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Induction of Nuclear Contour Irregularity DuringT-cell Activation
Via the T-cell Receptor/CD3 Complex
and CD2 Antigens
in the Presenceof Phorbol Esters
By Uwe Reinhold, Martin Herpertz, Sylvia Kukel, Iris Oltermann, Manfred Uerlich, and Hans-Wilhelm Kreysel
morphology in a lower percentage of cells in comparison
To determine whether specificT-cell activation pathways
could produce nuclear contour irregularity
in normal human with stimulation via the TCR/CD3 complex. In contrast,
of
mitogenic stimulation in vitro did not induce alterations
lymphocytes, purifiedT cells were stimulated in vitro and
nuclear morphologyin Tcells. lmmunoelectron microscopy
subsequently analyzed by electron microscopy. The degree
showed that nuclear contour irregularity induoed in vitro
of nuclear contour irregularity
was determined with the use
did not correlatewith surface-antigen expressionof T-cell
of a computerized planimeter. Stimulation
via the T-cell resubpopulations. The results indicatethat Sezary-like morceptor (TCR)/CD3 complex usinganti-CD3 monoclonal anphology is associatedwith cell activation in normal human
tibodies induced Setary-like morphology (nuclear contour
T cells.
(9%to 28%)of T cells
indices > 6.5) in a significant portion
0 1994 by The American Society
of Hematology.
derived from different normal donors. T-cell activation via
of Sezary-likenuclear
CD2 antigensshowedinduction
S
EZARY SYNDROME is a cutaneous T-cell lymphoma
(CTCL) characterized by a generalized exfoliative
erythroderma with lymphadenopathy.' The most characteristic feature of the disease is the presence of Skzary cells in
the peripheral blood (PB) and skin. The Skzary cell, first described in 1968 by Lutzner and Jordan? is a structurally
distinctive lymphoid cell that has a highly convoluted
cerebriform nucleus, a high nucleus-to-cytoplasm ratio, and
condensed chromatin along the nuclear membrane. Immunophenotypic analyses of malignant Shzary cells showed a
phenotype indistinguishable from that of mature T cells
with the exception of CD7 expre~sion.~
Shzary cells predominantly lack CD7 expression. Furthermore, cutaneous
infiltrates in CTCL lesions have been found to contain a
high number of CD7- T cell^.^.^ Recently, we identified a
subset of CD7- T cells in the normal human blood that may
represent the circulating counterpartof CD7- T cells found
in benign and malignant skin lesions.6
The cerebriform morphology of T cells in CTCLis poorly
understood. Several investigators have emphasized the nonspecificity of Skzary-like morphology because similar cells
have been described in benign inflammatory-skin diseases,
dermopathic lymphadenopathy, and a small percentage of
normal PB lymphocyte^.^"^ These data indicate that the
Skzary-like cell per se is not a malignant cell, but a reactive
cell that may be associated with skin inflammation. Investigators emphasizing the nonspecificity of the Skzary cell often quote thestudy from Yeckley et a]," who reported that
SCzary-like cellmorphology could be induced in normalPB
lymphocytes by stimulation with mitogens. However, other
groups could not reproduce this finding and, until now,
there was no study confirmingthe induction of Skzary-like
morphology in normal human lymphocytes bycell actiati ion.""^ We describe the differential induction of nuclear
contour irregularity in normal lymphocytes during T-cell
activation via specific T-cell surface structures that participate in signal transduction. Our results indicate that Skzarylike morphology is associated with cell activation in normal
human T cells.
MATERIALS ANDMETHODS
Ce//preparation. PB mononuclear cells (PBMC) were isolated
from whole blood of different normal healthy donors by Ficoll-HyB/&,
Vol83, No 3 (February 1), 1994: pp 703-706
paque density centrifugation (Seromed, Berlin, Germany). T-cellenriched populationswere purified from PBMC by passagethrough
a nylon-wool column (Wako Chem,Neuss, Germany). The proportion of residual CD 14' monocytes and CD 19' B cells was in every
case less than 1%. Purified T cells were distributed at I X 106/mLin
250-mL culture flasks in RPM1 1640 10%fetal calfserum. They
were cocultured with titrated amounts of anti-CD3 monoclonal antibody (MoAb) (OKT3; 250, 25, 2.5, 0.25, 0.025 ng/mL), 12-0tetradecanoyl-phorbol 13-acetate (TPA; I O ng/mL), anti-CD2
(TI I . 1 + TI 1.2; 200 ng/mL), anti-CD28 (1 pg/mL), phytohemagglutinin (PHA) (5Gg/mL), pokeweed mitogen (PWM) (5 &mL),
concanavalin A (ConA) (5 pg/mL), or combinations of these for
different periods of time (24,48, 120 hours). OKT3 was obtained
from Ortho Pharmaceutical (Heidelberg, Germany); TI I. I/TI 1.2
from Dianova (Hamburg,Germany); anti-CD28 from Janssen
Biochimica (Neuss, Germany); PHA, PWM and Con A from
Seromed; and TPA from Sigma (Munich, Germany). Cells were
harvested, washed, and prepared for electron microscopy.
Electron microscopy. Cells were fixed in 2.5% glutaraldehyde,
postfixed in 2% osmium tetroxide, dehydrated, and embedded in
Epon 8 12. Ultrathin sections were stained with uranyl acetate and
lead citrate and examined with a Zeiss EM9 electron microscope
(Zeiss, New York, NY). The degree of nuclear indentation was calculated by a nuclear contour index (NCI; NCI = nuclear perimeter/
The nuclear perimeter and area were measured by means
of a graphic tablet interfaced with a computer using an in-house
program (courtesy of W. Schneichel, University of Cologne, Cologne, Germany, unpublished). Cells in which the plane ofsection
grazed the nucleus were not analyzed. Morphometrically, a lymphocyte with NCI greater than 6.5 is defined to be a SCzary-like
cell. In each preparation a minimum of 1 0 0 cells was analyzed. For
+
From the Department ofDermatology, UniversityofBonn, Bonn,
Germany.
Submitted April 28. 1993; accepted September 9, 1993.
Supported by the Deutsche Forschungsgemeinschaft DFG (Grant
No. Re-690-3-1).
Address reprint requests ta Uwe Reinhold, MD, Department of
Dermatology,University
of Bonn, Sigmund-Freud-Strape25,
53105 Bonn, Germany.
The publication costs of this article were defrayed in part by page
charge payment.This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate thisfact.
0 1994 by The American Society
of Hematology.
0006-4971/94/8303-0115$3.00/0
703
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704
REINHOLD ET AL
Table 1. Effect of Various Stimuli on Nuclear Morphology
in T Lymphocytes
Stimulus
Mean NCI + SD
4.42 f 0.76
4.33 f 0.64
4.18 f 0 . 5 0
4.26 f 0.64
Anti-CD2
Anti-CD28
4.18~0.48
Anti-CD3 + TPA
5.46 f 1.48'
Anti-CD2 + TPA
4.84 2 9
0.86'
Anti-CD28 + TPA
4.49 f 0.71
5 . 1 7 r 1.14'
Anti-CD3 + CD2 + TPA
Anti-CD2 + CD28 + TPA
4.78 f 0.83'
Anti-CD3 + CD2 + CD28 +TPA 5.305 1.13'
PHA
4.42f 0.76
PWM
4.53 f 0.74
4.35 f 0.64
ConA
Medium
TPA
Anti-CD3
NCI
% Sezary-Like
Max.
Cells
6.60
6.01
6.14
8.33
6.18
10.32
8.62
6.77
8.55
8.05
8.94
6.60
7.69
6.80
1
0
0
1
0
22
1
13
9
13
1
2
1
~~
Purified T cells were stimulatedfor 5 days with various cell-activating
agents and subsequently analyzedby electron microscopy (see Materials and Methods). Anti-CD3was used at 2.5 ng/mL.
* Statistically significant (P < .05) in comparison with unstimulated
controls.
statistical analysis, Wilcoxon's two-sample test was used.
The level
ofsignificancewas P < .05.
Indirect i m ~ r r t l c ~ , ~cohl d~ t ~ ~ rnicro.scop.r.
rou
Cells activated in vitro in the presence of OKT3 (2.5 ng/mL)/TPA (10 ng/mL) were
harvested. prefixed with4% paraformaldehyde for 30 minutes. and
thereafter analyzed by indirect immunogold electron microscopy.'5
Cells ( I X IO') were incubated for I hour with MoAbs and washed
twice in phosphate-buffered saline containing 1% bovine serum albumin and 0.01% sodium azide. The following antibodies were
used: Leu-4 (CD3). Leu-3a (CD4). Leu-2a (CD8). all from Becton
Dickinson, Heidelberg. Germany: and 3 A I (CD7). fromBiozol,
Eching. Germany. Thereafter. cells wereincubated with a goat antimouse IgG + IgM antiserum labeled with 12-nm colloidal gold(Dianova) for 30 minutes at 4°C. washed and fixed in 2.5% glutaraldehyde.
postfixed
in osmium tetroxide. dehydrated. and resin
embedded. Control experiments were performed by incubating T
cells with isotype-specific mouse Ig followedby the gold-labeled
goat antimouse IgG + IgM. Ultrathin sections were stained with
uranyl acetate and lead citrate and examined by electron microscopy. A cell was considered to be reactive when several gold particles (at least three) were seen attached to the cell membrane. Cells
in control experiments did not show gold labeling. In each sample
at least 100 cells wereanalyzed.
not result in enhanced nuclear contour irregularity. On the
other hand. cell activation by immobilized OKT3 showed
similar inductionof Skzary-like morphology in comparison
with activation by soluble OKT3/TPA (not shown). Titration experiments showed that any concentration of OKT3
antibody sufficient to activate T cells in the presence ofTPA
was also sufficient to generate cells with Skzary-like morphology. Maximum of nuclear contour irregularity was
noted with 2.5 ng/mL OKT3. Kinetic studies showed maximum of nuclear contour irregularity after 120 hours ofculture. Increased numbers of Sbzary-like cells were not observed before 72 hours of stimulation. Several other T-cell
activating agents were tested for their capacity to induce
nuclear contour irregularity in T cells (Table I ). Cell activation in vitro via the CD2 pathway also induced a significant
numberofS6zary-likecells. However, thepercentageofSCzary-like cells induced by CD2 activation was lower in comparison with CD3 activation. Stimulation via CD28 did not
show any effect. In addition. no alteration of nuclear morphology was observed after T-cell stimulation with mitogens. Also, no significant difference in nuclear morphology
was detected when mitogenic activation was performed in
the presence of TPA.
To evaluate the phenotypeof Skzary-likecells induced by
cell activation in vitro. indirect-immunogold electron microscopy was performed. Purified T cells were activated in
vitro by OKT3/TPA and cells with NCI greater than 6.5
were subsequently analyzed for their expression of specific
T-cell antigens. The results show that T cells exhibiting
nuclear contour irregularity belong to both CD4' and CD8'
subpopulations. The CD4/CD8 ratio (2.4) in T cells with
NCI greater than 6.5 was in similar proportion to the total
number ofCD4' and CD8' T cells (2.6) in culture. Furthermore. the majority of SGzary-like cells expressed the CD7
antigen (>95?h). Stimulationof purified CD7- T cells in vi-
RESULTS
The electron-microscopic preparations of purified T cells
cultured i n medium without stimulating agents showed a
small number (52%)of cells with NCI greater than 6.5. In
contrast. preparations o f T cells activated in vitro by OKT3/
TPA contained a significant portion of cells that had acquired a nuclear contour irregularity, which would classify
them as a Sezary-like cell (NCI > 6.5) (Table I . Fig l ) . Our
results showed a wide variation in the number of Stzary-like
T cells induced in vitro. Among five different donors tested,
the numberof Skzary-like cells after activation with OKT3/
TPA was 9% to 28%. The highest NCI value observed was
I I .28. Cellincubation with soluble OKT3 or TPA alonedid
Fig 1. Electron micrographof a T cell stimulated in vitro via TCR/
CD3 complex showing nuclear contour irregularity (original magnification X 11,000; NCI = 8.09).
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705
SEZARY-LIKE MORPHOLOGY
tro by anti-CD3 showed induction of nuclear contour irregularity in a similar portionof cells as comparedwith unseparated Tcells.
DISCUSSION
In the present study, we have readdressed the question of
nuclear morphology in T cells after activation in vitro. We
show for the first time thatT cells with Stzary-like morphology (NCI >6.5) can be induced in vitro by incubation with
antibodies to CD3 and CD2 antigens in the presence of
phorbol esters. Antibodies to CD3 antigens were most
effective in this respect. Soluble anti-CD3 without phorbol
esters had no effect. On the otherhand, nuclear contour irregularity could be induced by cross-linked anti-CD3. This
indicates that inductionof Sizary-like cells via T-cellreceptor (TCR)/CD3complex or CD2 antigens is dependent on
secondary activation signals required for induction of proliferative responses in resting T cells. Stimulation with antiCD3 in the presence of phorbol esters mimics the T-cell interaction with a complex of antigen and MHC-encoded
protein and provides several intracellular signals including
Ca2+-fluxand protein kinase C activation.16 Several studies
indicate that these intracellular signals may interact with
certain cytoskeletal proteins that might, in turn, increase
nuclear contour irregularity in T cells." Consistent with the
findings of other investigators. we did not find significant
induction of Stzary morphology after stimulation with
PHA, which might be caused by less potent mitogenicity
of PHA or ConA in comparison with anti-CD3 reagents."
Simultaneous triggering of CD28 molecules, shown to cooperate with both the TCR/CD3-directed pathway and the
CD2-directed alternative pathway of T-cell activation," had
no additional effect on the degree of morphologic changes
induced in activated T cells. The observation that nuclear
contour irregularity is inducible by T-cell-specific activation in vitro is consistent with the finding that Sizary-like
cells have been found in a number of conditions associated
with antigen-specific T-cell activation in vivo. These include
contact dermatitis, reactive lymph nodes, and infiltrates of
patch-test
lmmunogold electron microscopy performed in this study showed that T cells exhibiting Sizary
morphology after TCR/CD3 stimulation expressed both
CD4andCD8 phenotypes. Furthermore, cerebriform T
cells were found in both CD7+ andCD7- T-cell subsets. Stzary cells in malignant lymphoma express the helper T-cell
phenotype and predominantly lack CD7 expression. Recently, we could identify a subset of helper T cells in normal
humans that specifically lack CD7 expression and, therefore, may represent the physiologic counterpart of malignant Sizarycells.632i
However, we found that normal CD7helper T cells do not exhibit nuclear contour irregularity.
Our present study shows the lack of phenotypic specificity
of nuclear contour irregularity and provides evidence that
there is no obligatory morphologic association between the
malignant Sizary cell and its possible physiologic counterpart.
The results of our study raise the question of possible discrimination between reactive Sdzary-like cells and malignant Shzary cells. To define additional diagnostic criteria to
differentiate CTCL from benign dermatoses alarge number
of ultrastructural studies were reported.22923
Willemze et a123
showed that most discriminating parameters for the differentiation between Sizary syndrome and benign forms of
erythroderma were: (1) a mean NCI value of 5.5 or more;
(2) more than 20% Stzary-like cells (lymphoid cells with a
NCI >6.5); or (3) the presence of cells with a NCI greater
than 11.5. The fulfillment of one or more of these criteria
was reported to be diagnostic for Sizary syndrome. Our results indicate thatcell activation of normal T cells in vitro by
TCR/CD3 stimulation may induce morphologic alteration
corresponding to thecriteria for malignant T cells in Skzary
syndrome, with the exception of an NCI greater than 1 I S.
However, other investigators found that the highest NCI
was nota good discriminating factor, because an NCI
greater than 11.5 was found in only a subgroup of Stzary
patients.24 On theotherhand,
lymphocytes with NCI
greater than I I .5 have been observed in benign cases.8Thus,
the computer-assisted morphometric criteria for the differentiation between malignant and reactive T cells with
nuclear contour irregularity must remain speculative. Further studiesshould clarify whether cell activation in vivo in
conditions other thanskin disease is also associated with the
occurrence of Stzary-like morphology in T cells.
ACKNOWLEDGMENT
We thank Martina lmpekoven for excellent technical assistance.
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1994 83: 703-706
Induction of nuclear contour irregularity during T-cell activation via
the T-cell receptor/CD3 complex and CD2 antigens in the presence of
phorbol esters
U Reinhold, M Herpertz, S Kukel, I Oltermann, M Uerlich and HW Kreysel
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