REVIEW ARTICLE

Histologic and Molecular Assessment of Human Thymus

Laura P. Hale, MD, PhD
Most work describing the histopathology of normal human thymus has focused on
pediatric thymus because of tissue availability and high thymopoietic activity.
However, pathologic examination of the thymus can provide information about
immune status that is relevant to the clinical care of patients of all ages. Under-
standing age-related changes in the relative abundance and composition of anatomic
compartments within the thymus is critical for evaluation of the thymus in normal
adults and patients with diseases that affect the thymus. The purpose of this review
is to acquaint diagnostic pathologists with some of the newer histologic, flow
cytometric, and molecular techniques for assessment of non-neoplastic thymus.
Diagnostic criteria are presented for assessment of thymic function and for deter-
mining the mechanisms underlying thymic hyperplasia. Accurate assessment of
thymic function is critical for the diagnosis and treatment of patients with complete
DiGeorge syndrome and can complement the clinical care of patients with a variety
of disorders that affect the immune system.
Ann Diagn Pathol 8: 50-60, 2004. © 2004 Elsevier Inc. All rights reserved.

E XAMINATION of the thymus is an important

part of pediatric autopsies, particularly those
with a history suggestive of immunodeficiency. Al-
though the thymus is rarely examined in adult
autopsies, it could potentially provide important
information regarding the patient’s immune status,
which might help improve the clinical care of fu-
ture patients. Thymus specimens are also submit-
ted to the surgical pathology service. Patients with
myasthenia gravis (MG) may undergo therapeutic
thymectomy, a procedure that can lead to remis-
sion of their autoimmune disease or to decreased
need for treatment with immunosuppressive
agents. Thymus biopsies may be obtained to rule
out malignancy in patients with symptomatic or
radiographically identified thymic enlargement or
masses. Portions of the thymus may be removed at
the time of cardiovascular surgery, either to expose
the operative site or to diagnose perceived discor-
dances in age and thymus cellularity. Thymus tis-
sues may also be removed at the time of surgery for
benign and malignant conditions of the thyroid or
From the Department of Pathology, Duke University Medical Center,
Durham, NC.
Address reprint requests to Laura P. Hale, MD, PhD, Box 3712,
Duke University Medical Center, Durham, NC 27710.
© 2004 Elsevier Inc. All rights reserved.
1092-9134/04/0801-0010$30.00/0
doi:10.1016/j.anndiagpath.2003.11.006
parathyroid, to ensure appropriate surgical mar-
gins.
It is of paramount importance to rule out the
presence of neoplasia in thymic specimens. Thymic
epithelial neoplasms (thymoma and thymic carci-
noma), lymphomas, and neuroendocrine or germ
cell tumors make up the majority of thymic neo-
plasms. Metastatic tumors are extremely rare. Ex-
cellent reviews are available regarding the diagnos-
tic histopathology and clinical outcome of thymic
neoplasms.1-7 However, beyond the presence or
absence of neoplasia, thymus tissues provide infor-
mation about immune status that is relevant to the
clinical care of patients of all ages. The purpose of
this review is to acquaint diagnostic pathologists
with some of the newer histologic and molecular
techniques for assessment of non-neoplastic thy-
mus tissues. Such techniques are critical for the
diagnosis and treatment of patients with complete
DiGeorge syndrome,8 and can complement the
clinical care of patients with a variety of disorders
that affect the immune system.
Age-Related Changes in Thymus Histology
and Function
The thymus has traditionally has been consid-
ered to be composed of a cortex and a medulla,
which contain developing thymocytes within a net-
work of thymic epithelial and stromal cells. Hassall
bodies composed of swirls of terminally differenti-
ated thymic epithelial cells9-12 are present in the

50 Annals of Diagnostic Pathology, Vol 8, No 1 (February), 2004: pp 50-60

 Assessment of Human Thymus
medulla and are a universally recognized histologic
indicator of the thymus. Because they both contain
thymic epithelial cells, the cortex and medulla can
also be referred to together as the thymic epithelial
space. The perivascular space is defined as the
portion of tissue that is within the thymic capsule
but outside of the thymic epithelial network.13,14 In
infant thymus, the perivascular space is essentially a
virtual space containing only the thymic blood ves-
sels and thus has largely been ignored. However,
the perivascular space becomes more prominent
with aging. Understanding the role of thymic epi-
thelial and perivascular compartments in T-cell
production (thymopoiesis) and immune reactions
is critical for accurate pathologic evaluation of non-
neoplastic adult thymus.
The thymic epithelial compartment is the site of
T-lymphocyte maturation within the thymus. Stem
cells migrate from the bone marrow to the thymus,
where they undergo rearrangement of their T-cell
receptor genes to create functional T-cell receptors
with unique specificities. Developing T cells inter-
act with thymic epithelium and/or stromal cells to
achieve positive selection of cells that recognize
antigen in the context of self-major histocompati-
bility complex antigens and negative selection to
delete cells with inappropriate autoreactivity. Fi-
nally, the thymocytes acquire cell surface markers
typical of mature T cells and are released into the
peripheral T-cell pool as new naive T cells.
The thymus is absolutely required to establish a
functional T-cell repertoire. Animal models of athy-
mia (eg, athymic “nude” mice with mutation of the
whn, winged helix-nude gene15) have been exten-
sively used in medical research because their pro-
found immunodeficiency prevents rejection of xe-
nogeneic cells and tissue. Human children with
complete DiGeorge syndrome have a developmen-
tal defect that results in total absence of thymus
tissue. These children are also profoundly immu-
nodeficient and typically die of infection before 2
years of age unless immune reconstituted by thymic
transplantation is achieved.16 Consistent with its
role in establishing the initial T-cell repertoire, the
human thymus is most active in T-cell production
early in life. Most work describing the histopathol-
ogy and function of normal human thymus has
focused on pediatric thymus because of tissue avail-
ability and high thymopoietic activity. However, the
thymus rapidly begins to undergo age-related de-
creases in activity, beginning in early childhood.
51
Although total thymic weight and volume remain
relatively constant,13 the portion of the thymus that
contains thymic epithelium and participates in pro-
duction of new T cells decreases progressively with
age (Fig 1).13,14,17,18 The thymic perivascular space
increases correspondingly, with prominent infil-
trates of peripheral lymphocytes and adipose tissue.
The density of lymphocytes in the perivascular
space is typically very similar to that of the adjacent
medulla. However, changes in proportion of thy-
mic epithelial and perivascular spaces can be high-
lighted using an immunostain (eg, cytokeratin) to
identify thymic epithelium. Use of a hematoxylin-
eosin counterstain (rather than hematoxylin
alone) with the cytokeratin immunostain is partic-
ularly useful for identifying changes in thymic com-
partments (Fig 1). Many of the lymphocytes
present within the thymic perivascular space have a
phenotype consistent with cytotoxic or memory T
cells (CD4 or CD8⫹, CD45RO [memory T cell]⫹,
TIA-1 [cytotoxic granule]⫹, CD38⫺)14 and thus
have migrated to the thymic perivascular space
from the periphery. A subset of B cells present in
the perivascular space have somatic mutations in
immunoglobulin genes, showing their previous
participation in germinal center reactions.19 High
endothelial venules that express the ligand for the
CD62L cell surface molecule used by lymphocytes
to home to lymph nodes are present in thymic
perivascular spaces that contain prominent infil-
trates of T and B lymphocytes.14 Therefore, the
adult thymus can be viewed as a chimeric organ,
with a thymic epithelial compartment that pro-
duces new T lymphocytes and a perivascular com-
partment that participates in recirculation of pe-
ripheral lymphocytes similar to a lymph node.20 As
aging progresses, lymphocyte infiltrates into the
perivascular space decrease and adipose tissue in-
filtration becomes prominent (Fig 1). However,
despite these age-related changes in thymic com-
position, we and others have shown that thymopoi-
esis continues to occur in the thymus of human
adults into late in life.14,21-23 Foci of immature
thymocytes undergoing development can be iden-
tified even in thymus tissues that grossly and micro-
scopically consist primarily of adipose tissue (Fig 2,
see below). Understanding age-related changes in
relative abundance and composition of thymic
compartments is critical to evaluating thymus his-
topathology of normal adults and patients with
diseases that affect the thymus.
52 Laura P. Hale

Figure 1. Age-related changes in the

human thymus. Total thymic size re-
mains relatively constant with aging,
but the fraction that contains thymic
epithelium and participates in thymo-
cyte production (pink) decreases pro-
gressively as shown. Thymus sections
stained with cytokeratin immunoperox-
idase plus hematoxylin-eosin demon-
strate corresponding changes in rela-
tive amounts of cortex (C), medulla (M),
and perivascular space (P) with aging.
(Low-power view.)

Figure 2. Aged thymus. The perivascular space contains abun-

dant adipose tissue and sparse infiltrates of mature lymphocytes
in this thymus from a 78-year-old woman. The oval region indi-
cated by the arrow contains a small focus of thymopoiesis, with
phenotypically immature thymocytes located within a light, lacy
network of thymic epithelial cells. (Scanning power.)

Figure 3. Histologic evaluation of thy-

mic function. Thymus from a 6-month-
old male infant shows robust thymopoi-
esis, with a light lacy cytokeratin
network, abundant CD1a⫹ mib-1⫹
small lymphocytes, and non-calcified,
non-cystic Hassall bodies (indicated by
arrowheads). (A) Hematoxylin-eosin.
(Scanning power.) (B) Cytokeratin im-
munoperoxidase (mAb clones AE1 ⫹
AE3). (Medium-power magnification.)
(C) CD1a immunoperoxidase (mAb
clone O10). (Medium-power magnifica-
tion.) (D) Ki-67 immunoperoxidase
(mAb clone mib-1). (Medium-power
magnification.) C, cortex; M, medulla.
 Assessment of Human Thymus
Histologic and Flow Cytometric Assessment of
Thymic Function
A large number of research studies in both ani-
mals and humans have determined the phenotype
of immature and mature thymocytes and periph-
eral lymphocytes and have established appropriate
cell surface markers that distinguish between them.
The most immature thymocytes (“triple negative
thymocytes”) do not express CD3, CD4, or CD8,
although they typically express other lymphocyte
markers such as CD45 and CD7. As these thymo-
cytes begin to undergo T-cell receptor gene rear-
rangement, they acquire cytoplasmic then cell sur-
face expression of CD3 and co-expression of CD4
and CD8. CD4⫹CD8⫹ “double positive” thymocytes
are located within the thymic cortex. Double posi-
tive thymocytes additionally express CD1a on their
surface (Fig 3C). They are undergoing rapid clonal
expansion and thus also react with mib-1 mAb that
recognizes the Ki-67 nuclear proliferation antigen
(Fig 3D). As double positive thymocytes complete
T-cell receptor gene rearrangement and undergo
positive and negative selection, they lose expres-
sion of both CD1a and Ki-67 and either CD4 or
CD8. The resulting CD4⫹ or CD8⫹ (“single posi-
tive”) thymocytes migrate into the thymic medulla
where they complete their maturation before they
are released to the periphery as naive mature T
cells. Knowledge of this developmental sequence
allows identification of ongoing thymopoiesis ei-
ther by multi-color flow cytometry or by immuno-
histologic analysis of tissue sections.
Multicolor flow cytometry is an ideal way to iden-
tify developing thymocytes within suspensions of
cells mechanically dissociated from thymic tissue.
Lymphocytes can be identified by their character-
istic forward scatter and side scatter properties.
Double positive immature thymocytes can then be
identified by their reactivity with both CD4 and
CD8 mAbs (Fig 4, upper right quadrant). The
CD4⫺CD8⫺ population (Fig 4, lower left quadrant)
contains the most immature triple negative thymo-
cytes as well as thymic B cells. Cells reactive with
either CD4 or CD8, but not both (Fig 4, upper left
and lower right quadrants), represent the more
mature medullary thymocytes as well as any periph-
eral T lymphocytes present. The majority (typically
80% to 85%) of lymphocytes present in pediatric
thymus have a CD4⫹CD8⫹ phenotype. Adult thy-
mus typically has a lower percentage of CD4⫹CD8⫹
53
Figure 4. Flow cytometric determination of thymocyte sub-
sets. Cells mechanically dissociated from thymus tissue from a
3-month-old female infant are reacted with fluorescently labeled
CD4 and CD8 mAbs and analyzed by multicolor flow cytometry.
The relative fluorescence with CD4-phycoerythrin (PE) versus
CD8-phycoerythrin-cyanin 5.1 (Cy5) antibodies for the lympho-
cytes gated is presented as a dot plot. Immature thymocytes
make up the bulk of the cells in this thymus with high thymo-
poietic activity. These cells co-express CD4 and CD8 surface
molecules and appear in the upper right quadrant of the graph.
lymphocytes because of the increased numbers of
thymic B cells (detected as CD4⫺CD8⫺ lympho-
cytes) and infiltration of CD4⫹ or CD8⫹ single
positive peripheral T cells into the thymic perivas-
cular space.14,19). Triple color flow cytometry may
be used for more precise identification of T-cell
subsets, using reactivity with CD3 mAb to identify
the lymphocyte population that will be analyzed for
reactivity with CD4 and CD8 mAbs.
Thymic tissues actively participating in thymopoi-
esis have a light lacy pattern of cytokeratin-positive
thymic epithelial cells that allows epithelial cells to
interact with multiple thymocytes (Fig 3B). The
presence of islands of condensed thymic epithelial
cells is characteristic of inactive thymus. Phenotyp-
ically immature (eg, CD1a⫹ mib-1⫹) thymocytes
are present within the thymic epithelial network in
active thymus (Fig 3C and D). In addition, we have
found that tissues with robust thymopoiesis that is
confirmed by flow cytometric or molecular meth-
ods also typically contain non-cystic, non-calcified
Hassall bodies (Fig 3). Thus thymic tissues with: (1)
a light lacy pattern of cytokeratin-positive thymic
epithelial cells; (2) CD1a⫹ mib-1⫹ small lympho-
54 Laura P. Hale
cytes; and (3) non-calcified, non-cystic Hassall bod-
ies, can be said to have histologic evidence for
thymopoiesis.
Molecular Analysis of Thymic Function
T-cell receptor gene rearrangement is a defining
event of T-cell development within the thymus.
Molecular techniques for assessing thymic function
can be divided into two types: those that analyze
thymic tissue directly for the presence of ongoing
T-cell receptor gene rearrangement and those that
analyze peripheral T cells for evidence of prior
T-cell rearrangement within the thymus.
To create T-cell receptors with unique specifici-
ties, concerted DNA breakage and repair occurs at
specific recombination signal sequences within the
T-cell receptor gene under the direction of a re-
combinase enzyme complex (Fig 5). A ligation-
mediated polymerase chain reaction (PCR) assay
can be used to directly quantitate the DNA breaks
that occur at specific loci within T-cell receptor
genes during thymocyte development. DNA is ex-
tracted from thymocytes or thymus tissue and a
double-stranded linker oligonucleotide is ligated to
all DNA breaks present in the sample. Breaks (now
tagged by the ligated linker) located adjacent to
T-cell receptor loci are identified by nested PCR
using primers specific for the linker and the T-cell
receptor locus of interest (Fig 5). Ligation-medi-
Figure 5. Ligation-mediated PCR de-
tects DNA breaks because of ongoing
T-cell receptor gene (TCR) rearrange-
ment. Breaks present in DNA isolated
from thymocytes are tagged by ligation
with a linker oligonucleotide (hatched
pattern). Breaks specifically associated
with recombinase-mediated TCR gene
rearrangement are identified by nested
PCR using primers (indicated by small
horizontal arrows) specific for both the
linker and for TCR gene sequences.
Ligation-mediated PCR is specific for
ongoing TCR rearrangement (lanes 1
and 3 represent DNA from two different
thymus donors). No signals are pro-
duced when the DNA lacks TCR loci
(lane 5 from bacterial DNA), contains
TCR loci that are not undergoing rear-
rangement (eg, spleen or tonsil DNA,
not shown), or when the ligation step is
omitted (lanes 2, 4, and 6).
ated PCR has been used to directly confirm the
presence of ongoing T-cell receptor gene rear-
rangement in adult thymus tissues.14
Surrogate measures of thymopoiesis have been
developed to allow assessment of thymic function
without the need for surgery to procure thymic
tissue. The DNA excised from the chromosome as
T-cell receptor gene rearrangement occurs within
the thymus is retained as an episome (DNA not
associated with a chromosome) within the develop-
ing T cell.24 These episomes, termed T cell recep-
tor excised circles or TRECs, cannot replicate and
are lost by dilution when the T cells containing
them undergo clonal expansion after encountering
antigen in the periphery. T cells that arise in the
periphery via clonal expansion are thus TREC-
negative. TREC-positive peripheral cells are either
newly developed T cells that have recently emi-
grated from the thymus or they are long-lived naive
T cells that have never encountered antigen or
undergone clonal expansion. TRECs present
within DNA derived from specific cell populations
(peripheral blood mononuclear cells [PBMCs],
CD3⫹ T cells, etc) can be quantitated by quantita-
tive competitive or real-time PCR assays.25 These
assays detect the sequence at the joint where the
two ends of the excised previously linear DNA have
been circularized to form the TREC episome (Fig
6). The number of TRECs per ␮g PBMC DNA has
 Assessment of Human Thymus 55

Figure 6. The TREC assay provides

a surrogate measure of thymic func-
tion. (A) T-cell receptor excision circles
are generated when a portion of the
TCR ␦ locus is excised before rear-
rangement of the TCR ␣ gene within
the thymus. A quantitative competitive
or real-time PCR assay that recognizes
unique sequences at the site of circu-
larization (horizontal arrow) can be
used to detect TRECs that are present
in PBMC. (B) The age-related de-
crease in the proportion of TREC⫹
PBMCs shown parallels the decrease
in thymus volume devoted to thymo-
cyte production as measured histologi-
cally.14,18 A portion of this data was
presented previously by Patel et al.31

been validated as a surrogate marker for thymopoi-
esis in aging normal patients (Fig 6),18 patients
with MG,18,26 and following immune reconstitution
therapy after human immunodeficiency virus
(HIV) infection,21,25,27-29 cancer chemotherapy
and umbilical cord blood transplantation,30 and
transplant of bone marrow or thymus to treat
congenital immunodeficiencies.8,31 The absolute
number and frequency of naive phenotype
(CD3⫹CD45RA⫹ CD62Lhigh) T cells in peripheral
blood is often measured by multicolor flow cytom-
etry in conjunction with TREC analysis because
most cells with this phenotype have undergone
thymic development. T cells generated by clonal
expansion in the periphery typically express the
RO isoform of CD45 recognized by the mAb
UCHL-1.
Because the thymus is responsible for establish-
ing the T-cell repertoire, its function can also be
assessed by examination of the diversity of T-cell
receptors present in peripheral blood T cells. Ran-
dom addition or subtraction of nucleotides at the
ends of rearranging T-cell receptor gene segments
through the action of terminal deoxynucleotide
transferase or deoxynucleotidases creates families
of T-cell receptors that share the same variable (V)
regions but have differing numbers of amino acids
in their antigen recognition sites (complementarity
determining regions), corresponding to each
group of three added or subtracted nucleotides.
The diversity of the T-cell repertoire can be deter-
mined by PCR measurement of V segment length.
cDNA derived from peripheral blood T cells is
amplified with fluorescently labeled primers that
recognize specific V segments and the constant
region of the T-cell receptor gene. Polymerase
chain reaction product length is determined using
DNA sequencing gels with fluorescent detection.
This assay has been called the “immunoscope”
technique.32 T-cell repertoires of normal diversity
have a Gaussian distribution of receptor lengths,
with an average of 6 to 10 peaks for each V segment
family (Fig 7C). Oligoclonal distributions with ⱕ 4
peaks or polyclonal non-Gaussian “skewed” distri-
butions (Figure 7A, B) are associated with immu-
nodeficiency and susceptibility to infection and/or
autoimmune phenomena.8,32
Thymic Hyperplasia
The non-malignant thymic enlargement that has
been classically termed “thymic hyperplasia” may
56 Laura P. Hale
Figure 7. Molecular (Immunoscope) analysis of T-cell receptor
repertoire diversity. RNA isolated from PBMC is reverse tran-
scribed to cDNA, then the regions encoding the variable antigen
binding (V␤) regions of the T-cell receptor ␤ chain are amplified
with primers specific for each V␤ family and the ␤ chain constant
(C␤) region. Polymerase chain reaction products are rendered
fluorescent using a fluorescent C␤ primer. Fluorescence intensity
as a function of PCR product size (in base pairs) is determined
using standard DNA sequencing equipment. Abnormal repertoires
in patients with deficient thymic activity may be oligoclonal (A) or
skewed polyclonal (B). Normal repertoires contain a Gaussian
distribution of receptor lengths (C). A full immunoscope analysis
includes 23 similar peak density histograms using a panel of 23 V␤
primers as described.32
occur because of two distinct mechanisms, with
different implications for patient treatment and
prognosis. Marked thymic enlargement may be
seen in settings of thymic reconstitution after che-
motherapy33 or after institution of highly active
antiretroviral therapy in HIV-infected patients.27-29
True thymic hyperplasia with active thymopoiesis is
characterized by expansion of the thymic epithelial
network by phenotypically immature thymocytes
and may be associated with decreased susceptibility
to opportunistic infections and subsequent normal-
ization of immune function. In contrast, thymic
enlargement may also occur because of increased
migration of mature T and B lymphocytes to the
perivascular space, adjacent to but outside of the
thymic epithelial network. If present, follicles with
germinal centers are also located within the
perivascular space (Fig 8).14,34 Hyperplasia of the
perivascular space typically reduces thymic produc-
tion of new T cells and may result in massive
thymus enlargement with minimal thymopoiesis.
These two distinct pathogenetic conditions may
appear similar on hematoxylin-eosin–stained sec-
tions, in part because the density of cells is similar
in the medulla and the adjacent perivascular space;
however, they can be readily distinguished using
cytokeratin immunostains. Additional immuno-
stains (CD1a, Ki-67) can be used to provide his-
topathologic confirmation of ongoing thymopoi-
esis. Adult patients with true thymic hyperplasia
may be at increased risk for autoimmune dis-
ease.28,35 Immunodeficient patients lacking thymo-
poiesis may need additional treatment to effect
immune reconstitution. Thus, it is important that
the anatomic diagnoses rendered on thymus spec-
imens reflect our understanding of the mecha-
nisms for non-neoplastic thymic enlargement.
Myasthenia gravis (MG) is an autoimmune dis-
ease that is typically caused by autoantibodies
against the acetylcholine receptor that disrupt neu-
romuscular transmission. For reasons that are still
not well understood, therapeutic thymectomy may
result in clinical improvement of MG symptoms
and allow tapering of immunosuppressive medica-
tions.36 Follicular thymic hyperplasia is the charac-
teristic lesion observed in non-neoplastic thymus
from patients with MG. The percentage of the
thymus that is composed of perivascular space is
thus increased in MG patients,14 with a correspond-
ing decrease in the thymic volume available for
production of new T cells. The hyperplasia in MG
thymus, including follicles with germinal centers, is
located totally in the perivascular space (Fig 8).
Thus, despite increased thymus cellularity com-
pared with age-matched normals, MG patients have
increased percentages of thymic perivascular
space14 and significantly decreased thymopoiesis as
measured by the TREC assay when compared with
age-matched normal patients.18,26 Myasthenia gra-
vis patients whose thymus tissues show histologic
evidence of robust thymopoiesis have decreased
TREC levels in peripheral blood lymphocytes fol-
lowing thymectomy. In contrast, MG patients
whose thymus tissues show minimal to no thymo-
poiesis have no change in TRECs following thymec-
tomy.26 Serial measurements of TREC levels in MG
patients at time points following thymectomy fur-
ther suggest that thymic activity also may regulate
the turnover of peripheral T cells. Following
thymectomy, naive phenotype T-cell levels remain
relatively constant, whereas memory phenotype T-
cell concentrations increase.26
Evaluation of Thymus from Pediatric Patients
With Suspected Immunodeficiency
The thymus from patients with severe combined
immunodeficiency is typically markedly hypoplastic
(⬍3 g; normal 12 ⫾ 5 g for 12-month-old male
 Assessment of Human Thymus 57

Figure 8. Follicular hyperplasia occurs in the perivascular

space. Combined cytokeratin immunoperoxidase (brown color)
and hematoxylin-eosin stains show that the germinal centers
characteristic of thymus from patients with MG occur within the
perivascular space, outside of the thymic epithelial network. C,
cortex; M, medulla; P, perivascular space. (Scanning power.)
infant).37,38 Severe combined immunodeficiency
thymus consists of compact lobules of tightly op-
posed thymic epithelial cells surrounded by a small
amount of adipose tissue (Fig 9A). Concentri-
cally arranged thymic epithelial cells (epithelial
“pseudo-rosettes”) may be present focally (Fig 9B).
CD1a⫹ small lymphocytes (ie, immature thymo-
cytes) are absent, although CD3⫹ cells may some-
times be observed. Corticomedullary distinction
and Hassall bodies are typically absent. The term
“thymic dysplasia” has been used to refer to the
histology of the thymus in primary immunodefi-
Figure 10. Stress and corticosteroid-induced thymic involu-
tion. (A) Thymus with mild stress involution due to endogenous
corticosteroids has a characteristic “starry sky” appearance.
The pale areas result from macrophage clearance of apoptotic
thymocytes. (Low-power magnification; inset, medium-power
magnification.) (B) Treatment with high-dose corticosteroids
results in depletion of cortical thymocytes and increased
numbers of medullary Hassall bodies. (Medium-power
magnification.)
ciencies such as severe combined immunodefi-
ciency. However, this term is confusing because
although the thymus is hypoplastic and has not
developed normally, it does not represent a pre-
neoplastic condition. The histologic picture of
post-natal thymic atrophy in association with immu-
nodeficiency differs from that of the primary thy-
mic hypoplasia observed in severe combined immu-
nodeficiency. In post-natal thymic atrophy, the

Figure 9. Primary thymic hypoplasia

versus post-natal thymic atrophy. The
thymus from patients with severe com-
bined immunodeficiency and primary
failure of thymic development (A, scan-
ning power) is small and composed
predominately of tightly opposed thy-
mic epithelial cells, with minimal adi-
pose tissue infiltration. Epithelial pseu-
dorosettes may be present (arrowhead
in B, medium power). In contrast, thy-
mus from a 2-year-old patient with
post-natal thymic atrophy (C, scanning
power) is large with abundant adipose
tissue and elongated collections of thy-
mic epithelial cells (D, medium power).
58 Laura P. Hale
thymus is typically of near normal size, with prom-
inent thymic vessels and large amounts of adipose
tissue (Fig 9C). Epithelial cells are typically ar-
ranged in elongated strands rather than compact
lobules, with absence of CD1a⫹ mib-1⫹ developing
T cells (Fig 9D).
The thymus of children without congenital im-
munodeficiencies may appear abnormal at autopsy
because of stress involution or corticosteroid treat-
ment.12,39 Endogenous corticosteroids produced in
response to the stress of serious illness or therapeu-
tic doses of corticosteroids cause apoptosis of im-
mature thymocytes,40,41 which are cleared by thy-
mic macrophages. Mild stress involution appears as
cleared areas within the normally densely packed
thymic cortex, the so-called “starry sky” pattern of
thymic involution (Fig 10A). As involution
progresses, the thymic cortex becomes progres-
sively depleted. The number of medullary Hassall
bodies may also increase in patients treated with
high dose corticosteroids (Fig 10B).12
Evaluation of Thymic Reconstitution
Most studies of patients undergoing therapy for
congenital immunodeficiencies, HIV infection, or
cancer have used radiographic determination of
thymus size and/or the TREC assay combined with
flow cytometric identification of CD3⫹CD45RA⫹
CD62Lhigh (naive) T cells to determine thymic
reconstitution status.27,30,33 The immunoscope as-
say has also been used to monitor normalization of
the T-cell receptor repertoire and to identify pa-
tients who have been successfully immunoreconsti-
tuted and thus are no longer at risk for opportu-
nistic infections.30,32 Few studies have combined
these assays with histologic examination of thymic
tissue. In cases where thymic biopsies were ob-
tained from two HIV⫹ patients treated with highly
active antiretroviral therapy, the thymus tissues
showed histologic evidence for thymopoiesis that
was concordant with elevated TREC levels and in-
creased numbers of naive phenotype peripheral
blood T cells following highly active antiretroviral
therapy.28
The transplantation of allogeneic thymic tissue
has been shown to restore normal immune func-
tion in some patients with complete DiGeorge syn-
drome and profound immunodeficiency.16 The ef-
ficacy of this treatment is currently being rigorously
studied in phase II clinical trials. As part of these
studies, biopsies of the thymic allograft are ob-
tained 2 to 3 months following transplantation to
assess graft survival and function. The principles
for histologic assessment of thymic function de-
scribed above have also proved useful for assessing
the success of the transplantation procedure, which
has profound implications for prognosis and clini-
cal care of these patients. Patients whose allograft
biopsies lack CD1a⫹ mib-1⫹ small lymphocytes and
show condensed thymic epithelium surrounded by
an infiltrate of CD3⫹ TIA-1⫹ activated cytotoxic T
cells do not develop immunoreconstitution. This
histologic picture is characteristic of impending
allograft rejection. In contrast, patients with a light
lacy cytokeratin network and CD1a⫹ mib-1⫹ small
lymphocytes at biopsy (Fig 11) typically develop
TREC⫹ peripheral blood cells and subsequent nor-
malization of the T-cell repertoire by immunoscope
analysis.8 Analyses of naive (CD45RA⫹CD62Lhigh)
T-cell numbers by flow cytometry, TRECs, and
T-cell repertoire by immunoscope analysis that
show lack of thymus-derived T cells can be used to
confirm the initial diagnosis of complete DiGeorge
syndrome. Following successful thymus transplan-
tation, thymus-derived T cells appear in the periph-
eral blood, with later normalization of the T-cell
repertoire.8
Summary
The thymus is absolutely required to initially
establish a functional T-cell repertoire in humans,
but begins to undergo age-related decreases in
activity, beginning in early childhood. Histologic
and molecular methods have been developed to
assess thymic activity as a function of age in normal
humans and in disease states that affect the thymus.
Although thymic volume and weight remain rela-
tively constant, the portion of the thymus contain-
ing thymic epithelium and participating in produc-
tion of new T cells decreases progressively with age.
The thymic perivascular space, defined as the por-
tion of the thymus within the thymic capsule but
outside the thymic epithelial network, increases
correspondingly with prominent infiltrates of pe-
ripheral lymphocytes and adipose tissue. However,
despite age-related changes in thymic composition,
thymopoiesis continues in normal human adults
into at least the eighth decade. Histologic and
molecular studies of thymic function have yielded
insights into the normal regulation of the T-cell
 Assessment of Human Thymus
Figure 11. Histologic confirmation of
thymopoiesis in a thymic allograft. A
frozen section biopsy of thymus tissue
obtained 3 months after grafting into
the quadriceps muscle of a patient with
DiGeorge syndrome shows robust
thymopoiesis (A). (Hematoxylin-eosin
stain.) Immunoperoxidase staining with
anti-cytokeratin mAbs (B) shows as
light lacy network of thymic epithelial
cells interacting with the CD1a⫹ (C),
mib-1⫹ (D) immature thymocytes that
are abundant in cortical regions of the
graft. (Medium-power magnification.)
repertoire and the pathogenesis of autoimmune
diseases. Determination of thymic function can im-
prove clinical decision-making in patients undergo-
ing immune reconstitution therapy for congenital
immunodeficiencies, HIV infection, or following
cancer chemotherapy.
Acknowledgment
The author would like to thank Steven Conlon for assistance
with figure preparation and Drs Barton F. Haynes and John
Whitesides for providing Figure 4. The data used to prepare
Figure 6B were provided by Drs Dhavel D. Patel, Rebecca H.
Buckley, Gregory D. Sempowski, and Barton F. Haynes. Dr M.
Louise Markert provided the immunostained sections used to
prepare Figure 11.
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