, for example, the B-cell follicles increase in size to respond
to mainly bacterial antigens, whereas in paracortical hyperplasia the T-cell compartment is enlarged in response to antigens
presented by dendritic cells (Fig. 5.5A and B).
The B-Cell Follicle
The B-cell follicle consists of a framework of follicular dendritic cells colonized by B cells, a specific subpopulation of
T cells, and tingible body macrophages (Fig. 5.6A and B). In
nonstimulated lymphoid tissue, only small, mainly round lymphocytes are embedded in this underlying follicular dendritic
cell network. These elementary B follicles are designated as
primary B follicles. Secondary B follicles arise as the result
of antigenic stimulation and can be distinguished from their
unstimulated counterparts by the presence of a well-developed
germinal center or follicle center. As a result, the secondary
follicle consists of the following compartments: the germinal
center, the follicle mantle, and sometimes the marginal zone.
In mesenteric lymph nodes, the spleen, and Peyer patches, this
­ arginal zone is common, but a clear-cut marginal zone is only
m
­occasionally demarcated in other lymph nodes (54); in these
the marginal zone B cells usually occur as a minor population,
­inconspicuously intermingled with the small lymphocytes of the
outer part of the mantle zone (54,55).
Newly formed germinal centers represent oligoclonal B-cell
populations (56–59), because on average each mature germinal
center is derived from only one to three B-cell clones. The germinal center reaction reaches its maximum by day 10 to 12 of
primary immune responses. Without further antigenic stimulation, germinal centers wane by 21 days after immunization.
In the germinal center at least two B-cell types are recognized
morphologically: small irregular cells (i.e., centrocytes) and
large cells (i.e., centroblasts) (Fig. 5.5B). Centrocytes are identified by an ample amount of clear cytoplasm and an irregular,
somewhat elongated nucleus with rather dense nuclear chromatin and inconspicuous nucleoli resulting in a paler staining
part of the germinal center. Centroblasts occupy the remaining
of the follicle center, which stains considerably more intensely
because its large cellular constituents are packed together in
a small area. Centroblasts have a small rim of cytoplasm and
a round nucleus with less condensed chromatin and several
nucleoli located along the nuclear membrane. Germinal centre
cells are characterized by the lack of BCL-2. Centroblasts and
centrocytes, typically lacking this cytoplasmic protein involved
in the protection against apoptosis, are programmed to die,
unless they are rescued by high-affinity interaction between
their antigen receptor and a given antigen. After apoptosis the
cell remnants accumulate in macrophages within the germinal
center, the tingible body macrophages, displaying the classic
phenotype of macrophages as they express neuron-specific
enolase, acid phosphatase, CD11b, CD14, CD68, and HLA-DR
(48,60).
Germinal center cells can also be recognized by their
expression of the nuclear phosphoprotein BCL-6 (61–63). With
its expression strictly confined to the follicle center, at least in
nonneoplastic lymphoid tissues, it has been speculated that
this transcription factor controls the proliferation and differentiation of B cells within the germinal center (64–66).
Following the differentiation scheme of B lymphocytes, IgD+
B lymphocytes that also express CD38 are the first ones to show
the features of a genuine germinal center cell. They are immunoreactive with antibodies directed against CD10, CD71, and
FAS. They are highly proliferative as can be demonstrated by
the expression of Ki-67. Two subpopulations of these IgD+CD38+
B lymphocytes are recognized. An IgM+ subset, called Bm2′,
FIGURE 5.5. A: A primary T-cell reaction is typically visible by light-appearing interdigitating dendritic cells admixed with T lymphocytes
(H&E stain, original magnification: 600× magnification). B: B-cell follicle with germinal centre, defining it as a secondary lymphoid follicle,
surrounded by the mantle zone. No marginal zone can be identified (H&E stain, original magnification: 125× magnification).
Orazi9781609136826-ch05.indd 6
6/11/2013 3:50:35 AM
Chapter 5 Normal Histology of the Lymphoid Tissues7
FIGURE 5.6. A: Immunohistochemical staining for CD79A of the germinal center of a B follicle of a reactive lymph node (original magnification: 250× magnification). Note the different levels of expression with weak staining by germinal centre cells, strong staining of the
mantle B cells and very strong staining of the plasma cells in the germinal center. B: Immunohistochemical staining for CD3 showing
staining of both paracortical and germinal center T cells.
may correspond to germinal center founder cells, because their
immunoglobulin variable genes (VH) carry few or no mutations.
The other IgD+CD38+ cells, which show no IgM expression, display an extraordinarily high number of somatic mutations in
their VH genes. Most of the IgD+CD38+ cells with a high number
of mutations undergo apoptosis, leaving only a small portion
of IgD+ germinal center cells provided with low to intermediate
numbers of mutations. The latter cells eventually differentiate
into mature plasma cells or long-lived memory B cells.
IgD−CD38+ cells represent the main germinal center cell
population, consistently expressing CD10, CD71, and FAS and
demonstrating intense proliferative activity, as well. Also these
germinal centre cells lack BCL-2 and thus are programmed to
die, unless they are selected by an appropriate signal, which
can be provided by a specific antigen or by CD40 ligand. The
two characteristic properties of these follicle center cells—
their impressive mitotic rate and their propensity to undergo
­apoptosis—should both be conferred by their elevated levels of
P53, MYC, BAX, and FAS.
Germinal centers harbor a significant number of CD2+, CD3+,
and CD4+-only T cells (Fig. 5.6B). These granular lymphocytes
co-express CD57 (67) and are CD45RA− and CD45RO+. Although
the specific activation marker CD69 can be demonstrated
on this particular subset of helper T cells, other surface molecules indicating cellular activation, such as CD25 (IL-2 receptor) and CD71 (transferrin receptor), are consistently absent.
This unusual phenotype correlates with the observation that,
on activation, these CD57+ T cells produce various cytokines
but never secrete IL-2, IL-4, interferon-γ, and tumor necrosis factor (67,68). This peculiar T-cell population may reach
its activated state through stimulation by antigen-presenting
B lymphocytes, because complex bidirectional interactions take
place between T lymphocytes on one hand and centroblasts
and centrocytes on the other. Various adhesive and costimulatory receptor-counterreceptor systems are involved, particularly CD80/CD86, CD40, LFA-1, and LFA-3 expressed on B cells
and CD28, CD40 ligand, CD54, and CD2 on the T-cell surface.
Although the primary follicle and the mantle zone of the secondary follicle are mainly composed of morphologically similar small lymphoid cells, the phenotype and function of these
lymphocytes is variable. IgD+CD38− B cells found in the follicle
mantle are small resting B cells expressing IgM, CD5, CD44,
and BCL-2 protein. They correspond to naive B cells as they
have not yet undergone somatic mutation. Based on the differential expression of CD23, two subpopulations of these mantle
Orazi9781609136826-ch05.indd 7
cells can be distinguished. The CD23− subset, designated as
Bm1, probably represents recently generated naive B cells,
and the acquisition of this surface marker by the remainder
of these B lymphocytes, Bm2, could reflect their selection by a
particular ligand.
The marginal zone, when present, is composed predominantly of typical marginal zone B cells, somewhat larger cells
with abundant clear cytoplasm, and an irregular bean-shaped
nucleus containing vesicular chromatin and an inconspicuous
nucleolus. By pure morphology, marginal zone B cells resemble monocytoid cells and centrocytes of the germinal center.
They were previously designated immature sinus histiocytes in
the lymph node and centrocyte-like cells in Peyer patches of
the small intestine. Nevertheless, marginal zone B cells have
a distinct phenotype. Studies demonstrating Ki-67 expression
in marginal zone cells (69) indicate that, in contrast with the
traditional point of view (70), they display proliferative activity. Marginal zone cells express pan-B-cell markers and surface
IgM but little or no IgD. They are not reactive with antibodies against surface CD5, CD10, and CD23. Phenotypic features
frequently used to accentuate these cells include their alkaline
phosphatase positivity and their expression of CD21 and CD25.
The unique positioning at the entrance of the antigenic influx
considerably facilitates their exposure to and interaction with
foreign antigens.
In addition to the characteristic marginal zone cell population, a variable number of different B lymphocytes is observed,
among which larger cells with immunoblast-like features and
plasma cells expressing cytoplasmic IgM can be recognized.
The diversity characterizing the cellular composition of the
marginal zone is reflected in the heterogeneity of marginal zone
cell lymphomas of the lymph node, the spleen, and extranodal
sites (69). Admixed with these peculiar lymphocytic elements,
other cells such as macrophages, granulocytes, and ordinary
small lymphocytes are also detected (71).
The cells of the follicle lay embedded in a cellular lacework
built up by follicular dendritic cells, a unique cell population
exclusively found in primary and secondary lymphoid follicles. These cells stand out by their ability to retain antigens
integrated in large immune complexes on their surface for a
prolonged period (72–74). By routine light microscopy alone,
it takes a considerable effort to identify these cells, because
only by their nucleus, which displays a very open chromatin
pattern in contrast with the rather condensed one observed
in the surrounding lymphoid cells, can they be distinguished
6/11/2013 3:50:38 AM
8
Knowles Neoplastic Hematopathology
from the surrounding cells. Nevertheless, immunohistologic and
­ultrastructural examinations of the B follicle allowed unequivocal detection of these peculiar cells and comprehensible description of their unique features.
Electron microscopy demonstrates that follicular dendritic
cells have one or more large, irregularly shaped nuclei with vesicular chromatin and long cytoplasmic dendrites connected by desmosomes, which together form an intricate network of delicate
processes seeded with lymphocytes. Along the slender cellular
protrusions, small globular structures or iccosomes, representing
immune complex–coated bodies, are observed. By visualizing the
immune complexes bound on their surface, phenotyping of follicular dendritic cells highlights the network they form.
All follicular dendritic cells express the monocytic marker
CD14, the three types of complement receptors—CD35 (CR-1),
the long isoform of CD21 (CR-2), and CD11b (CR-3)—and the
immunoglobulin Fc receptor CD32 (75,76). Displaying the latter receptors on their plasma membrane, the entire population
of follicular dendritic cells is provided with an efficient mechanism to trap passing antigen-antibody-C3 (Ag-Ab-C3) complexes. A subset of the follicular dendritic cells in the light zone
of the germinal center additionally expresses CD23, which is
the low-affinity receptor for IgE, and one of the ligands for
CD21, allowing them to bind complexes containing CD21 with
higher affinity and to interact with IgE.
The T-Cell Area
In contrast to the extensive studies that succeeded in unraveling almost the entire microarchitecture of its B-cell counterpart, the architecture of the T-cell area is less well appreciated.
Moreover, depending on the stage of the immune response or
the particular features of the antigen involved, the morphology
of the T-cell area may vary from a well-delineated nodule with
dendritic cells at the periphery to a less well-defined aggregate composed of a variable number of interdigitating dendritic
cells, with or without an admixture of Langerhans cells, and
T cells. Demarcation of the T-cell area is subject to considerable variation, and its precise cellular composition shows even
greater fluctuations, depending on the particular features of
the antigen involved and on the stage of the immune response.
In contrast with B lymphocytes, T cells cannot be activated
by soluble antigen; they require contact of their TCR with antigenic peptide presented on autologous MHC molecules—MHC
class I for CD8+ T cells and MHC class II for CD4+ T cells. At the
time of antigen recognition, numerous other cognate interactions occur, many of which serve to stabilize the interaction
between the antigen-presenting cell and the T lymphocyte.
Immunohistochemical studies have demonstrated that
CD80 and CD86 are expressed in the T-cell area, which harbors a specific dendritic cell population, the interdigitating dendritic cells. By means of their numerous cytoplasmic processes,
these cells establish a tridimensional network that envelops T
lymphocytes and creates a unique microenvironment for T-cell
activation and proliferation (77). In contrast with follicular
dendritic cells, for which well-developed desmosomes serve as
connection between the dendritic protrusions of different cells,
the cellular extensions of interdigitating dendritic cells join, as
their name indicates, by forming interdigitations. These cells
have abundant, pale-staining cytoplasm encompassing a large,
elongated, bizarre, but very characteristic nucleus. Its outline is
provided with several deep clefts and folds, and it contains very
delicate chromatin and inconspicuous nucleoli (78). These dendritic cells derive from bone marrow monocytes and display
quite similar light microscopic, ultrastructural, and phenotypic
features to Langerhans cells of the epidermis but lack Birbeck
granules, a specific, racquet-shaped cell organelle. Langerhans
cells are known to migrate to the lymph node. The resultant
image of Langerhans cells and interdigitating dendritic cells
Orazi9781609136826-ch05.indd 8
occurring side by side in an extended paracortex is p
­ articularly
prominent in dermatopathic lymphadenitis (79–81) (see Chapter 15). Dendritic cells express CD11c leukocyte integrin, the
DEC-205 multilectin receptor for antigen presentation, very
high levels of MHC class I and MHC class II products, and many
accessory molecules such as CD40, CD54, and CD86 (82). Moreover, they synthesize high levels of IL-2 (83). The dendritic cells
are particularly well equipped to stimulate the growth and activation of a variety of T lymphocytes, including CD8+ cytotoxic
T cells and CD4+ helper T cells. Westermann and his group
(84) demonstrated that, at least in rats, memory T cells migrate
through the T-cell area at a very high rate, and as they continuously recirculate, meanwhile surveying the surface of the interdigitating dendritic cells, they could eventually encounter their
specific antigen. Because mature interdigitating dendritic cells
and completely differentiated Langerhans cells have acquired
the appropriate accessory surface molecules on encounter with
their specific antigen presented by the aforementioned cells,
selected T lymphocytes undergo activation and eventually proliferate intensely. The specific state of the immune response
determines which T cells are predominant in the T-cell area.
In conclusion, the T-cell area of the lymph node contains
a diverse population of dendritic and T lymphocytes r­ eflecting
different stages in their development from immature cells to
potent, well-equipped professional antigen-presenting cells and
immunologic capable T cells.
THE SPLEEN
The spleen is an abdominal organ, situated in the left hypochondrium beneath the diaphragm. The weight of the spleen
varies considerably depending on the age, sex, size, and weight
of the individual. In general, a weight of 150 g is considered
normal.
Histology and Immunohistology
On its freshly sectioned surface, the two components of the
spleen can be distinguished even with the naked eye. Elongated
or rounded gray areas, measuring 0.2 to 0.7 cm in diameter and
called the white pulp, correspond microscopically to accumulations of lymphoid tissue. The reddish, soft mass that they are
embedded in, the red pulp, represents the entire vascular labyrinth that carries the blood along the splenic parenchyma. The
red pulp consists of pulp cords and sinuses. The red pulp cords
contain the arterial branches that gradually branch into arterioles and capillaries; the sinuses form a network that drains into
the veins. The spleen functions as an ingenious filter, intercalated in the bloodstream. Its entire structure is therefore based
on the vascular supply provided by the splenic artery. This
branch of the truncus celiacus perforates the splenic capsule
that completely surrounds the spleen at the hilus to give rise
to two smaller vessels, which further subdivide into segmental
arteries, each supplying one splenic segment.
The arterial branches, together with their concomitant vein
and lymphatics, form a vascular triad embedded in fibrous,
mainly collagenous tissue (85). The arteries end up as smaller
arterioles, which are no longer accompanied by venules and
collagenous fibers but are partially surrounded by a cuff of
lymphoid tissue, the T-cell areas. Subsequently, capillaries,
oriented perpendicularly to the arterioles, branch off and
terminate partially in a specialized vascular structure highly
characteristic for the spleen: the sheathed capillaries or periarteriolar macrophage sheaths.
At this level of the splenic vascularization, the endothelium
of the capillaries is replaced by concentrically arranged macrophages. Blood is forced through these sheathed capillaries and
reaches the sinuses through the cordal stroma of the red pulp
6/11/2013 3:50:38 AM
Chapter 5 Normal Histology of the Lymphoid Tissues9
and after having crossed the basal membrane lining the sinus
endothelium. Alternatively, blood can enter the perifollicular
zone, a distinct part of the red pulp immediately adjacent to the
white pulp that directly gives entrance to the sinuses (86). The
sinusoidal channels, covered by a flattened, elongated endothelial lining, form a blind ending system that debouch into the
veins, which parallel the arteries (85). As a whole, the sinuses
constitute a complex meshwork with many interconnections
and bulblike extensions inside the intersinus reticular tissue,
which are known as the cords of Billroth. These cords contain
reticulum cells, macrophages, and plasma cells, and the predominant population of CD8 positive T cells. Together with the
sinus labyrinth, they account for the main mass of the red pulp,
representing 75% of the splenic weight.
Most blood cells pass through the perifollicular zone, the
region bordering both the follicles and the T-cell areas, that
combined form the white pulp. Microanatomic data on this
region caused confusion because they resulted from studies
on spleens from various species, mainly rodents. In humans
it comprises sheathed capillaries; blood-filled, large flattened
spaces; terminal sinuses; and scattered B cells, T cells, and macrophages (86). Because the perifollicular zone drains directly
into the venous sinuses, most of the splenic blood flow is found
bypassing the filtration beds of the red pulp cords.
The white pulp of the spleen is composed of primary and
secondary B follicles and the T-cell areas that border or surround arterioles. The follicles are very similar to those in the
lymph node, the difference being the more extensive marginal
zone. In contrast to other species, there is no marginal zone
surrounding the T-cell areas. The T-cell areas differ from the
paracortex in the lymph node, since they consist mainly of CD4
positive cells. It is therefore likely that new B-cell follicles arise
in the red pulp, probably in the so-called nonfiltering areas.
These red pulp cord parts are devoid of sheathed capillaries
and contain CD8 positive T cells.
LYMPHOCYTE TRAFFICKING
Lymphocytes not only travel from bone marrow to thymus or
lymph nodes for maturation purposes but also circulate to be
able to react properly to antigens (87). Specific B and T cells
have specific routes for recirculation. For instance, lymphocytes that have matured at a specific extranodal site, that is,
the gut, will home to the stomach after recirculation, due to
down-regulation of L-selectin and CCR7 and expression a4b7
and CCR9 (88). Many of the factors that determine the recirculation process are known, but the picture is still far from
complete. The most important of the molecules are chemokine
receptors and adhesion molecules.
T lymphocytes enter the lymph node paracortex from the
blood by passing the high endothelial venules to encounter
antigen-presenting interdigitating dendritic cells or enter from
the lymph vessels through the marginal sinus after activation
by antigen in tissues. Crucial factors for transport through the
HEV are the chemokines CCL19 and CCL21 that can bind to
the chemokine receptor CCR7 and LFA1 on activated peripheral blood T lymphocytes. More recently, the importance of the
sphingosine-1-phosphate receptor signaling has been demonstrated to be important as well (88). The process is supported
by ICAM1 expression on paracortical stromal cells. B lymphocytes and follicular helper T cells expressing CCR5 and LFA1
are attracted by CXCL13, ICAM1, and VCAM1 on the follicular
dendritic cells (89). In the spleen, HEV are lacking, but similar
molecules are expressed on parts of the red pulp cord sinuses
(90). After encountering antigen-presenting cells in the T-cell
area, the T lymphocytes are trapped and activated, resulting
in the large expansion of the paracortical area one commonly
encounters in reactive lymph nodes.
Orazi9781609136826-ch05.indd 9
References
1. Kendall MD, Johnson HR, Singh J. The weight of the human thymus gland at
necropsy. J Anat 1980;131:483–497.
2. Rosai J, Levine GD. Nonneoplastic conditions of the thymus. In: Rosai J, Levine
G, eds. Tumors of the thymus, vol 13. Washington, DC: Armed Forces Institute of
Pathology, 1976:22–33.
3. Calder AE, Hince MN, Dudakov JA, et al. Thymic involution: where endocrinology
meets immunology. Neuroimmunomodulation 2011;18:281–289.
4. von Gaudecker B, Muller Hermelink HK. Ontogeny and organization of the stationary non-lymphoid cells in the human thymus. Cell Tissue Res 1980;207:287–306.
5. Nakahama M, Mohri N, Mori S, et al. Immunohistochemical and histometrical
studies of the human thymus with special emphasis on age-related changes in
medullary epithelial and dendritic cells. Virchows Arch B Cell Pathol Incl Mol
Pathol 1990;58:245–251.
6. Flores KG, Sempowski GD, Haynes BF, et al. Analysis of the human thymic perivascular space during aging. J Clin Invest 1999;104:1031–1039.
7. von Gaudecker B. The development of the human thymus microenvironment.
Curr Top Pathol 1986;75:1–41.
8. Wekerle H, Ketelsen UP, Ernst M. Thymic nurse cells. Lymphoepithelial cell
complexes in murine thymuses: morphological and serological characterization.
J Exp Med 1980;151:925–944.
9. Hiramine C, Nakagawa T, Hojo K. Murine nursing thymic epithelial cell lines
capable of inducing thymocyte apoptosis express the self-superantigen Mls-1a.
Cell Immunol 1995;160:157–162.
10. Drenckhahn D, von Gaudecker B, Muller Hermelink HK, et al. Myosin and actin
containing cells in the human postnatal thymus. Ultrastructural and immunohistochemical findings in normal thymus and in myasthenia gravis. Virchows Arch
B Cell Pathol Incl Mol Pathol 1979;32:33–45.
11. Kao I, Drachman DB. Thymic muscle cells bear acetylcholine receptors: possible
relation to myasthenia gravis. Science 1977;195:74–75.
12. Moll UM, Lane BL, Robert F, et al. The neuroendocrine thymus. Abundant occurrence of oxytocin-, vasopressin-, and neurophysin-like peptides in epithelial cells.
Histochemistry 1988;89:385–390.
13. Geenen V, Legros JJ, Franchimont P, et al. The neuroendocrine thymus: coexistence
of oxytocin and neurophysin in the human thymus. Science 1986;232:508–511.
14. Schmitt D, Monier JC, Dardenne M, et al. Location of FTS (facteur thymique serique)
in the thymus of normal and auto-immune mice. Thymus 1982;4:221–231.
15. von Gaudecker B. Functional histology of the human thymus. Anat Embryol (Berl)
1991;183:1–15.
16. Bhan AK, Reinherz EL, Poppema S, et al. Location of the T-cell and the
major histocompatibility complex antigens in the human thymus. J Exp Med
1980;152:771–782.
17. Naito T, Tanaka H, Naoe Y, et al. Transcriptional control of T-cell development. Int
Immunol 2011;23:661–668.
18. Billiard F, Kirshner JR, Tait M, et al. Ongoing Dll4-Notch signaling is required for
T-cell homeostasis in the adult thymus. Eur J Immunol 2011;41:2207–2216.
19. Spits H, Lanier LL, Phillips JH. Development of human T and natural killer cells.
Blood 1995;85:2654–2670.
20. Peschon JJ, Morrissey PJ, Grabstein KH, et al. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med
1994;180:1955–1960.
21. Conlon PJ, Morrissey PJ, Nordan RP, et al. Murine thymocytes proliferate in
direct response to interleukin-7. Blood 1989;74:1368–1373.
22. Watson JD, Morrissey PJ, Namen AE, et al. Effect of IL-7 on the growth of fetal
thymocytes in culture. J Immunol 1989;143:1215–1222.
23. Matsuzaki Y, Gyotoku J, Ogawa M, et al. Characterization of c-kit positive intrathymic stem cells that are restricted to lymphoid differentiation. J Exp Med
1993;178:1283–1292.
24. Rodewald HR, Kretzschmar K, Swat W, et al. Intrathymically expressed c-kit
ligand (stem cell factor) is a major factor driving expansion of very immature
thymocytes in vivo. Immunity 1995;3:313–319.
25. Veis DJ, Sentman CL, Bach EA, et al. Expression of the Bcl-2 protein in
murine and human thymocytes and in peripheral T lymphocytes. J Immunol
1993;151:2546–2554.
26. Raulet DH, Garman RD, Saito H, et al. Developmental regulation of T cell receptor
gene expression. Nature 1985;314:103–107.
27. Groettrup M, Ungewiss K, Azogui O, et al. A novel disulfide-linked heterodimer
on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein.
Cell 1993;75:283–294.
28. Saint Ruf C, Ungewiss K, Groettrup M, et al. Analysis and expression of a cloned
pre-T cell receptor gene. Science 1994;266:1208–1212.
29. Philpott KL, Viney JL, Kay G, et al. Lymphoid development in mice congenitally
lacking T cell receptor alpha beta-expressing cells. Science 1992;256:1448–1452.
30. Uematsu Y, Ryser S, Dembic Z, et al. In transgenic mice the introduced functional
T cell receptor beta gene prevents expression of endogenous beta genes. Cell
1988;52:831–841.
31. Crump AL, Grusby MJ, Glimcher LH, et al. Thymocyte development in major
histocompatibility complex–deficient mice: evidence for stochastic commitment
to the CD4 and CD8 lineages. Proc Natl Acad Sci U S A 1993;90:10739–10743.
32. von Boehmer H. Positive selection of lymphocytes. Cell 1994;76:219–228.
33. Nossal GJ. Negative selection of lymphocytes. Cell 1994;76:229–239.
34. Allen PM. Peptides in positive and negative selection: a delicate balance. Cell
1994;76:593–596.
35. Ashton Rickardt PG, Tonegawa S. A differential-avidity model for T cell selection.
Immunol Today 1994;15:362–366.
36. Anderson G, Owen JJ, Moore NC, et al. Thymic epithelial cells provide unique
signals for positive selection of CD4 +CD8+ thymocytes in vitro. J Exp Med
1994;179:2027–2031.
37. Mazda O, Watanabe Y, Gyotoku J, et al. Requirement of dendritic cells and B
cells in the clonal deletion of Mls-reactive T cells in the thymus. J Exp Med
1991;173:539–547.
38. Fend F, Nachbaur D, Oberwasserlechner F, et al. Phenotype and topography of
human thymic B cells: an immunohistologic study. Virchows Arch B Cell Pathol
Incl Mol Pathol 1991;60:381–388.
39. Isaacson PG, Norton AJ, Addis BJ. The human thymus contains a novel population of B lymphocytes. Lancet 1987;2:1488–1491.
6/11/2013 3:50:38 AM
10
Knowles Neoplastic Hematopathology
40. Spencer J, Choy M, Hussell T, et al. Properties of human thymic B cells. Immunology
1992;75:596–600.
41. Punnonen J, de Vries JE. Characterization of a novel CD2+ human thymic B cell
subset. J Immunol 1993;151:100–110.
42. Nieuwenhuis P, Stet RJ, Wagenaar JP, et al. The transcapsular route: a new way
for (self-) antigens to bypass the blood-thymus barrier? Immunol Today 1988;9:
372–375.
43. Bannister L, Kendall M. Lymphoid cells and tissues: thymus. In: Williams PL,
Bannister LH, Berry MM, et al., eds. Gray’s anatomy, 38th ed. New York:
Churchill Livingstone, 1995:1423–1431.
44. Fossum S, Ford WL. The organization of cell populations within lymph nodes: their
origin, life history and functional relationships. Histopathology 1985;9:469–499.
45. Sainte-Marie G, Peng FS, Belisle, C. Overall architecture and pattern of lymph
flow in the r at lymph node. Am J Anat 1982;164:275–309.
46. Kurokawa T, Ogata T. A scanning electron microscopic study on the lymphatic
microcirculation of the rabbit mesenteric lymph node: a corrosion cast study.
Acta Anat 1980;107:439–466.
47. Castenholz A. Architecture of the lymph node with regard to its function. In:
Grundmann E, Vollmer E, eds. Reaction patterns of the lymph node, vol 1. Berlin:
Springer-Verlag, 1990:1–32.
48. Wacker HH, Frahm SO, Heidebrecht HJ, et al. Sinus-lining cells of the lymph
nodes recognized as a dendritic cell type by the new monoclonal antibody Ki-M9.
Am J Pathol 1997;151:423–434.
49. Peters J, Krams M, Wacker HH, et al. Detection of rare RNA sequences by single
enzyme in situ RT-PCR: high resolution analysis of interleukin-6 mRNA in paraffin sections of lymph nodes. Am J Pathol 1996;150:469–476.
50. Umemoto E, Hayasaka H, Bai Z, et al. Novel regulators of lymphocyte trafficking
across high endothelial venules. Crit Rev Immunol 2011;31:147–169.
51. Kehrl JH, Hwang IY, Park C. Chemoattract receptor signaling and its role in lymphocyte motility and trafficking. Curr Top Microbiol Immunol 2009;334:107–127.
52. Förster R, Davalos-Misslitz AC, Rot A. CCR7 and its ligands: balancing immunity
and tolerance. Nat Rev Immunol 2008;8:362–371.
53. Matsuno K, Ueta H, Shu Z, et al. The microstructure of secondary lymphoid
organs that support immune cell trafficking. Arch Histol Cytol 2010;73:1–21.
54. van Krieken JH, von Schilling C, Kluin PM, et al. Splenic marginal zone lymphocytes and related cells in the lymph node: a morphologic and immunohistochemical study. Hum Pathol 1989;20:320–362.
55. van den Oord JJ, de Wolf Peeters C, Desmet VJ. The marginal zone in the human
reactive lymph node. Am J Clin Pathol 1986;86:475–479.
56. Liu YJ, Johnson GD, Gordon J, et al. Germinal centers in T cell–dependent
­antibody responses. Immunol Today 1992;13:17–21.
57. Jacob J, Kassir R, Kelsoe G. In situ studies of the primary immune response to
(4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding
cell populations. J Exp Med 1991;173:1165–1175.
58. Jacob J, Miller C, Kelsoe G. In situ studies of the antigen-driven somatic hypermutation of immunoglobulin genes. Immunol Cell Biol 1992;70:145–152.
59. Jacob J, Kelsoe G. In situ studies of the primary immune response to (4-hydroxy3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid
sheath-associated foci and germinal centers. J Exp Med 1992;176:679–687.
60. Kroese FGM, Timens W, Niewenhuis P. Germinal center reaction and B lymphocytes: morphology and function. In: Grundmann E, Vollmer E, eds. Reaction patterns of the lymph node, vol 1, part 1. Berlin: Springer-Verlag, 1990:116–117.
61. Allman D, Jain A, Dent A, et al. BCL-6 expression during B cell activation. Blood
1996;87:5257–5268.
62. Cattoretti G, Chang CC, Cechova K, et al. BCL-6 protein is expressed in germinalcenter B cells. Blood 1995;86:45–53.
63. Onizuka T, Moriyama M, Yamochi T, et al. BCL-6 gene product, a 92- to 98-kD
nuclear phosphoprotein, is highly expressed in germinal center B cells and their
neoplastic counterparts. Blood 1995;86:28–37.
64. Ye BH, Cattoretti G, Shen Q, et al. The BCL-6 proto-oncogene controls germinalcenter formation and Th2-type inflammation. Nat Genet 1997;16:161–170.
65. Dent AL, Shaffer AL, Yu X, et al. Control of inflammation, cytokine expression,
and germinal center formation by BCL-6. Science 1997;276:589–592.
Orazi9781609136826-ch05.indd 10
66. Pittaluga S, Ayoubi TA, Wlodarska I, et al. BCL-6 expression in reactive lymphoid
tissue and in B cell non-Hodgkin’s lymphomas. J Pathol 1996;179:145–150.
67. Velardi A, Mingari MC, Moretta L, et al. Functional analysis of cloned germinal
center CD4+ cells with natural killer cell–related features: divergence from typical
T helper cells. J Immunol 1986;137:2808–2813.
68. Bowen MB, Butch A, Parvin CA, et al. Germinal center T cells are distinct helperinducer T cells. Hum Immunol 1991;31:67–75.
69. Tierens A, Delabie J, Michiels L, et al. Marginal zone B cells in the human lymph
node and spleen show somatic hypermutation and display clonal expansion.
Blood 1999;93:226–234.
70. Liu YJ, Oldfield S, MacLennan IC. Memory B cells in T cell–dependent antibody
responses colonize the splenic marginal zones. Eur J Immunol 1988;18:355–362.
71. Kraal G. Cells in the marginal zone of the spleen. Int Rev Cytol 1992;132:31–74.
72. Kaplan MH, Coons AH, Deane HW. Localization of antigen in tissue cells. III. Cellular distribution of pneumococcal polysaccharides types II and III in the mouse.
J Exp Med 1950;91:15–29.
73. Szakal AK, Gieringer RL, Kosco MH, et al. Isolated follicular dendritic cells: cytochemical antigen localization, Nomarski, SEM, and TEM morphology. J Immunol
1985;134:1349–1359.
74. Nossal GJ, Abbot A, Mitchell J, et al. Antigens in immunity. XV. Ultrastructural
features of antigen capture in primary and secondary lymphoid follicles. J Exp
Med 1968;127:277–290.
75. Tew JG, Kosco MH, Burton GF, et al. Follicular dendritic cells as accessory cells.
Immunol Rev 1990;117:185–211.
76. Dijkstra CD, Van den Berg TK. The follicular dendritic cell: possible regulatory
roles of associated molecules. Res Immunol 1991;142:227–231.
77. Crivellato E, Baldini G, Basa M, et al. The three-dimensional structure of interdigitating cells. Ital J Anat Embryol 1993;98:243–258.
78. van der Valk P, Meijer CJ. The histology of reactive lymph nodes. Am J Surg
Pathol 1987;11:866–882.
79. van den Oord JJ, de Wolf-Peeters C, Desmet VJ, et al. Nodular alteration of the
paracortical area. An in situ immunohistochemical analysis of primary, secondary, and tertiary T nodules. Am J Pathol 1985;120:55–66.
80. van den Oord JJ, de Wolf Peeters C, de Vos R, et al. The paracortical area in
dermatopathic lymphadenitis and other reactive conditions of the lymph node.
Virchows Arch 1984;45:289–299.
81. Gould E, Porto R, Albores Saavedra J, et al. Dermatopathic lymphadenitis: the
spectrum and significance of its morphologic features. Arch Pathol Lab Med
1988;112:1145–1150.
82. Inaba K, Pack M, Inaba M, et al. High levels of a major histocompatibility complex
II–self peptide complex on dendritic cells from the T cell areas of lymph nodes.
J Exp Med 1997;186:665–672.
83. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature
1998;392:245–252.
84. Westermann J, Geismar U, Sponholz A, et al. CD4+ T cells of both the naive and
the memory phenotype enter rat lymph nodes and Peyer’s patches via high endothelial venules: within the tissue their migratory behavior differs. Eur J Immunol
1997;27:3174–3181.
85. van Krieken JH, te Velde J. Normal histology of the human spleen. Am J Surg
Pathol 1988;12:777–785.
86. Steiniger B, Barth P, Herbst B, et al. The species-specific structure of miroanatomical compartments in the human spleen: strongly sialoadhesin-positive macrophages occur in the perifollicular zone, but not in the marginal zone. Immunology
1997;92:307–316.
87. Matsuno K, Ueto H, Shu Z, et al. The microstructure of the secondairy lymphoid
organs that support immune cell trafficking. Arch Histol Cytol 2010;73:1–21.
88. Davis MD, Kehrl JH. The influence of sphingosine-1-phosphate receptor signaling on lymphocyte trafficking. Immunol Res 2009;43:187–197.
89. Mora JR, von Adrian UH. T-cell homing specificity and plasticity: new concepts
and future challenges. Trends Immunol 2006;27:235–243.
90. van Krieken JH, Te Velde J, Kleiverda K, et al. The human spleen; a histological
study in splenectomy specimens embedded in methylmethacrylate. Histopathology
1985;9:571–585.
6/11/2013 3:50:38 AM