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Institut de Recherche sur la Peau, Institut National de la Santé et de la Recherche Médicale U312, Hôpital Saint-Louis, Paris, France; and
Institut Pasteur, Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale U277, Département d’Immunologie, Paris, France
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identification of the T cells undergoing local expansions (i.e., the T cells activated locally by nevi Ags) is thus of primary importance in the analysis of the halo nevus phenomenon. A previous analysis of the TCR of T lymphocytes infiltrating the halo nevi revealed a local proliferation of T cell clones (7) The present work is aimed at approaching the in vivo diversity of the ß-chains of the TCR of the T cells present in nevi undergoing immune-mediated regression, which avoids the biases introduced by the in vitro cloning of T cells or the cloning of PCR products. For this purpose, we have used a PCR-based technology to describe the Vß, Jß, and complementarity-determining region 3 (CDR3)2 length usages of the T cells that infiltrate 13 halo nevi removed from eight patients. Two distinct populations of T cells infiltrating the halo nevi can be distinguished: 1) nonactivated or polyclonally activated T cells and 2) clonally expanded T cells. The ß-chains of the TCR of the T cells expanded in a halo nevus of a patient were also found to be associated with the T cells expanded in other halo nevi of the same patient but were not found in the blood. Finally, the amino acid sequences of the Ag recognition regions of the TCR ß-chains of the expanded T cells shared common amino acid patterns, suggesting that they may recognize the same or closely related antigenic epitope(s).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight Caucasian patients presenting 1–30 halo nevi were prospectively included in the present study. The patients showed no evidence of any overt disease. After approval of the research program by the local ethics committee, written informed consent was obtained from each patient. The diagnosis of halo nevus was confirmed by a histological analysis of skin biopsies. A total of 13 halo nevi (diameters ranging from 0.5 to 1 cm) were collected. Peripheral blood was taken from each patient, and PBLs were prepared by the Ficoll-Hypaque procedure. HLA class I and class II typing of patients A and K was determined by M. Abbal (Hôpital Purpan, Toulouse, France) using a PCR-based technology and allele-specific probes.
Immunohistochemical staining
A fragment of each biopsied halo nevus was frozen at -80°C immediately following surgery. Frozen sections were analyzed for the expression of CD3, CD4, CD8, S100, and HLA class II using mAbs purchased from Dako (Copenhagen, Denmark), Fas and Fas ligand mAbs (purchased from Immunotech, Marseille, France), perforin and granzyme B mAbs (purchased from Pharmacell, Paris, France), cutaneous lymphocyte-associated Ag (CLA) mAb (purchased from PharMingen, San Diego, CA), and HLA class I mAb (purchased from Sigma, St. Louis, MO) according to conventional procedures. Apoptotic cells were revealed using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate nick-end labeling procedure (Boehringer Mannheim, Mannheim, Germany).
RNA extraction and cDNA preparation
Total RNA was extracted as described previously (8) from 107 blood T cells or from the entire nevi, except for the fragment used for histology. mRNA was reverse-transcribed from PBLs and halo nevi using a technique described elsewhere (9).
TCR ß-chain analysis
The "immunoscope" technique has been extensively described elsewhere (10). Briefly, standardized amounts of cDNA (i.e., the product of the reverse transcription of 10 µg of total RNA) were PCR amplified using each of the 24 Vß-specific probes and a common Cß-specific probe. All primers used in the present study have been described elsewhere (11). Each Vß-Cß PCR product was analyzed by electrophoresis in an agarose gel. For the analysis of CDR3 length diversity, PCR-amplified products were subjected to five cycles of primer extension using an internal, fluorescent, Cß-specific probe (11). The labeled material was loaded on a sequencing gel and analyzed using an automatic sequencer (Applied Biosystems, Foster City, CA) equipped with a computer program (immunoscope), which enables the determination of the intensity of fluorescence of each band as well as its actual size. The results are depicted as peaks with surfaces that are proportional to the amount of material; the location of the peaks was determined by the length of the CDR3 region. Vß-Cß PCR products were also subjected to five cycles of primer extension using each of the 13 fluorescent Jß-specific probes (1.1–2.7). The resulting products were analyzed as described above. The size distribution of Vß-Cß and Vß-Jß is Gaussian in the case of nonactivated or polyclonally activated lymphocytes, whereas Ag-dependent proliferating T cells generated a non-Gaussian distribution, with expanded peaks corresponding to cells using a definite CDR3 length within a Vß-Jß combination (12).
Detection of melanoma-associated Ag mRNAs
The cDNA was PCR amplified with primers specific for the MAGE, BAGE, GAGE, and RAGE families, respectively, according to conventional procedures (13, 14, 15, 16). PCR products were analyzed by electrophoresis on an agarose gel. The mRNA coding for tyrosinase was used as an internal positive control.
Direct DNA sequencing
After separation of the PCR products by electrophoresis in a 2% agarose gel and electro-elution of the DNA fragments, direct sequencing of PCR products was conducted using the United Stated Biochemical-Amersham kit (Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A fragment of each halo nevus studied was sectioned and analyzed. None of the nevi showed signs of malignancy; however, all showed histological signs of regression that are associated with infiltration by lymphocytes, occasional macrophages, and neutrophils. Consequently, the regressing nevi retained for RT-PCR analysis fit the definition of halo nevi (3). Further immunohistochemical analysis confirmed earlier reports (4, 6), indicating that the infiltrate was composed of CD4+ T cells and CD8+ T cells. The CD4+/CD8+ ratio was in the 0.5–3 range depending upon the biopsy. CD8+ T cells were usually predominant. The majority of the T cells were granzyme B+, perforin+, and Fas ligand+ and were thus likely endowed with cytotoxic activity. Nearly all of the T cells present in the nevi were CLA+, a cell surface protein associated with skin homing (17). Melanocytes strongly expressed MHC class I but not class II molecules and underwent apoptosis as shown using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate nick-end labeling technique. Normal nevi did not show cell infiltration or apoptosis of melanocytes.
TCR repertoire of the 
ß T cells that infiltrate the halo nevi
is biased
Few T cells infiltrate the halo nevi (105 T cells/halo nevus on average), and they cannot be recovered easily from halo nevi, preventing direct FACS analysis. Thus, Vß usage was studied by RT-PCR. A total of 13 halo nevi were surgically removed from eight patients (A, K, C, G, L, B, P, and Y). Total RNA was immediately extracted from the samples and reverse transcribed. The resulting cDNAs were first amplified by PCR using each of the 24 Vß-specific probes and a common Cß-specific probe. PCR products were analyzed first by agarose gel electrophoresis. No Vß-Cß sequences could be amplified from normal nevi or from nevi C1 and L. In the other nevi, the different Vß-Cß sequences that could be amplified varied with the patients. They were Vß 1, 2, 3, 4, 5, 6B, 7, 8, 10, 14, 15, 16, 17, 18, 21, 22, and 23 in nevus A1; Vß 1, 2, 3, 4, 7, 8, 10, 15, and 17 in nevus A2; Vß 1, 2, 3, 4, 5, 6A, 6B 7, 8, 9, 14, 15, 16, 17, 18, and 19 in nevus K1; the same Vß in nevus K2 as in nevus K1, except Vß8; the same Vß in nevus K3 as in nevus K1, plus Vß 11 and 21; Vß8 in nevus C2; Vß7 in nevus C3; and Vß 6A and 7 in nevus G. Vß7 was used in nevus B. All Vß except for Vß12 and 13A were used in nevus P; Vß 3, 4, 6A, 7, 8, 14, and 18 were used in nevus Y. The absence of some Vß in the halo nevi cannot be attributed to sampling problems, because a complete Vß-Cß analysis can be conducted on as few as 1000 T cells (18). This absence is instead due to the extent of regression of the nevi studied. As a control of primer efficiencies and of the presence/absence of ß-chains, all 24 Vß-Cß families could be amplified out of the control PBMCs of each patient.
The Vß-Cß PCR products prepared from 10 halo nevi of five patients
(halo nevi A1, A2, K1, K2, K3, C1, C2, C3, G, and L) were further
analyzed for the distribution of CDR3 size by running primer extensions
on the Vß-Cß PCR products, using a common, fluorescently labeled,
internal Cß-specific primer. The labeled primer extension products
were separated on an automated sequencer. The results are depicted by
the pictograms of Fig. 1
. The Vß-Cß
patterns were found to be Gaussian in the blood of all patients. In
contrast, the Vß-Cß PCR products prepared from halo nevi displayed
diverse patterns. Most Vß-Cß combinations yielded a Gaussian signal
that was indicative of an absence of specific stimulation of the
corresponding T cells; other combinations (Vß15 in nevi A1,
A2, and K1; Vß1 in nevi K1, K2, and K3; Vß6A in nevus G; Vß6B in
nevus K3; Vß7 in nevi K1, K2, and K3; Vß17 in nevi K1, K2, and K3;
and Vß21 in nevus K3) yielded isolated peaks with a unique CDR3
length, revealing the presence of Ag-driven T cell proliferation. Thus,
the immune response to halo nevi is complex, and a limited number of T
cell clones is expanded, presumably due to the recognition of specific
Ag(s).
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A frequent feature of the halo nevus phenomenon is the simultaneous regression of several nevi in the same patient. To determine whether the same T cell repertoire is observed in distinct halo nevi of the same patient, we have determined the Vß-(CDR3)-Jß repertoire of the T cells present in several halo nevi by running primer extensions on the Vß-Cß PCR products that yielded single peaks in the immunoscope analysis. The procedure was conducted using each of the 13 Jß-specific, fluorescently labeled primers and was followed by an analysis of the products on a sequence gel and the subsequent determination of the nucleotide sequence of the PCR products.
Thus, we have analyzed the Vß-CDR3-Jß repertoire of the T cells infiltrating the two halo nevi removed from patient A, the three halo nevi removed from patient K, and the three halo nevi removed from patient C. The halo nevi of patients A and K presented a dense lymphocyte infiltrate, whereas the infiltrate in patient C had a very poor lymphocyte count.
In patient A, a single peak was detected in the Vß15-Cß PCR
products of the two halo nevi. Upon further analysis of the Vß15-Cß
PCR products using the 13 Jß-specific probes, Jß2.3 was found to be
predominantly used in the two halo nevi studied; it was associated with
a 10-aa long CDR3 region. In contrast, the Vß15-Jß2.3 repertoire of
the peripheral blood T cells of patient A showed a Gaussian pattern
(Fig. 2). The nucleotide sequence of the
Vß15-Jß2.3 PCR products obtained from the two halo nevi of patient
A was determined by direct sequencing (Fig. 3). The two nucleotide sequences were
found to be identical. Thus, T cells sharing the same ß-chain have
undergone expansion in two physically distinct halo nevi, a finding
that is strongly suggestive of a proliferative response to the same
antigenic peptide(s) presented by the same restriction element(s). A
probe specific for the Vß15-Jß2.3 CDR3 region of the ß-chain of
the TCR characteristic of the T cell population expanded in patient A
(clonotypic primer) was designed and used to search for T cells sharing
the same TCR ß-chain in the PBLs of patient A and also to determine
the relative frequency of these T cells in the halo nevi. Using
the clonotypic primer in run-off experiments on Vß15-Cß PCR
products, a single peak was observed in both halo nevi. The peak was
superimposable with that observed in the Vß15-Jß2.3 analysis (Fig. 4, solid line). No signal was observed in
the Vß15-Cß PCR products prepared from the PBLs of patient A (Fig. 4, broken line). The relative frequency of the T cells detected
by the clonotypic primers was assessed by determining the ratio of the
area of the peak of interest to the total area of all peaks (19). The T
cells bearing this particular ß-chain represented 16% and 11% of
the total T cells in the A1 and the A2 halo nevi, respectively, and
thus were largely predominant among other T cells present in the halo
nevi. Hence, they are expanded in the two halo nevi but not in blood.
Consequently, the expansion of T cells in the two halo nevi studied in
patient A must have occurred locally, within the nevus, and not at the
periphery due to some antigenic stimulation that would be irrelevant to
the immune-mediated regression of the nevi, such as an infection.
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(lines K1-K2 and K3). The Vß-Cß repertoire showed
that, among otherwise polyclonal profiles, the three halo nevi shared
three expanded T cell populations. Upon further Vß-Jß analysis,
three expanded T cell populations were identified in each halo nevus:
the first was characterized by a Vß17-Jß2.1 rearrangement and a
6-aa CDR3 length, the second by a Vß17-Jß2.3 rearrangement
and a 10-aa CDR3 length, and the third by a Vß7-Jß1.2 rearrangement
and a 8-aa CDR3 length (Fig. 5). We have
determined the nucleotide sequence of the Vß7-Jß1.2,
Vß17-Jß2.3, and Vß17-Jß2.1 PCR products prepared from each of
the three halo nevi of patient K. The three Vß7-Jß1.2 nucleotide
sequences were found to be identical, as were the three Vß17-Jß2.3
sequences and the three Vß17-Jß2.1 sequences (Fig. 3). Thus, three
different T cell populations characterized by usage of the same
ß-chain are found in the three halo nevi of the patient, presumably
indicating a local expansion of each population. A unique peak was also
observed in the Vß1-Cß combination of the three halo nevi of
patient K. However, the surface of the peak was very small compared
with that of the Vß7-Cß peak. Upon Jß analysis of the PCR
products, no significant peaks were observed. Finally, the analysis of
the T cell repertoire of the rare T cells infiltrating the three halo
nevi of patient C showed either a preferential usage of Vß7 (C3) or a
preferential usage of Vß8 (C2). No material could be amplified from
the third halo nevus (C1). Primer extension analysis of the PCR
products using Cß- and Jß-specific primers yielded a Gaussian
distribution. No peaks were detected, and the signals were beyond the
limits of detection. It is worth noting that the halo nevi of patient C
had been excised at the end of the regression process and were
associated with a very poor lymphocytic infiltrate.
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Some halo nevi exhibit moderate atypia (20). The abnormal expression in the halo nevi melanocytes of melanoma Ags that are not expressed in normal nevi could possibly explain their regression, as in spontaneously regressing melanomas. Consequently, we have searched for the expression of such Ags in the halo nevi on the basis of RT-PCR analysis of total mRNA. None of the halo nevi were found to express the melanoma-associated protein members of the MAGE, BAGE, GAGE, and RAGE families. Tyrosinase mRNA, used as an internal positive control, was amplified in all samples studied (data not shown). Thus, the peptide(s) involved in the halo nevus phenomenon are different from the tumor-associated Ags of the melanoma cells identified thus far.
Antigenic peptides presented by classical MHC class I or class II
molecules are primarily recognized by the CDR3 loop of the - and
ß-chains of the TCR, with little contribution from the CDR1 and CDR2
regions (21). The diversity of the -chain could not be studied for
lack of sufficient material. The CDR3ß sequences associated with all
of the T cells expanded in the halo nevi were found to use the Dß2.1
region. The in-frame use of part of the Dß2.1 region in sequences A,
K1, and K3 resulted in the presence of a recurrent motif (TS or
TSG) in the CDR3ß of these three clones (Table I). A TSG motif was also found in the
opposite direction, in the CDR3ß region of clone K2, which used an
out-of-frame Dß2.1 segment. The recurrence of the TS (TSG) motif may
indicate that the T cells under study recognize identical or closely
related peptides in the same or similar restriction context. In this
respect, the two patients shared the HLA-A1 allele: patient A was A1
(33), Cw6 (8), DR3 (10), and DQß (0205); patient K was A1, Cw7, DR1
(4), and DQ (5,3). Finally, none of the ß-chains of the nearly
400 TCR ß-chains identified in the melanoma-infiltrating T
cells or recognizing melanoma Ags (MAGE, BAGE, GAGE, RAGE,
MART-1, glycoprotein 100, tyrosinase, etc.) was found to
share any amino acid sequence homology with the A, K1, K2, or K3
sequences.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain that is suggestive of clonal T cell expansions (7). The
results are also similar to the ß-chain repertoire in
melanomas: a strictly local and clonal expansion of T cells has been
reported in regressive melanomas (2), and an expansion and accumulation
of specific T cells has been found in melanomas (25); the ß-chain
repertoire was found to be associated to clonal and oligoclonal
responses in progressive melanomas (26) and to different clones in the
regressive and progressive regions of primary malignant melanomas (27).
In addition to the oligoclonality of the T cell response in the halo
nevi, a few expansions of T cells sharing the same Vß-CDR3-Jß
nucleotide sequence were recurrently found in several halo nevi of the
same patient. Regardless of whether these cells share the same
rearranged TCR -chain, these proliferating T cells derive from a
small number of the same ß T cell clone(s) at most and
consequently probably recognize the same molecular target at different
sites in the skin. Because the nevi are not clonal in nature (28), the
Ag(s) are likely to be shared by all halo nevi of a given patient. In
addition, the recurrence of the TS or TSG motif in the CDR3 sequences
of the expanded T cells in different patients may be suggestive of the
recognition of related antigenic peptides. Aside from whether a
unique Ag or several Ags are recognized by this family of T cells, the
nature of the halo nevi Ag(s) remains unknown; however, it does not
belong to the proteins that are known thus far to be specifically
expressed by melanoma cells. The halo nevus Ag(s) might be an
autoantigen, part of the differentiation program of the normal nevi to
which the immune system is normally tolerant. Therefore, the
destruction of melanocytes in the halo phenomenon could tentatively be
viewed as the lysis of a distinctive cell type through an
autoimmune-like process mediated by an oligoclonal T cell response.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CDR, complementarity-determining region; CLA, cutaneous lymphocyte-associated Ag.
Received for publication July 6, 1998. Accepted for publication October 15, 1998.
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References |
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immunoreactivity in the inflammatory cell infiltrate of halo nevi. Br. J. Dermatol. 134:388.[Medline]
ß T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex. Science 274:209.This article has been cited by other articles:
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  L. P. Menasce, J. H. Shanks, V. S. Howarth, and S. S. Banerjee Regressed Cutaneous Malignant Melanoma Mimicking Lymphoma: A Potential Diagnostic Pitfall International Journal of Surgical Pathology, July 1, 2005; 13(3): 281 - 284. [Abstract] [PDF]
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 I. B. Bayer-Garner, D. Ivan, M. R. Schwartz, and J. A. Tschen The Immunopathology of Regression in Benign Lichenoid Keratosis, Keratoacanthoma and Halo Nevus Clin. Med. Res., May 1, 2004; 2(2): 89 - 97. [Abstract] [Full Text] [PDF]
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 D. Schrama, E. Fuchs, E.-B. Brocker, P. thor Straten, and J. C. Becker Identical T-cell Receptor Transcripts in Multiple Melanoma Metastases Cancer Res., October 15, 2002; 62(20): 5664 - 5667. [Abstract] [Full Text] [PDF]
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 S. Ingen-Housz-Oro, A. Bussel, B. Flageul, L. Michel, L. Dubertret, P. Kourilsky, G. Gachelin, H. Bachelez, and P. Musette A prospective study on the evolution of the T-cell repertoire in patients with Sezary syndrome treated by extracorporeal photopheresis Blood, August 28, 2002; 100(6): 2168 - 2174. [Abstract] [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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