HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Michalek, J. Articles by Hajek, R. Search for Related Content PubMed PubMed Citation Articles by Michalek, J. Articles by Hajek, R.

Detection and Long-Term In Vivo Monitoring of Individual Tumor-Specific T Cell Clones in Patients with Metastatic Melanoma¹

Jaroslav Michalek^2,*,, Ivo Kocak, Vuk Fait, Jan Zaloudik and Roman Hajek^*,

^* Cell Immunotherapy Center, Masaryk University, Brno, Czech Republic; Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas, TX 75390; Masaryk Memorial Oncology Institute, Brno, Czech Republic; and Department of Hematooncology, University Hospital, Brno, Czech Republic

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We investigated the presence of individual melanoma-specific T cell clones in patients with metastatic melanoma. Ten patients were examined for the presence of melanoma-reactive T cells using dendritic cells loaded with autologous tumor cells. Their specificity was tested using nonradioactive cytotoxicity test. Individual immunodominant T cell clones were identified by the clonotypic assay that combines in vitro cell culture, immunomagnetic sorting of activated IFN-⁺ T cells, TCR locus-anchored RT-PCR, and clonotypic quantitative PCR. All patients had detectable melanoma-reactive T cells in vitro. Expanded melanoma-reactive T cells demonstrated specific cytotoxic effect against autologous tumor cells in vitro. Three patients experienced objective responses, and their clinical responses were closely associated with the in vivo expansion and long-term persistence of individual CD8⁺ T cell clones with frequencies of 10^–6 to 10^–3 of all circulating CD8⁺ T cells. Five patients with progressive disease experienced no or temporary presence of circulating melanoma-reactive T cell clones. Thus, circulating immunodominant CD8⁺ T cell clones closely correlate with clinical outcome in patients with metastatic melanoma.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Adoptive immunotherapy using tumor-infiltrating lymphocytes, tumor-specific T cells, or tumor-specific vaccination strategies using dendritic cells (DC)³ loaded with tumor Ags demonstrated objective partial response in 30–50% of patients with metastatic melanoma (MM) and other solid tumors in the absence of serious vaccine toxicity (1, 2, 3, 4, 5, 6, 7, 8). Such tumor regression largely exceeds the rate of spontaneous regression of melanoma metastases and it is very likely due to the effect of adoptive immunotherapy. Because most of the patients demonstrate only a short-term response in various adoptive immunotherapy trials, the optimal number of tumor-reactive T cells that are believed to be central for the anticancer effect in vivo has not been well defined yet. This may be caused in part by our limited methodological approaches to monitor Ag-specific T cells in vivo and in part by changing the repertoire of tumor-specific T cell clones that occur in vivo at various stages of cancer treatment. Relatively limited information exists about the frequency of naive T cell precursors that are able to recognize a given Ag in association with MHC class I or MHC class II. In humans, for example, the relative frequency of CTL precursors that recognized a MAGE-3 peptide presented by HLA-A1 was estimated to be 3 x 10^–7 of the blood CD8⁺ cells (9). Assuming a mean CTL precursor frequency of 10^–6 of tumor Ag-specific CD8⁺ cells, a significant increase of this frequency, for example a 40-fold increase, translates into 0.00004 of the CD8⁺ cells following vaccination. This result is beyond the limit of detection of all immunological methods, including tetramer, unless the frequency of Ag-specific cells is increased through an in vitro restimulation assay (10). Direct methods can only detect substantially larger T cell populations with frequencies of >0.001 of Ag-specific T cells. Such responses are observed against viral peptides and quite frequently exceed even 0.01 of Ag-specific T cells in case of CMV or HIV peptides (11, 12). In cancer immunotherapy, there is no proof that a cancer vaccine has to stimulate a large number of T cells to initiate tumor rejection. Thus, we should take into account that T cell responses to tumor Ags may be low level and that negative results obtained with most ex vivo assays may not exclude the beneficial effect of tumor-specific T cells in vivo.

Previously, we were able to identify and quantify in vivo individual alloreactive and leukemia-reactive donor T cell clones in patients undergoing allogeneic HLA-matched hemopoietic stem cell transplantation who developed acute graft-vs-host disease (GVHD) and demonstrated their GVHD- and graft-vs-leukemia (GVL) specificity without prior knowledge of GVHD- and GVL-specific Ags (13, 14, 15). Using the clonotypic assay based on selection of Ag-reactive T cells and further molecular analysis of their TCR repertoire (14, 15), we were able to identify the most immunodominant Ag-specific T cells and detect them by clone-specific primers and probes quantitatively in vivo with a threshold frequency of 0.00001 T cells. Such a novel and sensitive approach to the detection of Ag-specific T cells does not require the prior knowledge of the particular Ag and can be used for long-term monitoring of individual tumor-specific T cells in cancer patients. In this study, we used the clonotypic assay in patients treated for MM and correlated their clinical outcome with individual tumor-specific T cell clone monitoring.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Patient characteristics and cell collection

Ten consecutive patients with MM (American Joint Committee on Cancer stage IV) were enrolled in the study and treated according to the Czech Oncology Society guidelines with dacarbazine or temozolomide, fotemustin, cisplatin, vinblastine, IFN-, and IL-2 at the Masaryk Memorial Oncology Institute (Brno, Czech Republic) from March 2001 to September 2005. Fresh PBMC as well as tumor cells were obtained as a part of routine diagnostic procedures at least 3 wk after the chemotherapeutic regimen was given. Tumor cells were obtained from lymph node metastases at the time of lymph node biopsy. Basic patient characteristics are described in Table I. All patients provided signed informed consent forms prepared in agreement with the Declaration of Helsinki and approved by the local Ethical Committee. As controls, PBMC from healthy individuals were used. Tumor cells were obtained from histopathologically verified tumor tissue by cutting into small peaces (<2 mm), cultivated shortly before harvest, and frozen at –80°C. PBMC were isolated using gradient centrifugation on Histopaque 1077 (Sigma-Aldrich) and either used immediately or stored deeply frozen at –80°C.

PBMC were incubated in 6-well culture plates in medium consisting of X-VIVO 10 (BioWhittaker) with 10% heat-inactivated human AB serum (Sigma-Aldrich), 80 U/ml DNase (Boehringer Mannheim), and 2 mM L-glutamine (Sigma-Aldrich) in an atmosphere of 5% CO₂ for 2 h. The nonadherent fraction was used for the generation of CTLs. After removing the nonadherent fraction, adherent cells were then cultured in the presence of 800 U/ml GM-CSF (Schering-Plough) and 100 ng/ml IL-4 (Sigma-Aldrich). On days 3 and 6, half of the culture was replaced with fresh complete medium and cytokines were added. Immature DC were obtained with typical morphological and phenotypic characteristics (CD80⁺, CD86⁺, CD83^low; data not shown). Melanoma cells were irradiated (60 Gy) and used as Ag pulsed to immature DC at a ratio of 1:1 on day 7. DC maturation was induced on day 8 using 1,000 U/ml TNF- (Bender MedSystems). CTLs were elicited from autologous PBMC using stimulation by Ag-loaded DC. Washed nonadherent lymphocyte-rich PBMC fraction was resuspended in medium consisting of X-VIVO 15, 50 mg/l gentamicin, 2 mM L-glutamine, 25 mg/ml HEPES (BioWhittaker), and 10% heat-inactivated human AB serum (Sigma-Aldrich). Ag-specific T cell priming was initiated by Ag-pulsed mature autologous DC added to primary cultures at a ratio (T cell:DC) of 20:1. Cells were incubated in tissue culture flasks at 37°C in 5% CO₂ for 7 days, and the restimulation with the same Ag-pulsed DC was performed in a ratio (T cell:DC) of 2:1 to obtain the highest yield of tumor-reactive T cells. Activated IFN--producing tumor-reactive T cells were harvested 24 h after restimulation using the Secretion Assay Cell Enrichment and Detection Kit (MACS Reagens; Miltenyi Biotec) as described previously (16).

Flow cytometric analysis

Cells were washed with PBS and incubated with FITC-conjugated, PE-conjugated, or allophycocyanin-conjugated Abs for 15 min on ice. Cell surface phenotype was analyzed by flow cytometry using anti-CD4-FITC, anti-CD8-FITC, anti-CD3-APC (Immunotech), and anti-IFN--PE mAbs (Miltenyi Biotec). After washing with cold PBS, cells were fixed with 1% paraformaldehyde (Sigma-Aldrich) and analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter).

Tumor-specific reactivity of CTLs in vitro

Harvested IFN--positive CTLs were cultured in complete medium containing X-VIVO 15 with 50 mg/l gentamicin, 2 mM L-glutamine, 25 mg/ml HEPES (BioWhittaker), 10% heat-inactivated human AB-serum (Sigma-Aldrich), and 500 IU/ml IL-2 (Proleukin; Chiron) for 3–4 wk. PHA (5 µg/ml) (Sigma-Aldrich) was added on day 1. Cultures were usually started with 10⁵ melanoma-reactive IFN--positive T cells, and when possible the single-cell culture was initiated in parallel. Complete culture medium was replaced twice weekly, and feeder cells (irradiated PBMC from healthy volunteers) were added once weekly starting at the second week of expansion. CTL assay with Calcein-AM (Molecular Probes) was performed as published elsewhere (17). This assay produces results comparable with standard Cr⁵¹ release assay but is nonradioactive and requires fewer cells. Briefly, autologous and allogeneic melanoma cells were labeled with Calcein-AM and used as target cells at various E:T ratios. Allogeneic PBMC and the ARH-77 myeloma cell line served as negative controls. HLA class I blocking was performed by using 10 µg/ml anti-HLA class I Ab (clone W6/32; Immunotech). Results were expressed as the mean of four replicates.

Identification of TCR CDR3 sequences

Clonotypic assay (14, 15) was used for identification of individual melanoma-specific T cell clones based on the analysis of unique DNA sequences of TCR CDR3. It was performed on sorted populations of activated IFN-⁺ melanoma-reactive T cells that were further separated immunomagnetically to CD3⁺CD4⁺ and CD3⁺CD4^– fraction by CD4-positive selection (Miltenyi Biotec). At least 97% of the CD3⁺CD4^– fractions were CD8⁺ T cells (data not shown). A total of 1 x 10⁵ IFN-⁺ T cells (either CD4⁺ or CD8⁺) was used for mRNA extraction (Oligotex Direct mRNA Mini Kit; Qiagen). Anchored RT-PCR was performed using a modified version of the SMART method (SMART Race cDNA Amplification Kit; BD Clontech) as described previously (15). TCRC primer (5'-GCTTCTGATGGCTCAAACACAGCGACCTC-3') was used to obtain TCR PCR products from the 5' end to the start of the TCRC region. The PCR product was ligated into the pGemT-Easy vector (Promega) and used for Escherichia coli transformation. Clones were defined by the presence of at least two identical DNA sequences (clonotypes) of the TCR CDR3. For each PCR product, at least 50 colonies were selected, amplified by PCR with vector-specific primers, and used for direct sequencing to obtain TCR CDR3 sequences corresponding to sorted, activated melanoma-reactive T cells.

In vivo quantitative monitoring of individual immunodominant T cell clones

The most immunodominant T cell clones were selected based on the frequency of their unique DNA sequence of the TCR CDR3 region (>10% of the same clonotype among all bacterial colonies sequenced). Clone-specific primers and probes were designed for the TCR CDR3 with Primer Express version 1.5. Clonotypic quantitative PCR (qPCR) was performed using an ABI PRISM 7700 on PBMC lysates or biopsy DNA as described previously (15). qPCR was a 50-cycle 2-step reaction using Platinum Taq (Invitrogen Life Technologies), with the annealing/extension phase at 65°C. A standard curve was plotted and template copies were calculated. Plasmid template for the qPCR standards was the same as that used in the sequencing of the CDR3. Each primer/probe set was tested against the target plasmids for the individual’s other primer/probe sets, and redesigned if necessary to ensure no cross-reactivity. Each set was also tested with PBMC derived from five different healthy donors, and we found no amplification with any of the 12 primer/probe sets. The high specificity and sensitivity (1 in 100,000 cells) of clonotypic qPCR has been shown previously (17, 18). Samples were analyzed in triplicate.

Statistical analyses

Correlations were performed by Spearman’s rank test, the Wilcoxon matched pairs test, and the Mann-Whitney U test using Prism 3.0 software.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Patients with MM have circulating melanoma-reactive CD4⁺ and CD8⁺ T cells

DC loaded with irradiated autologous tumor cells were used for activation of T lymphocytes in 10 patients with MM. Patient characteristics are shown in Table I. After two rounds of stimulation, low frequencies of IFN--producing CD4⁺ (0.9–3.2%, median 1.3%) and CD8⁺ (0.6–2.4%, median 1.0%) T cells were detected (Fig. 1A). Melanoma-reactive IFN--producing T cells were immunomagnetically separated. The percentage of activated CD3⁺IFN-⁺ T cells before magnetic separation was 1.8–4.4% (median 2.4%) and after the procedure reached 59.7–88.4% (median 76.2%) of all CD3⁺ T cells (Fig. 1B). Sorted CD3⁺IFN-⁺ T cells (0.52–2.39 x 10⁶ cells) were further expanded for 3–4 wk until at least 10⁸ viable T cells were reached. After the expansion, 96.7–99.5% (median 99.0%) of cells were CD3⁺ T cells with a prevalence of 72.8–86.4% (median 81.1%) CD3⁺CD8⁺ cells, remaining T cells were CD4⁺.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Sorting melanoma-reactive IFN-+ T cells. Melanoma-reactive IFN-+ T cells were sorted immunomagnetically. Representative data from patient no. 3 are shown. A, The presorted cell population (gated on CD3+ cells) with the presence of both CD3+CD4+ and CD3+CD8+ T cells. B, Enrichment of CD3+IFN-+ T cells.

A specificity of expanded melanoma-reactive CTLs was tested against the original autologous melanoma cells in five patients (nos. 1, 3, 4, 7, and 8) with sufficient numbers of remaining tumor cells. Unselected T cells showed specific cytotoxicity to autologous tumor cells in all five individuals, reaching 21–39% (median 33%) at 20:1 E:T ratio and 14–25% (median 21%) at 1:1 E:T ratio. Representative data from two independent experiments (patient nos. 1 and 3) are shown at Fig. 2A. In patient no. 3, we were also able to grow and expand individual T cell clones in 15 of 96 wells that were started as a single-cell suspension, and in 6 of 15 expanded clones we were able to confirm the presence of the same immunodominant clone (described in the next paragraph) as in the cell culture initiated with 10⁵ sorted cells. This particular clone demonstrated the same cytotoxic potential to autologous melanoma cells as the melanoma-reactive IFN-⁺CD3⁺CD8⁺ T cells expanded from 10⁵ cells in a single well. This particular clone from patient no. 3 recognized specifically autologous tumor cells but not the allogeneic tumor cells from patient no. 1 (Fig. 2A).

View larger version (22K): [in this window] [in a new window]   FIGURE 2. CTL assay. Expanded melanoma-reactive T cells demonstrate the specific cytotoxic effect to autologous melanoma tumor cells (Melanoma). Representative data from two different patients (nos. 1 and 3) are shown in A. In patient no. 3, single immunodominant CD8+ T cell clones (Clone 3A) demonstrated specific cytotoxicity to autologous melanoma cells but not to allogeneic melanoma cells from patient no. 1. Negligible response against autologous DCs (DC only) and no response to irrelevant Ags such as myeloma cell line (ARH77) and allogeneic PBMC (AlloPBMC) were noted. Abrogation of CTL activity by anti-human HLA class I Ab is shown in B.

Identification of immunodominant T cell clones in vitro

We next analyzed the clonal composition of the sorted IFN-⁺ melanoma-reactive T cells and defined them by the unique sequences of their TCR CDR3 region in patient nos. 1–8. TCR loci of the sorted cells were amplified by template switch-anchored RT-PCR, as described in Materials and Methods, to ensure that all melanoma-reactive TCR sequences were amplified without bias to particular TCR V families. Thus, the melanoma-reactive clonotypes were represented in the anchored RT-PCR product with a relative frequency reflecting that found in the original sorted cell population. Sequencing of 50 bacterial colonies containing TCRB CDR3 from each sort was performed to identify the number and frequency of individual T cell clones in each sorted population. We have previously demonstrated the specificity and sensitivity of this approach to analyze the clonality of HIV-, GHVD-, and GVL-specific T cells (15, 17). We have also demonstrated that only highly immunodominant T cell clones (forming at least 10% of all in vitro-identified clonotypes) are also detectable in vivo (15, 17). Table II shows the number of successfully sequenced bacterial colonies and the number of different melanoma-reactive T cell clones identified for each patient. The frequency and amino acid sequences of CDR3 regions that occurred in >10% of bacterial colonies in vitro are also shown. These are likely to represent the most "dominant" T cell clones and their presence in peripheral blood of melanoma patients were further studied in vivo. We observed considerable heterogeneity in the clonality of melanoma-reactive T cells among the patients. Notably, melanoma-reactive CD8⁺ T cells from patients nos. 4–8 who died due to rapid cancer progression were highly polyclonal, comprising between 28 and 35 (median 31) individual clonotypes, whereas melanoma-reactive CD8⁺ T cells from patients with at least some measurable clinical response were far more oligoclonal, comprising between 5 and 22 (median 9) individual clonotypes. Indeed, within these oligoclonal repertoires, only one or two CD8⁺ clones stood out as being clearly dominant in frequency for each patient with at least some treatment response. In one case (patient no. 3), we were able to identify a single immunodominant CD8⁺ T cell clone (Clone 3A, Table II) that represented 6 of 15 in vitro-expanded clones initiated from a single-cell culture. This particular clone was the same as the most immunodominant CD8⁺ T cell clone identified from the culture expanded in vitro from 10⁵ melanoma-reactive IFN-⁺ T cells in a single well, and we were able to monitor this clone in vivo for 24 mo as described in the next section.

Long-term persistence of immunodominant CD8⁺ T cell clones correlates with better clinical outcome

Having identified individual immunodominant CD8⁺ T cell clones in vitro, we next quantified the dominant clones at multiple time points after the patient was diagnosed with MM in peripheral blood. We have also examined the presence of these clones in tumor biopsies performed at the time of diagnosis and, in some cases, at the time of further metastatic spread of melanoma. We designed clone-specific PCR primers and probes specific for the TCR CDR3 region of each dominant melanoma-reactive clone, the sequence of which had been detected in at least 10% of bacterial colonies from the anchored RT-PCR product (Table II, bottom)—a total of 18 individual clones. We performed clonotypic quantitative real-time PCR on each sample from each recipient. Because a single T cell contains one productively rearranged TCR locus of a particular sequence, the copy number detected in each PCR was directly equivalent to the absolute number of melanoma-reactive clone cells present in the sample. Fig. 3 shows the levels of each immunodominant clone in the peripheral blood of seven patients characterized and numbered as shown in Table I. Notably, subjects 1–3 who are long-term survivors of MM have demonstrated a long-term persistence of the most dominant CD8⁺ T cell clones in vivo in their peripheral blood. In contrast, subjects 4–7 had only temporary or no (subject 8) detectable immunodominant CD8⁺ T cell clones in their peripheral blood. Quantification of absolute numbers of melanoma-reactive CD8⁺ T cell clones showed in general some variability reaching from the threshold levels of qPCR 0.000001–0.001 of CD8⁺ T cells in peripheral blood. It revealed a long-term persistence with minimal or mild expansion in long-term survivors but disappeared shortly after diagnosis of MM in nonsurvivors.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Long-term in vivo monitoring of immunodominant T cell clones. Individual immunodominant melanoma-reactive T cell clones identified in vitro at the time of metastatic dissemination (time 0) were quantitatively monitored in vivo in the peripheral blood of patient nos. 1–8. The number of clonotypic T cells is expressed as a percentage of all CD3+CD8+ T cells at the time of examination. No clonotypic T cells were detected in the peripheral blood of patient no. 8. The detection threshold for the clonotypic assay was 10–4%.

Finally, we examined the presence of immunodominant CD8⁺ clones in original tumor-infiltrated lymph node biopsies by the clone-specific PCR and demonstrated the presence of these clones in all survivors (subjects 1–3) but only in three of six nonsurvivors studied (in subjects 4, 6, and 7). Subjects 6 and 8 were rebiopsied for a new metastatic involvement (12 and 6 mo, respectively, after their initial lymph node biopsy), but none of their immunodominant CD8⁺ T cell clones were detected. Subjects 9 and 10 were not studied by the clone-specific qPCR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
By using DCs loaded with irradiated autologous tumor cells, we were able to identify circulating melanoma-reactive T cells in patients with MM. After stimulation, Ag-reactive T cells activate and produce IFN-. Both populations of CD4⁺ and CD8⁺ melanoma-reactive T cells were present. Such cells can be enriched immunomagnetically and further expanded in vitro (16, 19). To generate melanoma-specific CTLs for further study, we expand IFN-⁺ melanoma-reactive T cells that contain most of the immunodominant T cell clones. Despite the advanced stage of tumor spread, it was possible to expand functional melanoma-reactive T cells. For the most part, CD8⁺ CTLs were expanded and demonstrated a tumor-specific cytotoxic effect. Cytotoxicity against autologous melanoma cells was blocked by an anti-HLA class I Ab, indicating that the effect was most likely mediated through CD8⁺ HLA class I interaction. This finding confirms an important role for CD8⁺ CTLs in specific cytotoxicity that is in agreement with studies using a defined melanoma-specific peptide HLA class I-restricted Ag (9, 10).

In this study we identified in vitro, and quantified in vivo, individual immunodominant melanoma-reactive CD8⁺ T cell clones by developing an approach that combines in vitro cell culture, immunomagnetic sorting of activated IFN-⁺ T cells, TCR locus-anchored RT-PCR, and clonotypic qPCR. In published studies, the identification of clonal T cell responses has been accomplished by flow cytometry using mAbs directed against TCR V regions, by CDR3 spectratyping, or by microplate hybridization assay. These studies were able to demonstrate that the TCR repertoire is highly variable; for example, monoclonal, oligoclonal, or polyclonal CTL responses can be induced depending on the tumor Ag used (20, 21, 22). Interestingly, either monoclonal or polyclonal responses can be elicited by the same peptide derived from a MAGE-3.A1 tumor-specific Ag in different individuals (21, 22). Yet, using these techniques does not enable detailed analysis of individual T cell clones because, in the case of flow cytometry, the series of Abs are not complete enough to analyze all TCR V region families; in the case of spectratyping and microplate hybridization assay, TCR V region families, but not individual T cell clones within these families, can be identified (23, 24, 25). Our approach obviated skewing of the clonotype population by prolonged stimulation and propagation. Furthermore, the anchored RT-PCR step used to amplify TCR CDR3 regions of all T cell clones present in the sorted population avoided the incomplete coverage of, and bias to, particular TCR V families conferred by using TCR V-specific PCR primers. This approach required no prior knowledge of either the specific target Ag or the MHC-restricting element, and when combined with qPCR, it allowed for the sensitive and specific quantification of melanoma-reactive T cell clones in the patient at any point and in any blood or tissue sample. In agreement with spectratyping and microplate hybridization studies (23, 24, 25), we were able to detect oligoclonal or monoclonal expansions of melanoma-reactive CD8⁺ T cell clones. The sensitivity of clone-specific qPCR reached the threshold of 1 x 10^–6, thus allowing us to monitor low-frequency clones. It has been shown that the frequency of naive T cells recognizing melanoma-specific peptide Ags such as MAGE-A3_168–176, gp100, NA17, LAGE-1, or MAGE-A10 exists in vivo with the frequencies of 1 x 10^–7 to 1 x 10^–6 (10). The Melan-A/MART-1_28–36 is a remarkable exception with a very high naive T cell frequency of 5 x 10^–4 of CD8⁺ T cells (26). Recently, Speiser et al. (27) demonstrated that human CD8⁺ T cell clones specific to the Melan-A/MART-1 persisted for >1 year in a patient with melanoma, reaching up to 2.5% of the circulating CD8⁺ T cells. At the time of the melanoma-specific T cell clonal expansion, the disease stabilized but subsequently progressed with loss of Melan-A-specific T cell clones (27). In agreement with that study, we were able to demonstrate that the immunodominant CD8⁺ T cell clones in five patients with progressive disease were only temporarily present (or were undetectable in one case) in the circulation, and all of these patients subsequently died of their disease. Melanoma-reactive clones were also undetectable in tumor biopsies taken at the time of tumor progression shortly before death. In contrast, all three patients with objective responses had melanoma-reactive CD8⁺ T cells present in the circulation for 18–60 mo with frequencies of 10^–6 to 10^–3. These low-frequency CD8⁺ T cell clones can probably be expanded as a response to tumor growth or as a response to melanoma treatment. Such observation further confirms the importance of individual tumor-reactive CD8⁺ T cell clones in cancer survival. Thus, prolonged persistence of melanoma-reactive CD8⁺ T cell clones is associated with better survival.

	Acknowledgment

 
We thank Dr. Ellen S. Vitetta for helpful discussions and critical review of the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Grant Agency of the Czech Republic (no. 301/04/1387).

² Address correspondence and reprint requests to Dr. Jaroslav Michalek, Cell Immunotherapy Center, Masaryk University, Kamenice 5, Brno 61300, Czech Republic. E-mail address: jmichalek{at}fnbrno.cz

³ Abbreviations used in this paper: DC, dendritic cell; MM, metastatic melanoma; GVHD, graft-vs-host disease; GVL, graft-vs-leukemia; qPCR, quantitative PCR.

Received for publication November 29, 2006. Accepted for publication March 19, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Semino, C., L. Martini, P. Queirolo, G. Cangemi, R. Costa, A. Alloisio, G. Ferlazzo, M. R. Sertoli, U. M. Reali, G. B. Ratto, G. Melioli. 1999. Adoptive immunotherapy of advanced solid tumors: an eight year clinical experience. Anticancer Res. 19: 5645-5650. [Medline]
2. Falkenburg, J. H. F., A. R. Wafelman, P. Joosten, W. M. Smit, C. A. van Bergen, R. Bongaerts, E. Lurvink, M. van der Hoorn, P. Kluck, J. E. Landegent, et al 1999. Complete remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-reactive cytotoxic T lymphocytes. Blood 94: 1201-1208. [Abstract/Free Full Text]
3. Dudley, M. E., J. Wunderlich, M. I. Nishimura, D. Yu, J. C. Yang, S. L. Topalian, D. J. Schwartzentruber, P. Hwu, F. M. Marincola, R. Sherry, et al 2001. Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treatment of patients with metastatic melanoma. J. Immunother. 24: 363-373. [Medline]
4. Dudley, M. E., J. R. Wunderlich, P. F. Robbins, J. C. Yang, P. Hwu, D. J. Schwartzentruber, S. L. Topalian, R. Sherry, N. P. Restifo, A. M. Hubicki, et al 2002. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298: 850-854. [Abstract/Free Full Text]
5. Kurokawa, T., M. Oelke, A. Mackensen. 2001. Induction and clonal expansion of tumor-specific cytotoxic T lymphocytes from renal cell carcinoma patients after stimulation with autologous dendritic cells loaded with tumor cells. Int. J. Cancer 91: 749-756. [Medline]
6. Mulders, P., C. L. Tso, B. Gitlitz, R. Kaboo, A. Hinkel, S. Frand, S. Kiertscher, M. D. Roth, J. deKernion, R. Figlin, A. Belldegrun. 1999. Presentation of renal tumor antigens by human dendritic cells activates tumor-infiltrating lymphocytes against autologous tumor: implications for live kidney cancer vaccines. Clin. Cancer Res. 5: 445-454. [Abstract/Free Full Text]
7. Banchereau, J., B. Schuler-Thurner, A. K. Palucka, G. Schuler. 2001. Dendritic cells as vectors for therapy. Cell 106: 271-274. [Medline]
8. Banchereau, J., A. K. Palucka, M. Dhodapkar, S. Burkeholder, N. Taquet, A. Rolland, S. Taquet, S. Coquery, K. M. Wittkowski, N. Bhardwaj, et al 2001. Immune and clinical responses in patients with metastatic melanoma to CD34⁺ progenitor-derived dendritic cell vaccine. Cancer Res. 61: 6451-6458. [Abstract/Free Full Text]
9. Coulie, P. G., V. Karanikas, C. Lurquin, D. Colau, T. Connerotte, T. Hanagiri, A. Van Pel, S. Lucas, D. Godelaine, C. Lonchay, et al 2002. Cytotoxic T cell responses of cancer patients vaccinated with a MAGE antigen. Immunol. Rev. 188: 33-42. [Medline]
10. Coulie, P. G., P. van der Brugen. 2003. T-cell responses of vaccinated cancer patients. Curr. Opin. Immunol. 15: 131-137. [Medline]
11. Weekes, M. P., M. R. Wills, K. Mynard, A. J. Carmichael, J. G. Sissons. 1999. The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo. J. Virol. 73: 2099-2108. [Abstract/Free Full Text]
12. Gea-Banacloche, J. C., S. A. Migueles, L. Martino, W. L. Shupert, A. C. McNeil, M. S. Sabbaghian, L. Ehler, C. Prussin, R. Stevens, L. Lambert, et al 2000. Maintenance of large numbers of virus-specific CD8⁺ T cells in HIV-infected progressors and long-term non-progressors. J. Immunol. 165: 1082-1092. [Abstract/Free Full Text]
13. Michalek, J., R. H. Collins, B. J. Hill, E. S. Vitetta, D. C. Douek. 2001. The emergence of specific T cell clones during the generation of GVHD. Blood 98: 664a
14. Michalek, J., R. H. Collins, B. J. Hill, J. M. Brenchley, D. C. Douek. 2003. Pre-transplant identification of graft-versus-host specific T-cell clone and quantitative monitoring after allogeneic stem cell transplantation. Lancet 361: 1183-1185. [Medline]
15. Michalek, J., R. H. Collins, H. P. Durrani, P. Vaclavkova, L. E. Ruff, D. C. Douek, E. S. Vitetta. 2003. Definitive separation of graft-versus-leukemia and graft-versus-host specific CD4⁺ T cell clones by virtue of their T cell receptor locus sequences. Proc. Natl. Acad. Sci. USA 100: 1180-1184. [Abstract/Free Full Text]
16. Ocadlikova, D., L. Zahradova, L. Kovarova. 2006. Isolation and expansion of allogeneic myeloma-specific interferon- producing T cells for adoptive immunotherapy. Med. Oncol. 23: 377-384. [Medline]
17. Douek, D. C., M. R. Betts, J. M. Brenchley, B. J. Hill, D. R. Ambrozak, K. L. Ngai, N. J. Karandikar, J. P. Casazza, R. A. Koup. 2002. A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape. J. Immunol. 168: 3099-3104. [Abstract/Free Full Text]
18. Douek, D. C., J. M. Brenchley, M. R. Betts, D. R. Ambrozak, B. J. Hill, Y. Okamoto, J. P. Casazza, J. Kuruppu, K. Kunstman, S. Wolinsky, et al 2002. HIV preferentially infects HIV-specific CD4⁺ T cells. Nature 417: 95-98. [Medline]
19. Becker, C., H. Pohla, B. Frankenberger, T. Schuler, M. Assenmacher, D. J. Schendel, T. Blankenstein. 2001. Adoptive tumor therapy with T lymphocytes enriched through an IFN- capture assay. Nat. Med. 7: 1159-1162. [Medline]
20. Paczesny, S., J. Banchereau, K. M. Wittkowski, G. Saracino, J. Fay, A. K. Palucka. 2004. Expansion of melanoma-specific cytotoxic CD8⁺ T cell precursors in patients with metastatic melanoma vaccinated with CD34⁺ progenitor-derived dendritic cells. J. Exp. Med. 199: 1503-1511. [Abstract/Free Full Text]
21. Coulie, P. G., V. Karanikas, D. Colau, C. Lurquin, C. Landry, M. Marchand, T. Dorval, V. Brichard, T. Boon. 2001. A monoclonal cytotoxic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by gene MAGE-3. Proc. Natl. Acad. Sci. USA 98: 10290-10295. [Abstract/Free Full Text]
22. Godelaine, D., J. Carrasco, S. Lucas, V. Karanikas, B. Schuler-Thurner, P. G. Coulie, G. Schuler, T. Boon, A. Van Pel. 2003. Polyclonal CTL responses observed in melanoma patients vaccinated with dendritic cells pulsed with a MAGE-3.A1 peptide. J. Immunol. 171: 4893-4897. [Abstract/Free Full Text]
23. Verfuerth, S., K. Peggs, P. Vyas, L. Barnett, R. J. O’Reilly, S. Mackinnon. 2000. Longitudinal monitoring of immune reconstitution by CDR3 size spectratyping after T-cell-depleted allogeneic bone marrow transplant and the effect of donor lymphocyte infusions on T-cell repertoire. Blood 95: 3990-3995. [Abstract/Free Full Text]
24. Maslanka, K., T. Piatek, J. Gorski, M. Yassai, J. Gorski. 1995. Molecular analysis of T cell repertoires spectratypes generated by multiplex polymerase chain reaction and evaluated by radioactivity or fluorescence. Hum. Immunol. 44: 28-34. [Medline]
25. Matsutani, T., T. Yoshioka, Y. Tsuruta, S. Iwagami, T. Toyosaki-Maeda, T. Horiuchi, A. B. Miura, A. Watanabe, G. Takada, R. Suzuki, M. Hirokawa. 2000. Restricted usage of T-cell receptor -chain variable region (TCRAV) and T-cell receptor -chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation. Br. J. Haematol. 109: 759-769. [Medline]
26. Zippelius, A., M. J. Pittet, P. Batard, N. Rufer, M. de Smedt, P. Guillaume, K. Ellefsen, D. Valmori, D. Lienard, J. Plum, et al 2002. Thymic selection generates a large T cell pool recognizing a self-peptide in humans. J. Exp. Med. 195: 485-495. [Abstract/Free Full Text]
27. Speiser, D. E., P. Baumgaertner, C. Barbey, V. Rubio-Godoy, A. Moulin, P. Corthesy, E. Devevre, P. Y. Dietrich, D. Rimoldi, D. Lienard, et al 2006. A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination. J. Immunol. 177: 1338-1348. [Abstract/Free Full Text]

This article has been cited by other articles:

				V. Foltankova, E. Matejkova, M. Bartos, M. Dendis, D. Novotna, J. Mayer, and J. Michalek Molecular Identification of Individual T Cell Clones Specific for Graft- Versus-Leukemia Reaction Blood (ASH Annual Meeting Abstracts), November 16, 2008; 112(11): 1249 - 1249. [Abstract]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Michalek, J. Articles by Hajek, R. Search for Related Content PubMed PubMed Citation Articles by Michalek, J. Articles by Hajek, R.