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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1056-1063
IMMUNOBIOLOGY
From Institut National de la Santé et de la
Recherche Médicale (INSERM) 448, IM3, Paris XII, Hôpital
Henri Mondor, Créteil, France.
CDR3 of the functional rearranged T-cell receptor
variable
Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous
group of usually low-grade lymphomas primarily involving the
skin.1 Mycosis fungoides are characterized by skin invasion
of clonally-derived malignant CD4+ T lymphocytes that
phenotypically resemble mature T-helper cells. A more aggressive form
of CTCL occurs when the malignant cells become nonepidermotropic and
are associated with extracutaneous involvement. Sézary syndrome
(SS) is a more aggressive form of CTCL that is characterized by a
clonal expansion of CD4+/CD45RO+ T cells and
the appearance of these malignant T cells in the blood.2
The biology of the disease remains poorly understood, as it is
difficult to identify the malignant cell because of the lack of
specific cell surface markers. Thus, in cutaneous lesions it is
difficult to distinguish CD4+ CTCL from reactive
infiltrating CD4+ T lymphocytes.3-5 An approach
to this problem is to establish long-term cultured CTCL lines. The
functional characterization of these CTCL lines will allow
investigation of the mechanisms that contribute to the down-regulation
of the specific antitumor reactivity.6,7 Further, such CTCL
lines will represent a fundamental tool for the isolation of
characteristic megahistocompatability complex (MHC) class I peptides
that are important for the priming of antitumor
responses.8-10
Until now, only a small number of tumor T-cell lines have been
described, and they were derived from lesional skin or peripheral blood
of patients with SS, mycosis fungoides, or cutaneous large T-cell
lymphoma.11-15 However, so far no attempt has been made to
prove the identity of the cultured T-cell lines with the malignant T-cell clone detected in the skin or blood of CTCL patients. Recently, we reported a CD4+ T-cell line derived from CTCL
lesions.5 We found that the T-cell receptor variable In the present study, we report for the first time the characterization
of a novel long-term cultured CTCL line termed Pno that was developed
from a CTCL patient with a
CD3+V Patient
Tumor cell line
Monoclonal antibodies and flow cytometry studies Most mAbs were produced by our laboratory or were obtained through the exchanges of the Leukocyte Typing VI International Workshop on white cell differentiation antigens (T cell antigens, Kobe, Japan, November 10, 1996).16 We also obtained anti-TCR-V and anti-CD8 (Beckman-Coulter, Marseilles, France)
and the mAb L243 (Dr P. Le Bouteiller, INSERM, Toulouse, France), which
is reactive with monomorphic determinants shared by
HLA-DR, HLA-DP, and HLA-DQ. The mAbs were used as ascites
fluid and, when needed, were coupled to fluorescein isothiocyanate
(FITC) or biotin. Indirect immunofluorescence analysis was performed by
incubating 3 × 105 cells with each mAb for 30 minutes
at 4°C. The cells were then washed with phosphate-buffered saline
(PBS) supplemented with 1% bovine serum albumin (BSA) and incubated
with biotinylated affinity-purified goat antimouse immunoglobulin (Ig)
for an additional 30 minutes at 4°C.
CDR3 size analysis of V transcripts expressed by the Pno cell line
and malignant T lymphocytes freshly isolated from peripheral blood (PBLs), a run-off methodology was used.5 The
V and the C specific primers and the procedure used for CDR3 size
analysis have been reported previously.5 Briefly,
5 × 106 mononuclear lymphocytes from patient blood
and Pno lymphocytes were resuspended in 6 mol/L guanidium thiocyanate
buffer. Total RNA was then purified by CsCl gradient centrifugation.
For PBLs, total RNA was extracted using a modified guanidium
thiocyanate phenol/chloroform method (RNAzol B method). Complementary
DNA (cDNA) was prepared by a standard method using reverse
transcriptase (RT) and an oligo-dT primer. cDNA copies of 0.1 µg RNA
were amplified in 40 cycles of V/C polymerase chain reaction
(PCR) in 50, and 2-µL aliquots were copied in 1- to 5-cycle runoff
reactions primed with fluorescent-labeled (ABI
Fluorophore Fam; Applied Biosystems, Foster City, CA) oligonucleotides
specific for C or J fluorophores. Runoff products were then
subjected to electrophoresis on an ABI sequencer (Applied Biosystems)
in the presence of fluorescent size markers, and they were analyzed
with 672 Genescan software (Perkin Elmer SA, Courbevoie, France).
Directed sequencing of PCR products PCR products were purified using Qiagen columns (Qiaquick PCR purification kit; Qiagen, Hilden, Germany) and resuspended in 20 µL sterile water. The purified products were directly sequenced in both directions with a PRISM ready reaction DyeDeoxy Terminator cycle sequencing kit and a 373A DNA sequencer (Applied Biosystems).Proliferation assays The proliferative responses of the tumor cell line and patient PBLs to various stimuli were determined as described elsewhere18 by measuring the 3H-thymidine incorporation (cpm) of 50 × 103 responder cells. These tests were carried out in 96-well round-bottomed plates in 0.2 mL culture medium containing 10% inactivated human serum. Cytokines IL-2, IL-4, IL-7, and IL-13 ( gift of Sanofi-Synthélabo) and IL-15 and IL-12 (gift of Dr Chouaib, IGR, Villejuif, France) were used to test the proliferation of Pno lymphocytes and PBLs. The soluble mAbs used for proliferative responses with PMA (Sigma Biochemicals) were the mitogenic pair of anti-CD2 mAbs, CD2 × 11 and GT2 (ascites used as described elsewhere19); 5 µg/mL anti-CD3 mAb OKT3; and 2 µg/mL anti-CD28 mAb 4B10. For anti-CD3-induced proliferation, the wells were precoated for 2 hours at 37°C with 10 µg/mL OKT3 mAb and washed 3 times before the addition of cells with or without IL-2 or IL-7.Lymphokine assays We stimulated 1 × 105 cells per microwell with 2 ng/mL PMA with or without 5 µg/mL soluble purified anti-CD3 mAb in a final volume of 200 µL. The supernatants (100 µL per microwell) of the various cultures were harvested 24 hours later and stored at 80°C. The levels of IL-2, IL-4, IL-10, IFN- , and TGF-1
were determined by indirect sandwich enzyme-linked immunosorbent assays
(ELISAs) (Beckman-Coulter, France, and R&D Systems Europe, Abingdon,
England) employing the conditions recommended by the manufacturers. The assay of biological activity of TNF- was measured using the
WEHI 164 clone 13 cell line as described
elsewhere.20 The cells were cultured in RPMI 1640 medium
containing 5% fetal calf serum (FCS) at 5 × 103
cells per well in 96-well flat-bottomed plates. When the monolayer became confluent, actinomycin D and serial dilutions of either a known
concentration of recombinant TNF- (rTNF-) or experimental supernatants were added. After a 24-hour incubation, 125 µg MTT was
added to each well and incubated for 2 hours at 37°C. Following gentle aspiration of the supernatant, 100 µL SDS-DMF was added to
each well and vigorously shaken for 15 minutes. The absorbance at 570 nm (with reference at 650 nm) was then read, and the measurement for
the dilution series was plotted to produce dose-response curves in
which the OD read was proportional to the reciprocal of the concentration of biologically active TNF-.
Patient circulating tumor T lymphocytes and the long-term tumor
T-cell line Pno expressed TCR-V 22+- CD4+CD8+
phenotype (Figure 1). As very preliminary
studies indicated that in short-time cultures, the patient PBLs were
capable to proliferate in response to IL-7, we cultured the PBLs in 10 ng/mL IL-7. After expanding in vitro for 6 weeks, the cells stopped
dividing and began to decline, they were then plated at
3 × 104 cells per well into 96-microwell plates.
The cells started to grow again, thereby constituting the Pno cell
line. After 12 months of culture, the Pno cells were still
IL-7-dependent and exhibited logarithmic growth, with a doubling time
of 72 hours. Similar to the majority of freshly isolated patient PBLs,
the Pno cells expressed TCR-V22, CD4, and CD8 (because they
failed to react with an anti-CD8 mAb). In addition, both the Pno
cell line and PBLs expressed identical cell surface antigens such as
the MHC class I antigens, CD2, CD2R, CD28, CD95, CD122, and CD132.
However, they failed to express the MHC class II antigens, CD25, CD1a, CD1b, CD1c, and CD30 and the natural killer (NK) receptors (CD94, CD158a, and CD158b) (Table 1).
Patient circulating tumor T lymphocytes and the tumor T-cell
line Pno expressed an identical TCR-V gene segment in Pno tumor cells,
we used an RT-PCR approach and a panel of previously described V and
C or J primers. The TCR- chain structure determination of the
Pno cell line showed a unique TCR-V transcript corresponding to V22/J1.2 (Figure 2A). The
CDR3-size V distribution in PBLs was also examined. The results
obtained with the V22- and fluorescent C-specific primers gave a
dominant peak that was highly suggestive of clonal expansion (Figure
2B). Further, we found a unique V22/J1.2 rearrangement that had
an identical CDR3 size to the Pno cell line. The TCR-V chain
junctional region of the Pno cell line and the patient PBLs were then
further analyzed by cloning and sequencing amplified V22/J1.2
transcripts. Table 2 shows identical nucleic acid sequences of both the Pno cell line and the patient's circulating malignant lymphocyte junctional regions. These results indicate that the PBLs corresponded to a major
TCR-V22+CD4+CD8+ clonal
population that was identical to the long-term cultured IL-7-dependent
Pno T- cell line.
The tumor T-cell line Pno and patient circulating T lymphocytes proliferated in response to IL-7, IL-4, and IL-2 We compared the proliferative response of the Pno T cell line and patient PBLs to various cytokines. As expected, both the Pno cell line and the PBLs exhibited a strong dose-dependent proliferation in response to IL-7. In contrast, a weak proliferation was obtained with IL-2, and no proliferation was induced by IL-12, IL-13, and IL-15 (Figure 3A,B). It should be noted that the Pno cell line and the PBLs weakly differed in their ability to proliferate in response to IL-4, as the Pno cell line exhibited stronger proliferation in the presence of this cytokine. We next tested whether immobilized anti-CD3 mAbs were capable of increasing the Pno lymphocyte proliferation in response to IL-2 and IL-7. The results presented in Figure 4 show that triggering Pno CD3 molecules with specific mAbs resulted in an enhancement of IL-2- and IL-7-induced proliferative responses. It should be noted that immobilized anti-CD3 mAbs alone produced weak but significant proliferative responses in Pno lymphocytes. These results suggest that the CD3/TCR engagement in Pno lymphocytes was functional because it induced a proliferation and increased the response to exogenous cytokines including IL-2.
IL-7 prevented spontaneous apoptosis and maintained a high level of Bcl-2 expression in Pno lymphocytes We next studied the survival kinetics of Pno cells cultured in complete medium with or without IL-7 or IL-2. Annexin binding was used to detect the loss of asymmetry of the cell membrane and the appearance of phosphatidylserine residues at the cell surface, which are relatively early cellular events during apoptosis.21 The results presented in Figure 5 indicate that after 18 hours of culture without cytokines, one third of the Pno cells showed annexin V binding, and less than 2% were necrotic cells (propidium iodide uptake). Parallel cultures with IL-2 alone contained 15% of annexin V+ cells, whereas culture with IL-7 failed to contain significant apoptotic cells. Thus, only IL-7 was efficient to prevent Pno lymphocytes from spontaneous apoptosis. Changes in the levels of expression of Bcl-2 have been shown to be correlated with protection from apoptosis in various T-cell populations.22 Using flow cytometry to analyze the expression of Bcl-2 in single cells in the live gate, we found that Pno lymphocytes cultured with IL-7 gave a unique peak, with 100% positive cells (Figure 6). After 18 hours of culture without IL-7, Bcl-2 expression in viable cells was significantly reduced. These results indicate that the Pno lymphocytes were viable, were able to proliferate, and maintained high levels of Bcl-2 when they were cultured with IL-7. In contrast, in the absence of IL-7, the lymphocytes were apoptotic and expressed low levels of Bcl-2. It is of note that when Pno lymphocytes were cultured with IL-2 alone, the level of Bcl-2 was significantly reduced.
Triggering with anti-CD2, anti-CD3, and anti-CD28 mAbs induced Pno lymphocytes to proliferate and to express a Th3-type cytokine profile We studied the proliferation of Pno lymphocytes in response to a variety of activation stimuli. We found that the Pno lymphocytes failed to proliferate in response to soluble anti-CD3, anti-CD2 (antigen presenting cell-dependent [APC-dependent] mitogenic pair), and anti-CD28 mAb stimulation alone. In contrast, the combination of 2 ng/mL PMA with each of these soluble antibodies gave a significant proliferation (Figure 7). It must be noted that PMA alone, at a concentration of 2 ng/mL, induced a strong expression of CD25 molecules (Figure 8) and only a minor expression of lymphocyte proliferation (Figure 7). Thus, these results indicate that the CD3/TCR, CD2, and CD28 signaling pathways were functional. It should be noted that 4 ng/mL PMA was capable of inducing significant proliferation. We then studied the secretion of cytokines by the Pno lymphocytes and patient PBLs in response to a submitogenic dose of PMA alone or to the mitogenic combination of PMA/anti-CD3 mAb. The supernatants of the lymphocyte cultures were collected, and the amounts of IL-2, IL-4, IL-10, IFN-,
and TGF-1 were determined by ELISA, whereas the amount of TNF-
was determined using a biological assay. The results presented in Table
3 showed that 2 ng/mL PMA alone, which
induced very low proliferation (Figure 7), did not induce significant
amounts of cytokine production. In contrast, the combination of PMA and
soluble anti-CD3 mAb induced a significant production of TNF- and
TGF-1, whereas only small amounts of IL-2 and IL-4 and no IL-10 and
IFN- were detected in the supernatants. Interestingly, we found a
similar Th3-type cytokine profile with the patient PBLs stimulated with
PMA and anti-CD3 mAb.
Skin lesions in CTCL contain a heterogeneous lymphocytic infiltrate
composed of malignant T cells, which are most often CD4+,
and nonneoplastic tumor infiltrating T lymphocytes. We previously reported CD4+ cytotoxic tumor infiltrating lymphocytes
directed specifically against autologous malignant CD4+
cells.5 Thus, aside from the clonotypic TCR-V
Submitted February 4, 2000; accepted April 1, 2000.
Supported by grants from INSERM, Paris, France; Association de la Recherche contre le Cancer, Villejuif, France; Société Française de Dermatologie, Paris, France; Laboratoires La Roche Posay, LaRoche-Posay, France; and Académie de Médecine, Paris, France.
E.P. and M.B. contributed equally to this work.
Reprints: Armand Bensussan, INSERM 448, Faculté de Médecine de Créteil, 8 rue du Général Sarrail, 94010 Créteil, France; e-mail: bensussan{at}im3.inserm.fr.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Top
Abstract
Introduction
phenotype. It was named Pno and had been cultured for more than 1 year.
Both fresh and long-term-cultured tumor cells proliferated highly in
response to interleukin-7 (IL-7), and exogeneous IL-7 prevented Pno
lymphocytes from apoptosis and maintained high levels of
Bcl-2 expression. This unique malignant cloned lymphocyte
line was further used to carry out functional studies. The results indicated that the CD3/TCR structures expressed by the Pno lymphocytes were functional because an immobilized anti-CD3 monoclonal antibody (mAb) or the combination of a soluble anti-CD3 mAb with submitogenic doses of phorbol 12 
-bearing cloned lymphocytes.
J Immunol.
1990;144:1