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NEOPLASIA
From INSERM U448 and Service de Dermatologie de
l'hopital Henri Mondor, (AP-HP) Creteil, France; Dipartimento di
Medicina Sperimentale, Università di Genova, and Istituto
Nazionale per la Ricerca sul Cancro, Genova, Italy.
Tumor cells of patients with cutaneous T-cell lymphoma (CTCL)
have the cell surface phenotype of mature T-helper lymphocytes, and it
may be impossible to differentiate them from nonmalignant lymphocytes
in skin and blood. Until now, no specific cell membrane marker of CTCL
has been reported. In the current study, it is reported for the first
time that CTCL cells express the major histocompatibility complex class
I binding p140-killer cell immunoglobulin-like receptor, which has
been described on a minor subset of natural killer lymphocytes and on a
marginal circulating CD8+ T lymphocyte subset.
Interestingly, the molecular characterization of this KIR expressed by
CTCL allowed us to isolate a novel allelic form of p140-KIR3DL,
resulting in 4 amino acid substitutions, 3 in the extracellular
immunoglobulin-like domain of the protein and one in the cytoplasmic
region. This finding is likely to be important both for the
pathophysiology and for the clinical treatment of patients with CTCL.
(Blood. 2001;97:1388-1391) Cutaneous T-cell lymphomas (CTCL) are a
heterogeneous group of lymphomas primarily involving the
skin.1 Mycosis fungoides (MF) is characterized by skin
invasion of clonally derived malignant CD4+ T lymphocytes
that phenotypically resemble mature T-helper cells. A
more aggressive form of CTCL develops when the malignant cells become nonepidermotropic and are associated with extracutaneous involvement. Sézary syndrome (SS) is a more aggressive form of CTCL characterized by a clonal expansion of
CD4+/CD45RO+ T cells and the appearance of
these malignant T cells in the blood.2,3 The biology of
the disease remains poorly understood; 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 cells from reactive infiltrating CD4+
T lymphocytes.4-6 We previously reported a unique
CD4+ T-cell line derived from CTCL lesions.6
We demonstrated that the cell line and the in vivo tumor cells
expressed an identically sized, complementarily determining region 3 of
T-cell receptor (TCR)-V Here, we show that these 2 different CTCL lines express the
p140-KIR3DL2 inhibitory receptor for HLA-A alleles. Importantly, this
receptor was detected on freshly isolated tumor cells derived from the
same patients. Moreover, the p140 was also co-expressed by a major
subset of CD4+ lymphocytes in 7 other patients with SS, and
by tumor skin CD4+ cells in 2 additional patients with
advanced MF. The p140-KIR3DL2 is detected in healthy persons
on a minor natural killer (NK) cell subset8-10 and on rare
peripheral blood CD3+ CD8+
cells.11,12 P140 is a member of the killer
immunoglobulin-like receptor (KIR) family known to negatively modulate
NK-mediated cytotoxicity on recognition of different groups of HLA
class I alleles. In particular, p140 in normal NK cells has been shown to inhibit NK-mediated lysis after interaction with some HLA-A alleles,
such as HLA-A3 and HLA-A11. T cells obtained from skin in other
dermatologic diseases, such as inflammatory skin diseases and toxic
epidermal necrolysis, did not express this receptor.13 Thus, our current findings suggest that p140 represents a suitable marker on CD4+ cells for the identification of CTCL.
Patients
Isolation of tumoral lymphocytes
Long-term culture of tumor cell lines We established the long-term culture of Pno cell line (TCRV 22+, CD3+, CD4+,
CD8 +, major histocompatibility complex (MHC) class
I+, MHC class II ) in vitro from the
peripheral blood of the patient as previously described.6,7 The human leukocyte antigen (HLA) haplotype of patient Pno is HLA-A1, A2, and HLA-B18, B57. We demonstrated that
both the malignant clone circulating in the patient blood and the
derived cultured T-cell line were identical for their cell surface
phenotype and for their size and sequence of the TCR VDJ
region.7 The Cou-L cell line (TCRV13+) was
cultured in vitro with rIL-2 for more than 3 years. It corresponds to a
subclone of the CD4+ Cou-LS CTCL line previously
described.6 The Cou-L cell line, the original
TCRV13+, CD4+ Cou-LS CTCL, and the tumor
cells freshly isolated from the skin shared the same size and sequence
of the TCR VDJ region.6 The HLA haplotype of patient
Cou is HLA-A1, A2 and HLA-B5 (51) B35.
Monoclonal antibodies and flow cytometry studies One- and 2-color immunofluorescence analysis was performed as previously described.6 The monoclonal antibodies (mAbs) anti-CD3, anti-CD4, anti-MHC class I, and anti-MHC class II were produced locally. The anti-TCRV13+ mAb was purchased from BIOadvance
(Emerainville, France), and the anti-TCRV22+ mAb was
obtained from Beckman-Coulter (Marseille, France). The anti-CD8
2ST8.5H7 mAb was kindly provided by Dr E. L. Reinherz (DFCI, Boston,
MA). Q66 (IgM, anti-p140),8 AZ158 (IgG2a, recognizing both
p70-NKB1 and p140), Z27 (IgG1, anti-p70/NKB1), EB6 (IgG1, anti-p58.1/p50.1), GL183 (IgG1, anti-p58.2/p50.2), XA185 (IgG1, anti-CD94), and Z199 and Z270 (IgG2b and IgG2a respectively,
anti-NKG2A) were produced in one of our laboratories.11 Dr
B. Malissen (INSERM-CNRS, Marseille Luminy, France) provided the B9.4
(IgG2b, anti-CD8) mAb.
Biochemical characterization Then 20 × 106 cells were incubated (15' at 20°C) in 1 mL PBS pH 8 containing 250 µg EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce, Rockford, IL) and washed 3 times in washing buffer (10 mM Tris pH 8, 0.14 M NaCl). Cells were lysed in 1% NP-40 and immunoprecipitated with Sepharose-PA (Pharmacia Biotech, Piscataway, NJ)-coupled AZ158 (IgG2a, anti-p70/p140). Samples were analyzed by discontinuous SDS-PAGE either undigested or digested with N-glycosidase F (Boehringer Mannheim GmbH, Mannheim, Germany) and transferred to Immobilon P (Millipore, Bedford, MA). After staining the cells with Neutravidin (Pierce), we used the Renaissance Chemiluminescence Kit (NEN, Boston, MA) for detection. NK cell clones were obtained by limiting dilution as previously described.8RT-PCR analysis Total RNA was extracted from CTCL cell lines Pno and Cou-LCD8 using RNA-Clean System (AGS GmbH, Heidelberg, Germany). cDNA synthesis was performed using oligo-dT priming. Primers used for cDNA
amplification of the complete open-reading frame of KIR displaying 3 immunoglobulin-like domains (1395 bp) were the following:
5'CATGT(CT)GCTCA(CT)GGTCGTC (Ig3 UP) and 5'
GGTTTTGAGACAGGGCTG (Ig3 DOWN). Amplification was performed for 30 cycles (30 seconds at 94°C, 30 seconds at 55°C, 30 seconds at
72°C), followed by a 7-minute incubation at 72°C, using AmpliTAQ
(PerkinElmer Applied Biosystems, Foster City, CA). PCR products were
subcloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, CA).
DNA sequencing was performed using d-Rhodamine Terminator Cycle
Sequencing Kit and a 377 Applied Biosystems Automatic Sequencer
(PerkinElmer Applied Biosystems).
Transient transfections COS-7 cells were transfected with pcDNA3.1 TOPO-KIR3D cl.24 or with pCR3-cl.1.18 using Fugene 6 (Roche, Monza, Italy). Briefly, cells were seeded at 5 × 105/plate; 24 hours later they were incubated with 6 µg plasmid and 10 µL Fugene-6 reagent in Dulbecco minimum essential medium/10% fetal calf serum. After 48 or 72 hours, transfected cells were used for cytofluorometric analysis. Cell transfectants were stained with Q66 and AZ158 mAbs, followed by a phycoerythrin-conjugated goat antibody to mouse IgG2a or IgM and analyzed by flow cytometry using FACSort (Becton Dickinson, San Jose, CA).
CTCL cell lines are stained by mAbs to the p140 Two long-term CTCL tumor lines Pno (labeled with anti-TCR-V22
mAb)7 and Cou-L (labeled with anti-CD3 mAb)6
were analyzed for reactivity with different anti-KIR mAbs. We found
that both cell lines were reactive with mAbs Q66 (Figure
1) and AZ158 (not shown), both
recognizing p140. In contrast, these cell lines did not express other
inhibitory receptors specific for HLA class I molecules, including
p58.1, p58.2, p70 KIRs, and the CD94/NKG2A lectin-like receptor (data
not shown and 7).
Tumor T lymphocytes freshly isolated from patients with CTCL are stained by anti-p140 mAbs To determine whether p140 was expressed by freshly isolated tumor cells, we tested the reactivity of Q66 mAb with uncultured tumor cells isolated from the blood of SS patient Pno and from tumoral skin fragments of MF patient Cou. We found that most tumor cells were stained by this mAb (Figure 1). In particular, we observed that most TCRV22+ tumor lymphocytes isolated from the blood of
patient Pno were reactive with Q66 antibody and that most of the
TCRV13+ tumor lymphocytes isolated from the skin of
patient Cou were stained by the same antibody (Figure 1).
Next, we studied the phenotype of tumor T lymphocytes from the blood of
7 additional patients with SS, with malignant cells representing 10%
to 45% of circulating CD4+ lymphocytes, and tumor T
lymphocytes isolated from skin tumors of 2 other patients with a MF.
Remarkably, all patients tested exhibited a significant population
co-expressing CD4 and p140 (Table 1). It
should be noted that all Q66+ cells were included in the
CD4+ cell population (data not shown). Thus, the expression
of p140, which in healthy persons is restricted to subsets of
lymphocytes from the NK and CD8+ populations,6
appears to be a characteristic of CTCL tumor CD4+ T
lymphocytes, both in the skin and in the blood. As control, skin T
lymphocytes derived from another dermatologic disease, toxic epidermal
necrolysis, which were shown to contain small percentages of various
KIR-expressing T lymphocytes,13 failed to express p140
(data not shown).
Molecular characterization of the p140 receptor expressed by CTCL The Pno and Cou-L cell lines were surface labeled with biotin, and cell lysates were immunoprecipitated with an anti-p140 (AZ158 mAb). As shown in Figure 2, this mAb immunoprecipitated, from an NK clone and from the Pno and Cou-L cell lines, a molecule with a molecular mass of approximately 70 kd under reducing conditions. Treatment with N-glycosidase revealed a protein backbone of approximately 50 kd with a slightly higher mobility for Pno and Cou-L than the NK clone. These data suggested that the p140 inhibitory receptor expressed by these CTCL tumor cell lines could be almost similar to that previously detected on normal NK cells.8
Next, we determined whether the cDNA encoding the molecule recognized
by Q66 and AZ158 mAbs on Pno and Cou-L CTCL cell lines corresponded to
one of the already described cDNAs encoding p140. To this end, RT-PCR
was performed on RNA derived from these cell lines using a set of
primers able to amplify all cDNA encoding KIR with 3 immunoglobulin-like domains. From the Pno cell line, we isolated a
full-length cDNA, termed KIR3D cl.24. Comparison of its nucleotide
sequence with DNA sequences coding for all KIR characterized by 3 extracellular immunoglobulin-like domains revealed that KIR3D cl.24
represents a novel transcript for the p140 gene. In particular, its
nucleotide sequence displays 5 differences compared to the previously
described cl. 1.1 cDNA,8 resulting in 4 amino acid
substitutions in the mature protein (Figure
3). Three of the 4 substitutions are
found in the extracellular immunoglobulin-like domain (positions 20, 92, and 111 of the mature protein), whereas the other is located in the
cytoplasmic region (position 401). RT-PCR performed on Cou-L CTCL cell
line revealed 2 different allelic forms of p140, one corresponding to
cl. 1.1 cDNA and the other identical to KIR3D cl.24 (isolated from Pno
cell line). The cDNA derived from the CTCL cell lines were then
transiently transfected in COS-7 cells. As expected, all cell
transfectants were brightly stained by Q66 and AZ158 mAbs, whereas they
were unreactive with the p70/KIR3DL1-specific Z27 mAb used as a
negative control (data not shown). Finally, RT-PCR was performed on RNA extracted from PBL derived from 3 patients with SS (including patient
Pno) and from skin-derived T cells of one additional patient with MF.
In addition, in these samples one or another allelic isoform of p140
described above could be identified.
KIR3D cl.24 may represent an additional polymorphism in the p140 gene and could be considered a novel allelic form of p140 receptor. However, further investigation is required to assess the allelic nature of KIR3D cl.24 and to exclude the possibility that somatic mutations may play a role in the generation of KIR3D cl.24 sequence. On the other hand, this seems unlikely because, as mentioned above, KIR3D cl.24 sequence could be amplified from different unrelated patients.
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 specifically directed against the autologous malignant CTCL CD4+ cell line.6 However, because no tumor-restricted cell surface structure, aside from the clonotypic TCR expressed by tumor cells, has been identified on CTCL, it is difficult to use standard methods to distinguish malignant from nonmalignant, reactive CD4+ lymphocytes. In the current study, we report for the first time that tumor cells
from patients with MF and SS express p140/KIR3DL2. This receptor has
been identified in skin and blood tumor cells from patients with CTCL
and in 2 long-term culture CTCL lines. Two-color fluorescence analysis
indicated that p140 expression is restricted to T cells characterized
by a given TCR Previous studies indicated that this receptor was able to generate inhibitory signals on recognition of HLA-A3 and HLA-A11 alleles.8 It is of note, however, that p140 expression in the various patients analyzed is apparently independent of their own HLA class I haplotype. It is possible that the product of the novel transcript for p140 may recognize HLA-A alleles different from those reacting with the described p140 receptor. Additional studies will be required to verify this possibility. Nevertheless, the actual role of p140 in the pathophysiology of CTCL is an important feature to be studied by taking into account the potential role of this receptor in the tolerance to self.14 In conclusion, the current study demonstrates for the first time the expression of p140 in CD4+ CTCL cells and the isolation of a novel transcript encoding this receptor in tumor cells. This finding is likely to be an important new issue, both for the pathophysiology and the clinical management of patients with CTCL.
Submitted July 3, 2000; accepted October 12, 2000.
Supported by grants from Inserm, Paris XII University, ARC, Société Française de Dermatologie, Laboratoires La Roche Posay, Dermatologiques Evaux, and Académie de Médecine, Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanità, Ministero della Sanità, and Ministero dell' Università e della Ricerca Scientifica e Tecnologica, Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie, and by grant E.0892 from Telethon-Italy.
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.
Reprints: Martine Bagot, Faculté de Médecine, Inserm U448, 8 Avenue du Général Sarrail, 94010 Créteil, France; e-mail: martine.bagot{at}hmn.ap-hop-paris.fr.
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Willemze R, Kerl H, Sterry W, et al.
EORTC classification for primary cutaneous lymphomas: a proposal from the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer.
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Boumsell L, Bernard A, Reinherz EL, et al.
Surface antigens on malignant Sézary and T-CLL cells correspond to those of mature T cells.
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The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140 kD disulphide-linked dimer.
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1996;184:505-518 9. Döhring C, Scheidegger D, Samaridis J, Cella M, Colonna M. A human killer inhibitory receptor specific for HLA-A. J Immunol. 1996;156:3098-3101[Abstract]. 10. Long EO. Regulation of immune responses through inhibitory receptors. Annu Rev Immunol 1999;17:875-904[CrossRef][Medline] [Order article via Infotrieve]. 11. Moretta A, Biassoni R, Bottino C, et al. Major histocompability complex class I-specific receptors on human natural killer and T lymphocytes. Immunol Rev. 1997;155:105-117[CrossRef][Medline] [Order article via Infotrieve]. 12. Mingari MC, Moretta A, Moretta L. Regulation of KIR expression in human T lymphocytes: a safety mechanism which may impair protective T cell responses. Immunol Today. 1998;19:153-157[CrossRef][Medline] [Order article via Infotrieve]. 13. Le Cleach L, Delaire S, Boumsell L, et al. Blister fluid T lymphocytes during toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors. Clin Exp Immunol. 2000;119:225-230[CrossRef][Medline] [Order article via Infotrieve]. 14. Huard B, Karlsson L. KIR expression on self-reactive CD8+ T cells is controlled by T-cell receptor engagement. Nature. 2000;403:325-328[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  E. Fourmentraux-Neves, A. Jalil, S. Da Rocha, C. Pichon, S. Chouaib, G. Bismuth, and A. Caignard Two opposite signaling outputs are driven by the KIR2DL1 receptor in human CD4+ T cells Blood, September 15, 2008; 112(6): 2381 - 2389. [Abstract] [Full Text] [PDF]
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A. Marie-Cardine, D. Huet, N. Ortonne, N. Remtoula, S. L. Gouvello, M. Bagot, and A. Bensussan Killer cell Ig-like receptors CD158a and CD158b display a coactivatory function, involving the c-Jun NH2-terminal protein kinase signaling pathway, when expressed on malignant CD4+ T cells from a patient with Sezary syndrome Blood, June 1, 2007; 109(11): 5064 - 5065. [Full Text] [PDF]
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A. Munitz, I. Bachelet, R. Eliashar, A. Moretta, L. Moretta, and F. Levi-Schaffer The inhibitory receptor IRp60 (CD300a) suppresses the effects of IL-5, GM-CSF, and eotaxin on human peripheral blood eosinophils Blood, March 1, 2006; 107(5): 1996 - 2003. [Abstract] [Full Text] [PDF]
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P. Musette, L. Michel, F. Jean-Louis, M. Bagot, and A. Bensussan Polymorphic expression of CD158k/p140/KIR3DL2 in Sezary patients Blood, February 1, 2003; 101(3): 1203 - 1203. [Full Text] [PDF]
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M. Nikolova, P. Musette, M. Bagot, L. Boumsell, and A. Bensussan Engagement of ILT2/CD85j in Sezary syndrome cells inhibits their CD3/TCR signaling Blood, July 18, 2002; 100(3): 1019 - 1025. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), derived from a healthy donor, and the
cell lines Pno and Cou-L were surface labeled with biotin and
immunoprecipitated with anti-p140 mAb. Samples were treated (+) or not
(