Terminal deoxynucleotidyl transferase-positive cells in human tonsils.

Abstract

To study the possible cellular origin of recently recognized indolent terminal deoxynucleotidyl transferase (TdT)-positive T-lymphoblastic proliferations of the tonsils and oropharynx, we studied normal human tonsils for the presence of TdT-positive cells. TdT-positive cells were readily demonstrated in the tonsils from 15 children and adults by immunohistochemical staining. TdT-positive cells were distributed in discrete foci at the periphery of lobules of lymphoid tissue and adjacent to fibrous septa and had the morphologic features of small to medium-sized lymphocytes. Double-antibody staining indicated the TdT-positive cells had the phenotype of uncommitted early lymphoid precursors (CD3-, CD79a-, CD10-). Foci of TdT-positive cells were not identified in 6 reactive lymph nodes studied as controls. These studies indicate that tonsils, like bone marrow and thymus, are sites of lymphopoiesis. The presence of TdT-positive precursor cells in human tonsils may be a factor in the pathogenesis of recently described indolent T-lymphoblastic proliferations involving the tonsils and oropharynx. The presence of TdT-positive cells in human tonsils should not be misinterpreted as evidence of lymphoblastic lymphoma or leukemia.

Full text

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Hematopathology / TDT-POSITIVE TONSIL CELLS
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Am J Clin Pathol 2001;116:12-16 © American Society of Clinical Pathologists
Terminal Deoxynucleotidyl Transferase–Positive Cells in
Human Tonsils
James A. Strauchen, MD, and Lorraine K. Miller, PhD
Key Words: Terminal deoxynucleotidyl transferase; TdT; Tonsil; Lymphoblastic lymphoma
A b s t r a c t
To study the possible cellular origin of recently
recognized indolent terminal deoxynucleotidyl
transferase (TdT)-positive T-lymphoblastic
proliferations of the tonsils and oropharynx, we studied
normal human tonsils for the presence of TdT-positive
cells. TdT-positive cells were readily demonstrated in
the tonsils from 15 children and adults by
immunohistochemical staining. TdT-positive cells were
distributed in discrete foci at the periphery of lobules of
lymphoid tissue and adjacent to fibrous septa and had
the morphologic features of small to medium-sized
lymphocytes. Double-antibody staining indicated the
TdT-positive cells had the phenotype of uncommitted
early lymphoid precursors (CD3–, CD79a–, CD10–).
Foci of TdT-positive cells were not identified in 6
reactive lymph nodes studied as controls. These studies
indicate that tonsils, like bone marrow and thymus, are
sites of lymphopoiesis. The presence of TdT-positive
precursor cells in human tonsils may be a factor in the
pathogenesis of recently described indolent
T-lymphoblastic proliferations involving the tonsils
and oropharynx. The presence of TdT-positive cells in
human tonsils should not be misinterpreted as evidence
of lymphoblastic lymphoma or leukemia.
Terminal deoxynucleotidyl transferase (TdT) is a
template independent DNA polymerase characteristic of
lymphoid precursor cells.1,2TdT acts as a somatic mutagen in
these cells resulting in the generation of additional immuno-
logic diversity.3,4Detection of TdT by enzymatic,5immuno-
fluorescence,6,7or immunohistochemical8-10methods is
widely used as a marker for the neoplasms of precursor B-
and T-cell origin, specifically acute lymphoblastic leukemia
and lymphoblastic lymphoma (precursor B-lymphoblastic
leukemia/lymphoma and precursor T-lymphoblastic
lymphoma/leukemia in the Revised European-American clas-
sification of lymphoid neoplasms11). TdT-positive cells are
normally found in the thymus (cortical thymocytes) and in the
bone marrow (B- and T-precursor cells) but generally are not
recognized to occur in the peripheral lymphoid organs, eg,
tonsils, lymph nodes, and spleen, in the absence of lymphoid
neoplasia.9,10Strauchen12and others13have reported on the
occurrence of previously unrecognized TdT-positive, indolent,
T-lymphoblastic proliferations of undetermined clonality with
predilection for involvement of the tonsils and oropharynx.
To study the cellular origin of these TdT-positive prolif-
erations, we studied normal human tonsils for the presence of
TdT-positive cells, using immunohistochemical staining of
formalin-fixed, paraffin-embedded tissue. TdT-positive cells
were readily demonstrable in the tonsils from both children
and adults. The possible relation of these TdT-positive cells
to the pathogenesis of indolent T-lymphoblastic proliferations
of the oropharynx is considered.
Materials and Methods
Tissues
Tonsils were selected from 15 children and adults aged 1
to 40 years who had undergone tonsillectomy for benign

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Hematopathology / ORIGINAL ARTICLE
Am J Clin Pathol 2001;116:12-16 13
© American Society of Clinical Pathologists
hypertrophy. Lymph nodes with reactive hyperplasia were
selected from 6 children and adults aged 4 to 45 years as
controls. Tissues were fixed in 10% neutral buffered
formalin and processed for routine paraffin-embedding.
Immunohistochemical Analysis
Anti-TdT antibody (rabbit polyclonal) was obtained
from DAKO (Carpinteria, CA) and Supertechs (Bethesda,
MD). Anti-CD3 (rabbit polyclonal), and anti-CD79a were
obtained from DAKO. Anti-CD10 was obtained from Novo-
castra Laboratories (Newcastle upon Tyne, England). Anti-
Ki-67 (MIB-1) was obtained from Beckman Coulter
(Fullerton, CA). Tissue sections for immunohistochemical
analysis were deparaffinized in xylene and rehydrated
through graded ethanol to deionized water. Immunohisto-
chemical staining with anti-TdT (DAKO) was performed
following antigen retrieval with microwaving in a 10-
mmol/L concentration of citric acid at pH 6.0 for 15 minutes.
Staining was performed on an autostainer (DAKO) using the
EnVision+ System (DAKO) with peroxidase-diaminobenzi-
dine (DAB) chromogen. Immunohistochemical staining with
anti-TdT (Supertechs) was performed following antigen
retrieval with trypsinization. Negative controls included
absent first antibody, antibody diluent, and an irrelevant
rabbit antibody. A positive control (thymus) was included in
each staining. Double-antibody staining was performed
using the EnVision Doublestain System (DAKO) with
peroxidase-DAB and alkaline phosphatase–fast red chro-
mogens. Briefly, deparaffinized sections were rehydrated,
microwaved in a 10-mmol/L concentration of citric acid, pH
6.0, and incubated with anti-TdT antibody (DAKO) for 30
minutes at room temperature followed by the addition of
peroxidase-DAB chromogen. Sections were washed and
incubated with anti-CD3, anti-CD10, or anti-CD79a for 30
minutes at room temperature followed by the addition of
alkaline phosphatase–fast red chromogen. TdT-positive cells
were evaluated semiquantitatively by counting the number of
positive cells per high power (40× objective) field in the
areas with the most numerous positive cells.
Results
The tonsils from all 15 patients showed TdT-positive
cells distributed in discrete foci at the periphery of lobules of
lymphoid tissue and adjacent to fibrous septa ❚Image 1❚.
TdT-positive cells were not observed in the germinal centers
or tonsil epithelium. TdT-positive cells had the morphologic
features of small to medium-sized lymphocytes with round
to ovoid nuclei and scant cytoplasm ❚Image 2❚. TdT-positive
cells were found in the tonsils of adults and children but
were more numerous in children ❚Table 1❚. Two tonsils were
apparent outliers (Table 1); in these 2 cases, both tonsils
were studied; however, only the first was included in the
statistical analysis. Identical patterns of TdT staining were
observed with both antibodies; no staining was observed in
the absence of first antibody or with an irrelevant rabbit anti-
body control. The lymph nodes from 6 patients studied as
❚Image 1❚ Tonsil from a 4-year-old child showing a focus of
terminal deoxynucleotidyl transferase (TdT)-positive cells at
the periphery of a lobule of lymphoid tissue adjacent to a
fibrous septum (anti-TdT immunoperoxidase counterstained
with hematoxylin, original magnification ×200).
❚Image 2❚ Higher magnification of the tonsil in Image 1
showing terminal deoxynucleotidyl transferase (TdT)-positive
cells with the morphologic features of small to medium-sized
lymphocytes (anti-TdT immunoperoxidase counterstained
with hematoxylin, original magnification ×600).

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Strauchen and Miller / TDT-POSITIVE TONSIL CELLS
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Am J Clin Pathol 2001;116:12-16 © American Society of Clinical Pathologists
controls showed only rare, single TdT-positive cells in the
paracortex; foci of TdT-positive cells as in the tonsils were
not observed. Double-antibody immunohistochemical
studies were performed in 3 cases to define the phenotype of
the TdT-positive cells; approximately 50 TdT-positive cells
were evaluated in each. The TdT-positive cells were negative
for CD3, CD79a, and CD10 ❚Image 3❚. Staining for Ki-67
demonstrated numerous proliferating cells in areas
containing TdT-positive cells ❚Image 4❚.
Discussion
TdT is a marker for immature lymphocyte precursors of
B- and T-cell lineage and of lymphoblastic neoplasms.1,2
TdT-positive cells generally are considered to be restricted to
the central lymphoid organs, ie, the bone marrow and
thymus; however, small numbers may be detected in periph-
eral blood,14,15immature and fetal tissues,16,17and reactive
lymph nodes.9In the present article, we report on the occur-
rence of TdT-positive cells in human tonsils demonstrated by
immunohistochemical staining. The immunohistochemical
staining for TdT with affinity-purified antibody seems to be
specific. Although spurious staining for TdT has been
reported with indirect immunofluorescence with unpurified
antibody,18,19in the present study identical patterns of
staining were obtained using 2 different anti-TdT antibodies
with different antigen retrieval techniques. The results of the
present study confirm the detection of TdT activity in human
tonsil by biochemical assay.20TdT enzymatic activity was
identified in extracts of tonsils from 6 of 22 children under-
going routine tonsillectomy; the enzyme was identical to
reference TdT with respect to divalent cation and primer
requirements and inhibition by sulfhydryl inhibitors and
adenosine triphosphate, and was indistinguishable from acute
lymphoblastic leukemia–derived TdT by glycerol gradient
centrifugation and phosphocellulose chromatography.20
Double-antibody immunohistochemical studies were
performed to define the phenotype of the TdT-positive cells.
The TdT-positive cells did not stain for specific T-cell (CD3)
or B-cell (CD79a) antigens or CD10, suggesting an imma-
ture or uncommitted lymphoid precursor.21The phenotype
differs from TdT-positive cells in the thymus, which are
CD3+ T-cell precursors, and from TdT-positive cells in the
bone marrow, which are predominantly CD10+, CD79a+ B-
cell precursors.22The phenotype resembles, however, the
phenotype of TdT-positive cells in the peripheral blood that,
except for a small proportion expressing B-cell antigens and
common acute lymphoblastic leukemia antigen, lack specific
T- or B-cell antigens,14as well as the phenotype of TdT-posi-
tive bone marrow lymphoid stem cells, which lack lineage-
specific surface antigens.21
❚Table 1❚
Immunohistochemical Staining for Terminal Deoxynucleotidyl
Transferase (TdT)
Tissue/Sex/Age (y)TdT-Positive Cells per High-Power Field*
Tonsils
M/1
M/2
M/4
M/6
M/6
F/8
M/9
F/11
F/15
F/19
F/23
F/33
M/36
F/37
M/40
Mean
Lymph nodes
M/4
M/9
M/19
F/33
F/44
M/45
Mean
80.5
20.5
186.0
84.0
9.5/55.5†
161.5
118.5
223.0
107 .5
38.0
190.0/128.0†
8.0
61.0
57 .5
11.0
90.4 (±71.8)‡
1.0
3.0
2.5
13.0
8.0
4.5
5.3 (±4.4)‡
*Areas with most numerous TdT-positive cells.
†Both tonsils.
‡P < .01 by the Student t test. SD is given in parentheses.
❚Image 3❚ Double-antibody immunohistochemical stain with
anti–terminal deoxynucleotidyl transferase (TdT; brown) and
anti-CD79a (red) showing TdT-positive cells negative for the
B-cell antigen CD79a. TdT-positive cells also were negative
for the T-cell antigen CD3 and CD10 (not shown) (anti-TdT
immunoperoxidase with diaminobenzidine chromogen and
anti-CD79a immunoalkaline phosphatase with fast red
chromogen, original magnification ×600).

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Hematopathology / ORIGINAL ARTICLE
Am J Clin Pathol 2001;116:12-16 15
© American Society of Clinical Pathologists
The origin of the TdT-positive cells in the tonsils is
uncertain. The presence of numerous Ki-67–positive cells
indicates a high proliferative index suggesting active
lymphopoiesis. TdT messenger RNA and V(D)J recombina-
tion-associated genes are expressed in a fraction of mature
tonsil B cells in the germinal center centrocyte fraction.23
Under some conditions, TdT-positive T-cell precursors24and
T- and B-cell precursors25may be mobilized from the central
lymphoid organs and take up residence in peripheral
lymphoid organs. The derivation of the thymus from the
third and fourth pharyngeal pouches raises the speculation
that pharyngeal epithelium may share with thymic epithe-
lium tropism for bone marrow–derived T-cell precursors.
The TdT-positive cells in our study did not express T-cell
markers; however, uncommitted lymphoid precursors
capable of T-cell differentiation or early T-cell precursors
that have not yet acquired CD3 cannot be ruled out.14,21
The occurrence of TdT-positive T-cell precursors in the
tonsil may be related to the recently reported indolent T-
lymphoblastic proliferations involving the tonsils and
oropharynx.12,13These proliferations of undetermined clo-
nality exhibit a peculiarly indolent course, remaining con-
fined to the tonsils, oropharynx, and regional lymph nodes
for prolonged periods, even in the absence of therapy.13
These findings would not be inconsistent with a prolifera-
tion of TdT-positive lymphoid precursor cells intrinsic to the
tonsil.
TdT-positive cells are part of the lymphoid population of
the tonsil, which, like the bone marrow and thymus, seems to
be a site of postnatal lymphopoiesis. TdT-positive cells in the
tonsil should not be misinterpreted as lymphoblastic
lymphoma or leukemia.
From the Department of Pathology, Mount Sinai School of
Medicine, New York, NY.
Address reprint requests to Dr Strauchen: Dept of Pathology,
Mount Sinai School of Medicine, One Gustave Levy Place, New
York, NY 10029.
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A
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