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*
Department of Tumor Cell Biology, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark;
Department of Pathology, Herlev Hospital, Copenhagen, Denmark; and
Department of Dermatology, School of Medicine, Würzburg, Germany
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Abstract |
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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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Tumor-specific CTLs are present in the blood of melanoma patients. However, it remains unknown whether these cells actually take part in the immune response against melanoma and in more general terms whether the T cell response against melanoma occurs as a systemic or localized phenomenon. In this respect, powerful antitumor reactivity in the PBL in most cases does not correlate with tumor regression. The background for this enigma is not known, but it could possibly be related to the failure of melanoma-specific CTLs to localize to the tumor site(s).
In the present study, we have used a recently developed, highly sensitive technique based on RT-PCR and denaturing gradient gel electrophoresis (DGGE) to analyze for the presence of clonotypic T cells in six s.c. melanoma lesions from two patients. Our results demonstrate that the T cell infiltrate in melanoma is exceedingly heterogeneous and accommodates a much higher number of T cell clonotypes than previously anticipated. Semiquantitative RT-PCR analyses revealed that T cell clonotypes are present not only in overexpressed ß-variable (BV) regions, but also in (BV) regions expressed at low levels. Furthermore, in most cases specific T cell clonotypes were detected in a single lesion only, indicating that locally expanded clonotypic T cells dominate the T cell infiltrate in melanoma, and that these cells enter the periphery and home to other tumor sites only to a very limited extent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fresh s.c. melanoma lesions were received as biopsy material immediately after surgery performed at the Department of Plastic Surgery (University Hospital, Copenhagen, Denmark) (patient 1) or the Department of Dermatology (School of Medicine, Würzburg, Germany) (patient 2). Three lesions were excised from each of the patients from the following anatomical sites: Patient 1, right thorax (all lesions); patient 2: axilla (lesion 1), axilla (lesion 2), and forehead (lesion 3). Tumor lesions and blood samples were taken from the respective patients at the same timepoint. PBLs were used for tissue typing (patient 1: HLA-A1, A2, B8, B44, Cw3, Cw5, DR3, DR9; patient 2: HLA-A2, B15, B38; typing was only performed for HLA class I A and B molecules in this patient).
Immunohistology
Frozen sections were fixed in cold acetone for 10 min followed by removal of endogenous peroxidase with 0.03% H2O2 and blocking of collagenous elements with 10% species-specific serum in 1% BSA (Boehringer Mannheim, Mannheim, Germany)/PBS. Next, the Abs were overlayed onto serial sections at predetermined dilutions (usually 20 µg/ml), and the slides were incubated in a humid chamber for 30 min. With PBS washes between every step, a biotinylated link Ab was applied for 10 min followed by a streptavidin-linked enzyme (i.e., either peroxidase or alkaline phosphatase) for 10 min. Substrate was added after another wash, and the slides were incubated in the dark for 20 min. After a wash in PBS, the slides were counter stained, mounted, and viewed using an Olympus BH2 microscope (Hamburg, Germany) with photographic capabilities.
RT-PCR for quantitative TCRBV region analysis and DGGE
Tumor biopsies and blood samples were processed immediately after surgery. RNA was extracted using the Purescript Isolation Kit (Gentra Systems, Minneapolis, MN). cDNA synthesis and quantitation of TCR cDNA in the each sample were conducted as described previously (4). In brief, 2–5 µg of RNA were used in the synthesis of cDNA using SuperScript II reverse transcriptase (Life Technologies, Gaithersburg, MD) in a total volume of 50 µl 1x buffer (Life Technologies) containing 10 mM DTT. The RT reactions were primed with a mixture of oligo(dT) and random hexamers, and incubations were performed at 37°C for 30 min, 42°C for 30 min, and 72°C for 5 min. Using equal amounts of TCR template in all reactions, cDNA was amplified using separate primer panels for quantitative TCRBV region analysis (4) and TCR clonotype mapping by DGGE (5). The sequences of these primer panels are available upon request. Amplifications were conducted in a total volume of 25 µl containing 1x PCR buffer (50 mM KCl, 20 mM Tris (pH 8.4), 2.0 mM MgCl2, 0.2 mM cresol red, 12% sucrose, and 0.005% (w/v) BSA (Boehringer Mannheim)), 2.5 pmol of each primer, 40 mM of dNTP (Pharmacia LKB, Uppsala, Sweden), and 1.25 U of AmpliTaq polymerase (Perkin-Elmer Cetus, Emeryville, CA). The parameters used for amplification were 94°C for 60 s, 60°C for 60 s, and 72°C for 60 s for 32 (quantitative PCR) or 40 cycles (DGGE). Taq polymerase and dNTPs were added to the reaction tube at an 80°C step between the denaturation and annealing steps of the first cycle (Hot start).
For quantitative analysis, the constant region primer was end-labeled with 33P. Aliquots (10 µl) of PCR products were electrophoresed in a 2% NuSieve 3:1 agarose gel (FMC BioProducts, Rockland, ME) that was subsequently dried under vacuum and exposed to a Storage Phosphor Screen (Molecular Dynamics, Sunnyvale, CA). Quantitation was accomplished using ImageQuant software (6) and expressed as a mean percentage (±SD) of the sum of the total TCRBV signal detected.
For DGGE analysis,10-µl aliquots were loaded onto a denaturing gradient gel containing 6% polyacrylamide and a gradient of urea and formamide from 20% to 80%. Gels were run at 160 V for 4.5 h in 1x TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA) kept at a constant temperature of 58°C. After electrophoresis, the gel was stained with ethidium bromide and photographed under UV transillumination.
To analyze for the expression of tyrosinase, MART-1/Melan-A and glycoprotein 100 (gp100), RT-PCR was conducted with primers specific for these transcripts as described previously (7).
Sequencing
DNA bands that resolved at identical positions in the denaturing gradient gel were subjected to sequence analysis using the Thermo Sequenase cycle sequencing kit (Amersham Life Science, Cleveland, OH). In brief, bands were excised from the denaturing gradient gel, and DNA was eluted in H2O and reamplified. An aliquot (0.2 µl) of the PCR product was used as template in a 40-cycle sequencing reaction with 33P end-labeled ß-constant region sequencing primer. Gels were dried under vacuum and exposed to a Storage Phosphor Screen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBLs and three s.c. melanoma lesions from each of the two
patients were analyzed for the expression of TCRBV regions 1–24 by
semiquantitative RT-PCR. In each tumor sample, at least two BV regions
were expressed at significantly higher levels than in the peripheral
blood (Table I
). In patient 1, this
applied to BV2, BV4, BV8, BV14, and BV23; in patient 2, this applied to
BV3, BV5, BV8, BV19, BV20, and BV24. In patient 2, the BV regions BV3,
BV20, and BV24 were highly expressed in two lesions, and BV8 was highly
expressed in all three lesions. Major differences were detected at the
level of BV region expression between the different lesions from each
patient (e.g., patient 1, BV6, 2.8% in lesion 1 vs 10.1% in lesion 2;
patient 2, BV3, 10.6% in lesion 2 vs 3.4% in lesion 3). Thus, there
were not only differences in the relative expression of TCRBV regions
in TILs and PBLs, but also between the different melanoma lesions.
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To exclude the possibility that these variations of the TCRBV repertoire were a reflection of differences in the expression of MAA in the analyzed biopsies, RT-PCR was used to analyze for the expression of tyrosinase, MART-1, and gp100 mRNAs. This analysis revealed that all lesions were positive for expression of these transcripts (data not shown). For each of the three lesions, the presence of MART-1 and gp100 proteins was verified by immunohistochemical staining with mAb against these proteins (data not shown).
TCR clonotype mapping
PBLs and tumor lesions were analyzed for the presence of T
cell clonotypes by RT-PCR/DGGE-based TCR clonotype mapping. The
detection of clonally expanded T cells via this approach relies on the
fact that clonotypic transcripts have no junctional diversity and
therefore resolve at a fixed position in the denaturing gradient gel
(5). Analysis of the PBLs from both patients did not reveal any
distinct bands in the denaturing gradient gel (data not shown; also see
Fig. 2). Analysis of the tumor lesions, however, showed the presence of
multiple clonotypic TCR transcripts (with numbers ranging from 40 to
>60) covering the majority of the BV families 1–24 (Fig. 1 and Table I
). Notably, clonotypic
transcripts were detected in most BV regions, irrespective of the level
of expression. Even regions expressed at a level equaling <1%
contained clonotypic TCR transcripts (Table I). Conversely, clonotypic
TCR transcripts were absent in some BV regions expressed at high
levels, demonstrating that a high level of expression does not
necessarily imply the presence of T cell clonotypes (Table I).
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, most clonotypes were exclusively
present in a single lesion. More than fifty clonal TCR transcripts were
compared by DGGE; only six transcripts appeared to be accumulated in
two different lesions, and none of the clonotypes were present in all
three lesions from the same patient. Sequencing
A number of transcripts resolved at similar positions in
the gel, suggesting sequence identity (Fig. 2). These DNA bands were
subjected to sequence analysis. Of the six pairs of sequences with
identical migration distance, only four were indeed identical. It
should be noted that the two different BV4 sequences derived from
lesions 1 and 3 of patient 1 shared the same joining sequence and only
differed in the junctional region between the V and the J regions.
Immunohistochemical staining with mAbs specific for TCRBV regions
Staining of the metastatic lesions with mAbs specific for a number
of different TCRBV regions was performed to examine the distribution
pattern of T cells expressing these individual regions. Representative
results are shown in Fig. 3, where lesion
3 of patient 2 was stained with an Ab to either BV13 or BV14. These
regions were expressed at 5.1% and 3.6%, respectively, of the total
TCR expression, and both contained clonotypic transcripts (Table I). It
appears that positive cells were present as small clusters, which is in
agreement with the hypothesis that clonotypic expansion occurs in situ
(Fig. 3). In contrast, CD3-expressing cells were present throughout the
tumor (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have attempted to address some of these
questions by using the TCR clonotype mapping methodology. Analysis of
six melanoma lesions from two patients revealed the presence of
multiple clonotypic TCR transcripts in all cases, with numbers of
clonotypes ranging from 
40 forty to >60; furthermore, the majority
of the T cell clonotypes were limited to a single lesion only. Thus,
the T cell response against malignant melanoma seems to be very complex
and to involve a high number of localized T cell clones.
Initial studies have demonstrated the presence of a finite number of T cell clonotypes in lymphocytes infiltrating melanoma (12, 13, 14). These analyses have been conducted using quantitative RT-PCR of TCR V regions, and the subsequent analysis for clonality was limited to overexpressed regions. Recently, methods with considerably higher resolution (e.g., CDR3 size spectra analysis and single-strand conformational polymorphism) have been used to analyze T cell infiltrates in melanoma (15, 16). However, because these methods are rather labor-intensive when used at their full potential, they have often been used to analyze selected TCRBV regions only. The most intensive analysis to date estimated the number of T cell clonotypes in melanoma lesions to be in the range of 2–15 (11). The data presented here suggest that the complexity of the T cell response has been largely underestimated. However, the presence of clonal T cells in most BV regions irrespective of the level of expression suggests that a substantial fraction of in situ T cell clonotypes only proceed through a limited number of cell divisions. A possible explanation could be that specific T cells in situ do not reach the threshold level for full activation due to a low affinity of the TCR (17) or the absence of costimulatory signals. Alternatively, T cell clonotypes detected in regions expressed at low levels could be in the early phase of expansion.
In analogy with our findings, it was recently demonstrated that the numbers of virus-specific T cells during systemic infection were considerably higher than previously anticipated (18, 19, 20). Large clonal expansions of CD8 T cells have been detected in the peripheral blood during acute infection, with viral-specific T cells constituting as much as 40% of the T cells (18, 19, 21). Although a surprisingly high number of T cell clonotypes is present within the lymphocytes infiltrating melanoma, no clonotypes were detected in the blood of the patients, arguing against a major circulation of these clonotypes. This observation may reflect the difference between a localized incident (as in the case of a solid tumor) and a generalized incident (as in the case of a systemic viral infection). Indeed, during a localized viral infection, the specific T cell response is not of similar magnitude as during systemic infections, which is possibly due to the limited Ag load in the blood and lymph nodes (22). The notion of a localized T cell response against melanoma was recently corroborated by the demonstration of a functional dissociation between local and systemic T cell responses during peptide vaccination (23).
The thought that the T cell response against melanoma is localized was further substantiated by immunohistological studies. The melanoma lesions were infiltrated by numerous T cells throughout the tumor, whereas staining with Abs against specific TCRBV regions clearly demonstrated that the T cell clones expressing these regions were located in clusters of cells, suggesting a local clonal expansion. To further investigate whether clonally expanded T cells in situ were local by nature, we compared the clonotypes detected in the different lesions from each patient, with the aim of resolving whether specific T cell clones were identical (i.e., originated from the same clonal expansion). The results demonstrated that the preponderance of the clonotypes were unique (i.e., were present in a single lesion only). The in vivo accumulation of identical T cell clones in different metastases has been reported (11, 16, 24). The present study corroborates these findings, as a minority of the identified T cell clonotypes was present in two different lesions. Whether the capacity of these T cells to migrate to distant metastases is correlated to specialized functions (e.g., regulatory suppressive functions) is not known at the present stage. Furthermore, it is possible that the incidence of detecting identical clonotypes in different lesions would increase if it was possible to follow the infiltrate over time.
Our data indicate that locally expanded T cells execute the T cell response against melanoma, and that the majority of these T cell clonotypes do not recirculate to other metastatic lesions. It is possible that the majority of the clonotypic T cells involved in a cellular response are destined to die by apoptosis, whereas a minority possesses the capability to persist as long-term memory cells. The factors that influence the generation of memory T cells are poorly understood. Nevertheless, an important difference between effector T cells and memory T cells is related to their different growth capacities. The in situ T cell response against melanoma may not support the generation of memory T cells. Thus, the majority of the clonotypic T cells in TILs are effector T cells with a limited potential to proliferate, which implies that the T cells that leave the local environment are cells that have a limited growth capacity. Accordingly, these cells will not expand to a detectable level upon recognition of Ag in distant metastases. This would also explain the repeatedly observed inability to grow in vivo-expanded T cell clonotypes in vitro (25, 26).
The general conclusions from the present study are that the T cell infiltrate of malignant melanoma is exceedingly heterogeneous and composed of a much higher number of different T cell clonotypes than previously appreciated. Furthermore, our data indicate that the vast majority of clonotypic T cells in melanoma TILs, at least in the case of s.c. metastases, may be considered as being strictly localized.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Per thor Straten, Department of Tumor Cell Biology, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. E-mail address:
3 Abbreviations used in this paper: MAA, melanoma-associated Ags; TIL, tumor-infiltrating lymphocyte; DGGE, denaturing gradient gel electrophoresis; BV, ß-variable; gp, glycoprotein.
Received for publication January 7, 1999. Accepted for publication April 20, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß clonality by denaturing gradient gel electrophoresis (DGGE). Biotechniques 25:244.[Medline]
ß-variable regions among tumor-infiltrating lymphocytes in primary human malignant melanomas. Int. J. Cancer 56:78.[Medline]
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