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Sections of
* Oncology,
Surgery, and
Pathology, Departments of Oncology and Surgical Sciences and
Pediatrics, University of Padova, Padova, Italy; and
¶ Division of Medical Oncology, University Hospital, Padova, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain V
segment and the complementarity-determining region 3 (CDR3) length and
sequence revealed a large diversity in the TCR repertoire, with only
some of the clones showing a partial conservation in the CDR3. A
similar degree of diversity was found by analyzing a number of T cell
clones obtained after sorting a Melan-A-specific population derived
from PBLs of the same patient after in vitro culture with the
immunodominant epitope. Moreover, clonotypes found at one site were not
present in another, suggesting the lack of expansion and circulation of
one or more clonotypes. Taken together, these results buttress the
notion that the CTLs recognizing the immunodominant Ag of Melan-A
comprise a high number of different clonotypic TCR, of which only some
exhibit common features in the CDR3. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of studies addressing the complexity of TCR usage in T cell
populations reactive against Melan-A were hampered by the difficulty in
targeting the analysis to CTL directed against a single epitope. The
advent of tetramer technology combined with cell sorting opened new
perspectives, because Ag-specific T cells could be isolated and studied
at a clonal level. By means of this technique, TCR -chain V gene
segment (TCRBV) usage in purified CD8+ T cells
directed against the immunodominant peptide of Melan-A was analyzed in
bulk cultures of four patients (6); using a panel of mAbs,
the presence of a large repertoire of specific T cells was evidenced.
To take a closer look at the repertoire of T cells directed against
Melan-A, another study analyzed sorted cells from two melanoma patients
for their TCRBV chain and complementarity-determining region 3 (CDR3)
length by spectratyping and confirmed the extension of TCR repertoire
(7). This same group recently studied a single patient
vaccinated with the Melan-A26–35 peptide and
observed increased functional avidity and tumor reactivity in the
postimmune Melan-A-specific populations, as compared with the preimmune
blood sample (8).
Although the above studies clearly documented the high diversity of the Melan-A-specific T cells, none looked at TCR usage in several lesions and the blood from the same patient during disease progression. Only one case was reported in which the same antimelanoma T cell clone was found in two different metastatic sites (9); however, in this case the Ag was not identified.
We used HLA-A*0201/peptide tetramers to isolate Melan-A-specific T cells from TILs present in four metastases resected from a single melanoma patient, and compared them with those isolated from the peripheral blood of the same patient. The TCR repertoire of Melan-A/A2-specific CD8+ cells was analyzed to determine the degree of heterogeneity displayed by this T cell pool. In addition, CD8+/tetramer+ cells were cloned, each clone was analyzed functionally, their TCRBV segment examined, and the CDR3 sequenced.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was approved by the local Human Ethics Committee and performed following the patient’s informed consent. Fresh melanoma lesions were received as biopsy material immediately after surgery. In a follow-up period of 4 mo, four metastatic melanoma lesions were excised from a single HLA-A2+ patient at the following anatomical sites: nape (lesion A), arm muscle (lesion B), left arm (lesion C), and right arm (lesion D). Each lesion was divided into three parts: one was fixed in formaldehyde and processed for routine histopathology, one was snap frozen in liquid nitrogen and stored at -80°C until use for RNA extraction to ascertain Melan-A expression by RT-PCR, and the last was used for in vitro culture. To obtain TIL, biopsy material was finely minced with scalpels and then dissociated into a single cell suspension by incubation for 2 h at 37°C in sterile IMDM (Life Technologies, Paisley, Scotland) supplemented with DNase (30 U/ml), collagenase (1 mg/ml), and hyaluronidase (0.1 mg/ml). Cell suspensions were placed in tissue culture flasks (Falcon BD Labware, Franklin Lakes, NJ) containing IMDM supplemented with 0.24 mM Asn, 0.55 mM Arg, 1.5 mM Gln, 10% pooled human serum AB (PAA Laboratories Linz, Austria) (referred to as culture medium), and 200 U/ml human rIL-2 (rhIL-2), and cultured for 2–3 wk.
Melan-A-specific CTL clones were derived from TILs after cell sorting.
Cells stained by Melan-A/A2 tetramer, CD3, and CD8 mAbs were seeded at
1 cell/well in round-bottom 96-well microplates. They were then
stimulated by adding irradiated (50 Gy) allogeneic PBMC (8 x
104/well) as feeder cells as well as irradiated
(100 Gy) allogeneic HLA-A2 EBV-transformed B cells (2 x
104/well) incubated with the
Melan-A/A226–35 analog peptide ELAGIGILTV (10
µM) and washed. Culture medium contained rhIL-2 (100 U/ml), rhIL-4
(PeproTech EC, London, U.K.) (10 ng/ml), and rhIL-7 (PeproTech EC) (10
ng/ml). The CTL clones were restimulated each week by the addition of
feeder cells and peptide-pulsed HLA-A2 EBV-B cells, in medium with
rhIL-2 (50 U/ml); after 
3 wk, they were transferred into 2-ml wells
and maintained with weekly restimulations, alternating allogeneic
HLA-A2 PBMC and HLA-A2 EBV-B cells as peptide-presenting cells.
In vitro stimulation of PBMC
Two weeks after the last metastasis resection, PBMC were separated from heparinized blood, diluted 1/2 with RPMI 1640 medium, by centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech AB, Uppsala, Sweden), washed three times, and cryopreserved in IMDM supplemented with 10% human serum and 10% DMSO in liquid nitrogen. To use, PBMC were thawed and 5 x 106/ml were incubated for 1 h at room temperature with 1% human serum and 5 µM Melan-A/A226–35 analog peptide. Cells were then washed and plated at 4 x 106/2 ml in culture medium with rhIL-2 (20 U/ml), rhIL-4 (10 ng/ml), and rhIL-7 (10 ng/ml) in 24-well tissue culture plates. On day 7, the lymphocytes were incubated with the peptide as on day 0, washed, and resuspended in fresh culture medium. After 14 days of culture, lymphocytes were used for cytometric analysis and cell sorting. Melan-A/A2-specific CTLs were derived as described above.
Immunohistochemical analysis of Ag expression in tumor biopsies
Staining for Melan-A (Novocastra Laboratories, Newcastle upon Tyne, U.K.), CD20 (clone L26; DAKO, Glostrup, Denmark), CD3 (clone PS1; Novocastra Laboratories), CD4 (clone 1F6; Novocastra Laboratories), and CD8 (clone C8/144B; DAKO) was performed by a peroxidase-labeled avidin-biotin method on an automated stainer (DAKO autostainer). Paraffin tissue sections were mounted on amino-alkylsilane-treated glass slides and heated to optimize Ag retrieval. Endogenous peroxidase activity was blocked by incubation in hydrogen peroxide/methanol. Immunohistochemical staining involved the sequential application of diluted primary Ab, biotinylated goat secondary Ab, and then peroxidase-labeled avidin. The Ags were visualized by incubation with aminoethylcarbazole substrate in the presence of hydrogen peroxide. Nonimmune mouse IgG in place of specific Ab was used for negative control sections.
Tetramers, flow cytometry immunofluorescence analysis, and sorting
Soluble HLA-peptide tetramers were produced using a method
similar to that described previously (10). Briefly,
rHLA-A*0201 H chain, with a 15-aa substrate peptide for BirA-dependent
biotinylation at its C terminus, and human
2-microglobulin were produced as inclusion
bodies in Escherichia coli cells transformed with the
expression plasmid pET 23a+-HLA-A*0201 and pHN1 +
2-microglobulin (kindly provided by F.
Marincola, Clinical Center, National Institutes of Health, Bethesda,
MD) and peptide Melan-A26–35 A27L analog
(ELAGIGILTV). The inclusion bodies were purified and dissolved in
urea-denaturing buffer, as described (11). Monomeric
MHC-peptide complexes were formed by combining the HLA-A*0201,
2-microglobulin, and the peptide in an
arginine-folding buffer. The refolding reaction was dialyzed, and
concentrated on a Superdex 75 gel filtration column to purify the
folded protein (Amersham Pharmacia Biotech). Soluble purified complexes
were biotinylated using BirA enzyme (Avidity, Denver, CO). PE-labeled
tetramers were produced by mixing the biotinylated complexes with
Extravidin-PE (Sigma-Aldrich, St. Louis, MO), and were validated by
staining CTL clones with the appropriate specificity. Each tetramer
batch was titrated and used at the optimum concentration, ranging from
20 to 40 nM HLA-A2 H chain.
For surface Ag staining, lymphocytes were washed, resuspended in PBS with 1% human serum, and incubated for 15 min at room temperature with the HLA-A*0201 tetramer. Anti-CD3 Abs coupled to FITC (SK7; BD Biosciences, San Jose, CA) and anti-CD8 Abs coupled to PerCP (SK1; BD Biosciences) were then added, and after a further 15-min incubation at 4°C, the cells were washed and analyzed on an EPICS XL flow cytometer (Coulter Electronics, Hialeah, FL). Cell sorting was performed with a FACSVantage SE (BD Biosciences).
Cytotoxicity assay
Cytotoxic activity was measured using a chromium-release assay (12). Briefly, T2 cells (HLA-A*0201) were labeled with Na251CrO4 for 1 h at 37°C, washed, then pulsed with synthetic peptide ELAGIGILTV (2 µM) for 30 min at 37°C. After washing, the cells were added to varying numbers of effector cells in V-bottom microwells. Chromium release was measured in the supernatant harvested after 4 h of incubation at 37°C.
Analysis of TCRBV transcripts and sequencing of PCR products
Total RNA from CTL clones or from sorted populations was extracted using the Rneasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed at 42°C for 1 h with 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies) mixed with 4 µl 5x first strand buffer (Life Technologies), 2 µl 20 µM oligo(dT)15 (Promega, Madison, WI), 20 U RNasin (Promega), 2 µl 100 mM DTT (Life Technologies), 1 µl each dNTP at 25 mM each (Amersham Pharmacia Biotech), and diethyl pyrocarbonate-treated water, in a total volume of 20 µl. TCRBV usage was assessed by RT-PCR and sequencing. cDNA served as template for PCR amplification using panels of BV-specific upstream primers and one downstream BC primer, chosen on the basis of described panels of TCRBV region oligonucleotides (13) and alignments of TCR sequences available at the International Immunogenetics Database of M. P. Lefranc (Montpellier, France) (http://imgt.cines.fr). TCR BV-BC PCR products derived from clones or bulk cultures of sorted populations were purified with the Qiaquick PCR purification kit (Qiagen), and sequenced using the dye terminator cycle sequencing kit (ABI-PRISM; PerkinElmer, Foster City, CA), according to the manufacturer’s instructions, to obtain a complete identification of the CDR3.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a follow-up period of 4 mo, four metastatic lesions were
resected from a single HLA-A2-positive patient. The first two lesions
were resected from the nape of the neck and an arm muscle on the same
day, and the other two from the right and the left arms 4 mo later
(Table I
).
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, A and B).
Analysis of the lymphocytic infiltration revealed the predominance of
CD3+ cells (data not shown). T cell
immunophenotyping showed that CD4+ lymphocytes
were localized predominantly at the periphery of tumor nodules, while
the CD8+ lymphocytes were closely intermixed with
the tumor cells (Fig. 1, C and D).
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, all TIL populations contained
CD3+/CD8+/tetramer+
cells ranging from 2 to 11% in the four samples.
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To confirm the specificity of tetramer-positive cells,
CD3+/CD8+/tetramer+
cells were isolated by flow cytometry sorting. Cells gated in Fig. 2
were sorted according to tetramer staining and immediately cloned; they
were stimulated with irradiated HLA-A2+
EBV-transformed B cells that had been incubated with the Melan-A/A2
peptide, in the presence of irradiated allogeneic PBMC as feeder cells,
rhIL-2, and rhIL-4. A total of 56 T cell clones were derived that
showed growth in long-term cultures, and were stained by the Melan-A/A2
tetramer (Table I). These clones were also tested for their ability to
lyse specific target cells pulsed with the relevant peptide, as well as
melanoma cell lines expressing the Melan-A/A2 Ag. The results obtained
with 10 representative clones sorted from the TILs derived from the
nape of the neck (A) and the arm muscle (B) are
shown in Fig. 3. All the clones
efficiently lysed T2 cells pulsed with the Melan-A/A2 peptide, but did
not recognize T2 cells alone; they also recognized the melanoma cell
line PDO-35-MEL HLA-A2+ expressing the Melan-A
gene, albeit less efficiently than the peptide-pulsed T2 cells and with
a certain clone-to-clone variability. Similar results were obtained
with the other CD3+, CD8+,
and tetramer+ clones thus derived by cell sorting
from lesions C and D (data not shown). Taken together, these findings
provide functional evidence that the CTL clones derived from the TILs
are Melan-A/A2-specific cells endowed with cytotoxic activity against
tumor cell lines.
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As a first step to determine the repertoire of Melan-A/A2-specific
CD8+ cells present in the TIL populations after
in vitro expansion, we extracted RNA from the sorted Melan-A/A2
tetramer+ T cell populations derived from lesions
A and B and performed RT-PCR amplification using a panel of
subfamily-specific oligonucleotide primers complementary to the V and C
regions of the -chain of the TCR. This analysis showed the presence
of multiple clonotypic transcripts covering the majority of the BV
families (data not shown). To examine the TCRBV repertoire more
closely, we next analyzed the CDR3 at the clonal level using the RNA
obtained from CTL clones derived from TILs after sorting the
tetramer+ populations. Only 13 of the 56 clones
analyzed showed long-term expansion sufficient to obtain enough RNA for
the genetic analysis. RT-PCR was followed by sequence analysis of the
PCR product to determine the sequence of the CDR3 of the -chain of
the TCR. Among the sequenced clones, three came from lesion A, six from
lesion B, and four from lesions C and D (Table I). This analysis
revealed a wide diversity of BV usage by Melan-A/A2-specific
CD8+ cells not only among the four different
lesions, but also at the level of each single lesion. This diversity
was further confirmed when the CDR3 was analyzed: all the clones
differed from one CTL to another in terms of their amino acid
composition. We also observed that 8 of the 12 sequences analyzed were
recombined with the J1-5*01 segment, and that the sequences sharing
this J segment showed an overall CDR3 similarity, despite the usage of
different BV regions (Fig. 4A). In fact, besides the four
identical amino acids derived from the J segment and contributing to
the CDR3, these clonotypes shared from one to four other identical
amino acid residues, and six of these eight sequences had the same CDR3
length. Moreover, the clonotypes sharing the J1-5*01 segment came from
three different lesions. Concerning the clonotypes recombined with a
different J segment, the similarities in terms of amino acid
composition were more limited.
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TCRBV repertoire of Melan-A/A2-specific CTL clones derived from blood
To compare the repertoire of CTL directed against the Melan-A/A2
Ag present at the tumor site with that circulating in the peripheral
blood, we stimulated 4 x 106 PBMC from the
same melanoma patient with the Melan-A/A2 peptide in the presence of
rhIL-2, rhIL-4, and rhIL-7. PBMC were collected 2 wk after the last
metastasis excision. On day 15, after a second stimulation with the Ag
under the same conditions, we labeled the cells with the Melan-A/A2
tetramer and identified 16% tetramer+ cells in
the CD3+ CD8+ fraction
(data not shown). Triple-labeled cells were sorted, seeded at one
cell/well, and restimulated as above. We obtained 53 clones that were
stained by the Melan-A/A2 tetramer; the clones proved to be Ag specific
as they lysed T2 target cells pulsed with the Melan-A/A2 peptide as
well as a melanoma cell line expressing the Melan-A/A2 Ag, but they did
not recognize T2 cells alone (data not shown). We next extracted RNA
from 37 clones that showed long-term expansion for which we had enough
RNA for genetic analysis, and determined the CDR3 of the TCR -chain,
as described above. The nucleotide and predicted protein sequences
spanning the VDJ regions of these clones are shown in Fig. 4B. Among the 37 clones, we found 14 different clonotypes;
some were found in different copy number indicating that some clones
expanded in vitro. The 14 clonotypes showed an extensive usage of the
TCRBV region as well as recombination with several J regions; moreover,
they differed in CDR3 length, which ranged from 9 to 13 aa.
Interestingly, usage of BV and BJ segments was rather different
compared with that observed in the clones derived from the metastatic
lesions (Fig. 4A); moreover, none of the clonotypes matched
those derived from the TILs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50% of healthy donors (20), as it
comprises 10-3 of circulating phenotypically
naive CD8+ T cells (20, 21). To
date, this is the only known tumor Ag that can be detected ex vivo by
tetramers, without a round of in vitro stimulation (22).
These cells present a naive phenotype in healthy individuals, but may
develop an Ag-experienced one in some melanoma patients (15, 20). Staining with tetramer revealed that the repertoire against
this Ag not only has a higher frequency compared with other known tumor
Ags, but it is also highly diversified, as
tetramer+ cells are also stained by most of the
Abs directed against the different V-chain families
(6).
Our findings indicate a further level of complexity, because a high
degree of TCR diversity emerged from an extensive analysis of the
clonotypes recognizing this tumor Ag in different metastatic lesions
and the blood of our patient. Moreover, among the different clonotypes,
none was present in any other lesion or in the blood. A broadening of
the TCR repertoire was also demonstrated for a different tumor Ag,
gp100, in melanoma patients; in this case, repeated exposure to the
modified gp100:209–217 epitope enhanced immune competence by
increasing the frequency of immunogen-specific precursor T cells
through selection of higher affinity CTL (23). It is quite
striking to observe that the repertoire directed against a single
epitope includes such a high number of different TCRs. However, a
closer look at the CDR3s showed an overall similarity among most of the
clones derived from the TILs, while a more heterogeneous situation was
observed among the Melan-A/A2-specific clones derived from blood. If we
analyze the CDR3s in terms of hydrophobicity, it appears that the
majority of these sequences, from both blood and metastases, include a
central portion of variable length containing a 3-aa motif based on a
G-L-G sequence or amino acid substitutions with the same hydrophobic
characteristics, surrounded by two hydrophilic regions (Fig. 4, boxed residues). This central pattern might be important for the
recognition of the antigenic peptide, which, being hydrophobic, will
probably contact amino acids by hydrophobic interactions. Another
important amino acid in the CDR3 is a glutamic acid (Q), which was
present in all but one of the clones derived from TILs, and in 9 of 14
clones obtained from blood (Fig. 4, boxed residues). However, an
identification pattern of the Ag cannot easily be defined. Indeed,
preliminary findings obtained with a few of these clones in the CDR3 of
the 
-chain showed an elevated heterogeneity. Moreover, some of the
clones possessed two productively rearranged -chains composed of
different TCR AV and AJ segments (data not shown) that would have
increased the level of complexity of the clones recognizing a single
Melan-A/A2 epitope if they are, in fact, both expressed on the cell
surface.
The fact that clonotypes found at one site were not present at another argues against the possibility that Melan-A/A2-specific CTL might be expanded in vivo as a consequence of tumor-induced activation. The high frequency of precursors generally observed in blood might determine the presence of numerous different CTL against the immunodominant epitope of Melan-A in the metastatic lesions. To explain the high precursor frequency in blood, Loftus, Rivoltini and colleagues (24) advanced that the peptide sequence recognized by the CTL might resemble sequences occurring frequently in proteins of different origin. It was demonstrated that peptide sequences derived from viral, bacterial, and human proteins that conform to the HLA-A2-binding motif and possess features essential to recognition by anti-Melan-A CTL do indeed exist, therefore implying that epitope mimicry might play a role in modulating the CTL response against this Ag. A recent study traced the origin of these cells back to thymic selection and demonstrated that they are mostly generated by thymic output of a high number of precursors that undergo limited expansion in the periphery and remain functionally naive T cells in normal donors (25). In contrast to healthy individuals, the vast majority of melanoma patients has detectable accumulations of memory/effector Melan-A-specific T cells in metastatic tumor lesions (6, 15, 25), but only after vaccination with the Melan-A peptide it is possible to observe the expansion of some dominant clonotypes and the maturation of their functional avidity (8), thus suggesting that Melan-A-specific CTL can undergo clonal expansion upon appropriate stimulation.
Although our results are based only on the analysis of one patient, they provide direct evidence that a large Melan-A-specific T cell pool may also be maintained in the presence of high Ag load associated with tumor progression. The anti-Melan-A/A2 CTLs derived from our patient’s blood and metastatic lesions specifically recognized the exogenously supplied modified peptide as well as the endogenously processed natural peptide, thus excluding that tetramer-guided isolation of T cells might modify the natural T cell repertoire. However, as we did not determine the affinity of these CTL clones, we cannot rule out the possibility that those present within the metastatic lesions have a higher affinity. Moreover, the finding of tumor-specific CTLs at the tumor site does not reflect an ongoing cytotoxic response, irrespective of CTL affinity. In this regard, it would have been important to analyze the lymphocytes for surface expression of naive, effector, and memory-associated markers; however, the number of cells in the metastatic lesions was too small to be properly analyzed, and therefore culture in vitro was a mandatory step. To understand the meaning of tumor-specific CTLs at the tumor site, we would have to find one or more identical clonotypes in different sites in a single patient, thus demonstrating activation, proliferation, and circulation in the body. Although we cannot rule out the possibility that such CTL clones were present in our patient and that we lost them in culture as a result of an in vitro selection, it is clear that if such clones existed, they were not present in high copy number.
In conclusion, the original finding in this study is that a large pool of Melan-A-specific T cells, with a diverse TCRBV usage and a few common features in the CDR3, was present in different peripheral compartments of a single melanoma patient without evidence of Ag-driven selection or preferential homing at the metastatic tumor site; the relevance of this diverse T repertoire to the antitumor response remains to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Susanna Mandruzzato, Department of Oncology and Surgical Sciences, University of Padova, Via Gattamelata, 64, 35128 Padova, Italy. E-mail address: susanna.mandruzzato{at}unipd.it
3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; CDR3, complementarity-determining region 3; rhIL, human rIL; TCRBV, TCR -chain V gene segment.
Received for publication May 10, 2002. Accepted for publication July 23, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients. J. Immunol. 165:533.1-w29/V1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur. J. Immunol. 22:1261.[Medline]
secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases. J. Immunol. 166:7634. receptor. EMBO J. 7:3745.[Medline]
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), T2 cells pulsed with peptide
ELAGIGILTV (
), or melanoma cell line PDO-35-MEL (
) at the
indicated lymphocyte to target cell ratios. CTL clones were stained
with soluble Melan-A/A2 tetramer coupled to PE.